ABSTRACT
The role of pro-inammatory cytokine tumor necrosis factor-alpha (TNF-α) in human leishmaniasis is not fully understood. We analyzed the alterations in the plasma levels of TNF- α, soluble TNF receptor type 1 (sTNFR I), IL-17 and IL-22 in the volunteers with leishmaniasis. Blood samples were collected from patients with active cutaneous leishmaniasis (CL), the same CL patients after standard antimonial therapy as healed CL, active visceral leishmaniasis (VL) and healed VL volunteers. Levels of the cytokines were titrated on plasma samples by sandwich ELISA method. The mean level of TNF-α was significantly higher in active CL patients than healthy controls (P<0.001) and significantly reduced after treatment in the same volunteers (P<0.001). The mean level of sTNFR I was significantly higher in active CL patients than healthy controls (P<0.05). The mean level of IL-22 in plasma of the AVL patients was significantly higher than that of healthy control group (P<0.05). There is a negative correlation between the levels of TNF-α and sTNFR I and healing of CL. Measurement of cytokines in plasma samples is more feasible than cell culture in evaluation of immune response in human leishmaniasis.
ABSTRACT
Determination of the division histoty of T cells in vitro is helpful in the study of effector mechanisms against infections. Technique described here uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester [CFSE] to monitor the proliferation. In a cross sectional study, blood samples were collected from 7 volunteers with history of cutaneous leishmaniasis [CL] and one healthy control from endemic areas in Isfahan province who referred to the Center for Research and Training in Skin Diseases and Leprosy [CRTSDL], then CD4[+]/CD8[+] lymphocytes and CD14[+] monocytes were isolated from peripheral blood mononuclear cells [PBMC] using mAbs and magnetic nanoparticles. CFSE labeled CD4[+] or CD8[+] lymphocytes cultured with autologous monocytes in the presence of PHA, SLA, live Leishmania major or as control without stimulation. Cells were harvested after 7 days and were analyzed using flow cytometry. Five consecutive divisions were monitored separately. Stimulation of CD4[+] or CD8[+] lymphocytes from CL subjects with SLA showed a significant difference in proliferation comparing with unstimulated cells [P< 0.05]. The significant difference in the percentages of CD4[+] cells stimulated with SLA was revealed at different divisions for each subject. In CD8[+] lymphocyte, significant stronger stimulation of SLA was evident later in the proliferation process. The mean number of divisions in both CD4[+]/CD8[+] lymphocytes stimulated with SLA was significantly greater than when stimulated with live L. major [P=0.007 / P=0.012, respectively]. The percentage of divided cells might be calculated separately in each division. The cells remained active following CFSE staining and there is possibility of functional analysis simultaneously