Nasim Akhtar Ansari

Foxp3 and IL-10 expression correlates with parasite burden in lesional tissues of post kala azar dermal leishmaniasis (PKDL) patients.

Background Post kala-azar dermal leishmaniasis (PKDL), a sequel to visceral leishamaniasis (VL) in 5–15% cases, constitutes a parasite reservoir important in disease transmission. The precise immunological cause of PKDL outcome remains obscure. However, overlapping counter regulatory responses with elevated IFN-γ and IL-10 are reported. Methodology/Principal Findings Present study deals with ex-vivo mRNA and protein analysis of natural regulatory T cells (nTreg) markers (Foxp3, CD25 and CTLA-4) and IL-10 levels in lesion tissues of PKDL patients at pre and post treatment stages. In addition, correlation of nTreg markers and IL-10 with parasite load in tissue lesions was investigated. mRNA levels of nTreg markers and IL-10 were found significantly elevated in pre-treatment PKDL cases compared to controls (Foxp3, P = 0.0009; CD25 & CTLA-4, P<0.0001; IL-10, P<0.0001), and were restored after treatment. Analysis of nTreg cell markers and IL-10 in different clinical manifestations of disease revealed elevated levels in nodular lesions compared to macules/papules. Further, Foxp3, CD25 and IL-10 mRNA levels directly correlated with parasite load in lesions tissues. Conclusion/Significance Data demonstrated accumulation of nTreg cells in infected tissue and a correlation of both IL-10 and nTreg levels with parasite burden suggesting their role in disease severity in PKDL.

Interferon (IFN)–γ, Tumor Necrosis Factor–α, Interleukin-6, and IFN-γ Receptor 1 Are the Major Immunological Determinants Associated with Post–Kala Azar Dermal Leishmaniasis

Semiquantitative reverse-transcription polymerase chain reaction was used to analyze intralesional cytokine gene expression in 28 patients with post-kala azar dermal leishmaniasis (PKDL) and 14 patients with kala azar (KA). The data revealed mixed T helper cell type 1 (Th1) and T helper cell type 2 (Th2) responses, as reflected by elevated expression of interferon (IFN)-gamma , tumor necrosis factor (TNF)-alpha , transforming growth factor (TGF)-beta , interleukin (IL)-10, IL-6, and IL-4 mRNA, with minimal expression of IFN-gamma receptor 1 (IFN-gamma R1) mRNA in PKDL lesions, compared with that in normal skin tissue. In comparison with those in KA lesions, mRNA levels for IFN-gamma , TNF-alpha , and IL-6 were found to be significantly elevated in PKDL lesions, implying that these cytokines play an important role in PKDL pathogenesis. In the presence of elevated levels of IFN-gamma and TNF-alpha , interference with type 1 effector activity in PKDL may be due to minimal expression of the IFN-gamma R1 gene or the simultaneous presence of elevated levels of IL-10, IL-6, and TGF-beta , which have counteracting effects. After treatment, the restoration of IFN-gamma R1 at both mRNA and protein levels, coupled with down-regulation of counteracting cytokines, may facilitate the action of signals associated with IFN-gamma , yielding parasite clearance. Therefore, unfavorable clinical evolution in PKDL may not be due to the absence of an intralesional Th1 response but rather may be due to the presence of counteracting cytokines along with the down-modulation of IFN-gamma R1.

Evidence for involvement of Th17 type responses in post kala azar dermal leishmaniasis (PKDL).

Background Post kala-azar dermal leishmaniasis (PKDL), a dermal sequel of visceral leishmaniasis, caused by Leishmania donovani, constitutes an important reservoir for the parasite. Parallel functioning of counter acting immune responses (Th1/Th2) reflects a complex immunological scenario, suggesting the involvement of additional regulatory molecules in the disease pathogenesis. Methodology/Principal Findings In the present study, human cytokine/chemokine/receptor specific cDNA array technique was employed to identify modulations in gene expression of host immuno-determinants during PKDL, followed by evaluation of Th17 type responses by analyzing mRNA and protein expression of Th17 markers (IL-23, IL-17, RORγt) and performing functional assays using Leishmania antigen (TSLA) or recombinant (rec)IL-17. Array analysis identified key immuno-regulatory molecules including cytokines (TNF-α, IFN-γ, IL-10, IL-17), chemokines (MCP-1, MIP-1α), apoptotic molecules (FasL, TRAIL, IRF-1) and receptors (CD40, Fas). Up regulation in lesional expression of Th17 markers was observed during PKDL compared to control (IL-17 and IL-23, P = 0.0008; RORγt, P = 0.02). In follow-up samples, chemotherapy significantly down regulated expression of all markers. In addition, lesional expression of IL-17 was confirmed at protein level by Immuno-histochemistry. Further, systemic presence of Th17 responses (IL-17 and IL-23) was observed in plasma samples from PKDL patients. In functional assays, TSLA stimulated the secretion of IL-17 and IL-23 from PBMCs of PKDL patients, while recIL-17 enhanced the production of TNF-α as well as nitric oxide (NO) in PKDL compared to control (TNF-α, P = 0.0002; NO, P = 0.0013). Further, a positive correlation was evident between lesional mRNA expression of IL-17 and TNF-α during PKDL. Conclusion/Significance The results highlight key immune modulators in PKDL and provide evidence for the involvement of Th17 type responses in the disease pathogenesis.

Nested PCR Assay for Detection of Leishmania donovani in Slit Aspirates from Post-Kala-Azar Dermal Leishmaniasis Lesions

ABSTRACT A nested PCR assay to detect parasite DNA in slit aspirates from skin lesions of patients with post-kala-azar dermal lesihmaniasis (PKDL) is described. PCR results were positive in 27 of 29 (93%) samples by nested PCR assay, while only 20 of 29 (69%) were positive in a primary PCR assay. The nested PCR assay allowed reliable diagnosis of PKDL in a noninvasive manner.

Oral miltefosine in the treatment of post-kala-azar dermal leishmaniasis

mortality. Although severe staphylococcal infections may occur at any age, children are particularly vulnerable because of their immature immune system. Varicella lesions disrupt the skin barrier and allow bacteria entry into the deeper layers of the skin. It is also believed that VZV may alter host immunity, favouring secondary infections. Bullous varicella is a rare variant of the disease, usually seen in young children and S. aureus involvement in this condition has been reported. Our case was a 5-month-old girl who was admitted to hospital. She had been in direct contact with her sister, who had chickenpox. On admission, large bullae up to 40 mm in diameter were seen over the trunk, neck and face. Bullous disorders were considered. The next day (Fig. 1), typical polymorphic chickenpox lesions appeared and the child’s temperature was raised. Raised VZV IgM antibody titres confirmed current varicella infection. DNA of VZV was detected by PCR in bulla fluid. Skin culture revealed S. aureus infection, which was sensitive to most antibiotics excluding ampicillin. After combined treatment with intravenous cefuroxime and aciclovir, the varicella lesions and bullae resolved rapidly. Bullous onset of chickenpox as seen in our case is rare and represents a diagnostic problem. In our case, typical varicella vesicles were seen 24 h after the bullae had appeared; we did not see vesicles changing to bullae. A family history of chickenpox and simultaneous occurrence of both bullae and typical varicella lesions scattered throughout the skin on the second day assisted us to make the definitive diagnosis. In the literature, consensus has not been achieved on classification of this rather rare phenomenon. It is uncertain which of the two aetiological agents has the greater contribution to the formation of large blisters. Modern techniques including PCR have provided new insight into this problem, strongly supporting VZV involvement in the development of bullae, and this has also been shown in shingles (zoster), another disease of VZV infection. Bullous herpes zoster was reported in a child diagnosed with acute lymphoblastic leukaemia, when the vesicles enlarged rapidly, forming large bullae filled with fluid positive for VZV DNA. Individually, chickenpox in an unvaccinated population and S. aureus infections are widely seen in children, but bullous varicella is rather rare. Although the exact mechanisms by which large bullae are formed are not clear, some synergistic effect between the infectious agents must occur.

Evidence for involvement of TNFR1 and TIMPs in pathogenesis of post-kala-azar dermal leishmaniasis

Semi‐quantitative RT–PCR was exploited to analyse the intralesional cytokine gene expression in 14 post‐kala‐azar dermal leishmaniasis (PKDL) and 10 kala‐azar (KA) patients. The data provided evidence for both inflammatory and non‐inflammatory responses, as reflected by elevated tumour necrosis factor (TNF)‐α and interleukin (IL)‐10 in PKDL lesions compared with normal skin tissue (n = 6). The ratio of TNF‐α : IL‐10 message was 2·66 in PKDL cases, substantially higher than in KA (1·18). Investigation of TNF‐α receptors (TNFR1 and TNFR2) revealed a significant down‐regulation of TNFR1 transcript in both PKDL and KA compared with control. In the presence of elevated levels of TNF‐α transcript, interference with type 1 effector activity in PKDL may be due to minimal expression of the TNFR1 gene. Investigation of matrix metalloproteinases, known to be induced by TNF‐α, and the tissue inhibitors of matrix metalloproteinases (TIMPs), provided evidence for the roles of TIMP‐1 and TIMP‐3 in the pathogenesis of PKDL.

Cutaneous leishmaniasis in Nepal : Leishmania major as a cause

Nepal is endemic for visceral leishmaniasis; however, recently a solitary report of cutaneous leishmaniasis (CL) was recorded. The present report describes a CL case in a Nepalese woman, initially referred as a case of basal cell carcinoma. Histopathological examination, culture and PCR analysis of lesions revealed it to be a case of CL. Molecular methods, including kDNA PCR, ITS-1 PCR-RFLP and sequence analysis, revealed the species as Leishmania major. The patient was cured with sodium stibogluconate. This report suggests that the extent to which L. major causes CL in the Indian subcontinent needs to be monitored.

Elevated levels of IgG3 and IgG4 subclass in paediatric cases of kala azar

Visceral leishmaniasis (VL) or Kala azar (KA) is a systemic disease caused by the parasites of the Leishmania donovani complex. Control measures rely on treatment with antileishmanial agents, however, fraught with problems such as toxicity or drug resistance. The incidence rate is on the rise for children, for reasons yet undefined. Previously we have shown significantly elevated level of IL‐10 in children compared to adult KA cases. Here, antileishmanial antibody and C‐reactive protein (CRP) levels were investigated in paediatrics and adult patients of KA and post‐KA dermal leishmaniasis (PKDL). Level of IgG4 was significantly elevated in PKDL compared to KA, although total IgG and IgG1 were significantly lower. The antileishmania antibody levels of subclass IgG3 and IgG4 were found significantly elevated in paediatrics, however, levels of IgG, IgG1, IgG2 and CRP were comparable in paediatric and adult KA cases. In case of PKDL, levels of IgG and it subclass were similar in the two groups. No significant difference in antileishmanial antibody level was noticed between macular or polymorphic cases of PKDL. The differential antibody intensity in paediatric cases, together with significant level of circulating IL‐10, could be considered as a marker of differential disease susceptibility.

Immune response following miltefosine therapy in a patient with post-kala-azar dermal leishmaniasis

We report a case of post-kala-azar dermal leishmaniasis in which real-time PCR was exploited to measure changes in cytokine transcripts in lesion tissue before and after oral miltefosine treatment. Unlike antimonial therapy elevated levels of IFNgamma transcripts were noted, whereas TNFalpha, IL-10 and transforming growth factor-beta declined similar to that observed after therapy with antimonials. A significant increase in IFNgamma and CD40 levels seen after miltefosine therapy could enhance parasite clearance. The patient remained normal after 18 months of follow-up.