Immunohistochemical analysis of Foxp3 in tissue lesions of PKDL patients.

<p>Distribution of Foxp3 in dermal lesion tissue sections at pre treatment, post treatment stages and normal skin of healthy individuals. Panel A (10×), Panel B (40×) magnification.</p

Levels of cytokine nitric oxide and in PBMCs supernatants stimulated with TSLA or recIL-17.

<p>(a) IL-17 and IL-23 levels in PBMCs of PKDL pre (n = 8), post (n = 6) and control subjects (n = 6) stimulated with total soluble <i>Leishmania</i> antigen (TSLA). (b) Release of TNF-α (pg/ml) or (c) NO (ng/ml) from PBMCs of same set of subjects following incubation with TSLA (10 µg/ml) or recombinant IL-17 (50 ng/ml) for 72 h at 37°C. Cytokine levels were determined by ELISA and NO was quantified by Griess reagent method in culture supernatants. The concentrations shown are the values in the stimulated cultures minus the unstimulated controls. Individual values (pg/ml) are presented and the mean±SE are shown. **P<0.01, and ***P<0.001.</p

Ex vivo analysis of mRNA expression in different clinical manifestations of PKDL.

<p><i>Ex vivo</i> analysis of relative Treg markers and IL-10 mRNA levels in Nodular (N) (n = 12) and Macular/Papular (M/P) (n = 10) lesions in distinct forms of PKDL. ***P<0.001.</p

Correlation of mRNA expression of Treg markers and IL-10 with parasite load in PKDL.

<p>Relative mRNA levels of Treg markers and IL-10 with respect to parasite load within lesion tissues of PKDL patients (n = 15). Plot was created using logarithmic values for cycles over 18sRNA and parasite load. Correlation was calculated using Spearman/Pearson correlation test. Diagonal line represents linear regression.</p

mRNA expression of Th17 markers in PKDL.

<p>Relative mRNA levels of IL-17, IL-23 and RORγt was determined by QPCR in (a) tissues lesions at pretreatment (n = 19), post treatment (n = 12), or control tissues (n = 5) or (b) in paired samples (n = 12). The Y axis represents the number of cycles over endogenous control (18sRNA) required to detect the gene expression of interest. *P<0.05, **P<0.01, and ***P<0.001.</p

Validation of cDNA array results using real time PCR in tissue lesions of PKDL patients.

<p>Relative mRNA levels of IFN-γ, TNF-α, IL-6, IL-10, IL-12β, IL-17, MCP-1, CD40 and IRF-1 was determined by QPCR in tissues lesions of PKDL patients (n = 10) or control tissues (n = 7). The relative quantification of products was determined by the number of cycles over endogenous control (18sRNA) required to detect the gene expression of interest. **P<0.01, and ***P<0.001.</p

<i>Ex vivo</i> analysis of mRNA expression of Treg markers and IL-10 in PKDL.

<p>Relative mRNA levels of CD25, Foxp3, CTLA-4 and IL-10 in lesion tissues of PKDL patients determined by real time polymerase chain reaction at pretreatment (n = 25) or post treatment (n = 8), or control tissues (n = 5) (A) and in paired samples (n = 8) shown separately (B). **P<0.01, and ***P<0.001.</p

Plasma levels of IL-17 and IL-23 in PKDL.

<p>Plasma levels of IL-17 and IL-23 determined by ELISA (a) at pre (n = 25), post-treatment stage (n = 15), controls (n = 10) or (b) in paired samples (n = 12) (b). Individual values (pg/ml) are presented and the mean±SE are shown. **P<0.01, and ***P<0.001.</p

Genes showing altered expression in tissue lesions of PKDL (P) compared to human normal skin (HC).

<p>Genes showing altered expression in tissue lesions of PKDL (P) compared to human normal skin (HC).</p

Major characteristics of the study population.

<p>Abbreviations: M = Male, F = Female, PKDL = Post kala azar dermal leishmaniasis, KA = Kala azar.</p