Interleukin-10-dependent down-regulation of interferon-gamma response to Leishmania by Mycobacterium leprae antigens during the clinical course of a coinfection.

We have described a case of a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, two opposite polar forms of these spectral diseases. In the present follow-up study, we investigated the effect of the addition of Mycobacterium leprae antigens on interferon-gamma (IFN-γ) production in Leishmania antigen-stimulated cultures of peripheral blood mononuclear cells (PBMC) from this patient. For this purpose, PBMC cultures were stimulated with crude L. braziliensis and/or M. leprae whole-cell antigen extracts or with concanavalin A. In some experiments, neutralizing anti-human interleukin (IL)-10 antibodies were added to the cultures. IFN-γ and IL-10 levels in culture supernatants were measured by ELISA. During active leprosy, M. leprae antigens induced 72.3% suppression of the IFN-γ response to L. braziliensis antigen, and this suppression was abolished by IL-10 neutralization. Interestingly, the suppressive effect of M. leprae antigen was lost after the cure of leprosy and the disappearance of this effect was accompanied by exacerbation of mucosal leishmaniasis. Considered together, these results provide evidence that the concomitant lepromatous leprosy induced an IL-10-mediated regulatory response that controlled the immunopathology of mucosal leishmaniasis, demonstrating that, in the context of this coinfection, the specific immune response to one pathogen can influence the immune response to the other pathogen and the clinical course of the disease caused by it. Our findings may contribute to a better understanding of the Leishmania/M. leprae coinfection and of the immunopathogenesis of mucosal leishmaniasis.

Pattern of functional antibody activity against Haemophilus influenzae type b (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine

We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56 percent, 961/985) developed a strong anti-PRP IgG antibody response (¡Ý1.0 ¦Ìg/mL), while an anti-PRP IgM response was observed in 64.24 percent (634/985) of them (¡Ý0.15 ¦Ìg/mL). Only 18.88 percent (186/985) of the infants in the group with high PRP antibody IgG concentrations (¡Ý1.0 ¦Ìg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.

Pattern of functional antibody activity against Haemophilus influenzae type B (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine.

We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56%, 961/985) developed a strong anti-PRP IgG antibody response (>or=1.0 microg/mL), while an anti-PRP IgM response was observed in 64.24% (634/985) of them (>or=0.15 microg/mL). Only 18.88% (186/985) of the infants in the group with high PRP antibody IgG concentrations (>or=1.0 microg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.

Contrasting human cytokine responses to promastigote whole-cell extract and the Leishmania analogue receptor for activated C kinase antigen of L. amazonensis in natural infection versus immunization.

It is known that the same antigen can induce different immune responses, depending upon the way that it is presented to the immune system. The objective of this study was to compare cytokine responses of peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients and subjects immunized with a first-generation candidate vaccine composed of killed Leishmania amazonensis promastigotes to a whole-cell promastigote antigen extract (La) and to the recombinant protein LACK (Leishmania analogue receptor for activated C kinase), both from L. amazonensis. Thirty-two patients, 35 vaccinees and 13 healthy subjects without exposure to Leishmania, were studied. Cytokine production was assessed by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay. The interferon (IFN)-gamma levels stimulated by La were significantly higher and the levels of interleukin (IL)-10 significantly lower than those stimulated by LACK in the patient group, while LACK induced a significantly higher IFN-gamma production and a significantly lower IL-10 production compared with those induced by La in the vaccinated group. LACK also induced a significantly higher frequency of IFN-gamma-producing cells than did La in the vaccinated group. The contrast in the cytokine responses stimulated by LACK and La in PBMC cultures from vaccinated subjects versus patients indicates that the human immune response to crude and defined Leishmania antigens as a consequence of immunization differs from that induced by natural infection.

Detection of Th1/Th2 cytokine signatures in yellow fever 17DD first-time vaccinees through ELISpot assay.

Immunity to yellow fever (YF) is conferred by the interplay of humoral and cellular immune response. Despite the extensive literature on the humoral immune response to the YF vaccine virus, little is known about its cellular immune response to vaccination. The analysis of cytokine production by ex-vivo antigen-stimulated T cells has been considered as a valuable tool for understanding cellular immune response. Thus, we have analyzed two T(H)1/T(H)2 signature cytokines (IFN-gamma and IL-4) from 12 healthy first-time adults vaccinated with YF17DD virus. The cells, harvested on day 0 (before vaccination) and 7, 15 and 30 days after immunization were antigen-stimulated and analyzed by ELISpot. A significant increase in the number of spot-forming cells during the response to YF 17DD live virus stimulation by ELISpot assay was observed. IFN-gamma-and IL-4-producing cells were significantly increased on the 15th day after vaccination in all volunteers. These results presented herein are important for understanding the role of cytokines in the immune response to YF 17DD virus.

In vitro responses of human peripheral blood mononuclear cells to whole-cell, particulate and soluble extracts of Leishmania promastigotes.

Whole-cell and soluble extracts of Leishmania promastigotes have both been used as skin test antigens and have also been tested as vaccine candidates. However, the differences in antigenicity between soluble and particulate Leishmania fractions are not known. We evaluated in vitro responses of PBMC from 30 American tegumentary leishmaniasis (ATL) patients and seven noninfected donors to different antigen preparations from Leishmania promastigotes, namely Leishmania amazonensis and L. braziliensis whole-cell extracts, as well as soluble and particulate fractions of L. amazonensis. All Leishmania antigen preparations stimulated significantly higher proliferation and interferon (IFN)-gamma production (but not interleukin (IL)-10 production) in PBMC from the leishmaniasis patients than in cells from the control subjects. The L. braziliensis whole-cell extract stimulated significantly higher cell proliferation and IFN-gamma production than the L. amazonensis whole-cell extract in the group of patients but not in the control group. This result can be explained by the fact that the patients were infected with L. braziliensis. Again in the group of patients, the PBMC proliferative responses as well as the levels of IFN-gamma and IL-10 stimulated by L. amazonensis whole-cell extract were significantly greater than those elicited by the L. amazonensis soluble fraction but were not significantly different from those elicited by the L. amazonensis particulate fraction. We found a higher antigenicity of the particulate fraction as compared to the soluble fraction, what suggests that the antigens present in the particulate fraction account for most of the antigenicity of whole-cell Leishmania promastigote antigen extracts.

Relationship between apoptosis and thymocyte depletion in rabies-infected mice

The apoptosis of thymocytes from rabies-infected mice was investigated in a kinetic study covering the entire course of the infection. For this study, BALB/c mice (6-7-week old females) were inoculated intracerebrally with 100 LD50 of Challenge Virus Strain, a fixed rabies virus strain, and three animals were sacrificed per time point to remove thymuses. When thymocytes were fixed, stained with propidium iodide and analyzed by flow cytometry, a distinct subpopulation of cells was observed below the G0/G1 region, denoted as the A0 region. Cells in this region presented reduced fluorescence, and nuclear DNA fragmentation. The accumulation of cells in the A0 region, after infection, progressively increased, reaching 12 for unfractionated thymocytes, 62 for thymocytes from the 60 Percoll interface and 32 for thymocytes recovered at the 100 Percoll interface. This finding, observed only in thymocytes from infected mice, demonstrates a clear modification of chromatin condensation in these cells, suggesting the occurrence of an apoptotic process during rabies infection.