Studies in the Pathogenesis of Systemic Capillary Leak Syndrome

免疫失调可能是导致SCLS病理生理改变的原因之一。多发性骨髓瘤(MM)的癌前病变(MGUS)是一种未知意义的单克隆性丙种球蛋白(MGUS)，在大多数SCLS病例中，克隆性浆细胞群分泌在患者血清中可检测到的单克隆性免疫球蛋白(Ig，也称为副蛋白)。几名小细胞淋巴瘤患者中，MGUS进展为骨髓瘤或浆细胞白血病的患者在化疗后因其造血功能障碍而出现较少的毛细血管渗漏事件。这些发现表明，来自失调浆细胞群体的单克隆性副蛋白可能是观察到的病理生理结果的直接或间接来源。 我们正在对发病前和发病后的血细胞RNA转录组和SCLS血清/血浆的蛋白质组进行鉴定，以确定是否可以识别急性症状和/或病因的特定生物标记物。在过去的9年里，我们已经评估了超过65名在该方案下确诊为SCLS的患者。我们是SCLS在美国的主要转介中心。与缓解期血清相比，SCLS发作期血清循环通透性因子血管内皮生长因子(VEGF)、血管生成素-2(Angpt-2)、CXCL10、CCL2和IL-6升高。因此，诱导内皮细胞高通透性的血管生成蛋白和促炎细胞因子可能是导致小细胞淋巴瘤斑块周围一过性内皮屏障功能障碍的原因。 在2018财年，我们利用Somalogics技术对SCLS患者的急性期和恢复期血清进行了全面的蛋白质组筛选(1300种蛋白质)，从而扩展了这些发现。这项研究揭示了急性SCLS发作期间肾上腺髓质素的瞬时偏转，肾上腺髓质素是一种主要来自单核细胞和内皮细胞(ECs)的激素。肾上腺髓质素促进血管平滑肌松弛和内皮屏障的完整性。SCLS患者单核细胞表达的肾上腺髓质素高于健康对照组。这项研究的另一个发现是中性粒细胞颗粒蛋白的存在，这表明中性粒细胞在SCLS急性发作期间经历了激活。 SCLS中的一过性低血压休克和无症状性发作被认为是可逆性微血管屏障功能障碍引起的。将间歇性但非恢复期的SCLS血清应用于人微血管内皮细胞，可导致血管内皮细胞钙粘附素内化，内皮细胞间连接中断，肌动蛋白应激纤维形成，并在补充功能分析中增加通透性。因此，在急性发作期间，内皮细胞的收缩和附着连接的暂时减弱可能会使溶质和蛋白质泄漏到血管外间隙。 在2018财年，我们扩展了这些初步发现，以证明SCLS患者的皮肤微血管和内皮细胞系对常规炎症介质如血管内皮生长因子和组胺具有高反应性。目前的研究旨在确定导致通透性的信号通路中的异常，这可能是这些异常的原因。 特定基因缺陷在SCLS中的作用尚不清楚；例如，内皮细胞是否具有对常规刺激的高反应性。在SCL中没有一致的家族聚集性。使用Affymetrix单核苷酸多态(SNP)微阵列，我们首次对12名疾病受试者和18名对照的SCLS进行了全基因组SNP分析。从单核苷酸多态的无偏高密度图谱(SNPs)中，一个小的遗传区间3p25.3被确定为排名最高的候选易感基因座(p10-6)，优势比为41。对于如此小的样本量，顶级SCLS相关变种的优势比(7-41)和p值(10-4和10-6)都过大了。这些结果表明，一种罕见疾病的等位基因有很高的外显率，尚待鉴定。我们正在对患者内皮细胞的脱氧核糖核酸进行全基因组测序，以测试这一假设，即由于潜在的遗传缺陷，他们容易对其他普通的炎性应激因素做出夸大的反应(S)。 在18财年，我们利用近交系小鼠建立了一种SCLS模型。SJL/J小鼠的循环副蛋白类似于人类SCLS中的MGUS，在全身给药组胺后，它们表现出血管渗漏导致死亡的倾向。该性状定位于小鼠6号染色体上与人类3p25.3共线的一个隐性基因座，该基因与人类的SCLS有主要的遗传关联。大约50%的SCLS患者有引发SCLS急性发作的既往感染(通常是病毒)。我们发现，在流感感染后，SJL小鼠而不是其他近交系小鼠表现出血管泄漏，主要是皮肤和骨骼肌。目前的研究目的是缩小导致“SCLS样”表型的遗传间隔，以便确定可以在功能互补研究中询问的特定候选基因。

Immune dysregulation may contribute to the pathophysiologic findings seen in SCLS. A monoclonal gammopathy of unknown significance (MGUS, a premalignant precursor to multiple myeloma (MM), in which a clonal plasma cell population secretes monoclonal immunoglobulin (Ig, also referred to as a paraprotein) detectable in patient sera, is present in a majority of SCLS cases. Several patients with SCLS in whom MGUS evolved into myeloma or plasma cell leukemia experienced fewer capillary leak episodes after chemotherapy for their hematopoietic disorder. These findings suggest that the monoclonal paraprotein from the dysregulated plasma cell population may be the direct or indirect source of the pathophysiologic findings observed. We are characterizing the transcriptome of blood cell RNA and the proteome of SCLS serum/plasma, both pre- and post-attack, to determine whether specific biomarkers of acute symptoms and/or etiological factors can be identified. We have now evaluated more than 65 patients with a confirmed diagnosis of SCLS under this protocol in the last 9 years. We are the primary referral center in the U.S. for SCLS. Circulating permeability factors, vascular endothelial growth factor (VEGF), angiopoietin 2 (Angpt-2), CXCL10, CCL2, and IL-6, were elevated in episodic SCLS sera compared to remission sera. Thus, angiogenic proteins and proinflammatory cytokines that induce EC hyper-permeability may contribute to transient EC barrier dysfunction around SCLS flares. In FY18, we extended these findings by performing a comprehensive proteomic screen (1300 proteins) of acute and convalescent sera from SCLS patients using Somalogics technology. This study revealed transient deflections of adrenomedullin, a hormone derived primarily from monocytes and endothelial cells (ECs) during acute SCLS attacks. Adrenomedullin promotes vascular smooth muscle relaxation and endothelial barrier integrity. Monocytes from SCLS patients expressed more adrenomedullin than those from healthy controls. An additional finding of this study was the presence of neutrophil granule proteins, suggesting that neutrophils undergo activation during acute flares of SCLS. The transient episodes of hypotensive shock and anasarca in SCLS are thought to arise from reversible microvascular barrier dysfunction. Application of episodic but not convalescent SCLS sera to human microvascular ECs caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays. EC contraction and temporary attenuation of adherens junctions may thus permit leakage of solutes and proteins into the extravascular space during acute episodes. In FY18, we extended these initial findings to demonstrate that the skin microvasculature and endothelial cell lines from SCLS patients are hyper-responsive to routine inflammatory mediators such as VEGF and histamine. Current studies are aimed at identifying abnormalities in the signaling pathways leading to permeability that could account for these abnormalities. The role of specific gene defects in SCLS, if any, is unknown; e.g. whether the endothelium is genetically programmed for hyper-responsiveness to routine stimuli. There are no consistent familial aggregations in SCLS. Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. From unbiased high-density mapping of single-nucleotide polymorphisms (SNPs), a small genetic interval, 3p25.3, was identified as the highest-ranking candidate susceptibility locus (p 10-6) with an odds ratio of 41. Odds ratios (7-41) and p values (10-4 and 10-6) for the top SCLS-associated variants were outsized for such a small sample size. These results imply high penetrance for a rare disease allele that remains to be identified. We are performing whole genome sequencing of DNA from patient ECs to test the hypothesis that they are prone to exaggerated responses to otherwise mundane inflammatory stressors due to underlying genetic defect(s). In FY18, we utilized an inbred mouse strain to develop a model of SCLS. SJL/J mice have circulating paraproteins similar to MGUS in human SCLS, and they exhibit a propensity for vascular leak-induced mortality after systemic administration of histamine. This trait mapped to a recessive locus on mouse chromosome 6 syntenic with human 3p25.3, which harbors the primary genetic association with SCLS in humans. Approximately 50% of patients with SCLS have antecedent infections (typically viral) that trigger acute SCLS attacks. We found that SJL mice but not other inbred strains exhibit vascular leak, primarily in skin and skeletal muscle, following influenza infection. Current studies are aimed at narrowing the genetic interval responsible for the "SCLS-like" phenotype in order to identify specific gene candidates that can be interrogated in functional complementation studies.