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.

Níveis de carnitina na ração no desempenho corporal de tricogáster léri (Trichogaster leeri bleeker, 1852) / Carnitine levels in feed of body performance of the gouramis (Trichogaster leeri bleeker, 1852)

Foram avaliados os efeitos de carnitina na dieta de Trichogaster leeri, com peso inicial de 0,5±0,2g, distribuídos aleatoriamente e individualmente em aquários plásticos (1000mL), com renovação total da água a cada 48 horas. Cinco dietas isocalóricas, contendo 28 por cento de proteína bruta e 3100kcal de energia bruta, foram elaboradas com a suplementação de T1 = 0mg, T2 = 300mg, T3 = 600mg, T4 = 900mg e T5 = 1200mg de l-carnitina/kg de ração. As dietas foram administradas três vezes ao dia, ad libitum, durante 45 dias. Foram avaliados comprimento total e padrão, altura, peso final e tamanho de cauda, além do ganho de peso, conversão alimentar e fator de condição. Em todas as características analisadas, o tratamento de 900mg de carnitina/kg foi o mais eficiente no desempenho zootécnico para o T. leeri, e, após cálculos dos pontos máximos, sugere-se a concentração de 1.000mg/kg de ração. Observou-se diferença significativa para tamanho de cauda, altura do corpo, peso final e fator de condição.

The effects of carnitine were evaluated in diets fed to Trichogaster leeri, initial weight 0.5±0.2g and individually randomized into plastic containers (1000mL), with total renewal of water every 48 hours. Five isocaloric diets containing 28 percent crude protein and 3100 kcalof gross energy were made with the supplementation of: T1= 0 mg; T2= 300mg;T3= 600mg; T4= 900mg; and T5= 1200mg of l-carnitina/kg feed. The diets were administered three times daily ad libitum for 45 days. Total and standard length, height, weight and size of the tail end, in addition to weight gain, feed conversion ratio and condition factor were evaluated. In all parameters examined the treatment of 900 mg of carnitine/kg was the most efficient on the performance for the T. leeri, and after calculating the maximum points, a concentration of 1.000mg/kg feed is suggested. There was significant difference in tail length, body height, final weight and condition factors.

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.

Immunostimulatory effects of polar glycopeptidolipids of Mycobacterium chelonae for inactivated rabies vaccine.

Humoral and cellular immune responses were analyzed with Fuenzalida-Palacios rabies vaccine associated with pGPL-Mc, polar glycopeptidolipids extracted from Mycobacterium chelonae, aiming at its use as adjuvant. These results were compared to those obtained with BCG, a well-known immunostimulator, under the same conditions. Rabies vaccine plus pGPL-Mc (2.5 mg/kg) induced a significant increase in serum neutralizing activity, in vitro lymphocyte proliferation (spontaneous, specific and mitogen stimulation) and delayed type hypersensibility. In addition, pGPL-Mc, as well as BCG, enhanced the vaccine potency. Our results support further studies to encourage the use of pGPL-Mc as an immunostimulator of veterinary vaccines, before consideration for human vaccines.

[Immunogenicity of the avirulent strain RV194-2 of the rabies virus in mice]. / Imunogenicidade da cepa avirulenta RV194-2 do vírus rábico em camundongos.

RV194-2 rabies virus, an avirulent mutant of CVS strain, induces an inapparent infection limited to the central nervous system (CNS) in adult mice inoculated intracerebrally. This fact suggest that immune response of the host is able to eliminate the virus in CNS. For this reason, we have studied the induction of interferon and the humoral immune responses in BALB/c mice after RV194-2 inoculation. These mice presented high levels of interferon in the plasma and in the brain, with elevated levels of neutralizing antirabies antibodies. The 2-5A synthetase, an enzyme marker of interferon action, was analyzed in the brain of inoculated animals. Its enhancement in parallel to the interferon production in the brain, showed biochemical evidence that this interferon is active. Forty five days after RV194-2 virus inoculation, mice were protected against a challenge with the CVS virulent strain. The results presented herein show that RV194-2 strain has a high level of immunogenicity.

Alterations of erythrocyte morphology and lipid composition by hyperbilirubinemia.

Morphology and membrane lipid composition of erythrocytes from neonates (jaundiced and healthy) and adults (before and after incubation with bilirubin) were studied. The morphological index, expressing the relative proportions of the different stages of cell distortion, and the membrane cholesterol, phospholipids and cholesterol/phospholipids molar ratio, were determined. In jaundiced neonates a significant increase in the morphological index (P < 0.01) was found. After incubation with bilirubin, adult erythrocytes also showed an increase in the morphological index (P < 0.01). Hemolysis occurred under these conditions, and the red cell ghosts obtained (vesicles) showed a rounded morphology. Higher cholesterol/phospholipid ratio and lower phospholipid content were found in jaundiced neonates compared with healthy babies (P < 0.05) and adults (P < 0.01), as well as in the cells (P < 0.05) and vesicles (P < 0.01) obtained after bilirubin incubation. Bilirubin cytotoxicity may occur in a stepwise manner: deposition of bilirubin in membrane produces echinocytosis, which is followed by disintegration of the lipid bilayer with loss of phospholipids from the membrane.

Lymphocyte subsets and cell proliferation analysis in rabies-infected mice.

Alterations to lymphocyte subsets in the thymus and spleen of rabies-infected mice were investigated in a kinetic study covering the entire course of infection. Changes in the levels of thymic Thy1.2 cells were found to be proportional to total thymocyte depletion, while thymic CD4-/CD8- and B cell subsets were observed to increase in comparison to the total number of cells. The same was found to be true of CD4 and CD8 thymocytes, but only after the first clinical signs of disease had appeared. Meanwhile, drastic reductions in the number of CD4+/CD8+ thymocytes occurred during the course of infection. In the spleen, on the other hand, alterations to splenocyte subsets were not selective. Thymocytes expressing high levels of CD4 or CD8 markers were detected shortly before the death of the animals. Following the appearance of rabies symptomatology, lymphocyte proliferation decreased in comparison to the control. Thus, our results demonstrate that the thymus is the most injured lymphoid organ in cases of murine rabies infection.

Interferon production and immune response induction in apathogenic rabies virus-infected mice.

Pathogenic parental rabies virus strain CVS (challenge virus standard) and its apathogenic variant RV194-2 were shown to differ in their ability to induce interferon (IFN) and immune response of the host. After intracerebral inoculation, IFN and antibody production was higher in the RV194-2 virus-infected mice than in the CVS infection. The enhancement of 2-5A synthetase activity, an IFN-mediated enzyme marker, showed biochemical evidence that IFN is active in both apathogenic and pathogenic infections. On the other hand, spontaneous proliferation in vitro of thymocytes and splenocytes from CVS virus-infected mice was strongly inhibited in contrast to the RV194-2 infection. In the CVS infection, the thymocyte proliferation was more affected than the splenocyte proliferation. However, in the RV194-2 infection, the thymocyte proliferation was higher than of the splenocytes. These results suggest a better performance of T-cell response to the RV194-2 infection than to the CVS infection. This fact can be critical for an enhancement of antibody production in the apathogenic infection and subsequent virus clearance from the brain of RV194-2 virus-infected mice.

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.

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.