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The course of human infection with Leishmania braziliensis is variable, ranging from self-healing infection to chronic disease. It is therefore a useful system in which to study immunoregulatory aspects of leishmaniasis, including the effects of parasite antigens on host responses. In the present study, we report on the cloning of, expression of, and comparative analyses of patient immune response to two different L. braziliensis genes homologous to the genes for the eukaryotic 83- and 70-kDa heat shock proteins. rLbhsp83 contains a potent T-cell epitope(s) which stimulated peripheral blood mononuclear cells (PBMC) from all L. braziliensis-infected individuals to proliferate and to produce interleukin-2 (IL-2) gamma interferon, and tumor necrosis factor alpha. The elicitation of IL-4 and IL-10 mRNAs was found to differ depending on the portion of the rLbhsp83 used to stimulate PBMC. rLbhsp83a, which represents the nearly full-length protein, stimulated IL-10 but not IL-4 mRNA. In contrast, a approximately 43-kDa protein representing the C-terminal region of Lbhsp83 stimulated the production of IL-4 but not IL-10 mRNA. rLbhsp70 stimulated PBMC proliferation from patients with mucosal disease but, unlike rLbhsp83, did not stimulate PBMC from self-healing individuals. PBMC from mucosal patients were not stimulated by rHuhsp70 to either proliferate or produce cytokines. This suggests that the hyperresponsiveness of mucosal patient PBMC to Leishmania heat shock proteins does not involve an auto-immune phenomenon resulting from cross-reactivity with self hsp70. In general, although the cytokine profile of patient PBMC in response to both of these Leishmania heat shock proteins represents a mixed Th1-Th2 pattern, the levels of gamma interferon and IL-2 were significantly higher than those of the Th2 cytokines IL-4 and IL-10. Patients with active mucosal and cutaneous disease but not self-healing individuals had significant anti-immunoglobulin G antibody titers to both rLbhsp83 and rLbhsp70 but not to the homologous rHuhsp70. It therefore appears that differential patient immune responses to Leishmania hsp83 and hsp70 may be of particular significance in the induction of protective immune responses as well as in the development of tissue damage in cases with particularly strong hypersensitive reactions.

A chronic debilitating parasitic infection, viscerotropic leishmaniasis (VTL), has been described in Operation Desert Storm veterans. Diagnosis of this disease, caused by Leishmania tropica, has been difficult due to low or absent specific immune responses in traditional assays. We report the cloning and characterization of two genomic fragments encoding portions of a single 210-kDa L. tropica protein useful for the diagnosis of VTL in U.S. military personnel. The recombinant proteins encoded by these fragments, recombinant (r) Lt-1 and rLt-2, contain a 33-amino acid repeat that reacts with sera from Desert Storm VTL patients and with sera from L. tropica-infected patients with cutaneous leishmaniasis. Antibody reactivities to rLt-1 indicated a bias toward IgG2 in VTL patient sera. Peripheral blood mononuclear cells from VTL patients produced interferon gamma, but not interleukin 4 or 10, in response to rLt-1. No cytokine production was observed in response to parasite lysate. The results indicate that specific leishmanial antigens may be used to detect immune responses in VTL patients with chronic infections.

IL-12 is a pluripotent cytokine that interacts with NK and T cells to play a central role in the initiation and maintenance of Th1 responses and IFN-gamma production. Because of the interactive relationship between IL-12 and IFN-gamma response to infectious organisms, a study was undertaken to examine the role of IL-12 in the immune regulation of human visceral leishmaniasis (VL). Human (Hu) VL is associated with immune dysfunction and the appearance of IL-10 mRNA, not present in healed individuals. We found that PBMC from treated VL patients produced both IL-12 p40 and IFN-gamma in response to in vitro stimulation with Leishmania donovani. The production of both IL-12 p40 and IFN-gamma were interdependent and were abrogated by the addition of exogenous Hu rIL-10. In contrast, PBMC from active VL patients did not produce IL-12 p40 or IFN-gamma in response to L. donovani lysate. Neutralizing anti-IL-10 mAb led to the enhancement of IFN-gamma production by active VL PBMC cultured with L. donovani lysate, and this enhanced IFN-gamma production was blocked by anti-IL-12 mAb. The addition of exogenous Hu rIL-12 to PBMC from active VL patients resulted in the augmentation of IFN-gamma in response to L. donovani lysate. Therefore, treatment of active VL patient PBMC with anti-IL-10 or IL-12 shifted the response toward a Th1-type response with the production of IFN-gamma. These results indicate that IL-12 may play an important role in the regulation of the cellular immune responses in Hu VL.

The purpose of this study was to determine the effect of the Na+K+ATPase inhibitor sanguinarine on the refractory period of the porcine ventricle. Measurements of ventricular refractoriness and strength-interval curves were obtained before and after 0.5 to 4.0 mg/kg sanguinarine. The ventricular refractory period was found to be prolonged at all doses studied from a control of 276 +/- 12 msec to 318 +/- 24 msec at 4.0 mg/kg (p < 0.01). The strength-interval curve was shifted to increased values for ventricular refractoriness from 0.5 to 16.0 ma. No depression of arterial pressure or left ventricular dp/dt was observed. This study shows that sanguinarine prolongs ventricular refractoriness. This property may be useful in the treatment of ventricular arrhythmias.