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病原体贝塞登颗粒杆菌感染的决定因素 我们已经发表了一篇论文（Zarember et al.，2012），证明了G. Bethesdensis的正常和非CGD中性粒细胞，以及记录了高水平的抵抗血清补体和纯化的抗菌cathelicidin肽。 G. bethesdense表明该微生物在传播到其他部位之前可能在中性粒细胞中增殖。 我们目前正在探索正常受试者和CGD患者的人单核细胞和单核细胞衍生的巨噬细胞在杀死这种生物体中的作用，并预计在本财年结束前提交这项工作供出版。 2)G.白屈菜脂多糖（LPS） 在我们研究贝氏颗粒杆菌与免疫细胞的相互作用期间，我们发现该生物体对人类先天免疫系统具有显著的低刺激性，无论是在NADPH氧化酶的弱激活方面还是在细胞因子分泌的不良刺激方面。我们正在与格鲁吉亚大学复杂碳水化合物研究中心的Yossi Shiloach（NIDDK）和Russel Carlson合作，以完成这种生物体的非典型脂多糖（LPS）的纯化和结构表征，并确定它是否作为抗炎抑制性LPS。 3)贝塞登木甲醇脱氢酶 为了开发我们对贝塞登革菌感染的血清学检测（参见ZIA AI 000155 -36），我们从该微生物中纯化了甲醇脱氢酶。 使用离子交换和凝胶过滤，制备了高度富集的酶，并且正在进行生物化学测试以鉴定底物特异性，鉴定抑制剂，并且表明了最初认为的更广泛的底物特异性。在与Peter施泰因巴赫（NIH分子模型中心）的合作下，我们对G.白菊

1) To study the determinants of monocyte and macrophage host defenses against infection by the novel bacterial pathogen, Granulibacter bethesdensis We have published a paper (Zarember et al., 2012) that demonstrated killing of G. bethesdensis by normal and not CGD neutrophils as well as documenting a high level of resistance to serum complement and purified antimicrobial cathelicidin peptide. Normal, and especially CGD neutrophil apoptosis was delayed by G. bethesdensis suggesting that the organism might proliferate in neutrophils before dissemination to other sites. We are currently exploring the roles of human monocytes and monocyte-derived macrophages from normal subjects and CGD patients in killing this organism and anticipate submission of this work for publication before the end of the FY. 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 Russel 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.