Arginase activity in saliva, PBMCs and plasma.

<p>The activity of arginase was measured by enzymatic assay in (A) Saliva (VL patients: n = 16, VL/HIV patients: n = 13), (B) PBMCs (VL patients: n = 14, VL/HIV patients: n = 13) and (C) plasma (VL patients: n = 14, VL/HIV patients: n = 13). The straight line represents the median. NS = not significant.</p

Liver and kidney functions assays.

<p>GOT = glutamic-oxaloacetic transaminase, GPT = Glutamic-pyruvic transaminase, ALP = Alkaline phosphatase, BUN = blood urea nitrogen.</p

Phenotype of arginase expressing LDGs.

<p>PBMCs were isolated by density gradient from the blood of (A) one VL patient and (B) one VL/HIV patient and the phenotype of arginase-expressing cell was measured by flow cytometry. Data show the results of one representative experiment out of 8 independent experiments for the VL patients and 5 independent experiments for the VL/HIV patients.</p

BMI and upper arm circumference.

<p>(A) Measurements of BMI (VL patients: n = 25, VL/HIV patients: n = 14) and (B) circumference of the upper arm (VL patients: n = 17, VL/HIV patients: n = 13). The straight line represents the median. NS = not significant.</p

Parasite grade in spleen and bone marrow aspirates.

<p>The parasite grade was graded from 1+ to 6+ in smears from (A) spleen (VL patients: n = 16, VL/HIV patients: n = 11) and (B) bone marrow (VL patients: n = 10, VL/HIV patients: n = 3). The straight line represents the median.</p

Haematological data.

<p>Hct = hematocrit; Hb = haemoglobin.</p><p>Normal range: platelets (×10³) = 150–450; white blood cells (×10³) = 4.5–10.5; Hct (%) = 35–60; hb (g/dl) = 11–18.</p><p>The white blood cells were counted in total blood before Ficoll using a COULTER Ac•T diff Hematology Analyzer and are expressed as number of WBC (×10³) per µl of blood.</p

Clinical data.

<p>Spleen and liver size = measurement below left costal margin and right subcostal margin (respectively).</p

L-arginine levels and CD3ζ expression in CD4⁺ and CD8⁺ T cells.

<p>Plasma was isolated by centrifugation of the blood of controls (n = 11), VL patients (n = 11) and treated VL patients (n = 11) and (A) the levels of L-arginine were measured by HPLC. PBMCs were isolated by density gradient from the blood of controls (n = 10), VL patients (n = 10) and treated VL patients (n = 10) and the mean fluorescence intensity of CD3ζ was determined in CD4⁺ T cells (B) and CD8⁺ T cells (C). Box = interquartile range and median; whiskers = range. Statistical significance was determined by a two-tailed Mann-Whitney test. NS = not significant. Patients = VL patients before treatment; treated patients = VL patients after 3–4 weeks treatment.</p

Absolute numbers of LDGs, CD14⁺ cells and white blood cells.

<p>The white blood cell counts were determined after Ficoll using trypan blue exclusion and are expressed as cell number per ml of blood.</p

Phenotype and frequency of arginase-expressing cells in PBMCs.

<p>PBMCs were isolated by density gradient from the blood of controls (n = 14), VL patients (n = 17) and treated VL patients (n = 12). The phenotype of arginase-expressing cells was determined by flow cytometry. (A) Dot plot profile of CD15⁺ arginase⁺ cells, data show the results of one representative experiment out of 17 independent experiments; (B) dot plot profile of CD14⁺ arginase⁺ cells, data show the results of one representative experiment out of 17 independent experiments. (C) Percentages of CD15⁺arginase⁺ cells in the PBMCs of controls (n = 14), VL patients (n = 17) and treated VL patients (n = 12). Box  =  interquartile range and median; whiskers = range. Statistical significance was determined by a two-tailed Mann-Whitney test. NS = not significant. Patients = VL patients before treatment; treated patients = VL patients after 3–4 weeks treatment.</p