Effect Of Cytokines In Host Defense And Inflammation

2009年，我们继续将累积的患者纳入水疱性研究方案。该方案涉及在人类志愿者的前臂上形成水泡，以研究体内的炎症反应。今年，我们启动了一个项目，将外周血中性粒细胞的转录本与来自7名正常捐赠者的炎性渗出物(水泡)中性粒细胞进行比较。在这个项目中，我们与落基山实验室研究技术处(RML)合作，后者将为这些样本提供专家微阵列分析。我们正在增加我们研究池中的慢性GvHD患者的数量(现在总共有4名患者)，因为他们可以从Harry Malech博士的实验室的合作者那里获得。此外，在LCID的帮助下，我们现在已经研究了总共5名患有约伯综合征的患者，正在等待我们的承包商在SAIC-Frederick进行定量分析。(Zarember 20%的努力)。除了几名正常人外，我们还研究了三名慢性移植物抗宿主病患者和两名乔布斯综合征患者。更多的患者将被要求完全支持这些研究，以确定所观察到的细胞因子产生的失调是否具有统计学意义(Kol Zarember，20%的努力)。 2009年，我们继续研究启动和激活NADPH氧化酶以及内毒素(LPS)产生超氧化物的分子基础。NADPH氧化酶(NOX)是一种低聚合酶，通过产生细胞毒性超氧阴离子(O2.-)在中性粒细胞(PMN)介导的宿主防御中起关键作用。虽然体外和生化研究已经研究了这一重要的宿主免疫防御系统的组装和激活，但很少有研究研究NOX在除慢性肉芽肿疾病以外的原发免疫缺陷患者中的功能。我们研究了两种截然不同的免疫缺陷患者PMN中NOX的激活情况，这两种缺陷分别是白细胞介素1受体相关激酶4(IRAK4)缺陷和核因子kappaB(NEMO或IKK)缺乏。我们观察到脂多糖处理和fMLP激活的IRAK4缺乏的PMN产生O2-的功能受损，这与p47Phox的磷酸化降低和p47Phox、p67Phox、rac2和gp91Phox/NOX2的异常易位相关，表明TLR4信号转导到NOx激活途径需要IRAK4。与正常PMN相比，NEMO缺乏的PMN产生的O2.-也较少，对脂多糖和fMLP的反应也较少，转位到细胞膜的p47Phox和p67Phox也较少，但比IRAK4缺乏的细胞反应更快。IRAK4或NEMO缺陷型PMN中p38MAPK的磷酸化以及IRAK4缺陷型PMN中p21激活的蛋白激酶(PAK)的磷酸化水平降低，暗示了在调节LPS和fMLP对PMN超氧化物激活的信号转导途径(Anjali Singh，85%努力；Kol Zarember，10%努力)。 2009年，我们利用免疫荧光对肉芽肿的形成进行了一项研究，以探索肉芽肿的各种细胞和分子成分。利用这项技术，我们将展示肉芽肿中存在的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(如IL-1、IL-8、IL-10、转化生长因子和各种抗菌肽)，并可能对其形成起作用。我们已经在外周血涂片、福尔马林固定的石蜡包埋的棕黄色大衣小球和正常的福尔马林固定的石蜡包埋的脾和骨髓切片中验证了针对这些靶标的抗体，以确保以最小的背景/非特异性结合实现所需的结合。使用含有肉芽肿的CGD患者的肝、脾和肺组织的实验正在进行中，并正在努力从其他疾病患者(如炎症性肠病和结节病)获得含有肉芽肿的组织。(Soule，30%的努力)。 在过去的一年里，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方面的潜力，包括调节细胞因子的产生。MicroRNAs由500多个小的非编码RNA组成，通过抑制RNA翻译来调节基因的表达。它们被认为可以调节超过30%的基因组的表达，并且特定的microRNA种类已经被证明在中性粒细胞中是活跃的。中性粒细胞和单核细胞中的microRNA种类还没有进行全面的调查。我们已经使用了几种商业上可用的RT-PCR试剂盒来检测中性粒细胞中的特定microRNA，然而，由于要进行重大的实验来进行变异实验，这项工作变得复杂起来。对此，我们一直在与落基山实验室合作，对从正常对照组和X连锁(gp91缺失)和常染色体隐性遗传(p47缺失)CGD患者的纯化中性粒细胞制剂中提取的总RNA进行全RNA测序。这一信息本身将是新的，但也将促进未来涉及微RNA微阵列和传统RT-PCR的研究的发展。我们还开始了初步工作，研究将microRNA前体和抑制剂导入中性粒细胞的可行性，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讨论研究microRNA在髓系干细胞(CD34+细胞)中作用的可能性。有大量文献证明了microRNA在细胞和生物体水平上的发育作用。很可能microRNA在指导髓系干细胞的发育和分化方面是活跃的，但这一点还没有得到广泛的研究。

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.