Effect Of Cytokines In Host Defense And Inflammation

细胞因子在宿主防御和炎症中的作用

• 批准号: 7964281
• 负责人: JOHN I GALLIN
• 金额: $ 24.61万
• 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/7964281
• 关键词: Antibodies; B-Lymphocytes; Binding; Biochemical; Biological Assay; Biological Response Modifiers; Bone Marrow; Bulla; CD34 gene; Cells; Childhood; Chronic; Chronic Granulomatous Disease; Contractor; Development; Devices; Disease; Drug Design; Endotoxins; Ensure; Enzymes; Evolution; Experimental Models; Exudate; Fibroblasts; Forearm; Formalin; Functional RNA; Future; Gene Expression; Generations; Genome; Goals; Granuloma; Host Defense; Human; Human Volunteers; IL8 gene; Immune; Immunofluorescence Immunologic; Immunologic Deficiency Syndromes; In Vitro; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Interleukin-1; Interleukin-10; Job' s Syndrome; Laboratories; Link; Literature; Liver; MAP Kinase Gene; MAPK14 gene; Mediating; Mediator of activation protein; Membrane; MicroRNAs; Microarray Analysis; Molecular; Mutation; Myeloid Progenitor Cells; NADPH Oxidase; Nuclear; Organism; Paraffin Embedding; Pathway interactions; Patients; Phagocytes; Phosphorylation; Play; Preparation; Production; Protocols documentation; RNA; RNA Sequences; Regulation; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Sarcoidosis; Signal Transduction; Signal Transduction Pathway; Skin; Spleen; Stem Cell Development; Structure of parenchyma of lung; Superoxides; Surveys; System; TLR4 gene; Techniques; Technology; Tissues; Toll-Like Receptor Pathway; Translations; Variant; Work; antimicrobial peptide; base; chronic graft versus host disease; cytokine; cytotoxic; human IRAK4 protein; human subject; in vivo; inhibitor/antagonist; monocyte; neutrophil; neutrophil cytosol factor 67K; novel; p21 activated kinase; peripheral blood; research study; response

In 2009 we continued our accrual of patients into our blister study protocol. The protocol involves the formation of blisters on the forearms of human volunteers to study the inflammatory response in vivo. This year, we intiated a project comparing the transcriptomes of peripheral blood neutrophils with matched inflammatory exudate (blister) neutrophils from seven normal donors. For this project, we have teamed with the Rocky Mountain Laboraty Research Technologies Branch (RML) who will provide expert microarray analysis of these samples. We are increasing the number of chronic GvHD patients in our study pool (now a total of 4 patients) as they become available from our collaborators in Dr. Harry Malech's laboratory. Also, with the help of LCID, we have now studied a total of five patients with Job's syndrome and are awaiting quantitative analysis by our contractor at SAIC-Frederick. (Zarember 20% effort). In addition to several normals, we have also studied three patients with chronic graft-versus-host disease and two patients with Jobs Syndrome. Additional patients will be required to fully power these studies to determine whether the observed dysregulation of cytokine production is statistically significant (Kol Zarember, 20% effort). In 2009 we continued to study the molecular basis for priming and activation of the NADPH oxidase and superoxide production by endotoxin (LPS). The NADPH oxidase (NOX), an oligomeric enzyme, plays a key role in polymorphonuclear neutrophil (PMN)-mediated host defense by producing cytotoxic superoxide anion (O2.-). Whereas in vitro and biochemical studies have examined the assembly and activation of this important host immune defense system, few studies have examined the function of NOX in human patients with primary immunodeficiencies other than chronic granulomatous disease. We studied the activation of NOX in PMN from patients with two distinct immunodeficiencies, interleukin-1 receptor associated kinase 4 (IRAK4) deficiency and nuclear factor kappa (NF-κB) essential modulator (NEMO or IKKγ) deficiency. We observed impaired O2.- generation by LPS-treated and fMLP-activated IRAK4-deficient PMN that correlated with decreased phosphorylation of p47phox and subnormal translocation of p47phox, p67phox, Rac2, and gp91phox/Nox2 to the membranes indicating that TLR4 signaling to the NOX activation pathway requires IRAK4. NEMO-deficient PMN also generated less O2.- in response to LPS and fMLP and translocated less p47phox and p67phox to membranes than normal PMN but were more responsive than IRAK4-deficient cells. Decreased LPS and fMLP induced phosphorylation of p38 MAPK in both IRAK4- or NEMO-deficient PMN and of p21-activated kinases (PAK) in IRAK4-deficiency implicates additional signal transduction pathways in regulating PMN superoxide activation by LPS and fMLP (Anjali Singh, 85% effort; Kol Zarember 10% effort). In 2009 we initiatied a study of granuloma formation utilizing immunofluorescence to probe the various cellular and molecular components of granulomas. Using this technique, we will demonstrate the various types of immune cells (including neutrophils, monocytes, T and B cells, and fibroblasts) and immune mediators (such as IL-1, IL-8, IL-10, TGF, and various antimicrobial peptides) present in granulomas and potentially contributing to their formation. We have validated the antibodies against many of these targets in peripheral blood smears, a formalin fixed, paraffin embedded buffy coat pellet, and normal formalin fixed, paraffin embedded spleen and bone marrow sections to ensure that the desired binding is achieved with minimal background/non-specific binding. Experiments are underway using liver, spleen, and lung tissue from patients with CGD that contain granulomas and efforts are being made to obtain granuloma-containing tissue from patients with other diseases such as inflammatory bowel disease and sarcoidosis. (Soule, 30% effort). This past year we initiated another project to better understand the potential of microRNA in the regulation of phagocyte function, including regulation of production of cytokines. MicroRNAs comprise a class of over 500 small, non-coding RNA that regulate gene expression by inhibiting RNA translation. They are felt to regulate the expression of over 30% of the genome and specific microRNA species have been shown to be active in neutrophils. A complete survey of the microRNA species in neutrophils and monocytes has not been performed. We have used several commercially available RT-PCR kits to assay specific microRNA in neutrophils, however this has been complicated by significant experiment to experiment variation. In response to this, we have been collaborating with the Rocky Mountain Laboratory to perform whole RNA sequencing on total RNA isolated from purified neutrophils preparations from normal controls and patients with both X-linked (gp91-deficient) and autosomal recessive (p47-deficient) CGD patients. This information will itself be novel, but will also facilitate the development of future studies involving both microRNA microarrays and conventional RT-PCR. We have also begun preliminary work studying the viability of transfecting microRNA precursors and inhibitors into neutrophils to determine the effect of altering microRNA expression on neutrophil function. Finally, we have been speaking with other investigators about the possibility of studying the role of microRNA in myeloid stem cells (CD34+ cells). There is a large body of literature demonstrating the role of microRNA in development at the cellular and organism level. It is likely that microRNA are active in directing myeloid stem cell development and differentiation, but this has not been extensively studied.

2009年，我们将患者应计入我们的水泡研究方案。该方案涉及在人类志愿者的前臂上形成水泡，以研究体内炎症反应。 今年，我们将一个项目进行了一个项目，比较了外周血液中性粒细胞与来自七个正常供体的炎性渗出症（泡沫）中性粒细胞的转录组。 对于这个项目，我们与Rocky Mountain Laboraty Research Technologies Branch（RML）合作，他们将对这些样品进行专家的微阵列分析。 我们正在增加研究池中的慢性GVHD患者的数量（现在总共有4例），因为他们在哈里·莫勒克（Harry Malech）博士的实验室中获得了合作者。 另外，在LCID的帮助下，我们现在已经研究了五名Job综合征的患者，并正在等待我们在Saic-Frederick的承包商进行定量分析。 （Zarmerm 20％的努力）。除了几种正常状态外，我们还研究了三名患有慢性移植物抗宿主病和两名工作综合征患者的患者。 还需要其他患者为这些研究充分动力，以确定观察到的细胞因子产生失调是否具有统计学意义（Kol Zarember，20％的努力）。 在2009年，我们继续研究内毒素（LPS）启动和激活NADPH氧化酶和超氧化物的分子基础。 NADPH氧化酶（NOX）是一种低聚酶，通过产生细胞毒性超氧化物阴离子（O2.-），在多形核中性粒细胞（PMN）介导的宿主防御中起关键作用。尽管体外和生化研究已经检查了这种重要的宿主免疫防御系统的组装和激活，但很少有研究检查了除慢性肉芽肿性疾病以外的原发性免疫缺陷患者中NOX的功能。我们研究了具有两种不同免疫缺陷的患者的PMN激活NOX，白介素-1受体相关激酶4（IRAK4）缺乏症和核因子Kappa（NF-κB）必需调节剂（NEMO或IKKKγ）缺乏症。 We observed impaired O2.- generation by LPS-treated and fMLP-activated IRAK4-deficient PMN that correlated with decreased phosphorylation of p47phox and subnormal translocation of p47phox, p67phox, Rac2, and gp91phox/Nox2 to the membranes indicating that TLR4 signaling to the NOX activation pathway requires IRAK4.缺陷型PMN也对LPS和FMLP产生了较少的O2。-P47phox和P67Phox易于易位，而P67PHOX比正常PMN易于膜，但比IRAK4缺陷型细胞反应率更高。 LP和FMLP降低的IRAK4或缺乏型PMN和p21激活激酶（PAK）在IRAK4缺乏率中降低了p38 MAPK的磷酸化，这意味着在调节LPS和FMLP（Anjali singhermemers nocyhem nocyh，85％）中，在调节PMN超氧激活中的其他信号传递途径; 2009年，我们利用免疫荧光来探测肉芽肿的各种细胞和分子成分。 使用这种技术，我们将演示各种类型的免疫细胞（包括中性粒细胞，单核细胞，T和B细胞以及成纤维细胞）和免疫介质（例如IL-1，IL-8，IL-10，TGF和各种抗菌肽）中存在于肉芽核瘤中，并具有贡献的形式。 我们已经在外周血涂片，福尔马林固定，嵌入的石蜡嵌入的Buffy Coat Pellet和正常福尔马林固定的，石蜡嵌入的脾脏和骨髓切片中验证了许多此类靶标的抗体，以确保具有最小的背景/非特异性结合。 使用肝脏，脾脏和肺组织的CGD患者正在进行实验，这些患者含有肉芽肿，并正在努力从其他疾病患者（例如炎症性肠病和结节病）中获得含肉芽肿的组织。 （Soule，30％的努力）。 在过去的一年中，我们启动了另一个项目，以更好地了解microRNA在调节吞噬细胞功能的可能性，包括细胞因子的生产。 MicroRNA包含一类超过500个小的非编码RNA，这些RNA通过抑制RNA翻译来调节基因表达。 人们认为它们可以调节超过30％的基因组的表达，并且已显示特异性microRNA物种在中性粒细胞中活跃。 尚未对中性粒细胞和单核细胞中的microRNA物种进行完整的调查。 我们已经使用了几个可商购的RT-PCR试剂盒来测定中性粒细胞中的特定microRNA，但是通过实验变化的重要实验使其复杂化。 为此，我们一直与落基山实验室合作，对从正常对照组和患有X-Linked（GP91缺陷型）和常染色体隐性（P47缺乏症）的X-Linked患者（P47缺乏）患者的患者进行纯化的嗜中性粒细胞制剂进行整个RNA测序。 这些信息本身将是新颖的，但也将促进涉及MicroRNA微阵列和常规RT-PCR的未来研究的发展。 我们还开始研究研究中性粒细胞中转染microRNA前体和抑制剂的生存力的初步工作，以确定改变microRNA表达对中性粒细胞功能的影响。 最后，我们一直在与其他研究人员谈论研究microRNA在髓样干细胞（CD34+细胞）中的作用的可能性。 有大量文献证明了microRNA在细胞和生物水平上发育中的作用。 MicroRNA可能活跃于指导髓样干细胞的发育和分化，但这尚未得到广泛的研究。