Adult Opisthorchis viverrini Flukes in Humans, Takeo, Cambodia

(1). Sophisticated laboratory methods, although sensitive, are costly. The immunochromatographic strip test that uses recombinant K39 antigen (rK39), although satisfactory in India, is less sensitive in Africa, Latin America, and Mediterranean regions (2). Post–kala-azar dermal leishmaniasis (PKDL), a sequel to VL in India and Africa, is often confused with other skin diseases (3,4). Diagnosis of VL in dogs in Latin America and Mediterranean countries remains confusing because of rampant asymptomatic infections and elevated antibodies against Leishmania spp. (5)

Identification and glycobiological characterization of circulating immune complexes in patients with visceral leishmaniasis and post kala azar dermal leishmaniasis

321-328Here, we investigated the quantitative and qualitative differences in antibody classes and subclasses in serum immune complexes (ICs) of Visceral Leishmaniasis (VL), Post Kala-azar Dermal Leishmaniasis (PKDL) and different cross reactive diseases like Malaria, Leprosy, Vitiligo as compared to control subjects. IC levels were measured through a newly developed PEG ELISA, using L. donovani promastigote membrane antigen coated plate. Antibody classes and subclasses were identified using polyspecific sera and monoclonal antibodies, respectively. ICs were purified using polyethylene glycol (PEG) precipitation. Conditional logistic regression showed an association between IgG1-containing ICs and increased risk of PKDL (OR=75, P <0.05) and an association of IgG-containing ICs with VL (OR=621, P=0.001). PEG ELISA demonstrated almost 13-15 fold higher IgG containing ICs titers in VL as compared to control (P <0.001). The assay further established a significant (<i style="mso-bidi-font-style: normal">P <0.05) difference in the IgG containing ICs titers between VL and PKDL. The isolated ICs were further analyzed by subjecting them to one-dimensional PAGE and subsequently stained with combination of periodic acid schiff (PAS) with silver. A differential banding pattern between VL and PKDL was obtained. Four distinct bands with carbohydrate rich glycoconjugates were identified in PKDL ICs, which were absent in VL and control group. It suggests the scope for developing a novel differential diagnostic assay. </span

Status of circulating immune complexes, IL8 titers and cryoglobulins in patients with dengue infection

719-725Dengue, a serious viral infection caused by the mosquito vector, Aedes aegyptii, affects about 390 million people annually from more than 125 countries across the globe. However, until now, there is no reliable clinical or laboratory indicator to accurately predict the development of dengue severity. Here, we explored critical pathophysiological determinants like IL8, circulating immune complex (CIC) and cryoglobulin in dengue-infected patients for identification of novel dengue severity biomarker(s). Totally, 100 clinically suspected dengue cases were tested by NS1 ELISA and MAC ELISA for dengue virus aetiology. For control, 49 healthy volunteers were included. Blood profiling (complete hemogram and liver function test) of patient population were done using automated cell counter and standard auto analyzer based biochemical analysis. Serum CIC was quantified by PEG precipitation. Serum cryoglobulins were estimated by Folin assay. Levels of serum IL-8 were assessed by standard sandwich ELISA kits. Patient CIC were further characterized by SDS Gel electrophoresis. Forty per cent of the cases tested positive, of which 11 patients had severe clinical manifestation. The mean ±SEM of cryoglobulin concentration for DHF, DF, and HC were 1.30±0.31, 0.59±0.08 and 0.143±0.009 μg/μl, respectively. Thus, DHF and DF patients have shown 9- and 2.2-fold increase in cryoglobulin levels; and 18- and 5-fold increased CIC, respectively compared to HC patients. The mean ±SEM of CIC-PEG index for DHF, DF and HC were 491±41.22, 146±14.19 and 27.98±2.56, respectively. Raised levels of IL8 titers were also found in all 11 DHF patients. Peak levels of CIC, cryoglobulin and IL8 titers were associated with thrombocytopenia. SDS PAGE analysis of CIC from DHF revealed the presence of at least six protein bands that were not observed in samples from DF and HC. Prediction efficacy of IL8, CIC and cryoglobulin for DHF was determined using the receiver operator characteristic curve (ROC). The area under the curve was 1.00 for IL8, 0.99 for CIC and 0.74 for cryoglobulins. Overall, the results suggest that CIC, IL-8 and cryoglobulins may serve as important laboratory parameters to monitor dengue infection progression. </span

Systemic redox imbalance along with increased serum sialic acid is prevalent in patients with active vitiligo: A study from a tertiary care teaching hospital of eastern India

Background: Vitiligo is one of the common depigmenting disorders causing disfigurement and affecting the quality of life. Redox imbalance is known to play a contributory role in melanocyte destruction. Serum sialic acid (SA) is an important marker of the acute-phase response and is associated with oxidative protein damage. Aim: The aim of this study was to analyze the status of oxidative stress markers and serum SA in vitiligo patients and to correlate the same with disease activity. Materials and Methods: The different oxidative stress parameters namely superoxide dismutase (SOD), malondialdehyde (MDA), and serum SA were measured spectrophotometrically using standard biochemical methodologies in all the study subjects. Results: Serum SOD and MDA values were higher in patients with active vitiligo (n = 23) as compared to stable vitiligo (n = 20) and healthy controls (n = 20). The MDA/SOD ratio was higher in patients with active vitiligo (P<0.0001). Serum SA was increased in active vitiligo as compared to stable vitiligo and healthy controls (P<0.0001). Conclusion: This study indicates that patients with active vitiligo demonstrate enhanced MDA/SOD ratio and increased serum SA. The studied parameters can serve as an important tool to monitor disease activity in vitiligo

IL-10- and TGF-�-Mediated Susceptibility in Kala-azar and Post-kala-azar Dermal Leishmaniasis: The Significance of Amphotericin B in the Control of Leishmania donovani Infection in India1

Visceral leishmaniasis (VL) or kala-azar is known to be associated with a mixed Th1-Th2 response, and effective host defense requires the induction of IFN-� and IL-12. We address the role of the differential decline of IL-10 and TGF-� in response to sodium antimony gluconate (SAG) and amphotericin B (AmB), the therapeutic success of SAG and AmB in Indian VL, and the significance of IL-10 and TGF-� in the development and progression of post-kazla-azar dermal leishmaniasis (PKDL). In the active disease, PBMC from VL patients showed suppressed Ag-specific lymphoproliferation, IFN-� and IL-12 production, and elevation of IL-10 and TGF-�. Cure corresponded with an elevation in IFN-� and IL-12 production and down-regulation of IL-10 and TGF-�. Both CD4� and CD8� T cells were involved in IFN-� and IL-10 production. Interestingly, the retention and maintenance of residual IL-10 and TGF-� in some SAG-treated individuals and the elevation of IL-10 and TGF-� in PKDL, a sequel to kala-azar, probably reflects the role of these cytokines in reactivation of the disease in the form of PKDL. Contrastingly, AmB treatment of VL resulted in negligible TGF-� levels and absolute elimination of IL-10, reflecting the better therapeutic activity of AmB and its probable role in the recent decline in PKDL occurrences in India. Moreover, elucidation of immune responses in Indian PKDL patients revealed a spectral pattern of disease progression where disease severity could be correlated inversely with lymphoproliferation and directly with TGF-�, IL-10, and Ab production. In addition, the enhancement of CD4�CD25� T cells in active VL, their decline at cure, and reactivation in PKDL suggest their probable immunosuppressive role in these disease forms. The Journal of Immunology, 2007, 179: 5592–5603

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)-γ, interleukin (IL)-2, IL-4, and IL-10 being comparable to those in control subjects. Patients with VL had decreased IFN-γ 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

A defective oxidative burst and impaired antigen presentation are hallmarks of human visceral leishmaniasis

Purpose: Survival of the Leishmania parasite within monocytes hinges on its ability to effectively nullify their microbicidal effector mechanisms. Accordingly, this study aimed to delineate this biological niche in patients with visceral leishmaniasis (VL). Methods: In monocytes, the redox status, antigen presenting capacity, expression of Toll-like receptors (TLRs), co-stimulatory molecules (CD80/86) and generation of intracellular cytokines (IL-8, IL-1β, IL-10 and LAP-TGF-β1) was measured by flow cytometry, levels of circulating cytokines (IL-1β, IL-6, TNF-α, IL-8, IL-4, IL-13, IL-10 and GM-CSF) by ELISA and arginase activity by spectrophotometry. Results: Within monocytes, generation of an oxidative burst was markedly attenuated as evident by decreased generation of nitric oxide and reactive oxygen species, concomitant with raised levels of thiols. This was accompanied by lowered frequency of TLR₄⁺ monocytes, but the arginase activity remained unaltered. Pathogen persistence was enhanced by the predominance of anti-inflammatory cytokines within monocytes, notably IL-10. Alongside, development of adaptive immunity was severely attenuated as manifested by a pronounced impairment of antigen presentation and co-stimulation evident by down regulation of CD₅₄, HLA-DR and CD86. Treatment corrected the redox imbalance and reversed the impaired antigen presentation. Conclusions: In VL, monocyte functions were severely impaired facilitating parasite persistence; anti-leishmanial chemotherapy mediated parasite elimination through modulation of the macrophage microenvironment by restoring its redox status and antigen presenting capacity

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-γ 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-γ 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