Dynamics of immunosuppression in hamsters with experimental visceral leishmaniasis

Immunosuppression has been reported to occur during active visceral leishmaniasis and some factors such as the cytokine profile may be involved in this process. In the mouse model of cutaneous leishmaniasis using Leishmania (Leishmania) major, the Th1 response is related to protection while the Th2 response is related to disease progression. However, in hamsters, which are considered to be an excellent model for the study of visceral leishmaniasis, this dichotomy is not observed. Using outbred 45- to 60-day-old (140 to 150 g) male hamsters infected intraperitoneally with 2 x 10(7) L. (L.) chagasi amastigotes, we evaluated the immune response of spleen cells and the production of cytokines. We used 3 to 7 hamsters per group evaluated. We detected a preserved response to concanavalin A measured by index of proliferation during all periods of infection studied, while a proliferative response to Leishmania antigen was detected only at 48 and 72 h post-infection. Messenger RNA from cytokines type 1 (IL-2, TNF-α, IFN-γ) and type 2 (IL-4, IL-10 and TGF-β) detected by reverse transcriptase polymerase chain reaction and produced by spleen cells showed no qualitative difference between control non-infected hamsters and infected hamsters during any period of infection evaluated. Cytokines were measured by the DNA band intensity on agarose gel using the Image Lab 1D L340 software with no differences observed. In conclusion, the present results showed an antigen-dependent immunosuppression in hamsters with active visceral leishmaniasis that was not related to the cytokine profile.

Leishmania (Leishmania) chagasi-infected mice as a model for the study of glomerular lesions in visceral leishmaniasis

Renal involvement in visceral leishmaniasis (VL) is very frequent but the pathogenesis of this nephropathy is poorly understood. In previous studies using dogs with VL we have detected new immunopathological elements in the glomeruli such as T cells and adhesion molecules. Although Leishmania (Leishmania) chagasi-infected dogs and hamsters are considered to be good models for VL, their use is limited for immunopathologic studies. The use of isogenic mouse strains susceptible to L. (L.) chagasi infection was an alternative but, on the other hand, the renal lesions of these animals have not yet been characterized. Thus, our purpose in the present study was to characterize mice infected with L. (L.) chagasi as a suitable model to study VL nephropathy. Kidney samples were obtained from control mice (N = 12) and from BALB/c mice (N = 24) injected intraperitoneally with 20 million L. (L.) chagasi amastigotes 7, 15, and 30 days after injection and processed for histopathological studies and detection of IgG deposits. Glomerular hypercellularity was clearly visible and, upon Mason's trichrome and periodic acid methenamine silver staining, a pattern suggestive of mesangial proliferative glomerulonephritis was observed in mice with VL. Time-dependent IgG deposits were also seen in infected mice. We consider L. (L.) chagasi-infected mice to be a suitable model for studies of the immunopathogenesis of glomerular lesions in VL.

Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

Immunity and immunosuppression in experimental visceral leishmaniasis

Leishmaniasis is a disease caused by protozoa of the genus Leishmania, and visceral leishmaniasis is a form in which the inner organs are affected. Since knowledge about immunity in experimental visceral leishmaniasis is poor, we present here a review on immunity and immunosuppression in experimental visceral leishmaniasis in mouse and hamster models. We show the complexity of the mechanisms involved and differences when compared with the cutaneous form of leishmaniasis. Resistance in visceral leishmaniasis involves both CD4+ and CD8+ T cells, and interleukin (IL)-2, interferon (IFN)-gamma, and IL-12, the latter in a mechanism independent of IFN-gamma and linked to transforming growth factor (TGF)-á production. Susceptibility involves IL-10 but not IL-4, and B cells. In immune animals, upon re-infection, the elements involved in resistance are different, i.e., CD8+ T cells and IL-2. Since one of the immunopathological consequences of active visceral leishmaniasis in humans is suppression of T-cell responses, many studies have been conducted using experimental models. Immunosuppression is mainly Leishmania antigen specific, and T cells, Th2 cells and adherent antigen-presenting cells have been shown to be involved. Interactions of the co-stimulatory molecule family B7-CTLA-4 leading to increased level of TGF-á as well as apoptosis of CD4+ T cells and inhibition of macrophage apoptosis by Leishmania infection are other components participating in immunosuppression. A better understanding of this complex immune response and the mechanisms of immunosuppression in experimental visceral leishmaniasis will contribute to the study of human disease and to vaccine development.

Detection of immunoglobulin G in the lung and liver of hamsters with visceral leishmaniasis

Several organs are affected in visceral leishmaniasis, not only those rich in mononuclear phagocytes. Hypergammaglobulinemia occurs during visceral leishmaniasis; anti-Leishmania antibodies are not primarily important for protection but might be involved in the pathogenesis of tissue lesions. The glomerulonephritis occurring in visceral leishmaniasis has been attributed to immune complex deposition but in other organs the mechanism has not been studied. In the current study we demonstrated the presence of IgG in the lung and liver of hamsters with visceral leishmaniasis. Hamsters were injected intraperitoneally with 2 x 10(7) amastigotes of Leishmania (Leishmania) chagasi and the presence of IgG in the liver and lung was evaluated at 7, 15, 30, 45, 80 and 102 days postinfection (PI) by immunohistochemistry. The parasite burden in the spleen and liver increased progressively during infection. We observed a deposit of IgG from day 7 PI that increased progressively until it reached highest intensity around 30 and 45 days PI, declining at later times. The IgG deposits outlined the sinusoids. In the lung a deposit of IgG was observed in the capillary walls that was moderate at day 7 PI, but the intensity increased remarkably at day 30 PI and declined at later times of infection. No significant C3 deposits were observed in the lung or in the liver. We conclude that IgG may participate in the pathogenesis of the inflammatory process of the lung and liver occurring in experimental visceral leishmaniasis and we discuss an alternative mechanism other than immune complex deposition

CD4+ T cells participate in the nephropathy of canine visceral leishmaniasis

Renal involvement in visceral leishmaniasis (VL) is very frequent. The renal lesions of humans and dogs are similar but their pathogenesis has not been clearly elucidated. There is growing evidence that the cellular immune response is involved in the pathogenesis of immunologically mediated glomerulonephritis. Since T cells could participate in the pathogenesis of nephropathy, in the present study we investigated the possible involvement of CD4+ and CD8+ T cells in the nephropathy of canine VL. Six dogs naturally infected with Leishmania (Leishmania) chagasi from the endemic area in the Northeast of Brazil, the town of Teresina in the State of Piauí, were studied. An expressive inflammatory infiltrate of CD4+ T cells both in glomeruli and in interstitium was present in 4 animals and absent in 2. CD8+ T cells were detected only in one animal. CD4+ T cells alone were observed in 3 animals; when CD8+ T cells were present CD4+ T cells were also present. CD4+ T cells were observed in cases of focal segmental glomerulosclerosis, diffuse membranoproliferative glomerulonephritis, diffuse mesangial proliferative glomerulonephritis and crescentic glomerulonephritis. CD8+ T cells were present only in a case of crescentic glomerulonephritis. Leishmania antigen was detected in glomeruli and in interstitial inflammatory infiltrate in 4 animals and immunoglobulins were observed in 4 dogs. In this study we observed that T cells, in addition to immunoglobulins, are present in the renal lesion of canine VL. Further studies are in progress addressing the immunopathogenic mechanisms involving the participation of immunoglobulins and T cells in canine VL nephropathy

The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection

Monoclonal antibodies against low density lipoprotein with various degrees of oxidative modifications

Oxidative processes leading to the generation of oxidized low density lipoprotein (oxLDL) particles have been suggested to be an important factor in the pathogenesis of atherosclerosis. After initiation of the oxidative process, LDL undergoes a progressive protein and lipid fragmentation. To understand this process and the role of oxLDL in various diseases of inflammatory origin, we have generated mouse monoclonal antibodies against copper-oxidized human LDL. Mice were immunized intrasplenically and after one intravenous boost the spleen cells were fused with the Sp2/0 hybridoma fusion partner. The hybridoma clones obtained after selection and cloning were analyzed for reactivity against oxLDL with various degrees of copper-mediated oxidative modifications. Three hybridoma clones were purified and further characterized. The following observations were made: 1) the intrasplenic route of immunization, avoiding the use of mycobacterial adjuvants, yielded a high frequency of positive clones; 2) the individual hybridomas reacted against LDL with various degrees of oxidative modifications; 3) the monoclonal antibodies could be used in ELISA and to detect oxLDL in immunohistochemical tissue staining, and 4) the monoclonal antibodies also detected oxLDL from hamsters and rabbits. We conclude that these monoclonal antibodies could be useful to further investigate the role of oxLDL in inflammation and in the immune response.