Table 1.
Description of the study population by age, clinical and haematological parameters (Mean ± SD).
Fig 1.
Standard curve for quantification of Leishmania parasites by real-time PCR.
The standard curve was obtained by amplification of Leishmania DNA from 105 to 1 parasites. Linear regression analysis rendered the equation y = -3.189x+24.07 (R2 = 0.965). Correlation of disease manifestations with parasite load.
Table 2.
Results of Leishmania DNA quantification in blood samples and clinical parameters of VL patients.
Fig 2.
Correlation of parasite load with different clinical and hematological parameters of VL patients (n = 20).
Duration of illness (months, panel A), increase in spleen size (cm, panel B), white blood cell counts (cells/μl, panel C) and albumin levels (gm/dl, panel D) of VL patients were obtained from the hospital. Parasite loads (Parasites/ml) were determined by real-time PCR. Correlation was calculated using the Spearman/Pearson correlation test. Diagonal lines represent linear regression.
Fig 3.
Leishmania-specific cell-mediated immune response in VL patients and healthy controls (ECs and NECs).
PBMCs were isolated from VL patients (n = 10), ECs (n = 5) and NECs (n = 10). Cells (2×105/well) were stimulated with 12.5 μg/ml of LAg for 4 days and cytokines from supernatants were measured by ELISA. IL-10 and TGFβ levels (pg/ml) were compared between LAg-stimulated and unstimulated samples (only cell, OC) from VL patients as well as LAg-stimulated samples from ECs and NECs. Data are represented as mean ± SE. P values were calculated using non-parametric Mann-Whitney U test and Unpaired t test; P<0.05 was considered significant.
Fig 4.
Correlation of different cytokines in culture supernatants of VL patients (n = 10) with parasite load.
The unstimulated and LAg-stimulated levels (pg/ml) of IL-10 (panels A and B) and TGFβ (panels C and D) in PBMCs of VL patients were measured by ELISA, and parasite loads (Parasites/ml) were determined by real-time PCR. Correlation was calculated using Spearman/Pearson correlation test. Diagonal lines represent linear regression.
Fig 5.
Correlation of different T cell subtypes and CD4CD25FoxP3 with parasite load in VL patients.
Absolute numbers of CD3 (panel A), CD4 (panel B), CD8 (panel C), CD4CD25 (panel D), and CD4CD25hi (panel E) were determined by flow cytometry in whole blood samples of VL patients (n = 20). Percentages of CD4CD25FoxP3-positive cells (panel F) were calculated by flow cytometry in PBMCs of VL patients (n = 10). Parasite loads (Parasites/ml) were determined by real-time PCR. Correlation was calculated using Spearman/Pearson correlation test. Diagonal lines represent linear regression.
Fig 6.
Identification of cellular sources of IL-10 in PBMCs of VL patients (n = 10), ECs (n = 5) and NECs (n = 5).
Total PBMCs were freshly cultured in the presence of PMA (50 ng/μl), ionomycin (1 μg/μl) for 2 hrs and for additional 1 hr in presence of brefeldin A (10 μg/μl) before staining. (A) Percentages of CD25+ and CD25− cells among CD4+IL-10+ cells. (B) Percentages of CD25+ and CD25− cells among CD4+FoxP3+IL-10+ cells. Data are represented as mean ± SE. P values were calculated using Wilcoxon matched pairs signed rank test for paired samples; P<0.05 was considered significant. (C), (i)-(iv) Data showing one representative healthy control. (D), (i)-(iv) Data showing one representative active VL patient.
Fig 7.
Identification of cellular sources of TGFβ in PBMCs of VL patients (n = 10), ECs (n = 5) and NECs (n = 5).
Total PBMCs were freshly cultured in the presence of PMA (50 ng/μl), ionomycin (1 μg/μl) for 2 hrs and for additional 1 hr in presence of brefeldin A (10μg/μl) before staining. (A) Percentages of CD25+ and CD25− cells among CD4+TGFβ+ cells. (B) Percentages of CD25+ and CD25− cells among CD4+FoxP3+TGFβ+ cells. Data are represented as mean ± SE. P values were calculated using Wilcoxon matched pairs signed rank test for paired samples; P<0.05 was considered significant. (C), (i)-(iv) Data showing one representative healthy control. (D), (i)-(iv) Data showing one representative active VL patient.