The cytokine profile produced by peripheral blood mononuclear cells (PBMC) in response to leishmania antigens and the ability of interleukin-10 (IL-10) and transforming growth factor ␤ (TGF-␤) to modulate the immune response were evaluated in 21 mucosal leishmaniasis patients. Patients with mucosal disease exhibited increased gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) secretion and decreased IL-10 secretion compared to patients with classical cutaneous leishmaniasis. CD4 ؉ Th1 cells were the main source of IFN-␥ and TNF-␣ production in mucosal leishmaniasis patients. Evaluation of cytokine gene expression in PBMC of these patients showed that there was strong up-regulation of IFN-␥ transcripts upon stimulation with leishmania antigen, in contrast to the baseline levels of IL-10 mRNA. IL-10 suppressed IFN-␥ production by 48% in cell cultures from cutaneous leishmaniasis patients and by 86% in cell cultures from healthy subjects stimulated with purified protein derivative, whereas in similar conditions IL-10 suppressed IFN-␥ production by 19% in cell cultures from mucosal leishmaniasis patients stimulated with leishmania antigen. TGF-␤ suppressed IFN-␥ levels to a greater extent in healthy subjects than in mucosal leishmaniasis and cutaneous leishmaniasis patients. These data indicate that a poorly modulated T-cell response in mucosal leishmaniasis patients leads to production of high levels of proinflammatory cytokines, such as IFN-␥ and TNF-␣, as well as a decreased ability of IL-10 and TGF-␤ to modulate this response. These abnormalities may be the basis for the pathological findings observed in this disease.

Human infection with Leishmania braziliensis can lead to cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). We hypothesize that the intense tissue destruction observed in ML is a consequence of an uncontrolled exacerbated inflammatory immune response, with cytotoxic activity. For the first time, this work identifies the cellular sources of inflammatory and antiinflammatory cytokines, the expression of effector molecules, and the expression of interleukin-10 (IL-10) receptor in ML and CL lesions by using confocal microscopy. ML lesions displayed a higher number of gamma interferon (IFN-␥)-producing cells than did CL lesions. In both ML and CL, CD4؉ cells represented the majority of IFN-␥-producing cells, followed by CD8 ؉ cells and CD4 ؊ CD8 ؊ cells. The numbers of tumor necrosis factor alpha-positive cells, as well as those of IL-10-producing cells, were similar in ML and CL lesions. The effector molecule granzyme A showed greater expression in ML than in CL lesions, while inducible nitric oxide synthase did not. Finally, the expression of IL-10 receptor was lower in ML than in CL lesions. Thus, our data identified distinct cytokine and cell population profiles for CL versus ML patients and provide a possible mechanism for the development of ML disease through the demonstration that low expression of IL-10 receptor is present in conjunction with a cytotoxic and inflammatory profile in ML.

The clinical spectrum of leishmaniasis and control of the infection are influenced by the parasite-host relationship. The role of cellular immune responses of the Th1 type in the protection against disease in experimental and human leishmaniasis is well established. In humans, production of IFN-γ is associated with the control of infection in children infected by Leishmania chagasi. In visceral leishmaniasis, an impairment in IFN-γ production and high IL-4 and IL-10 levels (Th2 cytokines) are observed in antigen-stimulated peripheral blood mononuclear cells (PBMC). Moreover, IL-12 restores IFN-γ production and enhances the cytotoxic response. IL-10 is the cytokine involved in down-regulation of IFN-γ production, since anti-IL-10 monoclonal antibody (mAb) restores in vitro IFN-γ production and lymphoproliferative responses, and IL-10 abrogates the effect of IL-12. In cutaneous and mucosal leishmaniasis, high levels of IFN-γ are found in L. amazonensis-stimulated PBMC. However, low or absent IFN-γ levels were observed in antigen-stimulated PBMC from 50% of subjects with less than 60 days of disease (24 ± 26 pg/ml). This response was restored by IL-12 (308 ± 342 pg/ml) and anti-IL-10 mAb (380 ± 245 pg/ml) (P<0.05). Later during the disease, high levels of IFN-γ and TNF-α are produced both in cutaneous and mucosal leishmaniasis. After treatment there is a decrease in TNF-α levels (366 ± 224 pg/ml before treatment vs 142 ± 107 pg/ml after treatment, P = 0.02). Although production of IFN-γ and TNF-α might be involved in the control of parasite multiplication in the early phases of Leishmania infection, these cytokines might also be involved in the tissue damage seen in tegumentary leishmaniasis.Correspondence

Abstract. Mucosal leishmaniasis is characterized by an intense inflammatory reaction and tissue damage with few parasites in the lesion. On the basis of previous observations that suggest a possible role of tumor necrosis factor alpha (TNF-␣) in the pathology of this disease, an open-label study was performed to evaluate the efficacy of the treatment with an inhibitor of TNF-␣ (pentoxifylline) associated to antimony therapy in 10 patients with refractory mucosal leishmaniasis. Patients were treated with pentavalent antimony (20 mg per kilogram of body weight per day) plus orally administered pentoxifylline 400 mg 3 times daily for 30 days. Nine of 10 patients fulfilled the criteria for cure: they experienced complete reepithelization of the mucosal tissue 90 days after therapy and displayed no evidence of relapse at 1 year of follow-up. The TNF-␣ levels before therapy (776 Ϯ 342 pg/mL) fell to 94 Ϯ 57 pg/mL (P Ͻ 0.05) within 60 days after therapy. Our results indicate that pentoxifylline plus antimony therapy should be considered in all patients with mucosal leishmaniasis that is refractory to treatment.

In Corte de Pedra (CP), northeastern Brazil, Leishmania braziliensis causes three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared one L. major, two non-CP L. braziliensis, one CP L. amazonensis, and 45 CP L. braziliensis isolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CP L. braziliensis isolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CP's geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting that L. braziliensis genotypes may be accompanied by specific disease manifestation after infection.

The addition of pentoxifylline to Sb(v) in mucosal leishmaniasis reduces the healing time significantly and prevents the need for further courses of Sb(v).

Leishmania braziliensis is a parasite that can induce at least two clinical forms of leishmaniasis in humans: cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). In humans, the specific mechanisms that determine which form will develop following infection are not well established. In this study, peripheral blood mononuclear cells from 17 CL and 9 ML patients were compared both ex vivo and after culture with soluble leishmania antigen (SLA). Patients with ML presented a higher frequency of activated T cells as measured by ex vivo frequen-þ than those with CL. Moreover, after stimulation with SLA, patients with ML presented a higher frequency of TNF-a-producing CD4 þ and CD14 þ cells than CL individuals. While CL patients displayed a positive correlation between the frequency of IL-10 and TNF-a-producing monocytes, the ML patients did not. This lack of a positive correlation between IL-10-producing and TNF-aproducing monocytes in ML patients could lead to a less controlled inflammatory response in vivo. These results corroborate with a model of an exacerbated, unregulated, immune response in ML patients and point to key immunomodulatory leucocyte populations and cytokine networks that may be involved in the development of immunopathology in ML patients.

Lei shmaniasis has been documented in several countries, with an estimated prevalence of 12 million people and