• Clinical proteomics profiling for biomarker identification among patients suffering with Indian Post Kala Azar Dermal Leishmaniasis

Post Kala Azar Dermal Leishmaniasis (PKDL) is a non-fatal dermal sequel of Visceral Leishmaniasis (VL), caused by protozoan parasite Leishmania donovani. On the global scenario, PKDL in mainly restricted to distinct zones of South Asia (India, Nepal and Bangladesh) and East Africa, mainly Sudan. In India, the most prominent fatal form of leishmaniasis present is VL, affecting entirely the eastern region of the country. The clinical presentation of VL and PKDL differ substantially; where VL patients suffer from sustained fever, hepatospleenomegaly, weight loss and anemia, PKDL patients manifest dermal lesions in the form of macular, nodular and polymorphic. Although, compared to VL, the mortality rate of PKDL is significantly lower, yet it is observed as a stigmatizing disease that brings a substantial socioeconomic burden, further intensified by a hesitancy of patients, to obtain treatment, or due to noncompliance. Lesions, especially the polymorphic forms, are parasite-rich, concluding that PKDL plays a crucial role in the anthroponotic transmission of VL. PKDL patients with Macular lesions, having comparatively lower parasitic load, and thus lacks detection sensitivity with standard techniques like rK39 (73% sensitivity), Leishmanin skin test (54% sensitivity) and Histopathological studies with patients’ skin biopsy sample (7-33% sensitivity) and is often misdiagnosed as Vitiligo cases. In order to completely eliminate Kala-azar in Southeast Asia, proper identification of various routes of the disease is essential, which could efficiently distinguish between cured PKDL and active PKDL as well as active PKDL from other cross disease like Leprosy and Vitiligo. Till now very less work has been done towards biomarker identification for PKDL from patients’ plasma. Most of the methods for disease identification are based on highly invasive tissue biopsy followed by PCR/qPCR. Recently investigations from our group have demonstrated the presence of novel glycoproteins in the circulating immune complexes (CICs) from serum, as biomarkers for early identification of PKDL patients. An Immune complex (IC), sometimes called an antigen-antibody complex, is a molecule formed from the integral binding of an antibody to a soluble antigen. After an antigen-antibody reaction, the immune complexes can be subject to any number of responses, including complement deposition, opsonisation, phagocytosis, or processing by proteases. Accumulation of ICs leads to a broad spectrum of pro-inflammatory effects, including activation of the complement cascade and induction of cytokine secretion. These complexes may also be deposited in tissues and vessel walls, leading to inflammation, tissue damage and, ultimately, disease manifestations. Immune complexes also affect disease progression and outcome in various disorders through the induction of pro-inflammatory or anti-inflammatory cytokines. In the present study we hypothesize that the CICs’ proteome is altered among PKDL patients with different dermal manifestations (Macular and Polymorphic) as compared to Cured individuals (CR) and Healthy controls (HI). The aim of this current study is to identify differentially expressed CIC proteomic profile among different forms PKDL patients as compared to HI. The differential protein expression of specific unique peptide fragments, representing specific proteins, could be used as diagnostic biomarkers in early detection of both Macular and Polymorphic PKDL cases. In this prospective study, we included PKDL patients with Macular and/or Polymorphic lesion, Cured individuals and HI. Among the enrolled patients, blood samples were collected from 20 primarily confirmed PKDL patients with Macular lesions, 20 primarily confirmed PKDL patients with Polymorphic lesions, 12 Cured individuals previously suffering from PKDL and 12 Healthy individuals; collected during the period of 2015-2016, stored at -80°C. Next the CICs were purified from PKDL patients’ plasma and LC MS/MS analysis was performed using Q-ExactivePlusOrbitrap mass spectrometer. This work for the first time revealed the differential CICs proteome profile among different forms of PKDL patients as compared to their Cured and Healthy counterparts.

黑热病后皮肤利什曼病（Post Kala Azar Dermal Leishmaniasis, PKDL）是内脏利什曼病（Visceral Leishmaniasis, VL）的非致死性皮肤后遗症，由杜氏利什曼原虫（Leishmania donovani）这一原生动物寄生虫引发。在全球范围内，PKDL主要局限于南亚（印度、尼泊尔与孟加拉国）及东非的特定区域，尤以苏丹为典型。在印度，致死性最强的利什曼病类型为内脏利什曼病，其流行范围覆盖该国东部全境。二者的临床表现差异显著：内脏利什曼病患者会出现持续性发热、肝脾肿大、体重减轻与贫血症状，而PKDL患者则表现为斑疹、结节及多形性皮肤损伤。尽管相较于内脏利什曼病，PKDL的死亡率显著更低，但该病仍属于带有污名化色彩的疾病，会带来沉重的社会经济负担；而患者的治疗犹豫或治疗依从性不佳，又进一步加重了这一负担。尤其是多形性皮损，其寄生虫载量较高，这表明PKDL在内脏利什曼病的人源传播过程中发挥着关键作用。伴有斑疹皮损的PKDL患者寄生虫载量相对较低，因此采用rK39检测（灵敏度73%）、利什曼素皮肤试验（灵敏度54%）以及皮肤活检样本的组织病理学研究（灵敏度7%~33%）等标准检测技术时，检出灵敏度不足，这类患者常被误诊为白癜风（Vitiligo）病例。为彻底消除东南亚地区的黑热病，明确该病的各类传播途径至关重要，这不仅可以精准区分治愈性PKDL与活动性PKDL，还能将活动性PKDL与麻风病（Leprosy）、白癜风等其他易混淆疾病区分开来。截至目前，针对PKDL患者血浆的生物标志物筛选研究仍较为匮乏，当前多数疾病诊断方法均依赖于创伤性极强的组织活检，后续结合聚合酶链式反应/定量聚合酶链式反应（PCR/qPCR）开展检测。近期本团队的研究证实，血清循环免疫复合物（circulating immune complexes, CICs）中存在新型糖蛋白，可作为PKDL患者的早期诊断生物标志物。免疫复合物（Immune complex, IC），有时又称抗原-抗体复合物，是由抗体与可溶性抗原紧密结合形成的分子复合物。抗原-抗体结合反应发生后，免疫复合物可引发多种免疫应答过程，包括补体沉积、调理作用、吞噬作用或蛋白酶加工处理。免疫复合物的蓄积可引发广泛的促炎效应，包括补体级联反应激活与细胞因子分泌诱导。此类复合物还可沉积于组织及血管壁，引发炎症反应、组织损伤，最终导致疾病相关症状出现。免疫复合物还可通过调控促炎或抗炎细胞因子的分泌，影响多种疾病的病程进展与转归。本研究提出如下假说：与治愈个体（Cured individuals, CR）及健康对照（Healthy controls, HI）相比，伴不同皮肤表现（斑疹型与多形性）的PKDL患者的CIC蛋白质组存在差异。本研究的目的在于，相较于健康对照，明确不同类型PKDL患者的CIC蛋白质组差异表达特征。代表特定蛋白质的特异性独特肽段的差异表达情况，可作为斑疹型与多形性PKDL的早期诊断生物标志物。本前瞻性研究纳入了伴斑疹型和/或多形性皮损的PKDL患者、治愈个体以及健康对照。本研究共采集20例经初步确诊的斑疹型PKDL患者、20例经初步确诊的多形性PKDL患者、12例既往罹患PKDL的治愈个体以及12名健康个体的血液样本，样本采集于2015-2016年间，保存于-80℃环境中。随后从PKDL患者的血浆中纯化得到CICs，并采用Q-Exactive Plus Orbitrap质谱仪进行液相色谱-串联质谱（LC-MS/MS）分析。本研究首次揭示了不同类型PKDL患者相较于其治愈个体与健康对照的CIC蛋白质组差异特征。