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，即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)，在大多数SCLS病例中存在。几名小细胞淋巴瘤患者中，MGUS进展为原始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。 我们正在通过检测单抗Ig在体外血管病理发展中的作用来探索SCLS的分子机制。使用来自SCLS患者的单抗Ig，我们将确定它是否与特定的细胞类型、靶抗原(如果有)结合，以及由此产生的对内皮细胞的影响(直接或间接)。使用2D凝胶电泳和质谱仪，我们已经鉴定出似乎与SCLS患者的免疫球蛋白结合的蛋白质，但不是健康对照。对潜在副蛋白靶标的验证性研究正在进行中。 我们还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了鉴定，以确定是否可以识别急性症状的特定生物标志物。在过去的5年里，我们已经在NIH临床中心评估了近40名使用该方案的患者。我们是全球主要的SCLS研究转介中心。 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。应用发作性SCLS血清，但在补充功能分析中，无论是纯化的免疫球蛋白部分还是从缓解期患者获得的血清，都不会引起血管内皮细胞钙粘附素内化、内皮细胞连接中断、肌动蛋白应激纤维形成和通透性增加。循环通透性因子血管内皮生长因子(VEGF)和血管生成素2(Ang2)在SCLS发作期血清中升高，而在缓解期血清中未见升高。这些结果表明，体液因子如血管内皮生长因子和血管紧张素2参与了小细胞淋巴瘤的一过性内皮收缩，提示了这一高度致命性疾病的分子机制。目前的研究目标是检测来自SCLS患者和对照组的内皮细胞对通透性介质的反应。 在2014财年，我们使用详细的机制研究对一名具有非典型疾病特征的受试者进行了表征。微血管屏障功能分析证实了我们的非典型患者对IVIG的临床抵抗，并提示他的发作是由急性期细胞因子IL-8和TNF-α驱动的。因此，我们的数据表明，SCLS可能有不同的临床表现形式，在SCLS患者组中，不同的细胞因子可能介导了毛细血管渗漏。 在2014财年，我们使用Affymetrix单核苷酸多态性(SNP)微阵列，对12名疾病受试者和18名对照进行了第一次全基因组范围的SCLS SNP分析。对其中9名患者的基因组DNA进行外显子组捕获测序，作为SNP芯片发现和从头数据生成的验证。我们确定了SCLS的候选易感基因座。鉴定了一种近交系小鼠(SJL/J)，其表型与SCLS相似，包括对多种通透性因子(包括组胺、5-羟色胺和缓激肽)的自发血管超敏反应，这些通透性因子是人类内皮高通透性的重要介质，以及以血清副蛋白存在为特征的B细胞肿瘤。在SJL小鼠中，一个控制全身血管对透明质酸超敏反应(SHS)的隐性基因被定位在小鼠6号染色体上与人类3p25.3同线的区间，该基因与人类的SCLS具有主要的遗传关联。这个区间包含几个候选基因，这些候选基因将在SCLS队列中进行分析。

Immune dysregulation may contribute to the pathophysiologic findings seen in SCLS. A monoclonal gammopathy of unknown significance (MGUS, which is 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 frank 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 exploring the molecular mechanisms of SCLS by examining the function of the monoclonal Ig in the development of vascular pathology in vitro. Using monoclonal Ig from SCLS patients, we will determine whether it binds to a specific cell type, target antigen (if any), and resulting effect (direct or indirect) on endothelial cells. Using 2D gel electrophoresis and mass spectrometry, we have identified proteins that appear to bind to IgG from subjects with SCLS, but not healthy controls. Confirmatory studies of potential paraprotein targets are underway. We are also 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 can be identified. We have now evaluated nearly 40 patients at the NIH clinical center under this protocol in the last 5 years. We are the primary worldwide referral center for research on SCLS. The transient episodes of hypotensive shock and anasarca in SCLS are thought to arise from reversible microvascular barrier dysfunction. Application of episodic SCLS sera, but neither the purified immunoglobulin fraction nor sera obtained from patients during remission, to normal human microvascular endothelial cells caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays. Circulating permeability factors, vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2), were elevated in episodic SCLS sera but not in remission sera. These results suggest that humoral factors such as VEGF and Ang2 contribute to transient endothelial contraction in SCLS, suggesting a molecular mechanism for this highly lethal disorder. Current studies are aimed at examined responses of endothelial cells derived from subjects with SCLS and controls to permeability mediators. In FY 2014, we characterized a subject with atypical disease features using detailed mechanistic studies. Microvascular barrier function assays corroborated our atypical patient's clinical resistance to IVIG and suggested that his episodes were driven by acute phase cytokines IL-8 and TNF-alpha. Thus, our data suggest that SCLS may have clinically varying forms of presentation and that within the group of patients with SCLS, different cytokines may mediate capillary leak. In FY 2014, 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. Exome capture sequencing was performed on genomic DNA from nine of these patients as validation for the SNP-chip discoveries and de novo data generation. We identified candidate susceptibility loci for SCLS. An inbred mouse strain was identified (SJL/J), whose phenotype bears similarity to SCLS, including spontaneous vascular hypersensitivity to a variety of permeability factors including histamine (HA), serotonin, and bradykinin, important mediators of endothelial hyperpermeability in humans, and B cell neoplasms characterized by the presence of serum paraproteins. A recessive locus in SJL mice controlling systemic vascular hypersensitivity to HA (Shs) was mapped to an interval on mouse chromosome 6 syntenic with human 3p25.3, which harbors the primary genetic association with SCLS in humans. This interval contains several gene candidates, which will be analyzed in the SCLS cohort.