Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

在2020财年，我们继续研究了慢性肉芽肿病（CGD）患者中出现的病原体--贝塞登颗粒杆菌。 根据已发表的病例，CGD患者感染该微生物的病死率为30%。 然而，以前的研究表明，这种微生物的长期持续存在而没有临床上明显的疾病也可能发生在一些患者中。 为了更好地了解这种微生物的发病机制，我们从这10例报告病例中的9例中收集了分离株，并进行了全基因组测序以及各种旨在解剖这种微生物的基因型/表型特征的实验室研究。 在分离株之间可以看到显著的遗传多样性，包括高达11%的开放阅读框对于特定分离株是独特的，尽管16 S rDNA序列>99%相同。 基因组岛的详细分析正在进行中，有证据表明几个噬菌体整合事件可能影响表型。 我们分离出一种假定的荚膜多糖，似乎是致命的分离物所特有的，并通过MS和NMR分析这种材料，以确定其组成。 用这种多糖免疫家兔，并通过免疫印迹、免疫沉淀和其他正在进行的方法评价所得血清，目的是确定这种材料是否有助于产生它的菌株的毒力。 COVID-19疫情对该项目的影响重大，实验室活动减少。 尽管如此，通过与LCIM内的其他小组在过去几个月的合作，我们已经进行了研究的血清和血浆中的重要炎症介质的浓度超过175例记录感染的SARS-CoV 2和发现特定的生物标志物与临床结果的相关性。 这些结果正在准备中，以便在20财年结束前提交。 在2020财年，我们完成了一份手稿，该手稿描述了血浆凝溶胶蛋白（pGSN）的实验室研究，pGSN是一种丰富的血浆蛋白，通过隔离促炎物质（如肌动蛋白和血小板活化因子）来减少全身炎症。 我们测量了慢性肉芽肿病（CGD）患者的样本中的pGSN，并检测到与健康对照相比显著较低的浓度。 这一发现可能部分解释了这种疾病的过度炎症特征，并提出了一个问题，即提供凝溶胶蛋白是否有助于控制这些患者的炎症反应，特别是在感染期间。

During FY20, we continued our studies of Granulibacter bethesdensis, emerging pathogen in patients with chronic granulomatous disease (CGD). Based on published cases, infection of CGD patients with this organism has a case fatality rate of 30%. Previous studies have shown, however, that long-term persistence of this organism without clinically apparent disease may also occur in some patients. To better understand pathogenesis by this organism, we have collected isolates from 9 of these 10 reported cases and performed complete genome sequencing as well as a variety of laboratory studies aimed dissecting genotype/phenotype characteristics of this organism. Remarkable genetic diversity may be seen between isolates including up to 11% of open reading frames being unique to a specific isolate despite >99% identical 16S rDNA sequences. Detailed analysis of genomic islands is underway with evidence of several bacteriophage integration events that may impact phenotypes. We isolated a putative capsular polysaccharide that appears to be unique to lethal isolates and are analyzing this material by MS and NMR to identify its composition. Rabbits were immunized with this polysaccharide and resulting sera evaluated by immunoblotting, immunoprecipitation, and other ongoing approaches with an aim toward determining if this material contributes to the virulence of strains producing it. The impact of the COVID19 pandemic on this project has been significant in terms of decreased laboratory activities. Nevertheless, through collaborations with other groups within LCIM during the past several months, we have performed studies on the concentrations of important inflammatory mediators in serum and plasma from over 175 patients with documented infections by SARS-CoV2 and discovered correlations of particular biomarkers with clinical outcomes. These results are being prepared for submission before the end of FY20. During FY20 we completed a manuscript that is now in press that describes laboratory studies of plasma gelsolin (pGSN), an abundant plasma protein that decreases systemic inflammation by sequestering pro-inflammatory substances such as actin and platelet activating factor. We measured pGSN in samples from patients with Chronic Granulomatous Disease (CGD) and detected a significantly lower concentration compared to healthy controls. This finding may, in part, explain the excessive inflammation characterizing this disease and raising the question of whether or not providing gelsolin might help control inflammatory responses in these patients, particularly during infections.