Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

1)研究单核细胞和巨噬细胞宿主抵抗新型细菌致病菌贝氏杆菌感染的决定因素。 我们最近的出版物(Zarember等人，感染和免疫，2012)表明，慢性肉芽肿病(CGD)患者的中性粒细胞杀灭贝氏革兰氏菌是有缺陷的，这种革兰氏阴性病原菌对补体和阳离子抗菌肽具有显著的耐药性。在2013财年，Jessica Chu(博士后IRTA)完成了一项研究，证明CGD人类单核细胞和单核细胞来源的巨噬细胞无法正常杀死白念珠菌，这种微生物可以在CGD巨噬细胞中存活。本文还证明了经IFN-γ处理的单核细胞对微生物的杀灭作用与残留的中性粒细胞NADPH氧化酶活性呈正相关。这一发现对使用干扰素(Actimune)治疗CGD患者具有潜在的诊断意义。 2)赤霉菌脂多糖(LPS) 在研究颗粒菌与免疫细胞相互作用的过程中，我们发现该菌对人类先天免疫系统具有明显的低刺激作用，无论是对NADPH氧化酶的弱激活还是对细胞因子分泌的刺激都很弱。我们正在与佐治亚大学复合碳水化合物研究中心的Yossi Shiloach(NIDDK)和Russell Carlson合作，完成这种生物的非典型内毒素(LPS)的纯化和结构表征，并确定它是否具有抗炎抑制内毒素的作用。 3)G.bethedensis甲醇脱氢酶 为了发展我们对白纹伊蚊感染的血清学检测(见ZIA AI000155-36)，我们从该微生物中纯化了甲醇脱氢酶。利用离子交换和凝胶过滤，制备了高度浓缩的酶，并正在进行生化测试以鉴定底物专一性，鉴定抑制剂，S显示出比最初认为的更广泛的底物专一性。与Peter Steinbach(NIH分子建模中心)合作，我们已经对G.bethedensis MDH的结构进行了建模。

1) To study the determinants of monocyte and macrophage host defenses against infection by the novel bacterial pathogen, Granulibacter bethesdensis Our recent publication (Zarember et al., Infection and Immunity, 2012) demonstrated that killing of G. bethesdensis by neutrophils from patients with chronic granulomatous disease (CGD) was defective and that this Gram-negative pathogen was remarkably resistant to complement and to cationic antimicrobial peptides. During FY13, Jessica Chu (Postdoctoral IRTA) completed a study demonstrating that CGD human monocytes and monocyte-derived macrophages were unable to kill G. bethesdensis normally and that this organism could persist in CGD macrophages. This paper also demonstrated a positive correlation between microbial killing by IFNgamma-treated monocytes and residual neutrophil NADPH oxidase activity. This finding has potential diagnostic implications in the use of IFN (Actimune) to treat CGD patients. 2) G. bethesdensis Lipopolysaccharide (LPS) During our studies of the interaction of Granulibacter bethesdensis with immune cells, we found that this organism is remarkably hypostimulatory of the human innate immune system, both in terms of weak activation of the NADPH oxidase and poor stimulation of cytokine secretion. We are collaborating with Yossi Shiloach (NIDDK) and Russell Carlson of the University of Georgia Complex Carbohydrate Research Center to complete the purification and structural characterization of the atypical lipopolysaccharide (LPS) of this organism and determine whether it acts as an anti-inflammatory inhibitory LPS. 3) G.bethesdensis Methanol Dehydrogenase In order to develop our serological testing for G.bethesdensis infection (see ZIA AI000155-36), we purified Methanol Dehydrogenase from this organism. Using ion exchange and gel filtration, highly enriched enzyme was prepared and biochemical testing is underway to identify substrate specificity, identify inhibitors, and s indicate a much broader substrate specificity that originally thought. In collaboration with Peter Steinbach (NIH Center for Molecular Modeling), we have modeled the structure of G. bethesdensis MDH.