Interleukin-12 augments a Th1-type immune response manifested as lymphocyte proliferation and interferon gamma production in Leishmania infantum-infected dogs

The dog is the major reservoir for human visceral leishmaniasis caused by Leishmania infantum. Interleukin-12 is considered to have an essential role in the development of both innate and adaptive immunity to Leishmania spp. and other intracellular pathogens. This study focused on the influence of IL-12 in experimental and natural canine visceral leishmaniasis. Responses of peripheral blood mononuclear cells to IL-12, interleukin-10 and Leishmania soluble antigen were evaluated in L. infantum experimentally infected oligosymptomatic beagles, uninfected beagles, naturally infected polysymptomatic dogs, and their matched uninfected controls. Leishmania soluble antigen induced strong peripheral blood mononuclear cells proliferation both in experimentally infected dogs (median stimulation index [SI]=15.01), and in naturally infected dogs (SI=8.86), but not by cells from the control groups. IL-12 addition further enhanced cell proliferation in naturally (SI=14.95), but not in experimentally infected animals. Peripheral blood mononuclear cells from experimentally infected dogs were able to produce significant amounts of IFN-gamma (3.39 ng/ml) upon LSA stimulation, but no such production was detected in cells from naturally infected or control animals. Interestingly, addition of IL-12 reversed the inhibitory effect of LSA on IFN-gamma production by cells from polysymptomatic naturally infected dogs and the uninfected beagles (4.84 and 7.45 ng/ml, respectively), and further increased IFN-gamma production by peripheral blood mononuclear cells from experimentally infected oligosymptomatic dogs (29.28 ng/ml). IFN-gamma mRNA expression correlated well with IFN-gamma production. Addition of IL-10 to Leishmania soluble antigen stimulated peripheral blood mononuclear cells inhibited proliferation and IFN-gamma production in experimentally infected dogs. Thus, the ability of IL-12 to augment IFN-gamma production by peripheral blood mononuclear cells from dogs with experimental or natural symptomatic canine visceral leishmaniasis makes it a good candidate for cytokine therapy in dogs that are refractory to current therapy.     

T cell response of asymptomatic Leishmania chagasi infected subjects to recombinant leishmania antigens.

In areas of Leishmania chagasi transmission the ability to control leishmania infection is associated with IFN-gamma production. In visceral leishmaniasis down-regulation of T cell responses is mediated by interleukin-10 (IL-10). In this study we evaluated the lymphoproliferative response, IFN-gamma and IL-10 production on lymphocyte cultures stimulated with recombinant leishmania antigens in subjects with asymptomatic L. chagasi infection. There was a statistically significant difference in the lymphoproliferative response of the subjects with asymptomatic infection as compared to patients with visceral leishmaniasis and healthy subjects with respect to crude antigens (p<0.01), gp-63 (p<0.05) and hsp-70 (p<0. 01), as well as between asymptomatic L. chagasi infected subjects and patients with visceral leishmaniasis with respect to the response to all antigens tested. The IFN-gamma production observed in the group with asymptomatic infection with all the three recombinant antigens tested was higher (p<0.01) than that observed in patients with visceral leishmaniasis and in healthy subjects. Furthermore, in individuals with asymptomatic infection, IL-10 levels in cultures stimulated with recombinant antigens were very low. This study shows that lymphocytes from individuals with asymptomatic L. chagasi infection are able to recognize recombinant leishmania antigens with production of a cytokine that is associated with leishmania killing.     

Schistosoma mansoni antigens alter the cytokine response in vitro during cutaneous leishmaniasis

Schistosoma mansoni infection or associated products are able to down-modulate the type 1 CD4+ T cell inflammatory response characteristic of autoimmune diseases. In this study, we evaluated how S. mansoni antigens altered the immune response that was induced by the soluble Leishmania antigen (SLA) from cutaneous leishmaniasis (CL) patients. Cytokines were measured from the supernatants of peripheral blood mononuclear cell cultures stimulated with SLA. This was performed using the sandwich enzyme linked immunosorbent assay technique in the presence or absence of S. mansoni recombinant antigens Sm29, SmTSP-2 and PIII. The addition of S. mansoni antigens to the cultures resulted in the reduction of interferon gamma (IFN-γ) levels in 37-50% of patients. Although to a lesser extent, the antigens were also able to decrease the production of tumour necrosis factor-alpha (TNF-α). We compared patients that either had or did not have reduction in IFN-γ and TNF-α production in cultures stimulated with SLA in the presence of S. mansoni antigens. We found that there was no significant difference in the levels of interleukin (IL)-10 and IL-5 in response to S. mansoni antigens between the groups. The antigens used in this study down-modulated the in vitro proinflammatory response induced by SLA in a group of CL patients through a currently undefined mechanism.     

Role of costimulatory molecules in immune response of patients with cutaneous leishmaniasis

T cell-mediated immunity is critical in resistance against Leishmania parasites, and T cell activation requires signals provided by costimulatory molecules. Herein we evaluated the role of costimulatory molecules on cytokine production and T cell surface molecule expression by peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis (CL) patients. PBMC from CL patients were stimulated with soluble Leishmania antigen (SLA, 10 microg/ml), in the presence or absence of soluble CTLA4-Ig to block CD28-B7 interaction or in the presence or absence of anti-human CD40L to block CD40-CD40L interaction. Supernatants were harvested to evaluate tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), transforming growth factor beta (TGF-beta) and interferon gamma (IFN-gamma) production by ELISA. Cells were harvested after 48 h of culture, stained for specific activation markers and analyzed by flow cytometry. Results show that the blockade of CD28-B7 interaction by CTLA4-Ig downmodulated IFN-gamma, IL-10, and TNF-alpha secretion by PBMC from CL patients. No alteration was detected on either TGF-beta production or the expression of CTLA44 or CD25 on CD4+ and CD8+ T cells. When the CD40-CD40L interaction was blockade using anti-CD40L, we did not observe changes in cytokine production or in surface molecule expression. The blockade of the CD28-B7 interactions by CTLA4-Ig also did not alter cytokine production in volunteers immunized against tetanus toxoid (TT). Taken together, these data suggest that the interaction of CTLA4 and CD28-B7 is a TGF-beta-independent mechanism that specifically downmodulates the immune response in cutaneous leishmaniasis patients.     

Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis

Mucosal leishmaniasis is arguably the most morbid sequelae of cutaneous leishmaniasis. The importance of early diagnosis for effective therapy, coupled with the difficulty of diagnosing the disease parasitologically, prompted this investigation of humoral immune markers of mucosal disease. Promastigote soluble antigens of Leishmania braziliensis, isolated from cutaneous and mucosal lesions, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis; antigens were identified by immunoblotting with parasite-specific IgG antibody-positive sera of patients with mucosal disease (n = 18) and cutaneous disease (n = 23). For antigens of the cutaneous parasite WR 2095, mucosal sera generally reacted intensely to antigens of 75, 66, and 45 kDa and weakly to 48-50-kDa antigens, whereas cutaneous sera generally detected weakly the first 3 antigens and intensely the latter doublet. The data suggest that the transition from the cutaneous antigenic profile to a mucosal antigenic profile could be used to predict mucosal disease in approximately half of mucosal patients. An additional finding was that antibodies present in the sera of patients with mucosal disease labeled a 66-kDa peptide of normal human lip mucosa more intensely than did cutaneous sera. Autoimmune processes stimulated by the reaction of IgG, originally directed against the 66-kDa of L. braziliensis, to the 66-kDa antigen of mucosal tissue may contribute to the clinical presentation of mucosal leishmaniasis.     

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.     

Native and recombinant herpes simplex virus type 1 envelope proteins induce human immune T-lymphocyte responses.

The abilities of whole herpes simplex virus type 1 (HSV-1) antigen (HSV-ag) and purified HSV-1 native and recombinant envelope proteins to stimulate in vitro T-lymphocyte responses were compared in patients with recurrent herpes labialis. Immunochemically purified preparations of native glycoproteins B, C, and D (ngB, ngC, ngD) from cultured HSV-1 as well as expressed recombinant plasmid preparations of gD (rgD-1t, rgD-45K) elicited lymphocyte proliferation (LT) and production of gamma interferon (IFN-gamma) and interleukin-2 (IL-2) only in seropositive individuals. The IFN-gamma induced by rgD-1t correlated with the time to the next herpetic lesion in 19 volunteers followed to recurrence (r = 0.69, P less than 0.008), although the magnitude and frequency of LT and IFN-gamma responses were lower with either recombinant or native purified antigens than with the whole-virus antigen. Combinations of ngB plus ngD or ngB plus ngC plus ngD stimulated more IFN-gamma, equivalent to whole-virus-antigen responses. Recombinant-derived human IL-2 also specifically increased LT and IFN-gamma responses in antigen-driven cultures. ngD stimulated IL-2 and LT responses similar to those of whole-virus antigen and higher than those of ngC. HSV-ag and ngB induced significantly higher titers of total IFN than could be accounted for by IFN-gamma; this was not seen for the other antigens, which induced only IFN-gamma. HSV-ag-driven Leu 2a-, plastic-nonadherent blood cells, unlike whole peripheral blood mononuclear cells, showed evidence of an increase and then a decline in the frequency of HSV-responsive cells after a lesion recurrence. These studies suggest that HSV-1 envelope proteins are capable of stimulating an immune T-helper-cell response which is associated with the prevention of human herpes simplex lesion recurrence. Although the whole virus probably contains additional important antigens, increasing concentrations or combinations of certain purified glycoproteins or the addition of nonspecific enhancers of T-lymphocyte function can drive in vitro immune responses to the same level as the complete set of viral antigens.     

T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia) braziliensis

Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL) patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma) production in comparison to CCL patients. On the other hand, interleukin (IL-10) production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma) and regulatory mechanisms (IL-10). The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10) and effector cytokines (IFN-gamma), leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.     

Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. In this study, we demonstrate for the first time that a recombinant stage-specific hydrophilic surface protein of Leishmania donovani, recombinant hydrophilic acylated surface protein B1 (HASPB1), is able to confer protection against experimental challenge. Protection induced by rHASPB1 does not require adjuvant and, unlike soluble Leishmania Ag + IL-12, extends to the control of parasite burden in the spleen, an organ in which parasites usually persist and are refractory to a broad range of immunological and chemotherapeutic interventions. Both immunohistochemistry (for IL-12p40) and enzyme-linked immunospot assay (for IL-12p70) indicate that immunization with rHASPB1 results in IL-12 production by dendritic cells, although an analysis of Ab isotype responses to rHASPB1 suggests that this response is not sufficient in magnitude to induce a polarized Th1 response. Although both vaccinated and control-infected mice have equivalent frequencies of rHASPB1-specific CD4(+) T cells producing IFN-gamma, vaccine-induced protection correlates with the presence of rHASPB1-specific, IFN-gamma-producing CD8(+) T cells. Thus, we have identified a novel vaccine candidate Ag for visceral leishmaniasis, which appears to operate via a mechanism similar to that previously associated with DNA vaccination.     

Human peripheral blood mononuclear cells stimulated by Schistosoma mansoni antigens: association between protein tyrosine kinases, mitogen-activated protein kinases and cytokine production

Concerning schistosomiasis, little is known about the intracellular signaling response of human peripheral blood mononuclear cells (PBMC) to Schistosoma mansoni antigens. To understand the critical role of protein tyrosine kinases (PTKs) in PBMC activation by S. mansoni antigens, we investigated how inhibition of PTKs by genistein, a tyrosine kinase inhibitor, affects proliferation, cytokine production and activation of mitogen-activated protein kinases (MAPKs). Our studies showed that PTKs have an important role in proliferation of PBMC from chronic schistosomiasis patients as cells stimulated with S. mansoni soluble antigens in the presence of genistein had an impaired proliferation. In contrast, PTK inhibition failed to cause any effect on MAPKs activity. We also evaluated the cytokine production for interleukin (IL)-2, interferon gamma (IFN-gamma), and IL-10 in culture supernatants of PBMC treated with or without PTKs inhibitor. Our results show that PBMC from chronic patients produced a high amount of IL-10 when stimulated with soluble egg antigen preparation (SEA), however, the amount produced of IL-2 and IFN-gamma was not significant. In the presence of PTKs inhibitor only the production of IL-10 was decreased. The findings suggest that PTKs are involved on signal transduction pathway for PBMC activation, but may not be an absolute requirement for all signaling responses to S. mansoni antigens.     

KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.

The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection.     

The human cytokine response to Leishmania major early after exposure to the parasite in vitro

Leishmaniasis is caused by the protozoan parasite Leishmania spp. In murine leishmaniasis, a T helper cell type-I (Th1) response, characterized by the secretion of interferon (IFN)-gamma is necessary for clearing the infection. whereas a Th2 response, accompanied by the production of interleukin (IL)-5, can exacerbate the disease. Moreover, the early cytokine milieu is thought to play an important role in determining the outcome of infection. In human leishmaniasis little is known about this early cytokine response. Because of this, we cocultured human peripheral blood mononuclear cells (PBMC) with Leishmania major in vitro and measured the production of IFN-gamma, IL-5, and IL-10. We also treated PBMC cultures with various cytokines and neutralizing anticytokines. We found that the principal cytokine produced was IFN-gamma and that its production was regulated by IL-10 and IL-12. In contrast, only low levels of Th2 cytokines such as IL-5 were produced. Therefore, the Th1-Th2 dichotomy that exists in inbred strains of mice does not appear to apply to the response of humans to L. major. Rather, Th2 cytokines may play a role in regulating IFN-gamma production.     

Antigen-induced human interferon-gamma production. Differential dependence on interleukin 2 and its receptor

Once stimulated with Toxoplasma gondii or cytomegalovirus (CMV) antigens, peripheral blood mononuclear cells from healthy seropositive donors secrete comparable levels of interferon-gamma (IFN-gamma). Both antigens also stimulated specific production of interleukin 2 (IL-2), a lymphokine believed to be important in IFN-gamma generation. T. gondii antigen, however, induced ninefold more IL-2 than did CMV antigen suggesting different mechanisms for antigen-stimulated IFN-gamma production. Therefore, we examined for both antigens 1) the cellular sources of IL-2 and IFN-gamma, 2) the kinetics of IL-2 production and IL-2 receptor (IL-2R) expression, and 3) the effect of antibodies to IL-2 and IL-2R on IFN-gamma secretion. For both antigens, IL-2 and IFN-gamma secretion was T4+ cell-dependent. T. gondii antigen induced high levels of IL-2 at 24 hr which increased further at 48 hr, and IFN-gamma production was strongly inhibited by antibodies to both IL-2 (90 +/- 2%) and IL-2R (80 +/- 5%). In contrast, CMV antigen stimulated low levels of IL-2 at 24 hr which declined still further by 48 hr, and CMV-stimulated IFN-gamma generation was appreciably less well inhibited by antibodies to IL-2 (47 +/- 2%) and IL-2R (31 +/- 8%). These results suggest the possibility of two mechanisms for antigen-induced IFN-gamma production--one primarily dependent on and the other largely independent of IL-2 and its receptor. Both mechanisms, however, require the activity of sensitized T4+ cells.     

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.     

BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice

Cellular immune responses are required for protective immunity against Leishmania chagasi. Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens. Previous studies demonstrated that the L. chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L. chagasi infection. To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1). Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L. chagasi infection than LCR1 protein alone. BCG-LCR1 administered intraperitoneally did not protect. Splenocytes from mice immunized s.c. with either BCG-LCR1 or BCG-pMV261 and then infected with L. chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice. We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine.     

Antigen-stimulated lymphokines from patients with cutaneous leishmaniasis induce monocyte killing of Leishmania major intracellular amastigotes

The specific immune response of patients with cutaneous leishmaniasis including the ability of their lymphokines to enhance the monocytes' leishmaniacidal activity was studied. In 16 patients with cutaneous leishmaniasis, their concanavalin A-induced lymphocyte proliferative responses, interferon-gamma and interleukin 2 activities and the ability of their concanavalin A-induced lymphokines to kill monocyte intracellular amastigotes were not different from normal controls. Antigen-stimulated lymphocyte cultures showed that 13 of 13 patients had an increased lymphocyte proliferative response; 11 of 16 produced interleukin 2 and 12 of 13 produced interferon-gamma; in addition, 10 of 11 of these antigen-induced supernatants increased the monocytes' killing of Leishmania major amastigotes. Antibody levels to parasite membrane antigens determined by radioimmunoassay showed that 8 of 13 titers were greater than 10 and 4 of 13 titers were 2 to 10 times higher than control. Our findings demonstrate that patients with cutaneous leishmaniasis elicit a specific immune response to L. major antigens and part of this response is the production of lymphokine capable of promoting monocyte killing of intracellular amastigotes.     

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.     

Macrophages at intermediate stage of maturation produce high levels of IL-12 p40 upon stimulation with Leishmania

IL-12 is one of the main cytokines driving the immune response to a resistant phenotype in leishmaniasis and in several other diseases involving intracellular microbes. In this study, we investigated IL-12 production by mononuclear phagocytes at several developmental stages when stimulated with Leishmania major, L. amazonensis or L. chagasi. Bone marrow cells were cultured for 4-6 days in vitro in the presence of M-CSF, GM-CSF or IL-3. After density separation, only cells banding at the 40-50% Percoll interface, but not those at 20-40% or 50-80% interfaces, produced large amounts of IL-12 p40 when stimulated with LPS or live Leishmania promastigotes. However, only low levels of IL-12 p70 were produced under these conditions. The high IL-12 p40-producing cells could be similarly derived from mouse strains with different susceptibility to Leishmania. Quantitative analysis of monocyte/macrophage lineage marker expression, in combination with positive and negative selection, led to the conclusion that the high IL-12 p40-producing cells are macrophages at an intermediate stage of maturation between immature and fully differentiated cells, expressing ER-HR3 but only low levels of the mature markers, scavenger receptor and CD11b/Mac-1. They do not express any of the precursor markers CD31/ER-MP12, Ly-6C/ER-MP20 or ER-MP58. Because recruitment of monocytes to an infection site and its draining lymph node is a general phenomenon, the notion that, developing from these monocytes, a population of mononuclear phagocytes at an intermediate maturation stage has the capacity to synthesize large amounts of IL-12 p40 has significant bearing on our understanding of immune regulation in leishmaniasis and also in infections by other pathogens.     

Atypical mucocutaneous leishmaniasis caused by Leishmania braziliensis in an acquired immunodeficiency syndrome patient: T-cell responses and remission of lesions associated with antigen immunotherapy.

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5%) most of them with CD8+ phenotype (33%). Very low CD4+ cells (2.2%) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells.