Nilay Kanti Das

Increased Levels of Interleukin-10 and IgG3 Are Hallmarks of Indian Post-Kala-Azar Dermal Leishmaniasis

BACKGROUND Post-kala-azar dermal leishmaniasis (PKDL), an established sequela of visceral leishmaniasis (VL), is proposed to facilitate anthroponotic transmission of VL, especially during interepidemic periods. Immunopathological mechanisms responsible for Indian PKDL are still poorly defined. METHODS Our study attempted to characterize the immune profiles of patients with PKDL or VL relative to that of healthy control subjects by immunophenotyping, intracellular cytokine staining of peripheral blood mononuclear cells, and enzyme-linked immunosorbent assay for serum cytokines and immunoglobulin G (IgG) subclasses. RESULTS Patients with PKDL had significantly raised percentages of peripheral CD3+CD8+ cells compared with control subjects, a difference that persisted after cure. Patients with PKDL showed an intact response to phytohemagglutinin, with the percentages of lymphocytes expressing interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10 being comparable to those in control subjects. Patients with VL had decreased IFN-gamma and IL-2 expression, which was restored after cure, and increased IL-10 expression, which persisted after cure. In their response to Leishmania donovani antigen, patients with PKDL showed a 9.6-fold increase in the percentage of IL-10-expressing CD3+CD8+ lymphocytes compared with control subjects, and this percentage decreased with treatment. Patients with PKDL had raised levels of IgG3 and IgG1 (surrogate markers for IL-10), concomitant with increased serum levels of IL-10. CONCLUSIONS IL-10-producing CD3+CD8+ lymphocytes are important protagonists in the immunopathogenesis of Indian PKDL.

Post-kala-azar dermal leishmaniasis - an overview.

Post‐kala‐azar dermal leishmaniasis (PKDL) is a dermal sequela of visceral leishmaniasis (VL), reported mainly from two regions – Sudan in eastern Africa and the Indian subcontinent, with incidences of 50–60% and 5–10%, respectively. Importantly, patients with PKDL are considered as reservoirs of VL, linking its eradication to effective control of PKDL. The etiopathogenesis of PKDL is presumably due to an immunological assault on latent dermal parasites. Immunological markers include IL‐10, whose expression in skin and plasma of Sudanese patients with VL predicted onset of PKDL. Cell‐mediated immune responses, notably restoration of IFN‐γ production by antigen‐stimulated lymphocytes are well documented in Sudanese PKDL, but remain ambiguous in the Indian form; recently, antigen‐specific IL‐10‐producing CD8+ lymphocytes have been implicated in pathogenesis. In Indian PKDL, upregulation of intralesional IFN‐γ and TNF‐α is counterbalanced by IL‐10 and TGF‐β together with downregulated IFN‐γ R1. Although IL‐10 curtails excessive IFN‐γ‐mediated reactivity and ensures parasite survival, its cellular source remains to be confirmed, with infiltrating regulatory T cells (Tregs) being a likely candidate. Future functional investigations on Tregs and their interaction with lesional effector lymphocytes would be indispensable for development of immunomodulatory therapies against Leishmania infection.

Pathogenesis, clinical features and pathology of chronic arsenicosis

Arsenicosis is a multisystem disorder, with virtually no system spared from its vicious claw; though its predominant manifestations are linked to cutaneous involvement. Cutaneous effects take the form of pigmentary changes, hyperkeratosis, and skin cancers (Bowens disease, squamous cell carcinoma, and basal cell epithelioma). Peripheral vascular disease (blackfoot disease), hypertension, ischemic heart disease, noncirrhotic portal hypertension, hepatomegaly, peripheral neuropathy, respiratory and renal involvement, bad obstetrical outcome, hematological disturbances, and diabetes mellitus are among the other clinical features linked to arsenic toxicity. The effects are mediated principally by the trivalent form of arsenic (arsenite), which by its ability to bind with sulfhydryl groups present in various essential compounds leads to inactivation and derangement of body function. Though the toxicities are mostly linked to the trivalent state, arsenic is consumed mainly in its pentavalent form (arsenate), and reduction of arsenate to arsenite is mediated through glutathione. Body attempts to detoxify the agent via repeated oxidative methylation and reduction reaction, leading to the generation of methylated metabolites, which are excreted in the urine. Understandably the detoxification/bio-inactivation process is not a complete defense against the vicious metalloid, and it can cause chromosomal aberration, impairment of DNA repair process, alteration in the activity of tumor suppressor gene, etc., leading to genotoxicity and carcinogenicity. Arsenic causes apoptosis via free radical generation, and the cutaneous toxicity is linked to its effect on various cytokines (e.g., IL-8, TGF-beta, TNF-alpha, GM-CSF), growth factors, and transcription factors. Increased expression of cytokeratins, keratin-16 (marker for hyperproliferation) and keratin-8 and -18 (marker for less differentiated epithelial cells), can be related to the histopathological findings of hyperkeratosis and dysplastic cells in the arsenicosis skin lesion.

Arsenicosis: diagnosis and treatment.

Diagnosis of arsenicosis relies on both clinical and laboratory criteria, but principally it can be diagnosed on the basis of its cutaneous manifestations. Cutaneous manifestations (melanosis, keratosis, and cutaneous cancers) are essential clues in the diagnosis, and trained dermatologists or arsenic experts are able to clinically confirm a case even without laboratory backup. Although systemic manifestations are not considered as diagnostic hallmarks, yet their presence serves as important telltale signs in arriving at the diagnosis. In countries where laboratory facilities are available, measuring the level of arsenic in drinking water (consumed in the last 6 months), urine, hair, and nails is of immense value. Newer biomarkers of arsenic exposure are being explored to provide early information about arsenic intoxication, of which urinary porphyrin level, blood metallothionein have shown promising results. Controlling the problem of arsenicosis depends on various factors, of which the most important is cessation of intake of arsenic-contaminated water. Deep wells, traditional dug wells, treatment of surface water, rainwater harvesting, and removing arsenic from the contaminated water by arsenic removal plant or arsenic treatment unit are the available options for providing arsenic-free drinking water. The role of nutrition and antioxidants in preventing the onset of symptoms of arsenicosis is also of importance. Nonspecific therapies (e.g., keratolytics for hyperkeratosis) cannot also be ignored and serve as palliative measures. The persons affected need to be followed up at regular intervals to detect the onset of cancers (if any) at the earliest. Role of counseling and education should never be underestimated since absence of public awareness can undermine all efforts of mitigation measures.

Enhanced Lesional Foxp3 Expression and Peripheral Anergic Lymphocytes Indicate a Role for Regulatory T Cells in Indian Post-Kala-Azar Dermal Leishmaniasis

Indian post-kala-azar dermal leishmaniasis (PKDL) is a low-frequency (5-10%) dermal sequela of visceral leishmaniasis (VL) caused by Leishmania donovani; importantly, affected individuals are speculated to be parasite reservoirs. Insight into its immunopathogenesis could translate into rational immunomodulatory therapeutic approaches against leishmaniases. In patients with PKDL (n=21), peripheral lymphocytes were analyzed for surface markers, intracellular cytokines, and lymphoproliferative responses using flow cytometry. In lesional tissue biopsies (n=12), expression of counter-regulatory cytokines (IFN-gamma and IL-10) and the T-regulatory transcription factor forkhead box protein 3 (Foxp3) was analyzed using reverse transcriptase-PCR, along with immunohistochemical detection (n=8) of CD3 and Foxp3 positivity. In patients with PKDL, circulating CD8(+)CD28(-) and antigen-induced IL-10(+)CD3(+) lymphocytes were increased and receded with treatment. CD8(+) lymphocytes showed impaired proliferative responses to L. donovani antigen (LDA) and phytohemagglutinin, which were reinstated after treatment. At presentation, the upregulated lesional IFN-gamma and IL-10 messenger RNA (mRNA), Foxp3 mRNA, and protein were curtailed after treatment. In Indian patients with PKDL, increased frequency of the CD8(+)CD28(-) phenotype, enhanced antigen-specific IL-10 production, and accompanying anergy of circulating lymphocytes suggest their regulatory nature. Furthermore, the concomitantly elevated lesional expression of Foxp3 suggests their possible recruitment into the lesional site, which would sustain disease pathology.

Miltefosine Effectively Modulates the Cytokine Milieu in Indian Post Kala-Azar Dermal Leishmaniasis

BACKGROUND The increasing incidence of unresponsiveness to antimonials in leishmaniasis prompted the use of newer drugs such as miltefosine. Miltefosine influences macrophage effector functions, but its effect on patients with post kala-azar dermal leishmaniasis (PKDL) has not been evaluated. METHODOLOGY The immunomodulatory activity of miltefosine was evaluated in patients with PKDL by studying the expression of activation markers (CD14 and CD16) and costimulatory molecules (CD80 and CD86) on circulating monocytes, levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 6, interleukin 1β, and interleukin 8) and anti-inflammatory cytokines (interleukin 10, transforming growth factor β, interleukin 4, and interleukin 13) in serum and peripheral blood mononuclear cell culture supernatants, and serum nitrite and arginase activity. RESULTS Miltefosine on circulating monocytes upregulated expression of CD16 and CD86 and reduced that of CD14. Miltefosine also induced a significant increase in circulating levels of pro-inflammatory cytokines with a concomitant decrease in anti-inflammatory cytokines. Its macrophage activating potential was evidenced by its ability to decrease serum arginase activity and increase serum nitrite. CONCLUSIONS Miltefosine increased the proportion of monocytes that have a pro-inflammatory phenotype, which was accompanied by an enhanced secretion of pro-inflammatory cytokines and increased levels of serum nitrite. The decrease in anti-inflammatory cytokine levels and serum arginase activity collectively indicated that miltefosine triggered a robust T-helper 1 response that facilitated parasite elimination.

M2 Polarization of Monocytes-Macrophages Is a Hallmark of Indian Post Kala-Azar Dermal Leishmaniasis

Abstract The high level of functional diversity and plasticity in monocytes/macrophages has been defined within in vitro systems as M1 (classically activated), M2 (alternatively activated) and deactivated macrophages, of which the latter two subtypes are associated with suppression of cell mediated immunity, that confers susceptibility to intracellular infection. Although the Leishmania parasite modulates macrophage functions to ensure its survival, what remains an unanswered yet pertinent question is whether these macrophages are deactivated or alternatively activated. This study aimed to characterize the functional plasticity and polarization of monocytes/macrophages and delineate their importance in the immunopathogenesis of Post kala-azar dermal leishmaniasis (PKDL), a chronic dermatosis of human leishmaniasis. Monocytes from PKDL patients showed a decreased expression of TLR-2/4, along with an attenuated generation of reactive oxidative/nitrosative species. At disease presentation, an increased mRNA expression of classical M2 markers CD206, ARG1 and PPARG in monocytes and lesional macrophages indicated M2 polarization of macrophages which was corroborated by increased expression of CD206 and arginase-1. Furthermore, altered vitamin D signaling was a key feature in PKDL, as disease presentation was associated with raised plasma levels of monohydroxylated vitamin D3 and vitamin D3- associated genes, features of M2 polarization. Taken together, in PKDL, monocyte/macrophage subsets appear to be alternatively activated, a phenotype that might sustain disease chronicity. Importantly, repolarization of these monocytes to M1 by antileishmanial drugs suggests that switching from M2 to M1 phenotype might represent a therapeutic opportunity, worthy of future pharmacological consideration.

Diagnosing leprosy: revisiting the role of the slit-skin smear with critical analysis of the applicability of polymerase chain reaction in diagnosis

Background  Diagnosing leprosy is challenging, especially in early‐stage cases, and the need for a sensitive diagnostic tool is urgent. Polymerase chain reaction (PCR) holds promise as a simple and sensitive diagnostic tool, but its usefulness in the Indian context requires further evaluation. Slit‐skin smear (SSS) remains the conventional method of leprosy detection. Hence, this study was undertaken to evaluate and compare the diagnostic efficacy of PCR versus that of SSS.

Cutaneous adverse drug reaction profile in a tertiary care out patient setting in Eastern India

Background: Cutaneous adverse drug reactions (CADR) are the most frequent of all manifestations of drug sensitivity and manifest with varied and diverse morphology. Aims: To study the prevalence and clinical spectrum of CADR among patients attending outpatient department (OPD) in a tertiary care hospital. Materials and Methods: An observational study was undertaken over a 1-year period in dermatology OPD of a tertiary care teaching hospital in Eastern India. Patients presenting with suspected drug-related cutaneous lesions were included if drug identity could be ascertained. Clinical profiling was done. Drug history was recorded in a format specified in Indian National Pharmacovigilance Programme and causality assessment carried out as per World Health Organization-Uppsala Monitoring Centre (WHO-UMC) criteria. Results: Commonest CADR in our study was morbilliform eruption (30.18%), followed by fixed drug eruption (24.52%), Stevens–Johnson syndrome (SJS)-Toxic epidermal necrolysis (TEN) and overlap of two (24.50%), exfoliative dermatitis (7.54%), urticaria (5.6%), phototoxic drug reaction (3.8%), pityriasis rosea-like eruptions (1.89%), and severe mucositis (1.80%). Drugs implicated were sulfonamides (17%), fixed-dose combinations of fluoroquinolones with nitroimidazoles (11.30%), analgesics (11.30%), antiepileptics (11.30%), beta-lactam antibiotics (9.40%), fluoroquinolones alone (7.50%), allopurinol (7.50%), and azithromycin (5.70%). Reaction latency varied from 1 to 43 days. Causality assessment was certain and probable for 18.9% and 41.5% of the reactions, respectively, and reactions were serious in 33.96% (95% confidence interval 21.21-46.71%). Conclusions: Cutaneous adverse drug reaction profile in this study is similar in many ways to studies conducted earlier in India. Incidence of life-threatening reactions like SJS-TEN was higher compared with studies conducted abroad. Reaction time and lesion patterns are helpful in identifying an offending drug in the setting of multiple drug therapy.

Evaluation of serological markers to monitor the disease status of Indian post kala-azar dermal leishmaniasis

Post kala-azar dermal leishmaniasis (PKDL), a dermal sequel of visceral leishmaniasis presents with macular or polymorphic lesions. As immunological variations between these two forms have not been delineated, we evaluated levels of antileishmanial total Ig, IgG and its subclasses, IgM, IgE, IgG avidity, cytokines IL-10, IL-4, IL-13 and expression of CD19. The levels of Ig and IgG in polymorphic PKDL were higher than macular PKDL, while significant curtailment in levels of Ig, IgM and IgG following treatment was evident only in polymorphic PKDL. With regard to IgG subclasses, IgG1 and IgG3 were significantly raised in polymorphic PKDL, whereas in macular PKDL only IgG1 was elevated; treatment decreased levels of IgG1, IgG2 and IgG3 only in polymorphic PKDL; IgE levels were raised in both groups but no marked alterations occurred following treatment. The avidity of IgG was higher in polymorphic PKDL and correlated with duration of disease. IL-10 was higher in polymorphic PKDL and decreased significantly after treatment, whereas in macular PKDL IL-4 predominated. Taken together, in PKDL the humoral immune response was greater in the polymorphic variant than the macular form suggesting that serological markers may have a role in monitoring polymorphic PKDL.