Inflammation and Therapy for Respiratory Virus Infection

正在进行的研究重点是探索急性呼吸道病毒感染的病理炎症反应，并利用这些信息来制定创造性的策略，以规避这种疾病的致命后遗症的特点。 我们报告两个已发表的主要研究和第三个合作工作： 我们的第一份报告介绍了我们正在进行的益生菌乳酸菌的免疫调节能力的评估。 我们先前表明，野生型小鼠通过鼻内接种活的或热灭活的乳杆菌属物种被完全（100%）保护免受致命的后遗症感染的致命病原体，小鼠肺炎病毒（PVM），一种反应，这是与减少表达的促炎细胞因子和减少病毒恢复。我们扩展了这些发现，证明了当PVM攻击延迟长达5个月时，40%的用活乳酸杆菌致敏的小鼠存活。这种对PVM感染的强大和持续的抵抗力是由先前与其他无关微生物的相互作用引起的，是异源免疫的一个深刻的例子;我们试图了解这种反应的性质和独特特征。我们发现，鼻内接种L。reuteri引起与促炎介质（CXCL 1、CCL 3、CCL 2、CXCL 10、TNF-α和IL-17 A）相关的快速、短暂的中性粒细胞募集，但不引起Th 1细胞因子;事实上，经典的Th 1细胞因子干扰素-γ对乳杆菌引发促进的存活没有贡献。利夫湖在肺组织中检测到的reuteri被迅速清除，并且在接种后24小时未检测到。与此相反，L.罗伊氏肽聚糖（PGN）和L.分别在接种后24和48 h检测罗伊氏菌基因组DNA（gDNA）。与活菌相比，鼻内接种分离的L。罗伊氏gDNA不引起嗜中性粒细胞募集，对病毒恢复和病毒相关的CCL 3产生的影响最小，并且不提供针对病毒感染的负面后遗症的保护。孤立的PGN引起中性粒细胞募集和促炎细胞因子，但并没有促进持续生存，在随后的PVM感染。总体而言，进一步评估导致乳酸杆菌介导的异源免疫的反应可能会提供新的抗病毒预防方式的见解。 在我们的第二份报告中，我们继续对康奈尔大学兽医学院的合作者在野外分离的新型肺炎病毒病原体进行评估。 原始的新分离株PnV-Ane 4在小鼠肺中复制并可在感染状态下从小鼠肺中重新分离，但与小鼠肺炎病毒（PVM）株J3666相比引起最小的死亡率。我们以前评估了10个新的PnV分离株的生殖和生理反应。与表征的PVM相比，所有PnV的G/糖蛋白序列包括延长的氨基末端，并表明分为A组和B组。虽然我们观察到BALB/c小鼠对存活剂量的反应中细胞因子产生和中性粒细胞向肺的募集存在显著差异，（50 TCID 50单位）代表性A组（114378-10-29-KY-F）和B组（7968-11- 0 K）PnV，我们没有观察到PnV/PnV之间的正选择（dN>dS）的证据，PVM/PnV或PVM/PVM G/糖蛋白或F/融合蛋白序列对。 这些新的变异，虽然满足科赫的假设，在小鼠中，仍然是不同的，并提供了有趣的途径，探索独特的病毒决定簇的反应。 最终报告是与纽约州立大学北部医科大学（锡拉丘兹，NY）Joseph Domachowske博士实验室合作的成果。 这是一项临床研究，重点是病毒诱导的炎症对呼吸道合胞病毒（RSV）疾病发病机制的贡献。该研究的目的是确定与幼儿更严重形式疾病相关的特定介质。 1998年9月至2008年5月因呼吸道症状在Golisano儿童医院急诊科就诊的5岁以下儿童有资格入组。从所有合格患者中采集鼻咽（NP）冲洗样本，并记录临床数据。如果NP清洗样品仅对RSV呈阳性，则将个体纳入本研究。入组研究的患者按疾病严重程度分层，定义为轻度（未住院）、中度（住院）或重度（需要入住ICU）。使用Luminex人30重测定法测定NP洗涤液中单个炎性生物标志物的浓度。851名患者符合研究标准，其中包括268名（31.5%）轻度，503名（59.1%）中度和80名（9.4%）重度疾病。正如预期，疾病严重程度与年轻、早产、心脏或肺部疾病、RSV A组感染以及白细胞介素（IL）-2R，IL-6，CXCL 8，肿瘤坏死因子（TNF）-，干扰素（IFN）-，CCL 3，CCL 4和CCL 2浓度升高直接相关。此外，该研究确定了严重RSV疾病的几种新的和机制上重要的炎症生物标志物，包括IL-1，IL-1-RA，IL-7，表皮生长因子（EGF）和肝细胞生长因子（HGF）。

Ongoing research focuses on the exploration of pathologic inflammatory responses to acute respiratory virus infection and the use of this information to develop creative strategies to circumvent these lethal sequelae characteristic of this disease. We report on two published primary studies and a third collaborative work: Our first report features our ongoing evaluation of the immunomodulatory capacity of probiotic Lactobacillus species. We showed previously that wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus species were fully (100%) protected against the lethal sequelae of infection with the virulent pathogen, pneumonia virus of mice (PVM), a response that is associated with diminished expression of proinflammatory cytokines and diminished virus recovery. We extended these findings with a demonstration that 40% of the mice primed with live Lactobacillus survived when PVM challenge was delayed for as long as 5 months. This robust and sustained resistance to PVM infection resulting from prior interaction with an otherwise unrelated microbe is a profound example of heterologous immunity; we seek to understand the nature and unique features of this response. We found that intranasal inoculation with L. reuteri elicited rapid, transient neutrophil recruitment in association with proinflammatory mediators (CXCL1, CCL3, CCL2, CXCL10, TNF-alpha and IL-17A) but not Th1 cytokines; indeed, the classic Th1 cytokine, interferon-gamma, does not contribute to survival promoted by Lactobacillus-priming. Live L. reuteri detected in lung tissue underwent rapid clearance, and was undetectable at 24h after inoculation. In contrast, L. reuteri peptidoglycan (PGN) and L. reuteri genomic DNA (gDNA) were detected at 24 and 48h after inoculation, respectively. In contrast to live bacteria, intranasal inoculation with isolated L. reuteri gDNA elicited no neutrophil recruitment, had minimal impact on virus recovery and virus-associated production of CCL3, and provided no protection against the negative sequelae of virus infection. Isolated PGN elicited neutrophil recruitment and proinflammatory cytokines but did not promote sustained survival in response to subsequent PVM infection. Overall, further evaluation of the responses leading to Lactobacillus-mediated heterologous immunity may provide insight into novel antiviral preventive modalities. In our second report, we continued with our ongoing evaluation of novel pneumovirus pathogens isolated in the wild by our collaborators at Cornell University College of Veterinary Medicine. The original novel isolate, PnV-Ane4, replicated in and could be re-isolated in infectious state from mouse lung but elicited minimal mortality compared to pneumonia virus of mice (PVM) strain J3666. We previously assessed phylogeny and physiologic responses to 10 new PnV isolates. The G/glycoprotein sequences of all PnVs include elongated amino-termini when compared to the characterized PVMs, and suggest division into groups A and B. While we observed significant differences in cytokine production and neutrophil recruitment to the lungs of BALB/c mice in response to survival doses (50 TCID50 units) of representative group A (114378-10-29-KY-F) and group B (7968-11-OK) PnVs, we observed no evidence for positive selection (dN>dS) among the PnV/PnV, PVM/PnV or PVM/PVM G/glycoprotein or F/fusion protein sequence pairs. These novel variants, while fulfilling Koch's postulates in mice, remain distinct, and provide interesting avenues for exploring responses to unique virus determinants. The final report is a contribution to a collaborative work with the laboratory of Dr. Joseph Domachowske, SUNY Upstate Medical University, Syracuse, NY. This is a clinical study that focuses on the contribution of virus-induced inflammation to the pathogenesis of respiratory syncytial virus (RSV) disease. The aim of the study was to identify specific mediators that are associated with more severe forms of illness in young children. Children less than 5 yrs of age who were seen in emergency department of Golisano Childrens' Hospital for respiratory symptoms from September 1998 to May 2008 were eligible for enrollment. Nasopharyngeal (NP) wash samples were collected from all eligible patients, and clinical data were recorded. Individuals were included in this study if NP wash samples were positive for RSV only. Patients enrolled in the study were stratified by disease severity, defined as mild (not hospitalized), moderate (hospitalized), or severe (requiring ICU stay). Concentrations of individual inflammatory biomarkers in NP wash fluids were determined using the Luminex human 30-plex assay. 851 patients met study criteria, which included 268 (31.5 percent) with mild, 503 (59.1 percent) with moderate, and 80 (9.4 percent) with severe illness. As expected, illness severity was directly associated with young age, prematurity, heart or lung disease, infection with RSV group A, and elevated concentrations of interleukin (IL)-2R, IL-6, CXCL8, tumor necrosis factor (TNF)-α, interferon (IFN)-α, CCL3, CCL4, and CCL2. In addition, the study identified several novel and mechanistically important inflammatory biomarkers of severe RSV disease, including IL-1β, IL1-RA, IL-7, epidermal growth factor (EGF), and hepatocyte growth factor (HGF).