Endogenous peroxidase activity was quenched by immersion of the sections in methanol containing 2% H2O2 for 30 min, and non-specific binding was blocked by immersion learn more of the sections in Tris buffered saline (TBS)
containing 2% BSA. Single antigen staining was carried out with antibodies against myeloperoxidase (MPO; DakoCytomation) and IL-8 (Invitrogen), at dilutions of 1 : 600, and with antibody against inducible nitric oxide synthase (iNOS) (R&D systems, Minneapolis, MN), at a dilution of 1 : 300, in TBS for 45 min. All steps of the procedure were preceded by washes with TBS containing 0·05% Tween-20. After colour development with permanent red chromogen, the sections were counterstained with haematoxylin, dehydrated and mounted. Negative controls comprised omission of the primary antibody and its replacement with TBS. The differences between
experimental groups were analyzed using the Student’s paired and unpaired t-tests. All data are presented as mean ± SE and a difference in mean values was considered significant when the P-value was < 0·05. Correlations between continuous variables were evaluated using Spearman’s correlation test. To avoid the potential dependency between variables related to multiple lesions from the same individual, only one lesion (randomly selected) per patient was included in the statistical analysis. Intralesional expression of messenger RNA (mRNA) for IFN-γ, tumour necrosis Navitoclax molecular weight factor-α (TNF-α), IL-1β, IL-8, IL-10 and IL-4 was analyzed by reverse transcription–polymerase chain reaction in patients with CL (n = 31) and in healthy controls (n = 6). Transcripts of IFN-γ, TNF-α, IL-1β, IL-8 and IL-10 were
expressed in lesions of all the CL patients, while IL-4 was detected in 77·4% (24/31) of biopsies. The levels of expression of all cytokines were significantly Alectinib ic50 elevated in CL lesions, compared with those in control tissues (P < 0·001 for all cytokines) (Fig. 1). IL-1β was expressed at a very high level compared with other cytokines in all the samples. A strong correlation was found in the expression of IFN-γ with IL-8 (r > 0·7) and IL-10 (r > 0·8), and between TNF-α and IL-8 (r > 0·8). The strongest correlation was observed in the expression of IL-10 with TNF-α and IL-8 and between IFN-γ with TNF-α (r > 0·9, Table 1). Paired samples were collected from nine patients at post-treatment stage for comparative analysis of cytokine mRNA levels. A significant decrease in the levels of expression of mRNA for IFN-γ, TNF-α, IL-1β, IL-8, IL-10 and IL-4 was noticed after treatment (P < 0·05 for levels of all cytokines) (Fig. 2a). IL-8 is a chemoattractant and recruits the accumulation of PMNs at inflammatory sites,17 whereas MCP-1, also a chemoattractant, contributes not only to the recruitment of macrophage into Leishmania-infected skin but also to macrophage activation via the production of NO.