Clinical Studies Of Abnormal Host Defense

宿主防御异常的临床研究

• 批准号: 7964198
• 负责人: JOHN I GALLIN
• 金额: $ 18.97万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964198
• 关键词: Amphotericin B; Antibiotics; Antifungal Agents; Aspergillosis; Aspergillus; Aspergillus fumigatus; Atherosclerosis; B-Lymphocytes; Binding; Biomedical Research; Biometry; Blood; Blood Vessels; Calcium; Candida albicans; Cardiac; Catabolism; Cell Line; Cell physiology; Cells; Chelating Agents; Child; Chronic Granulomatous Disease; Clinical; Clinical Protocols; Clinical Research; Collaborations; Complement; Coronary artery; CpG dinucleotide; Cryptococcus neoformans; Cyclic Neutropenia; Cytoplasmic Granules; Data; Defect; Deletion Mutation; Development; Disease; Disease model; Enrollment; Enzyme Stability; Exhibits; Extracellular Domain; Extramural Activities; Family; Fluconazole; Frameshift Mutation; Functional disorder; Genes; Genotype; Germination; Goals; Growth; Growth Factor; Heart Diseases; Host Defense; Host Defense Mechanism; Human; Human Herpesvirus 4; Hyphae; IRAK4 gene; Imaging Techniques; Immune System Diseases; Immune system; Immunocompromised Host; Infection; Infectious Agent; Inflammation; Interferon Type II; Iron; Iron Chelating Agents; Itraconazole; Job' s Syndrome; Ketoconazole; Laboratories; Lactoferrin; Leukocyte Adhesion Deficiency; Leukocyte-Adhesion Deficiency Syndrome; Leukocytes; Limulus; Link; Lipid A; Longitudinal Studies; Measures; Microbe; Missense Mutation; Modeling; Molecular; Monitor; Mothers; Mus; Mutation; Mycoses; NADP; NADPH Oxidase; Nonsense Mutation; Nucleotides; Organism; Oxidases; Patients; Phagocytes; Pharmaceutical Preparations; Phenotype; Physiological; Population; Prevalence; Prevention; Production; Property; Proteins; Protocols documentation; RNA Processing; Reactive Oxygen Species; Recording of previous events; Recruitment Activity; Recurrence; Resistance; Resources; Role; Sampling; Statistical Methods; Superoxides; Survival Analysis; Syndrome; Testing; Transmembrane Domain; Tryptophan; Tryptophan Metabolism Pathway; United States National Institutes of Health; Work; Writing; antimicrobial peptide; chediak-higashi syndrome; cohort; combinatorial; cytokine; disease phenotype; disorder subtype; falls; fungus; human CYBA protein; immune function; in vivo; killings; mortality; neutrophil; pathogen; response

The LHD has a long history of investigating patients with abnormalities of phagocytic cell function. These studies include the delineation of clinical, functional, and molecular defects of patients with neutrophil specific granule deficiency, chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), the syndrome of hyperimmunoglobulinE and recurrent infections (Jobs Syndrome) and IRAK4 deficiency. Large cohorts of these patients have been recruited over the years and represent a unique national resource for biomedical research at the NIH. Currently we follow over 150 patients with CGD, about 40 patients with Jobs syndrome, and 30 patients with other phagocyte dysfunction syndromes, including LAD, cyclic neutropenia, neutrophil specific granule deficiency, Chediak-Higashi syndrome, IRAK4 deficiency and NEMO deficiency. We now have EB virus transformed B cells from most of our patients and we have been pleased to share these B cell lines with other intramural or extramural colleagues. We continue to monitor and expand these cohorts of patients who serve as models for long term studies of the clinical consequences of the immune dysfunction in humans. In 2009 we continued our studies of the importance of lactoferrin in protecting against Aspergillus fumigatus infection, the most common infectious cause of mortality in CGD patients today. We found that while CGD PMN are unable to kill Aspergillus hyphae, their ability to arrest the growth of conidia was identical to that of normal PMN showing a role for nonoxidative mechanisms in host defenses against this organism. We then showed that the neutrophil secretory product, lactoferrin, inhibits conidia germination by sequestering iron, a critical growth factor. We have continued studying the growth inhibitory properties of iron chelating drugs against Aspergillus fumigatus, alone and in combination with first line antibiotics such as amphotericin B, itraconazole, and fluconazole and demonstrated antifungal synergy in some combinations. Combinatorial fungal effects against A. fumigatus conidia were synergistic for ketoconazole with either ciclopirox or deferiprone, lactoferrin with amphotericin B, and fluconazole with deferiprone. Desferoxamine enhanced fungal growth. With the biostatistics branch, we developed a new statistical method for the analysis of synergy. Further studies have demonstrated significant antifungal activity of some iron chelators against other fungi (Cryptococcus neoformans, Candida albicans) suggesting an important new avenue for prevention and treatment of fungal infections common to immunocompromised patients. We are actively working with June Kwon-Chung (LID) to test whether these drugs work in vivo in murine aspergillosis. (Kol Zarember, 20% effort). In 2009 we completed our gene sequencing data and phenotype correlates in 259 patients with chronic granulomatous disease (CGD) representing 221 distinct families, a cohort large enough to provide sufficent statistical power to compare specific CGD subtypes. We have also analyzed 40 X-linked heterozygous carriers in 34 families and 9 mothers who have children with spontaneous mutaions in gp91phox, but do not display heterozygosity. This cohort included unreported mutations in gp91phox (44 mutations), in p67phos (9 mutations), and in p22phox (1 mutation). Missense and frameshift mutations in gp91phox are randomly distributed throughout the protein and are generally family-specific. However, nonsense mutations are more discrete, preferentially localized to CpG dinucleotides "hotspots" that are common to as many as 9 separate families. Furthermore, 4 of the patients with spontaneous CGD exhibit de novo mutations at CpG dinucleotides. Analysis of gp91phox mutations indicates that missense mutations with the carboxyl terminal FAD and NADPH nucleotide binding domains almost completely abrogate the activity of the oxidase, but often do not alter the expression and/or stability of gp91phox. Muations in the amino terminal transmembrane region of gp91phox, particularly the extracellular domains, are more permissive, retaining some activity of the oxidase, but generally with diminished expression and/or stability of the enzyme. Survival analysis of the major CGD genotypes indicates that patients with missense mutations in gp91phox and patients with mutations in p47phox exhibit survival significantly better than patients with nonsense, frameshift, RNA processing, and deletion mutations in gp91phox mutations. Moreover, patients with missense mutations in gp91phox are not significantly different from the survival curve of the general US population. These data indicate that CGD phenotype can be used to predict NADPH oxidase function and survival in patients with CGD. In other related studies in a few gp91phox deficient CGD patients it appears that patients with missense mutations are responsive to interferon gamma in terms of superoxide production suggesting mutational analysis may predict interferon gamma responsiveness. Over the past year, the laboratory has expanded its study of Granulibacter bethesdensis, a recently described bacterial pathogen of CGD patients. Granulibacter is remarkably hypostimulatory of the innate immune system, both in terms of weak activation of the NADPH oxidase and poor stimulation of cytokine secretion. Ongoing efforts to purify and characterize what apperas to be an atypical lipolysaccharide (LPS) of this organism will expand in coming months. We have isolated a fraction that appears to represent the Lipid A of this organism that has strongly stimulatory activity in the limulus LPS test but fails to activate human PMN in keeping with our findings that this microbe may avoid host defenses by using molecular stealth. We have also found that G. bethesdensis is remarkably resistant to complement and antimicrobial peptides. In collaboration with LCID, we are evaluating internalization and killing of Granulibacter by normal and CGD leukocytes. Further studies examining the transcriptional responses of the pathogen to attachk by the host and thost cells in response to the pathogen are underway. (Zarember 50% effort). In 2009 we studied the role of tryptophan metabolism in CGD. Mouse CGD models were recently implicated to have defective tryptophan catabolism as a major regulator of inflammation in CGD. We attempted to validate these findings in human clinical samples and discovered that unlike CGD mice, human CGD patients do not display this defect in tryptophan catabolism. (Zarember, 10% effort). In addition to these projects, in 2009 we have written a clinical protocol that will enroll patients to study the development of atherosclerotic disease in patients with immune system disorders. Atherosclerosis, the major cause of heart disease, is thought to relate to dysregulated inflammation. in the cardiac blood vessels and possibly results from over production of reactive oxygen species (ROS). We hypothesize that CGD patients, who have absent production of reactive oxygen species by their phagocytes may be protected from developing atherosclerosis. The primary endpoint of this study is to determine the prevalence of atherosclerosis in these and other patients with in-born disorders of immune function. The primary endpoint will be assessed using imaging techniques to measure coronary artery calcium scores and the presence of absence of soft plaque. Secondary endpoints include physiologic markers such as blood pressuer as well as circulating biomakers associated with heart disease. We expect to enroll patients into thei protocol in the fall of 2009. (Soule 15 % effort).

LHD在研究吞噬细胞功能异常的患者方面有悠久的历史。这些研究包括嗜中性粒细胞特异性颗粒缺乏症患者的临床，功能和分子缺损的描述，慢性颗粒疾病（CGD），白细胞粘附缺乏（LAD），高免疫球蛋白和复发性感染（工作综合症）的综合征，综合症综合症（工作综合症）和伊拉克综合症。这些年来，这些患者的大量人群已被招募，代表了NIH的独特国家生物医学研究资源。目前，我们关注150多名CGD患者，约40例工作综合征患者以及30例其他吞噬细胞功能障碍综合症患者，包括LAD，环状中性粒细胞减少症，中性粒细胞特异性颗粒缺乏症，Chediak-Higashi综合征，IRAK4缺乏症和Nemo缺乏症。现在，我们拥有EB病毒从大多数患者中转化的B细胞，我们很高兴与其他壁内或壁外同事共享这些B细胞系。我们继续监视和扩展这些同类的患者，这些患者是长期研究人类免疫功能障碍的临床后果的模型。 2009年，我们继续研究乳铁蛋白在预防曲霉感染中的重要性，这是当今CGD患者死亡率最常见的传染病。我们发现，尽管CGD PMN无法杀死曲霉的菌丝，但它们阻止分生孢子的生长与正常PMN的生长能力相同，在宿主防御剂中对这种生物体的防御措施表现出了非氧化机制的作用。然后，我们表明中性粒细胞分泌产物乳铁蛋白通过隔离铁（一种关键生长因子）抑制分生孢子发芽。我们一直在研究单独并与第一线抗生素（如两性霉素B，伊曲康唑和氟康唑，并在某些组合中表现出抗真菌性的Synergy），并与第一线抗生素（如两性霉素B，Itraconazole，Itraconazole和Fluconazole）相结合，研究铁螯合药物的生长抑制特性。对烟曲霉分生孢子的组合真菌作用与酮康唑与千洛酮或脱氟酮，带有两性霉素B的乳糖铁和氟康唑与脱氟酮是协同作用。去铁胺增强了真菌的生长。在生物统计学分支的情况下，我们开发了一种用于协同分析的新统计方法。进一步的研究表明，某些铁螯合剂对其他真菌（隐孢子虫，白色念珠菌）的显着抗真菌活性，这表明预防和治疗免疫功能低下患者常见的真菌感染的重要新途径。我们正在与June Kwon-Chung（LID）积极合作，以测试这些药物在鼠曲霉病中是否在体内起作用。 （Kol Zarember，20％的努力）。 在2009年，我们完成了259例代表221个不同家族的慢性肉芽肿性疾病（CGD）患者的基因测序数据和表型，这是一个足够大的同胞，可以提供足够的统计能力来比较特定的CGD亚型。 我们还分析了34个家庭中的40个X连接的杂合载体和9名母亲在GP91Phox中具有自发性突变的孩子，但不显示杂合性。该队列包括GP91Phox（44个突变），p67phos（9个突变）和p22phox（1个突变）中的未报告突变。 GP91Phox中的错义和移码突变在整个蛋白质中随机分布，通常是家族特异性的。但是，胡说八道的突变更加离散，优先将其定位于CPG二核苷酸“热点”，这些点数通常9个单独的家庭常见。此外，有4例自发CGD患者在CPG二核苷酸中表现出从头突变。 GP91Phox突变的分析表明，具有羧基末端FAD和NADPH核苷酸结合结构域的错义突变几乎完全消除了氧化酶的活性，但通常不会改变GP91Phox的表达和/或稳定性。 GP91Phox的氨基末端跨膜区域中的嵌入，尤其是细胞外结构域，更宽松，保留了氧化酶的某些活性，但通常会降低酶的表达和/或稳定性。 对主要CGD基因型的生存分析表明，p47phox的GP91Phox和突变患者的生存率明显好于胡说八道，Frameshift，RNA处理和GP91Phox突变中的缺失突变的患者。 此外，GP91Phox中错义突变的患者与美国普通人群的生存曲线没有显着差异。 这些数据表明，CGD表型可用于预测CGD患者的NADPH氧化酶功能和存活。 在一些GP91Phox缺乏CGD患者的其他相关研究中，似乎错义突变的患者就超氧化物的产生而对干扰素伽马有反应，这表明突变分析可能预测了干扰素伽马反应性。 在过去的一年中，该实验室扩大了对肉芽杆菌贝塞斯丁斯（Bethesdensis）的研究，这是最近描述的CGD患者的细菌病原体。颗粒杆菌对先天性免疫系统的极不受欢迎，这是因为NADPH氧化酶的激活和细胞因子分泌的刺激不良。 正在进行的净化和表征哪些Apperas是该生物体的非典型脂糖糖（LPS）的努力，将在未来几个月内扩大。 我们分离了一个似乎代表该生物体的脂质A的部分，该生物具有在Limulus LPS测试中具有强烈刺激活性的，但无法激活人PMN，这与我们的发现相吻合，即这种微生物可以通过使用分子隐身来避免宿主防御。 我们还发现，贝塞斯丁氏菌对补体和抗菌肽具有明显抗性。与LCID合作，我们正在评估正常和CGD白细胞对颗粒杆菌的内在化和杀死。 进一步研究了病原体对宿主和Thost细胞对病原体的转录反应的进一步研究正在进行中。 （Zarmerm 50％的努力）。 2009年，我们研究了色氨酸代谢在CGD中的作用。 最近，小鼠CGD模型牵涉到具有缺陷的色氨酸分解代谢作为CGD炎症的主要调节剂。 我们试图在人类临床样本中验证这些发现，并发现与CGD小鼠不同，人CGD患者在色氨酸分解代谢中没有显示出这种缺陷。 （Zarember，10％的努力）。 除这些项目外，在2009年，我们还编写了一项临床方案，该方案将招募患者研究免疫系统疾病患者的动脉粥样硬化疾病的发展。 动脉粥样硬化是心脏病的主要原因，被认为与炎症失调有关。在心脏血管中，可能是由于活性氧（ROS）的过度产生而导致的。 我们假设没有通过吞噬细胞产生活性氧的CGD患者可以受到保护，以免产生动脉粥样硬化。这项研究的主要终点是确定这些和其他具有免疫功能疾病的患者的动脉粥样硬化的患病率。 将使用成像技术评估主要终点，以测量冠状动脉钙评分和不存在软斑块的存在。 次要终点包括生理标记物，例如血液压力机以及与心脏病相关的循环生物制造商。 我们希望在2009年秋天将患者纳入THEI方案。（Soule 15％的努力）。