Studies in the Pathogenesis of Systemic Capillary Leak Syndrome

免疫功能失调可能导致SCLS的病理生理学改变。MGUS是多发性骨髓瘤（MM）的癌前病变，其特征在于分泌单克隆免疫球蛋白（IG，也称为副蛋白）的克隆浆细胞群。MGUS在大多数SCLS病例中可检测到。 几名MGUS演变为骨髓瘤或浆细胞白血病的SCLS患者在化疗后因造血功能障碍发生毛细血管渗漏的次数较少。 这些结果表明，从失调的浆细胞群的单克隆副蛋白可能是直接或间接的来源观察到的病理生理学结果。目前的研究主要集中在使用高通量蛋白质阵列鉴定SCLS单克隆IG的靶抗原。 我们还表征了血细胞RNA的转录组和SCLS血清/血浆的蛋白质组，包括发作前和发作后，以确定是否可以鉴定急性SCLS症状和/或病因的特定生物标志物。在过去的12年里，我们已经评估了超过75例确诊为SCLS的患者。我们是SCLS在美国的主要转诊中心。与缓解期血清相比，发作性SCLS血清中的循环渗透因子、血管内皮生长因子（VEGF）、血管生成素2（Angpt-2）、CXCL 10、CCL 2和IL-6升高。这些发现表明，血管生成蛋白和促炎细胞因子诱导内皮细胞（EC）的高渗透性可能有助于SCLS耀斑周围的短暂EC屏障功能障碍。 SCLS中短暂发作的水肿性休克和全身水肿被认为是由可逆性微血管屏障功能障碍引起的。在补充功能测定中，将间歇性但非恢复期SCLS血清应用于人微血管EC引起血管内皮钙粘蛋白内化、内皮间连接破坏、肌动蛋白应力纤维形成和通透性增加。因此，EC收缩和粘附连接的暂时衰减可能允许溶质和蛋白质在急性发作期间泄漏到血管外空间中。在21财年，我们继续研究SCLS患者的皮肤微血管和内皮细胞系对常规炎症介质（如VEGF和组胺）具有高反应性这一发现的潜在机制。初步数据表明，SCLS细胞有一个主要的代谢异常，使他们更容易受到这些介质。 SCLS中特定基因缺陷的作用（如果有的话）尚不清楚;例如，内皮细胞是否遗传性编程为对常规刺激的高反应性。在SCLS中没有一致的家族聚集性，并且对患有SCLS的无关成人和几个亲子三人组的全外显子组测序未能揭示统一的外来病因。使用Affyssin单核苷酸多态性（SNP）微阵列，我们在12名疾病受试者和18名对照者的队列中进行了SCLS的首次全基因组SNP分析。从单核苷酸多态性（SNPs）的无偏高密度作图，一个小的遗传间隔，3p25.3，被确定为最高级别的候选易感性位点（p10 -6）的优势比为41。 对于如此小的样本量，最高的SCL相关变异的比值比（7-41）和p值（10-4和10-6）过大。这些结果意味着一种罕见疾病等位基因的高等位率仍有待确定。目前，我们正在对患者的DNA进行全基因组测序，以测试以下假设：由于潜在的遗传缺陷，他们倾向于对其他普通炎症应激源产生过度反应。 在2023财年，我们报告了2例SCLS患者在自体干细胞移植治疗骨髓瘤后症状缓解或治疗需求减少，表明单克隆丙种球蛋白病在特发性全身毛细血管渗漏综合征发病机制中的作用。 在2023财年，我们发现了一名有肺炎球菌菌血症和SCLS病史的患者，该患者携带IRAK 4杂合子功能缺失变体，并伴有TLR信号传导缺陷。 最后，在2023财年，我们继续与PharmAbcine，Inc.合作。研究单克隆抗体4 E2，其靶向Angpt 1/2受体Tie 2。4 E2是一种新型激动性抗体，其结合Tie 2受体并促进小鼠肿瘤模型中渗漏血管的稳定。4 E2独立于Angpts激活SCLS患者来源的内皮细胞中的Tie 2，并在稳态下强化屏障功能，并抑制炎症应激源诱导的渗透性。4 E2降低了由全身组胺激发诱导的SCLS的SJL/J小鼠模型的死亡率，并减少了与呼吸道病毒感染相关的血管渗漏。这些发现支持Tie 2失调在ISCLS中的关键作用，并强调了这种灾难性疾病的可行治疗方法。

Immune dysregulation may contribute to the pathophysiologic findings seen in SCLS. A MGUS, a premalignant precursor to multiple myeloma (MM), is a condition characterized by a clonal plasma cell population that secretes monoclonal immunoglobulins (Ig, also referred to as a paraprotein). MGUS is detectable 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. Current studies are focused on identifying the target antigen of SCLS monoclonal Ig, if any, using high-throughput protein arrays. 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 SCLS symptoms and/or etiological factors can be identified. We have now evaluated more than 75 patients with a confirmed diagnosis of SCLS under this protocol in the last 12 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. These findings suggest that angiogenic proteins and proinflammatory cytokines that induce endothelial cell (EC) hyper-permeability may contribute to transient EC barrier dysfunction around SCLS flares. 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 fiscal year 21, we continued to study mechanisms underlying the discovery that the skin microvasculature and endothelial cell lines from SCLS patients are hyper-responsive to routine inflammatory mediators such as VEGF and histamine. Preliminary data suggest that SCLS cells have a primary metabolic abnormality that renders them more susceptible to these mediators. 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, and whole exome sequencing of unrelated adults with SCLS and several parent-child trios failed to reveal a uniform exotic etiology. 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. Currently, we are performing whole genome sequencing of DNA from patients to test the hypothesis that they are prone to exaggerated responses to otherwise mundane inflammatory stressors due to underlying genetic defect(s). In FY23, we reported that two patients with SCLS had either remission of symptoms or reduced treatment requirements following autologous stem cell transplantation for myeloma, suggesting a role for the monoclonal gammopathy in idiopathic systemic capillary leak syndrome pathogenesis. In FY23, we identified a patient with a history of pneumococcal bacteremia and SCLS, who carried a heterozygous loss of function variant in IRAK4 with associated defects in TLR signaling. Finally, in FY23 we continued a collaboration with PharmAbcine, Inc. to study a monoclonal antibody, 4E2, which targets the Angpt1/2 receptor, Tie2. 4E2 is a novel agonistic antibody that binds to Tie2 receptor and promotes stabilization of leaky blood vessels in tumor models in mice. 4E2 activated Tie2 in SCLS patient-derived endothelial cells independently of Angpts and fortified barrier function at homeostasis, and dampened permeability induced by inflammatory stressors. 4E2 reduced mortality in the SJL/J mouse model of SCLS induced by systemic histamine challenge and diminished vascular leakage associated with respiratory viral infection. These findings support a critical role for Tie2 dysregulation in ISCLS and highlight a viable therapeutic approach to this catastrophic disorder.