信息编号11340201至11340250间共50条。
☉ 11340202:Complement slows heart cells
The complement activation product C5a prevents heart cells from contracting, according to Niederbichler and colleagues on page 53. This finding may help explain the cardiac dysfunction commonly seen in patients with septic shock—a disease caused by bacterial infections that is characterized in part by the robust activation of the complement system. A central component of septic shock is decreased heart function, which deprives tissues of oxygen and nutrients and can progress to fatal multiorgan failure. Past studies have shown that a "myocardial depressant factor" is released into the serum during sepsis, with most studies attributing the depressant activity to proinflammatory cytokines released by immune cells responding to the infection. Robust activation of the complement cascade is another hallmark of bacterial sepsis. Complement is a series of plasma proteins that helps destroy infecting microbes and attracts immune cells to sites of infection. C5a activates immune cells, such as neutrophils, which can then attack and kill the invading bacteria. But too much C5a can be deleterious, as blocking the protein or its receptor increases survival in rodent models of septic shock. This group previously showed that excessive C5a somehow prevents the activation of MAP kinase signaling in neutrophils, which jams up downstream signals and robs the cells of their ability to destroy bacteria. The receptor for C5a (C5aR) is also expressed on airway cells, including bronchial epithelial cells and smooth muscle cells, but a link between complement and sepsis-induced cardiac dysfunction had not been directly investigated. Niederbichler and colleagues now show that cardiomyocytes, which help control heartbeat, also express C5aR—possibly to increase blood flow in response to infection or stress. But as with neutrophils, too much C5a was bad for cardiomyocytes. Treatment of cardiomyocytes with high concentrations of C5a impaired their ability to contract. Blocking the interaction between C5a and its receptor in rats relieved the sepsis-induced drop in blood pressure. The authors are now investigating the molecular pathways that link C5aR signaling to the contractile machinery of the cell; they suspect that excessive C5a might induce a signaling paralysis in cardiomyocytes similar to that seen in neutrophils....查看详细 (2359字节)

☉ 11340203:Heart cells keep the rhythm
G-CSF treatment restores the expression of connexin 43 on cardiomyocytes near areas of heart attack–induced damage (arrows). Cytokine therapy post–heart attack restores the expression of gap junction proteins and thus restores electrical connections between heart cells, according to a new study by Kuhlmann and colleagues (page 87). These heart cells, with better electrical connections, are able to pump with a more normal rhythm. Restoring the function of damaged heart tissue is a new approach to the treatment of myocardial infarctions, or heart attacks. The scar tissue from heart attacks creates a roadblock for electrical conduction and can cause the heart to beat erratically. Indeed, erratic heartbeats—or arrhythmias—are the primary cause of death in patients who have had heart attacks. Heart attack treatments range from blood-thinning and cholesterol-lowering drugs, which reduce the likelihood of developing blocked arteries, to implanted defibrillators that shock erratically beating hearts into line. But the idea of repairing damaged heart tissue is new. One promising repair approach involves the intravenous injection of two cytokines—G-CSF (granulocyte colony-stimulating factor) and SCF (stem cell factor)—which mobilize stem cells from the bone marrow (BM). Early studies suggested that these stem cells travel to the damaged heart and differentiate into functional heart cells called cardiomyocytes. Other studies, however, found no evidence that stem cells can become cardiomyocytes. Kuhlmann and colleagues now confirm that G-CSF/SCF treatment in mice with heart damage causes BM-derived cells to enter the heart, but they show that virtually none of these cells become bona fide heart cells. The benefit of this treatment—which improved the heart's pump function and protected against induced arrhythmias—instead correlated with enhanced expression of the gap junction protein connexin 43 on the surface of heart cells adjacent to the injured tissue. The expression of connexin 43, which provides the electrical connection between neighboring cells, is decreased after heart injury, thus reducing the conductive capacity between cells. The connexin-inducing effect of G-CSF might be direct, as cardiomyocyte expression of the G-CSF receptor was increased in response to injury. But this possibility is difficult to test in vitro, as mouse cardiomyocytes grow poorly in culture. The authors are also investigating G-CSF–independent signaling pathways that induce the expression of connexin 43 in other cell types. hvanepps@rockefeller.edu...查看详细 (2607字节)
☉ 11340205:Complaints against doctors in child protection work have increased fivefold
London The threat of complaints and ruined reputations is driving paediatricians away from fulfilling their role in child protection, leaving vulnerable children at risk of abuse, says the Royal College of Paediatrics and Child Health. A survey of college members, to which almost 80% of the country's 6072 paediatricians replied, has shown that the number of complaints made against doctors involved in child protection has risen fivefold in the last seven years. And while complaints have risen, paediatricians have turned their backs on child protection work. Currently 30% of posts where paediatricians take a lead in child protection are unfilled. The catalyst for the increase in complaints has been the publicity surrounding two high profile cases, said Professor Neil McIntosh, vice president of science and research at the college. The first was that of paediatrician Professor David Southall, who was suspended in 1999 while North Staffordshire Hospital NHS Trust investigated complaints about his research and child protection work ( BMJ 2000;320: 9) but was reinstated two years later after no evidence was found against him ( BMJ 2001;323: 885). The second was that of an accused mother, Sally Clarke, who was convicted of smothering her two infants in 1999 but was freed by the Court of Appeal last year ( BMJ 2003;326: 304). Professor McIntosh said: "The incredible response rate to the survey reflects how agonised paediatricians feel about this issue. The fact is that paediatricians are ducking out of child protection because of the flak they get from the media." Between 1995 and 2003 the number of complaints against paediatricians rose from 20 a year to 100 a year, the survey shows. Seventy eight of the 732 complaints detailed are still ongoing, but of the remainder only 3% have been upheld. However, the effect of the publicity has eroded the public's confidence in the profession and led to a reluctance among paediatricians to take a lead role in cases of suspected child abuse, said Dr McIntosh. "Complaints in such cases are hot news, but the doctor involved cannot comment both because of issues of patient confidentiality and because the case is sub judice," said Dr McIntosh. "This not does not prevent comment by family or the media. By the time the complaint has been shown worthless the reputation of the paediatrician has been damaged; the issue is no longer newsworthy. Retractions and apologies are almost unknown." The college plans to examine a sample of the complaints more closely to see what they were for and how they were dealt with. Dr McIntosh hopes this will help to identify the problem areas of child protection work so that the college can develop supportive measures to encourage paediatricians to take on responsibilities for children at risk. However, this is likely to take at least nine months, he said. Penny Mellor, a campaigner who supported parents in recent high-profile cases including that of Angela Cannings, said there was no orchestrated campaign against paediatricians. "There are a really small handful of five paediatricians that we have concerns about," she said on BBC Radio's Today programme. "As to the rest of the world of paediatrics, we do not have any concerns with their work." A Department of Health spokesperson said they recognised child protection work could be extremely stressful for staff. "Paediatricians play an essential role in child protection. A consultant has a clear responsibility to make a referral to social services in any case where he or she has 'reasonable cause to suspect that a child is suffering or likely to suffer significant harm'."...查看详细 (3709字节)

☉ 11340206:噪声性听觉损伤防治的研究进展
[摘要] 噪声是一种普遍存在于各种职业环境下的有害因素，强噪声可导致机体出现噪声性听觉损伤甚至噪声性耳聋，预防和治疗噪声性听觉损伤的研究一直备受国内外学者的关注。根据噪声性听觉损伤(NIHL)的特点和机制，国内外学者对噪声防治的研究主要包括个体护耳器防护和应用相关药物进行防治等。近年来，关于噪声习服和噪声易感性现象的研究为噪声性听觉损伤的防治提供了新的思路。本文综述了国内外在噪声防治领域的新进展，重点介绍了噪声性听觉损伤的易感者筛选以及易感性相关基因的研究方法...查看详细 (10762字节)
☉ 11340207:噪声性听觉损伤的特点与机制研究进展
[摘要] 噪声是一种常见的危害人类身心健康的环境因素，在噪声作业环境中更是普遍存在，军事作业人员的听觉系统受噪声影响尤为多见。关于噪声性听觉损伤的研究一直是很多学科的工作重点。本文主要介绍噪声性听觉损伤的特点和机制的研究进展。噪声性听觉损伤主要表现为听觉敏感度下降、听阈提高，其形式主要有暂时性听阈偏移和永久性听阈偏移2种;噪声对听觉器官的损伤主要表现为毛细胞、听神经和听觉中枢的病理生理改变。关于噪声性听觉损伤的机制目前主要有3种学说:机械损伤学说、代谢学说和血管学说...查看详细 (10985字节)

☉ 11340208:消化性溃疡的药物治疗及复发
[关键词] 消化性溃疡;药物治疗;复发 随着消化性溃疡(peptic ulcer，PU)病因学研究的深入和针对病因开展的治疗药物的不断开发，其治疗效果日益提高，但消化性溃疡愈合后的复发也已经引起广泛的关注。本文对其药物治疗及愈合后复发的研究现状综述如下。 1 药物治疗 1.1 H 2 受体阻止剂 西咪替丁和雷尼替丁是目前应用最广的第1代H 2 受体阻断剂，疗效良好。两药对溃疡病疼痛的缓解率、4～8周的溃疡愈合率、首次疗程及维持治疗的失败率、停药后的复发率大致相同或接近...查看详细 (3961字节)
☉ 11340209:细胞因子抗辐射作用研究进展
[关键词] 细胞因子;辐射;研究进展 由于世界核安全形势和各种放射治疗的需要，辐射损伤的防治仍是当前研究的热点之一。常用的辐射防护药物均有一定的毒副作用，严重限制了它们的应用。国内外多年的研究结果说明细胞因子具有较独特的抗辐射作用，合理的细胞因子联用会提高疗效或抵消细胞因子单用时存在的局限性和副作用。本文对近年来国外细胞因子抗辐射作用的研究状况作一综述，旨在为寻求更为高效低毒的辐射防护药物奠定基础...查看详细 (17181字节)
☉ 11340210:前髓细胞白血病核小体研究概况
[摘要] 前髓细胞白血病核小体(PML-NBs)是真核生物细胞的细胞核亚单位之一。本文讨论了这一细胞核组织的成分、结构及其参与的许多重要的细胞生命活动，包括细胞周期、细胞衰老凋亡、病毒感染、细胞应激等，它与细胞基因组中的高转录活性区域相关。PML-NBs由于拥有众多功能各异的蛋白，其功能形成了一个复杂的网络，许多未知的领域还有待深入研究。 [关键词] 前髓细胞白血病核小体;转录;细胞周期;凋亡 哺乳动物细胞核是一个复杂的细胞器...查看详细 (14923字节)

☉ 11340211:支气管动脉栓塞术治疗肺结核大咯血12例疗效分析
[关键词] 支气管;动脉栓塞术;肺结核;咯血 近年来经皮穿刺行支气管动脉栓塞术治疗大咯血在国内外已有较多的应用，我院自2002年以来，开展用明胶海绵颗粒栓塞支气管动脉，成功治疗12例肺结核伴大咯血患者。现报告如下。 1 资料与方法 1.1 一般资料 本组12例，男8例，女4例，年龄28～55岁，基础疾病均为肺结核病，肺结核病程最短为4年，最长为22年，均未经过正规的抗痨治疗...查看详细 (3206字节)
☉ 11340212:直肠癌术中骶前静脉丛大出血防治体会
[摘要] 目的: 总结直肠癌根治术骶前静脉丛大出血预防和术中处理方法。 方法: 回顾性总结分析16例直肠癌根治术发生骶前静脉丛大出血情况，采用明胶海绵、图钉按压或骨蜡嵌入、纱布绷带填塞等方法压迫止血。 结果: 经用上述方法止血后均未再出血，效果满意。 结论 :癌肿浸润、术式估计不足及手术操作不当是造成出血的主要原因。为避免大出血，应在直视下操作，沿骶前筋膜前进行，动作细心轻柔，解剖层次清楚。如果出血时止血困难...查看详细 (4901字节)
☉ 11340213:蔗糖铁联合基因重组人红细胞生成素治疗肾性贫血临床观察
[摘要] 目的: 比较静脉用铁剂蔗糖铁(维乐福)和口服铁剂右旋糖酐铁分别与基因重组人红细胞生成素(EPO)联合应用治疗伴有缺铁的维持性血液透析肾性贫血患者的有效性和安全性。 方法: 随机选取40例血液透析患者分为静脉组和口服组，静脉组20例，第1周将蔗糖铁20mg溶于0.9%生理盐水200ml中透析，以30～60滴/min缓慢滴入，每周2次，第2周以后每次用蔗糖铁100mg，每周2次，方法同上，直至完成补铁总量...查看详细 (4422字节)
☉ 11340214:针刺加超短波治疗急性胃痉挛66例
[关键词] 急性胃痉挛;针刺;超短波 急性胃痉挛属中医“胃痛”范畴，以上腹胃脘部急性绞痛、拒按为主，可伴有恶心、脘闷、呕吐等症。笔者自2002年以来，应用针刺加超短波治疗急性胃痉挛66例，取得满意疗效。现报告如下。 1 临床资料 本组病例均为门诊患者，共66例，其中男41例，女25例;年龄最小22岁，最大60岁;病程最短1h，最长24h。临床均排除阑尾炎、胃肠穿孔等器质性疾病...查看详细 (1986字节)

☉ 11340215:纤维支气管镜对上呼吸道癌变临床诊断观察
[摘要] 目的: 探讨纤维支气管镜对上呼吸道癌变的诊断效果。 方法: 通过对115例患者进行纤维支气管镜复查，并与X线片诊断结果进行比较。 结果: 对115例患者X线片诊断后再行纤维支气管镜复查，发现10例与X线诊断有误差，误诊率为8.70%。 结论: 常规X检查不易发现部分上呼吸道病变，易漏诊、误诊，在诊断不明的情况下，应常规行纤维支气管镜检查。 [关键词] 纤维支气管镜;上呼吸道;诊断 2002年5月至2006年6月...查看详细 (3016字节)
☉ 11340216:纤维支气管镜在治疗创伤性肺不张患者中的应用
[关键词] 纤维支气管镜;胸创伤;开胸手术;肺不张 肺不张是严重胸外伤及开胸手术患者常见的并发症，对患者心肺功能影响较大，尤其是年老体弱患者，直接关系到患者能否顺利恢复，严重者将发生呼吸衰竭而危及生命。对于这类患者，如果临床上给予雾化吸入、协助翻身、拍背、鼻导管吸痰、刺激咳嗽排痰等方法仍不能顺利排痰的，宜采用纤维支气管镜吸痰、肺灌洗，清除支气管内积血、血凝块，刺激呛咳，促进肺功能恢复。我院2004年6月至2006年6月应用纤维支气管镜治疗严重胸外伤及开胸术后并发肺不张的患者28例...查看详细 (5356字节)
☉ 11340217:氯胺酮复合麻醉在老年人大面积烧伤切削痂术中的应用
[关键词] 氯胺酮;异丙酚;复合麻醉;老年人;烧伤;切削痂术 我院于2000年6月至2005年12月收治大面积火焰烧伤老年患者32例，行切削痂植皮术时，采用氯胺酮复合异丙酚、咪唑安定麻醉，取得良好效果。现报道如下。 1 资料与方法 1.1 一般资料 患者共32例，男28例，女4例;年龄60～78岁。根据美国麻醉医师协会病情估计分级(ASA)，Ⅰ～Ⅱ度烧伤面积30%～87%...查看详细 (3080字节)
☉ 11340218:腮腺多形性腺瘤部分腮腺切除术式的临床观察
[摘要] 目的: 探讨腮腺多形性腺瘤部分腮腺切除的可行性手术方式。 方法: 回顾性分析1998年1月至2002年12月我院收治的60例腮腺多形性腺瘤患者行腮腺部分切除的手术方式。 结果: 58例患者中，其肿瘤最大直径小于4cm者行肿瘤及周围部分腮腺切除术;对2例最大直径超过4cm的肿瘤行腮腺全切除术。术后均未发生永久性面神经麻痹。3例术后出现术侧暂时性面神经麻痹，3例出现腮腺瘘，10例出现Frey's综合征...查看详细 (4965字节)

☉ 11340219:力尔凡联合化疗治疗中晚期恶性肿瘤疗效观察
[摘要] 目的: 观察力尔凡单用或与化疗药物联合治疗中晚期恶性肿瘤的效果。 方法: 30例已确诊的Ⅲ、Ⅳ期恶性肿瘤患者。全身治疗:力尔凡单药治疗10例，皮下注射力尔凡5mg+1ml0.2%盐酸利多卡因，连用5d;静脉滴注力尔凡10mg+生理盐水250ml，每天1次，14d为1周期，休息1周后进行第2个周期，连用3个周期;力尔凡和化疗药物同时应用。局部治疗:为恶性胸、腹水的患者胸腹灌注和动脉介入治疗。 结果: 30例患者...查看详细 (4477字节)
☉ 11340220:颅内血肿微创清除术治疗脑出血26例临床分析
[摘要] 目的: 探讨颅内血肿微创清除术治疗脑出血的治疗效果。 方法: 回顾性分析26例脑出血患者，经CT定位，采用微创颅内血肿粉碎穿刺针配合生化酶血肿液化技术清除颅内血肿。 结果: 23例患者意识和肢体肌力不同程度恢复，其中治愈11例，好转12例，死亡3例。 结论: 颅内血肿微创清除术操作简便，手术创伤小，安全且疗效可靠，是一种有效治疗颅内血肿的方法，尤其适合基层医院推广应用。 [关键词] 微创清除术;颅内血肿;脑出血 脑出血是神经科常见的危急重症...查看详细 (6329字节)
☉ 11340221:Controlling the Spread of HIV/AIDS in the Indian Subcontinent
1 Tajen UniversityYanpu, Taiwan, Republic of China,2 National Sun Yat-sen UniversityKaohsiung, Taiwan, Republic of China The article on HIV/AIDS infection by Singh and colleagues outlines an alarming fact about the spread of this deadly virus in Nepal [1]. We would like to add that more assertive campaigns are necessary to curb the spread of infection in the Indian subcontinent before it's too late. In the year 2000 alone, a total of 5.3 million people were infected with HIV worldwide [2]. Since the epidemic started two decades ago, HIV/AIDS has killed 22 million people globally. India, Indochina, and the former Soviet republics have seen the most rapid rise of HIV incidence in recent years. AIDS experts have raised alarm bells over its spread in the Asia-Pacific region, and called for a united effort to control it. The Joint United Nations Programme on HIV/AIDS (UNAIDS) estimates about 5 million people in India alone are infected. The first report of HIV/AIDS infection in India was in 1986, and since then the virus has spread rapidly throughout the country. Both HIV serotypes 1 and 2 exist in India, and HIV-1 C is the most common subtype reported. Sexual transmission of HIV is the predominant route of transmission in India [3]. According to the Ministry of Health in New Delhi, only 3% of Indians use condoms for birth control, since the tradition and culture dictate that women undergo sterilization or take birth control pills. Prostitution plays a major role in spreading the disease among the heterosexuals in urban areas. Although Mumbai appears to be the main locus for AIDS, rapid spread has occurred through other major cities as well. Migration of people from cities to rural areas is so rapid that the disease may already be out of control in many areas. The blood screening tests conducted at most hospitals in rural India are not adequate to confirm presence of the virus, making blood transfusion unsafe. The National AIDS Control Organization (NACO), the apex body for controlling AIDS in India, has reported a high incidence (8.2%) of blood donors who are HIV-positive among healthy blood donors in urban areas [4]. AIDS is a sexually transmitted disease, and as long as people are educated thoroughly and warned about the dangerous consequences of unsafe sex, there is less to fear. Unfortunately, the intervention program launched by the NACO had very little impact in controlling the spread of the epidemic in India [4]. The current educational programs are often restricted to the passive dissemination of information through posters, media, and display of safe-sex billboards behind automobiles. More aggressive efforts are, therefore, needed to reach out to each and every rural/urban community throughout India to combat the spread of the disease. The state and central government agencies must build specialized shelters for people with HIV/AIDS. More funds must be spent for effective AIDS awareness campaigns, research, routine screening tests, and treatment. According to the Asia Pacific Network of People Living with AIDS, a considerable number of people were refused treatment or delayed provision of treatment or health services after being diagnosed with HIV/AIDS. Breaches of confidentiality by health workers were common in Asian countries. Within families and communities, women were discriminated against more than men—including ridicule, harassment, and physical assault—and they were often forced to change their place of residence because of their HIV status [5]. Although politicians and policymakers are increasingly committed to AIDS prevention and control efforts in countries such as India, a multidisciplinary approach such as early identification and treatment of sexually transmitted diseases, promotion of condom usage, rapid blood screening to test for HIV in rural areas, public awareness campaigns, poverty eradication, and development of prevention interventions have to be considered for effective control of the spread of this virus in the Indian subcontinent. Moreover, people from all walks of life must take an active role to promote AIDS awreness and prevention across the Indian subcontinent. It is time for the local and regional celebrities, such as political leaders, movie stars, and beauty pageant winners, in the Indian subcontinent to get involved in helping people with HIV and in educating the public, which would certainly raise awareness among the rural public more quickly than current efforts. It is time to remember how the late Princess of Wales reached out to people with AIDS, shook hands to console them, and also raised millions of dollars for their welfare. Countries in the Indian subcontinent have experienced and handled the outbreak of deadly epidemics in the past [6], and we hope that AIDS can also be controlled and eradicated eventually in the near future. References Singh S, Mills E, Honeyman S, Suvedi BK, Pant NP (2005) HIV in Nepal: Is the violent conflict fuelling the epidemic PLoS Med 2:e216 DOI: 10.1371/journal.pmed.0020216. Joint United Nations Programme on HIV/AIDS. (2002) AIDS epidemic update. Geneva: Joint United Nations Programme on HIV/AIDS. Godbole S, Mehendale S (2005) HIV/AIDS epidemic in India: Risk factors, risk behaviour and strategies for prevention and control. Indian J Med Res 121:356–368. Choudhury N, Ayagiri A, Ray VL (2000) True HIV seroprevalence in Indian blood donors. Transfus Med 10:1–4. Paxton S, Gonzales G, Uppakaew K, Abraham KK, Okta S, et al. (2005) AIDS-related discrimination in Asia. AIDS Care 17:413–424. Karlen A (1995) Man and microbes. Disease and plagues in history and modern times New York: Simon and Schuster. 266 p....查看详细 (5753字节)
☉ 11340222:急性脑膨出在重型颅脑损伤术中的预防与处理
[摘要] 目的: 探讨急性脑膨出在重型颅脑损伤开颅术中出现的原因及预防对策。 方法: 对86例重型颅脑损伤开颅术中急性脑膨出的原因、对策及预后进行回顾性分析。 结果: (1)迟发性血肿42例，其中同侧脑内血肿8例，硬膜外血肿6例，对侧硬膜外血肿10例，硬膜下血肿6例，脑内血肿8例，大脑纵裂血肿4例;(2)急性弥漫性脑肿胀22例;(3)外伤性大面积脑梗死10例;(4)复合伤患者8例，合并胸、腹部及四肢骨折，有明显的低血压和低血氧症状;(5)术中操作不当...查看详细 (4723字节)

☉ 11340223:The Statistical Significance of Suffering
1 GDNF 4 Parkinson'sWashington, District of Columbia, United States of America Musa Mayer makes several good points about the importance of enrolling people with life-threatening conditions in clinical trials in order to identify new treatments and speed the pipeline along for the greater good [1]. However, the idea that clinical trial enrollment suffers when seriously ill individuals are provided compassionate use of treatments is myopic; one does not negate the other. In many cases, persons who seek compassionate use of medications are ineligible for the clinical trials Mayer would want them to enroll in, and will likely die or suffer considerably before the experimental treatment they are seeking is approved for the public. In a world of limited resources, we need to ask, how do we encourage enrollment in clinical trials to develop treatments and cures that will benefit people in the future, while humanely treating those who are ineligible for these trials and suffer right now The first step is to understand that clinical trial enrollment and compassionate-use programs are not competing interests today, as they perhaps were in the 1980s and 1990s. The next step is to educate the public, not only about the importance of enrollment in clinical trials, but about their rights as informed participants in the noble process of science. Mayer's perspective [1] fails to consider the ultimate goal of clinical trials: to relieve human suffering. It serves no one's interest to demand an all-or-nothing approach to scientific progress. As Einstein said, “Not everything that can be counted counts, and not everything that counts can be counted.” Reference Mayer M (2005) When clinical trials are compromised: A perspective from a patient advocate. PLoS Med 2:e358 DOI: 10.1371/journal.pmed.0020358....查看详细 (1834字节)
☉ 11340224:Correction/Clarification about FDA Review Documents
1 Portland VA Medical Center and Oregon Health and Science UniversityPortland, Oregon, United States of America Emma Veitch cites my PLoS Medicine Essay [1] about how the Food and Drug Administration's (FDA's) review of documents can serve as a source of clinical trials data, but she follows it up with the statement, “However, it is difficult to have confidence in data released by sponsors when the data have not been subjected to external, independent peer review. Furthermore, this information is not integrated with other data, or indexed” [2]. While I agree with the second assertion, the first assertion—that the data are not subjected to external, independent peer review—is off the mark. FDA reviews are indeed external and independent to the sponsor. These reviews are conducted not by the sponsors but by physicians and scientists employed by the United States government. True, the data originate with the sponsor. However, once the sponsor submits data to the FDA, a level of rigor and scrutiny is applied to them that is arguably higher than what occurs in the typical journal manuscript review process. First, FDA reviewers typically revisit the original protocol submitted before the study was conducted in order to verify that the sponsor has not engaged in hypothesizing after the results are known (“HARKing”) [3]. By contrast, journal reviewers typically do not have access to the original protocol. As a result, they must trust that HARKing has not occurred, a dubious assumption in view of recent data [4]. Second, FDA statistical reviewers obtain the raw data from the sponsor, and determine whether the sponsor's findings can be replicated. By contrast, journal reviewers typically have access to only the summary statistics reported (perhaps selectively) to them by the authors or the sponsors. Consequently, reviewers can only speculate whether they could replicate the findings. As a result, I believe that the FDA review process warrants a higher level of confidence than the conventional journal manuscript review process. References Turner EH (2004) A taxpayer-funded clinical trials registry and results database. PLoS Med 1:e60 DOI: 10.1371/journal.pmed.0010060. Veitch E PLoS Medicine Editors (2005) Tackling publication bias in clinical trial reporting. PLoS Med 2:e367 DOI: 10.1371/journal.pmed.0020367. Kerr NL (1998) HARKing: Hypothesizing after the results are known. Pers Soc Psychol Rev 2:196–217. Chan AW, Hrobjartsson A, Haahr MT, Gotzsche PC, Altman DG (2004) Empirical evidence for selective reporting of outcomes in randomized trials: Comparison of protocols to published articles. JAMA 291:2457–2465....查看详细 (2709字节)
☉ 11340225:筋膜切开术在创伤外科中的应用(附13例报告)
[摘要] 目的: 观察筋膜切开减压术在创伤外科患者中的应用效果。 方法: 采用肌肉筋膜切开和肌间隔切开使筋膜腔减压。 结果: 共治疗13例因不同外伤所致不同部位的筋膜间隙综合征患者，均挽救了生命并保住了肢体，治疗效果满意。 结论: 筋膜切开减压是抢救和治疗筋膜间隙综合征确切有效的方法，早期作出诊断和及时进行筋膜切开减压是治疗的关键。 [关键词] 筋膜间隙综合征;筋膜切开术;创伤 筋膜间隙综合征和挤压综合征是创伤外科常见的较为严重的并发症...查看详细 (5716字节)
☉ 11340226:Editor's Reply
1 PLoS Clinical TrialsCambridge, United Kingdom Erick Turner appropriately points out the high levels of rigor applied during regulatory authorities' review of clinical trial data [1]. However, the statement beginning “However, it is difficult to have confidence in data released by sponsors…” [2] was not intended to highlight the release of review documents by the Food and Drug Administration (FDA), but rather the publication of summary clinical trial data on sponsors' own Web sites, which does seem to lack an integral peer-review mechanism. I support efforts to make Drugs@FDA more systematic and comprehensive, an initiative which can sit comfortably alongside peer-reviewed journal publication. References Turner E (2005) Correction/clarification about FDA review documents. PLoS Med 2(12):e422. Veitch E the PLoS Medicine Editors (2005) Tackling publication bias in clinical trial reporting. PLoS Med 2:e367 DOI: 10.1371/journal.pmed.0020367....查看详细 (979字节)
☉ 11340227:Paclitaxel Modulates TGFβ Signaling in Scleroderma Skin Grafts in Immunodeficient Mice
1 Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America,2 Division of Rheumatology, Johns Hopkins University, Baltimore, Maryland, United States of America Background Systemic sclerosis (SSc) is characterized by excessive fibrosis and obliterative vascular lesions. Abnormal TGFβ activation is implicated in the pathogenesis of SSc. Aberrant TGFβ/Smad signaling can be controlled by stabilization of microtubules with paclitaxel. Methods and Findings SSc and healthy human skin biopsies were incubated in the presence or absence of paclitaxel followed by transplantation into severe combined immunodeficient mice. TGFβ signaling, fibrosis, and neovessel formation were evaluated by quantitative RT-PCR and immunohistochemical staining. Paclitaxel markedly suppressed Smad2 and Smad3 phosphorylation and collagen deposition in SSc grafts. As a result, the autonomous maintenance/reconstitution of the SSc phenotype was prevented. Remarkably, SSc grafts showed a 2-fold increase in neovessel formation relative to normal grafts, regardless of paclitaxel treatment. Angiogenesis in SSc grafts was associated with a substantial increase in mouse PECAM-1 expression, indicating the mouse origin of the neovascular cells. Conclusion Low-dose paclitaxel can significantly suppress TGFβ/Smad activity and lessen fibrosis in SCID mice. Transplantation of SSc skin into SCID mice elicits a strong angiogenesis—an effect not affected by paclitaxel. Although prolonged chemotherapy with paclitaxel at higher doses is associated with pro-fibrotic and anti-angiogenic changes, the findings described here indicate that low-dose paclitaxel may have therapeutic benefits for SSc via modulating TGFβ signaling. Academic Editor: Tom Huizinga, Leiden University Medical Centre, The Netherlands These authors contributed equally to this work. These authors contributed equally to this work. Introduction Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive deposition of extracellular matrix (ECM) proteins and obliterative vascular lesions in the skin and internal organs [1]. Although the causative factors of this disease remain to be characterized, the main pathobiological features of SSc comprise three interactive components: autoimmune attack, vascular damage, and a lesion in fibroblasts [2,3]. The fibrotic process consists of massive deposition of connective tissue, mostly collagens, which is frequently responsible for the failure of many organs in patients with SSc. Consequently, an array of antifibrotic agents has been developed to: (1) reduce synthesis, excretion, or polymerization of collagen fibrils; (2) enhance collagenase activity; and (3) neutralize cytokines capable of stimulating collagen synthesis, such as TGFβ (transforming growth factor-beta), interleukin-4, and interleukin-6. To date, several of these antifibrotic agents have been tested in clinical trials [4]. These include D-penicillamine, colchicine, interferon gamma, and relaxin. Unfortunately, none of these medicines have any proven efficacy in retarding the fibrotic process [5]. TGFβ is a multifunctional regulatory cytokine that is involved in a large number of cellular activities. TGFβ induces matrix accumulation and tissue fibrosis associated with SSc [4]. In addition, TGFβ promotes endothelial cell apoptosis (a property that may be amplified by the presence of anti-endothelial cell autoantibodies found in SSc), and inhibits smooth muscle cell apoptosis. It also regulates T lymphocyte–mediated immune reactions [6]. Thus, TGFβ is believed to play a central role in the pathogenesis of SSc by activating tissue fibroblasts directly or indirectly through endothelial cells, by regulating lymphocyte function, and by affecting endothelial and smooth muscle cell survival and thrombus formation. Therefore, it is conceivable that inhibition of the aberrant TGFβ signaling may be a promising therapeutic strategy for SSc. TGFβ initiates its diverse cellular responses by binding to and activating specific cell-surface receptors that have intrinsic serine/threonine kinase activity. The activated TGFβ receptors stimulate the phosphorylation of receptor-regulated Smad2 and Smad3 proteins (R-Smads), which in turn form complexes with Smad4 that accumulate in the nucleus and regulate the transcription of target genes. Inhibitory Smad7 acts in an opposing manner to the R-Smads, inhibiting TGFβ signaling [7]. We have previously demonstrated that endogenous Smad-2, 3, and 4 bind microtubules (MTs) in several cell lines, and the binding provides a negative regulatory mechanism to control TGFβ activity. Disruption of the MT network by chemical agents, such as nocodazole and colchicine, leads to ligand-independent Smad nuclear accumulation and transcription of TGFβ-responsive genes, and increases TGFβ-induced Smad activity [8]. We have also found that inhibitory Smad7 is selectively decreased, and we and others have shown that R-Smad3 is increased, in SSc skin fibroblasts, resulting in uncontrolled TGFβ activity that may be, at least in part, responsible for the aberrant ECM deposition observed in SSc [9]. Indeed, in a hybrid human–severe combined immunodeficient (SCID) mouse skin xenotransplant model, we were able to maintain/reconstitute the SSc phenotype using skin biopsies from SSc patients, indicating that the altered balance between inhibitory Smad7 and R-Smads, due to Smad7 deficiency and Smad3 up-regulation, may represent an intrinsic and persistent defect in tissue fibroblasts that can maintain or even induce SSc lesions autonomously in the absence of altered circulatory or systemic factors [9]. The aim of this study was to determine if MT stabilization with low-dose paclitaxel could inhibit TGFβ/Smad signaling, ameliorating the fibrotic changes associated with SSc. Methods Participant Characteristics Skin biopsy (6-mm punch) was obtained in an area above the elbow considered to have grossly intact skin thickness as determined by clinical palpation of patients with SSc and in the same location of control participants. Thirty-two patients, of whom 26 were female and 6 were male, aged 38–64 y of age (average 48 y) with diffuse cutaneous SSc were studied. The median duration of skin disease was 5.5 y (2–10 y). Concomitant treatment of SSc patients included the immunosuppressant mycophenolate mofetil, angiotensin-converting enzyme inhibitors, calcium channel blockers, and proton-pump inhibitors. These patients were recruited from the Johns Hopkins University Scleroderma Center. All patients met the American College of Rheumatology criteria for the diagnosis of SSc [10]. Patients with overlap syndromes (e.g., lupus) were excluded. Twelve normal participants, including nine women and three men with an average age of 44 y (range 33–58 y) were also analyzed. All patients and volunteers gave their informed consent, and the study was approved by the Johns Hopkins University Human Subjects Institutional Review Board Committee. Skin Transplantation SCID mice (C.B-17/lcrHsd-scid) were purchased from Jackson Laboratory (Bar Harbor, Maine, United States). Mice aged 6–8 wk and weighing 18–22 g were used for transplantation. Briefly, the skin biopsy patch was trimmed into an oval shape depleted of fat tissue, and placed in 2 mM paclitaxel (Taxol; Sigma, St. Louis, Missouri, United States) or in PBS for 30 min at 4 °C. The tissue was rinsed with PBS just before transplantation. On the dorsolateral surface of the recipient SCID mouse, an oval graft bed approximately 6 mm in diameter was created to fit the graft, leaving the deep fascia layer intact. The trimmed skin patch was transplanted onto the graft bed by suturing the skin patch into the defect with 8–0 suture around the margin of the patch. A total of 25 transplants, including 17 SSc grafts and eight normal grafts, were included in the study (Table 1). The grafts, together with a small ring of the native skin, were harvested at 30 d following transplantation. Nineteen non-transplanted biopsy samples (15 SSc and four normal) were included as nonsurgical controls. Upon sacrifice, the underside of the skin was photographed for angiogenesis study, and the tissue was then divided into two segments: One segment was fixed in 10% neutral buffered formalin and embedded in paraffin, and the other segment was stored in RNAlater (Ambion, Austin, Texas, United States) for RNA expression studies. All animals were cared for in compliance with the “Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals,” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NO86 to 23, revised 1985). Immunohistochemistry and Histology SSc and normal specimens were processed by 10% formalin-fixation and paraffin-embedding. Immunohistochemistry (IHC) for Smad2, Smad3 (Santa Cruz Biotechnology, Santa Cruz, California, United States), phospho-Smad2 (Upstate Biotechnology, Lake Placid, New York, United States), phospho-Smad3 (a generous gift from Dr. Peter ten Dijke, Leiden University, Leiden, The Netherlands), collagen-1 (Santa Cruz Biotechnology), and PAI-1 (American Diagnostica, Greenwich, Connecticut, United States) was performed using Vector's ABC kits (Vector Laboratories, Burlingame, California, United States) on 3-μm consecutive serial sections. Briefly, after deparaffinization, slides were quenched in 3% H2O2 for 10 min to block endogenous peroxidase and washed in PBS. Sections were incubated with the primary antibody for 1 h and then with biotinylated secondary antibody followed by ABC reagents. Color development was achieved by incubating diaminobenzidine (DAB) as a substrate. Slides were counterstained with Mayer's hematoxylin. Preincubation of the primary antibody with specific blocking peptides or substitution of the primary antibody with an irrelevant IgG served as negative controls. Smad2- and Smad3-positive cells were counted in at least six high-power fields in each sample by two independent observers (CMD and XL). A minimum of 500 cells was counted. Percent positive cells were calculated as the number of positive cells/total number of cells × 100. Cells positive for phospho-Smad2 and phospho-Smad3 were counted in a similar fashion. Among the Smad2- and Smad3-positive cells, the percentage of those stained for phospho-Smad2 and phospho-Smad3 was calculated as the number of phospho-Smad2– and phospho-Smad3–positive cells/the number of cells positive for Smad2 and Smad3, respectively, × 100. Sections in series with IHC were stained with H & E (hematoxylin/eosin), and Verhoeff's van Gieson elastin and Mason trichrome (VVM). Each section was examined for the presence, extent, and distribution of collagen, elastic fibers, and other matrix proteins. Angiogenesis Assessment The number of microvessels was counted from three to five randomly selected high-power fields (40× magnification) in histology slides stained with H & E. Neovessel formation was also evaluated macroscopically by counting the number of vessels on the underside of the grafts and skin biopsies in a 6-mm field—the entire graft. Angiogenic activity was compared among different groups, including SSc skin biopsy, SSc grafts, normal skin biopsy, and normal skin grafts. TaqMan Real-Time Reverse Transcription-PCR RNA was isolated from the skin using RNeasy Mini kit (Quiagen, Chatsworth, California, United States). One μg total RNA was used for the synthesis of first strand cDNA using the SUPERSCRIPT Preamplification System (Life Technologies, Rockville, Maryland, United States). PCR was optimized for the quantitation of alpha2(I) collagen (COL1A2; mouse and human share the same sequence), human PECAM-1 (platelet endothelial cell adhesion molecule-1), and mouse PECAM-1 with specific primers and probes. A sequence detector (ABI Prism 7700, PE Applied Biosystems, Foster City, California, United States) was used to measure the amplified product in direct proportion to the increase in fluorescence emission continuously during the PCR amplification. For each sample, a threshold cycle (Ct) value was calculated from each amplification plot, representing the PCR cycle number at which the fluorescence was detectable above an arbitrary threshold. To normalize Ct of the target gene copies to 18S rRNA, ΔCt was calculated as Ct (target) Ct (18S rRNA). For each sample, the level of COL1A2, human PECAM-1, and mouse PECAM-1 was calculated as 2ΔCt. Each sample was tested in triplicate and repeated twice. Statistical Analysis Data are presented as mean ± SEM. Analysis of variance was performed to compare differences among different groups. A p-value 0.05; (Figure1A–1F). Minimal, if any, effects of paclitaxel on Smad2 activity in normal skin grafts were detected (normal grafts + paclitaxel, 26% ± 18% versus normal grafts, 21% ± 13%, p > 0.05). Furthermore, the level of total Smad2 and Smad7 (data not shown) remained unchanged. Strong nuclear staining for phospho-Smad2 (A) and total Smad2 (B) is observed in an SSc skin graft. Smad2 phosphorylation (C) is rare in normal graft, which expresses abundant Smad2 (D). Smad2 phosphorylation is suppressed with paclitaxel (Taxol) treatment (E), without affecting total Smad2 (F) in SSc skin graft. Tx, transplantation. A similar increase in Smad3 phosphorylation was detected in SSc specimens compared to normal skin tissue (83% ± 11% versus 20% ± 15%, p 0.05). Furthermore, the expression level of total Smad3 was also downregulated by paclitaxel treatment in SSc (81.5% ± 10% versus 57. 5% ± 12%, p = 0.05) (Figure S1A–S1F). There was no detectable effect of paclitaxel on Smad3 activation in normal skin grafts (30% ± 16% versus 25% ± 11%, p > 0.05). These findings indicate that preincubation of SSc skin with paclitaxel can effectively offset Smad7 deficiency and Smad3 up-regulation–induced augmented TGFβ signaling in SSc skin grafts, without affecting the total Smad2 and Smad7 expression level. Moreover, when TGFβ signaling is not perturbed in normal skin grafts, paclitaxel does not exert detectable effects. The progressive accumulation of ECM in the skin and internal organs is a hallmark of SSc, of which collagen type I is the major constituent. Indeed, collagen type I metabolites have been used as markers to evaluate disease activity in SSc [11]. To examine whether paclitaxel treatment affected collagen deposition in SSc skin grafts, we performed quantitative TaqMan real-time reverse transcription-PCR (TRT-PCR) for COL1A2. Instructively, the expression of COL1A2—a gene whose promoter contains multiple Smad-binding elements (SBE)—was reduced by 4.5-fold (p < 0.01) with paclitaxel treatment in SSC grafts, reaching a level that approximated COL1A2 mRNA expression in normal skin grafts. By contrast, paclitaxel had hardly any effects on COL1A2 mRNA expression in normal skin grafts (Figure 2). TRT-PCR analysis shows that the expression of COL1A2 is reduced 4.5-fold with paclitaxel (Taxol) treatment in SSC grafts, reaching a level equivalent to that of normal skin grafts. By contrast, paclitaxel has no effects on COL1A2 expression in normal skin grafts. To confirm the TRT-PCR findings, we performed histological VVM staining to evaluate total collagen deposition and IHC to examine collagen-1 expression level. As shown in Figure 3, there was extensive deposition of total collagen and other ECM proteins, including elastic fibers, in the entire dermis of SSc grafts, which was markedly reduced with paclitaxel treatment. Strong, intense collagen-1 staining was demonstrated in untreated SSc grafts, relative to much weaker staining in paclitaxel-treated SSc tissue, which was comparable to normal skin grafts treated with and without paclitaxel (Figure 3). These data indicate that low-dose paclitaxel prevents the maintenance/reconstitution of the SSc phenotype in SCID mice, and this effect may be mediated by stabilizing MT-Smad complex and subsequent inhibition of TGFβ/Smad signaling. VVM staining shows abundant, thick collagen bundles in the SSc graft (A). The amount of collagen is markedly reduced, and the collagen fibers become finer with paclitaxel (Taxol) treatment (B), comparable to that seen in the normal skin graft (C). Similarly, IHC staining demonstrates intense collagen-1 staining in the SSc graft (D), which is substantially decreased with paclitaxel treatment (E) to a level similar to that seen in the normal skin graft (F). Paclitaxel Does Not Affect the Enhanced Local Angiogenesis in SSc Skin Grafts SSc skin lesions are characterized by obliterative microvascular lesions and decreased capillary density, suggesting excessive endothelial injury and/or a deregulated, insufficient angiogenic response [12]. Three basic mechanisms could account for the defective vascular repair in SSc: (1) lack of signals produced by the skin to recruit progenitor cells from the bone marrow, (2) appropriate skin recruitment signals but failure of the bone marrow to mount an adequate repair process, and (3) appropriate skin recruitment signals and adequate bone marrow endothelial progenitor cell (EPC) supply but excessive destruction of EPCs upon their mobilization by immune system. To determine if the defective angiogenic response might be related to lack of signals produced by the skin to recruit progenitor cells from the bone marrow and whether paclitaxel would adversely affect the angiogenic process in SCID mice, we examined neovessel formation following SSc and normal skin transplantation. The EPC supply from the mouse bone marrow is assumed to be nonlimiting. Macroscopic examination of the underside of the 6-mm skin grafts revealed more pronounced angiogenesis in SSc grafts than normal skin transplants (Figure 4A–4D). There were on average 30 vessels in the SSc graft in three high-power fields (40×), as compared with 15 vessels in normal skin grafts (p < 0.01) (Figure 4E). To further confirm these findings and determine the origin of the neovascular cells, we performed TRT-PCR for human and mouse PECAM-1, an endothelial cell marker. If bone marrow failure and/or autoimmunity-mediated EPC destruction, but not secretion of mobilizing factors by the skin, represented the major bottleneck for a defect repair process in SSc, an aggressive angiogenic response with mouse progenitor cells and, therefore, increased mouse PECAM-1, in the SSc grafts would be expected. As shown in Figure 4F, mouse PECAM-1 was almost undetectable in SSc and normal skin biopsies before transplantation, reflecting the specificity of the primers and probe for the mouse gene. Substantial increase in mouse PECAM-1 mRNA expression was observed in SSc and normal skin grafts. The amplitude of the increase in mouse PECAM-1, however, was significantly greater in SSc than in normal grafts (p < 0.01). Human PECAM-1 was expressed in much lower levels in SSc than in normal skin tissue (p < 0.01) before transplantation, indicative of vascular deficiency in SSc. The level of human PECAM-1 did not change significantly before and after transplantation (Figure 4G). Collectively, these data indicate that the neovascular cells are of recipient mouse origin and that exhaustion of EPC supply from the patient's bone marrow and/or autoimmunity-mediated targeting and destruction of EPCs after mobilization may contribute to the vascular lesion associated with SSc. Macroscopic analysis of the vessels in the underside of normal (A and B) and SSc (C and D) skin grafts reveals more pronounced angiogenesis in SSc grafts; paclitaxel (Taxol) treatment has no effect on angiogenesis at the macroscopic level (B and D versus A and C). Microscopic analysis shows that the number of vessels in three high-power fields of the SSc grafts is greater than that of normal skin grafts; paclitaxel has no effect on angiogenesis at the microscopic level (E). TRT-PCR for mouse PECAM-1 demonstrates a substantial increase in mouse PECAM-1 mRNA expression in SSc and normal skin grafts regardless of paclitaxel treatment; the amplitude of the increase is significantly greater in SSc than in normal skin grafts (F). Human PECAM-1 is expressed in much lower levels in SSc than in normal skin tissues, indicative of vascular deficiency in SSc; the lack of difference in the expression level of human PECAM-1 in SSc and normal skin tissues before and after transplantation indicates that the neovascular cells are derived from the recipients (G). The putative activity of paclitaxel to induce endothelial cell or EPC apoptosis and/or to inhibit the proliferation of these cells with subsequent blockade of angiogenesis represented a potential untoward side effect for the use of paclitaxel in the treatment of SSc. To exclude this possibility, we compared the number of vessels in SSc and normal skin grafts treated with and without paclitaxel. As shown in Figure 4A–4E, treatment with paclitaxel did not adversely affect the angiogenic process. A similar increase in neovessel formation was observed in paclitaxel-treated versus nontreated SSc grafts, as compared with pre-transplantation skin biopsies from the same patients. Furthermore, paclitaxel incubation had little, if any, effect on the expression level of PECAM-1 mRNA, in particular mouse PECAM-1, in SSc and normal skin grafts (Figure 4F and 4G). These findings support the notion that low-dose paclitaxel can be used to modulate TGFβ/Smad signaling and treat the fibrotic lesion, without adversely affecting the vascular component of SSc pathobiology. Discussion In the present study, we found that the autonomous maintenance/reconstitution of the SSc phenotype in the hybrid SCID mouse transplant model was substantially prevented by pre-transplant incubation of the SSc skin with 2 mM paclitaxel (taxol), an MT-stabilizing agent. Furthermore, SSc grafts showed a 2-fold increase in neovessel formation, as compared with normal skin grafts, regardless of paclitaxel treatment. The angiogenic process in SSc grafts was associated with a substantial increase in mouse, but not human, PECAM-1 expression, pointing the origin of the neovascular cells to the recipient mice, perhaps the bone marrow. The angiogenesis data suggest that the skin of patients with SSc has preserved ability to trigger and support an angiogenic response. Collectively, these findings indicate that low-dose paclitaxel may potentially help keep in check the fibrotic process associated with SSc, without adversely affecting the vascular component of the disease. Members of the TGFβ superfamily play a central role in fibrosis, contributing to the influx and activation of inflammatory cells and fibroblasts and their subsequent elaboration of ECM [4]. TGFβ propagates its signal mainly via a signal transduction network involving receptor serine/threonine kinases at the cell surface and their substrates, the Smad proteins. Upon phosphorylation and oligomerization, R-Smads move into the nucleus to regulate transcription of target genes [7]. Recent findings indicate that the aberrant ECM synthesis by cultured SSc fibroblasts is due, at least in part, to the constitutively enhanced activation of the TGFβ signaling, which may result from the elevated levels of TGFβ receptor type I, inappropriate overexpression/activation of Smad2 and Smad3, and/or decreased Smad7 expression. Indeed, evidence obtained from Smad3-deficient mice shows that TGFβ-induced pro-fibrotic activities are mainly mediated by Smad3 [13]. Smad3-deficient inflammatory cells and fibroblasts do not respond to the chemotactic effects of TGFβ and do not auto-activate TGFβ [14,15]. Furthermore, Smad3-deficient mice are resistant to radiation-induced cutaneous fibrosis, bleomycin-induced pulmonary fibrosis, carbon tetrachloride–induced hepatic fibrosis, and glomerular fibrosis induced by type 1 diabetes caused by streptozotocin [16,17]. We have demonstrated that Smad7, the inhibitory Smad specific for TGFβ signaling, is selectively decreased, whereas Smad3 expression is increased in SSc fibroblasts. TGFβ signaling events, including phosphorylation of Smad2 and Smad3 and transcription of PAI-1 gene, are increased in SSc fibroblasts, relative to normal fibroblasts. Importantly, the imbalance between Smad7 and Smad3 itself can maintain or induce the SSc phenotype in SCID mice [9]. Furthermore, we have previously shown that MTs serve as a negative regulator for TGFβ/Smad signaling by forming a complex with endogenous Smad2, Smad3, and Smad4, sequestering the R-Smads away from the TGFβ receptor in several cell types [8]. Stabilization of MTs by low-dose paclitaxel can dampen to a normal level the exacerbated TGFβ signaling due to MT instability and block TGFβ-induced inhibition of myogenesis in C2C12 myoblasts [18]. In the present study, we provide evidence indicating that transient incubation of SSc skin with paclitaxel before transplantation into SCID mice substantially suppressed the phosphorylation of Smad2 and Smad3, two homologous Smad proteins that transduce signals from TGFβ and activin. Remarkably, paclitaxel treatment efficiently blocks the autonomous reconstitution and maintenance of the SSc phenotype in SCID mice. These data are consistent with our previous observations, supporting the notion that TGFβ/Smad signaling is regulated by the dynamic stability of MTs, which is sensitive to low-dose MT stabilizing agents, like paclitaxel. Prolonged chemotherapeutic treatment with paclitaxel has been associated with scleroderma-like changes, albeit in only a small fraction of patients. It is noteworthy that the anti-tumor effect of paclitaxel is mediated via the inhibition of cell proliferation and requires a much higher dosage. The inhibition of TGFβ/Smad signaling, however, can be achieved with very-low-dose paclitaxel. We and others have demonstrated that low-dose paclitaxel had minimal, if any, detectable effects on cell proliferation and other cellular activities, including fibrosis. Intriguingly, low-dose paclitaxel has been shown to inhibit collagen-induced arthritis and other autoimmune disorders in various animal models[19,20]. The low-dose paclitaxel treatment in our human–SCID mouse skin transplant model resulted in marked inhibition of TGFβ/Smad signaling, as evidenced by the decreased phosphorylation of Smad2 and Smad3, and lessened fibrosis. Our data indicate that under our experimental conditions in the SCID mouse, low-dose paclitaxel does not induce scleroderma skin changes. This does not, however, refute the potential linkage between higher doses of paclitaxel used for cancer therapy in humans and skin fibrosis. The structural and functional vascular and microvascular abnormalities, including Raynaud's phenomenon, represent one of the most important pathological features of SSc [12]. Indeed, microvascular damage and consequent loss of blood supply is found in all involved organs and leads to underperfusion and chronic ischemia, which may play an important role in organ dysfunction and even in the pathogenesis of in SSc. The fibrotic changes may represent a default pathway resulting from vascular failure. Endothelial apoptosis caused by viral infection, immune reactions to viral or environmental factors, reperfusion injury, or anti-endothelial antibodies, is considered a precipitating event in the genesis of vascular lesions in SSc [21]. Recent evidence, however, indicates that vascular repair, particularly that mediated via adult stem cells/EPCs from the bone marrow, plays an important role in maintaining vascular homeostasis and angiogenesis in a variety of disease states [22,23]. It is hypothesized that the vessel wall can deal fairly well with multiple circulating and local noxious stimuli as long as the bone marrow–derived repair capacity remains intact [24]. Indeed, many autoimmune processes might target the repair pathways that are needed to maintain the homeostasis of involved tissues [25]. Adequate vascular repair entails adequate supply of competent progenitor cells in, and their efficient mobilization from the bone marrow, as well as the effective homing to, and subsequent differentiation of these progenitor cells within the vessel wall. Any dysregulation in these processes can tilt the balance of vascular repair and injury in favor of injury and vascular lesion formation. It was postulated that the inadequate angiogenic response in SSc was due to reduced expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), and their receptors. It was recently shown, however, that both VEGF and its receptors (VEGFR1 and VEGFR2) were up-regulated in SSc skin specimens compared with healthy controls [26]. In addition, VEGF protein was significantly increased in blood samples from patients with SSc, reaching levels observed in patients with numerous malignant diseases [27]. Thus, there appears to exist a proper, if not increased, activation of the VEGF/VEGF-receptor axis—key to EPC mobilization—in patients with SSc [26]. One could not, however, rule out the possibility that other, yet to be identified, factors might be missing from the skin and blood of SSc patients, factors that are required to mount and support a successful angiogenic response. The human skin–SCID mouse transplantation model provides a remarkable opportunity to determine whether lack of signals from the skin of SSc patients to recruit EPCs from the bone marrow might play a role in the vascular lesion formation in SSc, since the bone marrow of the SCID mice is considered intact in terms of EPC supply and there is an absence of immune-mediated destruction of EPCs. Extensive analysis of vascular formation following SSc and normal skin transplantation both at microscopic and macroscopic levels revealed a robust angiogenic response in the SSc grafts, at least twice that seen in normal skin grafts. TRT-PCR for human and mouse PECAM-1 demonstrated a substantial increase in mouse PECAM-1 mRNA expression in SSc and normal skin grafts. Furthermore, the amplitude of the increase in mouse PECAM-1 was significantly greater in SSc than in normal grafts. In contrast, human PECAM-1 was expressed in much lower levels in SSc than in normal skin tissues before transplantation, indicative of vascular deficiency in SSc, and the expression level of human PECAM-1 did not change before and after transplantation in SSc and normal skin tissues, indicating that the neovascular cells are derived from the mouse recipients rather than the human donors. These data indicate that the signals from SSc skin, if anything, are stronger in stimulating the mobilization of EPCs from the bone marrow. Moreover, the robust angiogenic activity observed in SSC relative to normal grafts argues against the possibility that the angiogenic response in the SCID mouse is solely due to a wound-healing effect in response to grafting procedures. It remains to be determined, however, if the EPC supply in the bone marrow or autoimmunity-induced targeting and destruction of EPCs after their mobilization or both serve as the culprit in undermining the vascular repair process, contributing to vascular lesion formation in SSc. Using an animal model of atherosclerosis, we have demonstrated that exhaustion of selected progenitor cell populations, including EPCs and their supporting cells, a process that is accelerated by risk factors, can lead to the inability of the bone marrow to mount a successful vascular repair process, contributing to the initiation and progression of atherosclerotic vascular lesion formation [22,28]. Circulating EPCs and CD34+/KDR+ precursor cells were reduced in patients with atherosclerotic coronary artery disease. The reduction of these cells represented significant risk factors for atherosclerosis, even after adjusting for most classic risk factors, including age, sex, hypertension, diabetes, smoking, family history, and low-density lipoprotein cholesterol levels. Furthermore, factors that reduce cardiovascular risk, such as statins or exercise, elevate EPC levels, which contribute to enhanced endothelial repair. Hence, a reduced circulating EPC level has been proposed as a significant risk factor for cardiovascular disease. There is conflicting evidence regarding the level of circulating EPCs in SSc patients. Specifically, Kuwana et al. [25] showed that the levels of circulating EPCs, defined by the expression of CD34, CD133, and VEGFR2, were decreased in SSc patients. By contrast, Del Papa et al. [29] found that circulating EPCs—cells positive for CD34 and CD133—were increased in SSc patients, particularly in the early stages of the disease. Although the discrepancy between these studies might be due to the differing definitions of EPCs and different disease stages of SSc patients, it underscores the idea that further investigation is warranted in delineating the relative contribution of inadequate bone marrow EPC supply and excessive destruction of circulating EPCs to the imbalance between vascular injury and repair in SSc. The established mechanism that confers the antitumor effects of paclitaxel relates to its antiproliferative and antiangiogenic activity when used at large doses and for a prolonged period of time [30]. Indeed, the putative antiangiogenic effect of paclitaxel represented our primary concern for its use in the treatment of SSc. Remarkably, when paclitaxel- and sham-treated SSc and normal grafts were analyzed for their angiogenic response at microscopic and macroscopic levels, similar numbers of vessels were observed, indicating that low-dose paclitaxel does not affect neovessel formation that is likely mediated via EPCs recruited from the bone marrow. In conclusion, we have demonstrated that SSc skin treated with low-dose paclitaxel can significantly suppress the exacerbated TGFβ/Smad activity of SSc skin and lessen the fibrotic changes upon transplantation into SCID mice. We have found that transplantation of SSc skin into SCID mice elicits effective angiogenesis—an effect that is significantly stronger than with normal skin grafting. Importantly, the neovascular cells are almost exclusively derived from the recipients, perhaps originating from the mouse bone marrow. These observations should shed light on SSc disease pathogenesis and provide evidence for the development of novel therapeutic strategies. The fact that low-dose paclitaxel suppresses fibrosis without dysregulating angiogenesis suggests that fibrosis might be the result of a “default” pathway that develops autonomy once the SSc tissue becomes depleted of blood vessels. Supporting Information Strong nuclear staining for phospho-Smad3 (A) and total Smad3 (B) is observed in an SSc skin graft. Smad3 phosphorylation (C) is rare in normal graft, which also expresses a low level of total Smad3 (D). Smad3 phosphorylation and perhaps total Smad 3 are suppressed with paclitaxel (Taxol) treatment in SSc (E and F). (9.5 MB TIF). Acknowledgments This work was supported by a grant from the Scleroderma Research Foundation to PJGC. The authors wish to thank Ms. Ederick Forbes for her technical assistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: CD had the original idea for the study, and designed and oversaw the study. XL and SZ performed most of the experimental procedures and data analysis and participated in the study design. TW performed some experiments. LH and FMW recruited the patients and collected the skin biopsies. PJGC helped develop the concept of the study, and coordinated and supervised the experiments. XL, PJGC, and CD contributed to the writing of the report. Patient Summary Background Systemic sclerosis or scleroderma (SSc) is the name for a group of progressive diseases, all of which involve the abnormal growth of connective tissue. SSc is triggered when the body's immune system turns against the body, causing abnormal production of collagen that can be limited to the skin or extend to the blood vessels and internal organs. Why Was This Study Done Various genes have been suggested as being involved in the production of collagen. One of the key genes in this pathway, and in SSc, is TGFβ, which affects the activity of a number of other genes. A part of a cell's internal structure, known as microtubules, affects TGFβ activity. Previous work has shown that microtubules can be affected by drugs, such as the one in this study, paclitaxel, which stabilizes the microtubules. What Did the Researchers Do and Find They looked at the effect of paclitaxel on human skin samples that had been taken from people with SSc, or from people with normal skin, and then transplanted into mice. This transplantation is a good way of studying the effects of drugs in this disease without having to test them directly on people. The researchers found that paclitaxel affected the activity of TGFβ and the related genes, and ultimately decreased the amount of collagen that would usually be found in the skin of people with SSc. What Do These Findings Mean These findings suggest that paclitaxel would be worth investigating further as a useful drug in the treatment of people with SSc. One concern, however, is that in people who have been treated with high doses of this drug for other conditions, such as some types of cancer, the opposite effect of that shown here has been found: increased collagen has been produced. Therefore, before it can be certain that this drug is safe to use, further work will need to be done to determine what the different effects of the drug are at high and low doses, and what a safe dose is in humans. Where Can I Get More Information Online MedlinePlus has many links to pages of information on SSc: The Scleroderma Foundation is a non-profit organization based in the United States that provides information on scleroderma for patients, and supports research: The Scleroderma Research Foundation, which helped support this work: Patient Summary Background Systemic sclerosis or scleroderma (SSc) is the name for a group of progressive diseases, all of which involve the abnormal growth of connective tissue. SSc is triggered when the body's immune system turns against the body, causing abnormal production of collagen that can be limited to the skin or extend to the blood vessels and internal organs. Why Was This Study Done Various genes have been suggested as being involved in the production of collagen. One of the key genes in this pathway, and in SSc, is TGFβ, which affects the activity of a number of other genes. A part of a cell's internal structure, known as microtubules, affects TGFβ activity. Previous work has shown that microtubules can be affected by drugs, such as the one in this study, paclitaxel, which stabilizes the microtubules. What Did the Researchers Do and Find They looked at the effect of paclitaxel on human skin samples that had been taken from people with SSc, or from people with normal skin, and then transplanted into mice. This transplantation is a good way of studying the effects of drugs in this disease without having to test them directly on people. The researchers found that paclitaxel affected the activity of TGFβ and the related genes, and ultimately decreased the amount of collagen that would usually be found in the skin of people with SSc. What Do These Findings Mean These findings suggest that paclitaxel would be worth investigating further as a useful drug in the treatment of people with SSc. One concern, however, is that in people who have been treated with high doses of this drug for other conditions, such as some types of cancer, the opposite effect of that shown here has been found: increased collagen has been produced. Therefore, before it can be certain that this drug is safe to use, further work will need to be done to determine what the different effects of the drug are at high and low doses, and what a safe dose is in humans. Where Can I Get More Information Online MedlinePlus has many links to pages of information on SSc: The Scleroderma Foundation is a non-profit organization based in the United States that provides information on scleroderma for patients, and supports research: The Scleroderma Research Foundation, which helped support this work: References LeRoy EC (1992) A brief overview of the pathogenesis of scleroderma (systemic sclerosis). Ann Rheum Dis 51:286–288. Kissin EY, Korn JH (2003) Fibrosis in scleroderma. Rheum Dis Clin North Am 29:351–369. Kahaleh MB, LeRoy EC (1999) Autoimmunity and vascular involvement in systemic sclerosis (SSc). Autoimmunity 31:195–214. Branton MH, Kopp JB (1999) TGF-beta and fibrosis. Microbes Infect 1:1349–1365. Wigley FM, Sule SD (2001) Novel therapy in the treatment of scleroderma. Expert Opin Investig Drugs 10:31–48. Pollman MJ, Naumovski L, Gibbons GH (1999) Vascular cell apoptosis: Cell type-specific modulation by transforming growth factor-beta1 in endothelial cells versus smooth muscle cells. Circulation 99:2019–2026. Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425:577–584. Dong C, Li Z, Alvarez R Jr, Feng XH, Goldschmidt-Clermont PJ (2000) Microtubule binding to Smads may regulate TGF beta activity. Mol Cell 5:27–34. Dong C, Zhu S, Wang T, Yoon W, Li Z, et al. (2002) Deficient Smad7 expression: A putative molecular defect in scleroderma. Proc Natl Acad Sci U S A 99:3908–3913. Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. (1980) Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 23:581–590. Scheja A, Wildt M, Wollheim FA, Akesson A, Saxne T (2000) Circulating collagen metabolites in systemic sclerosis. Differences between limited and diffuse form and relationship with pulmonary involvement. Rheumatology (Oxford) 39:1110–1113. Sgonc R (1999) The vascular perspective of systemic sclerosis: Of chickens, mice and men. Int Arch Allergy Immunol 120:169–176. Lakos G, Takagawa S, Chen S, Ferreira AM, Han G, et al. (2004) Targeted disruption of TGF-beta/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma. Am J Pathol 165:203–217. Yang X, Letterio JJ, Lechleider RJ, Chen L, Hayman R, et al. (1999) Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta. EMBO J 18:1280–1291. Feinberg MW, Shimizu K, Lebedeva M, Haspel R, Takayama K, et al. (2004) Essential role for Smad3 in regulating MCP-1 expression and vascular inflammation. Circ Res 94:601–608. Zhao J, Shi W, Wang YL, Chen H, Bringas P, et al. (2002) Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 282:L585–593. Flanders KC (2004) Smad3 as a mediator of the fibrotic response. Int J Exp Pathol 85:47–64. Zhu S, Goldschmidt-Clermont PJ, Dong C (2004) Transforming growth factor-beta-induced inhibition of myogenesis is mediated through Smad pathway and is modulated by microtubule dynamic stability. Circ Res 94:617–625. Brahn E, Tang C, Banquerigo ML (1994) Regression of collagen-induced arthritis with taxol, a microtubule stabilizer. Arthritis Rheum 37:839–845. Cao L, Sun D, Cruz T, Moscarello MA, Ludwin SK, et al. (2000) Inhibition of experimental allergic encephalomyelitis in the Lewis rat by paclitaxel. J Neuroimmunol 108:103–111. LeRoy EC (1996) Systemic sclerosis. A vascular perspective. Rheum Dis Clin North Am 22:675–694. Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, Wang T, Gregg D, et al. (2003) Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 108:457–463. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, et al. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41–49. Goldschmidt-Clermont PJ (2003) Loss of bone marrow-derived vascular progenitor cells leads to inflammation and atherosclerosis. Am Heart J 146:S5–12. Kuwana M, Okazaki Y, Yasuoka H, Kawakami Y, Ikeda Y (2004) Defective vasculogenesis in systemic sclerosis. Lancet 364:603–610. Distler O, Distler JH, Scheid A, Acker T, Hirth A, et al. (2004) Uncontrolled expression of vascular endothelial growth factor and its receptors leads to insufficient skin angiogenesis in patients with systemic sclerosis. Circ Res 95:109–116. Distler O, Del Rosso A, Giacomelli R, Cipriani P, Conforti ML, et al. (2002) Angiogenic and angiostatic factors in systemic sclerosis: Increased levels of vascular endothelial growth factor are a feature of the earliest disease stages and are associated with the absence of fingertip ulcers. Arthritis Res 4:R11. Edelberg JM, Tang L, Hattori K, Lyden D, Rafii S (2002) Young adult bone marrow-derived endothelial precursor cells restore aging-impaired cardiac angiogenic function. Circ Res 90:E89–E93. Del Papa N, Colombo G, Fracchiolla N, Moronetti LM, Ingegnoli F, et al. (2004) Circulating endothelial cells as a marker of ongoing vascular disease in systemic sclerosis. Arthritis Rheum 50:1296–1304. Thatte U, Bagadey S, Dahanukar S (2000) Modulation of programmed cell death by medicinal plants. Cell Mol Biol (Noisy-le-grand) 46:199–214....查看详细 (47702字节)
☉ 11340228:鼻内窥镜下鼓膜修补术的临床观察(附12例报告)
[关键词] 鼻内窥镜;鼓膜;修补术 鼓膜修补术作为一种恢复中耳生理功能、增进听力的手术，常规在显微镜下完成。1999年1月至2005年1月，我院在鼻内窥镜引导下对12例(15耳)鼓膜穿孔患者进行鼓膜修补术，临床效果良好。现报告如下。 1 材料与方法 1.1 临床资料 共12例15耳，其中，男8例(10耳)，女4例(5耳);年龄18～57岁，平均34岁。病程5个月至11年...查看详细 (2947字节)
☉ 11340229:Persistent Amyloidosis following Suppression of Aβ Production in a Transgenic Model of Alzheimer Disease
1 Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America,2 Division of Biology, California Institute of Technology, Pasadena, California, United States of America,3 Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America,4 Mouse Cancer Genetics Program, National Cancer Institute Frederick Cancer Research and Development Center, Frederick, Maryland, United States of America,5 Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America,6 Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America Background The proteases (secretases) that cleave amyloid-β (Aβ) peptide from the amyloid precursor protein (APP) have been the focus of considerable investigation in the development of treatments for Alzheimer disease. The prediction has been that reducing Aβ production in the brain, even after the onset of clinical symptoms and the development of associated pathology, will facilitate the repair of damaged tissue and removal of amyloid lesions. However, no long-term studies using animal models of amyloid pathology have yet been performed to test this hypothesis. Methods and Findings We have generated a transgenic mouse model that genetically mimics the arrest of Aβ production expected from treatment with secretase inhibitors. These mice overexpress mutant APP from a vector that can be regulated by doxycycline. Under normal conditions, high-level expression of APP quickly induces fulminant amyloid pathology. We show that doxycycline administration inhibits transgenic APP expression by greater than 95% and reduces Aβ production to levels found in nontransgenic mice. Suppression of transgenic Aβ synthesis in this model abruptly halts the progression of amyloid pathology. However, formation and disaggregation of amyloid deposits appear to be in disequilibrium as the plaques require far longer to disperse than to assemble. Mice in which APP synthesis was suppressed for as long as 6 mo after the formation of Aβ deposits retain a considerable amyloid load, with little sign of active clearance. Conclusion This study demonstrates that amyloid lesions in transgenic mice are highly stable structures in vivo that are slow to disaggregate. Our findings suggest that arresting Aβ production in patients with Alzheimer disease should halt the progression of pathology, but that early treatment may be imperative, as it appears that amyloid deposits, once formed, will require additional intervention to clear. Academic Editor: Adriano Aguzzi, Zürich University, Switzerland ¤ Current address: Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, Florida, United States of America Introduction Over a decade ago the amyloid cascade hypothesis predicted that increased levels of amyloid-β (Aβ) peptide lead to secondary pathologies that ultimately culminate in the onset of Alzheimer disease (AD) [1]. Early support for this hypothesis came from genetic studies linking early-onset AD to mutations in the amyloid precursor protein (APP), from which Aβ is derived, and presenilins 1 and 2, which are interchangeable components of a endoprotease complex that releases Aβ from APP (for review see [2,3]). If, as predicted, overproduction of Aβ initiates the cascade of events leading to disease, then therapeutic strategies that lower Aβ levels should either arrest or reverse the progression from peptide to dementia. Early evidence from clinical trials of antibody-mediated clearance, one of the first Aβ-lowering approaches tested in humans, suggested that treatments designed to reduce amyloid burden may indeed be beneficial. Although the trials were halted because of adverse effects in a subset of volunteers [4,5], further analysis of several patients found evidence that amyloid pathology, and to a lesser degree cognitive function, was improved in proportion to the patient's titer of Aβ-specific antibody [6,7]. While this approach is promising, constant exposure to antibodies that recognize an epitope highly enriched in the brain may have unexpected side effects that will limit its long-term use. An alternative approach that is being actively pursued for future treatment of AD seeks to lower Aβ levels by limiting its production from the precursor protein APP. Peptide Aβ is released from APP by the action of two enzymes, the β-APP cleaving enzyme 1 (BACE1) and γ-secretase, which cleave the holoprotein at the N- and C-termini of Aβ, respectively. Several inhibitors of γ-secretase have already been produced [8,9], and small molecule inhibitors of β-APP cleaving enzyme 1 are currently being developed [10,11]. The long-term effectiveness of this approach in either humans or model systems, however, has not been reported. Although loss of β-APP cleaving enzyme 1 function can prevent the development of plaques in transgenic mouse models for AD (F. Laird, H. Cai, P. C. Wong, personal communication), it is not known whether the brain can clear pre-existing amyloid deposits once production of Aβ has been suppressed. Clearly, the amyloid-lowering approach should be rigorously examined in animal models before these reagents are tested in patients. However, the chemical secretase inhibitors most likely to reach human trials are still in development. Therefore, we developed a mouse model of Alzheimer-type amyloid that expresses a controllable APP transgene. This system, commonly known as the tet-off system, can be regulated by analogs of tetracycline administered in food or water [12,13]. The strong expression levels produced with the tet-off vectors, combined with the ability to reduce this expression by several orders of magnitude with tetracycline [14], allowed for a stringent test of how a highly effective pharmaceutical inhibitor of Aβ production would impact the progression of amyloid pathology and whether reversal of these lesions might be possible following such treatment. Methods Transgene Construction We created a tetracycline-responsive chimeric mouse/human APP695Swedish/Indiana (swe/ind) vector by replacing the promoter region of the moPrP.XhoI vector (also known as pPrPpE1/E2,3sal [15]) with the tetracycline-responsive promoter of pTetSplice (Life Technologies, Rockville, Maryland, United States), and then ligating mouse APP with a humanized Aβ domain (mo/huAPP695) cDNA into the new vector. We began by cloning the tetracycline-responsive promoter (bp 6–481) from pTetSplice by PCR using primers that added external BamHI and NotI sites to the 5′ end and a BamHI site to the 3′ end, while destroying XhoI and BamHI sites within the promoter (forward: GCC GGA TCC GCG GCC GCC GTC GAG TTT ACC ACT CCC TAT C; reverse: GCC GGA TCC ACT CTA GAA GAT CCC CGG GTA CCG). We then isolated the moPrP.XhoI intron by amplification with primers that added an external BamHI site to the 5′ end of exon 1 and ran through the Asp718 site of exon 2 (forward: GCC GGA TCC GAT CAG CAG ACC GAT TCT GG; reverse: GCC GGT ACC ACT AGG AAG GCA GAA TGC). This 2-kb intron fragment was cloned into a TA cloning vector (Invitrogen, Carlsbad, California, United States), then excised by Asp718 digestion and ligated to the 6.8-kb Asp718 fragment of moPrP.XhoI containing exon 2, exon 3, the 3′ UTR, and pBluescript to generate an intermediate vector with all three exons and a central intron but no promoter. This vector was then opened at the BamHI site introduced by the intron cloning primer, and ligated to the 0.5-kb BamHI-cut tetracycline promoter fragment. This ligation generated a 9.3-kb vector encoding the tetracycline promoter from pTetSplice with two exons, one intron, and the original 3′ UTR of the moPrP.XhoI vector, all carried in the pBluescript cloning vector. We incorporated the Swedish (KM570/571NL) and Indiana (V617F) mutations into the mo/huAPP695 cDNA (in BS-KS) by PCR using a four-primer strategy: first, two partially overlapping products were generated in separate reactions using primers that encode the desired mutations (Swedish forward: GGA GAT CTC TGA AGT GAA TCT GGA TGC AGA ATT CCG/Indiana reverse: GGG TGA TGA AAA TCA CGG TTG C; Indiana forward: CAA CCG TGA TTT TCA TCA CCC TGG/M13 reverse). The two PCR products were ligated, digested with BglII and ApaI and cloned back into the original mo/huAPP695-BS-KS vector. Finally, the new APP695swe/ind was subcloned into the XhoI site of the moPrP-tetP vector from above to complete the construct. Pronuclear Injection, Screening of Founders, and Maintenance of the Lines The moPrP-tetP-mo/huAPP695swe/ind vector was linearized and the pBluescript domain excised by digestion with NotI. The purified vector was injected into the pronucleus of fertilized eggs from C57BL/6J × C3HeJ F1 matings. Founder animals were screened for the presence of the transgene by three-way PCR using the S36 and PrP-S/PrP-AS primers described below. Transgene-positive founders were bred to animals expressing the tetracycline transactivator (tTA) under control of the calcium-calmodulin kinase IIα (CaMKIIα) promoter obtained from Jackson Laboratory [16] (Bar Harbor, Maine, United States; stock # 3010; B6;CBA-TgN[Camk2a-tTA]1Mmay). The colony was thereafter maintained by crossing single transgenic tTA and APP offspring for each of the four APP lines. All mice were provided fresh food and water ad libitum. Animal protocols were approved by both the Johns Hopkins University and the California Institute of Technology Institutional Animal Care and Use Committees. Doxycycline Administration Doxycycline (dox) was administered through commercially available dox-containing chow (BioServ, Frenchtown, New Jersey, United States). The chow contained 200 mg/kg of antibiotic; based on estimated consumption of 5 g per mouse per day, the expected dose to each animal was 1 mg dox per day. The average 25-g animal therefore received 40 μg of dox per gram body weight per day. Chow was changed 1–2 times per week to prevent breakdown of the antibiotic. Genotyping Offspring were genotyped for the presence of each transgene by PCR amplification of genomic DNA extracted from a 5-mm tail biopsy. Tails were heated to 95 °C for 45 min in 250 μl of 50 mM NaOH, vortexed, then neutralized with an equal volume of 0.5 M Tris-HCl (pH 5.5). Debris was sedimented by centrifugation, and 3 μl of supernatant was used for amplification. Genotyping for APP and tTA transgenes was performed in the same PCR reaction, using five separate primers. APP was amplified using forward primer S36 located in the 3′ end of the APP cDNA (CCG AGA TCT CTG AAG TGA AGA TGG ATG) and reverse primer PrP-AS-J located in the 3′ UTR of the vector (CCA AGC CTA GAC CAC GAG AAT GC). The tTA transgene was detected using a primer set that amplified across its two subdomains with tet forward located within the Tn10 tetracycline repressor (CGC TGT GGG GCA TTT TAC TTT AG) and tet reverse within the HSV1 VP16 (CAT GTC CAG ATC GAA ATC GTC). All reactions, whether transgene-positive or not, amplified a segment of the endogenous prion protein gene as a control for DNA quality using a forward primer, PrP-S-J, specific to the mouse PrP open reading frame (GGG ACT ATG TGG ACT GAT GTC GG) and a reverse primer, PrP-AS-J, shared by the 3′ UTR of the endogenous PrP gene and the transgene vector. Amplification reactions were run for 37 cycles at 94 °C for 30 s, 64 °C for 1 min, and 72 °C for 1 min. All samples, transgenic and wild-type, gave a 750-bp product from the endogenous PrP gene. The APP transgene yielded an additional band at 400 bp; the tTA product fell in between at 480 bp. Immunoblotting/Quantitation Frozen cortical or whole forebrain tissue was homogenized by sonication in five volumes of phosphate-buffered saline (PBS) with 5 mM EDTA and protease inhibitors (Mammalian cell cocktail, Sigma, St. Louis, Missouri, United States), using a probe sonicator set to 50% output (TEKMAR, Cincinnati, Ohio, United States). After dilution with an equal volume of PBS/EDTA/protease inhibitor, the samples were centrifuged briefly and the supernatant used for analysis. Fifty micrograms (6E10 and CT15) or 5 μg (22C11) of brain homogenate was loaded per lane onto 7.5%, 10%–20%, or 4%–20% Tris-HCl PAGE gels (Bio-Rad Laboratories, Hercules, California, United States) and electrophoresed for several hours in 1× Tris-glycine–sodium dodecyl sulfate (1×TG-SDS) buffer (6E10 and 22C11; Amresco, Solon, Ohio, United States) or 1× Tris-tricine-SDS buffer (CT15; Invitrogen, Carlsbad, California, United States). Proteins were transferred overnight to 0.45-μm Optitran nitrocellulose (Schleicher and Schuell, Keene, New Hampshire, United States) in 1× TG buffer (Amresco). Blots were blocked in PBS containing 5% nonfat dry milk powder, and incubated for 3 h at room temperature in blocking solution with one of the following antibodies: mouse monoclonal 22C11 (kind gift of Konrad Beyreuther and Andreas Weidemann; [17]) diluted 1:1,000, mouse monoclonal 6E10 (Signet Laboratories, Dedham, Massachusetts, United States) diluted 1:2,500, rabbit polyclonal anti-superoxide dismutase 1 (m/hSOD1) [18] diluted 1:2,500 to 1:4,000, or rabbit polyclonal CT15 (kind gift of Ed Koo; [19]) diluted 1:1,000. Subsequently, the blots were washed with PBS containing 0.1% Tween-20, and then incubated with either goat anti-mouse– or goat anti-rabbit–HRP conjugated secondary antibody diluted 1:1,000 in blocking solution. After several additional rinses in PBS with 0.1% Tween-20, blots were developed with enhanced chemiluminescence reagent and imaged with the Bio-Rad Molecular Imager FX system. Staining intensity within each lane was quantified using the Quantity One image analysis software (Molecular Imager FX, Bio-Rad Laboratories). Background was calculated from across the image and subtracted from the entire file. The signal intensity for each band (corrected signal intensity × pixel number) was then calculated using the Volume report tool. Slot Blot mRNA Analysis Five micrograms per sample of total RNA extracted from fresh-frozen brain, liver, kidney, heart, lung, spleen, and skeletal muscle was vacuum-filtered through 0.45-μm Optitran nitrocellulose. After several washes through the manifold with 10× SSC, blots were UV-cross-linked and probed with a radiolabeled ～350-bp BglII–XhoI cDNA fragment of mo/huAPP695 cDNA. After hybridizing overnight at 65 °C in 1% BSA/1 mM EDTA/0.5 M sodium phosphate buffer (pH 7.2)/7% SDS [20], the blots were washed twice at 65 °C for 30 min each in 0.1% BSA/1 mM EDTA/40 mM sodium phosphate buffer (pH 7.2)/5% SDS before two final 30-min washes at 65 °C with 1 mM EDTA/40 mM sodium phosphate buffer (pH 7.2)/1% SDS. Blots were wrapped wet and exposed to phosphorscreens overnight at room temperature. Amyloid Histology Mice were euthanized by ether inhalation and brains removed for immersion fixation in 4% paraformaldehyde/1× PBS. After 48 h in fixative at 4 °C, brains were transferred to PBS, dehydrated in alcohols, treated with cedarwood oil and methylsalicylate, and embedded in paraffin for sectioning. Hirano silver stain Silver impregnation histology was performed on 10-μm paraffin-embedded sections by Hirano's modification of the Bielschowsky method [21]. Briefly, sections were deparaffinized through xylene and alcohols into tap water before being placed into fresh 20% silver nitrate solution for 20 min. After being washed thoroughly with distilled water, slides were immersed in 20% silver nitrate solution titrated with fresh ammonium hydroxide. After 20 min, slides were washed with ammonia water before being individually developed with 100 μl of developer (20 ml of 37% formaldehyde, 100 ml of distilled water, 50 μl of concentrated nitric acid, and 0.5 g of citric acid) added to 50 ml of titrated silver nitrate solution. Slides were then rinsed in tap water, fixed in 5% sodium thiosulfate, and dehydrated through alcohols and xylene. Thioflavin-S staining Following deparaffinization of sections through xylene and alcohols, amyloid impregnation with thioflavin-S was performed according to the Guntern modification of the standard protocol. Slides holding 10-μm paraffin sections were washed twice in distilled water, then immersed for 5 min in a 0.25% potassium permanganate solution, followed by 5 min in a 1% potassium metabisulfate/1% oxalic acid solution. After this preparation, slides were placed into a filtered aqueous 0.02% thioflavin-S solution (Chroma-Gesellschaft Schmid, Kongen, Germany) for 8 min. Excess stain was removed by two brief rinses in 80% ethanol, then two in distilled water, after which slides were finished in aqueous mounting medium for florescence photomicrography. Ubiquitin, glial fibrillary acidic protein, and Aβ immunohistochemistry Prior to immunostaining, slides were deparaffinized by oven heating followed by immersion in xylene. After rehydration through graded alcohols into tap water, endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in methanol. Slides were microwaved for 5–7 min in water, cooled for 5 min, then washed in TBS. Nonspecific staining was blocked for 1 h with 3% normal goat serum and 0.1% Triton-X 100 in TBS. Slides were then placed into primary antibody (rabbit anti-Aβ peptide polyclonal antibody, Zymed Laboratories, South San Francisco, California, United States; rabbit anti-ubiquitin and rabbit anti–glial fibrillary acidic protein (GFAP) polyclonal antibodies, Dako, Carpinteria, California, United States) diluted 1:500 in TBS with 2% normal goat serum and incubated overnight at room temperature. After being washed of excess primary antibody with several changes of TBS, slides were incubated with either the Vectastain Elite anti-rabbit secondary system (anti-Aβ; Vector Laboratories, Burlingame, California, United States) or peroxidase/anti-peroxidase reagents (anti-ubitquitin and anti-GFAP; Sternberger Monoclonals, Lutherville, Maryland, United States) according to the manufacturers' directions. Antibody binding was visualized with diaminobenzidene, and sections were counterstained with hematoxylin. Filter Trap Assay An aliquot of each cortical homogenate used for Western blotting above was partially solubilized by the addition of SDS to a final concentration of 1%. Serial 1:2 dilutions were made with 1× PBS/1% SDS, and 100 μl of each dilution was then vacuum-filtered through a pre-wet 0.22-μm cellulose acetate membrane (Schleicher and Schuell) [22]. Each well was washed several times with PBS, after which blots were incubated overnight with polyclonal anti-Aβ antibody (Zymed Laboratories) diluted 1:600 in a blocking solution of 1× TBS/5% nonfat dry milk powder. After washing the blots three times for 10 min each in 1× TBS/0.1% Tween-20, the membrane was incubated for 1 h with HRP-conjugated protein A (Sigma) diluted 1:5,000 in blocking solution. The membranes were again washed three times with 1× TBS/0.1% Tween-20, before antibody binding was detected with enhanced chemiluminescence (PerkinElmer, Boston, Massachusetts, United States). Digital images of each blot were captured with a Molecular Imager FX gel documentation system, and the intensity of Aβ staining was quantified using Quantity One image analysis software. Aβ ELISA An aliquot of cortical homogenate generated for Western analysis described above was subjected to a three-step sequential extraction using PBS, 2% SDS, and 70% formic acid (FA). At each step, the sample was sonicated in appropriate buffer and centrifuged at 100,000g for 30 min (1- to 1.5-mo samples) or 60 min (6- to 12-mo samples) at 4 °C as previously described [23–25]. The supernatant was removed for analysis, and the pellet was sonicated in the next solution in sequence. The 2% SDS extracts were diluted in EC buffer, and the FA extracts neutralized with 1M Tris-phosphate buffer (pH 11) then diluted with EC buffer prior to testing. Human Aβ was measured in each fraction using BAN50 for capture (epitope Aβ1–16) and BA27 and BC05 for detection (Aβ40 and Aβ42, respectively) (Takeda Chemical Industries, Osaka, Japan). Total Aβ (mouse + human; 1- to 1.5-mo samples only) was measured in each fraction using BNT77 for capture (epitope Aβ11–28) and BA27 and BC05 for detection. All values were calculated as picomoles per gram based on the initial weight of cortical tissue. Activity Monitoring Daily basal activity was studied in 28 CaMKIIα-tTA × tet-APPswe/ind line 107 mice between 4 and 5 mo of age. Animals were separated into individual cages immediately before the start of each experiment (n = 3–6 per genotype untreated, 2–5 per genotype dox-reared). The cages were placed inside activity-monitoring frames designed to count every time the animal passed through one of three photobeams spanning the width of the cage (San Diego Instruments, San Diego, California, United States). Experiments were started midway through the light phase of the day, and data were collected in 1-h bins for the following 48 h. Testing rooms were maintained on the same 13:11 h day:night cycle as the main animal housing areas and were closed to entry during the experiment. Statistical Analyses Statistical analyses of protein expression, ELISA data, and filter trap assays were performed by ANOVA with Tukey's honest significant difference post-hoc test applied to significant main effects or interactions (Statistica 6.0, StatSoft, Tulsa, Oklahoma, United States). In cases of positively skewed data distribution, log10(x + 0.5) transformation was applied to the raw data before submitting them to ANOVA. Results We used the tet-off transgene system to express a double mutant version of chimeric mo/huAPP695 (swe/ind KM570, 571NL, and V617F) from a tetracycline-responsive promoter [12,13]. Transgenic APP expression was activated by crossing the APPswe/ind mice to animals producing tTA under control of the CaMKIIα promoter [16]. After initial screening of founders, we identified four lines of tet-APPswe/ind mice that produced very high levels of transgene product in offspring coexpressing tTA (Figures 1 and Figure S1). Compared to a standard APP transgenic line used for previous amyloid studies by our laboratory (line C3–3; [15,26,27]), we estimated that the four controllable lines produce transgenic APP protein at 10- to 30-fold over endogenous levels (Figure S1). This estimate was confirmed by direct comparison of APP levels in nontransgenic and tet-off APP mice using an antibody that recognizes both endogenous APP (and amyloid precursor-like protein 2) and the transgenic protein (monoclonal antibody 22C11; Figure 1D). (A) Western blotting for transgenic APP using the human-specific 6E10 antibody shows expression of full-length protein in forebrain tissue from young predeposit double transgenic animals (line 107) and its suppression following dox treatment. Untreated animals show high levels of transgene expression; protein levels drop dramatically in animals acutely treated with dox for 2 wk. A faint, but detectable band of full-length protein remains in acutely treated animals that can be eliminated in mice born and raised on dox. (B) Immunoblotting with the N-terminal antibody 22C11 to detect both transgenic and endogenous protein shows that dox treatment reduces APP/APLP to levels found in nontransgenic mice. (C) Measurement of signal intensity from the Western blot in (A) shows transgenic APP levels are decreased more than 95% by dox in both acutely and chronically treated animals (97.2% for 4 wk + 2 wk dox, 98.0% for reared on dox versus 4 wk untreated; ANOVA effect of treatment group F4,15 = 85.55, p 0.9, Tukey post-hoc test). (D) Measurement of signal intensity from the Western blot in (B) shows total APP/APLP levels in dox-treated tTA/APP mice are significantly lower than in 4-wk-old untreated mice (ANOVA effect of treatment group F4,15 = 84.41, p 0.9, Tukey post-hoc test). , p 0.3, Tukey post-hoc test). (B) In the young animals tested here prior to the formation of visible amyloid deposits, most Aβ is extracted into the SDS fraction (84% and 76% of all transgene-derived Aβ40 and Aβ42, respectively). As in the PBS-soluble fraction, Aβ levels in the SDS fraction are significantly lowered by dox treatment compared to untreated animals (ANOVA effect of group F4,24 = 197.57 and 163.48, p 0.8, Tukey post-hoc test). (C) The FA-soluble fraction already contains a small but significant pool of aggregated Aβ42 in untreated animals by 4 wk of age (p 0.9, Tukey post-hoc test), consistent with poor turnover of aggregated Aβ species. (D) Measurements of total Aβ, including both endogenous and transgene-derived peptides, show that animals born and raised on dox harbor Aβ levels identical to nontransgenic animals (p > 0.9, Tukey post-hoc test, effect of group ANOVA F4,24 = 39.13 and 35.29, p 0.8, Tukey post-hoc test), but leaves a small amount of nonsuppressed Aβ42. The residual Aβ42 observed in acutely treated young animals derives from uncleared aggregates extracted in the SDS and FA fractions. , p 95%) of transgenic APPswe/ind levels in the dox-treated animals was confirmed by immunoblot (Figure 3). To ensure that the transgene could be suppressed as rapidly in 6-mo-old mice with fulminant pathology as it can in young, predeposit animals, we treated an additional set of 6-mo-old animals with dox for 1 wk prior to harvest. Importantly, both APPswe/ind and APP–C-terminal fragment levels were fully suppressed after only 1 wk of treatment, indicating that the in vivo half-life of APPswe/ind and its processed C-terminal fragments are relatively short (Figure 3D). (A) Cortical homogenates from 6- to 12-mo-old animals used for pathology studies described below (line 107) were immunoblotted with human-specific antibody 6E10 to examine transgene suppression following 3 or 6 mo of dox treatment. The blot was co-immunostained for endogenous superoxide dismutase 1 (SOD1) as a control for loading. (B) Quantitation of signal intensity from the Western blot shown in (A). Transgenic APP levels are significantly suppressed following 3 or 6 mo of dox treatment (96.9% and 97.6%, respectively). , p 0.9, Tukey post-hoc test). Single transgenic tTA samples were included as negative controls and showed no signal above background. , p 99%) was extracted into the SDS and FA fractions (Figure 5A and 5B). Consistent with the filter trap results presented above, there were no significant differences in SDS- or FA-soluble Aβ between the 6 mo untreated cohort and either the 6 mo + 3 mo treated or 6 mo + 6 mo treated cohorts. However, brains of both 6 mo + 3 mo and 6 mo + 6 mo treated cohorts contained roughly twice as much PBS-soluble Aβ40 as untreated 6-mo-old mice (Figure 5C). Levels of Aβ42 showed a similar trend, but did not reach statistical significance. In fact, levels of PBS-soluble Aβ40 and Aβ42 in the 6 mo + 3 mo and 6 mo + 6 mo treated cohorts were most similar to that of the 9 mo untreated cohort, suggesting that age, as opposed to synthetic rate (which would be negligible in the treated animals), may determine the fraction of PBS-soluble Aβ in these animals. Aβ levels in untreated 6- and 9-mo-old tTA/APP line 107 mice (shown in Figure 4) were compared to those in 9- and 12-mo-old animals treated with dox from the age of 6 mo. Single transgenic APP samples were included as negative controls. Cortical homogenates were fractionated by sequential multi-step extraction with PBS, 2% SDS, and 70% FA followed by human-specific Aβ ELISA to measure transgene-derived peptide in each fraction. Aβ40 is shown in white, Aβ42 in black. (A and B) Most Aβ in the brains of plaque-bearing mice is extracted into the FA and SDS fractions. Consistent with amyloid burden (Figures 4 and Figure S3), SDS- and FA-extracted Aβ levels in untreated 9-mo-old mice were significantly higher than in untreated 6-mo-old mice (Tukey post-hoc test applied to significant effect of group ANOVA for SDS and FA fractions F3,18 = 4.72–12.92, p 0.2 compared to 6 mo untreated mice, Tukey post-hoc test). , p 0.5, Tukey post-hoc test). , p < 0.05; , p < 0.005; , p < 0.001 versus 9-mo-old untreated mice, Tukey post-hoc test. (962 KB TIF). Amyloid histology in cortical (first and third rows) and hippocampal (second and fourth rows) sections from untreated tTA/APP mice shows a dramatic progression of pathology between 6 and 9 mo of age. Suppression of transgenic APP expression arrests this progression, although without any sign of plaque clearance (6 mo + 3 mo dox). Hirano silver stain (top panels); thioflavin-S (bottom panels). (5.8 MB PSD). Acknowledgments We thank Patrick Tremblay for helpful advice on the tet system at a critical time in the project, and Mark Mayford for sharing the CaMKIIα-tTA mice through Jackson Laboratory. We also thank Fraser Moss for saving several immunoblots with last-minute shipments, Andy Groves for many thoughtful discussions, Neil Segil for generously sharing his laboratory and equipment, Beth Olson, Natasha Bouey, and Yolanda Jackson for outstanding animal care, Debbie Swing for expert microinjection, and Dave Fromholt for genotyping and dissection. We gratefully acknowledge Takeda Chemical Industries for providing antibodies BAN50, BA27, and BC05, Konrad Beyreuther and Andreas Weidemann for providing 22C11 antibody, and Ed Koo for sharing CT15 antibody. This work was supported by grants from the Johns Hopkins Alzheimer's Disease Research Center (JLJ), the National Alliance for Research on Schizophrenia and Depression (Young Investigator Award [JLJ]), the Rose Hills Foundation (JLJ), the Alzheimer's Association (Zenith Award [DRB]), the National Institute of Aging (K01 AG26144–01 [JLJ], P50 AGO5146–20 [DRB], R01 AG006656–16 [SGY], and P01 AG015453 [SGY]), the National Institute of Neurologic Disease and Stoke (R01 NS 047225 [DRB]), and the National Cancer Institute (NAJ and NGC). The funding agencies generously provided for research supplies, animal care, and salary support; the funders of this work had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: JLJ and DRB designed the study and wrote the manuscript, JLJ, HHS, VG, JCW, LHY, SGY and DRB performed experiments, NAJ and NGC generated transgenic founders, HAL assisted with data interpretation, and AVS performed statistical analyses. Patient Summary Background Patients with Alzheimer disease (AD) have elevated levels of a small protein called amyloid-β peptide that sticks together to form what are known as amyloid plaques in their brains. This peptide is normally made at low levels in healthy individuals, and is made when a larger protein called amyloid precursor protein (APP) is cut down in size. New treatments are now being developed that will decrease the amount of Aβ produced from APP. However, it is not clear whether lowering the production of Aβ will allow the brain to heal itself by clearing the amyloid plaques. The answer to this question may be important for deciding when Aβ-lowering drugs should be started, and may also determine how effective they are in reversing the mental symptoms of AD. What Did the Researchers Do and Find Because new drugs designed to lower Aβ levels are still in development, they are not available for testing in animal models of the disease. Instead, basic questions about the effectiveness of this type of treatment must be answered using systems that mimic how the drugs work. To do this, the authors created mice that produce too much APP and that develop the same amyloid lesions as do human patients with AD. Unlike normal mice, these mice also carried a “switch” gene that allowed the researchers to turn off APP by feeding the mice special food. Turning off APP in these mice had the same effect as treating them with Aβ-lowering drugs, and so the researchers were able to ask what happened to the amyloid plaques after Aβ production was shut down. They showed that lowering Aβ production prevents the amyloid lesions from getting worse as the disease progresses. This means that treatment with Aβ-lowering drugs may be able to stop the disease from filling the brain with plaques. However, the researchers also found that the amyloid lesions that had formed before treatment was started remained intact throughout the experiment. What Do These Findings Mean These results indicate that treatments designed to lower the production of Aβ may be an important part of future AD treatment, as this approach seems to prevents additional amyloid plaques from forming in the mouse brain. However, by itself, this strategy may not be able to rid the brain of plaques that have already formed in the brain before treatment is started. The findings suggest that early treatment may be important for this approach to succeed. Where Can I Get More Information Online MedlinePlus has several Web pages of information on Alzheimer disease: The ADEAR Center of the US Government's National Institute on Aging also has information on Alzheimer disease: The Alzheimer's Association Web site contains information on both caregiving and research: Patient Summary Background Patients with Alzheimer disease (AD) have elevated levels of a small protein called amyloid-β peptide that sticks together to form what are known as amyloid plaques in their brains. This peptide is normally made at low levels in healthy individuals, and is made when a larger protein called amyloid precursor protein (APP) is cut down in size. New treatments are now being developed that will decrease the amount of Aβ produced from APP. However, it is not clear whether lowering the production of Aβ will allow the brain to heal itself by clearing the amyloid plaques. The answer to this question may be important for deciding when Aβ-lowering drugs should be started, and may also determine how effective they are in reversing the mental symptoms of AD. What Did the Researchers Do and Find Because new drugs designed to lower Aβ levels are still in development, they are not available for testing in animal models of the disease. Instead, basic questions about the effectiveness of this type of treatment must be answered using systems that mimic how the drugs work. To do this, the authors created mice that produce too much APP and that develop the same amyloid lesions as do human patients with AD. Unlike normal mice, these mice also carried a “switch” gene that allowed the researchers to turn off APP by feeding the mice special food. Turning off APP in these mice had the same effect as treating them with Aβ-lowering drugs, and so the researchers were able to ask what happened to the amyloid plaques after Aβ production was shut down. They showed that lowering Aβ production prevents the amyloid lesions from getting worse as the disease progresses. This means that treatment with Aβ-lowering drugs may be able to stop the disease from filling the brain with plaques. However, the researchers also found that the amyloid lesions that had formed before treatment was started remained intact throughout the experiment. What Do These Findings Mean These results indicate that treatments designed to lower the production of Aβ may be an important part of future AD treatment, as this approach seems to prevents additional amyloid plaques from forming in the mouse brain. However, by itself, this strategy may not be able to rid the brain of plaques that have already formed in the brain before treatment is started. The findings suggest that early treatment may be important for this approach to succeed. 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☉ 11340230:腹腔镜胆囊切除术后患者肝功能和免疫功能的变化
[摘要] 目的: 观察腹腔镜胆囊切除术(LC)对患者肝功能和免疫功能的影响，为LC后患者的治疗与护理提供依据。 方法: 选择26例胆囊良性病变且肝功能正常需行胆囊切除术者为研究对象，分别在LC前1d、术后第1、3、7天测定肝功能指标(包括ALT、AST、TBIL、ALB)和免疫功能指标(包括CD3、CD4、CD8、NK细胞的百分比、免疫球蛋白IgA、IgM、IgG及补体C3、C4含量)。 结果: ALT、AST、TBIL在术后第1天较术前升高...查看详细 (5917字节)
☉ 11340231:Local Literature Bias in Genetic Epidemiology: An Empirical Evaluation of the Chinese Literature
1 Department of Rheumatology, Shandong Provincial Hospital, Jinan50021, Shandong, China,2 Clinical and Molecular Epidemiology Unit, Department of Hygiene and Epidemiology, University of Ioannina School of Medicine, Ioannina, Greece,3 Institute for Clinical Research and Health Policy Studies, Department of Medicine, Tufts-New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts, United States of America,4 Biomedical Research Institute, Foundation for Research and Technology–Hellas, Ioannina, Greece Background Postulated epidemiological associations are subject to several biases. We evaluated whether the Chinese literature on human genome epidemiology may offer insights on the operation of selective reporting and language biases. Methods and Findings We targeted 13 gene-disease associations, each already assessed by meta-analyses, including at least 15 non-Chinese studies. We searched the Chinese Journal Full-Text Database for additional Chinese studies on the same topics. We identified 161 Chinese studies on 12 of these gene-disease associations; only 20 were PubMed-indexed (seven English full-text). Many studies (14–35 per topic) were available for six topics, covering diseases common in China. With one exception, the first Chinese study appeared with a time lag (2–21 y) after the first non-Chinese study on the topic. Chinese studies showed significantly more prominent genetic effects than non-Chinese studies, and 48% were statistically significant per se, despite their smaller sample size (median sample size 146 versus 268, p T polymorphism and risk of coronary heart disease: a meta-analysis. JAMA 288:2023–2031. Engel LS, Taioli E, Pfeiffer R, Garcia-Closas M, Marcus PM, et al. (2002) Pooled analysis and meta-analysis of glutathione S-transferase M1 and bladder cancer: A HuGE review. Am J Epidemiol 156:95–109. Tarnow L, Gluud C, Parving HH (1998) Diabetic nephropathy and the insertion/deletion polymorphism of the angiotensin-converting enzyme gene. Nephrol Dial Transplant 13:1125–1130. Marcus PM, Vineis P, Rothman N (2000) NAT2 slow acetylation and bladder cancer risk: A meta-analysis of 22 case-control studies conducted in the general population. Pharmacogenetics 10:115–122. Krontiris TG, Devlin B, Karp DD, Robert NJ, Risch N (1993) An association between the risk of cancer and mutations in the HRAS1 minisatellite locus. N Engl J Med 329:517–523. Noble EP (1998) The D2 dopamine receptor gene: A review of association studies in alcoholism and phenotypes. Alcohol 16:33–45. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55:74–108. Li HZ, Rosenblood L (1994) Exploring factors influencing alcohol consumption patterns among Chinese and Caucasians. J Stud Alcohol 55:427–433. Easterbrook PJ, Berlin JA, Gopalan R, Matthews DR (1991) Publication bias in clinical research. Lancet 337:867–872. Dickersin K, Min YI (1993) Publication bias: The problem that won't go away. Ann N Y Acad Sci 703:135–148. Munafo MR, Clark TG, Flint J (2004) Assessing publication bias in genetic association studies: Evidence from a recent meta-analysis. Psychiatry Res 129:39–44. Agema WR, Jukema JW, Zwinderman AH, van der Wall EE (2002) A meta-analysis of the angiotensin-converting enzyme gene polymorphism and restenosis after percutaneous transluminal coronary revascularization: Evidence for publication bias. Am Heart J 144:760–768. Cohen J (1994) The earth is round (P-less-than .05). Amer Psychol 49:997–1003. Ioannidis JP, Polycarpou A, Ntais C, Pavlidis N (2003) Randomised trials comparing chemotherapy regimens for advanced non-small cell lung cancer: Biases and evolution over time. Eur J Cancer 39:2278–2287. Gelernter J, Goldman D, Risch N (1993) The A1 allele at the D2 dopamine receptor gene and alcoholism. A reappraisal. JAMA 269:1673–1677. Ioannidis JP (2005) Contradicted and initially stronger effects in highly cited clinical research. JAMA 294:218–228. Keavney B, McKenzie C, Parish S, Palmer A, Clark S, et al. (2000) Large-scale test of hypothesised associations between the angiotensin-converting-enzyme insertion/deletion polymorphism and myocardial infarction in about 5000 cases and 6000 controls. International Studies of Infarct Survival (ISIS) Collaborators. Lancet 355:434–442. Ioannidis JP (2005) Why most published research findings are false. PLoS Medicine 2:e124. Salanti G, Amountza G, Ntzani EE, Ioannidis JP (2005) Hardy-Weinberg equilibrium in genetic association studies: An empirical evaluation of reporting, deviations, and power. Eur J Hum Genet 13:840–848. Bogardus ST Jr, Concato J, Feinstein AR (1999) Clinical epidemiological quality in molecular genetic research: The need for methodological standards. JAMA 281:1919–1926. Attia J, Thakkinstian A, D'Este C (2003) Meta-analyses of molecular association studies: Methodologic lessons for genetic epidemiology. J Clin Epidemiol 56:297–303. Ioannidis JP, Rosenberg PS, Goedert JJ, O'Brien TR (2002) International Meta-analysis of HIV Host Genetics. Commentary: Meta-analysis of individual participants' data in genetic epidemiology. Am J Epidemiol 156:204–210. Ioannidis JP, Bernstein J, Boffetta P, Danesh J, Dolan S, et al. (2005) A network of investigator networks in human genome epidemiology. Am J Epidemiol 162:302–304. De Angelis C, Drazen JM, Frizelle FA, Haug C, Hoey J, et al. (2004) Clinical trial registration: A statement from the International Committee of Medical Journal Editors. N Engl J Med 351:1250–1251....查看详细 (43654字节)
☉ 11340232:Genetic Prediction of Future Type 2 Diabetes
1 Department of Clinical Sciences, Diabetes and Endocrinology, Lund University, University Hospital Malm, Malm, Sweden,2 School of Mathematical Sciences, Chalmers University of Technology, Gothenburg, Sweden,3 Department of Medicine, Division of Diabetology, Helsinki University Hospital, Helsinki, Finland,4 Folkhlsan Research Center, Institute of Genetics, Helsinki, Finland Background Type 2 diabetes (T2D) is a multifactorial disease in which environmental triggers interact with genetic variants in the predisposition to the disease. A number of common variants have been associated with T2D but our knowledge of their ability to predict T2D prospectively is limited. Methods and Findings By using a Cox proportional hazard model, common variants in the PPARG (P12A), CAPN10 (SNP43 and 44), KCNJ11 (E23K), UCP2 (866G>A), and IRS1 (G972R) genes were studied for their ability to predict T2D in 2,293 individuals participating in the Botnia study in Finland. After a median follow-up of 6 y, 132 (6%) persons developed T2D. The hazard ratio for risk of developing T2D was 1.7 (95% confidence interval [CI] 1.1–2.7) for the PPARG PP genotype, 1.5 (95% CI 1.0–2.2) for the CAPN10 SNP44 TT genotype, and 2.6 (95% CI 1.5–4.5) for the combination of PPARG and CAPN10 risk genotypes. In individuals with fasting plasma glucose ≥ 5.6 mmol/l and body mass index ≥ 30 kg/m2, the hazard ratio increased to 21.2 (95% CI 8.7–51.4) for the combination of the PPARG PP and CAPN10 SNP43/44 GG/TT genotypes as compared to those with the low-risk genotypes with normal fasting plasma glucose and body mass index A) with increased risk of T2D and impaired insulin secretion [24–27], whereas other studies have reported reduced risk of T2D [28]. Increased expression of UCP2 in pancreatic islets is associated with increased uncoupling and thereby decreased ATP production required for insulin secretion [29]. In this study, we tested variants in a number of candidate genes for T2D for their ability to predict diabetes in 2,293 individuals without diabetes participating in the Botnia prospective study in western Finland. Methods Study Participants The Botnia study is a family-based study aiming to identify genes increasing susceptibility to T2D [30,31]. Details of the study cohort and sampling strategy have been presented earlier [31]. In brief, individuals with T2D from the area of five health-care centers in western Finland were invited to participate, together with their family members [31]. An oral glucose tolerance test (OGTT) was performed for all participants aged 18–70 y who had fasting plasma glucose concentration (FPG) lower than 11 mmol/l. Participants without diabetes, either family members of T2D patients or control participants (spouses without first or second degree family history of diabetes), between 18–70 y were offered prospective visits every 2–3 y. During the study period (which started in 1990 and was closed for this analysis in 2002), 1,869 relatives of T2D patients from 577 extended pedigrees (approximately three persons per pedigree) and 424 controls without family history of diabetes participated in at least OGTTs with a median follow-up of 6 y (range 2–12 y). Of the participants in both these groups, 1,569 had normal glucose tolerance and 724 had impaired fasting glucose and/or impaired glucose tolerance at baseline. Carriers of mutations causing maturity onset diabetes of the young (n = 20) were excluded from the present study. Glucose tolerance was defined according to the current World Health Organization criteria [32]. All participants gave informed consent, and the local ethics committee approved the study. Anthropometric Measurements and Assays The participants' weight, height, waist and hip circumference, and blood pressure were measured as previously reported [30,31]. Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters) squared. All participants participated in a 75-g OGTT after a 12-h overnight fast. Fasting blood samples were drawn for the measurement of high density lipoprotein cholesterol, triglyceride, and free fatty acid concentrations, and at 10, 0, 30, 60, and 120 min for the measurement of plasma glucose and serum insulin. Insulin resistance was estimated as homeostasis model assessment index (HOMAIR) using a computer-based model [33] and β-cell function as the ratio of incremental insulin to glucose responses during the first 30 min of the OGTT (ΔI/ΔG = ΔI30 min fasting/ΔG30 min fasting); this index is also called the insulinogenic index. The disposition index was used to adjust insulin secretion for the degree of insulin resistance (insulinogenic index/HOMAIR). Genotyping Genotyping of SNPs was performed with a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method and agarose gel electrophoresis for IRS1 G972R, or using the Multiplex SNaPshot kit (Applied Biosystems, Stockholm, Sweden) for single base pair extension on ABI 3100 (Applied Biosystems) for CAPN10 SNP43 and 44, or with an allelic discrimination assay-by-design method on ABI 7900 (Applied Biosystems) for KCNJ11 E23K, IRS1 G972R, and UCP2 866G>A (Table S1). By randomly regenotyping 10%–20% of the samples, we achieved concordance rates of 99% for PPARG P12A, CAPN10 SNP44, KCNJ11 E23K, IRS1 G972R, and UCP2 866G>A, and 98% for CAPN10 SNP43. All genotypes were in Hardy–Weinberg equilibrium except CAPN10 SNP44, which showed a moderate deviation from equilibrium (p = 0.035). Genotyping errors are an unlikely explanation for this deviation from equilibrium, because in genotyping 1,880 samples of CAPN10 SNP44 using two different methods (allelic discrimination and single base extension) the concordance rate was 99%. Statistical Analyses Variables are presented as median (interquartile range). A χ2 test was used for comparison of group frequencies. Survival analysis was used to estimate the effect of genetic variants (risk and non-risk genotypes defined from previous studies) on the risk of developing T2D and shown by Kaplan–Meier survival curves as the distribution of age at onset (the proportion of individuals developing T2D at certain age). The risk of developing T2D was expressed as a hazard function (the negative slope divided by the survival curve) using an age-adjusted Cox proportional hazard regression model [34]. The hazard function is the (conditional) probability for the development of diabetes during a time interval divided by the length of that time interval, for an individual that is diabetes-free at the start of the time interval. The relative effect is presented as the ratio between the hazard functions (hazard ratio [HR]) of the two groups. HRs quantify the effect size of both discrete variables (carriers versus non-carriers) and continuous variables (used in the interaction analysis below where HR measures the effect of an increase in one unit of the continuous variable). All survival analyses were stratified for gender and adjusted for family history of diabetes and BMI (when appropriate). The information that an individual did not or did belong to a nuclear family with at least one other affected member was coded as zero or one, respectively, and used as a covariate in the Cox regression analyses. All survival analyses were performed with a robust variance estimate to adjust for within family dependence extended to the large pedigrees. In using a robust variance estimate we treated each pedigree (instead of each individual) as an independent entity for calculating the variance of the estimates. Expected risk genotypes were defined according to earlier reports (PP genotype of PPARG, GG genotype of CAPN10 SNP43, TT genotype of CAPN10 SNP44, EK/KK genotypes of KCNJ11, GR/RR genotypes of IRS1, GG genotype of UCP2). However, the risk TT genotype of CAPN10 SNP44 was in opposite direction compared to other studies [13,14] and selected based upon a previous report from the Botnia study [15] showing that the combination of the TT genotype of SNP44 and the GG genotype of SNP43 in CAPN10 was significantly more frequent in patients with T2D than in control individuals. Therefore, in the present study we refer to the TT genotype of CAPN10 SNP44 as an at-risk genotype. Individuals with missing data were excluded from the analyses. Analyses of interaction between effect of phenotype (P) defined as insulin secretion (disposition index) and insulin action (HOMAIR) and genotype (G) (1 = risk and 0 = non-risk) on age at onset of T2D were performed using the following Cox proportional hazards model: h(t) = h0(t)exp(β1P + β2G + β3PG), in which h(t) is the hazard function and h0(t) is the baseline hazard function, with β1 and β2 measuring the univariate effects and β3 measuring the interaction. If there is an interaction (β3 ≠ 0), the HR for carriers and non-carriers of the risk genotype will not be the same. Thus, in different genotype carriers the HR of T2D associated with x units increase/decrease in the phenotypic value (P + x) is HR = exp(β1 + β3)x for the risk genotype carriers and HR = exp(β1x) for the non-risk genotype carriers. A logistic regression analysis was applied to explore the relationship between FPG and BMI, with genetic factor (defined as 1 = risk and 0 = non-risk) as dependent variable and FPG, BMI, and an interaction term as covariates. All statistical analyses were performed using Number Crunching Statistical Systems version 2004 (NCSS, Kaysville, Utah, United States), R (www.r-project.org, and Stata (StataCorp, College Station, Texas, United States). Two-sided p-values of less than 0.05 were considered statistically significant. Results In total, 2,293 persons (1,051 men and 1,242 women) were included in the study (Table 1). Of them, 1,078 (47%) had non-normal FPG (≥ 5.6 mmol/l), 280 (12.3%) had BMI ≥ 30 kg/m2, and 160 (7%) had both elevated FPG and BMI ≥ 30 kg/m2. Of the 2,293 persons included, 132 (6%) (67 men and 65 women; 40 with normal and 92 with abnormal glucose tolerance) developed diabetes during the follow-up period of 6 y (converters). PPARG The allele and genotype frequencies of the PPARG P12A polymorphism were similar to those previously reported in Caucasians [4], with 73.3% of participants carrying the risk PP genotype (Table 2). Of all individuals who developed T2D, 109 (82.6%) had the PP genotype, which also significantly increased the risk of subsequent T2D (HR 1.7, p = 0.016) (Figure 1; Table 3). Because we have previously shown that a family history of diabetes, non-normal FPG (≥ 5.6 mmol/l), and BMI ≥ 30kg/m2 identify individuals at high risk of T2D [31], we now tested whether the PPARG risk genotype could replace family history in this prediction. In fact, the incidence of T2D was increased in carriers of the PP genotype with elevated FPG and high BMI as compared with the PA/AA genotype carriers without any other risk factors (22.9% versus 1.5%, p A variant. The GG genotype was associated with a modestly increased risk of T2D (HR 1.4, p = 0.049) (see Figure 1; Table 3). This risk was not influenced by BMI and FPG at baseline. However, the GG genotype was also associated with increased risk of developing T2D in patients with earlier onset of diabetes (HR 2.0, 95% CI 1.2–3.3, p = 0.0057) (Table 4). Furthermore, the GG genotype was also more frequent among patients with earlier than late onset of T2D (60.3% versus 39.7%, p = 0.042; χ2 test and odds ratio 2.5, 95% CI 1.2–5.3, p = 0.016; logistic regression analyses adjusted for gender, BMI, and family history of diabetes). None of the other tested genotypes predicted significantly earlier onset of T2D. IRS1 Twenty-three (17.6%) converters carried the RR/RG genotypes of the IRS1 G972R polymorphism. Whereas the R allele had no independent effect on T2D risk, it increased the risk of T2D in a dominant fashion (RR or RG versus GG) in participants with elevated FPG and BMI ≥ 30 kg/m2 to 9.3 (95% CI 3.6–23.9, p A (rs659366). Acknowledgments This work was supported by grants from the Sigrid Juselius Foundation, European Community (Genomic Integrated Force for Type 2 Diabetes, grant QLG2-Ct-1999–0546), Folkhlsan Research Foundation, the Swedish Research Council, Academy of Finland, the Lundberg Foundation, the Novo Nordic Foundation, the European Federation for the Study of Diabetes (Sankyo Pharma), the Diabetes Research Foundation, the Albert Phlssons Foundation, Crafoord Foundation, and the Anna-Lisa and Sven-Eric Lundgren Foundation. We thank the patients for their participation and the Botnia research team, as well as Malin Svensson and Barbro Gustavsson for excellent technical assistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Botnia Study Group Investigator from Vasa, Finland: Mikael Nissen. Investigators from Jakobstads Health Center, Jakobstads Hospital, and Folkhalsan stanlid, Jakobstad, Finland: Bo Isomaa, Leena Sarelin, and Carola Svenfelt. Investigators from Korsholms Health Center, Korsholm, Finland: Ulla-Britt Bjrk, Nils Holmstrm, and Jessica Strand. Investigators from Malax Health Center, Malax, Finland: Lisbeth kerman and Inga-Britt Stenback. Investigators from Nrpes Health Center, Nrpes, Finland: Bjrn Forsen, Monika Gullstrm, Maja Hggblom, and Susann Sderback. Investigators from Vasa Health Center, Vasa, Finland: Kaj Lahti, Marianne Nyman, and Sonja Paulaharju. Investigators from Department of Medicine, Helsinki University Central Hospital; Folkhalsan Research Center, Department of Genetics, and Research Program fr Molecular Medicine, University of Helsinki, Helsinki, Finland: Seija Heikkinen, Paula Kokko, Merja Lahtinen, Mikko Lehtovirta, and Virve Lundgren. Investigators from Department of Medicine, Helsinki University Central Hospital; Research Program for Cardiovascular Diseases, University of Helsinki, Helsinki, Finland: Hannele Hilden and Marja-Riitta Taskinen. Investigators from Department of Clinical Sciences, Diabetes, and Endocrinology, Lund University, Malm University Hospital, Malm, Sweden: Esa Laurila and Margareta Svensson. Author Contributions: VL extracted, genotyped, and analyzed the data, and drafted the report. PA and DA were responsible for the statistical analyses, MOM and MS for genotyping, and CS and TT for the phenotype data. LG designed the study and supervised all parts of the work including drafting the final report. All researchers took part in the revision of the report and approved the final version. Patient Summary Background Type 2 diabetes, also known as adult onset or non-insulin-dependent diabetes, is increasing in frequency around the world. Many different factors work together to make someone more likely to develop diabetes, including factors in their environment—for example, the food they eat—and in their family background—the genes they inherited from their parents. Many studies have been done looking at which genes are associated with diabetes, but few have tried to see whether it is possible to predict who will get diabetes in future from looking at a person's genes before any symptoms develop. Why Was This Study Done These authors wanted to look at changes in five genes previously shown to be associated with diabetes in a group of people who were to be followed prospectively—that is, from before they developed diabetes—and see if it was possible to predict who would get diabetes. What Did the Researchers Do and Find They studied 2,293 people in Finland who were family members or spouses of people with type 2 diabetes, but who themselves did not have diabetes. They followed these people for up to 12 years, starting in 1990. In total, 132 of these individuals (6%) developed diabetes during this time. They found that changes in two of the genes, PPARG (which is involved in how the body regulates fat tissue) and CAPN10 (which is involved in modifying certain proteins), were associated with people having a higher chance of getting type 2 diabetes. This chance was increased substantially when the participants already had slightly raised blood glucose, and a high body mass index. What Do These Findings Mean In some people, it does seem possible to use certain genes to predict whether a person will develop type 2 diabetes. However, environmental factors are also very important, and any risk is much increased in people who are already overweight. Where Can I Get More Information Online MedlinePlus has many links to pages of information on diabetes: The Finnish Diabetes Association has information on diabetes in general and more specifically for Finland: Patient Summary Background Type 2 diabetes, also known as adult onset or non-insulin-dependent diabetes, is increasing in frequency around the world. Many different factors work together to make someone more likely to develop diabetes, including factors in their environment—for example, the food they eat—and in their family background—the genes they inherited from their parents. Many studies have been done looking at which genes are associated with diabetes, but few have tried to see whether it is possible to predict who will get diabetes in future from looking at a person's genes before any symptoms develop. Why Was This Study Done These authors wanted to look at changes in five genes previously shown to be associated with diabetes in a group of people who were to be followed prospectively—that is, from before they developed diabetes—and see if it was possible to predict who would get diabetes. What Did the Researchers Do and Find They studied 2,293 people in Finland who were family members or spouses of people with type 2 diabetes, but who themselves did not have diabetes. They followed these people for up to 12 years, starting in 1990. In total, 132 of these individuals (6%) developed diabetes during this time. They found that changes in two of the genes, PPARG (which is involved in how the body regulates fat tissue) and CAPN10 (which is involved in modifying certain proteins), were associated with people having a higher chance of getting type 2 diabetes. This chance was increased substantially when the participants already had slightly raised blood glucose, and a high body mass index. What Do These Findings Mean In some people, it does seem possible to use certain genes to predict whether a person will develop type 2 diabetes. However, environmental factors are also very important, and any risk is much increased in people who are already overweight. Where Can I Get More Information Online MedlinePlus has many links to pages of information on diabetes: The Finnish Diabetes Association has information on diabetes in general and more specifically for Finland: References Zimmet P, Alberti KG, Shaw J (2001) Global and societal implications of the diabetes epidemic. Nature 414:782–787. Bonadonna RC (2004) Alterations of glucose metabolism in type 2 diabetes mellitus. An overview. Rev Endocr Metab Disord 5:89–97. Chan JM, Rimm EB, Colditz GA, Stampfer MJ, Willett WC (1994) Obesity, fat distribution, and weight gain as risk factors for clinical diabetes in men. Diabetes Care 17:961–969. Altshuler D, Hirschhorn JN, Klannemark M, Lindgren CM, Vohl MC, et al. (2000) The common PPARgamma Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes. Nat Genet 26:76–80. Barroso I, Luan J, Middelberg RP, Harding AH, Franks PW, et al. (2003) Candidate gene association study in type 2 diabetes indicates a role for genes involved in β-cell function as well as insulin action. PLoS Biol 1:e20 DOI: 10.1371/journal.pbio.0000020. Florez JC, Hirschhorn J, Altshuler D (2003) The inherited basis of diabetes mellitus: Implications for the genetic analysis of complex traits. Annu Rev Genomics Hum Genet 4:257–291. Laukkanen O, Pihlajamaki J, Lindstrom J, Eriksson J, Valle TT, et al. (2004) Common polymorphisms in the genes regulating the early insulin signalling pathway: Effects on weight change and the conversion from impaired glucose tolerance to type 2 diabetes. The Finnish Diabetes Prevention Study. Diabetologia 47:871–877. Parikh H, Groop L (2004) Candidate genes for type 2 diabetes. Rev Endocr Metab Disord 5:151–176. Lindi VI, Uusitupa MI, Lindstrom J, Louheranta A, Eriksson JG, et al. (2002) Association of the Pro12Ala polymorphism in the PPAR-gamma2 gene with 3-year incidence of type 2 diabetes and body weight change in the Finnish Diabetes Prevention Study. Diabetes 51:2581–2586. Memisoglu A, Hu FB, Hankinson SE, Liu S, Meigs JB, et al. (2003) Prospective study of the association between the proline to alanine codon 12 polymorphism in the PPARgamma gene and type 2 diabetes. Diabetes Care 16:2915–2917. Andrulionyte L, Zacharova J, Chiasson JL, Laakso M (2004) Common polymorphisms of the PPAR-gamma2 (Pro12Ala) and PGC-1alpha (Gly482Ser) genes are associated with the conversion from impaired glucose tolerance to type 2 diabetes in the STOP-NIDDM trial. Diabetologia 47:2176–2184. Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, et al. (2000) Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet 26:163–175. Weedon MN, Schwarz PE, Horikawa Y, Iwasaki N, Illig T, et al. (2003) Meta-analysis and a large association study confirm a role for calpain-10 variation in type 2 diabetes susceptibility. Am J Hum Genet 73:1208–1212. MSong Y, Niu T, Manson JE, Kwiatkowski DJ, Liu S (2004) Are variants in the CAPN10 gene related to risk of type 2 diabetes A quantitative assessment of population and family-based association studies. Am J Hum Genet 74:208–222. Orho-Melander M, Klannemark M, Svensson MK, Ridderstrale M, Lindgren CM, et al. (2002) Variants in the calpain-10 gene predispose to insulin resistance and elevated free fatty acid levels. Diabetes 51:2658–2664. Gloyn AL, Weedon MN, Owen KR, Turner MJ, Knight BA, et al. (2003) Large-scale association studies of variants in genes encoding the pancreatic beta-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) confirm that the KCNJ11 E23K variant is associated with type 2 diabetes. Diabetes 52:568–572. Laukkanen O, Pihlajamaki J, Lindstrom J, Eriksson J, Valle TT, et al. (2004) Polymorphisms of the SUR1 (ABCC8) and Kir6.2 (KCNJ11) genes predict the conversion from impaired glucose tolerance to type 2 diabetes. The Finnish Diabetes Prevention Study. J Clin Endocrinol Metab 89:6286–6290. Florez JC, Burtt N, De Bakker PI, Almgren P, Tuomi T, et al. (2004) Haplotype structure and genotype-phenotype correlations of the sulfonylurea receptor and the islet ATP-sensitive potassium channel gene region. Diabetes 53:1360–1368. Marchetti P, Lupi R, Federici M, Marselli L, Masini M, et al. (2002) Insulin secretory function is impaired in isolated human islets carrying the Gly(972)→Arg IRS-1 polymorphism. Diabetes 51:1419–1424. Jellema A, Zeegers MP, Feskens EJ, Dagnelie PC, Mensink RP (2003) Gly972Arg variant in the insulin receptor substrate-1 gene and association with type 2 diabetes: A meta-analysis of 27 studies. Diabetologia 46:990–995. Zeggini E, Parkinson J, Halford S, Owen KR, Frayling TM, et al. (2004) Association studies of insulin receptor substrate 1 gene (IRS1) variants in type 2 diabetes samples enriched for family history and early age of onset. Diabetes 53:3319–3322. Florez JC, Sjogren M, Burtt N, Orho-Melander M, Schayer S, et al. (2004) Association testing in 9,000 people fails to confirm the association of the insulin receptor substrate-1 G972R polymorphism with type 2 diabetes. Diabetes 53:3313–3318. van Dam RM, Hoebee B, Seidell JC, Schaap MM, Blaak EE, et al. (2004) The insulin receptor substrate-1 Gly972Arg polymorphism is not associated with type 2 diabetes mellitus in two population-based studies. Diabet Med 21:752–758. Wang H, Chu WS, Lu T, Hasstedt SJ, Kern PA, et al. (2004) Uncoupling protein-2 polymorphisms in type 2 diabetes, obesity, and insulin secretion. Am J Physiol Endocrinol Metab 286:E1–E7. Sasahara M, Nishi M, Kawashima H, Ueda K, Sakagashira S, et al. (2004) Uncoupling protein 2 promoter polymorphism 866G/A affects its expression in beta-cells and modulates clinical profiles of Japanese type 2 diabetic patients. Diabetes 53:482–485. D'Adamo M, Perego L, Cardellini M, Marini MA, Frontoni S, et al. (2004) The 866A/A genotype in the promoter of the human uncoupling protein 2 gene is associated with insulin resistance and increased risk of type 2 diabetes. Diabetes 53:1905–1910. Bulotta A, Ludovico O, Coco A, Di Paola R, Quattrone A, et al. (2005) The common 866G/A polymorphism in the promoter region of the UCP2 gene is associated with reduced risk of type 2 diabetes in Caucasians from Italy. J Clin Endocrinol Metab 90:1176–1180. Krempler F, Esterbauer H, Weitgasser R, Ebenbichler C, Patsch JR, et al. (2002) A functional polymorphism in the promoter of UCP2 enhances obesity risk but reduces type 2 diabetes risk in obese middle-aged humans. Diabetes 51:3331–3335. Chan CB, De Leo D, Joseph JW, McQuaid TS, Ha XF, et al. (2001) Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion: Mechanism of action. Diabetes 50:1302–1310. Groop L, Forsblom C, Lehtovirta M, Tuomi T, Karanko S, et al. (1996) Metabolic consequences of a family history of NIDDM (the Botnia study): Evidence for sex-specific parental effects. Diabetes 45:1585–1593. Lyssenko V, Almgren P, Anevski D, Perfekt R, Lahti K, et al. (2005) Predictors of and longitudinal changes in insulin sensitivity and secretion preceding onset of type 2 diabetes. Diabetes 54:166–174. World Health Organization. (1999) Definition, diagnosis, and classification of diabetes mellitus and its complications. Report of a WHO consultation. Part 1: Diagnosis and classification of diabetes mellitus Geneva: World Health Organization. Levy JC, Matthews DR, Hermans MP (1998) Correct homeostasis model assessment (HOMA) evaluation uses the computer program. Diabetes Care 21:2191–2192. Klein JP, Moeschberger ML (2003) Survival analysis: Techniques for censored and truncated data, 2nd ed New York: Springer. 536 p. Deeb SS, Fajas L, Nemoto M, Pihlajamaki J, Mykkanen L, et al. (1998) A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nat Genet 20:284–287. Stumvoll M, Haring H (2002) Reduced lipolysis as possible cause for greater weight gain in subjects with the Pro12Ala polymorphism in PPARgamma2 Diabetologia 45:152–153. Nicklas BJ, van Rossum EF, Berman DM, Ryan AS, Dennis KE, et al. (2001) Genetic variation in the peroxisome proliferator-activated receptor-gamma2 gene (Pro12Ala) affects metabolic responses to weight loss and subsequent weight regain. Diabetes 50:2172–2176. Luan J, Browne PO, Harding AH, Halsall DJ, O'Rahilly S, et al. (2001) Evidence for gene-nutrient interaction at the PPARgamma locus. Diabetes 50:686–689. Baier LJ, Permana PA, Yang X, Pratley RE, Hanson RL, et al. (2000) A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. J Clin Invest 106:R69–R73. O'Rahilly S, Spivey RS, Holman RR, Nugent Z, Clark A, et al. (1987) Type II diabetes of early onset: A distinct clinical and genetic syndrome Br Med J 294:923–928. Esterbauer H, Schneitler C, Oberkofler H, Ebenbichler C, Paulweber B, et al. (2001) A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans. Nat Genet 28:178–183. Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, et al. (2004) Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. N Engl J Med 350:1838–1849. Nichols CG, Koster JC (2002) Diabetes and insulin secretion: Whither KATP Am J Physiol Endocrinol Metab 283:E403–E412....查看详细 (46246字节)

☉ 11340233:白内障超声乳化术后缺血性视神经病变的临床分析
[关键词] 白内障;超声乳化术;缺血性视神经病变 白内障超声乳化手术具有切口小，副反应少，术后散光小的特点，是当前最常用的白内障术式之一，但其手术并发症也引起临床医生的重视。缺血性视神经病变是其并发症之一，严重影响视功能。2005年1月至2006年1月，我院行白内障超声乳化手术后并发缺血性视神经病变7例，现将其发病原因及治疗方法总结如下。 1 临床资料 患者共7例(7只眼)...查看详细 (4159字节)
☉ 11340234:医学研究单位加强科研经费管理的做法与体会
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☉ 11340235:Underutilization of Aspirin Persists in US Ambulatory Care for the Secondary and Primary Prevention of Cardiovascular Disease
1 Program on Prevention Outcomes and Practices, Stanford Prevention Research Center, Stanford University School of Medicine, Stanford, California, United States of America Background Despite the proven benefits of aspirin therapy in the primary and secondary prevention of cardiovascular disease (CVD), utilization rates of aspirin remain suboptimal in relation to recommendations. We studied national trends of aspirin use among intermediate- to high-risk patients in the US ambulatory care settings and compared the priority given to aspirin versus statins for CVD risk reduction. We also examined patient and health care provider contributors to the underuse of aspirin. Methods and Findings We used the 1993–2003 US National Ambulatory Medical Care Survey and National Hospital Ambulatory Medical Care Survey to estimate aspirin use by cardiovascular risk. Physician-noted cardiovascular diseases defined high risk. Intermediate risk was defined as having diabetes mellitus or multiple major risk factors. The proportion of patient visits in which aspirin was reported increased from 21.7% (95% confidence interval: 18.8%–24.6%) in 1993–1994 to 32.8% (25.2%–40.4%) in 2003 for the high-risk category, 3.5% (2.0%–5.0%) to 11.7% (7.8%–15.7%) for visits by patients diagnosed with diabetes, and 3.6% (2.6%–4.6%) to 16.3% (11.4%–21.2%) for those with multiple CVD risk factors. Beginning in 1997–1998, statins were prioritized over aspirin as prophylactic therapy for reducing CVD risk, and the gaps remained wide through 2003. In addition to elevated CVD risk, greater aspirin use was independently associated with advanced age, male gender, cardiologist care, and care in hospital outpatient departments. Conclusion Improvements in use of aspirin in US ambulatory care for reducing risks of CVD were at best modest during the period under study, particularly for secondary prevention, where the strongest evidence and most explicit guidelines exist. Aspirin is more underused than statins despite its more favorable cost-effectiveness. Aggressive and targeted interventions are needed to enhance provider and patient adherence to consensus guidelines for CVD risk reduction. Academic Editor: Colin Baigent, Radcliffe Infirmary, United Kingdom Introduction Cardiovascular disease (CVD), including myocardial infarction and stroke, is the leading cause of morbidity and mortality in the United States. A broad array of randomized trials have demonstrated the benefits of low doses of aspirin (75–325 mg) [1,2] for both the primary [3–7] and secondary prevention [8–11] of CVD. Most trials demonstrate a 15%–40% reduction in cardiovascular events with chronic aspirin use. Aspirin is unequivocally recommended as a secondary prevention strategy in non-contraindicated patients with known CVD [12,13]. As for primary prevention, the American Diabetic Association recommends regular aspirin for men and women with diabetes mellitus (DM) who are older than 40 y or have additional cardiovascular risk factors [14]. In addition, aspirin is indicated for apparently healthy individuals without CVD or DM but otherwise with an increased cardiovascular risk, which is defined as a 3% or greater risk in 5 y by the US Preventive Services Task Force [2] or a 10% or greater risk in 10 y by the American Heart Association [1]. However, the latest results from the Women's Health Study [7] suggest that careful ascertainment of the absolute benefit and risk on a case-by-case basis is essential to deciding on the use of aspirin therapy in men and, even more so, in women who have showed no clinical manifestations of CVD or diabetes. Despite the proven benefits of aspirin therapy for reducing cardiovascular risk, aspirin use falls considerably short of recommendations. National surveys of the prescribing of cardiac medications found that aspirin use in visits by patients with coronary heart disease (CHD) increased significantly from 5% in 1980 to 32% in 1995, but then remained unchanged or even declined in subsequent years [15,16]. The Third National Health and Nutrition Examination Survey (also called NHANES III) data showed that among patients with DM, only 37% of those with CHD and 13% of those with risk factors for CHD were regular aspirin users [17]. While aspirin underutilization is also present in other countries [18,19], some evidence suggests that the problem is more prominent in the US. For instance, outpatient use of aspirin for secondary prevention ranged from approximately 40% to 90% in many European countries, in comparison to approximately 24% in the US [15,20–23]. Greater aspirin use is associated with middle to older age (55–75 y old), male gender, diagnosis of hyperlipidemia, smoking, having medical insurance, revascularization or coronary angioplasty, and use of other medications [24–28]. Despite ample evidence of aspirin underutilization, research on national trends of outpatient aspirin use by CVD risk category is limited. Using two companion national datasets on ambulatory care in the US, our study tracked changes from 1993–2003 in reported aspirin use by CVD risk status, distinguishing between secondary and primary prevention. Multiple reasons may account for the widespread aspirin underutilization, one being lower priority assigned to aspirin therapy compared to other medications available for CVD risk reduction. To explore this possibility, we examined the priority given to aspirin in comparison to statins. We also examined patient and physician contributors to shortfalls in aspirin use. Methods The Stanford University Institutional Review Board determined that this study was exempt from “human subjects” requirements. Data Sources We obtained annual data 1993–2003 from the National Ambulatory Medical Care Survey (NAMCS) and the Outpatient Department component of the National Hospital Ambulatory Medical Care Survey (NHAMCS). The National Center for Health Statistics provides complete descriptions of both surveys and yearly data at http://www.cdc.gov/nchs/about/major/ahcd/ahcd1.htm. These surveys, particularly NAMCS, have been validated against other data sources [29,30] and utilized in past research on aspirin use for CVD risk reduction [16,25]. In brief, NAMCS captured health-care services provided by private office-based physicians, while NHAMCS captured services offered at hospital outpatient departments. The sampling universe for NAMCS was office-based, patient-care physicians in 15 specialty strata from the master files maintained by the American Medical Association and American Osteopathic Association. The sampling frame for NHAMCS included short-stay (shorter than 30 d) hospitals, or general-specialty (medical or surgical) or children's general hospitals. Both surveys utilized multistage probability sampling procedures, which enable researchers to generate nationally representative estimates. Between 1993 and 2003, annual participation rates among physicians selected for NAMCS averaged 70%, while the participation rate of selected hospitals with outpatient departments was 90% in NHAMCS. Standard encounter forms were completed for a systematic random sample of patient visits during randomly assigned reporting periods. Yearly encounter forms varied slightly between NAMCS and NHAMCS and were revised every 2 y. We based this study on variables common to NAMCS and NHAMCS over time, including patient demographics, visit characteristics, reasons for visit (up to three), diagnoses (up to three), and new and continuing medications (up to five in 1993–1994, six in 1995–2002, and eight in 2003). Item nonresponse rates were mostly 5% or less in both surveys for all years. Patients CHD risk categorization We defined four mutually exclusive categories of CVD risk based on the presence of specific diagnoses and risk factors. The presence of CHD, myocardial infarction, stroke or transient ischemic attack, peripheral vascular disease, claudication, or angina defined high CVD risk. In the absence of known CVD, visits by patients with DM who were older than 40 y or had additional risk factors (i.e., hypertension, smoking, dyslipidemia, and/or albuminuria) were defined as intermediate risk. The remaining patients were defined in a second intermediate risk category if they met either of the following criteria: (1) Two or more major CVD risk factors (i.e., hypertension, smoking, and/or dyslipidemia) among men age 45–54 and women age 55–64; or (2) One or more risk factors among men older than 55 and women older than 65. Patient visits ineligible for any of the former three categories were considered low risk. The absence of data elements such as family history of premature CHD or levels of high-density lipoprotein cholesterol precluded more accurate risk stratification according to the Framingham risk scoring [31]. Patient visit characteristics We included the following patient visit characteristics: patient age, gender, race/ethnicity, health care insurance status, visit status, US census region, metropolitan area status, and physician specialty. Health care insurance was classified as private/commercial insurance, public insurance (i.e., Medicare and Medicaid), and other insurance (e.g., workers' compensation and self-pay). Visit status distinguished first-time visits from return visits to a physician's practice. Physician specialty was available only from NAMCS, which contributed more than 90% of the total weighted visits for each of the study years. We categorized physician specialties into cardiology, internal medicine, general and family practice, and a category encompassing all others. Measures Our primary analytic goals were to assess the probability of aspirin use by CVD risk and its relationship to patient visit characteristics. The probability of aspirin use was defined as the proportion of non-contraindicated patient visits in which aspirin or a therapeutically equivalent medication was reported as a new or continuing medication. Measuring the probability of use by CVD risk provided a means to estimate the gaps between current practice and evidence-based medicine regarding aspirin therapy. We defined aspirin therapy as reported use of generic or brand name forms of aspirin, clopidogrel, ticlopidine, or non-narcotic combination analgesics containing aspirin. The number of patient visits in which clopidogrel or ticlopidine was reported is too small to allow their use over time being tracked separately. Oral anticoagulants are not considered aspirin equivalents and are not recommended for the primary or secondary prevention of CVD in a vast majority of patients. Moreover, judging the appropriateness of using or avoiding aspirin for someone who is already on anticoagulant therapy required more clinical detail than our data sources can provide. Therefore, we felt it was appropriate to exclude patients on anticoagulant therapy. We were unable to assess patients' use of over-the-counter aspirin if it was not reported on the encounter form. We excluded visits by patients younger than 21 y and those with bleeding tendency, gastrointestinal bleeding, anticoagulant therapy, or clinically active hepatic disease. Statistical Analyses Statistical analyses accounting for sampling weights and the complex sample designs of NAMCS and NHAMCS were performed using SAS for Windows software (SAS Institute, Cary, North Carolina, United States) and SAS-callable SUDAAN software (RTI, Research Triangle Park, North Carolina, United States). The unit of analysis in both surveys was the patient visit. Comparisons of NAMCS and NHAMCS suggested limited differences on key outcome measures. We therefore combined the two surveys to obtain a wider range of outpatient settings and a broader socioeconomic spectrum of patients seeking ambulatory care. Also, to minimize random fluctuations between years, we analyzed data in 2-y groupings, except for 2003, for depicting temporal trends in aspirin use. The SAS SURVEYMEANS procedure was performed, which generated national estimates of the probability of aspirin use by CVD risk and corresponding 99% confidence intervals (CIs). We chose to report 99% CIs in following National Center for Health Statistics analytical guidelines and also as a conservative measure to avoid over-interpretation of the findings. Chi-square tests were performed using PROC CROSSTAB in SUDAAN to examine the association of aspirin use with each patient visit characteristic based on combined 1993–2003 NAMCS and NHAMCS data. The independent effect of each patient visit characteristic on aspirin use after controlling for all other characteristics was assessed with a multivariate logistic regression model using PROC RLOGISTIC in SUDAAN. The model produced adjusted odds ratios and 99% CIs. Results The volume of outpatient visits by patients identified as being at elevated risk for future CVD events, particularly those at intermediate risk, rose markedly over the study period. The number of high-risk patient visits increased by 33% from 44.2 (99% CI, 41.0–47.4) million in 1993–1994 to 58.8 (54.0–63.6) million in 2001–2002. The number of intermediate-risk patient visits in which a diagnosis of DM was noted more than doubled, from 40.5 (37.1–43.9) million to 83.3 (77.4–89.3) million, and for those with multiple risk factors the increase was 57%, from 70.2 (65.7–74.7) million to 110.4 (102.8–118.0) million. The number of low-risk patient visits rose by 23%, from 975.4 (962.6–988.2) million to 1.20 (1.18–1.22) billion. In 2003, the number of patient visits in each of the four risk categories was 29.5 (22.5–36.6) million for high-risk patients, 39.9 (32.0–47.9) million for intermediate-risk patients, 55.8 (45.5–66.2) million for those with multiple risk factors, and 626.9 (537.1–716.7) million for those with low risk. Trends over time showed improved, though still substantially suboptimal, aspirin use in the high and intermediate risk categories, with sustained improvements seen beginning in 1999–2000 (Figure 1). The probability of aspirin use among patient visits in 1993–1994 was 21.7% (18.8%–24.6%) for the high-risk category, 3.5% (2.0%–5.0%) for the diabetic, intermediate-risk category, and 3.6% (2.6%–4.6%) for the other intermediate-risk category. The probabilities for these three risk categories fluctuated somewhat but remained essentially unchanged through 1999–2000. Increases were observed in 2001–2002 and persisted in 2003. The probability of aspirin use in 2003 was 32.8% (25.2%–40.4%) for the high-risk category, 11.7% (7.8%–15.7%) for the diabetic, intermediate-risk category, and 16.3% (11.4%–21.2%) for the other intermediate-risk category. Aspirin use remained 1%–3% among low-risk patient visits. To explore the relative priority assigned to aspirin and statins, we examined trends in the co-prescribing of the medications. For this series of analyses, the number of visits by patients with DM was relatively small and were therefore grouped with those with known CVD to compose the high-risk category. Both aspirin and statins were used more frequently when the other therapy was present; however, improvements over time were more evident for statin use among aspirin-treated patient visits than for aspirin use among statin-treated patients. Specifically, the proportion of visits by high-risk patients on aspirin while a statin was used declined modestly from 36.5% (24.9%–48.2%) in 1993–1994 to 25.6% (20.1%–31.1%) in 1999–2000 but then rebounded to 43.9% (35.1%–52.8%) in 2003 (Figure 2). In contrast, statin use among visits by high-risk patients on aspirin grew successively from 11.6% (7.4%–15.7%) to 54.3% (45.7%–63.0%) (Figure 2). Of visits by intermediate-risk patients, the probability of aspirin use when on a statin increased from 6.0% (1.4%–10.6%) in 1993–1994 to 33.8% (21.5%–46.0%) in 2003, while the probability of statin use when on aspirin rose from 8.8% (2.2%–15.3%) to 48.1% (35.2%–61.0%) (Figure 3). Vertical bars indicate 99% CIs. Vertical bars indicate 99% CIs. The association of greater aspirin use with higher CVD risk was confirmed by multivariate logistic regression (Table 1). After adjusting for patient visit characteristics and the number of medications reported, aspirin use was over four time as likely among visits by high-risk patients and approximately two times as likely among visits by patients with multiple risk factors as it was among low-risk patient visits. The odds ratio was marginally significant for the diabetic, intermediate-risk category. The significance of increases in aspirin use over time did not sustain in the multivariate logistic regression. As for patient visit characteristics, lower probability of aspirin use was found among 20- to 44-y-olds (versus those 45 y or older), women (versus men), visits to noncardiologists (versus visits to cardiologists), return visits (versus first-time visits), and visits to private physician offices (versus visits to hospital outpatient departments). Finally, the probability of aspirin use was positively associated with the number of medications reported (odds ratio, 1.71; 99% CI, 1.65–1.76). Discussion This study documents national trends in the probability of aspirin use by CVD risk category among patient visits to office-based physicians and hospital outpatient departments. Some improvements were observed over time in the use of aspirin for both the secondary and primary prevention of CVD. However, the magnitude of those improvements is minimal relative to the substantial gaps between clinical practice and evidence-based recommendations. The gaps observed with secondary prevention are particularly concerning, given the existence of conclusive clinical evidence and unequivocal practice guidelines. The use of aspirin among primary prevention patients, including those with diabetes, also appears to be suboptimal, but additionally may reflect uncertainty about the evidence. Our analysis also suggests that despite aspirin's more favorable cost-effectiveness, statins have been prioritized ahead of aspirin as therapy for reducing CVD risk. While ample evidence attests to the underuse of aspirin in reducing risks of CVD, this study uniquely provides an 11-y trajectory of aspirin use in US outpatient settings and reveals that improvements have been at best modest. The magnitude of improvements seems particularly small in the context of often-repeated national guidelines and abundant clinical evidence supporting aspirin use for the prevention of CVD, particularly in patients with known CVD. Even in 2003, aspirin use was reported in only one-third of the visits by patients having CVD, which points to widespread under-appreciation of aspirin as an efficacious and cost-effective secondary prevention therapy. The usage was 12% among visits by diabetics, a group at increased cardiovascular risk. This was lower than the 16% found among visits by patients with multiple major cardiovascular risk factors for whom evidence supporting prophylactic aspirin therapy is less definitive. The continued increases in aspirin use since 1999–2000 may reflect heightened awareness of the benefits of aspirin in reducing cardiovascular morbidity and mortality, mediated through intensified dissemination of national guidelines and clinical trial findings [2,9–11,32]. We did not find evidence of aspirin overuse in low-risk patients. Compared with clinical practice in Europe [22,23], our study results add support to the observation that underuse of aspirin is more problematic in the US. The genesis of this gap is likely multifactorial and open to postulation. For instance, US physicians may face greater pressure than their European colleagues to prescribe newer medications as a result of less-restrictive regulations on drug advertising. Also, direct-to-consumer advertising has been shown to change patient-physician relationships and physician prescribing behavior. The widespread aspirin underutilization could be partly due to uncertainties in risk assessment. Health care providers show little consistency as to how much risk of excess bleeding is acceptable, which may partly account for the variability in aspirin prescribing [33]. Data indicate that aspirin use is linked to approximately 2.5%–4.5% of the annual upper gastrointestinal events (symptomatic ulcers) and 1%–1.5% of serious complications, such as severe bleeding, perforation, and obstruction [34]. These risk estimates should be evaluated in the context of average reductions of 1540% in cardiovascular events when aspirin is used as a preventive therapy [3,10,11,14,35]. Accurate risk assessment can be difficult at the individual patient level, especially when discrepancies arise between verbal and written medical history information [36]. Aspirin resistance may also limit the rates of aspirin use. However, the frequency of aspirin resistance is less well known and may range from 5% to 60% [37]. In some patients it may be dose related. Lee et al. [38] indicate that even a low-dose aspirin of 100 mg or less may increase aspirin resistance in patients with coronary artery disease. Past research also suggests that physicians may assign lower priority to aspirin therapy than to other cardiovascular risk-lowering therapies [25,26,36], and our evaluations of the co-prescription of aspirin and statins support this assessment. We found that aspirin and statin use was significantly higher when the other therapy was present; however, the incremental use became progressively greater for statins over time. Beginning in 1997–1998, statin use in the presence of aspirin transcended aspirin use in the presence of statins for both the high- and intermediate-risk categories, and the gaps remained wide through 2003. These results suggest that even though statins themselves may be underused, aspirin is given even lower priority for lowering cardiovascular risk. These findings are intriguing because both therapies reduce cardiovascular risk by similar magnitudes but differ vastly in cost; statins are prioritized despite the far greater cost-effectiveness of aspirin [39–41]. Also, secondary analyses of clinical trial data indicate that aspirin and statins used in combination may be more effective at reducing the relative risk of CVD events than when used alone [42]. Statins are newer and more intensely advertised than aspirin, which may partly explain the preferential use of these drugs. Lipid-lowering medications already ranked the fifth most promoted drug class in the US in 1998 [43]. Statins are proven effective for both the primary and secondary prevention of CVD, whereas the effectiveness of aspirin in primary prevention is less certain. Also, while they are increasingly used as a prophylactic treatment, statins are still most commonly prescribed to people with hyperlipidemia. In contrast, use of aspirin is not specific to any risk factor in the prevention of CVD and therefore may be neglected by many physicians who are trained to perform in an overly acute-care-centered health care system. In addition, statins may be perceived to have a more favorable side-effect profile than aspirin, which has been shown to increase the risk of severe gastrointestinal and cerebral hemorrhage [34]. Finally, our comparison of aspirin and statin use is confounded by the likelihood of underreporting of over-the-counter aspirin use by participating physicians and clinical staff. In agreement with previous findings, lower aspirin use is associated with female gender, younger age, noncardiologist care, and care in the private office setting [15,16,27,28,44,45]. The appropriateness of prophylactic aspirin therapy among women, particularly those under 65 y of age, is yet to be determined in light of the new evidence from the Women's Health Study [7]. However, variations of aspirin use by physician specialty and type of health care setting raise questions about equity in the process of care. As a result of high penetration of managed care, patients are increasingly less likely to see a specialist such as cardiologist, unless referred by their primary care provider [45]. Primary care providers, including those who practice in private offices, are expected to adhere more diligently to practice guidelines in this area that was previously the domain of specialists. Our findings should be interpreted in the context of several limitations of the data sources used. Both NAMCS and NHAMCS are designed to produce national estimates on the basis of patient visits, and they provided no way to link multiple visits by the same patient. The per-patient visit nature of our analysis may lead to overestimation of aspirin use, particularly for high-risk patients, due to more frequent visits by sicker patients and indiscriminate reporting of sporadic and long-term use of aspirin. Individuals who have visited an ambulatory care facility may differ from those who fail to do so or do so less frequently. However, observed aspirin use may underestimate actual administration due to its low-cost, over-the-counter availability, although participating physicians and clinical staff are instructed to record nonprescription medication. In an attempt to indirectly gauge the potential of underreporting of aspirin use, Stafford [16] studied the reporting of multivitamin use during pregnancy and nonprescription analgesic use for osteoarthritis, and concluded that these surveys capture a reasonably substantial proportion of nonprescription medication use. By limiting the number of medications reported to six or fewer, some medications, particularly those perceived as less critical for the treatment of primary diagnoses, may not be reported. When we compared patient visits in which the maximum number of medications were reported with visits in which fewer were reported, we found no differences in the likelihood of aspirin use. If aspirin is, in fact, under-reported, less clinical attention and priority may be given to aspirin use compared to other therapies. While these data limitations present certain difficulties in interpreting the absolute usage of aspirin, they should have limited impact on our trend analysis. The extent of under-reporting may have attenuated over time due to increased awareness of its effectiveness in cardiovascular risk reduction, which could partly explain the increasing trends in use that we observed. We have no reason to believe that under-reporting varies so substantially by patient visit characteristics that it could have confounded our multivariate logistic analysis. In conclusion, improvements in aspirin use for reducing risks of CVD among US outpatients are at best modest, and substantial treatment gaps persist, particularly in secondary prevention, for which definitive evidence of benefits is available. Aspirin is more underused than statins despite its more favorable cost-effectiveness. Marked changes in clinical practice are unlikely to occur unless more aggressive, innovative means are implemented to enhance health care provider and patient adherence to consensus guidelines on aspirin therapy to prevent CVD events. In particular, targeted interventions may be warranted in patient subpopulations in which aspirin use is lower than average, including women, young adults, and ethnic minorities. Targeted continuing medical education for primary care providers especially in solo or small-group practices, may introduce greater consistency into practice by specialty and practice setting. Acknowledgments This research was sponsored by an unrestricted grant from Bayer Pharmaceutical Corporation. Additional support was provided by a research award from the Agency for Healthcare Research and Quality (R01-HS11313). Both funding sources had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: RSS obtained study funding. RSS and JM designed the study. RSS, VM, and JM analyzed the data and contributed to writing the paper. Patient Summary Background Aspirin is known to be effective in lessening the chance of heart attack, stroke, or other cardiovascular diseases that may occur when blood vessels are blocked by blood clots. Therefore, guidelines recommend that certain groups of people take aspirin regularly either to prevent such clots forming in the first place, or after such a clot has formed to prevent further clots. However, aspirin may increase the chance of bleeding in some people; hence it is important that the benefits of taking aspirin are balanced against possible side effects. Why Was This Study Done The researchers wanted to investigate temporal patterns of aspirin use among patients who would potentially benefit from taking it, and ask whether there were any particular reasons—for either patients or their health care providers—that influenced such use. What Did the Researchers Do and Find They used data over 11 years from two nationwide surveys in the US that study prescribing patterns in outpatients. Some improvements were observed between 1993 and 2003 in the use of aspirin among patients with known CVD and those without. However, the magnitude of those improvements is minimal relative to the substantial gaps between clinical practice and evidence-based recommendations. From 1997 to 1998 onward, statins were used more frequently compared with aspirin as prophylactic therapy for reducing cardiovascular disease risk. Greater aspirin use was seen most frequently in people of advanced age, who were male, who were being cared for by cardiologists (rather than general physicians or other specialists), and who were being seen in hospital outpatient departments (rather than private practices). What Do These Findings Mean Although there is very good evidence that aspirin is particularly useful when given after a cardiovascular event—so-called secondary prevention—there were only modest increases in the use of aspirin in this period. Aspirin is less frequently used than statins, despite its greater cost-effectiveness. Innovative interventions are needed to enhance patients' and health care providers' understanding of and adherence to the guidelines that have been developed on reducing the risk of cardiovascular disease. Where Can I Get More Information Online MedlinePlus has information on aspirin and related drugs: Omni is a UK-based free catalog of hand-selected and evaluated Internet resources in health and medicine, including a page of links on aspirin: Patient Summary Background Aspirin is known to be effective in lessening the chance of heart attack, stroke, or other cardiovascular diseases that may occur when blood vessels are blocked by blood clots. Therefore, guidelines recommend that certain groups of people take aspirin regularly either to prevent such clots forming in the first place, or after such a clot has formed to prevent further clots. However, aspirin may increase the chance of bleeding in some people; hence it is important that the benefits of taking aspirin are balanced against possible side effects. Why Was This Study Done The researchers wanted to investigate temporal patterns of aspirin use among patients who would potentially benefit from taking it, and ask whether there were any particular reasons—for either patients or their health care providers—that influenced such use. What Did the Researchers Do and Find They used data over 11 years from two nationwide surveys in the US that study prescribing patterns in outpatients. Some improvements were observed between 1993 and 2003 in the use of aspirin among patients with known CVD and those without. However, the magnitude of those improvements is minimal relative to the substantial gaps between clinical practice and evidence-based recommendations. From 1997 to 1998 onward, statins were used more frequently compared with aspirin as prophylactic therapy for reducing cardiovascular disease risk. Greater aspirin use was seen most frequently in people of advanced age, who were male, who were being cared for by cardiologists (rather than general physicians or other specialists), and who were being seen in hospital outpatient departments (rather than private practices). What Do These Findings Mean Although there is very good evidence that aspirin is particularly useful when given after a cardiovascular event—so-called secondary prevention—there were only modest increases in the use of aspirin in this period. Aspirin is less frequently used than statins, despite its greater cost-effectiveness. Innovative interventions are needed to enhance patients' and health care providers' understanding of and adherence to the guidelines that have been developed on reducing the risk of cardiovascular disease. 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☉ 11340236:DC-SIGN Induction in Alveolar Macrophages Defines Privileged Target Host Cells for Mycobacteria in Patients with Tuberculosis
1 Institut Pasteur, Unite de Genetique Mycobacterienne, Paris, France,2 Hpital Necker-Enfants-Malades, AP-HP, Service de Pneumologie et d'Allergologie Pediatrique, Paris, France,3 Hpital Saint-Louis, AP-HP, Service de Pneumologie, Paris, France,4 Hpital Saint-Louis, AP-HP, Service de Microbiologie, Paris, France,5 Institut Pasteur, Groupe Virus et Immunite, Paris, France,6 Centre National de la Recherche Scientifique, URA 2172, Paris, France Background Interplays between Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB) in human and host professional phagocytes, namely macrophages (Ms) and dendritic cells (DCs), are central to immune protection against TB and to TB pathogenesis. We and others have recently shown that the C-type lectin dendritic cell–specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN; CD209) mediates important interactions between mycobacteria and human monocyte-derived DCs (MoDCs) in vitro. Methods and Findings In order to explore the possible role of DC-SIGN in M. tuberculosis infection in vivo, we have analysed DC-SIGN expression in broncho-alveolar lavage (BAL) cells from patients with TB (n = 40) or with other non-mycobacterial lung pathologies, namely asthma (n = 14) and sarcoidosis (n = 11), as well as from control individuals (n = 9). We show that in patients with TB, up to 70% of alveolar Ms express DC-SIGN. By contrast, the lectin is barely detected in alveolar Ms from all other individuals. Flow cytometry, RT-PCR, and enzyme-linked immunosorbent assay analyses of BAL-derived fluids and cells indicated that M. tuberculosis infection induces DC-SIGN expression in alveolar Ms by a mechanism that is independent of Toll-like receptor-4, interleukin (IL)-4, and IL-13. This mechanism most likely relies on the secretion of soluble host and/or mycobacterial factors that have yet to be identified, as both infected and uninfected bystander Ms were found to express DC-SIGN in the presence of M. tuberculosis. Immunohistochemical examination of lung biopsy samples from patients with TB showed that the bacilli concentrate in pulmonary regions enriched in DC-SIGN-expressing alveolar Ms in vivo. Ex vivo binding and inhibition of binding experiments further revealed that DC-SIGN–expressing alveolar Ms constitute preferential target cells for M. tuberculosis, as compared to their DC-SIGN counterparts. In contrast with what has been reported previously in MoDCs in vitro, ex vivo DC-SIGN ligation by mycobacterial products failed to induce IL-10 secretion by alveolar Ms, and IL-10 was not detected in BALs from patients with TB. Conclusion Altogether, our results provide further evidence for an important role of DC-SIGN during TB in humans. DC-SIGN induction in alveolar Ms may have important consequences on lung colonization by the tubercle bacillus, and on pulmonary inflammatory and immune responses in the infected host. Academic Editor: Paul Klenerman, Oxford University, United Kingdom These authors contributed equally to this work. These authors contributed equally to this work. Introduction Interactions between Mycobacterium tuberculosis, the airborne agent of tuberculosis (TB) in human and host phagocytes, namely macrophages (Ms) and dendritic cells (DCs), are central to anti-mycobacterial immunity and to TB pathogenesis [1,2]. In particular alveolar Ms constitute a primary niche for the tubercle bacillus [3]. At the molecular level, interactions between the bacillus and host phagocytes rely on a variety of cellular receptors, among which the C-type lectin DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN; CD209) has recently received particular attention. Initially described as an HIV gp120 receptor [4,5], DC-SIGN has afterwards been shown to allow monocyte-derived DCs (MoDCs) to recognize a variety of microbes, including viruses, parasites, and bacteria [6]. A set of recent reports has established that DC-SIGN plays a key role in mycobacteria interactions with DCs, at least in vitro [7,8]. Indeed, DC-SIGN allows human MoDCs to recognize members of the M. tuberculosis complex through lipoarabinomannan (LAM), an abundant lipoglycan of the mycobacterial envelope [7,9], and through other molecules, such lipomannan, arabinomannan, and the 19 kDa antigen [8]; and DC-SIGN ligation by LAM partially inhibits the maturation of bacterial lipopolysaccharide (LPS)-stimulated MoDCs and potentiates the secretion of the anti-inflammatory cytokine interleukin (IL)-10 by these cells [8]. DC-SIGN interactions with mycobacteria or mycobacterial products may thus be of benefit for either the pathogen, by down-modulating DC functions, or for the host, by limiting tissue inflammation and immunopathology [11–13]. It is noteworthy that DC-SIGN is expressed by human pulmonary DCs [7,14], and mycobacteria-derived antigens have been detected in DC-SIGN+ DCs in lymph nodes from patients with TB, suggesting that interactions between the lectin and the bacillus may occur during the natural course of infection [7]. In order to further our understanding of the role of DC-SIGN in vivo, we have analysed the expression of the lectin in broncho-alveolar lavage (BAL) cells from patients with TB, as compared to cells from other patients. Methods Patients and Samples Individuals studied (n = 74; mean age 17.6 ± 19.3 y; median age 10.5 y; 54% males) included patients with TB (n = 40), sarcoidosis (n = 11), or asthma (n = 14). Other individuals had alveolar proteinosis (n = 1), tracheal obstruction (n = 1), tracheal surgery (n = 1), inactive TB (n = 5), and lymphoma (n = 1). These individuals (n = 9) are considered as controls in the rest of the study. All individuals were HIV negative and non-smokers. None were receiving anti-mycobacterial therapy and/or corticosteroids at the time of biopsy, except for the treatment of asthma. TB was diagnosed by smear observation and/or bacterial culture and/or clinical symptoms. Asthma was defined as a history of recurrent wheezing episodes and daily use of asthma medication. The diagnosis of sarcoidosis was based on previously described criteria [16]. Inactive TB refers to patients who had a previous history of TB (which was successfully treated) without reactivation. BAL fluids were performed as previously described [17] for diagnostic purpose. Blood mononuclear cells from healthy volunteers (Etablissement Franais du Sang, Paris, France) and from TB patients with smear observation were isolated by Ficoll-Paque (Pharmacia, Uppsala, Sweden) centrifugation. Only fluids in surplus were used in this study, according to institutional guidelines. Alveolar Ms Isolation, Treatment, and Infection BAL cells were washed and cultured in RPMI-1640 (Invitrogen, Carlsbad, California, United States) supplemented with 10% heat-inactivated foetal calf serum (FCS; Dutscher, Brumath, France) and 2 mM L-glutamine in Petri dishes for 1 h at 37 °C before removing nonadherent cells. When required, cells were treated with IL-4 (10 ng/ml, R&D Systems), tumor necrosis factor (TNF)-α (50 ng/ml; R&D Systems, Minneapolis, Minnesota, United States), Escherichia coli–derived LPS (100 ng/ml; Sigma, St. Louis, Missouri, United States), or a green fluorescent protein (GFP)-expressing strain of M. tuberculosis H37Rv (pEGFP plasmid was a kind gift from G. R. Stewart, University of Surrey, United Kingdom) at a multiplicity of infection (MOI) of one bacterium per cell. THP1 Cell THP1 (ATCC TIB-202) cells were transduced with a lentiviral DC-SIGN–encoding vector [18]. DC-SIGN–expressing cells were positively selected by flow cytometry–based cell sorting and cultivated, as THP1 cells, in RPMI-1640 supplemented with 10% heat-inactivated FCS and 2 mM L-glutamine. Flow Cytometry Cells were treated and analysed as previously described [19]. The following monoclonal antibodies (mAbs) were used: anti-CD11b-PE, anti-CD11c-PE, anti-CD16-PE, anti-CD32-PE, anti-CD40-PE, anti-CD64-FITC, and anti-CD206-PE (all from Beckman Coulter, Allendale, New Jersey, United States); anti-CD1a-PE, anti-CD11b-APC, anti-CD14-PE, anti-CD83-PE, anti-CD86-PE, anti-CD123-PE-Cy5, and anti–human leukocyte antigen (HLA)-DR-PE (all from BD Biosciences, San Diego, California, United States); anti-CD209 (DC-SIGN)-FITC and anti-PE (clone 120507; R&D Systems); anti-TLR2-PE, anti-TLR4-PE, and anti-TLR9-PE (all from eBioscience, San Diego, California, United States); and anti-BDCA-1-APC, anti BDCA-2-PE, and anti-BDCA-3-PE (all from Miltenyi Biotech, Bergisch Gladbach, Germany). Isotype controls were all purchased from BD Biosciences. Fluorescence was analyzed using FACScalibur and CellQuest Pro software (BD Biosciences). Binding Experiments Total BAL cells were pre-incubated for 30 min at 37 °C, according to a previously published procedure [20], in RPMI-1640 containing 10% FCS and eventually containing isotype controls, either a mix of PE-conjugated and unconjugated anti-DC-SIGN antibodies (clones 120507 and 1B10, a kind gift from A. Amara, Institut Pasteur, Paris, France) or a mix of APC-conjugated and unconjugated anti-CD11b antibodies (clones M1/70 [BD Biosciences] and 2LPM19c1 [Dako, Glostrup, Denmark]). All antibodies were used at 10 μg/ml. Pre-incubation at 37 °C in the presence of antibodies resulted in partial internalization of the corresponding receptors but did not decrease cell-associated fluorescence, because a mix of unlabelled and fluorescently labelled antibodies was used during this step (data not shown). Cells were then infected with GFP-expressing M. tuberculosis H37Rv, at a multiplicity of infection of five bacteria per cell, for 4 h at 4 °C to allow binding without phagocytosis. Cells were then washed in RPMI-1640 and further stained with PE-conjugated anti-DC-SIGN and APC-conjugated anti-CD11b antibodies for 30 min at 4 °C. Cells were then analyzed by flow cytometry. Binding to THP1 and THP1::DC-SIGN cells was realized as previously described for DC-SIGN–expressing HeLa cells [7]. Inhibition of binding was realized using anti–DC-SIGN antibodies at 10 μg/ml. Enzyme-Linked Immunosorbent Assay BAL fluids were centrifuged for 15 min at 1000 × g and supernatants were concentrated three to eight times using Ultra-15 and YM-5 concentrators (Amicon; Millipore, Billerica, Massachusetts, United States). Concentrated BAL fluids were analyzed for IL-4, IL-10, and IL-13 using high-sensitivity enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems. The minimal detectable dose of IL-4, IL-10, and IL-13 was 0.11 pg/ml, 60% of M. tuberculosis binding to DC-SIGN–expressing Ms (Figure 4A and 4B). The antibodies had a slight positive effect on mycobacterial binding to CD11b+DC-SIGN cells, which was likely due to more mycobacteria available for binding to these cells in the presence of the antibody (Figure 4A and 4B). Conversely, anti-CD11b antibodies could inhibit M. tuberculosis binding to DC-SIGN alveolar Ms of almost 50% in average, whereas they had only a very minor effect on binding to CD11b+DC-SIGN+ cells (Figure 4A and 4B). In order to confirm that DC-SIGN constitutes a major M. tuberculosis receptor in the context of a M cell and in the presence of other mycobacterial receptors, THP1 human Ms were transduced with a lentivirus-based DC-SIGN-encoding vector and were used in M. tuberculosis–binding experiments. M. tuberculosis was found to bind to DC-SIGN–expressing THP1 Ms by greater than 10-fold more than to THP1 cells (Figure 4C). Anti-DC-SIGN antibodies could fully inhibit mycobacterial binding to DC-SIGN-expressing cells, whereas they had virtually no effect on binding to THP1 cells. Altogether, these results indicate that DC-SIGN expression renders Ms, and in particular alveolar Ms in the lungs of patients with TB, more susceptible to infection than their DC-SIGN counterparts. Confocal microscopy examination of alveolar Ms infected with GFP-expressing M. tuberculosis and subsequently immunostained for DC-SIGN, showed, as in MoDCs [7], a marked recruitment of the lectin at the site of bacterial attachment and in the nascent phagosome (Figure 4D, upper and middle panels), followed by exclusion of the receptor from the mycobacterial vacuole once the bacillus was engulfed, most likely as a result of receptor recycling (Figure 4D, lower panels). (A) Alveolar Ms from a patient with TB were infected with GFP-expressing M. tuberculosis, in the absence (; upper left panel) or the presence of control isotype (upper right panel), anti-CD11b (lower left panel), or -DC-SIGN (lower right panel) blocking antibodies. In the upper panels, cells were then stained with fluorescent PE-conjugated anti-DC-SIGN and APC-conjugated anti-CD11b antibodies. In lower panels, fluorescent antibodies were added together with blocking antibodies (same clones). (B) Proportion of GFP+ cells in DC-SIGN (open bars) and DC-SIGN+ (grey bars) alveolar Ms as calculated from (A) using BALs from two patients with TB. THP1 Ms expressing or not expressing DC-SIGN (THP1::DC-SIGN) were used in a binding experiment with M. tuberculosis H37Rv, in the presence or absence of anti-DC-SIGN antibodies. (D) Confocal microscopy examination of adherent DC-SIGN+ cells infected with GFP-expressing M. tuberculosis for various times. DC-SIGN Does Not Mediate IL-10 Secretion in Alveolar Ms Besides its function in pathogen recognition and phagocytosis, DC-SIGN has been proposed to play a role in triggering intracellular signals and cytokine secretion. In particular, it has previously been reported that DC-SIGN ligation by LAM synergizes with TLR4 ligation by LPS to potentiate IL-10 secretion by MoDCs [8]. We sought to determine whether this might also be the case in alveolar Ms, which also express TLR4 (see Figure 2C). Alveolar Ms from three patients with TB were purified by adherence and stimulated for 18 h with LPS alone or LPS and M. tuberculosis–derived LAM, in the presence or absence of anti-DC-SIGN or control isotype antibodies. Although basal IL-10 production differed among M preparations, treatment of the cells with LPS resulted in a slight increase in IL-10 secretion in all cases, as revealed by ELISA (Table 2). Treatment of the cells with LAM alone had virtually no effect on IL-10 production. By contrast with what has been reported in MoDCs [8], LAM did clearly not synergize with LPS to induce production of the cytokine by alveolar Ms (Table 2). This was confirmed by measuring IL-10 in BAL fluids from patients with TB or with other pathologies (Table 3). IL-10 was detected in some samples but not systematically in samples from patients with TB that contained DC-SIGN-expressing Ms. Discussion Our study reveals the presence of a novel DC-SIGN–expressing subset of alveolar Ms in the lungs of patients with TB. In patients with TB, this M subset represents up to 70% of total CD11b+ Ms. By contrast, in other patients with non-tuberculous lung diseases, as well as in control individuals, DC-SIGN Ms represent only 3% on average of CD11b+ Ms. Our results strongly suggest that DC-SIGN+ Ms arise from induction of the DC-SIGN gene during infection. IL-4 and IL-13, two cytokines known to induce DC-SIGN expression in monocytes [22], are unlikely to account for DC-SIGN expression by alveolar Ms in patients with TB, because these cytokines were either not detected (IL-13) or detected in variable amounts and independently of pathology (IL-4) in BAL fluids from a number of different patients. Recently, DC-SIGN induction has been demonstrated in human circulating blood monocytes treated with TLR agonists [23]. This is unlikely to be the case here because we have shown that LPS treatment of DC-SIGN alveolar Ms obtained from individuals without TB could not induce DC-SIGN expression in these cells (see Figure 3B). Inflammation alone is unlikely to induce DC-SIGN, because alveolar Ms from patients with asthma or sarcoidosis were mostly DC-SIGN, whereas these diseases are characterized by a marked inflammatory response in the lungs. The fact that both M. tuberculosis–infected and uninfected alveolar Ms up-regulate DC-SIGN during ex vivo infection (see Figure 3B) is of interest, as it strongly suggests that soluble factor(s) from the host and/or the microbe can induce DC-SIGN in bystander DC-SIGN Ms. These factors will have to be defined in future studies. In particular, the role of M. tuberculosis–derived lipids and proteins that are secreted by the bacterium prior to uptake by host cells and from inside infected cells [24,25] will have to be investigated. In addition, the possible role of host factors, such as IL-15, will have to be examined in this respect. Indeed, IL-15 has recently been shown to induce DC-SIGN expression in human monocytes in vitro [23], and alveolar Ms from patients with TB have been reported to produce this cytokine ex vivo [26]. So far, mycobacterial receptors on Ms have been mostly characterized using model Ms, namely mouse bone marrow–derived or human monocyte–derived Ms [27]. Very few reports are available on mycobacterial receptors on alveolar Ms, and all of them used cells from healthy individuals or laboratory animals. The main mycobacterial receptors identified on human alveolar Ms are CRs, especially CR4 and CR3 on human cells [28–30], the surfactant protein A receptor [31], and the mannose receptor [32]. Other studies in murine alveolar Ms confirmed these findings and added other receptors, such as scavenger receptors, to the list [33]. However, to our knowledge, ours is the first study of M. tuberculosis receptors on alveolar Ms in patients with TB. Our results suggest a novel scenario of alveolar M infection during TB. In this scenario, CRs likely mediate most of cell infection in a naive host, and DC-SIGN–expressing Ms become privileged target cells for the bacillus once the infection is established. Furthermore, DC-SIGN induction in bystander cells may be of advantage for the tubercle bacillus to increase its intracellular territory inside the infected host. Apart from pathogen binding, DC-SIGN may play a role in signal transduction. In particular, DC-SIGN ligation by the mycobacterial lipoglycan LAM has been reported to potentiate TLR-4–mediated IL-10 secretion by LPS-stimulated MoDCs [8]. However, treatment of LPS-stimulated alveolar Ms with LAM did not result in an increase of IL-10 production, which remained relatively low in both stimulated and untreated cells. Moreover, IL-10 was not detected in BAL fluids from patients with TB (Table 3). This is in accordance with previous studies reporting that IL-10 is detected in comparable amounts in BALs and lung biopsies from TB patients, and from healthy individuals [34]. IL-10 production is known to be low in Ms as compared to in DCs. The discrepancy between our results, that show no role of DC-SIGN in IL-10 secretion, and those from Geijtenbeek et al. [8], who reported such a role, likely relies on the different cell types used in the two studies. Our results do not exclude the possibility that DC-SIGN may participate in IL-10 production by DCs in patients with TB, especially in the lymph nodes. This possibility will have to be further explored and may have important local consequences, including down-modulation of the local inflammation due to infection, either directly or by driving T lymphocytes toward a regulatory phenotype [35,36]. In conclusion, our study reveals that during the natural course of TB in human lungs, soluble host and/or mycobacterial factor(s) induce DC-SIGN expression by alveolar Ms, which renders the cells highly prone to infection by the tubercle bacillus. DC-SIGN induction in alveolar Ms may have important consequences on lung colonization by M. tuberculosis, as well as on host immune and inflammatory responses, which will require further investigation in cell and animal models, as well as in patients with TB. Acknowledgments We thank D. Ensergueix (Paris) for technical assistance in histology. Confocal microscopy was realised at Dynamic Imaging Platform at Institut Pasteur. We thank G. R. Stewart, A. Amara, and J. Nigou for providing pEGFP, 1B10 antibody, and ManLAM, respectively. This research project has been co-financed by Institut Pasteur and the European Commission, within the 6th Framework Programme, contract no. LSHP-CT-2003–503367. The text represents the authors' views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information. LT is a fellow of the European Commission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: LT and ON designed the study with the help of NPT, ABL, JLH, PS, PHL, JdB, AT, and BG. NPT, ABL, and JLH performed clinical sampling. LT, PC, OS, and ON performed the experiments. LT and ON wrote the paper with the contribution of NPT, ABL, JLH, OS, PS, PHL, JdB, AT, and BG. Patient Summary Background Tuberculosis (TB) is one of the most common major infectious disease today. It is estimated that two billion people—or one-third of the world's population—are chronically infected without active symptoms. Nine million new cases of active disease are diagnosed annually, resulting in two million deaths, mostly in developing countries. TB is predominantly a lung disease. It is caused by a microbe called Mycobacterium tuberculosis, which infects lung cells. Patients with active disease easily infect others through coughing, sneezing, and spitting. Why Was This Study Done Most of what we know about how Mycobacterium infects lung cells during the early stages of TB comes from animal studies or studies in healthy volunteers. In this study, the researchers wanted to examine whether lung cells from patients with TB were different from those of healthy people or those with different lung diseases, and what this might tell us about the way the infection spreads in the lung. In particular, they looked at the surface of the lung cells, because this is the part directly involved in the first contact with Mycobacterium. What Did the Researchers Do and Find They studied 74 individuals: 40 had TB, 25 had other inflammatory lung diseases, and nine had neither active TB nor lung inflammation and served as healthy “controls.” The patients underwent a procedure (called bronchoalveolar lavage) that washes out some of the secretions and cells from the lower respiratory tract. The researchers then analysed the cells in different ways. They concentrated on a type of cell called a macrophage (the natural target of Mycobacterium) and found that macrophages from patients with TB had much more of a particular protein called DC-SIGN on their surface than macrophages from patients with other diseases or from the control individuals. They then took macrophages from a control individual (which thus had very low levels of DC-SIGN) and infected them with Mycobacterium under laboratory conditions. The researchers found that shortly after infection, not only the infected cells but also some of their neighbours started to display DC-SIGN on their surface. The researchers also found that having DC-SIGN on the surface made uninfected cells much more susceptible to infection. What Does This Mean The results suggest that DC-SIGN has an important function in amplifying TB infection in the lung. In the long run, understanding how Mycobacterium infects patients and either makes them ill or establishes a chronic infection without acute symptoms should help with the development of new or better ways to prevent infection or treat disease. Where Can I Find More Information Online The following Web sites provide information on tuberculosis. World Health Organization pages on TB: TB Vaccine Cluster page: Tuberculosis.net, a source for TB teaching materials: Wikipedia pages on TB: MedlinePlus pages on TB: Patient Summary Background Tuberculosis (TB) is one of the most common major infectious disease today. It is estimated that two billion people—or one-third of the world's population—are chronically infected without active symptoms. Nine million new cases of active disease are diagnosed annually, resulting in two million deaths, mostly in developing countries. TB is predominantly a lung disease. It is caused by a microbe called Mycobacterium tuberculosis, which infects lung cells. Patients with active disease easily infect others through coughing, sneezing, and spitting. Why Was This Study Done Most of what we know about how Mycobacterium infects lung cells during the early stages of TB comes from animal studies or studies in healthy volunteers. In this study, the researchers wanted to examine whether lung cells from patients with TB were different from those of healthy people or those with different lung diseases, and what this might tell us about the way the infection spreads in the lung. In particular, they looked at the surface of the lung cells, because this is the part directly involved in the first contact with Mycobacterium. What Did the Researchers Do and Find They studied 74 individuals: 40 had TB, 25 had other inflammatory lung diseases, and nine had neither active TB nor lung inflammation and served as healthy “controls.” The patients underwent a procedure (called bronchoalveolar lavage) that washes out some of the secretions and cells from the lower respiratory tract. The researchers then analysed the cells in different ways. They concentrated on a type of cell called a macrophage (the natural target of Mycobacterium) and found that macrophages from patients with TB had much more of a particular protein called DC-SIGN on their surface than macrophages from patients with other diseases or from the control individuals. They then took macrophages from a control individual (which thus had very low levels of DC-SIGN) and infected them with Mycobacterium under laboratory conditions. The researchers found that shortly after infection, not only the infected cells but also some of their neighbours started to display DC-SIGN on their surface. The researchers also found that having DC-SIGN on the surface made uninfected cells much more susceptible to infection. What Does This Mean The results suggest that DC-SIGN has an important function in amplifying TB infection in the lung. In the long run, understanding how Mycobacterium infects patients and either makes them ill or establishes a chronic infection without acute symptoms should help with the development of new or better ways to prevent infection or treat disease. Where Can I Find More Information Online The following Web sites provide information on tuberculosis. World Health Organization pages on TB: TB Vaccine Cluster page: Tuberculosis.net, a source for TB teaching materials: Wikipedia pages on TB: MedlinePlus pages on TB: References Flynn JL, Chan J (2001) Immunology of tuberculosis. Annu Rev Immunol 19:93–129. Kaufmann SH (2001) How can immunology contribute to the control of tuberculosis Nat Rev Immunol 1:20–30. Russell DG (2001) Mycobacterium tuberculosis Here today, and here tomorrow. Nat Rev Mol Cell Biol 2:569–577. 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☉ 11340237:Prolonged Activation of Virus-Specific CD8+T Cells after Acute B19 Infection
1 Institution for Medicine, Infectious Disease Unit, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden,2 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom,3 Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, Oxford University, Oxford, United Kingdom,4 Department of Virology, John Radcliffe Hospital, Oxford, United Kingdom,5 Division of Clinical Virology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden,6 Nuffield Orthopaedic Centre NHS Trust, Oxford, United Kingdom Background Human parvovirus B19 (B19) is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously. Methods and Findings The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA–peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%–0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low. Conclusion This is the first example to our knowledge of an “acute” human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation—analogous to that for cytomegalovirus infection—is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development. Academic Editor: Paul Moss, University of Birmingham, United Kingdom These authors contributed equally to this work. These authors contributed equally to this work. Introduction Human parvovirus B19 (B19) is a ubiquitous, single-stranded DNA virus. The 5.6-kb genome codes for only three major proteins, the two overlapping capsid proteins VP1 and VP2 and the non-structural protein NS1. B19 targets immature erythroid cells in the bone marrow after respiratory transmission. Common manifestations of the infection are the benign febrile illness erythema infectiosum followed by an acute arthropathy (in approximately 5% of children and up to 50% of adult infections) that spontaneously resolves within 3 wk. Some adult patients who develop chronic arthritis can fulfil the diagnostic criteria of rheumatoid arthritis [1,2]. Other severe manifestations include transient aplastic crisis in individuals with increased red cell turnover, and chronic anaemia in immunocompromised patients. Furthermore, infection during pregnancy is a major cause of fetal death. Clinical resolution of acute infection is associated with the emergence of antiviral IgG, which is maintained lifelong [3]. However, although antibodies are of importance, evidence for an important role for cellular immune responses in the control of B19 infection is also emerging. In some individuals with apparently intact antibody responses, virus replication continues long term [4]. Readily detectable CD8+ T cell responses in three asymptomatic seropositive individuals have also been observed [5]. CD8+ T cells play an essential role in the control of viral infections by direct killing of virus-infected cells and through cytokine secretion. After a primary viral infection, nave antigen-specific T cells expand clonally and undergo several differentiation stages, followed by a contraction phase mediated by apoptosis. Expression of the chemokine receptor CCR7 on memory T cells has been used to divide this population into central and effector memory subsets. CCR7+ “central” memory lymphocytes mostly home to secondary lymphoid organs and have a high proliferative capacity in response to antigen re-encounter. CCR7 “effector” memory cells home to non-lymphoid organs and are capable of rapid effector function [6]. Peptide–major histocompatibility complex multimers can be used to further quantify and phenotype T cells bearing T cell receptors of appropriate peptide/human leukocyte antigen (HLA) specificity [7,8]. Amongst “effector” memory populations, a spectrum of phenotypes exists, as assessed by surface expression of markers such as CD27, CD28, and CD57, and intracellular expression of perforin. Amongst the viral infections studied, cytomegalovirus (CMV) is associated with the emergence of the largest long-term memory populations with the most “mature” phenotype, CD27/CD28 low, CD57 high, and often perforin positive [9]. The factors that ultimately determine the phenotype and function of these populations of lymphocytes are not fully understood, but continued exposure to antigen is thought to be important. Persistent infections such as hepatitis C virus (HCV), Epstein–Barr virus (EBV), and HIV also show antiviral populations that are “effector” (CD62L or CCR7 low), but show lower levels of maturation than CMV-specific responses [7]. Its small stable genome and clear seroconversion illness make B19 an ideal viral infection in which to study the evolution of antigen-specific T cells longitudinally. Most other significant viruses in humans are either highly variable (HCV and HIV) or have extremely large genomes (EBV and CMV). In our previous analyses, we observed a surprisingly robust response to B19 in three individuals with an asymptomatic seropositive state [5]. We aimed in this study to define the origins of such responses and track a range of antiviral populations during and after acute disease. Methods Study Participants Eleven previously healthy immunocompetent adults presenting to their general practitioner with symptoms of fever, arthralgia, fatigue, and rash were prospectively identified (B19 IgM-positive) at the Departments of Clinical Virology at the Oxford Radcliffe Hospitals, Oxford, United Kingdom, and Karolinska University Hospital, Stockholm, Sweden. The two cohorts were ascertained and studied independently. Clinical details of the patients are shown in Table 1. The timing of blood samples is given from onset of symptoms for both cohorts. B19 DNA in serum was detected by nested PCR amplifying 284 bp in the NS1 gene with a sensitivity of 103 DNA copies/ml (Table 1) [5]. In addition, five healthy B19 IgG-positive, B19 IgM/DNA-negative healthy laboratory volunteers were studied as a “remotely infected” cohort. One gave a history of acute rash and arthritis 10 y previously; the remainder had no history suggestive of B19 infection and were likely infected in childhood. No interferon-γ (IFNγ)– or B19-specific T cells were detected in B19 IgG/IgM/DNA-negative healthy controls (data not shown). Peripheral blood mononuclear cells (PBMCs) were separated from heparinized blood samples within 8 h of sampling, by gradient centrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) or Lymphoprep (Fresenius Kabi Norge, Halden, Norway). DNA was extracted from PBMCs using a QIAamp DNA Mini kit (VWR International, Stockholm, Sweden) or PURGENE DNA purification kit (Gentra Systems, Minneapolis, Minnesota, United States). B19 IgM and IgG were tested using a commercial EIA (Biotrin International, Dublin, Ireland). Ethical approval for the study was obtained from the local ethical committee at Huddinge University Hospital, Karolinska Institutet, Stockholm, Sweden, and from the Oxfordshire Clinical Research Ethics Committee (CO2.113), Oxford, United Kingdom, and informed patient consent was obtained. HLA Tissue Typing HLA class I genotyping was performed using multiplex PCR on DNA extracted from PBMCs (ABC SSP Unitray, Dynal Biotech, Oslo, Norway). Generation of HLA–Peptide Multimeric Complexes HLA–B19 peptide multimeric complexes were constructed as previously described [10] and are listed in Table 2. Briefly, recombinant β2 microglobulin and HLA heavy chain (modified by deletion of the transmembrane–cytosolic tail and addition of a BirA enzymatic biotinylation site) were expressed in BL21pLysS Escherichia coli. The resulting inclusion bodies were purified and solubilized in urea, and refolded by limiting dilution. Peptides were purchased (Biopeptide, San Diego, California, United States) or synthesized by F-moc chemistry and were of greater than 95% purity. The refolded complex was then concentrated, buffer exchanged, biotinylated with BirA enzyme, and purified by fast protein liquid chromatography. HLA class I peptide complexes were stored at 80 °C. Before staining, the monomers were tetramerized with phycoerythrin or Alexa 647–labeled streptavidin (Molecular Probes, Eugene, Oregon, United States) at a molar ratio of 1:4. In addition, a Pro5 Pentamer containing the HLA-B40 TEADVQQWL peptide conjugated to allophycocyanin was purchased (ProImmune, Oxford, United Kingdom). Specificity of the MHC monomers was confirmed using T cell lines established in vitro. When tested against non-HLA-matched cytotoxic T lymphocyte (CTL) lines, no population was detected (<0.02%; data not shown). FACS Analysis Cryopreserved PBMCs were thawed and washed twice with RPMI-1640 supplemented with 10% fetal calf serum, L-glutamine, penicillin, streptomycin, and Hepes buffer at pH 7.5. In preliminary experiments, freshly isolated PBMCs were stained in parallel with the same results. PBMCs (2.5 × 105 cells) were stained with the respective major histocompatibility complex multimer and incubated for 20–30 min at 37 °C. After two washes with PBE (2 mM EDTA and 0.05% BSA, in PBS [pH 7.4]), cells were co-stained with the appropriate monoclonal antibodies for 15 min on ice, and fixed in 1%–2% formaldehyde. Monoclonal antibodies used were directly conjugated and purchased ( Becton-Dickinson, Stockholm, Sweden). Four-colour FACS was performed using fluorochrome-coupled anti-human CD3-, CD8-, CD27-, CD28-, CD38-, CD45RA-, CD45RO-, CD57-, CD62L-, CCR7-, and perforin-specific antibodies. For perforin staining, the cells were permeabilized for 15 min using permeabilizing solution (Becton Dickinson, Palo Alto, California, United States) before staining with perforin monoclonal antibody. Cell acquisition was performed with a four-colour FACS by using a FACSCalibur with CellQuest software (Becton Dickinson). T Cell Lines and Functional Assays PBMCs were pulsed with 50 μM of the respective epitope and cultured at 2 × 106 cells/ml in 24-well plates for 12–18 d. At day three, 10 units/ml of IL-2 was added. Half of the medium was replaced each third day with fresh medium containing 10 units/ml of IL-2. Ex Vivo IFNγ ELISpots IFNγ ELISpots were performed as described previously [11]. Briefly 2.5 × 105 PBMCs were stimulated in triplicates with peptide pools/PHA (Sigma, St. Louis, Missouri, United States). Synthetic peptides, 15-mer, overlapping by ten amino acids spanning the entire B19 protein sequence were used in pools of ten at a final concentration of 10 μM. IFNγ responses were confirmed using individual peptides, with optimal epitopes and HLA restriction identified by synthesis of truncated peptides, prepulsing, and extensive washing of HLA-matched and -mismatched target cells, and synthesis of HLA–peptide multimeric complexes [12]. Using these techniques we confirmed previous epitopes in two independent cohorts, and identified two new epitopes shown in Table 2, the nonamer FYT restricted by HLA-A24 and FPG restricted by HLA-B35. The latter is in addition to the HLA-B35 epitope described previously [5]. Intracellular Cytokine Staining Short-term stimulation of PBMCs was carried out using either peptide pools or individual peptides as previously described [13]. Epitope HLA restriction required the prepulsing of matched and mismatched PBMCs (from B19 seronegative individuals) with peptide at 20 μM for 1 h at 37 °C. Cells were washed and added to CTLs at a 10:1 ratio for 1 h before the addition of Brefeldin A. Chromium Release Assay CTLs were set up as described above. The cytolysis was performed by killing of chromium-labelled targets as described previously by Nixon et. al. [14] using LBL721.220 transfected with HLA-A0201 as target cells. Results B19-Specific CD8+ T cells Expand and Persist at High Frequency Following Resolution of Acute Symptomatic Infection Eleven adults with acute B19 infection (five in Stockholm cohort and six in Oxford cohort) and five remotely infected seropositive individuals were studied. The clinical details, symptom duration, and HLA types are shown in Table 1. Surprisingly, in both cohorts (studied independently) the frequency of B19-specific (multimer-positive) CD8+ T cells in peripheral blood samples continued to increase for many months following symptom resolution. Figure 1A shows representative staining from patient O3. The frequency of A24 FYT tetramer-staining CD8+ T cells increases up to 15 mo after symptom presentation. Patient O3 presented with rash and arthritis; however, symptoms resolved within 5 wk and clinical examination was entirely normal at the time of the second and subsequent venesections. Figure 1B and 1C show the levels of multimer-positive CD8+ T cells for the Oxford and Stockholm cohorts of acutely infected individuals over time. Only one sample was obtained for patient O6; all other individuals were studied 2–7 times. All acutely infected individuals showed B19-specific CD8+ T cell percentages ranging from 0.09% to 4.5% total CD8+ T cells. The levels rose for at least the first 4 mo and frequently persisted for 12–32 mo after symptom onset. The frequencies at 22 mo (or nearest time point) were significantly greater than at first sampling (Willcoxon rank sum test, p = 0.0020), and than those observed for remotely infected seropositive individuals (Figure 1B; Mann–WhitneyU test, p = 0.0022). (A) Representative A24 FYT tetramer staining of individual O3\′s PBMCs. Plots are gated on live CD8+ lymphocytes stained directly ex vivo. Percentages shown are those of tetramer-positive CD8+ T cells. Time points indicated refer to the number of months after first symptoms reported. Symptoms in this individual lasted 5 wk. (B) Frequency of B19-specific responses over time for six acutely infected individuals in the Oxford cohort (O1–O5) and five remotely infected individuals (OR1–OR5). In one case, two epitopes were studied. (C) Frequency of B19-specific responses over time for five acutely infected individuals in the Stockholm cohort (S1–S5). In two cases, two epitopes were studied. B19 DNA could be detected by nested PCR in serum for different time periods (see Table 1). No B19 DNA could be amplified from individuals in the remotely infected group. IgG levels rose quickly and were maintained at a stable level in patients studied (data not shown). B19-Specific CD8+ T Cells Remain Activated Following Resolution of Acute Symptomatic Infection In order to understand the origins and potential pathogenic role of virus-specific CD8+ T cells during and after acute infection, we studied the activation phenotype of these cells ex vivo. Figure 2 shows examples of T cell phenotypic marker expression at early and late sample time points for three different patients using three different B19 peptide–HLA multimers. Figure 2A shows that HLA-B40 TEA pentamer-positive CD8+ T cells from individual S2 are largely perforin positive and CD62L negative, and that these changes become more pronounced from the early (4 mo) to late (21 mo) time points. Figure 3 shows sequential phenotypic data for all of the acutely and remotely infected Oxford cohort. The upper left panel shows that all acutely infected patients showed increases in perforin expression on their B19-specific T cells over the first year following symptom development. By contrast, the seropositive remotely infected individuals showed low levels of perforin expression on tetramer-positive cells. These differences were statistically significant (Mann–Whitney U test, p = 0.0079). Figure 4 shows sequential data from two individuals, S1 and S2, of the Stockholm cohort, each studied with two different HLA–peptide multimers at five or six time points after acute presentation. Percentages shown are the frequency of marker-positive cells amongst multimer-positive cells (plots gated on live CD8+ lymphocytes ex vivo). (A) Patient S2 B40 TEA pentamer staining at 4 mo (left) and 21 mo (right), showing perforin and CD62L levels. (B) Patient O3 A24 FYT tetramer staining at 2 mo (left) and 20 mo (right), showing CD27 and CD28 staining. (C) Patient O5 A2 GLC tetramer staining at 4 mo (left) and 18 mo (right), showing CD38 and CD57 staining. Remotely infected individuals show a less mature/activated phenotype but express only low levels of CCR7 and CD62L (Oxford cohort). The y-axis show the frequency of marker-positive cells amongst tetramer-positive CD8+ cells, while the x-axis show the number of months after symptom onset. Data are shown in perforin, CD38, CD57, CD27, CD28, and CCR7: data are derived from six acutely infected individuals in the Oxford cohort (O1–O3, O5, and O6; insufficient cells available for O4) and five remotely infected individuals (OR1–OR5). (A) Blood from patient S1 was stained with the two tetramers A2 LLH and A2 GLC. (B) Blood from patient S2 was stained with tetramers A2 GLC and the Pro5 B40 TEA pentamer. The top panels show the frequency of tetramer-positive cells over time for the two different responses in each individual (data equivalent to those in Figure 1C). The subsequent panels show the frequency of B19-specific CD8+ cells positive for perforin, CD38, CD57, and CD62L in both patients. CD62L expression was found to be low at all time points in the acutely infected cohort as well as in the remotely infected cohort (see Figure 2A, lower panels; Figure 4, bottom panels; data not shown). Figure 3 also shows that, for the Oxford acutely infected cohort, CCR7 expression was low and fell over the study period. B19-specific CD8+ T cells from four of the five remotely infected individuals had low CCR7 expression (see Figure 3, bottom right panel). Persistent CD38 Expression Following B19 Infection Suggests Ongoing Low-Level Antigenic Stimulation All acutely infected individuals maintained high levels of CD38 expression for more than 10 mo after symptom onset, as shown for the Oxford cohort in Figures 2C and 3 and for two individuals of the Stockholm cohort in Figure 3. Strikingly, for one patient, O3, there was a dramatic fall in CD38 expression at 27 mo compared to the 20-mo time point. Figure 4 shows the sequential CD38 expression of multimer-positive T cells of two specificities for Stockholm patients S1 and S2. S1 shows fluctuations over a period of over 32 mo; S2 shows a gradual decline in CD38 expression from high levels over a comparable period. CD57 expression levels increased over time in almost all acutely infected individuals. This is shown for one Oxford individual in Figure 2C, for the Oxford acutely infected cohort in Figure 3 (bottom left panel), and for two of the Stockholm cohort in Figure 4. Thus, for individual S2 (Figure 4) a high level of CD57 expression was maintained at 30 mo, the last time point tested. By contrast, Figure 3 (bottom left panel) shows that remotely infected individuals have low levels of CD57 expression on their B19-specific CD8+ T cells. T cells 20 mo post-infection in acutely infected individuals expressed significantly higher CD57 than in the remotely infected cohort (Mann–Whitney U test, p = 0.0043). Differing Patterns of CD45 Isoform Expression Follow Acute B19 Infection CD45 isoform expression showed variation between individuals. Most acutely infected patients from the Stockholm and Oxford cohorts expressed high levels of CD45RO within a few months of symptom onset, with variable downregulation over time (data not shown). In the remotely infected cohort, three individuals expressed high levels of CD45RO on tetramer-positive cells. Two of these individuals, however, had only just over 50% of their tetramer-positive cells expressing CD45O (data not shown). CD45RA was downregulated in some patients in the Oxford cohort, whereas in the Stockholm cohort the B19-specific CD8+ cells during the entire study period generally expressed high frequency of CD45RA (data not shown). B19-Specific CD8+ T Cells Have Efficient Effector Function (A) Left panel shows that PBMCs from acutely infected patient O1 secrete IFNγ ex vivo after 18 h of FYTPLADQF peptide stimulation. Negative control (zero spots) and two peptide-stimulated wells from an ELISpot plate are shown. Numbers represent IFNγ-secreting cells per 250,000 PBMCs. Right panel shows A24 FYT tetramer staining of PBMCs at same time point, displaying the number of tetramer-positive cells expressed as a percentage of CD8+ T cells. (B) Tetramer staining of patient S2\′s PBMCs ex vivo (left) and after short-term TEADVQQWL peptide stimulation in vitro (right). (C) Ex vivo IFNγ ELISpot results for remotely infected individual OR3. Mean and standard deviations of triplicates are shown. Cells were stimulated for 18 h with no peptide, GLCPHCINV, or TEADVQQWL. (D) 51Cr release assay using HLA-A2-restricted GLCPHCINV-specific CTLs from individual OR1. PBMCs were stimulated for 14 d with GLCPHCINV peptide and cytolysis was tested against HLA-A0201-transfected LBL.721.220 target cells at various effector-to-target-cell (E:T) ratios. Lastly, we studied the function of B19-specific CD8+ T cells from remotely infected individuals (who in all cases had no symptoms attributable to possible B19 infection for at least 5 y). Figure 5C shows that these cells were capable of direct ex vivo IFNγ release on 18 h culture with GLCPHCINV or TEADVQQWL peptides. A T cell line derived by culture of OR1 PBMCs with peptide in vitro was capable of HLA-A0201-restricted B19-peptide-specific cytolysis (Figure 5D). Discussion In this study we observed, to our knowledge for the first time, a striking pattern of evolving, long-lived CD8 immune responses against B19 in 11 adults with primary B19 infection. Although the symptoms of this virus are generally short-lived and the virus is not classically regarded as persistent, the immune responses showed a sustained activated state many months after initial infection. This pattern was observed in a range of patients in two different clinical centres and appears to represent a new and distinct style of host–virus relationship. This is the first example to our knowledge of an “acute” human viral infection inducing persistent activated CD8 T cell responses. The CD8+ T cell responses tracked here were mapped using a comprehensive screening system facilitated by the compact and stable viral genome of B19. The virus has only one NS gene, which appears to be the major target of CD8+ T cell responses during acute disease, with a range of epitopes identified [5,12]. HLA-A2-restricted epitopes were commonly targeted, but, interestingly, no clear-cut dominance of one over the other was consistently seen, in contrast to infections such as CMV and HIV [15,16]. CTL responses to HLA-A2 and non-HLA-A2 epitopes showed similar kinetics, frequencies, and phenotypes. Thus, future studies might reasonably track defined epitopes, rather than requiring individual mapping, as is the case in, for example, HCV [17–19]. We used, to our knowledge for the first time, HLA–peptide multimeric complexes to detect CD8+ T cell responses during acute B19 infection. Surprisingly, these continued to increase in magnitude at later time points, long after resolution of acute symptoms. In some cases, these responses reached high levels in blood, and were sustained over many months. Even responses of lower frequency appeared to show this delayed expansion. This differs to responses to almost all other human viruses studied in such detail. Responses to HIV and HCV are strong in acute infection, but typically decline as virus is controlled [20]. EBV-specific responses to latent antigens may increase over time [21]. Few data exist on acute CMV infection in immunocompetent humans, but in murine infection, a phenomenon of “memory inflation” is seen for some but not all epitopes [22–24]. Here, responses showed gradual accumulation over time after a short acute response and a lag period of about 8–10 wk after acute infection. The current study did not have the resolution to determine whether B19-specific responses were biphasic in this manner, although in the mouse such phenomena may differ substantially between different virus preparations, doses, and experimental settings. The B19-specific T cell populations underwent contraction 1.5–2 y after acute infection. The kinetics of this contraction were not defined in this study, but “remotely infected” individuals, who had no recent history of infection, and in some cases may have been primarily infected as long ago as 30 y previously, showed smaller populations of B19-specific T cell populations . However, as we have noted previously, B19-specific CTLs are readily detectable—often much larger than equivalent responses to viruses such as influenza and comparable to some EBV-specific responses [5]. A single individual who had a documented B19 infection 10 y previously showed populations of a size and phenotype (see below) similar to the other remotely infected persons. In addition to a sustained and prolonged expansion of antiviral responses, we also observed continued maturation of B19-specific CD8+ T cells in the cohorts of acutely infected individuals studied. This expansion was consistent across a range of markers, all of which have been linked to the evolution of antiviral “effector” memory cells against persistent virus infections. Consistent with these data is the finding of sustained CD8 effector function of the Swedish cohort over time, as evidenced by IFNγ production in response to viral peptides [12]. Antiviral T cell responses to CMV have been extensively studied and are typically regarded as exhibiting a “mature” phenotype associated with loss of expression of co-stimulatory molecules CD27 and CD28, sustained loss of lymph node homing markers CD62L and CCR7, and generally positive expression of intracellular perforin [7,25–27]. A particular feature of these cells is sustained expression of CD57, which is considered to be a marker of terminally differentiated cells [20]. Although there are substantial differences between individuals, many groups report re-expression of CD45RA on such highly differentiated cells [28–31]. All of these features were clearly reproduced in the B19-specific responses tracked in the months following acute infection. Interestingly, the gradual evolution of responses from a CD28+ to CD28 status, in concert with changes in other markers, could be clearly tracked over time. These phenotypic changes have not been extensively investigated in human CMV, but in murine CMV, CD28 loss does appear to occur relatively early, and to be subsequently maintained in the immunodominant populations [32]. It is generally considered likely, although not proven, that such marker evolution represents a maturation pathway, driven by restimulation in vivo with antigen. The nature or duration of the encounter, coupled with the survival of antigen-specific cells, may lead to the typical appearances of T cells specific for different persistent virus infections [7,25]. The findings that not only are the cells phenotypically mature but also strongly activated in vivo (CD38+), is consistent with continuous encounter with antigen over the post-infection period. This period of restimulation appears to be sustained, but, unlike in CMV, appears to wane over time, perhaps after 1.5–2 y. Eventual disappearance of antigen would be consistent with the relatively less differentiated phenotype seen in remotely infected individuals. Nevertheless, although such populations are smaller in size, less activated, and less mature, they retain a CD62L-low phenotype. Murine T cell populations that exist in the absence of antigen typically show slow reversion to a CD62L-high state [33], even in the case of CMV [32]. Thus antigenic drive may be reduced but would appear to be still sufficient to maintain an “effector” memory T cell population. The striking features of the T cell responses to B19 infection indicate persistence of antigen long after the resolution of acute infection. The status of the virus in the post-acute period is not fully understood. Direct nucleic acid analysis has revealed loss of detection in blood after 3 wk in infected volunteers [3], although this may be prolonged in some cases [4]. Nested PCR analysis in our study revealed the presence of B19 DNA in blood at early time points during acute infection, but such assays were negative at time points 6–12 mo after infection, when T cell populations remained activated. It is possible that B19 persists, in the blood and other sites including bone marrow, joints, or skin. Detection of B19 DNA in the bone marrow of asymptomatic remotely infected volunteers has been reported [3]. The finding of parvovirus in skin remains restricted to a single study of a specific genotype distinct from the genotype 1 strains found in our study participants [34]. Low-level, contained replication at a tissue site for weeks or months after infection does seem like the most likely explanation for the immune responses seen, and more sensitive quantitative PCR assays are being validated to address this question. It is also possible that viral antigen is retained, for example, on follicular dendritic cells, following the extremely high burden seen acutely. Alternatively subgenomic particles may be generated in the post-acute period, in the absence of full viral replication. Parvovirus is not thought to establish true latency or integrate into the host genome, so the mechanism behind this low-level persistence remains to be explored. The relationship between joint or bone marrow pathology and the T cell responses seen is not clear. Indeed, since the most active CD8+ T cell responses were seen at stages where joint symptoms had resolved, these T cell responses are unlikely to be directly involved. It remains an open and interesting question, however, whether these or perhaps CD4+ T cells are involved in the prolonged inflammatory arthritis seen in a proportion of cases. A previous study did identify an HLA association with B19-induced arthritis syndrome, suggesting a significant role for cellular immune responses [1]. All our patients presented—as is common in adults—with joint symptoms, and in principle an acutely infected but asymptomatic group would provide an ideal comparison to address such a question. It is also possible that the CTL responses seen might be involved in immune-mediated pathology in the bone marrow. Although the virus itself may lead to direct death of the critical progenitor cells, in other settings vigorous T cell responses can also contribute importantly to marrow suppression through lytic and non-lytic pathways [35]. In summary, this is the first demonstration to our knowledge of a virus not thought to cause true or classical persistent infection leading to a persistent activated CD8+ T cell response. Responses of this quantity and quality (i.e., CD27 and CD28 low and CD57 and perforin high) have only previously been seen for infection with CMV, a virus known to establish persistent infection. Our data suggest that B19 persists in some form after acute infection, and provokes sustained activated CD8+ T cell responses, which might ultimately play a role in viral clearance. Defining the role of CTLs in this setting will be of value not only in expanding further our understanding of the role of T cells in acute and persistent viral infections but also in vaccine design and in immunotherapy, as has been applied to treatment of EBV and CMV infections in immunosuppressed individuals. B19 is a small virus and attracts relatively little attention from clinicians and immunologists [36], but clearly attracts a great deal of attention from the immune system. Now that progress has been made in the definition of the kinetics and antigenic targets of these immune responses, further studies in specific clinical settings where the virus remains a significant problem could readily redress this balance. Acknowledgments This study was financially supported by the Medical Research Council UK, the Tobias Foundation, the Swedish Cancer Foundation, and the specific Programme for Research and Technological Development “Quality of Life and Management of Living Resources, Human Parvovirus Infection: Towards Improved Understanding Diagnosis and Therapy” (QLK2-CT-2001–00877) of the Swedish Medical Research Council, the Wellcome Trust, and the Commission of the European Communities. However, the study does not necessarily reflect the views of these funders and in no way anticipates the European Commission's future policy in this area. We are also grateful to Mr. Tim Rostron for assistance with tissue typing. We would also like to thank the patients and volunteers who donated blood for the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: PK, TT, and PB designed the study. AI, VK, ON, and AL performed the experimental work. AI and VK analyzed the data. KJ and KB enrolled patients. AI, VK, PK, TT, and PB contributed to writing the paper. Patient Summary Background Parvovirus B19 is a small virus that is very common. Usually it causes mild symptoms of fever and a typical “slapped cheek” rash on the face, which may then spread; in around one in 20 children, and one in two adults it can also cause joint pain. Other, more serious complications occur in people whose red cells do not last as long as usual, for example, people with sickle cell disease; these people can get a severe aplastic anemia—the bone marrow stops making blood completely for a time. The virus can also cause fetal death if a woman contracts it while pregnant. Why Was This Study Done It is not clear how the body's defenses—immune system—work to clear this virus. There is some evidence that in some people the virus might continue to divide in the body for a long time after the first infection. The authors wanted to study one part of the immune system—T cells—in people who had just recently been infected with the virus and compare the findings with people who had had the infection a long time previously. What Did the Researchers Do and Find They compared findings from two groups of people: 11 who had recently had the infection (six from Oxford and five from Stockholm) and five who had had the virus many years previously. They found that over the year following the infection, one particular type of T cell continued to increase in numbers and responsiveness to the B19 virus, despite the fact that the patients' clinical symptoms had gotten better. What Do These Findings Mean It seems the virus remained in the patients' bodies for a considerable time after they appeared to have recovered, and the virus continued to stimulate T cells to respond to it. These results may be useful in designing a strategy to develop a vaccine for this virus. Where Can I Get More Information Online The Health Protection Agency in the United Kingdom has a Web page of information on parvovirus: MedlinePlus also has links to further information: Patient Summary Background Parvovirus B19 is a small virus that is very common. Usually it causes mild symptoms of fever and a typical “slapped cheek” rash on the face, which may then spread; in around one in 20 children, and one in two adults it can also cause joint pain. Other, more serious complications occur in people whose red cells do not last as long as usual, for example, people with sickle cell disease; these people can get a severe aplastic anemia—the bone marrow stops making blood completely for a time. The virus can also cause fetal death if a woman contracts it while pregnant. Why Was This Study Done It is not clear how the body's defenses—immune system—work to clear this virus. There is some evidence that in some people the virus might continue to divide in the body for a long time after the first infection. The authors wanted to study one part of the immune system—T cells—in people who had just recently been infected with the virus and compare the findings with people who had had the infection a long time previously. What Did the Researchers Do and Find They compared findings from two groups of people: 11 who had recently had the infection (six from Oxford and five from Stockholm) and five who had had the virus many years previously. 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☉ 11340238:Maternal Malaria and Gravidity Interact to Modify Infant Susceptibility to Malaria
1 MOMS Project, Malaria Antigen Discovery Program, Seattle Biomedical Research Institute, Seattle, Washington, United States of America,2 Gates Malaria Partnership, London School of Hygiene and Tropical Medicine, London, United Kingdom,3 National Institute for Medical Research, Muheza Designated District Hospital, Muheza-Tanga, Tanzania,4 Statistical Center for HIV/AIDS Research & Prevention, Department of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America,5 Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America Background In endemic areas, placental malaria due to Plasmodium falciparum is most frequent and severe in first-time mothers, and increases the risk of infant mortality in their offspring. Placental malaria may increase the susceptibility of infants to malaria parasitemia, but evidence for this effect is inconclusive. Methods and Findings During 2002–2004, we monitored parasitemia in 453 infants, including 69 who were born to mothers with placental malaria, in a region of northeastern Tanzania where malaria transmission is intense. We used a Cox proportional hazards model to evaluate the time from birth to first parasitemia, and a generalized estimating equations logistic regression model to evaluate risk of any parasitemia throughout the first year of life. Compared with infants whose mothers did not have placental malaria at delivery (“PM-negative”), offspring of mothers with placental malaria at delivery (“PM-positive”) were 41% more likely to experience their first parasitemia at a younger age (adjusted hazard ratio [AHR] = 1.41, 95% confidence interval [CI] 1.01–1.99). The odds of parasitemia throughout infancy were strongly modified by the interaction between placental malaria and gravidity (p for interaction = 0.008, Type 3 likelihood ratio test). Offspring of PM-negative primigravidae had lower odds of parasitemia during infancy (adjusted odds ratio [AOR] = 0.67, 95% CI 0.50–0.91) than offspring of PM-negative multigravidae, and offspring of PM-positive primigravidae had the lowest odds (AOR = 0.21, 95% CI 0.09–0.47). In contrast, offspring of PM-positive multigravidae had significantly higher odds of parasitemia (AOR = 1.59, 95% CI 1.16–2.17). Conclusion Although parasitemia is more frequent in primigravid than multigravid women, the converse is true in their offspring, especially in offspring of PM-positive women. While placental malaria is known to increase mortality risk for first-born infants, it surprisingly reduced their risk of parasitemia in this study. Placental malaria of multigravidae, on the other hand, is a strong risk factor for parasitemia during infancy, and therefore preventive antimalarial chemotherapy administered to multigravid women close to term may reduce the frequency of parasitemia in their offspring. Academic Editor: Robert Snow, Centre for Geographic Medicine Research Coast, Kenya Introduction The hallmark of pregnancy malaria due to Plasmodium falciparum is the accumulation of infected erythrocytes (IEs) in the placenta [1]. Placental IEs are a distinct parasite form that binds to chondroitin sulfate A (CSA) on syncytiotrophoblast and in intervillous spaces [2]. Placental IEs do not adhere to CD36, a ubiquitous receptor on the microvascular endothelium that commonly supports adhesion of IEs from non-pregnant individuals [2]. Adhesion to CSA allows parasites to sequester in the placenta, where dense accumulations can often occur with little or no parasitemia detectable in the peripheral blood [3]. Because CSA-binding parasites do not commonly infect non-pregnant individuals, women lack immunity to this parasite form prior to the first pregnancy [4]. In areas of stable malaria transmission, women acquire antibodies against placental parasites over successive pregnancies as a consequence of repeated exposures [4,5]. Maternal antibodies against placental IEs are associated with reduced risk of maternal parasitemia and improved pregnancy outcomes [6,7]. Because immunity is acquired over successive pregnancies, susceptibility to malaria is greatest during the first pregnancy and diminishes with increasing gravidity. Similarly, placental inflammation and the sequelae of pregnancy malaria, such as severe maternal anemia and low birth weight, are most frequent during first pregnancies [8–10]. Pregnancy malaria is estimated to cause tens of thousands or hundreds of thousands of infant deaths each year. However, these estimates are extrapolated from the incidence of malaria-related outcomes, such as low birth weight and maternal anemia that increase infant mortality risk [11,12], and the estimates sometimes differ from the real benefits observed in chemoprophylaxis trials. Low birth weight caused by pregnancy malaria has been estimated to cause 6% of infant deaths in sub-Saharan Africa [12], while antimalarial chemoprophylaxis delivered to pregnant women during the third trimester reduced infant mortality in The Gambia by 18% and 4% among offspring of primigravid and multigravid women, respectively [13]. In Malawi, chemoprophylaxis during pregnancy reduced infant mortality by 3%–5% [14]. The incomplete effectiveness of antimalarial regimens used in chemoprophylaxis studies [14] and the poor sensitivity of maternal peripheral blood slides or placental impression slides for detecting placental malaria (PM) [15] complicate studies that estimate the consequence of PM on infant outcomes. Few studies have directly examined the effects of pregnancy malaria on infant health. A study conducted in southern Cameroon found no significant difference in the frequency of malaria between infants born to PM-positive mothers (46.5%) and infants born to PM-negative mothers (38.5%) during the first two years of life (χ2 = 0.24, p = 0.60) [16]. However, the age-specific prevalence of P. falciparum parasitemia was consistently higher between 4 and 18 mo of age among infants born to mothers with PM. The overall malaria-free survival rates were not significantly different between the two groups of infants, although a considerable decrease was observed between 5 and 8 mo of age among infants born to placenta-infected mothers [16]. In a subset analysis of the same infant cohort, cord serum reactivity against two CSA-binding parasite isolates (but not reactivity against other parasites) was related to a younger age at first parasitemia in the infant (p 2,500 parasites per 200 white blood cells), vomiting that precluded oral medications, or prostration. Protocols for procedures used in this study were approved by the International Clinical Studies Review Committee of the Division of Microbiology and Infectious Diseases at the US National Institutes of Health, and ethical clearance was obtained from the Institutional Review Boards of Seattle Biomedical Research Institute and the National Institute for Medical Research in Tanzania. Analytical Population Infants whose data are reported in this study were enrolled between September 2002 and September 2003, and were followed throughout the first year of life. During the recruitment period, 495 mothers gave consent to participate and delivered at the Muheza Designated District Hospital. Excluded from the analyses were ten twin infants, four stillbirths, three early neonatal deaths, and six neonates who were not examined beyond 4 wk of age because their families moved out of the study area. Infants with any evidence of HIV infection (mother seropositive on voluntary testing [n = 7], infant presented with suggestive signs or symptoms [n = 8], or suffered HIV/AIDS-related death [n = 4] during follow-up) were also excluded (n = 19 total). During the period of this study, HIV-seropositivity rates were 7.5% among women who agreed to undergo antenatal testing. After exclusions, a total of 453 infants remained for analyses presented in this report. Statistical Analysis We examined the frequency distribution of maternal and infant characteristics according to PM status (parasitemia or no parasitemia) or gravidity (primigravid, secundigravid, or multigravid) using a chi-squared test for categorical variables and Student's t-test for continuous measurements. The Mann-Whitney test was used to assess differences in parasite density among women with PM. Median time to first parasitemia was obtained using Kaplan-Meier survival analysis and assessed with the log rank test. Follow-up time was calculated in weeks from birth until date of first positive diagnosis of parasitemia, date of death, date last known to be alive, September 30, 2004 (end of follow-up period), or 54 wk of age, whichever occurred first. Cox regression analysis was used to determine whether the risk of first parasitemia was associated with PM at delivery. The dependent variable was time between birth and first positive diagnosis measured in weeks. The following variables were considered to be potential confounders a priori and were included in the model: gravidity (primigravid, secundigravid, multigravid), transmission season at time of birth (high or low), and bed net use in a household. According to the incidence of parasitemia among 3- to 12-mo-old infants in Muheza (unpublished data), the high transmission seasons in both 2003 and 2004 occurred from May through October, and the low transmission seasons occurred from November through April. Residence location was grouped into six categories by geographical area and distance to nearest health center. In order to allow for a different baseline hazard for each location, we used a stratified Cox regression model [19]. All variables were assessed for conformity to the proportional hazards assumption using the global p (PH) statistic based on the Schoenfeld residuals. The likelihood ratio test was used to assess for effect modification. Information on bed net use was missing for almost 14% of the study population, and the bed net data were therefore analyzed using a missing indicator, by assigning values of yes, no, and unknown. To assess the effect of PM at delivery on the probability of infant parasitemia throughout infancy, generalized estimating equations logistic regression models were constructed with a first-order autoregressive working correlation structure (the analysis with exchangeable and m-dependent correlation structure produced nearly identical results for the data) and model-based standard errors [20]. This method allowed full use of the data while accounting for the correlation of repeated measurements over time. The multivariate model incorporated the main effect of PM at delivery, as well as gravidity and other confounding factors including bed net usage, age and transmission season at time of bloodsmear, location of residence, and transmission season at time of birth. A quadratic term for age ((age)2) was also included in the model because there appeared to be a curvilinear relationship between age and parasitemia. Offspring of secundigravidae versus multigravidae had similar probability of parasitemia during infancy (p > 0.98 and p > 0.38 when the mother was PM-positive or PM-negative, respectively), and therefore were combined for the final analysis. We assessed for effect modification between PM and gravidity by including an interaction term in the regression model and tested for significance using a Type 3 likelihood ratio test. Because the interaction term was significant (p = 0.008), results are presented stratified by primigravidae (no previous pregnancies) and secundi/multigravidae (one or more previous pregnancies). Treatment intensity (number of antimalarial treatments per week) was also assessed as a potential effect modifier in the different gravidity groups. Offspring of the four groups (PM-positive primigravid, PM-positive multigravid, PM-negative primigravid, PM-negative multigravid) did not significantly differ in the frequency of antimalarial treatments using the Kruskal-Wallis test (p = 0.20). Therefore, treatment intensity would not have a significant bias effect on the assessment of the relationship between gravidity, PM status, and other factors, and was not included in the final models. All p-values are two-sided, and confidence intervals (CIs) were calculated at the 95% level. Statistical significance was set at p ≤ 0.05. Data analyses were conducted using STATA version 8.0 (Stata Corporation, College Station, Texas, United States), Statview version 5.0, and SAS version 9.0 software (SAS Institute, Cary, North Carolina, United States). Results PM was identified in 69 (15.2%) of the mothers. PM-positive women were more likely to be younger and to be experiencing first-time pregnancies than PM-negative women (Table 1). Among primigravid and secundigravid women, the frequency of PM was similar (24.2% and 23.6%, respectively), whereas only 5.6% of multigravid women had PM at delivery. The mean placental parasite density among PM-positive women was significantly higher for primigravidae (7.1% of red cells infected), compared with secundigravidae (2.2%) and multigravidae (1.6%) combined (Mann-Whitney, tied p = 0.03). Although the mean placental parasite density of PM-positive women did not differ significantly between birth months, the four highest density placental parasitemias occurred in three primigravidae (35.0%, 52.8%, and 76.5% parasitemias) and one secundigravida (23.3% parasitemia) during the high transmission seasons. Reported usage of sulfadoxine-pyrimethamine for IPT against malaria during pregnancy was high (69.7%), but did not significantly differ between PM-positive and PM-negative mothers. Reported IPT usage was also similar among different gravidity groups (63%, 73%, and 72% for primigravidae, secundigravidae, and multigravidae, respectively), as was bed net usage (62%, 59%, and 58%, respectively). Residence area was not associated with gravidity group (chi-squared test, p = 0.12). Offspring of PM-positive mothers were similar to those of PM-negative mothers by several measures, including gender, birth month, and residence (Table 1). Of the 445 infants whose birth weights were available, the mean birth weight was 3.2 kg (range 2.0–4.5 kg). Twenty-six (5.8%) weighed less than 2.5 kg, which is the standard criterion for low birth weight. Infants of PM-positive mothers weighed significantly less on average at birth than those of PM-negative mothers (Table 1). The overall mean duration of follow-up of infants was 50 wk (range 4–54) and did not differ by PM status at delivery (p = 0.67). Sixty-three (13.9%) infants were lost to follow-up between 4 and 54 wk of age (moved or parent[s] withdrew consent), including six of the 69 infants born to PM-positive mothers. Thirteen infants died before reaching 54 wk of age; three (4.4%) of those from PM-positive mothers and ten (2.6%) of those from PM-negative mothers. Risk of First Parasitemia Two hundred and sixty (57.4%) infants in the study cohort experienced at least one episode of parasitemia by the age of 54 wk. Of the 193 infants who did not experience parasitemia, 138 (71.5%) were followed for at least 1 y. The median time to first parasitemia was 38 wk (95% CI 34–42) with a 75% probability of remaining free of parasitemia up to 18 wk of age. Of the 63 infants lost to follow-up, 17 (27.0%) presented with parasitemia before dropping out of the study. The median time to first parasitemia for infants born to PM-positive mothers was 32 wk (95% CI 21–38) compared with 39 wk (95% CI 35–46) for offspring of PM-negative mothers (Figure 1). The effect of PM on the time to first parasitemia in the offspring appeared to vary across gravidity groups (Figure 2). For offspring of primigravidae, the median time to first parasitemia for infants born to PM-positive mothers was 34 wk (95% CI 23–52) compared with 38 wk (95% CI 30–49) for infants born to PM-negative mothers (p = 0.48). Among offspring of secundigravidae, the median time to first parasitemia for infants born to PM-positive mothers was 27 wk (95% CI 14–54) compared with 52 wk (95% CI 38–54) for infants born to PM-negative mothers (p = 0.06). Among offspring of multigravidae, the median time to first parasitemia for infants born to PM-positive mothers was 22 wk (95% CI 10–38) compared with 37 wk (95% CI 32–46) for infants born to PM-negative mothers (p = 0.01). The graph shows the age at first parasitemia in offspring of PM-positive mothers (solid line) versus PM-negative mothers (dashed line). Infants born to PM-positive mothers experience their first parasitemia at a significantly younger age than infants of PM-negative mothers (log rank, p = 0.02). The graphs show the unadjusted (left panels) and adjusted (right panels) age at first parasitemia in first-born (A and B), second-born (C and D), or third-born or subsequent (E and F) offspring of PM-positive mothers (solid lines) versus PM-negative mothers (dashed lines). PM is associated with a significantly younger age at the time of first parasitemia among offspring of multigravidae (log rank, p = 0.01), but not among offspring of primigravidae (log rank, p = 0.48) or secundigravidae (log rank, p = 0.06). Adjustment estimates using a Cox model did not change statistical significance in any gravidity group. Using stratified Cox regression, the estimated hazard ratio of first parasitemia for infants born to PM-positive mothers was 1.41 (95% CI 1.01–1.99) times that of infants born to PM-negative mothers, after adjustment for gravidity, transmission season at time of birth, area of residence, and bed net usage (Table 2). Although the test for interaction between gravidity and placental infection was not statistically significant (likelihood ratio test, p = 0.27), the effect of PM on infant risk appeared to vary across gravidity groups after adjustment for potential confounders (Figure 2B, 2D, and 2F). Among offspring of multigravid women, the risk of first parasitemia for infants born to PM-positive mothers was more than twice that of infants born to PM-negative mothers, and remained significant after adjustment for transmission season at time of birth, area of residence, and bed net usage (adjusted hazard ratio [AHR] = 2.20, 95% CI 1.15–4.20). The risk of first parasitemia among offspring of primigravid or secundigravid women was not significantly associated with PM after adjustment for potential confounding factors (AHR = 1.15, 95% CI 0.66–2.00, AHR = 1.15, 95% CI 0.61–2.17, respectively). Risk of Any Parasitemia The age-specific prevalence of parasitemia during infancy differed by gravidity and PM status of the mother regardless of transmission season (Figure 3). Blood-slide positivity was observed more frequently among offspring of PM-positive multigravidae than among offspring of PM-positive primigravidae, in all age groups above 2 months of age. This relationship was observed in analyses of all bloodsmears collected from infants in either the low transmission (Figure 3C) or high transmission season (Figure 3D). These graphs show age-specific parasite positivity of all blood slides obtained from infants born to primigravid women (dashed lines) versus secundigravid or multigravid women (solid lines). The frequency of parasitemia among offspring of PM-negative mothers (A and B) or offspring of PM-positive mothers (C and D) is presented according to whether the slides were collected during the low-transmission season (A and C) or high-transmission season (B and D). Low transmission of malaria around Muheza occurs from November through April, and high transmission occurs from May through October. Parasitemia was more frequent among the offspring of secundigravid and multigravid women versus primigravid women when PM was present at delivery. This relationship was observed in all age groups except neonates, and was observed in both low- (C) and high- (D) transmission seasons. Figures represent frequency of positivity for all slides collected during the study period, including slides collected subsequent to treatment. Results from generalized estimating equations logistic regression analysis of the interaction between gravidity and PM on the odds of parasitemia are presented in Table 3. After adjustment for age, transmission season at birth and at time of bloodsmear, bednet usage, and residence, offspring of PM-negative primigravid women had significantly reduced odds of parasitemia during the first year of life (adjusted odds ratio [AOR] = 0.67, 95% CI 0.50–0.91), and the odds were lowest among offspring of PM-positive primigravid women (AOR = 0.21, 95% CI 0.09–0.47). In contrast, PM at delivery in multigravid women was associated with significantly increased odds of parasitemia in their offspring (AOR = 1.59, 95% CI 1.16–2.17). Discussion In this prospective cohort study of 453 mother–offspring pairs, both PM and maternal gravidity influenced the risk of P. falciparum parasitemia during infancy. Overall, offspring of PM-positive mothers experienced their first parasitemia at a significantly younger age than offspring of PM-negative mothers, and the risk of early first parasitemia was highest among offspring of PM-positive multigravidae. The risk of any parasitemia during infancy was strongly modified by an interaction between PM and gravidity. Offspring of PM-negative primigravidae had a decreased risk of parasitemia, while offspring of PM-positive primigravidae had the lowest risk. In contrast, offspring of PM-positive multigravidae had an increased risk of parasitemia. Our results are the first to identify an effect of maternal gravidity to modify malaria risk in offspring during infancy. An earlier study in southern Cameroon observed an increased age-specific prevalence of P. falciparum in infants born to PM-positive mothers [16], but did not assess the effect of gravidity. Surprisingly, in the present study, PM decreased malaria risk in offspring of primigravidae but increased risk in offspring of other gravid groups. The opposing effects of PM in different gravid groups observed in the present study may explain why earlier studies failed to find significant relationships between PM and malaria risk during the first two years of life [16]. Because PM had been suggested to increase susceptibility during infancy [16,21], we expected offspring of PM-positive primigravidae to be at highest risk, since PM is most frequent and placental parasite densities are highest during primigravid pregnancies. In Malawi, high placental parasite densities were associated with increased risk of cord blood parasitemia [22], supporting the conjecture that congenital malaria could account for the relationship between PM and susceptibility of infants [17]. Although we expected PM to increase susceptibility in offspring of primigravid women to the greatest degree, we observed the opposite pattern. Our data are consistent with those of an earlier study [17] showing that cord blood levels of antibody against CSA-binding parasites were positively related to child susceptibility to malaria. Antibody against CSA-binding parasites is highest in multigravid women [4], and we find that offspring of multigravid women have the highest risk of parasitemia. The results from the earlier study were interpreted to suggest that antibody against CSA-binding parasites reflected a recent maternal parasitemia that may induce immunologic tolerance or congenital malaria [17], or else reflected an increased exposure to malaria for both the mother and the offspring [23]. However, neither interpretation would fully explain our finding that an interaction between gravidity and PM modifies infant susceptibility to parasitemia. PM during the antenatal period may be influencing infant outcomes, separate from any effect of neonatal exposure occurring at delivery, and could explain the reduced risk of parasitemia among offspring of PM-negative primigravidae. In areas of stable transmission, primigravid women are parasitemic more frequently [24], and experience higher parasite densities, more severe inflammatory responses [8], and greater clinical sequelae [13,25] than women in other gravid groups. In the current cohort, 24.2% of primigravid women had PM with a mean parasite density of 7.1% at the time of delivery, compared with 5.6% of multigravid women with a mean parasite density of 1.6%, reflecting the susceptibility of primigravid women. Although we cannot exclude the possibility that an intrinsic effect of gravidity may modify malaria susceptibility of offspring, we favor the explanation that PM during the antenatal period is modulating the susceptibility of the offspring, because PM at delivery is having a similar (but more profound) effect. We propose that differences in the load of malarial antigen or the intensity of the inflammatory response may account for the differences observed between gravid groups, but this remains to be determined. Factors such as area of residence, season of observation, and HIV may increase the risk of parasitemia in both mother and offspring, and therefore may confound an analysis of maternal parasitemia and infant susceptibility. However, our adjusted analyses suggest that these confounding factors would not fully explain the results. Although the incidence of parasitemia during infancy differed significantly between villages in this study (unpublished data), the frequency of PM did not (Table 1). Nor did the frequency of PM vary significantly by birth month (Table 1). While infants born during the low transmission season had a higher risk of parasitemia (presumably due to the seasonality of transmission), the increased frequency of parasitemia observed in offspring of PM-positive secundi- and multigravidae was apparent in both high and low transmission seasons (Figure 3), suggesting that the relationship between PM and infant susceptibility in the different gravidity groups was independent of seasonality. The intensive longterm follow-up of infants was likely to identify the majority of infants with HIV infection, as progression to disease is rapid in infants, especially African infants [26]. Eleven of the 12 offspring of PM-positive multigravidae included in our analyses had been followed for 18 mo or more, and none of the 12 experienced signs or symptoms of HIV infection. In sum, confounding factors associated with risk of parasitemia do not fully explain the gravidity-dependent relationships observed in this study, and specifically run counter to the observation that offspring of PM-positive primigravidae have a decreased risk of parasitemia. Behavioral and environmental factors may vary with gravidity and could account for some gravidity-dependent relationships. An increased number of children in multigravid households would possibly attract more mosquitoes or increase the proportion of mosquitoes that are infective, thereby increasing malaria exposure among offspring of multigravidae. However, this would not explain why PM at delivery decreases malaria risk in offspring of primigravidae. Conversely, the increased frequency of fever in primigravid women may cause them to consume more antimalarials, which could be transferred to the fetus or infant and reduce infant parasitemia. However, reported usage of antimalarials during pregnancy was similar among the different gravidity groups. Furthermore, PM was associated with a risk of parasitemia throughout infancy, and is unlikely to be accounted for by ingestion of antimalarials at term. Our findings suggest that PM decreases susceptibility in the offspring of primigravidae, and increases susceptibility in the offspring of multigravidae. Among primigravidae, the pronounced inflammatory response to PM could confer protection, for example by reducing parasitemia [27] and thereby preventing congenital transmission. Alternatively, sensitization of the fetal or neonatal immune system may be modulating immunity in a way that modifies susceptibility to parasitemia, such as by inducing regulatory T (Treg) cells, which have been related to malaria susceptibility in rodents [28,29]. Inflammatory signals inhibit the development of Treg cells [30], and thus the inflammatory responses that are common in primigravid but not multigravid women with PM could lead to differential effects of sensitization in their offspring. Separately, studies in Kenya have shown that antibodies against the P. falciparum MSP-1 antigen were lower among offspring of PM-positive mothers [21], and it will be valuable to determine whether anti-MSP-1 antibody responses in infants vary according to both PM and maternal gravidity. Future studies should seek to better understand congenital transmission of P. falciparum, as well as the effect of placental inflammation to modulate the infant's antibody and cellular responses against the parasite. The findings of the present study establish a conundrum. PM at delivery greatly reduced the odds of parasitemia in offspring born to primigravid women, yet earlier studies reported that PM was the strongest risk factor for severe morbidity [31–34] and mortality of first-born infants [13]. More research is needed to understand the impact of PM on malaria-related and other clinical outcomes during infancy. The gravidity-dependent pattern of infant susceptibility identified here may provide a useful tool to study the relationship between the frequency of P. falciparum parasitemia and disease in young children. In summary, PM decreases the risk of parasitemia among offspring of primigravidae but increases the risk among offspring of multigravidae. Traditionally, pregnancy malaria has been thought to affect the offspring primarily through low birth weight or maternal anemia, which are risk factors for neonatal and infant mortality. The present findings indicate that pregnancy malaria may also have prolonged effects on immunity and susceptibility to malaria in the offspring, and that these effects are strongly influenced by maternal gravidity. Further investigation of these relationships with assessment of other potential confounders, as well as their impact on infant morbidity and mortality, is needed with larger cohort studies. A preventive dose of antimalarial chemotherapy delivered to secundigravid or multigravid women in the last 1–2 wk before term may reduce P. falciparum parasitemia in their offspring, but more research is needed to predict its effect on malaria-related disease and death. The benefits of malaria prevention during pregnancy may extend beyond its effect on birth outcomes, and should be studied in randomized clinical trials with careful attention to PM status at delivery and outcomes during early childhood. Acknowledgments The authors gratefully acknowledge the participation of the mothers and their infants in the MOMS Project, and the work of the MOMS Project staff, including assistant medical officers, nurses, village health workers, laboratory technicians and microscopists, and data entry personnel. Medical care for the infants participating in the project was provided by Muheza Designated District Hospital. Jason Wendler designed the MOMS database, and Peter Gilbert and E. Robert Greenberg provided statistical advice. The MOMS Project is supported by grants from US National Institutes of Health (R01 AI 52059 from NIAID and TW 05509 from Fogarty International Center) and the Bill & Melinda Gates Foundation. This research was presented November 8, 2004 at the 53rd annual meeting of the American Society of Tropical Medicine and Hygiene in Miami Beach, Florida (Abstract #25). The views expressed in this paper do not necessarily reflect those of the US Department of Defense. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: The study was designed by TKM, MF, and PED. Clinical studies were coordinated by TKM, laboratory studies were coordinated by GJD, MF, and PED, and data management was coordinated by JLL. Statistical analysis was performed by MCB, JLL, and XL. MCB and PED wrote the report with assistance from the other authors. Patient Summary Background In areas where malaria is common, most adult women have acquired some level of immunity against the malaria parasite. However, during pregnancy, many experience a new type of malaria infection in their placenta. This placental malaria is particularly severe during first-time pregnancy. Pregnant women with placental malaria often give birth to babies with a low birth weight, and these babies have an increased risk of dying as infants. Why Was This Study Done The connection between placental malaria and low birth weight is well established. However, it is not clear whether placental malaria makes it more likely for infants to get infected with malaria parasites themselves, and this is the question the researchers addressed in this study. What Did the Researchers Do and Find They studied a total of 453 infants, of which 69 were born to mothers with placental malaria. Of those 69 infants, 31 were born to first-time mothers, and 38 to mothers who had given birth before. The researchers then followed those infants for a year and checked how often during that time they had parasites in their blood. Surprisingly, the researchers found that children followed one of two opposing patterns, depending on their birth order. Children from first-time mothers were less likely to have parasites in their blood, especially children whose mothers had placental malaria. Children from other mothers were more likely to have parasites in their blood, especially children whose mothers had placental malaria. What Does This Mean These results suggest that both the number of previous pregnancies and placental malaria affect an infant's chances of developing malaria. It might be that in first-time mothers placental malaria stimulates the infant's immune system and this somehow protects the baby against malaria, but at present this is just speculation. Also, the study is relatively small, and the connections between the number of previous pregnancies, placental malaria, and the chances of an infant getting malaria need to be tested in other studies. Where Can I Find More Information Online The following Web sites provide information about malaria. Wikipedia pages on malaria: WHO pages on malaria, which contain a section on malaria and pregnancy: MedlinePlus pages on malaria: UNICEF page on malaria with links: Patient Summary Background In areas where malaria is common, most adult women have acquired some level of immunity against the malaria parasite. However, during pregnancy, many experience a new type of malaria infection in their placenta. This placental malaria is particularly severe during first-time pregnancy. Pregnant women with placental malaria often give birth to babies with a low birth weight, and these babies have an increased risk of dying as infants. Why Was This Study Done The connection between placental malaria and low birth weight is well established. However, it is not clear whether placental malaria makes it more likely for infants to get infected with malaria parasites themselves, and this is the question the researchers addressed in this study. What Did the Researchers Do and Find They studied a total of 453 infants, of which 69 were born to mothers with placental malaria. Of those 69 infants, 31 were born to first-time mothers, and 38 to mothers who had given birth before. The researchers then followed those infants for a year and checked how often during that time they had parasites in their blood. Surprisingly, the researchers found that children followed one of two opposing patterns, depending on their birth order. Children from first-time mothers were less likely to have parasites in their blood, especially children whose mothers had placental malaria. Children from other mothers were more likely to have parasites in their blood, especially children whose mothers had placental malaria. What Does This Mean These results suggest that both the number of previous pregnancies and placental malaria affect an infant's chances of developing malaria. It might be that in first-time mothers placental malaria stimulates the infant's immune system and this somehow protects the baby against malaria, but at present this is just speculation. Also, the study is relatively small, and the connections between the number of previous pregnancies, placental malaria, and the chances of an infant getting malaria need to be tested in other studies. Where Can I Find More Information Online The following Web sites provide information about malaria. Wikipedia pages on malaria: WHO pages on malaria, which contain a section on malaria and pregnancy: MedlinePlus pages on malaria: UNICEF page on malaria with links: References Garnham PCC (1938) The placenta in malaria with special reference to reticulo-endothelial immunity. Trans R Soc Trop Med Hyg 32:13–48. Fried M, Duffy PE (1996) Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta. Science 272:1502–1504. Clark HC (1915) The diagnostic value of the placental blood film in aestivo-autumnal malaria. J Exp Med 22:427–444. Fried M, Nosten F, Brockman A, Brabin BJ, Duffy PE (1998) Maternal antibodies block malaria. Nature 395:851–852. Ricke CH, Staalsoe T, Koram K, Akanmori BD, Riley EM, et al. (2000) Plasma antibodies from malaria-exposed pregnant women recognize variant surface antigens on Plasmodium falciparum-infected erythrocytes in a parity-dependent manner and block parasite adhesion to chondroitin sulfate A. J Immunol 165:3309–3316. Duffy PE, Fried M (2003) Antibodies that inhibit Plasmodium falciparum adhesion to chondroitin sulfate A are associated with increased birth weight and the gestational age of newborns. Infect Immun 71:6620–6623. Staalsoe T, Shulman CE, Bulmer JN, Kawuondo K, Marsh K, et al. (2004) Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria. Lancet 363:283–289. Walter P, Garin JF, Blot P, Philippe E (1981) Placenta et paludisme—Etude morphologique, parasitologique, et clinique. J Gynecol Obstet Biol Reprod 10:535–542. Meuris S, Piko BB, Eerens P, Vanbellinghen AM, Dramaix M, et al. (1993) Gestational malaria: Assessment of its consequences on fetal growth. Am J Trop Med Hyg 48:603–609. Fried M, Muga RO, Misore AO, Duffy PE (1998) Malaria elicits type 1 cytokines in the human placenta: IFN-gamma and TNF-alpha associated with pregnancy outcomes. J Immunol 160:2523–2530. Murphy SC, Breman JG (2001) Gaps in the childhood malaria burden in Africa: Cerebral malaria, neurological sequelae, anemia, respiratory distress, hypoglycemia, and complications of pregnancy. Am J Trop Med Hyg 64:57–67. Guyatt HL, Snow RW (2004) Impact of malaria during pregnancy on low birth weight in sub-Saharan Africa. Clin Microbiol Rev 17:760–769. Greenwood AM, Armstrong JR, Byass P, Snow RW, Greenwood BM (1992) Malaria chemoprophylaxis, birth weight and child survival. Trans R Soc Trop Med Hyg 86:483–485. Steketee RW, Wirima JJ, Campbell CC (1996) Developing effective strategies for malaria prevention programs for pregnant African women. Am J Trop Med Hyg 55:95–100. Rogerson SJ, Mkundika P, Kanjala MK (2003) Diagnosis of Plasmodium falciparum malaria at delivery: Comparison of blood film preparation methods and of blood films with histology. J Clin Microbiol 41:1370–1374. Le Hesran JY, Cot M, Personne P, Fievet N, Dubois B, et al. (1997) Maternal placental infection with Plasmodium falciparum and malaria morbidity during the first 2 years of life. Am J Epidemiol 146:826–831. Cot M, Le Hesran JY, Staalsoe T, Fievet N, Hviid L, et al. (2003) Maternally transmitted antibodies to pregnancy-associated variant antigens on the surface of erythrocytes infected with Plasmodium falciparum: Relation to child susceptibility to malaria. Am J Epidemiol 157:203–209. Ellman R, Maxwell C, Finch R, Shayo D (1998) Malaria and anaemia at different altitudes in the Muheza district of Tanzania: Childhood morbidity in relation to level of exposure to infection. Ann Trop Med Parasitol 92:741–753. Cox DR, Oakes D (1984) Analysis of survival data New York: Chapman and Hall. 201 p. Liang KY, Zeger SL (1986) Longitudinal data analysis using generalized linear models. Biometrika 73:13–22. Bonner PC, Zhou Z, Mirel LB, Ayisi JG, Shi YP, et al. (2005) Placental malaria diminishes development of antibody responses to Plasmodium falciparum epitopes in infants residing in an area of western Kenya where P. falciparum is endemic. Clin Diagn Lab Immunol 12:375–379. Redd SC, Wirima JJ, Steketee RW, Breman JG, Heymann DL (1996) Transplacental transmission of Plasmodium falciparum in rural Malawi. Am J Trop Med Hyg 55:57–60. Hviid L, Staalsoe T (2004) Malaria immunity in infants: A special case of a general phenomenon Trends Parasitol 20:66–72. Cannon DSH (1958) Malaria and prematurity in the western region of Nigeria. Br Med J 877–878. Watkinson M, Rushton D (1983) Plasmodial pigmentation of placenta and outcome of pregnancy in West African mothers. Br Med J 287:251–254. Chakraborty R (2005) HIV-1 infection in children: A clinical and immunologic overview. Curr HIV Res 3:31–41. Stevenson MM, Riley EM (2004) Innate immunity to malaria. Nat Rev Immunol 4:169–180. Hisaeda H, Maekawa Y, Iwakawa D, Okada H, Himeno K, et al. (2004) Escape of malaria parasites from host immunity requires CD4+ CD25+ regulatory T cells. Nat Med 10:29–30. Long TT, Nakazawa S, Onizuka S, Huaman MC, Kanbara H (2003) Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice. Int J Parasitol 33:175–183. Piccirillo CA, Shevach EM (2004) Naturally-occurring CD4+CD25+ immunoregulatory T cells: Central players in the arena of peripheral tolerance. Semin Immunol 16:81–88. van Eijk AM, Ayisi JG, Ter Kuile FO, Misore AO, Otieno JA, et al. (2002) Malaria and human immunodeficiency virus infection as risk factors for anemia in infants in Kisumu, western Kenya. Am J Trop Med Hyg 67:44–53. Reed SC, Wirima JJ, Steketee RW (1994) Risk factors for anemia in young children in rural Malawi. Am J Trop Med Hyg 51:170–174. Cornet M, Le Hesran JY, Fievet N, Cot M, Personne P, et al. (1998) Prevalence of and risk factors for anemia in young children in southern Cameroon. Am J Trop Med Hyg 58:606–611. Dolan G, ter Kuile FO, Jacoutot V, White NJ, Luxemburger C, et al. 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☉ 11340239:Heritability of Malaria in Africa
1 School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom,2 Department of Pathology, University of Cambridge, Cambridge, United Kingdom,3 Kenya Medical Research Institute/Wellcome Trust Programme, Centre for Geographic Medicine Research, Coast, Kilifi District Hospital, Kilifi, Kenya,4 Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, United Kingdom,5 Department of Paediatrics, John Radcliffe Hospital, Oxford, United Kingdom Background While many individual genes have been identified that confer protection against malaria, the overall impact of host genetics on malarial risk remains unknown. Methods and Findings We have used pedigree-based genetic variance component analysis to determine the relative contributions of genetic and other factors to the variability in incidence of malaria and other infectious diseases in two cohorts of children living on the coast of Kenya. In the first, we monitored the incidence of mild clinical malaria and other febrile diseases through active surveillance of 640 children 10 y old or younger, living in 77 different households for an average of 2.7 y. In the second, we recorded hospital admissions with malaria and other infectious diseases in a birth cohort of 2,914 children for an average of 4.1 y. Mean annual incidence rates for mild and hospital-admitted malaria were 1.6 and 0.054 episodes per person per year, respectively. Twenty-four percent and 25% of the total variation in these outcomes was explained by additively acting host genes, and household explained a further 29% and 14%, respectively. The haemoglobin S gene explained only 2% of the total variation. For nonmalarial infections, additive genetics explained 39% and 13% of the variability in fevers and hospital-admitted infections, while household explained a further 9% and 30%, respectively. Conclusion Genetic and unidentified household factors each accounted for around one quarter of the total variability in malaria incidence in our study population. The genetic effect was well beyond that explained by the anticipated effects of the haemoglobinopathies alone, suggesting the existence of many protective genes, each individually resulting in small population effects. While studying these genes may well provide insights into pathogenesis and resistance in human malaria, identifying and tackling the household effects must be the more efficient route to reducing the burden of disease in malaria-endemic areas. Academic Editor: Simon Foote, Royal Melbourne Hospital, Australia Introduction While a growing number of genes have been described that are associated with protection from infection and severe disease due to Plasmodium falciparum malaria, the contribution of each gene, or of all the genes combined, relative to the many environmental factors that also influence malarial risk, has rarely been estimated [1,2]. Putting genetic and environmental factors into perspective will inform the design and interpretation of intervention studies aimed at reducing the burden of malarial disease and will help to rationalise research priorities. It is difficult to estimate the overall contribution of genetic factors (“heritability”) to the between-person variation in the incidence of infectious diseases in the field because it requires both longitudinal data on individual patients sufficient to obtain adequate measures of their risk and also the identification of genetic relatedness between these patients. Furthermore, because related individuals often share a common environment (such as a house), and environmental factors play a major role in the risk of infectious diseases, environmental and genetic effects are inseparable in most field study designs (for example, those that have pairs of full-sibs, each pair living in a different house [1,3–5]). In order to separate these effects, it is necessary to study sets of individuals of varying genetic relatedness who live together in the same house: information on genetically related individuals who live in different households also helps. In this study, we make use of all known genetic relationships within and between houses in order to estimate the heritability of disease risk. This is done, essentially, by regressing the correlation between individuals in their disease incidence on the degree of genetic relationship between them. For example, if the correlation in incidence between full-sibs (who share half their genes) is 0.2, or among half-sibs (who share one quarter of their genes) is 0.1, then the heritability is estimated to be 0.4 [6]. Here we use a generalised version of this principle [7] (the so-called animal model that is widely used in animal breeding) that takes account of all degrees of genetic relatedness to determine the relative contributions of host genetics and other factors to the risk of malaria and other diseases in children living in a malaria-endemic area on the coast of Kenya. Methods Data Collection We analysed data from two separate studies, one addressing “mild,” uncomplicated clinical malaria, and the second addressing malaria resulting in admission to hospital, as described in detail previously [8,9]. Briefly, in study 1, the mild disease cohort study, conducted between October 1998 and September 2003, we monitored the incidence of fevers in 640 children 10 y old or younger who were residents of the Ngerenya area of Kilifi District, using active weekly surveillance in the community. The coastal community is made up of a group of nine closely related ethnic groups, broadly known as the Mijikenda, two of which dominated the study populations here. We defined malaria as a measured fever (axillary temperature > 37.5 °C) or a reported history of fever within the preceding 48 h in conjunction with a slide positive for blood-stage asexual P. falciparum parasites at any density, and nonmalarial fevers as those in which the blood slide was negative for malarial parasites. We used verbal interview of mothers or key representatives of the household to obtain information on genetic relationships between study children, their parents, and sometimes their grandparents, and to identify genetic links between households (for example, sisters married into different households). From this information, we created a three-column “pedigree” list (one for the individual, two for their parents) of all individuals identified as being related to at least one child in the study. This list was then used to build up a square matrix (“the relationship matrix,” see below) containing coefficients of relationship among all pairs of individuals in the study and among their parents or more distant ancestors where such links were identified [10]. A household typically comprised a group of 3–6 adjacent houses, each occupied by one woman and her children whose husbands were full-sib or half-sib brothers and who sometimes had more than one wife. Thus, within each household, the children generally formed several full-sib, half-sib, and first cousin groups. In study 2—the birth cohort study—we monitored the incidence of admission to hospital with malaria and other diseases through passive surveillance of a birth cohort of 2,914 children 5 y old or younger, residing in a wider geographical area that encompassed Ngerenya (study 1) within 16 km of Kilifi District Hospital between May 1992 and December 1997. The birth cohort was recruited from a continuous demographic surveillance system used to monitor child survival. For the purpose of this analysis, we classified admissions into three categories: (i) malaria, (ii) other infectious diseases (such as acute respiratory infections, meningitis, measles, or gastroenteritis), or (iii) accidents. In this paper we use the term “hospitalised malaria” to refer to hospital admissions with malaria, but do not distinguish between the severity of disease within this class (for example, cerebral malaria, severe malarial anaemia, or neither of these). In this study, we identified full-sibs, but did not record other information on genetic relationships. Data Analysis For study 1, we calculated the annual incidence of malarial and nonmalarial fevers for each child separately as the number of episodes divided by the total number of weeks of surveillance, multiplied by 52. We performed this calculation both for each child over the entire period spent in the study (method 1), and for each age year the child spent in the study (for example as an 8-, 9-, and 10-y-old [method 2]). We excluded records for 3 wk following treatment for an episode of malaria. We also excluded data from patients with less than 30 records per year in order to standardise the measurement variation in incidence and, in the case of infants, to minimise the influence of maternally acquired immunity. Finally, we analysed data, including and excluding data from extreme children who suffered more than ten episodes of malaria or more than ten episodes of nonmalarial fever per year, on the basis that these were possibly manifestations of additional health problems. As the distribution of malaria incidence data was skewed, and because we expected heterogeneous variances across different age groups and years associated with their different means, we analysed our data both before and after applying or log10(x + 1) transformations. We analysed our incidence data using a mixed linear model that incorporated genetic relationships to partition the total variation in disease incidence into its genetic, household, systematic, and other causes. The fixed effects fitted were age range (0–2, 3–5, 6–8, 9–10-y-olds, using the average for the entire study period; this effect was only fitted in method 1), year (one level for each year of study based on the average for the record in question), sex (male, female), use of mosquito nets over beds at night (yes, no, or unknown), ethnic group (Giriama, Chonyi, or other), and haemoglobin S genotype (wild type, HbAA; heterozygote, HbAS). The random effects fitted were for household and additive genetic value of each child. We separated additive genetic from other effects pertaining to an individual (nongenetic “permanent environmental,” or nonadditive genetic) by incorporating an “additive genetic relationship matrix” into our model [7]. This matrix, built from the pedigree list described above, contained the expected degree of genetic relationship between all children in the study (for example, for full-sibs) and all their relatives. By incorporating this matrix into the model, the analysis essentially regresses the covariance among relatives in the trait under analysis onto their degree of genetic relatedness: this provides an estimate of the heritability based on all pairs of observations on related individuals from across the whole spectrum of relatedness. The model was fitted using restricted maximum likelihood procedures and the DFREML package [11]. We calculated the contribution of each of the fixed effects, and their sum (f2), to the total variation in the trait (phenotypic variance, VP) from the ANOVA table, by dividing the type 3 sum of squares by the total sum of squares. Contributions of additive genetics (h2) and household (c2) were calculated from the ratio of their estimates of variance (VA and VC) to VP. Approximate standard errors of h2 and c2 were calculated from the information matrix around the maximum of the likelihood surface [12]. We analysed our data in two ways. First, we analysed the average incidence over the entire study period (1 record per child) using the above model (method 1). However, since different children spent different periods of time in the study, and because variances might differ across ages and years in association with their different means, we were concerned that the model's assumption of homogeneous errors would be violated. Therefore, we also performed a second analysis using repeated records on the same child, one for each full age year recorded (method 2), in which we fitted a multivariate model treating each age year as a separate, but correlated, trait (that is, 11 traits for age years 0–10). This method thus allowed h2 and c2 to vary across age groups: it also yielded estimates of genetic and environmental correlations in incidence between age groups. Estimates of h2 and c2 and their standard errors of annual incidence were pooled across age groups, using appropriate weights for the number of observations in each age group. In study 2, our outcomes of interest were rates of admission to hospital with malaria, other infectious diseases, and accidents. We excluded data from children who were alive at the end of the study but who were absent from the district for more than 5 mo during the study period. We included data from children who died during their hospital stay, but excluded data from those who either died outside hospital or at less than 1 mo of age. Given the binary nature of the data, we used threshold models for these analyses. These models assume that there is an underlying normally distributed “liability” trait, with a threshold level above which the disease is manifested. These took three forms. First (method 3), we compared the incidence of malaria admissions to hospital among affected full-sibs to that in the general, unrelated, population: a difference in incidence among relatives reflects a genetic component in the trait on the underlying scale and thus allows an estimation of its heritability [13]. This method does not allow for incorporation of fixed effects into the analysis, or for separation of h2 from c2, but does provide an easy way of calculating an upper limit to heritability from incidence data. In method 4, we analysed the data on the observed (binary) scale under a model fitting random effects for sibship and household, and fixed effects for age, sex, ethnic group, and bednet use. In this model, we did not fit year as it was heavily confounded with age in this study, and no information was available on haemoglobin S genotype. The estimates of h2 and c2 on the observed scale were then transformed to the underlying scale [14]. In method 5, we fitted the same model as in method 4, but data were analysed on the probit scale using general linear modelling procedures [15]. For method 3, data from all children were used in order to obtain an accurate estimate of incidence in the general population. However, in methods 4 and 5, data from children not in sibships were excluded because they contained no relevant information. Standard errors of h2 and c2 estimates were calculated based on method 4 [16]. To determine whether genetic and household effects could be adequately separated from the data structure encountered in these studies, we simulated phenotypic data according to the observed pedigree and household structure, assuming a range of values of additive genetic and household variances between 0.1 and 0.5, a phenotypic variance of 1, and either a normal (study 1) or binary distribution (study 2) of the trait. Ten replicate datasets per parameter combination were generated and then analysed under a model fitting household and an additive genetic effect per person as random effects. Results The study design, and estimates of h2, c2 and f2 from both these studies, and from a previous similar study conducted in Sri Lanka [2], are summarised in Table 1. As estimates in study 1 did not change by more than 0.05 as a result of transforming the data or excluding values of greater than ten episodes per year, only the estimates based on untransformed, uncensored data are shown. Study 1 The final analysis of the mild disease cohort study included data from 640 children living in 77 different households with a total of 1,727 annual age year records (2.7 per child). We identified the parents of 602 of these children (177 fathers, 222 mothers) and one or both grandparents of 119. The total pedigree included 1,590 individuals. An average household comprised 8.3 children fathered by an average of 2.3 men, themselves brothers, half-brothers, or first cousins, each with an average of 1.14 wives. Thus there were typically three groups of full-sibs per household who also formed groups of first cousins or half-sibs. The ethnic composition of this population was 84% Giriama, 10% Chonyi, and 6% other Mijikenda. The incidence of nonmalarial fevers decreased rapidly from birth and averaged 1.9 episodes per child per year over the ages 0–10 y (Figure 1). In contrast, the incidence of malaria increased until age 3 y, but then remained stable until 10 y of age, the overall average being 1.6 episodes per child per year. Because superinfection (new infections in people who are already infected) is common in malaria, and the total number of fevers remained approximately constant between these ages, it is probable that our case definition more accurately represented the prevalence of blood-stage parasites than it did the incidence of new infections. Less than 5% of these infections resulted in hospital admission. In Figure 2, we have partitioned the total variation in the incidence of mild malaria and nonmalaria fevers, when averaged over the entire study period (method 1), into its components. As sex and bednet usage each explained less than 0.3% of this variation, we have omitted them from the figure. Ethnic group and HbAS each accounted for around 2% of the variation in malarial fevers and less than 0.4% in nonmalarial fevers, even when additive genetics and household were not included in the model. Age explained a lower proportion of the variation in malarial fevers than nonmalarial fevers, reflecting the age-incidence patterns for each (see Figure 1). Averaging the estimates from methods 1 and 2, additive genetics (h2) explained 24(± 11)% and 39(± 12)% of the variation in malarial and nonmalarial fevers, respectively: the corresponding estimates for household effects (c2) were 29(± 6)% and 9(± 4)%, respectively. Colour coding for all four charts follows that in the top left using method 1. Although phenotypic variances correlated positively with mean incidence at each age, there was no obvious change in h2 with age. Genetic correlations between incidence at consecutive ages averaged 0.40 and 0.46 for malaria and nonmalaria, respectively. The corresponding “environmental” (residual) correlations were 0.01 and 0.02, and phenotypic correlations were 0.36 and 0.26. Phenotypic, genetic, and environmental correlations between the incidence of malarial and nonmalarial fevers within age years were 0.01, 0.23 and 0.29, respectively, the latter no doubt reflecting the fact that these two traits represent opposite sides of the same coin. When averaged over years, the corresponding values were 0.37, 0.61, and 0.38, suggesting that children share susceptibilities to both types of fever for both genetic and nongenetic (but not household) reasons. The mean correlations between age groups in household effects for malaria and nonmalaria were 0.81 and 0.55, respectively, indicating that household effects were consistent across age groups, especially for malaria. Study 2 During the study, 2,914 children remained resident, their average age at the end of the study or at death (and hence length of time under surveillance) being 4.1 y (range 1 mo–5.7 y). Overall, 33% of these children were admitted to the hospital at least once during the study period. Forty-eight percent of all admissions were due to malaria, while a further 26%, 9%, and 2% of admissions were due to acute respiratory infections, gastrointestinal infections, and accidents, respectively. The incidence of malaria and other infections decreased rapidly with age (Figure 1). The average incidence of hospitalised malaria over the 4.1 y was 0.054 per child per year (compared to 1.64 per year in children ≤5 y old in study 1). The average age at first admission with malaria was 1.6 y (range 5 wk–5.3 y), and the average age of all first admissions was 1.4 y (4 wk–5.3 y). Case-fatality rates for hospitalised malaria and other illnesses were 2.6% and 2.3%, respectively. The ethnic composition of the population in study 2 was broadly similar to that in study 1, being 77% Giriama, 13% Chonyi, and 10% other Mijikenda. Estimates of h2 and c2 for nonmalarial infections in this study were respectively lower and higher than for malaria, reversing the pattern seen for mild infections in study 1 (see Figure 2; Table 1). On the other hand, in the case of both mild and hospitalised disease, fixed effects accounted for more of the variation in nonmalarial infections than in malaria. Our estimates of h2, c2 and f2 for accidents were 0, 0, and 4%, respectively, although the incidence was too low for these to be reliable. The simulation study showed that estimates of h2 and c2 were unbiased by the data structure (that is, by confounding between genetic groupings and household) but that, as expected from some confounding, the sampling correlations between them were 0.5 for study 1 and 0.7 for study 2. This means that if, for sampling reasons, the true h2 was overestimated by 0.2 (that is, two standard errors), c2 would be underestimated by 0.05 in study 1 and by 0.07 in study 2. These simulations also showed that our estimates of standard errors were reasonable. Discussion Our analyses of data from two independent studies conducted on the coast of Kenya, and a third study conducted in Sri Lanka [2], each focussed on a different part of the wide spectrum of disease severity in malaria, and using a variety of statistical methods, suggest that host genetic factors generally account for about a quarter of the total variation in the susceptibility of individuals to malarial disease (Figure 2; Table 1). The Sri Lankan study differed from the Kenyan studies in that it had a much lower transmission intensity and hence disease incidence, none of which was severe, a high prevalence of Plasmodium vivax in addition to P. falciparum, a study population consisting mainly of adults, and an entirely different genetic composition to that of Kenya. Nevertheless, the results from both studies are in broad agreement, suggesting that substantial genetic variability for resistance to infection and disease severity is maintained in human populations that have been exposed to the disease for a very long time. We could only attribute a small proportion of this variation to the best known of the malaria resistance genes, HbS and α-thalassaemia. For example, in theory [6] we expect that HbAS, which roughly halves the incidence of mild malaria (equivalent to half a standard deviation in incidence) and is found at a frequency of 0.15 in our population [17,18], would only explain around 2.5% of the total variation (2.4% additive genetic and 0.06% dominance variation) in incidence of mild clinical malaria, a figure close to that derived through observation in our studies (Figure 2). Similarly, we would anticipate that the mutant that causes α-thalassaemia, which is found in our population at an allele frequency of 0.43, and which reduces the incidence of uncomplicated malaria by around 0.4 of a standard deviation in homozygotes and a little more than half this in heterozygotes (T. N. Williams, personal communication), would account for only 2% (1.9% additive and 0.1% dominance) of the total phenotypic variation [6]. For hospitalised malaria, the corresponding values are 0.6% for HbAS [18] and 0.7% for α-thalassaemia [19]. Thus, on their own, even the most prominent of the known malaria resistance genes make only minor contributions to the total impact of host genetics. These examples highlight the fact that, as shown by an increasing number of studies conducted both in the field and in the laboratory [20–23], malaria resistance is under complex, multigenic control, with each individual gene having a relatively small epidemiological impact. The heritability estimates we report here are almost certainly conservative for several reasons. First, we anticipate that our assessment of paternity will have been subject to error, reducing our observed relative to true estimates by a factor of (1 p)2 where p measures the misclassification of paternity [24]. Second, our models only estimated the contributions of genes that act additively: the effects of genes with nonadditive effects, such as dominance or epistasis—the latter of which has already been demonstrated for some known resistance genes [25]—will not contribute to the heritability estimates reported here. Although statistical models are available that allow estimation of nonadditive genetic variance, much larger sets of suitably structured data would be required to obtain reliable estimates. Finally, there is growing evidence to suggest that variability in parasite virulence genes interacts with host genetic polymorphisms [26]: this form of host genetic variability is not represented in the additive genetic heritabilities estimated here. Our studies suggest that host genetic factors also affect susceptibility to nonmalarial fevers and, to a lesser extent, nonmalarial infections leading to hospital admission. Indeed, the high genetic correlation between the average rate of malarial and nonmalarial fevers (61%) suggests that susceptibility to a range of childhood infections might be mediated via mechanisms with a common genetic basis. High heritabilities of the immune response to some antigens from malaria parasites and other infectious pathogens [3,27,28,] may indicate genetic control of a generalised immune response to pathogens. This does not rule out the possibility that some individual genes may have specific, but opposing effects on resistance to malaria versus other pathogens, thus perhaps helping to maintain the remarkable degree of genetic variation for disease resistance observed in this heavily selected population. A striking result from this study was the amount of variation that could be attributed to household, particularly in the incidence of mild malaria and nonmalarial hospitalised infections. Children living in the 10% most malarious houses had about twice as many malaria infections (2.39 per year) as those in the 10% least malarious houses (1.14 per year). As our measure of malaria incidence almost certainly includes superinfections, this between-house variability probably reflects variation in transmission intensity due to spatial variation in breeding sites for mosquitoes and other household-related factors such as insecticide and repellant use [29]. We could not attribute the between-household variation in transmission intensity to bednet use, as also found in a second study in this area [29]. Even though untreated bednets in good condition are protective in this study area, damaged nets are not [30]. In the present analysis, where we considered whether or not bednets of any kind were being used, we did not find a protective effect. Socioeconomic factors such as quality of building and surrounds, nutrition, education, and access to health care may also play a part in explaining between-household differences in both malarial and nonmalarial infections. Clearly, identifying and improving factors relating to the risk of individual households would go a long way towards relieving the burden of disease in children living under such conditions. This study shows that despite the inherent stochasticity in malaria transmission, the average risk of malaria in children living in our study area is in large part due to factors that are predetermined, both at the genetic and nongenetic level. While manipulation of the host's genes or their products may not yet seem plausible, determining how specific genes control the protective response may, ultimately, lead to a better understanding of the mechanisms of pathogenesis and host resistance. In the meantime, tackling household-related factors would seem to be a more tractable option for disease control. Acknowledgments We are grateful to all study participants and their families and to the clinical and medical officers, nursing, field, and laboratory staff of the Kenya Medical Research Institute (KEMRI)/Wellcome Trust Programme–Coast, for their help with data collection. The study received financial support through a programme grant awarded to RWS, KM, and C. R. J. C. Newton by the Wellcome Trust. MJM is supported by a fellowship from the Royal Society of London, and TMW, RWS, and KM are supported by fellowships from the Wellcome Trust. We thank W. G. Hill and I. M. S. White for statistical advice and J. A. Rowe and D. J. Weatherall for comments on the manuscript. This paper is published with the permission of the director of KEMRI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: TWM, KM, and TNW conceived and conducted the mild disease cohort study, and pedigree data were collected by MJM and TWM. RWS and TNW designed and conducted the birth cohort study. Statistical analyses were conducted by MJM. Patients were enrolled by TWM, RWS, and TNW. MJM and TNW wrote the paper. Patient Summary Background Humans exposed to malaria get infected and sick to varying degrees, and some of that variation is due to differences in genetic makeup between individuals. Because the disease has killed humans for thousands of years, selection over time has increased protective variants of human genes in regions where malaria was and is common. One well-known example is the sickle-cell variant of the hemoglobin gene, which protects against severe malaria and is more common in people of African descent. Why Was This Study Done Most research has focused on identifying the specific genes whose variants confer susceptibility to or protection from malaria. Some have been identified, but it is also clear that there are many genes involved, most of which contribute a small amount to the overall picture. In this study, the researchers wanted to estimate how much all genetic factors taken together, relative to the many environmental factors that also affect malaria risk, influence the number and severity of malaria cases. What Did the Researchers Do and Find The researchers recruited and studied groups of children in rural Kenya. To estimate the overall contribution of genetic factors, they needed to observe the children over a long enough time that they would get a sense of an individual's malaria risk. In addition, they needed to know the genetic relatedness among the children, and the living arrangements needed to be such that children of different degrees of genetic relatedness (full siblings, half siblings, first cousins, etc.) shared a common environment. They found that genetic differences among people accounted for about 25% of the variability in malaria risk. This was less than the contribution by “household factors,” which accounted for around 30% of the total variability. Some of these household factors are known ones, such as insecticide use, but most of the 30% in risk variability was due to unidentified household factors. What Do These Findings Mean The risk of malaria is strongly predetermined by genetic and nongenetic factors. However, while understanding how specific genes affect malaria risk will ultimately lead to a better understanding of the disease and improve prevention and treatment, genetic factors are not the biggest contributors to malaria. Therefore, in the short term, focusing on identifying and improving household-related factors is more likely to reduce the burden from the disease. Where Can I Get More Information Online General information on the disease from the World Health Organization and links to many other sites: The Wellcome Trust's malaria pages, which include a section on malaria and people that discusses genetic factors: The malaria pages of the Centers for Disease Control and Prevention, which contain a section on geographic distribution and epidemiology: Patient Summary Background Humans exposed to malaria get infected and sick to varying degrees, and some of that variation is due to differences in genetic makeup between individuals. Because the disease has killed humans for thousands of years, selection over time has increased protective variants of human genes in regions where malaria was and is common. One well-known example is the sickle-cell variant of the hemoglobin gene, which protects against severe malaria and is more common in people of African descent. Why Was This Study Done Most research has focused on identifying the specific genes whose variants confer susceptibility to or protection from malaria. Some have been identified, but it is also clear that there are many genes involved, most of which contribute a small amount to the overall picture. In this study, the researchers wanted to estimate how much all genetic factors taken together, relative to the many environmental factors that also affect malaria risk, influence the number and severity of malaria cases. What Did the Researchers Do and Find The researchers recruited and studied groups of children in rural Kenya. To estimate the overall contribution of genetic factors, they needed to observe the children over a long enough time that they would get a sense of an individual's malaria risk. In addition, they needed to know the genetic relatedness among the children, and the living arrangements needed to be such that children of different degrees of genetic relatedness (full siblings, half siblings, first cousins, etc.) shared a common environment. They found that genetic differences among people accounted for about 25% of the variability in malaria risk. This was less than the contribution by “household factors,” which accounted for around 30% of the total variability. Some of these household factors are known ones, such as insecticide use, but most of the 30% in risk variability was due to unidentified household factors. What Do These Findings Mean The risk of malaria is strongly predetermined by genetic and nongenetic factors. However, while understanding how specific genes affect malaria risk will ultimately lead to a better understanding of the disease and improve prevention and treatment, genetic factors are not the biggest contributors to malaria. Therefore, in the short term, focusing on identifying and improving household-related factors is more likely to reduce the burden from the disease. Where Can I Get More Information Online General information on the disease from the World Health Organization and links to many other sites: The Wellcome Trust's malaria pages, which include a section on malaria and people that discusses genetic factors: The malaria pages of the Centers for Disease Control and Prevention, which contain a section on geographic distribution and epidemiology: References Jepson A, Banya W, Sisay-Joof F, Hassan-King M, Bennett S, et al. (1995) Genetic regulation of fever in Plasmodium falciparum malaria in Gambian twin children. J Infect Dis 172:316–319. Mackinnon MJ, Gunawardena DM, Rajakaruna J, Weerasinghe S, Mendis KN, et al. (2000) Quantifying genetic and nongenetic contributions to malarial infection in a Sri Lankan population. Proc Natl Acad Sci U S A 97:12661–12666. Stirnadel HA, Al-Yaman F, Genton B, Alpers MP, Smith TA (2000) Assessment of different sources of variation in the antibody responses to specific malaria antigens in children in Papua New Guinea. Int J Epidemiol 29:579–586. Garcia A, Dieng AB, Rouget F, Migot-Nabias F, Le Hesran JY, et al. (2005) Role of environment and behaviour in familial resemblances of Plasmodium falciparum infection in a population of Senegalese children. Microbes Infect 6:68–75. Ranque S, Safeukui I, Poudiougou B, Traore A, Keita M, et al. (2005) Familial aggregation of cerebral malaria and severe malarial anemia. J Infect Dis 191:799–804. Falconer DS, Mackay TFC (1996) Introduction to quantitative genetics, 4th ed Harlow (United Kingdom): Longman. 464p. Lynch M, Walsh B (1998) Genetics and analysis of quantitative traits Sunderland (Massachusetts): Sinauer Associates. 990p. Snow RW, Howard SC, Mung'ala-Odera V, English M, Molyneux CS, et al. (2000) Paediatric survival and re-admission risks following hospitalization on the Kenyan Coast. Trop Med Int Health 5:377–383. Nyakeriga AM, Troye-Blomberg M, Chemtai AK, Marsh K, Williams TN (2004) Malaria and nutritional status in children living on the coast of Kenya. Am J Clin Nutr 80:1604–1610. Henderson CR (1975) Use of relationships among sires to increase accuracy of sire evaluation. J Dairy Sci 58:1731–1738. Meyer K (1998) DFREML user notes for programmes to estimate variance components for individual animal models by restricted maximum likelihood, version 3.0 [computer program] Armidale (Australia): University of New England. 29p. Kendall M, Stuart A (1979) Inference and relationship. Volume 2, The advanced theory of statistics, 4th ed New York: Macmillan. 749p. Falconer DS (1965) The inheritance of liability to certain diseases, estimated from the incidence among relatives. Ann Hum Genet 29:51–76. Robertson A, Lerner IM (1949) The heritability of all-or-none traits: Viability of poultry. Genetics 34:411. SAS Institute. (1997) GLIMMIX: A SAS macro for fitting generalized linear mixed models using PROC MIXED and the Output Delivery System (ODS), version 8.1 [computer program] Cary (North Carolina): SAS Institute. Hill WG, Smith C (1977) Estimating heritability of a dichotomous trait. Biometrics 33:230–236. Williams TN, Mwangi TW, Wambua S, Alexander ND, Kortok M, et al. (2005) Sickle cell trait and the risk of P. falciparum malaria and other diseases. J Infect Dis 192:178–186. Williams TN, Mwangi TW, Roberts DJ, Alexander ND, Weatherall DJ, et al. (2005) An immune basis for malaria protection by the sickle cell trait. PLoS Medicine 2:e128DOI:10.1371/journal.pmed.0020128. Williams TN, Wambua S, Uyoga S, Macharia A, Mwacharo J, et al. (2005) Both heterozygous and homozygous α+-thalassemia protect against severe and fatal Plasmodium falciparum malaria on the coast of Kenya. Blood 106:368–371. Abel L, Dessein AJ (1997) The impact of host genetics on susceptibility to human infectious diseases. Curr Opin Immunol 9:509–516. Burt RA, Baldwin TM, Marshall VM, Foote SJ (1999) Temporal expression of an H2-linked locus in host response to mouse malaria. Immunogenetics 50:278–285. Fortin A, Stevenson MM, Gros P (2002) Susceptibility to malaria as a complex trait: Big pressure from a tiny creature. Hum Mol Genet 11:2469–2478. Frodsham A, Hill AVS (2004) Genetics of infectious diseases. Hum Mol Genet 13:R187–R194. Van Vleck LD (1970) Misidentification in estimating the paternal-sib correlation. J Dairy Sci 53:1469–1474. Williams TN, Mwangi TW, Wambua S, Peto TEA, Weatherall DJ, et al. (2005) Negative epistasis between the malaria-protective effects of α-thalassaemia and the sickle cell trait. Nat Genet In press. Kwiatkowski DP (2005) How malaria has affected the human genome and what human genetics can teach us about malaria. Am J Hum Genet 77:192. Jepson A, Sisay-Joof F, Banya W, Hassan-King M, Frodsham A, et al. (1997) Genetic linkage of mild malaria to the major histocompatibility complex in Gambian children: Study of affected sibling pairs. BMJ 315:96–97. Stirnadel HA, Beck HP, Alpers MP, Smith TA (1999) Heritability and segregation analysis of immune responses to specific malaria antigens in Papua New Guinea. Genet Epidemiol 17:16–34. Snow RW, Peshu N, Forster D, Bomu G, Mitsanze E, et al. (1998) Environmental and entomological risk factors for the development of clinical malaria among children on the Kenyan coast. Trans R Soc Trop Med Hyg 92:381–385. Mwangi TW, Ross A, Marsh K, Snow RW (2003) The effects of untreated bednets on malaria infection and morbidity on the Kenyan coast. Trans R Soc Trop Med Hyg 97:369–372....查看详细 (40470字节)
☉ 11340240:Thyrotoxicosis and Pregnancy
Petros Perros is in the Endocrine Unit, Freeman Hospital, Newcastle upon Tyne, United Kingdom. The Learning Forum section editors are Susan Lightman and William Lynn. DESCRIPTION of CASE A 35-year-old woman became aware of a swelling in her neck about ten weeks before referral. She had been trying to conceive for ten months. She had had a missed abortion five months earlier. Because of the neck swelling, her family doctor arranged thyroid function tests, which were in the thyrotoxic range on two occasions five weeks apart: serum free thyroxine, 26 and 28 pmol/l (normal range, 11–23); serum free tri-iodothyronine, 10.9 and 11 pmol/l (normal range, 3.5–6.5); serum thyroid-stimulating hormone (TSH), <0.05 mU/l (normal range, 0.3–4.1). Her previous medical history included a partial thyroidectomy for thyrotoxicosis at the age of 24. Other than a goitre, she had no symptoms except increased appetite and a slight tremor, which she had been aware of for about eight weeks. Following the missed abortion, she had two normal menstrual periods. Her only medication was folic acid supplements. She worked part-time and had a two-and-a-half-year-old child. On examination she was of average weight. Her hands were warm and moist. There was a fine tremor. A previous thyroidectomy scar was noted. The right lobe of the thyroid was palpable and felt smooth. There was a bruit over the right thyroid lobe on auscultation. She had lid retraction and lid lag but no other signs suggestive of thyroid-associated ophthalmopathy (TAO) (Figure 1). Her pulse rate was 100 beats per minute and regular. Her blood pressure was 150/70 mm Hg. The rest of the examination was normal. Note that there are no signs of TAO, but the patient has minimal upper lid retraction (the upper lid should normally be halfway between the limbus and the pupil). What Is the Cause of Her Thyrotoxicosis Thyrotoxicosis is no more than a descriptor for a pattern of biochemical abnormalities. Before considering treatment, it is the clinician's task to define the underlying cause, as an accurate diagnosis is an essential guide to the most appropriate treatment (Box 1). The most likely causes in this case were Graves disease, thyroiditis, toxic multinodular goitre (TMNG), and toxic adenoma. The hallmark of TMNG or toxic adenoma is the presence of one or more palpable thyroid nodules. In this case the patient had previously undergone a partial thyroidectomy and a vascular thyroid remnant was palpable on the right thyroid lobe. Post-partum thyroiditis occurs within 12 months of childbirth; a variant of this condition occurs after miscarriage. In this patient's case post-partum thyroiditis was unlikely because her previous pregnancy was 2.5 years earlier; however, the miscarriage five months earlier may have been relevant. Viral thyroiditis is usually preceded by an upper respiratory tract infection and the thyroid gland is tender to touch; the absence of these features makes viral thyroiditis unlikely. “Silent” thyroiditis may present in this way and was a possibility here. Laboratory tests that may help differentiate between the different causes of thyrotoxicosis include a radiolabelled technetium or iodide thyroid scan (Figure 2), and measurement of anti–thyroid peroxidase (TPO) antibodies, TSH receptor antibodies, and inflammatory markers (Table 1). The thyrotoxic phase of thyroiditis is usually followed by spontaneous euthyroidism and in some cases hypothyroidism. Repeating thyroid function tests within a few weeks of the first set may identify cases of thyroiditis. (A) Normal. (B) Graves disease: diffuse increased uptake in both thyroid lobes. (C) TMNG: “hot” and “cold” areas of uneven uptake. (D) Toxic adenoma: increased uptake in a single nodule with suppression of the surrounding thyroid. (E) Thyroiditis: decreased or absent uptake. (Image: Giovanni Maki) In this case the prolonged time course of thyrotoxicosis, the presence of a vascular thyroid remnant, the persistently thyrotoxic thyroid function tests, and the elevated serum levels of TSH receptor antibodies (62 U/l; reference range, 0–10) were in favour of a diagnosis of recurrent Graves disease. What Are the Effects of Thyrotoxicosis on Fertility and Risk of Abortion Menstrual irregularities occur in about 20% of thyrotoxic women [1]. Infertility is common in women with thyrotoxicosis even when they maintain ovulatory cycles [1]. Thyrotoxicosis also increases the risk of miscarriage to 26% [2]. How Should This Patient Be Treated There are three treatment options for thyrotoxicosis due to Graves disease: radioiodine (131I) therapy, thyroidectomy, and anti-thyroid drugs [3]. 131I therapy is safe and effective, but pregnancy should be deferred for 4–6 months after treatment as there are theoretical risks of fetal abnormalities. Most national regulatory authorities recommend avoidance of close contact with adults for a few days and with children and pregnant women for 2–3 weeks. 131I therapy was not appropriate for this patient because she wished to proceed with pregnancy as soon as possible and she had a two-and-a-half-year-old child, who would be difficult to care for after 131I therapy. A second thyroidectomy is worthy of consideration, but involves general anaesthesia and a period of recuperation of a few weeks and therefore disruption of family and professional life. The risks of damage to the recurrent laryngeal nerves and parathyroid glands after a second thyroidectomy are considerably greater than after a first operation and are of the order of 5%–10%. Because of these considerations, thyroidectomy was not felt to be a suitable option. Anti-thyroid drugs (carbimazole, methimazole, and propylthiouracil) restore euthyroidism within a few weeks of initiation of treatment [4]. Minor side effects (such as skin rashes) occur in about 5% of cases. Agranulocytosis is rare (～0.4%), but the consequences are life threatening and all patients on anti-thyroid drugs must be made aware of this complication (Box 2). All anti-thyroid drugs have been used and are acceptable in pregnancy. Congenital anomalies have been reported in association with anti-thyroid drugs, but the increase in risk above background is very marginal. The risks of aplasia cutis and choanal and oesophageal atresia may be slightly lower with propylthiouracil than with other anti-thyroid drugs (choanal and oesophageal atresia, scalp defects, minor facial anomalies, and psychomotor delay compose an embryopathy implicated with methimazole use). But because the evidence is inconclusive and the additional risk minimal, all three drugs are widely used in pregnancy. The lowest dose of anti-thyroid drug that maintains euthyroidism should be used in women who wish to become or are already pregnant, in order to avoid fetal hypothyroidism and fetal goitre formation. In this case propylthiouracil was used initially, at a dose of 50 mg four times per day. The patient was advised to take contraceptive measures until euthyroidism. Four weeks later her thyroid function tests had improved: serum free thyroxine, 13 pmol/l; serum total tri-iodothyronine, 2.5 nmol/l (normal range, 1.34–2.73); serum TSH, <0.05 mU/l. The dose of propylthiouracil was reduced to 25 mg four times per day, and the patient was advised that she could start trying to conceive. What Are the Risks of TAO TAO is a complication that many patients fear. It can be disfiguring and difficult to treat [3]. If there are no clinical features of TAO at presentation, the risk of developing it in future is approximately 15%. Smoking is an important predisposing factor. As this patient was a non-smoker the probability of developing TAO is less than 10%. What Monitoring Is Required during Pregnancy The dose of anti-thyroid drug usually needs to be decreased during pregnancy, and often Graves disease remits completely and the medication can be withdrawn. This is probably due to the overall immunosuppressive effect of pregnancy. Monitoring of free thyroid hormone concentrations is of paramount importance during pregnancy and should be performed every 4–6 weeks, or more frequently if thyroid status is changing. The biochemical target is to achieve and maintain maternal serum free thyroxine levels at or slightly above the upper limit of normal, using the lowest dose of anti-thyroid drug possible. TSH receptor antibodies should be measured in the third trimester because positivity is predictive of neonatal thyrotoxicosis [5]. When the mother (as in this case) has a functioning thyroid gland or remnant in situ, maternal thyroid function mirrors that of the fetus. If there are concerns about fetal thyrotoxicosis (e.g., because maternal hyperthyroidism proves difficult to control), fetal heart rate monitoring should be undertaken. A persistent fetal tachycardia greater than 160 beats per minute is suggestive of fetal thyrotoxicosis. In cases where fetal thyrotoxicosis is diagnosed, monitoring of fetal growth and fetal goitre by ultrasound is imperative. In most cases the fetus can be treated satisfactorily by adjusting the dose of anti-thyroid drug in the mother and by following the fetal response clinically and by ultrasound. What Are the Risks to the Fetus in a Woman with Graves Disease Poor control of maternal hyperthyroidism is associated with significant obstetric complications including miscarriage (26%), low birth weight, prematurity, (pre-)eclampsia, and possibly congenital malformations [6]. After the fetal thyroid matures (from 20 weeks of gestation onwards), maternal TSH receptor antibodies may act on the fetal thyroid to cause fetal thyrotoxicosis and goitre. The risk of fetal thyrotoxicosis is about 1% of all pregnancies in women with Graves disease, and if untreated, fetal mortality may be as high as 24%. Overtreatment may lead to hypothyroidism in the fetus, which is associated with subtle neurocognitive deficits later on in life, particularly if the hypothyroidism occurs in the first trimester [7]. Fetal goitre can develop as a result of fetal thyrotoxicosis or fetal hypothyroidism and in severe cases can obstruct labour. What Are the Risks of Recurrence of Thyrotoxicosis after Delivery The risk of relapse of maternal thyrotoxicosis is high in the post-partum period (up to 80%), and close monitoring is required. Anti-thyroid drugs can be used safely during breastfeeding [8]. Prenatal Counselling of Women with Graves Disease Pregnancy is a common concern among women of childbearing age who are receiving treatment for Graves disease. Some women may elect to have definitive treatment before pregnancy, which can be either a thyroidectomy or 131I therapy. The advantage of these treatment options is that the risk of maternal thyrotoxicosis during pregnancy is reduced, if not eliminated. Fertility is not affected by 131I therapy for thyrotoxicosis, but pregnancy should be deferred for 4–6 months after 131I therapy, although the basis of this recommendation is largely empirical. The risk of fetal and neonatal thyrotoxicosis is not eliminated by previous thyroidectomy or 131I therapy. The most important advice to women who have a previous history of thyroid dysfunction is to work with their practitioner to ensure that thyroid function tests are normal at the time of conception and throughout pregnancy. DISCUSSION Aetiology of Graves Disease Graves disease is an autoimmune condition and is mediated by stimulatory autoantibodies to the TSH receptor. There is a significant genetic component to the aetiology of Graves disease, although environmental factors and stress also seem to confer risk [3]. Typically, the thyroid gland of patients with Graves disease is diffusely enlarged and vascular. Toxic Multinodular Goitre TMNG may also run in families. The pathogenesis is unknown. The disease begins with the formation of a single or few colloid nodules, and over a period of several years these become larger and more numerous. Some nodules are functioning and gradually acquire autonomy. With the passage of time serum TSH declines and may become undetectable until at a later stage serum free thyroid hormones rise. The hyperthyroidism of TMNG is usually mild and tends to occur in middle life or later. Toxic adenomas are benign neoplasms of the thyroid that are autonomous. In some cases they arise because of somatic mutations that lead to constitutive activation of the TSH receptor. As with TMNG, the hyperthyroidism tends to be mild. Thyroiditis Thyroiditis is due to an inflammatory process affecting the thyroid epithelium. Unlike other causes of thyrotoxicosis, there is no increased synthesis of thyroid hormones; instead, stored thyroid hormones in colloid are released into the circulation because of the leaky epithelium. The thyrotoxic phase of thyroiditis may be followed by a hypothyroid phase a few weeks later, but as a rule the patient recovers and euthyroidism ensues without any intervention. Thyroiditis may occur after a viral infection (referred to as subacute or De Quervain thyroiditis), in which case the patient typically has a viral sore throat, the thyroid is tender, and inflammatory markers (erythrocyte sedimentation rate and C-reactive protein) are raised. “Silent” thyroiditis is autoimmune and characterised by positive anti-TPO antibodies. Investigating the Cause of Thyrotoxicosis In many cases of thyrotoxicosis the aetiology will be apparent from information that can be obtained from the history and clinical examination. In cases where there is doubt, additional investigations are indicated. Direct measurement of TSH receptor antibody levels is not widely available, but can be very valuable as modern assays are highly sensitive and specific. TSH receptor antibodies can occasionally be positive in post-partum thyroiditis (this seems to be particularly rare in Europe, though reported in North America and Japan), and in cases of doubt a thyroid scan showing no uptake of radioisotope is diagnostic of thyroiditis [9]. TSH receptor antibody measurement is indicated in pregnancy to assess the risks of fetal and neonatal thyrotoxicosis. Anti-TPO antibodies occur in a significant proportion of the normal population, and this limits the use of this test. High concentrations of anti-TPO antibodies are present in silent and post-partum thyroiditis. Radioisotope scans are useful in identifying the cause of thyrotoxicosis (Figure 2), but should be avoided in pregnancy. Treatment of Thyrotoxicosis The treatment of thyrotoxicosis depends on the underlying cause. Anti-thyroid drugs are effective in Graves disease, TMNG, and toxic adenoma (Table 2), but not in thyroiditis because the latter is not associated with increased de novo synthesis of thyroid hormones. After a course of anti-thyroid drug treatment, remission may be expected in Graves disease as a result of the immunosuppressive effect of anti-thyroid drugs on synthesis of TSH receptor antibodies, but relapse is the rule in cases of TMNG or toxic adenoma. 131I therapy is effective for Graves disease, TMNG, and toxic adenoma. 131I therapy is ineffective in thyroiditis because iodine uptake is reduced or absent in this condition (Figure 2). Most patients with Graves disease develop permanent hypothyroidism after 131I therapy, whereas most patients with TMNG and toxic adenoma do not. 131I therapy is associated with a small risk of exacerbation of new development of TAO, particularly in smokers. Thyroiditis may require symptomatic treatment with beta blockers during the thyrotoxic phase. The type of thyroidectomy (subtotal versus total) for Graves disease as primary treatment has been the subject of controversy for some years. The argument in favour of total thyroidectomy is that the risk of recurrence of the thyrotoxicosis is eliminated, and that if performed by skilled thyroid surgeons the probability of hypoparathyroidism and vocal cord palsy is no greater than for a subtotal thyroidectomy [10]. A subtotal thyroidectomy, on the other hand, provides the best chance of any treatment for Graves disease for long-term euthyroidism without the need for thyroxine or other treatments for thyrotoxicosis. The choice of treatment for Graves disease should be tailored to the needs of the individual patient, but also depends on local facilities, surgical expertise, and patient choice. Box 1. Causes of Thyrotoxicosis Common causes of thyrotoxicosis in a young female Graves disease Thyroiditis Toxic multinodular goitre Toxic adenoma Iodine excess Other rare causes of thyrotoxicosis Hyperemesis gravidarum Choriocarcinoma TSH-producing pituitary adenoma Iatrogenic thyrotoxicosis Factitious thyrotoxicosis Struma ovarii Metastatic follicular thyroid cancer Thyroid hormone resistance syndrome Box 1. Causes of Thyrotoxicosis Common causes of thyrotoxicosis in a young female Graves disease Thyroiditis Toxic multinodular goitre Toxic adenoma Iodine excess Other rare causes of thyrotoxicosis Hyperemesis gravidarum Choriocarcinoma TSH-producing pituitary adenoma Iatrogenic thyrotoxicosis Factitious thyrotoxicosis Struma ovarii Metastatic follicular thyroid cancer Thyroid hormone resistance syndrome Box 2. Patient Information Leaflet Used by the Author to Remind Patients Receiving Anti-Thyroid Drugs of the Potential Complication of Agranulocytosis “You have been started on a drug called Carbimazole/Methimazole/Propylthiouracil to control the activity of your thyroid gland. This is important treatment and Carbimazole/Methimazole/Propylthiouracil is a well established drug that has been used for many years. The great majority of people treated with Carbimazole/Methimazole/Propylthiouracil have no problems whatsoever. “Some people occasionally develop a rash—if this happens please consult your doctor as soon as possible; you need not discontinue the drug unless he/she tells you to do so. “More rarely, Carbimazole/Methimazole/Propylthiouracil affects white cells in the blood, in which case you would be likely to develop a very severe sore throat and to feel ill with a fever. If this happens while you are on Carbimazole/Methimazole/Propylthiouracil treatment you must attend either your family doctor or the hospital on the same day, to have your blood count checked. Take no more tablets until the blood count has been checked. If your white blood count is normal you can carry on with the Carbimazole/Methimazole/Propylthiouracil. If your white blood count is abnormal your family doctor or the hospital will need to deal with this problem urgently. “Please keep this with you in case you need to show it to your doctor.” Box 2. Patient Information Leaflet Used by the Author to Remind Patients Receiving Anti-Thyroid Drugs of the Potential Complication of Agranulocytosis “You have been started on a drug called Carbimazole/Methimazole/Propylthiouracil to control the activity of your thyroid gland. This is important treatment and Carbimazole/Methimazole/Propylthiouracil is a well established drug that has been used for many years. The great majority of people treated with Carbimazole/Methimazole/Propylthiouracil have no problems whatsoever. “Some people occasionally develop a rash—if this happens please consult your doctor as soon as possible; you need not discontinue the drug unless he/she tells you to do so. “More rarely, Carbimazole/Methimazole/Propylthiouracil affects white cells in the blood, in which case you would be likely to develop a very severe sore throat and to feel ill with a fever. If this happens while you are on Carbimazole/Methimazole/Propylthiouracil treatment you must attend either your family doctor or the hospital on the same day, to have your blood count checked. Take no more tablets until the blood count has been checked. If your white blood count is normal you can carry on with the Carbimazole/Methimazole/Propylthiouracil. If your white blood count is abnormal your family doctor or the hospital will need to deal with this problem urgently. “Please keep this with you in case you need to show it to your doctor.” Learning Points Thyrotoxicosis is not a diagnosis, merely a biochemical result. An accurate clinical diagnosis encompassing the aetiology is imperative for optimal management. The most common cause of thyrotoxicosis in women of childbearing age is Graves disease. Thyrotoxicosis impairs fertility, and thyroid status should be assessed in women with secondary infertility or recurrent abortions. Three treatments are available for thyrotoxicosis due to Graves disease: anti-thyroid drugs, 131I therapy, and thyroidectomy. The right treatment is that which suits the patient's individual circumstances best. 131I therapy is an absolute contraindication in pregnancy. Anti-thyroid drugs may be used safely, and the dose should be titrated to the minimum dose that maintains normal maternal thyroid hormone levels. The hyperthyroidism of Graves disease usually remits after the first trimester, and anti-thyroid drugs can be withdrawn; however, relapse of maternal thyrotoxicosis in the post-partum period is common. Uncontrolled maternal hyperthyroidism can lead to fetal thyrotoxicosis with devastating effects on the fetus. Fetal thyrotoxicosis can be treated satisfactorily by appropriate manipulation of the maternal dose of anti-thyroid drug and careful fetal monitoring. Learning Points Thyrotoxicosis is not a diagnosis, merely a biochemical result. An accurate clinical diagnosis encompassing the aetiology is imperative for optimal management. The most common cause of thyrotoxicosis in women of childbearing age is Graves disease. Thyrotoxicosis impairs fertility, and thyroid status should be assessed in women with secondary infertility or recurrent abortions. Three treatments are available for thyrotoxicosis due to Graves disease: anti-thyroid drugs, 131I therapy, and thyroidectomy. The right treatment is that which suits the patient's individual circumstances best. 131I therapy is an absolute contraindication in pregnancy. Anti-thyroid drugs may be used safely, and the dose should be titrated to the minimum dose that maintains normal maternal thyroid hormone levels. The hyperthyroidism of Graves disease usually remits after the first trimester, and anti-thyroid drugs can be withdrawn; however, relapse of maternal thyrotoxicosis in the post-partum period is common. Uncontrolled maternal hyperthyroidism can lead to fetal thyrotoxicosis with devastating effects on the fetus. Fetal thyrotoxicosis can be treated satisfactorily by appropriate manipulation of the maternal dose of anti-thyroid drug and careful fetal monitoring. References Krassas GE, Perros P (2002) Reproductive function in patients with thyroid diseases. Hot Thyroidology 2. Abramson J, Stagnaro-Green A (2001) Thyroid antibodies and fetal loss: An evolving story. Thyroid 11:57–63. Weetman AP (2000) Graves' disease. N Engl J Med 26 343:1236–1248. Cooper DS (2005) Antithyroid drugs. N Engl J Med 352:905–917. Laurberg P, Nygaard B, Glinoer D, Grussendorf M, Orgiazzi J (1998) Guidelines for TSH-receptor antibody measurements in pregnancy: Results of an evidence-based symposium organized by the European Thyroid Association. Eur J Endocrinol 139:584–586. Davis LE, Lucas MJ, Hankins GD, Roark ML, Cunningham FG (1989) Thyrotoxicosis complicating pregnancy. Am J Obstet Gynecol 160:63–70. Pop VJ, Vulsma T (2005) Maternal hypothyroxinaemia during (early) gestation. Lancet 365:1604–1606. American Academy of Pediatrics Committee on Drugs. (2001) Transfer of drugs and other chemicals into human milk. Pediatrics 108:776–789. Muller AF, Drexhage HA, Berghout A (2001) Postpartum thyroiditis and autoimmune thyroiditis in women of childbearing age: Recent insights and consequences for antenatal and postnatal care. Endocr Rev 22:605–630. Palit TK, Miller CC 3rd, Miltenburg DM (2000) The efficacy of thyroidectomy for Graves' disease: A meta-analysis. J Surg Res 90:161–165....查看详细 (24542字节)
☉ 11340241:潜艇艇员经鱼雷发射管脱险训练的医学保障
[关键词] 艇员;鱼雷发射管;脱险训练;医学保障 由于执行任务海域辽阔，海上水文气象条件、地域、疆界等因素的影响，无论何种原因导致潜艇失事，都可能使救援部队舰船的就位、实施救援受到一定限制。因此，潜艇艇员应熟练掌握自救技术。经鱼雷发射管脱险是比较常用的艇员自救方法之一。为了提高艇员脱险技术水平，2005年我部进行了潜艇艇员经鱼雷发射管脱险训练，我部医院顺利完成了医学保障任务。 1 对象与方法 1.1 参训者 24名参训人员参加了海上实艇脱险训练...查看详细 (4443字节)
☉ 11340242:通信兵16PF人格特征与工作绩效关系的研究
[摘要] 目的: 探讨通信兵的人格特征与工作绩效的关系，为通信兵岗位的心理选拔提供依据。 方法: 采用卡特尔16种人格因素量表(16PF)对160名男性通信兵进行了测试，就其量表分数与常模、工作绩效分数进行比较和分析。 结果: 通信兵在16种人格因素的12种上与常模分数具有显著差异;不同次元人格类型的通信兵在工作绩效上有显著差异;四种二元人格类型以及心理健康、专业成就和成长能力与工作绩效有显著相关;心理健康分数、创造力分数和恃强性因素分数对工作绩效有显著的预测作用...查看详细 (7839字节)
☉ 11340243:Biocrimes, Microbial Forensics, and the Physician
Steven E. Schutzer is a physician-scientist in the Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, United States of America. Bruce Budowle is Senior Scientist in the Laboratory Division, Federal Bureau of Investigation, Quantico, Virginia, United States of America. Ronald M. Atlas is in the graduate school of University of Louisville, Louisville, Kentucky, United States of America. Attending physicians, regardless of their specialty or setting of practice, may suspect or learn that their patient has been attacked with a biological agent. In such cases, it is important to be aware of the interactions that may occur with law enforcement, and of the role that the evolving science of microbial forensics [1] may play in the investigation. Physicians are in key positions to preserve critical evidence and, thereby, contribute to the chain of custody, and, at the same time, offer suggestions to help develop the field of microbial forensics. This article provides guidance to physicians who believe that one of their patients is a victim of an act of bioterrorism or of another biocrime, and who are compelled by law, or with the patient's consent, wish to assist law enforcement in an investigation. In this regard, there are instructive lessons that can be learned from cases of past biocrimes and from analogies to more familiar cases of sexual assault and child abuse (Table 1). Recent Biocrimes The most widely publicized bioterrorism event in the United States was the US anthrax mail attacks of 2001. In this case, an astute physician diagnosed the index case of systemic anthrax [2] that set off national panic and a federal investigation that is still ongoing. Highly publicized in Europe was the assassination of a Bulgarian exile in London using ricin, a toxin extracted from castor beans, [3,4] which was delivered to him using an umbrella. Less publicized are other biocrimes such as the case of a laboratory worker in Texas intentionally infecting hospital co-workers with Shigella dysenteriae. Biocrimes are much less likely to occur than many infectious diseases, such as HIV/AIDS. In fact, it is often the abundance of naturally occurring infections that may make the detection of a biocrime difficult. However, there are also documented cases of non-bioterrorism biocrimes (Box 1) [5,6], which far exceed the number of documented bioterrorism acts, as well as many hoaxes where physicians and clinical laboratories were involved in determining if there was a real threat to exposed individuals. Box 1. Examples of Non-Bioterror Biocrimes Intentional Salmonella typhi food contamination in France from 1910 to1918 A Yersinia pestis attack by injection in 1933 in India Deliberate use of HIV-infected blood and secretions to inflict harm Source: [5,6]. Box 1. Examples of Non-Bioterror Biocrimes Intentional Salmonella typhi food contamination in France from 1910 to1918 A Yersinia pestis attack by injection in 1933 in India Deliberate use of HIV-infected blood and secretions to inflict harm Source: [5,6]. (Reprinted cover image from [30] with permission from Elsevier) A biocrime is similar to an assault crime, except, instead of a gun or knife, the weapon is a pathogen or a toxin. In the US, acts of bioterrorism are federal crimes that are governed by different responses by law enforcement and public health agencies than those that govern other biocrimes [7]. Most biocrimes and their subset of bioterrorism cases will involve public health agencies because of the nature of a disease threat to the public. The numerous hoaxes that are biocrimes include white powders found in letters that proclaim the presence of anthrax, and threatening notes claiming ricin contamination of baby food. Ricin currently appears to be a prevalent bioweapon, particularly as a tool for extortion. These potential ricin threats demonstrate the impact of bioterrorism on patient care: physicians had to monitor patients who might have ingested the poisoned food and, hence, were distracted from caring for other patients [8]. Hoaxes can be challenging for the physician, who must distinguish between symptoms and signs that could be toxin-related and those that are just variants of normal health. This challenge was compounded in a recent case where there were trace findings of an inactive form of ricin in baby food. Nonetheless, a hoax is also a crime, and the physician should not discard any evidence simply because material appears innocuous. As environmental biothreat sensors expand into the workplace and public places, they will be relied on as sentinels for possible biothreat releases. In the US, sensors for anthrax are being placed in some postal offices and at some major public events, and air monitoring is being carried out in major cities and transit systems (the BioWatch system). When such sensors indicate a possible bioterrorist attack, even if the signal is later found to be a false signal, the public will react, and are likely to seek out their own physicians for medical diagnostics, therapy, and advice. Potential cases that follow a public alert warrant evaluation, collection of patient samples, and possibly institution of prophylactic treatments until an alert is deemed a false alarm. Such a situation occurred in March 2005 following a presumptive positive detection of anthrax in a US Department of Defense mailroom. This situation necessitated treating over 900 potential victims with antibiotics as a precaution. However, despite the newer sensor technologies, physicians will likely remain the best and definitive authorities on the presence of an infection. Reporting a Biocrime Patients who believe that they have been a victim of a biocrime generally want both a medical and law enforcement response—that is, they want medical treatment, and they want the perpetrator to be found, prosecuted, and punished. Requirements for how physicians and other members of the health-care system initially report a suspected biocrime are governed at the local level (but regulations are ever-changing, and it is important to check current information at the time it is needed). Many US state regulations mandate that diagnostic laboratories report preliminary isolates of certain microbes to public health authorities, who, in turn, are sometimes compelled to notify law enforcement, depending on the isolate. Certain states have laws that mandate that a physician report specific diseases or unusual clinical manifestations to public health authorities and, in some cases, directly to law enforcement. The latter requirement is analogous to what is expected when there is suspicion of child abuse or gunshot wounds, and failure to notify authorities is itself a crime. The regulations of the Health Insurance Portability and Accountability Act (HIPAA) of 1996 established national standards for health-care transactions as well as security and privacy policy for health-related information, and physicians should be aware of these standards. However, even though these regulations severely restrict the release of patient health–related information, they still permit physicians and health-care facilities to release otherwise protected health information in instances of suspected crimes or threats to public health [9,10]. Given the limited guidance for reporting suspected biocrimes, physicians could face several dilemmas. For example, some patients may not want to report a crime or a disease condition, yet may have reasonable concerns. At the beginning of the HIV/AIDS epidemic, when no treatment was available, a diagnosis of HIV infection caused many patients to fear discrimination and loss of employment, a situation that persists in many parts of the world. Similar questions may arise in bioterrorism events about insurance coverage if an event is deemed an act of war. Communication between physician and patient should help the patient understand the pros and cons of notifying law enforcement of a suspected biocrime, including whether withholding notification could place others at risk. At the very least, discussion can strengthen the doctor–patient relationship. Fear and embarrassment of reporting potential false alarms to law enforcement or public health authorities may also be a concern for physicians and patients. But if a suspicion is not reported, a critical situation may go unrecognized and continue to worsen. Early notification to law enforcement authorities may provide valuable time and direction for investigative leads. It is expected that there will be many more negatives (false alarms) than positives when alerting law enforcement. Early reports to public health authorities may stem an epidemic. It is a misconception that you must wait for a firm diagnosis before reporting a potential case to authorities. There are many other misconceptions about biocrimes (Table 2). Guidance in the US concerning the reporting of suspicions of biocrimes is provided by the Centers for Disease Control and Prevention (CDC; http://www.cdc.gov), the Federal Bureau of Investigation (FBI; http://www.fbi.gov), and the Department of Homeland Security (DHS; http://www.dhs.gov) (Table 2). A joint statement by the FBI, the CDC, and the DHS advises calling the FBI and public health authorities if a suspicious situation arises [11]. Local public health departments are advised to notify the FBI before notifying the CDC. Specifically, “the FBI must be notified for any case of smallpox or pulmonary anthrax, uncommon agent or disease, an illness caused by a microorganism with markedly atypical features, an illness due to aerosol or food or water sabotage, as opposed to a usual transmission route, one or more clusters of illnesses that remain unexplained after a preliminary investigation; deliberate chemical, industrial, radiation or nuclear release” [12]. Calls or online tips should be directed to the FBI (https://tips.fbi.gov). Interpol and the World Health Organization are also developing response plans to help the public and physicians respond to suspected biocrimes and acts of bioterrorism (http://www.who.int/topics/bioterrorism/en). The Physician's Role in Collecting Evidence Although finding the perpetrator of a crime is a law enforcement function, the actions of attending physicians can help with microbial forensics—the scientific discipline dedicated to analyzing evidence from a biocrime or an act of bioterrorism, and that seeks to authenticate a piece of the puzzle for attribution. Implicit in the term attribution is the identification of the responsible party or the exclusion of the innocent [13]. Many physicians are familiar with the treatment of sexual assault victims, and the need to collect and preserve evidence when the patient consents. Sexual assault analysis kits have been validated to preserve semen, saliva, hair, blood, and skin. They also provide instructions on how to maintain a chain of custody to ensure that there has been no tampering with the evidence. Chain of custody is the process that assures integrity of the evidence, and ensures that there is documentation of the time the evidence is handled and each individual handling or examining the evidence. Courses exist in crime scene investigation, evidence collection, and chain of custody of the evidence in suspected sexual assault cases, and there are often well-trained support personnel who can assist patients and physicians. Such evidence collection guidance and support structures are not well-developed for biocrimes. In contrast to typical human DNA forensic investigations, with microbial forensics, a chain of custody might not be enacted at the initial stage of medical diagnostics. Good diagnostic practices could permit samples to be used as supporting evidence in a criminal investigation. In some cases, samples can be obtained subsequently under a stricter chain-of-custody process. Law enforcement authorities can assist with such documentation processes. Microbial Forensics Microbial forensics includes the full scope of forensic evidence, such as analyses of microbes, materials used to prepare, stabilize, and deliver the toxin or pathogen, and fingerprints, hair, fiber, and pollen [1,14]. The laboratory analyses used for microbial forensics may include molecular sequencing, microbiological cultures, biochemistry, electron microscopy, crystallography, and mass spectrometry. These analyses go well beyond those used for medical diagnoses and epidemiologic investigations [15]. They require, however, the same substances used by the physician for diagnostics, for example, body fluid samples and microbial cultures. In this regard, the physician and the clinical laboratory have critical roles in the collection and initial analyses of samples for microbial forensics. In the 2001 anthrax letter attacks, the preservation of the initial and subsequent isolates enabled microbial forensic methods to identify the strain in the attacks as the Ames strain of Bacillus anthracis. Analyses were based initially on a method to identify variable-number tandem repeat sequences, and later on, whole genome sequencing. Comparisons with existing strains in culture collections narrowed the likely source to a laboratory as opposed to being obtained directly from nature [16–18]. Fortunately, the initial strain from the Florida patient (the index case) and strains isolated from other victims, as well as spores from the letters, were preserved for future analyses. Microbial forensic analysis is now able, with the help of specialized facilities such as the Institute for Genome Research (TIGR), to determine the whole genome sequence of the approximately 5 million bases of B. anthracis to identify the polymorphisms that may be signatures of the bioweapon [17]. A major thrust of microbial forensics will, therefore, be the analysis of nucleic acids that can relate the genome of the pathogen to specific sources. This analysis is analogous to human DNA forensic analysis, which is being widely used to prosecute criminals and to exonerate the innocent [19]. But there are important differences between the analyses of microbial genomes and those used in human DNA forensics. Because of the sheer number of potential pathogens that could be employed as a weapon, identifying genetic markers for microbes is a more daunting task than identifying human DNA. In the case of human identification, only one species is involved, and it is often possible to identify an individual person. Viruses and most bacteria are haploid. Microbes primarily reproduce asexually, but can also evolve by recombination, horizontal gene transfer, and gene duplication. Therefore, statistical methodologies and interpretation will require different tools than are currently used for comparing and estimating the rarity of (diploid) human DNA profiles [20,21]. Nevertheless, obstacles due to genetic complexity can be reduced by obtaining samples as early as possible. If physicians suspect a biocrime, they should take steps to ensure the preservation of the diagnostic samples so that they are not prematurely destroyed. Physicians may also advise the patient to preserve additional material that may prove useful for a criminal investigation. Just as in sexual assaults, in a suspected biocrime, the patient's personal articles may carry traditional forensic evidence that is of equal value to the information revealed by the microbe itself. Unlike sexual assault evidence, in a suspected biocrime, procedures used to preserve one particular microbe may be deleterious for other microbes and for physical evidence (such as fingerprints, culture media, isotopes, hair, and environmental material). In addition, procedures useful for preserving one microbe may be insufficient to preserve another that may be unknown at the time. History and Physical Examination The physician's record of the patient's history and physical examination is evidence that can be expected to be part of any public health and forensic investigation (and subsequent legal proceedings) in either a true attack or a hoax. The physician's ability to interpret the clinical history and physical examination may go beyond differential diagnoses—for example, it can help establish timelines of exposure and of the evolution of disease, which will have forensic and public health implications. The physician is positioned to assist in identification and collection of evidence, and initiate the chain of custody that protects the integrity of the evidence (see supporting online material of [1]; [14,22]) or, at a minimum, maintains good medical practice, akin to that used for transfusion of blood products. The Case of Louisiana v. Schmidt The case of Louisiana v. Schmidt, in which HIV-infected blood was used as the weapon in an attempted murder [23,24], is instructive for the microbial forensics system. A vial of HIV-infected blood was found in the office of a suspect, a gastroenterologist. The challenge for microbial forensics was to provide evidence that this was, or was not, the source of the victim's HIV infection. HIV, an RNA virus, undergoes rapid mutation, so any direct genetic comparison of the donor source and the recipient (the victim) is complicated. In this case, analysis focusing on both rapidly and more slowly mutating genes of HIV proved to be useful. Examination of strains from the vial, the victim, and control samples (known samples from other patients with HIV residing in the same geographic region as the victim) revealed that the viral RNA from the victim was more closely aligned to that from the vial in the suspect's office than to isolates from other patients in the area. The clinical history and clinical laboratory data obtained by attending physicians provided supporting evidence of the uninfected status of the victim prior to this injection. The victim's prior HIV-negative status was documented by blood donation screenings and negative-HIV tests of sexual partners, prior to the injection. The evidence was presented in a US criminal court. Based on the composite epidemiologic and microbial forensic evidence presented, a conviction for attempted murder was obtained. This case illustrates several points. Sample collection and documentation by the attending physician are paramount to the biocrime investigation. If an attending physician is suspicious about an acquired infection, especially with an organism that is known to mutate rapidly, more frequent sampling and preservation of those samples are important. The sample may contain other clues (the victim, in this case, also was allegedly injected with blood that was hepatitis C positive). These samples could be helpful both epidemiologically, if there were an outbreak, and forensically, if there were an intentional incident. Analysis of these patient samples and other specimens may determine who was the source, and who was the victim. This case showed that even though the earliest isolates were not obtained, when the possibility of a biocrime was considered, there was still sufficient time to obtain valuable specimens, even with this rapidly mutating virus. The case also illustrates that microbial evidence can be informative, but it is rarely the sole deciding evidence. When considered in conjunction with other evidence—in this situation, epidemiological and clinical data—the case was very strong. Instructive lessons can also be found by reviewing other cases in the literature [5], such as the laboratory technician who poisoned her co-workers with a laboratory stock of Shigella dysenteriae type 2 in muffins [25], and the poisoning of salad bars to skew an election for political gain [26]. Although our primary focus has been on the role that the practicing physician can play, it is important to remember that medical examiners or coroners can also serve as sentinels for discovering acts of bioterrorism and biocrime, as well as collecting pertinent microbial forensic evidence [27]. They have the statutory authority to investigate deaths that are sudden, suspicious, violent, and unattended. Moreover, the medical examiner or coroner may encounter victims that were never examined by practicing physicians. Autopsies can be crucial for diagnosis of unknown infections and for acquiring evidence for subsequent criminal investigations [1,19]. For example, in 1979, in Sverdlovsk, USSR, at least 66 people died during an anthrax outbreak. The official Soviet government position was that the victims were infected by eating contaminated meat. Autopsy data were inconsistent with the proclaimed cause of death, and, instead, supported the proposition that the disease was inhalational anthrax due to an accidental aerosol emission from a secret military weapons facility [28]. Close working relationships should be developed between the medical examiner/coroner and public health and law enforcement entities to alert one another of possible outbreaks (whether natural or intentional) as soon as possible. For suspected attacks, the medical examiner/coroner should immediately collect case-specific death investigation information and establish a chain of custody. Fortunately, medical examiners and coroners have a long-established relationship with law enforcement. However, if there are questions regarding notification or evidence collection, the medical examiner/coroner can contact the proper public health and law enforcement entities (see Table 2). Conclusions Physicians and other health-care providers are positioned to recognize suspicious situations and alert public health and law enforcement officials. This alone may be the most important step physicians can take (Box 2). Box 2. Measures That a Physician May Take toward Securing Evidence in Cases of Biocrimes Maintain primary role in caring for the patient, even at the risk of compromising evidence collection. Discuss the situation with the patient, including options for interaction with and disclosure to public health and law enforcement officials. If permitted by patient consent, or if required by law, alert as early as possible public health authorities and law enforcement, who can provide the necessary expertise or guidance to collect and preserve evidence. Do not assume one agency will notify the other in a time-sensitive period. Ensure that notification has occurred. Maintain well-documented medical records because documentation of history, physical examination, and patient course may constitute evidence. Obtain samples that may serve as evidence early, frequently, and under a defined chain-of-custody process. Once a biocrime is suspected, ensure that the clinical laboratory does not discard microbial isolates, but preserves them for forensic analyses or transfers them under a chain-of-custody procedure (along with accessory material such as the transport tube initially used to transport microbial isolates from patient to laboratory). Law enforcement and public health personnel participating in the Laboratory Response Network can provide this assistance. Box 2. Measures That a Physician May Take toward Securing Evidence in Cases of Biocrimes Maintain primary role in caring for the patient, even at the risk of compromising evidence collection. Discuss the situation with the patient, including options for interaction with and disclosure to public health and law enforcement officials. If permitted by patient consent, or if required by law, alert as early as possible public health authorities and law enforcement, who can provide the necessary expertise or guidance to collect and preserve evidence. Do not assume one agency will notify the other in a time-sensitive period. Ensure that notification has occurred. Maintain well-documented medical records because documentation of history, physical examination, and patient course may constitute evidence. Obtain samples that may serve as evidence early, frequently, and under a defined chain-of-custody process. Once a biocrime is suspected, ensure that the clinical laboratory does not discard microbial isolates, but preserves them for forensic analyses or transfers them under a chain-of-custody procedure (along with accessory material such as the transport tube initially used to transport microbial isolates from patient to laboratory). Law enforcement and public health personnel participating in the Laboratory Response Network can provide this assistance. In cases of biocrimes, physicians may interact with nonmedical authorities—who often do not fully appreciate that a trusting doctor–patient relationship is crucial for proper care and healing, and that information should be private. It is helpful to inform such officials about the importance of the doctor–patient relationship at the outset so they can be sensitive to the obligations of physicians. Just as with a sexual assault case, once there is recognition of the possibility of a bioterrorism act or other biocrime, the physician should discuss the entire situation with the patient, explaining what can be done with the consent of the patient and what actions physicians are mandated to take to comply with public health and legal requirements. This communication will likely strengthen the patient's relationship with the physician. Within the context of microbial forensics, if the patient consents, or the law requires it, the physician can facilitate preservation of evidence. To the extent possible, earlier, more, and serial sampling of evidence is best. Physicians can ultimately serve their patients by acting, in the traditional role, as a healer, and by working with public health and law enforcement entities to help prevent further attacks and to achieve justice. As with sexual assaults, identification and conviction of the attacker can bring closure and provide a degree of security to the patient, who can then evolve from being a victim to being a survivor [29]. Physicians and their colleagues are likely to have creative ideas to contribute to the field of microbial forensics. Their input is encouraged and welcomed. Acknowledgments The authors are indebted to Stephen A. Morse (CDC), Barbara K. Richardson (Mt. Sinai Medical Center, New York), and Suzanne Atkin (University of Medicine and Dentistry of New Jersey—New Jersey Medical School) for their suggestions and critical review of the manuscript. References Budowle B, Schutzer SE, Einseln A, Kelley LC, Walsh AC, et al. (2003) Public health. Building microbial forensics as a response to bioterrorism. Science 301:1852–1853. Bush LM, Abrams BH, Beall A, Johnson CC (2001) Index case of fatal inhalational anthrax due to bioterrorism in the United States. N Engl J Med 345:1607–1610. Marks JD (2004) Medical aspects of biologic toxins. Anesthesiol Clin North America 22:509vii–32. Mayor S (2003) UK doctors warned after ricin poison found in police raid. BMJ 326:126. Carus WS (1999) Bioterrorism and biocrimes: The illicit use of biological agents in the 20th century center for counterproliferation research. Washington (D.C.): National Defense University. Budowle B, Murch RS, Chakraborty R (2005) Microbial forensics: The next forensic challenge. Int J Legal Med In press. Department of Health and Human Services. (2003) Select agents and toxins. Washington (D. C.): Department of Health and Human Services. Title 42, Code of Federal Regulations, part 73. Health Talk. (2004) FDA responds to ricin baby food contamination. San Francisco: Health Talk. Annas GJ (2003) HIPAA regulations—A new era of medical-record privacy N Engl J Med 348:1486–1490. Hodge JG Jr, Brown EF, O'Connell JP (2004) The HIPAA privacy rule and bioterrorism planning, prevention, and response. Biosecur Bioterror 2:73–80. Federal Bureau of Investigation, Department of Homeland Security, Centers for Disease Control and Prevention. (2004) Guidance on initial responses to a suspicious letter/container with a potential biological threat. Atlanta: Centers for Disease Control and Prevention. Centers for Disease Control and Prevention. (2001) Local health officer is informed of a bioterrorist incident or threat. Atlanta: Centers for Disease Control and Prevention. Murch RS (2003) Microbial forensics: Building a national capacity to investigate bioterrorism. Biosecur Bioterror 1:117–122. United States Federal Bureau of Investigation Laboratory Division. (1999) Handbook of forensic services. Yeskey K, Morse SA (2003) Physician recognition of bioterrorism-related diseases. In: Roy MJ, editor. Physician's guide to terrorist attack Totowa: Humana Press. pp 39–46. Keim P, Smith KL (2002) Bacillus anthracis evolution and epidemiology. Curr Top Microbiol Immunol 271:21–32. Read TD, Salzberg SL, Pop M, Shumway M, Umayam L, et al. (2002) Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science 296:2028–2033. Pearson T, Busch JD, Ravel J, Read TD, Rhoton SD, et al. (2004) Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing. Proc Natl Acad Sci U S A 101:13536–13541. Lander ES, Budowle B (1994) DNA fingerprinting dispute laid to rest. Nature 371:735–738. Budowle B (2004) Genetics and attribution issues that confront the microbial forensics field. Forensic Sci Int 146:S185–S188. Budowle B, Chakraborty R (2004) Genetic considerations for interpreting molecular microbial forensic evidence. In: Doutremepuich C, Morling N, editors. Progress in forensic genetics 10 Amsterdam: Elsevier. pp 56–58. American Society of Crime Laboratory Directors/Laboratory Accreditation Board. (2004) Laboratory management and operations. Garner (North Carolina): American Society of Crime Laboratory Directors/Laboratory Accreditation Board. Metzker ML, Mindell DP, Liu XM, Ptak RG, Gibbs RA, et al. (2002) Molecular evidence of HIV-1 transmission in a criminal case. Proc Natl Acad Sci U S A 99:14292–14297. Heitpas J, McMullen LK, Mindell DP, Hanson HL, Rice CM (2005) Keeping track of viruses. In: Breeze RG, Budowle B, Schutzer SE, editors. Microbial forensics San Diego: Academic Press. pp 55–97. Kolavic SA, Kimura A, Simons SL, Slutsker L, Barth S, et al. (1997) An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination. JAMA 278:396–398. Torok TJ, Tauxe RV, Wise RP, Livengood JR, Sokolow R, et al. (1997) A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA 278:389–395. Nolte KD, Hanzlick RL, Payne DC, Kroger AT, Oliver WR, et al. (2004) Medical examiners, coroners, and biologic terrorism: A guidebook for surveillance and case management. MMWR Recomm Rep 53:1–27. Jackson PJ, Hugh-Jones ME, Adair DM, Green G, Hill KK, et al. (1998) PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: The presence of multiple Bacillus anthracis strains in different victims. Proc Natl Acad Sci U S A 95:1224–1229. Hampton HL (1995) Care of the woman who has been raped. N Engl J Med 332:234–237. Breeze R, Budowie B, Schutzer S (2005) Microbial Forensics. San Diego: Academic Press. 448 p....查看详细 (31645字节)
☉ 11340244:A Systematic Analytic Approach to Pandemic Influenza Preparedness Planning
Daniel J. Barnett and Ran D. Balicer made an equal contribution to the development of this manuscript. Daniel J. Barnett is at the Johns Hopkins Center for Public Health Preparedness, Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America. Ran D. Balicer is in the Epidemiology Department, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. Daniel R. Lucey is in the Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, District of Columbia, United States of America. George S. Everly, Jr., is at the Johns Hopkins Center for Public Health Preparedness, Department of Psychiatry, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America. Saad B. Omer is in the Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America. Mark C. Steinhoff is in the Department of International Health and the Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, and the Department of Pediatrics, Johns Hopkins School of Medicine. Itamar Grotto is in the Epidemiology Department, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. The prospect of a pandemic with avian influenza is an urgent concern for public health leaders worldwide [1]. As pathogenic avian influenza A (H5N1) strains (Figure 1) continue to spread in East Asia, with recently reported expansion to Siberia and westward regions in Russia [2,3] as well as to migratory birds [4,5], the risk for reassortment of avian and human strains increases. Evidence cited by the World Health Organization in May 2005 suggests that H5N1 may be adapting to humans, thus potentially setting the stage for the next influenza pandemic [6]. The viruses are gold, and the MDCK cells are green. (Photo: CDC/C. Goldsmith, J. Katz, and S. Zaki) Animal data suggest that the current H5N1 strain appears to be even more deadly than the original 1997 Hong Kong avian influenza, a finding that correlates well with the observed human case fatality rates [7]. As of August 5, 2005, there have been 112 human cases of H5N1 in East Asia resulting in 57 deaths (case fatality rate = 51%) [8,9]. Also concerning are recent findings that in China and Indonesia the virus has infected pigs, a possible “mixing vessel” for both avian and human influenza viruses, thus providing an opportunity for reassortment from which a pandemic human strain could emerge [10,11]. Research suggesting that cats could host or transmit the H5N1 infection [12] adds to a worrisome picture of multispecies transmission that can elevate the risk of reassortment [9]. This epizootic outbreak in Asia is not expected to wane in the short term [9]. Influenza pandemics can have devastating impacts. The Spanish flu of 1918 was particularly destructive (Figures 2 and 3), resulting in a higher death total in less than two years than in all of World War I [13]. Although earlier accounts suggested the mortality from the 1918 pandemic was 20 million to 40 million, more recent assessments including new estimates from Africa and Asia suggest that a more realistic figure is 50–100 million [14]. The high rates of infection with the pandemic virus meant that even an average case fatality rate lower than 3% resulted in this large number of deaths [13,15]. A 1918-type influenza pandemic today is projected to cause 180–360 million deaths globally (including 1.7 million deaths in the United States) [1], with transmission of the disease lasting at least two years [16]. (Photo: Image “NCP 1603,” National Museum of Health and Medicine, Armed Forces Institute of Pathology, Washington, D.C.) (Photo: Image “Reeve 3143,” National Museum of Health and Medicine, Armed Forces Institute of Pathology, Washington, D.C.) The Next Pandemic: “Inevitable, and Possibly Imminent” In light of recent episodes of human infection with H5N1 virus, the World Health Organization reiterated its 1997 call for all countries to prepare for the next pandemic, which it termed “inevitable, and possibly imminent” [17], and updated its own pandemic plan in April 2005 [18]. In the United States, it has been argued that of the 12 disaster scenarios recently assessed by the US Department of Homeland Security, pandemic influenza is the most likely and perhaps the most deadly [19]. A draft form of the US pandemic influenza plan was made public in August 2004 [20], and an updated plan is anticipated by September 2005. The urgent need for comprehensive pandemic influenza planning is profound: an influenza pandemic starting today may have major international consequences, including global economic and political destabilization, an overwhelming of health care resources, and panic [21]. Current international plans [18,22], while useful, could benefit from enhanced detail [21] and organization; moreover, pandemic influenza plans have usually been national in scope and, in most countries, are only in a draft form and lack legal status [23]. The Haddon Matrix An analytic approach for traffic safety injury epidemiology and prevention was developed by Dr. William Haddon, Jr. in the 1960s [24], and has since been termed “the Haddon matrix.” This matrix provides a multidimensional approach to understanding the contributing factors to injury before, during, and after an event [25]. The current version of the matrix is a grid with four columns, or axes, that represent contributing factors to injury (host, agent/vector, physical environment, sociocultural environment) and three rows that correspond to the time phases of a given form of injury (pre-event, event, and post-event) [26]. By compartmentalizing an injury into dimensions of time and contributing factors, the matrix can break a complex problem into more manageable segments. For each of the 12 cells, a decision analysis or prioritization can be used to select policies or actions with greatest feasibility or influence [27]. Although the Haddon matrix may seem unfamiliar to some infectious disease scientists, it incorporates familiar analytic elements in a systematic way. The four columns represent the classical epidemiologic triad of host, agent, and environment (physical and sociocultural). The three rows are equivalent to primary, secondary, and tertiary prevention of disease outbreaks. Indeed, Haddon himself used his analytic matrix to describe an outbreak of polio [24], and this matrix has been recently applied to other public-health emergency preparedness challenges such as SARS [28]. Applying the Matrix to Pandemic Influenza Preparedness Comprehensive public health emergency preparedness and response efforts require effective pre-event (preventive), event (mitigation), and post-event (consequence management) strategies. By identifying the factors that may modify the outcome in each of these phases, one can prescribe the appropriate measures necessary to tackle each factor. To this end, we specifically applied the Haddon matrix to pandemic influenza planning and response (Table 1), systematically identifying relevant factors in each phase (pre-event, event, post-event) and on each axis (human, agent/vector, physical environment, sociocultural environment). We then identified factors that may be associated with opportunities for public health intervention, and marked these factors in bold within the matrix (consistent with an approach described by Runyan [27]). The table shows that in all phases of an influenza pandemic, opportunities for public health intervention include a number of contributing human, physical environment, and sociocultural factors, but generally not agent/vector factors, since viruses generally cannot be modified easily as injury-causing devices. Importantly, the pre-event, event, and post-event rows of the matrix reflect the phase of a pandemic in which public health preparedness and response measures will take their effects; however, planning for each of these measures must occur before the pandemic begins. The use of the Haddon matrix in the table as an analytic and planning tool for pandemic influenza is illustrated below by its application to readiness efforts in two different countries: Thailand, focusing on pre-event factors; and Israel, focusing on event factors. We chose Thailand as an example because of its regional susceptibility and the proactive nature of its anti-H5N1 planning efforts to date. We selected Israel as an example of a country outside of East Asia that has taken steps to address this potential global crisis. For both countries, we demonstrate the application of the matrix by addressing selected factors within each axis. Pandemic Influenza Planning in Thailand Thailand has had experience with H5N1 infections in both humans and animal populations—including chickens, ducks, birds, fighting roosters, and tigers—since January 2004. By October 2004, a total of 17 patients with H5N1 infection were identified, of whom 12 had died. The initial success of Thailand's national program against H5N1 avian influenza that began in the autumn of 2004 is evidenced by the fact that no human cases have been found between October 2004 and August 2005. Thus, Thailand's experience may offer practical lessons in preparing for an avian influenza-related human pandemic. Through the lens of pre-event Haddon matrix factors, one can identify the strengths in Thailand's preparedness efforts, as well as opportunities for further enhancements. Selected examples of the pre-event axes for Thailand's pandemic influenza readiness efforts are described below. Pre-event human factors Thailand has developed surveillance and laboratory testing algorithms for influenza-like-illness in humans and animals, including definitions for “suspect,” “probable,” “confirmed,” “excluded,” and “on investigation” cases of H5N1. With written guidance from national authorities [29], public health workers, veterinary health workers, village health volunteers, and others [30] participated in an ongoing surveillance campaign nationwide beginning in October 2004 [31]. Pre-event risk communication to at-risk populations are also important. In the scenario of pandemic influenza, effective pre-event risk communication can reduce event-phase risk communication barriers [32]. An array of appropriate information on avian influenza and potential pandemic human influenza has been disseminated by the Thai Ministry of Public Health [33]. Pre-event agent/vector factors Strain pathogenicity to its avian and human hosts is the major pre-event agent/vector factor. Most cases of human H5N1 infection have resulted from contact with infected chickens, fighting roosters, or ducks [9], with some ducks possibly being asymptomatic [34]. Regarding human pathogenicity, an autopsy of a patient from Thailand, one of the few involving H5N1 infection [35,36] reported that the virus can replicate in the human intestine as well as the lung [37] perhaps helping to explain the finding of diarrhea in some patients in Thailand and Vietnam [37–41]. Pre-event physical environment factors Thailand has established a multifaceted communication system, including websites for human and animal-related H5N1 updates and standard protocols. Provincial health offices were directed by the Ministry of Public Health to form Surveillance and Rapid Response Teams at the provincial and district levels [42]. Hospital infection control infrastructure and protocols are also crucial. Patients meeting criteria for possible H5N1 infection “should be isolated and placed in a single room according to the standard precautions of the Ministry of Public Health” [42]. Even if the patient's initial rapid test for influenza A is negative “the patient must be treated with antivirals immediately” [42] in an effort to increase survival [41]. The availability of avian strain-specific vaccines is another significant factor. Webster and Hulse observed that Thailand's investigation of flu vaccines for “open range” (noncommercial) poultry represents a “prudent” policy shift that should be replicated in other countries in East Asia [43]. H5N1 vaccine studies in humans have not yet been initiated in Thailand. Pre-event sociocultural factors One of the most significant factors is political and social willingness to acknowledge and report disease dissemination. Initially, the Thai government was criticized for underplaying the existence and the magnitude of avian influenza in Thailand [44] but it has since taken significant proactive steps to address this urgent challenge. Between January 2004 and July 2005, a total of 59 official reports on surveillance for Highly Pathogenic Avian Influenza have been submitted to the World Organization for Animal Health by Thailand [45], and detailed reports were promptly published [35,36,38,41,46–48]. On September 28, 2004, the first media report of a probable case of person-to-person transmission appeared in Thailand [49] and was rapidly published [36]. On September 29 of that year, a national campaign against the H5N1 virus was declared by the Prime Minister of Thailand, with involvement by the Thai Cabinet [50,51]. These resulting efforts seem to have had a substantial impact, as detailed above. Budget (preparedness resource allocation) is also important. The Thai National Strategic Plan for Avian Influenza and Plan for Pandemic Preparedness 2005–2007 was initiated with a budget of 4,026 million Thai baht (～US$105 million) [52,53]. Thailand has been reported recently to have approved funding for the future purchase of up to 100,000 treatments of oseltamivir [54]. An in-place culling policy played a significant role. The culling of ducks (with farmer compensation) reduced the flocks that were positive for H5N1 from around 40% infected in October 2004 to almost undetectable levels in March 2005 [43]. Collaboration between human and veterinary health authorities was vital. Efforts are ongoing to closely link public health and animal health responses to H5N1 [52,53]. Surveillance combines epidemiologically linked testing for animals and humans [55]. In addition, Thailand interacts frequently with the World Health Organization regarding clinical H5N1 issues, and with the World Organization for Animal Health in reporting on animal surveillance for H5N1 [56]. Pandemic Influenza Planning in Israel Applying the various influencing factors listed in the event phase of the Haddon matrix to the unique Israeli setting leads to several important insights regarding local pandemic preparedness, as shown in the following examples. Event human factors Israel has not initiated, as of yet, training activities for health care professionals directed specifically at pandemic preparedness, although such activities are planned to take place. Nevertheless, Israeli health care professionals, particularly frontline health care workers, are well experienced with terrorism-related mass casualty emergencies. Continuous training of the various components of the health care system for bioterrorism threats likely serves to enhance these workers' ability to deal with naturally occurring epidemic threats; these health care teams were shown to have increased likelihood of reporting to duty during a crisis [57]. Simulation-assisted medical training may be useful in increasing health care workers' compliance with personal protective equipment and infection control protocols, as has been shown in the preparation of Israeli medical teams to respond to chemical warfare casualties [58]. Upcoming tabletop exercises will test and refine current national contingency plans, while full-scale drills may be required to test certain practical and logistical aspects of antiviral drug dissemination. Event agent/vector factors Agent/vector factors listed in the matrix are expected to determine much of the local impact of the pandemic, but they generally cannot be influenced by preparedness and mitigation efforts. As these factors will remain unknown until the first stages of the pandemic, Israeli preparedness planners have taken into account a wide range of scenarios with different attack and mortality rates [59] in addressing issues such as surge capacity. For instance, a highly transmissible pandemic may render isolation and quarantine efforts largely futile [60] while a less transmissible strain, as witnessed in previous pandemics [61] may enable a containment approach more similar to that taken during the SARS epidemic (while accounting for considerable differences such as the incubation time or the impact of infectious asymptomatic cases). A highly pathogenic strain, perhaps more pathogenic than the 1918 strain (considering current case fatality rates of H5N1 human cases), will require the unparalleled ability to rapidly mobilize medical equipment and personnel to meet the increased demands for care in both primary and secondary care facilities. However, a less pathogenic strain may require measures similar to those taken during severe seasonal influenza epidemics. Event physical environment factors The availability of an effective immunization will be crucial. The importance of the recently published successful preliminary results of phase-I human H5N1 vaccine trials cannot be overestimated [62]. Nevertheless, both the safety and efficacy of the new vaccine remain to be assessed, and the effectiveness of this vaccine against a reassortant pandemic strain is currently difficult to predict. Research efforts to produce active or passive immunization that will be universally effective against any influenza strain are currently underway in Israel and elsewhere. Once available, such modalities hold great promise for mitigation of future pandemics in their first stages [63]. Another type of immunotherapy that may be considered during an event is the use of immunoglobulins isolated from recovered patients to treat the ill or protect the exposed. Stockpiled antivirals and antibiotics are important to Israel's preparedness. The Israeli Ministry of Health has successfully used cost-benefit analyses [59] to persuade decision makers to invest the funds necessary for the rapid creation of a national antiviral stockpile, and several strategies for the use of these drugs during the pandemic are considered [64]. The antiviral oseltamivir was found to be effective in mice against the newest strains of avian influenza currently sweeping through East Asia, suggesting that higher doses and prolonged courses of this drug may be required [7]. These findings, if validated in humans, may need to be factored into stockpiling planning efforts. Event sociocultural factors Israel has ensured that a legal and ethical framework for implementation of response measures exists. Including pandemic influenza in the list of “dangerous communicable diseases” defined by Israeli law will allow the Ministry of Health to uphold extreme measures such as involuntary quarantine and isolation, if needed. Prioritizing target groups for antiviral drugs and vaccines, expected to be in short supply, requires the addressing of complex ethical, legal, social, and political considerations. The choice of which groups to prioritize would derive, in part, from the prioritizing of the various goals in using these drugs. If the focus is on reducing all mortality, different groups may be prioritized than if the main attempt is to reduce social disruption. A national ethics committee was recently appointed to address these issues. Conclusions By offering phase-specific insights into pandemic influenza planning, the Haddon matrix bridges injury-prevention epidemiology with global infectious disease preparedness and response. In the process, this analytic tool sheds light on opportunities for prevention, mitigation, and consequence management strategies to address a global public health threat. In the face of the challenges described, the Haddon matrix analysis of pandemic influenza planning in Thailand and Israel reflects its applicability as a systematic tool for identifying urgent national and international pandemic avian influenza readiness needs. The scalability of the matrix also allows its use at the level of a county or city, as well as within institutions. At each of these levels, the matrix may facilitate the enhancement of preparedness plans, needs assessments, best practice identification, and resource distribution strategies. Although the national examples above have selectively focused on pre-event factors in Thailand and event factors in Israel, the Haddon matrix can be also used to augment existing post-event phase plans. For example, the psychology of post-event reactions [65] must be addressed through ongoing mental health support and follow up and by effective post-event risk communication. The public health infrastructure may face the dual challenge of helping populations, including health care providers themselves, to be psychologically prepared for the next wave of a pandemic—perhaps worse than the first wave, as was the case in the 1918 pandemic [13]—while trying to recover from the first wave. The Haddon matrix has limitations that must be recognized to ensure appropriate implementation. Importantly, the matrix is not a stand-alone planning tool; rather, the results of any Haddon-matrix–based analysis must be operationalized in the form of policies and procedures to achieve their desired effects on the factors included in the matrix. Moreover, the matrix is not static; the contents within its cells can and should be modified according to changing disease dynamics and situational challenges to maintain its usefulness in an evolving crisis. Furthermore, even before a crisis, the choice of contents for each cell is not absolute, and open to the subjective interpretation of those who are preparing the matrix. Consequently, the table presented in this article should be regarded as a starting planning framework, not a final checklist. Also, while many of the items in the Haddon matrix cells may be measurable, the matrix itself is only a planning instrument—not an evaluation tool. The known potential for an avian influenza pandemic offers not only challenges but also unprecedented opportunities for advance planning at all levels of public health in the international community [66]. This planning window may be rapidly closing, however [21]. As an efficient yet comprehensive analytic approach, the Haddon matrix lends itself to the types of rapid and complex decision making necessary to plan for and respond more effectively to an urgent pandemic health threat. References Osterholm MT (2005) Preparing for the next pandemic. 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☉ 11340245:Breaking Up(Amyloid) Is Hard to Do
Sam Gandy is at the Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America. Frank L. Heppner is at the Institute of Neuropathology, University Hospital Zurich, University of Zurich, Zurich, Switzerland. Standard “Alzheimerology” lore holds that the insolubility of amyloid plaques and neurofibrillary tangles was a great impediment to elucidating the molecular composition of each of these structures. Roughly 77 years passed between Alois Alzheimer's description of the clinical and pathological features of the illness suffered by Auguste D. and the now classical reports from George Glenner, Colin Masters, and Konrad Beyreuther describing partial solubilization, Edman degradation, and primary amino acid sequencing of the Aβ peptide (the protein that accumulates into amyloid plaques) [1–3]. Peter Davies (who discovered the cholinergic deficiency in Alzheimer disease during that same interval [4]) has long joked that one easy way to purify plaques and tangles is to allow the brain from an affected person to autolyze and liquefy completely (“on a summer sidewalk,” according to one colorful variation), whereupon the macro structure of the organ would disintegrate, leaving behind only fibrous clumps and twists. There is little wonder, then, that the strategy of treating Alzheimer disease with “plaque busting” drugs was relatively slowly embraced: 20 years passed between the availability of amyloid aggregation assays and the clinical trials of the first specific antiamyloid-aggregation compounds. (“Aggregation” is essentially equivalent to clumping, and amyloid clumping can be monitored in a test tube since floating clumps cause light to disperse in a measurable fashion). Conventional wisdom was nihilistic, holding that therapeutic dissolution of amyloid deposits was probably too slow to be approachable. People love to see a dogma challenged, and the availability of mouse models of Alzheimer disease enabled Brad Hyman's group to show, in 2001, that deposits of human Aβ in the transgenic mouse brain were surprisingly dynamic, forming and, unexpectedly, dissolving over a timescale of days [5]. These data injected new optimism into the pursuit of antiamyloid strategies, and by 2005, over 30 discrete compounds or combinations were in development [6]. Old Worries Resurface Now, with publication of a study by David Borchelt's group in this issue of PLoS Medicine [7], old worries about the efficiency of plaque clearance resurface. David Borchelt, Joanna Jankowsky, and their colleagues report the development of Aβ plaque-forming transgenic mice in which pathology is driven by brain-specific overexpression of a mutated form of the human amyloid precursor protein (APP). The innovation here is that human APP expression due to a genetic trick is extinguishable by adding the antibiotic tetracycline to the mouse food (“tet-off” APP mice). As rightly contended by these authors, the ability to abolish human APP gene expression—instantly and completely—can be conceptually envisioned as equivalent to the most effective antiamyloid strategy imaginable: in other words, a best-case scenario from the point of view of drug efficacy. The rationale was to see how long human amyloid deposits would persist in a plaque-laden mouse brain once new accretion ceased (i.e., once tetracycline switched off new mutated human APP production). The results are arguably applicable to every antiamyloid strategy delivered to patients impaired by Alzheimer disease, since all are believed to enter therapy with at least some existing brain plaque burden. In this tet-off paradigm, unlike the paradigm used earlier by Hyman and colleagues, amyloid pathology was allowed to accumulate, and then human APP expression was completely shut off. Borchelt and colleagues were unable to detect any change in brain amyloid load for at least six months after complete cessation of Aβ biogenesis. The Borchelt data dovetail well with recent biophysical data, proving that the thermodynamic barrier to redissolution of amyloid fibrils is very high indeed (Figure 1) [8]. In this issue of PLoS Medicine, Borchelt and colleagues demonstrate in the living amyloid-laden mouse brain that Aβ plaques are cleared very slowly, even if synthesis of new Aβ precursor molecules is extinguished using a tet-off system [7]. In the recent, relevant, but independent, X-ray crystallography study [8], Nelson et al. envisioned the free-energy plot shown above as a graphic description of the kinetics of transition from monomeric Aβ to fibrillar Aβ (ΔGformation). For the reverse reaction, Nelson et al. envision the ΔGdissolution as the large free-energy barrier to spontaneous solubilization of amyloid fibrils. Presumably, it is this ΔGdissolution that underlies the slow disappearance of brain plaques in the Borchelt study in transgenic mice. (Illustration: Sapna Khandwala, adapted from [8]) Implications for Therapy and Prevention If this mouse model represents the best-case scenario, what are the realistic hopes of success for anti-Aβ therapies in the treatment of human Alzheimer disease Several points come to mind. First, the recent proposal that oligomeric/dodecameric forms of Aβ (also known as Aβ-derived diffusible ligands [ADDLs]) are the real culprits in mediating Aβ neurotoxicity [9] provides some hope that ADDLs, not Aβ plaques, represent the most important Aβ biophysical form that must be purged in order to cause a therapeutic benefit. Borchelt and colleagues have not yet determined what happens to ADDL levels in the brains of their tet-off APP mice, but such data are eagerly anticipated. Second, Borchelt and colleagues' data are consistent with the evidence that all model antiamyloid strategies are superior when initiated long before onset of amyloidosis. Such evidence fuels a new initiative of the United States Alzheimer's Association aimed not at treating but at preventing Alzheimer disease (http://www.alz.org/maintainyourbrain). Interventional strategies early in life should not only prevent amyloid pathology, but also take advantage of the greater regenerative capacity of the human brain at younger ages. The wisdom of preventing Alzheimer disease is further galvanized by the appreciation over the last five years of evidence that many modifiable diet and lifestyle factors (body mass index, cholesterol levels, control of diabetes and blood pressure, and mental and physical exercise) may modulate risk for late-life degenerative dementia [10]. The Challenges Ahead Significant challenges lie ahead in understanding how these risks and pathologies are intertwined, but the aging of the baby boom population promises an unprecedented epidemic of dementia if effective interventions are not discovered soon. In the US, the population of individuals age 65 years and over is the fastest growing segment in society, and one-half of individuals over 85 experience dementia [11]. On a practical level, this means that almost everyone will be either a patient or a caregiver in the near future. Disasters such as tsunamis and hurricanes—as painful, dramatic, and expensive as they are—pale in comparison to the cataclysm our aging societies will face if current trends in the epidemiology of this dementing illness continue on unchecked. Acknowledgments This work was supported by United States National Institutes of Health grants NS41017 (to SG) and NS046006 (to FLH). References Alzheimer A, Stelzmann RA, Schnitzlein HN, Murtagh FR (1995) An English translation of Alzheimer's 1907 paper, “Uber eine eigenartige Erkankung der Hirnrinde.”. Clin Anat 8:429–431. Glenner GG, Wong CW (1984) Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120:885–890. Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, et al. (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A 82:4245–4249. Davies P (1979) Biochemical changes in Alzheimer's disease-senile dementia: Neurotransmitters in senile dementia of the Alzheimer's type. Res Publ Assoc Res Nerv Ment Dis 57:153–166. Bacskai BJ, Kajdasz ST, Christie RH, Carter C, Games D, et al. (2001) Imaging of amyloid-beta deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy. Nat Med 7:369–372. Kwon MO, Fischer F, Matthisson M, Herrling P (2004) List of drugs in development for neurodegenerative diseases. Neurodegenerative Dis 1:113–152. Jankowsky JL, Slunt HH, Gonzales V, Savonenko AV, Wen J, et al. (2005) Persistent amyloidosis following suppression of Aβ production in a transgenic model of Alzheimer disease. PLoS Med 2:e355 DOI: 10.1371/journal.pmed.0020355. Nelson R, Sawaya MR, Balbirnie M, Madsen A, Riekel C, et al. (2005) Structure of the cross-beta spine of amyloid-like fibrils. Nature 435:773–778. De Felice FG, Vieira MN, Saraiva LM, Figueroa-Villar JD, Garcia-Abreu J, et al. (2004) Targeting the neurotoxic species in Alzheimer's disease: Inhibitors of Abeta oligomerization. FASEB J 18:1366–1372. Marx J (2005) Neuroscience. Preventing Alzheimer's: A lifelong commitment Science 309:864–866. Hebert LE, Scherr PA, Beckett LA, Albert MS, Pilgrim DM, et al. (1995) Age-specific incidence of Alzheimer's disease in a community population. JAMA 273:1354–1359....查看详细 (9583字节)
☉ 11340246:Human Health Risks from Low-Level Environmental Exposures: No Apparent Safety Thresholds
Donald T. Wigle is at the McLaughlin Centre for Population Health Risk Assessment, Institute of Population Health, University of Ottawa, Ottawa, Canada. Bruce P. Lanphear is at the Cincinnati Children's Environmental Health Center, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America. Recent developments in environmental epidemiology offer the promise of strengthening human health protection. Regulatory agencies responsible for protecting human health from environmental hazards assess data on relationships between exposure levels and adverse health effects to develop limits for contaminant levels in air, water, food, soil, house dust, and consumer products. Most regulatory agencies assume that there is no safe level of exposure to carcinogens and use linear dose-response models to estimate human health risks at low exposure levels. In contrast, regulators usually assume that a threshold, or “safe,” exposure level exists for noncarcinogens. Risk Assessment In conducting risk assessments to characterize potential adverse health effects of human exposures to environmental hazards [1], regulators depend on experimental animal studies in the absence of adequate epidemiologic data. These studies are critical to uncover health effects before human exposure occurs (e.g., premarket testing of a new chemical) whereas epidemiologic studies can be used to directly evaluate health effects among exposed persons. The difficulty of directly measuring health risks at very low exposure levels can be an important limitation of both epidemiologic and toxicologic studies. Sources of uncertainty in conventional animal studies include: (1) the much shorter exposure period compared to humans, (2) testing is often limited to adult (but not pregnant, newborn, or sexually immature) animals, (3) use of genetically homogeneous animals (with loss of the ability to detect potentially heightened risks among genetically diverse subgroups, such as exist in human populations), (4) the use of very high doses of test chemicals (e.g., administration of high doses of a teratogenic toxicant to pregnant animals may cause early pregnancy loss before birth defects can be readily observed), (5) small numbers of test animals, and (6) the need to extrapolate across species to humans [2]. For instance, neurotoxic effects of prenatal or early-life exposure to lead, polychlorinated biphenyls, and methylmercury in humans occur at intake levels about three orders of magnitude lower than those predicted from rodent data [3]. The role of potential biases and crude exposure indices in producing uncertainties in epidemiologic studies has been reduced, to some degree, by the increasing use of improved study methodologies, e.g., the use of exposure and susceptibility biomarkers. There are higher risks per unit exposure dose at low exposure levels. The following case studies for four of the most widespread and extensively studied environmental hazards show that (1) there is no apparent threshold for health risks with dose-response relationships over exposure ranges far below those generally used in animal studies, and, in some cases, (2) there are higher risks per unit of exposure dose at low exposure levels. Case Studies Lead Lead is a potent neurotoxin capable of causing severe childhood brain damage at blood lead levels only 2- to 3-fold higher than those that cause no overt symptoms. Overt lead poisoning has been recognized for centuries, but there was no convincing evidence of IQ deficits at relatively low-level lead exposure until 1979 [4]. Noting the lack of a lead exposure threshold for impaired cognitive function and heme synthesis, the Environmental Protection Agency has not specified a safe exposure level. The United States Centers for Disease Control and Prevention do not recommend public health or medical actions for children unless their blood lead level exceeds 0.48 μM (10 μg/dl), a level about 100-fold higher than that estimated for pre–Industrial-Age children [5]. Epidemiologic studies of children in several countries found inverse relationships between IQ and blood lead levels over a range extending below 0.48 μM, with no evidence of a threshold [6]. In a recent study of over 4,000 children, scores on four cognitive test subscales (math, reading, block design, digit span) were inversely associated with current blood lead levels, even in analyses restricted to those with blood lead levels less than 0.48 μM (20 μ/dl are shown. (Figure by authors, adapted from [11]). Tobacco smoke There is convincing epidemiologic evidence that prenatal maternal active smoking impairs fetal growth. A US prospective study demonstrated an inverse nonlinear relationship between term birth weight and third-trimester smoking intensity, with larger birth weight decrements at low maternal smoking intensities [12]. It now appears that even low-level exposure to environmental tobacco smoke (ETS), or “passive smoking,” can reduce fetal growth. In a Finnish study of nonsmoking women, the risk of preterm birth was dose-related to self-reported prenatal maternal ETS exposure intensity and maternal hair nicotine levels [13]. Glutathione-S-transferase (GST) enzymes detoxify many chemicals, including polycyclic aromatic hydrocarbons and certain other toxicants present in tobacco smoke. A Korean study of nonsmoking women found that combined maternal ETS exposure and null polymorphisms of two GST genes involved in tobacco smoke metabolism (GSTT1 and GSTM1) were associated with birth weight deficits [14]. A US study of over 4,000 children age 6 to16 years found inverse dose-response relationships between serum cotinine (the major metabolite of nicotine) and scores on reading, math, and visuospatial reasoning independent of several potential confounders [15]. Importantly, the dose-response relationship between reading scores and serum cotinine was stronger among children with cotinine concentrations below 0.5 ng/ml compared to those with higher levels. Radon An expert committee recently concluded that the most plausible relationships between low-level ionizing radiation and mutations, chromosome aberrations, and cancer are linear, with no threshold [16]. The high radon levels in the air of some underground mines cause lung cancer among occupationally exposed men, the risk being a linear function of cumulative radiation dose [17]. A pooled analysis of eight epidemiologic studies of underground miners showed that the excess risk of lung cancer per unit of cumulative radon exposure was greater at lower exposure levels [18]. Among men with the same cumulative radon exposure, therefore, prolonged exposure at low levels is more hazardous than shorter exposures at higher levels. Indoor air radon levels vary widely in homes and other buildings. Average cumulative radon doses from lifetime residential exposures are about 10-fold lower than those among exposed miners. Despite the relatively low average radon levels in homes, combined analysis of 17 epidemiologic studies showed that persons with time-weighted average residential radon exposures of 150 Bq/m3 (the current level above which the Environmental Protection Agency recommends actions to confirm radon levels and sources and the need for remedial measures such as ventilation) had a 24% (95% CI 11%–38%) increased lung cancer risk [19]. Thus, directly measured lung cancer risk at relatively low radon levels in the general population is consistent with an estimate based on linear extrapolations of risks for miners with much higher average exposures [20]. Chlorination disinfection by-products in drinking water During disinfection of drinking water, chlorine reacts with naturally occurring organic material and produces many by-products of disinfection, including the trihalomethanes (THMs) chloroform, bromodichloromethane, dibromochloromethane, and bromoform, that are known animal carcinogens. Based on a risk assessment of kidney tumors in rats chronically exposed to high chloroform doses, Health Canada concluded that the human lifetime cancer risk associated with drinking water containing THMs at 100 μg/l (the current Canadian THM drinking water guideline) would be negligible [21]. However, a recent pooled analysis of six epidemiologic studies of human bladder cancer with over 8,000 subjects showed that men exposed to THM levels above 1 μg/l had a 24% increased bladder cancer risk compared to less exposed men, representing an excess lifetime bladder cancer risk of about seven per 1,000 [22]. This risk is much higher than those usually designated as negligible (regulatory agencies have variably defined negligible risk as a lifetime excess risk of 106 to 105). Thus, a risk assessment of THMs based on carcinogenicity of chloroform in animals may greatly underestimate human cancer risk. Conclusion In contrast with animal studies, epidemiologic studies can be used to assess health risks at exposure levels prevalent in human populations. Findings from some of the most thoroughly studied and widely dispersed environmental contaminants indicate that there is no apparent safe exposure level. Indeed, in some cases, there are greater risks for a given exposure at the relatively low exposure levels most prevalent in human populations. Environmental chemicals should be thoroughly evaluated for toxicity before they are marketed [23], but when available, epidemiologic data should preferentially be used to develop environmental standards and to assess the adequacy of existing standards based on experimental animal studies. The public depends on decision makers, scientists, and regulators to restrict exposure to widespread toxins that have known or suspected serious potential health effects. We hold that risk assessments should not assume thresholds for noncarcinogens as well as carcinogens, especially for toxins shown in epidemiologic data to exhibit no apparent threshold and those not yet adequately tested for developmental toxicity. The four major toxins reviewed here are widely dispersed in the environment and emerging evidence indicates that exposures must be virtually eliminated to protect human health. It would be imprudent to assume that there are not other widely distributed environmental toxins or chemicals with the potential to cause adverse human health effects at exposure levels currently considered to be “low.” References National Academy of Sciences. (1983) Risk assessment in the federal government: Managing the process. National Academy of Sciences. (2000) Scientific frontiers in developmental toxicology and risk assessment. Washington (District of Columbia): National Academy Press. 354 p. Rice DC, Evangelista de Duffard AM, Duffard R, Iregren A, Satoh H, et al. (1996) Lessons for neurotoxicology from selected model compounds: SGOMSEC joint report. Environ Health Perspect 104:S205–S215 Suppl 2. Needleman HL, Gunnoe C, Leviton A, Reed R, Peresie H, et al. (1979) Deficits in psychologic and classroom performance of children with elevated dentine lead levels. N Engl J Med 300:689–695. Mushak P (1993) New directions in the toxicokinetics of human lead exposure. Neurotoxicology 14:29–42. Schwartz J (1994) Low-level lead exposure and children's IQ: A meta-analysis and search for a threshold. Environ Res 65:42–55. Lanphear BP, Dietrich K, Auinger P, Cox C (2000) Cognitive deficits associated with blood lead concentrations <10 microg/dL in US children and adolescents. Public Health Rep 115:521–529. Wasserman GA, Factor-Litvak P, Liu X, Todd AC, Kline JK, et al. (2003) The relationship between blood lead, bone lead and child intelligence. Neuropsychol Dev Cogn C Child Neuropsychol 9:22–34. Canfield RL, Henderson CR Jr, Cory-Slechta DA, Cox C, Jusko TA, et al. (2003) Intellectual impairment in children with blood lead concentrations below 10 microg per deciliter. N Engl J Med 348:1517–1526. Bellinger DC, Needleman HL (2003) Intellectual impairment and blood lead levels. N Engl J Med 349:500–502. Lanphear BP, Hornung R, Khoury J, Yolton K, Baghurst P, et al. (2005) Low-level environmental lead exposure and children's intellectual function: An international pooled analysis. Environ Health Perspect 113:894–899. England LJ, Kendrick JS, Gargiullo PM, Zahniser SC, Hannon WH (2001) Measures of maternal tobacco exposure and infant birth weight at term. Am J Epidemiol 153:954–960. Jaakkola JJ, Jaakkola N, Zahlsen K (2001) Fetal growth and length of gestation in relation to prenatal exposure to environmental tobacco smoke assessed by hair nicotine concentration. Environ Health Perspect 109:557–561. Hong YC, Lee KH, Son BK, Ha EH, Moon HS, et al. (2003) Effects of the GSTM1 and GSTT1 polymorphisms on the relationship between maternal exposure to environmental tobacco smoke and neonatal birth weight. J Occup Environ Med 45:492–498. Yolton K, Dietrich K, Auinger P, Lanphear BP, Hornung R (2005) Exposure to environmental tobacco smoke and cognitive abilities among U.S. children and adolescents. Environ Health Perspect 113:98–103. National Council on Radiation Protection and Measurements. (2001) Evaluation of the linear-nonthreshold dose-response model for ionizing radiation. Bethesda (Maryland): National Council on Radiation Protection and Medicine. Report nr 136 263 p. Lubin JH, Boice JD, Edling C, Hornung RW, Howe GR, et al. (1995) Lung cancer in radon-exposed miners and estimation of risk from indoor exposure. J Natl Cancer Inst 87:817–827. Hornung RW (2001) Health effects in underground uranium miners. Occup Med 16:331–344. Pavia M, Bianco A, Pileggi C, Angelillo IF (2003) Meta-analysis of residential exposure to radon gas and lung cancer. Bull World Health Organ 81:732–738. National Academy of Sciences. (1999) Health effects of exposure to radon. BEIR VI. Washington (District of Columbia): National Academy Press. 516 p. Health Canada. (1996) Guidelines for Canadian drinking water. Ottawa (Canada): Minister of Supply and Services Canada. Villanueva CM, Cantor KP, Cordier S, Jaakkola JJ, King WD, et al. (2004) Disinfection byproducts and bladder cancer: A pooled analysis. Epidemiology 15:357–367. Lanphear BP, Vorhees CV, Bellinger DC (2005) Protecting children from environmental toxins. PLoS Med 2:e61 DOI: 10.1371/journal.pmed.0020061....查看详细 (16129字节)
☉ 11340247:女性医护人员的海上救护心理评估
[摘要] 目的: 了解女性医护人员实施海上救护的心理特征与状态。 方法: 使用SCL-90和卡特尔16种人格因素量表(Cattell's16PF)对56例海上医疗救护船女性医护人员登船前、登船后2周进行问卷调查。 结果: 女性医护人员SCL-90中的总分、躯体化、强迫、抑郁、焦虑、偏执等因子分数显著高于对照组，Cattell's16PF中的稳定性、敏感性、怀疑性和紧张性等人格特质与对照组有显著差异(P 1 对象与方法 1.1 调查对象 选取2002～2005年实施海上救护的女性医护人员56人...查看详细 (4503字节)
☉ 11340248:Tweaking Microtubules to Treat Scleroderma
Jacob M. van Laar is Associate Professor and Tom W.J. Huizinga is Professor of Experimental Rheumatology in the Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands. Systemic sclerosis (SSc; also referred to as “scleroderma”) is a rare but debilitating autoimmune disease clinically characterized by skin thickening and signs and symptoms of vasculopathy, which can involve the heart, lungs, kidneys, and gut. The disease spectrum can range from limited to diffuse disease, depending on the distribution of skin involvement, specificity of autoantibodies, and type of organ involvement. Patients with extensive skin thickening and organ dysfunction in particular are at risk of premature mortality [1]. The disease poses a challenge for the treating clinician, as no proven therapy exists that improves outcome, although recent data indicate that cyclophosphamide-based regimens may be effective in a subset of patients with early disease [2]. The etiology of SSc remains enigmatic, and few genetic and environmental predisposing factors have been identified. Pathogenesis of SSc Nevertheless, important aspects of its pathogenesis have been elucidated, particularly those related to progressive fibrosis, which is one of the hallmarks of the disease. Transforming growth factor β (TGFβ) is a pivotal cytokine in this process; it is a pleiotropic cytokine that induces matrix accumulation, regulates lymphocyte function and promotes endothelial cell apoptosis. Binding of TGFβ to the type II TGFβ receptor triggers its heterodimerization with, and activation of, type I TGFβ receptor. This activation results in a downstream signaling cascade with phosphorylation of specific receptor-regulated Smad (R-Smad) proteins (Smad2/3), which partner with Smad4 after dissociation from the TGFβ receptor (Figure 1). Smad2/3–Smad4 oligomers migrate to the nucleus, recruit other gene regulatory proteins, and activate transcription of specific target genes. In the absence of ligand stimulation, Smads reside predominantly in the cytoplasm; translocation of the activated R-Smad–Smad4 complex into the nucleus is a key step in signal transduction. Step 1: TGFβ binding to a type II receptor causes the receptor to recruit and phosphorylate a type I receptor. Step 2: phosphorylated type I receptor recruits and phosphorylates Smad2 or Smad3, upon which the Smads open up and expose a dimerization surface. Step 3: phosphorylated Smad2 or Smad3 dissociates from the receptor and oligomerizes with inhibitory Smad4. Step 4: the Smad2/3–Smad4 complex migrates to the nucleus, recruits other gene regulatory proteins (blue), and activates transcription of specific target genes. Skin fibroblasts from patients with SSc express relatively high levels of TGFβ receptor, and contain high concentrations of R-Smad3 in the nucleus, while inhibitory Smad7 is functionally defective [3–5]. These and other data suggest that TGFβ signaling is constitutively activated in SSc fibroblasts, thus contributing to aberrant extracellular matrix synthesis. The important role of Smads in fibrosis is illustrated by the finding that Smad3-deficient mice are resistant to different forms of fibrosis. Not surprisingly, the TGFβ/Smad axis has been identified as a therapeutic target in fibrotic conditions such as SSc. A New Study in a Mouse Model of SSc A study published in this issue of PLoS Medicine by Liu et al. [6] shows that, in a hybrid human SSc skin–severe combined immunodeficient mouse xenotransplant model, stabilizing microtubules using paclitaxel (Taxol; a powerful anticancer agent and angiogenic inhibitor isolated from the bark of the Pacific yew tree) reduces production of phosphorylated Smad2/3 and expression of COL1A2 (one of the genes involved in production of collagen, whose promoter contains multiple Smad-binding elements). The end result is to lessen fibrosis histologically. The study takes advantage of an important animal model for scleroderma, the engraftment of SSc skin samples in immunodeficient mice. These samples have previously been shown to retain their phenotype and abnormal Smad expression [7]. The study also builds on previous work that has shown that microtubules provide a negative feedback loop in TGFβ signaling in cell lines by forming a complex with endogenous Smad2, Smad3, and Smad4, sequestering R-Smads away from the TGFβ receptor [8]. Taken together, these studies suggest that modulating TGFβ/Smad signaling with paclitaxel may be an effective means to treat skin fibrosis. The Role of Other Signaling Cascades However, recent data indicate that other signaling cascades are also perturbed [9], and it is, therefore, conceivable that the beneficial effects of paclitaxel on scleroderma skin thickening are not solely due to changes in TGFβ/Smad signaling. One of the read-outs of fibrogenesis in the study of Liu et al. is reduced expression of COL1A2, an essential gene involved in the biosynthesis of collagen. However, this process is complex: extensive posttranslational modification of the COL1A2 gene product occurs during the fibrotic process in which many key enzymes such as telopeptide lysyl hydroxylase are involved [10]. Future studies should address the effect of paclitaxel on the expression of the wide array of enzymes involved in fibrosis by genome-wide expression studies in patients treated with paclitaxel or ex vivo on scleroderma skin samples. Next Steps By contrast, scleroderma-like changes in patients with cancer have been ascribed to the use of taxanes, including paclitaxel [11]. Whether, as suggested by Liu et al., this paradoxical effect on skin relates to the use of low doses in the mouse model described by them rather than the high doses used in patients with cancer remains to be determined, but the point underscores the need for further studies. Further work is also needed on the in vivo effects of paclitaxel on the vasculature and immune abnormalities in SSc patients, which are difficult to evaluate using scleroderma skin grafts in immunodeficient mice. At the low doses used in the studies by Liu et al. no antiangiogenic effect was found. Clearly, there is a delicate balance between microtubule stabilizing and destabilizing forces in scleroderma, which paclitaxel may alter. These findings suggest, however, that a small pilot study of such therapy in selected patients with diffuse SSc, though a daring endeavor, may be worth the risk. References Medsger TA Jr (2004) Classification. Prognosis. In: Clements PJ, Furst DE, editors. Systemic sclerosis, 2nd ed London: Lippincott Williams and Wilkins. pp 17–28. Clements Ph, Furst DE, Silver RM, Tashkin DP, Roth MD, et al. (2005) The Scleroderma Lung Study shows the beneficial effects of cyclophosphamide over placebo in systemic sclerosis patients with active alveolitis. Arthritis Rheum 52:S257. Kawakami T, Ihn H, Xu W, Smith E, LeRoy C, et al. (1998) Increased expression of TGF-beta receptors by scleroderma fibroblasts: Evidence for contribution of autocrine TGF-beta signaling to scleroderma phenotype. J Invest Dermatol 110:47–51. Mori Y, Chen SJ, Varga J (2003) Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts. Arthritis Rheum 48:1964–1978. Asano Y, Ihn H, Yamane K, Kubo M, Tamaki K (2004) Impaired Smad7-Smurf-mediated negative regulation of TGF-beta signaling in scleroderma fibroblasts. J Clin Invest 113:253–264. Liu X, Zhu S, Wang T, Hummers L, Wigley FM, et al. (2005) Paclitaxel modulates TGFβ signaling in scleroderma skin grafts in immunodeficient mice. PLoS Med 2:e354 DOI: 10.1371/journal.pmed.0020354. Lakos G, Takagawa S, Chen SJ, Ferreira AM, Han G, et al. (2004) Targeted disruption of TGF-β/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma. Am J Pathol 165:203–217. Zhu S, Goldschmidt-Clermont PJ, Dong C (2004) Transforming growth factor-β-induced inhibition of myogenesis is mediated through Smad pathway and is modulated by microtubule dynamic stability. Circ Res 94:617–625. Pockwinse SM, Rajgopal A, Young DW, Mujeeb KA, Nickerson J, et al. (2005) Microtubule-dependent nuclear-cytoplasmic shuttling of Runx2. J Cell Physiol. E-pub ahead of print. Van der Slot AJ, Zuurmond AM, Bardoel AF, Wijmenga C, Pruijs HE, et al. (2003) Identification of PLOD2 as telopeptide lysyl hydroxylase, an important enzyme in fibrosis. J Biol Chem 278:40967–40972. Farrant PBJ, Mortimer PS, Gore M (2005) Scleroderma and the taxanes. Is there really a link Clin Dermatol 29:360–362....查看详细 (8679字节)
☉ 11340249:海训对女性医护人员身心不良影响的分析及预防
[摘要]:目的 掌握和分析海训对女性医护人员身心的不良影响。 方法: 采用自行设计的问卷调查表进行问卷调查。 结果: 月经失调、皮肤晒伤和皮肤虫咬伤是海训对女性医护人员身心的常见不良影响，其中月经失调是最为常见的，占70.2%。 结论: 针对海训对女性医护人员身心不良影响的原因，可通过合理安排训练项目、改善驻地环境、进行健康宣教等措施进行预防和治疗，以提高海训质量。 [关键词] 海训;不良影响;预防;女性医护人员 海训是航海训练中的一个重要项目...查看详细 (3783字节)
☉ 11340250:Selection Bias in Meta-Analyses of Gene-Disease Associations
Jin Ling Tang is the director of the Chinese Cochrane Centre Hong Kong Branch, based in the Faculty of Medicine, the Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, The People's Republic of China. Many studies have too small a sample size for their findings to be conclusive, but large studies are expensive and time-consuming. Meta-analysis is an alternative to conducting large studies in tackling the problem of small sample size, by combining available small studies to increase the total sample size. Since the 1980s, meta-analysis has been widely used in summarizing results from clinical trials of medical interventions and has also recently gained increasing attention in studying gene-disease associations. However, selection bias may occur in meta-analyses due to the inability to identify and include all conducted and relevant studies. Such selection bias can cause exaggerated or even false-positive gene-disease associations [1]. Failure to include all relevant studies is largely caused by selective publication of studies with certain results (publication bias), and the inability to identify studies published in languages other than English (language bias). Selection bias has been well recognized in meta-analyses of clinical trials [2–4]. Less is known about selection bias in meta-analyses of studies of gene-disease associations; such studies generally address weak associations and thus are particularly vulnerable to biases. A Study of the Chinese Literature In their study published in this issue of PLoS Medicine, Pan and colleagues compared genetic studies conducted in mainland China with those from other places [5]. The researchers identified 12 gene-disease associations and compared a total of 161 Chinese studies and 309 non-Chinese studies. The Chinese studies were on average smaller in sample size than non-Chinese studies and appeared in the literature a few years after the first non-Chinese studies. Chinese studies in general reported a stronger gene-disease association and more frequently a statistically significant result. These two characteristics were more likely to occur in Chinese studies identified through PubMed than in those accessible only locally. These findings suggest a variation or heterogeneity in the strength of the gene-disease association (often expressed in an odds ratio) observed between Chinese and non-Chinese studies. These studies are primarily case-control studies. Many factors may contribute to the variation in the estimate of odds ratio across such studies, such as the genetic make-up of the population studied, the type of patients included, the selection of controls, the quality of the study design, and the quality of the laboratory work. These factors could lead to either over- or under-estimation of the true odds ratio. However, it is difficult to conceive that any single factor, or combination of these factors, could consistently cause the exaggerated odds ratio in Chinese studies in all the topics (gene-disease associations) examined by Pan and colleagues. Selective publication is therefore a very likely and worrying explanation for their findings. Implications for Clinical Practice and Research Selective publication can cause publication bias, which in turn could lead to false gene-disease associations in meta-analyses. It would be a disaster if a genetic screening program (in which healthy people are tested for a gene and offered a treatment if they test positive) were based on such a false association. Even if such a false gene-disease association were only subjected to further related investigations, this would be a waste of valuable resources for medical research. Selective publication of positive studies in China and a few other Asian countries has been observed in clinical trials of acupuncture [6,7]. However, selective publication by no means exists in only the Chinese literature. It is probably a common phenomenon in the entire field of biomedical research. Given the fact that positive studies are more likely to be published than negative ones, and given the pressure on researchers worldwide to publish in indexed journals (especially in international journals with high impact factors), selective publication is likely to continue in the foreseeable future. As compared with English-speaking countries, selective publication is perhaps more likely to occur in non-English-speaking countries where there are a small number of indexed journals to publish local studies. Addressing the Problem Journals accessible through PubMed or other major biomedical databases are unlikely to have the same mechanism of selection for publication as local journals that are less accessible to researchers outside the country, such as the Chinese journals. Thus, meta-analyses that include only internationally accessible studies (which is, currently, often the case for meta-analyses) are likely to have language or location bias. Meta-analyses that included only local studies could be even worse, as implied by Pan and colleagues' study. Inclusion of every study published worldwide would probably still not totally solve the problem, as many studies are never published or their publication is delayed. Odds ratios thus estimated would normally be an over-estimate. Registration of studies is ideal and has been widely advocated for clinical trials [8]. Before such registration becomes universal practice, it would be important for journals, in selecting papers for publication, to emphasize the quality of the study rather than the size and direction of the odds ratio and the p-value of the statistical test. However, such an emphasis on quality (rather than the size and direction of the odds ratio) would not be much help to researchers who are currently doing meta-analyses. Current researchers must strive to not only identify relevant studies but also examine the possibility of publication bias in the results. Although better tools have yet to be developed [9,10], current methods for detection and adjustment for publication bias in meta-analyses of clinical trials would be useful for meta-analyses of gene-disease associations [1,11,12]. References Thornton A, Lee P (2000) Publication bias in meta-analysis: Its causes and consequences. J Clin Epidemiol 53:207–216. Easterbrook PJ, Berlin JA, Gopalan R, Matthews DR (1991) Publication bias in clinical research. Lancet 337:867–872. Dickersin K, Min YI, Meinert CL (1992) Factors influencing publication of research results: Follow-up of applications submitted to two institutional review boards. JAMA 267:374–378. Egger M, Davey-Smith G (1998) Bias in location and selection of studies. BMJ 316:61–66. Pan ZL, Trikalinos TA, Kavvoura FK, Lau J, Ioannidis JPA (2005) Local literature bias in genetic epidemiology: An empirical evaluation of the Chinese literature. PLoS Med 2:e334 DOI: 10.1371/journal.pmed.0020334. Vickers A, Goyal N, Harland R, Rees R (1998) Do certain countries produce only positive results A systematic review of controlled trials. Control Clin Trials 19:159–166. Tang JL, Zhan SY, Ernst E (1999) Review of randomised controlled trials of traditional Chinese medicine. Br Med J 319:160–161. 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