Immunomodulatory activities associated with β-glucan derived from Saccharomyces cerevisiae

In this study we investigated the effect of β-glucan derived from Saccharomyces cerevisiae on fungicidal activity, cytokine production and natural killer activity. Spleen and peritoneal cells from female C57BL/6 mice, previously injected (24 or 48 h) with 20 or 100 μg of glucan by i.p. route, were assayed. In vivo β-glucan administration primed spleen cells for a higher production of IL-12 and TNF-α when S. aureus was used as a stimulus. In addition, β-glucan increased NK spleen cells activity against YAC target cells. Some immunomodulatory activities not yet described for β-glucan were observed in this work. © 2005 Institute of Physiology, Academy of Sciences of the Czech Republic.

Detection and neutralization of venom by ovine antiserum in experimental envenoming by Bothrops jararaca

In this study we optimized an enzyme-linked immunosorbent assay (ELISA) to evaluate bothropic venom levels in biological samples. These samples were obtained by two distinct protocols. In the first one, Swiss mice were injected with 1 LD 50 of Bothrops jararaca (B. jararaca) venom and 15 minutes later, animals were treated with ovine antibothropic serum. Blood and spleen homogenate samples were obtained 6 hours after antiserum therapy. Ovine antibothropic serum significantly neutralized venom levels in serum and spleen. In the second protocol, BALB/c mice were injected with 1 LD 50 of bothropic venom by either intraperitoneal (IP) or intradermal (ID) route and venom levels were evaluated 1, 3 and 6 hours after, in blood, spleen homogenates and urine. Serum and splenic venom levels were significantly higher in animals envenomed by IP route comparing with animals envenomed by ID route. Higher venom levels were also detected in urine samples from animals envenomed by IP route. However, these differences were not statistically significant. These results demonstrated that the optimized ELISA was adequate to quantify venom levels in different biological samples. This assay could, therefore, substitute the in vivo neutralizing assay and also be useful to evaluate the severity of human and experimental envenomations.

Invasin of Yersinia pseudotuberculosis is not a polyclonal activator of mouse B lymphocytes

It is known that the invasin molecule of Yersinia pseudotuberculosis stimulates human peripheral B cells in vitro. In this work we evaluated the in vivo role of invasin as polyclonal activator of B lymphocytes in the mouse experimental model, by comparing strains of Y. pseudotuberculosis expressing invasin and isogenic inv mutants. Swiss mice were infected intravenously with two strains expressing invasin (YpIII pIB1 and an isogenic virulence plasmid-cured strain, YpIII) and with two invasin mutant strains (Yp100 pIB1 and Yp100, plasmid-cured). Spleen cells were sampled on days 7, 14, 21 and 28 after infection. Immunoglobulin (Ig)-secreting spleen cells were detected by protein A plaque assay and specific antibodies were detected in sera by ELISA. The virulent strain YPIII pIB1 (wild type) did not provoke polyclonal activation of B lymphocytes in vivo. In general, fewer Ig-secreting spleen cells of all isotypes were found in the infected animals than in the control animals. Specific IgG antibodies were detected in the sera of animals infected with all strains. The peak response occurred on the 21 st day post-infection, and the Yp100 strain provoked the highest level of these antibodies. We concluded that invasin is not a polyclonal activator of murine B cells.

DNA damage and nitric oxide synthesis in experimentally infected Balb/c mice with Trypanosoma cruzi

This study aimed to evaluate whether experimental Chagas disease in acute phase under benznidazole therapy can cause DNA damage in peripheral blood, liver, heart, and spleen cells or induce nitric oxide synthesis in spleen cells. Twenty Balb/c mice were distributed into four groups: control (non-infected animals); Trypanosoma cruzi infected; T. cruzi infected and submitted to benznidazole therapy; and only treated with benznidazole. The results obtained with the single cell gel (comet) assay showed that T. cruzi was able induce DNA damage in heart cells of both benznidazole treated or untreated infected mice. Similarly, T. cruzi infected animals showed an increase of DNA lesions in spleen cells. Regarding nitric oxide synthesis, statistically significant differences (p < 0.05) were observed in all experimental groups compared to negative control, the strongest effect observed in the T. cruzi infected group. Taken together, these results indicate that T. cruzi may increase the level of DNA damage in mice heart and spleen cells. Probably, nitric oxide plays an important role in DNA damaging whereas benznidazole was able to minimize induced T. cruzi genotoxic effects in spleen cells. © 2006 Elsevier Inc. All rights reserved.

Immune response to Mycobacterium bovis-an5 infection in genetically selected mice (selection IV-A)

Mice genetically selected for high (H) and low (L) antibody production (Selection IV-A) were used as murine experimental model. The aim of the present work was to evaluate the macrophagic activity and to characterize the immune response in Mycobacterium bovis-AN5 infected mice (3×10 7 bacteria). The response profile previously observed in such strains was not similar to that obtained during M. bovis infection; however, it corroborated works carried out using Selection I, which is very similar to Selection IV-A regarding infection by M. tuberculosis and Bacillus Calmette-Guérin (BCG). Considering bacterial recovery, LIV-A mice showed higher control of the infectious process in the lungs than in the spleen, whereas HIV-A mice presented more resistance in the spleen. With respect to macrophagic activity, hydrogen peroxide (H2O 2) was probably not involved in the infection control since there was an inhibition in the production of this metabolite. Nitric oxide (NO) and TNF-α production seemed to be important in the control of bacterial replication and varied according to the strain, period and organ. Evaluation of the antibody production indicated that the multi-specific effect commonly observed in these strains was not the same in the response to M. bovis. Antibody concentrations were higher in LIV-A than in HIV-A mice at the beginning of the infection, being similar afterwards. Such data were compared with delayed-type hypersensitivity (DTH), which was more intense in HIV-A than in LIV-A mice, indicating that antibody production is independent of the capability to trigger DTH reactions and that cellular and humoral responses to M. bovis antigens show a polygenic control and an independent quantitative genetic regulation. Differences were observed among organs and metabolites, suggesting that different mechanisms play an important role in this infection in natural heterogeneous populations, indicating that NO, TNF-α and Th1 cytokines are involved in the infection control.

Pattern of macrophage activation in yersinia-resistant and yersinia-susceptible strains of mice

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFβ-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-γ and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-β1 and IL-4 production by BALB/c mice and to an increase in the IFN-γ levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.

Produção deficiente de citocinas Th1 em camundongos BALB/c jovens

O objetivo do presente estudo foi comparar a produção de IFN-γ, IL-12 e IL-4 entre camundongos jovens (5, 12 e 19 dias de idade) e adultos (30 dias de idade). As avaliações foram feitas por estimulação, in vitro, de células esplênicas com Concanavalina A (ConA) , Staphylococcus aureus (S. aureus) e lipopolissacarídeo (LPS). Diferentes concentrações de cada estímulo foram testadas e os sobrenadantes das culturas foram coletados após 48 horas de incubação e as concentrações de IFN-γ, IL-12 e IL-4 determinadas por ELISA. Células de camundongos jovens e adultos produziram níveis igualmente elevados de IFN-γ após estímulo com ConA. Somente animais adultos produziram IFN-γ em resposta ao estímulo com S. aureus. Em culturas estimuladas com LPS, a produção desta citocina foi baixa e similar nos animais jovens e significativamente elevada nos animais adultos. Somente células de animais adultos estimuladas com S. aureus foram capazes de produzir IL-12. O único estímulo capaz de induzir níveis detectáveis de IL-4 foi ConA, sendo que estes níveis foram mais elevados nos animais com 12 e 19 dias de idade em comparação com animais neonatos e adultos. A diminuição das doses ótimas dos estímulos não mudou o perfil de produção de cada citocina nos animais jovens. Estes resultados permitem concluir que a idade afeta a produção de citocinas: ocorre maior produção de IL-4 em camundongos jovens e maior produção de IL-12 e IFN-γ em animais adultos. Estas informações são importantes devido ao papel destas citocinas na polarização das respostas imunes nos sentidos Th1 e Th2. Palavras-chave: camundongo; citocina; interferon-gama; interleucina-4; interleucina-12.

Análise histopatológica de órgãos linfóides de bovinos e camundongos intoxicados experimentalmente por Baccharis coridifolia: Caracterização imunoistoquímica de linfócitos B e T

Baccharis coridifolia is one of the most important poisonous plants to cattle in the South of Brazil. The plant provokes necrotic lesions in the lymphoid tissues and in the gastrointestinal tract of cattle. Experimental administration to mice produces most of the lesions seen in the lymphoid tissues of cattle. This study was conducted to search possible differences in the susceptibility of T and B lymphocyte subpopulations. Lymph nodes, spleen, thymus and gut-associated lymphoid tissue (GALT) of cattle and mice experimentally poisoned were evaluated. The results were evaluated based on cell populations affected or remaining in the organs. Immunostaining for B lymphocytes (anti-BLA-36) identified the germinal center of follicles of the lymph node, spleen and GALT in both species. Immunostaining for T lymphocyte (anti-CD3) identified the paracortical area of the germinal centers of the lymph nodes and GALT, the periarteriolar area of the spleen, and the whole thymus both in cattle and mice. Experimentally poisoned cattle and mice shows necrosis of the germinal center of secondary follicles of the lymph nodes, spleen and GALT, where necrotic cells were immunostained for B and less often for T lymphocyte. Necrotic cells in the paracortical region of the lymph node were less often and were immunostained. Necrotic lesions of the thymus were seen only in mice, with positively stained for T lymphocyte. The distribution of the lesions in the lymphoid tissues and the immunostaining in necrotic cells suggested that the active principles of the plant are cytotoxic to B and T cells.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background: Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods: In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results: DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion: The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. © 2009 Santos et al; licensee BioMed Central Ltd.

Ocorrência de leishmaniose em gatos de área endêmica para leishmaniose visceral

Despite the description of several cases of feline leishmaniasis around the world, little information is available about the importance of the cat as a reservoir of the disease. The aim of the present study was to determine the occurrence of leishmaniasis in cats from an endemic area for visceral leishmaniasis in Brazil. Two hundred cats were included in this study. Infection was evaluated through the presence of amastigotes in stained smears from fine-needle aspirates of lymph nodes, bone marrow, spleen and liver, and by antibody reactivity against Leishmania chagasi using indirect ELISA. Our results showed a prevalence of infection in 14.5% (31/200) of the feline population studied, with 4% (8/200) of positivity by parasitological diagnosis and 11.5% (23/200) by serology.

Avaliação clínica e parasitológica de cães naturalmente infectados por Leishmania (Leishmania) chagasi submetidos a tratamento com antimoniato de meglumina e alopurinol

Aiming to assess the efficacy of the treatment, to verify the occurrence of possible disease relapses and to search for the presence of parasites after the treatment, seven dogs naturally infected by Leishmania sp., were submitted to a treatment with meglumine antimoniate and allopurinol. For this, lymph node and bone marrow aspiration biopsies were carried out at seven moments. After the end of the six-month observation period all dogs were submitted to euthanasia. Then, spleen and liver imprints and in vitro cultures were carried out to search for amastigote forms of the parasite. All animals presented remission of the symptoms and during all the observation period no dog presented relapse of the disease, although amastigote forms of the parasite were observed in two of the animals at the end of the experiment. Thus, it was possible to conclude that the treatment promotes clinical healing but it does not eliminate the parasites completely.

Evaluation of Agaricus blazei in vivo for antigenotoxic, anticarcinogenic, phagocytic and immunomodulatory activities

The development of various types of cancer results from the interaction among endogenous, environmental and hormonal factors, where the most notable of these factors is diet. The aim of the present study was to determine the antigenotoxic, anticarcinogenic, phagocytic and immunomodulatory activities of Agaricus blazei. The test antigenotoxicity (Comet Assay) and anticarcinogenic (Test of Aberrant Crypt Foci) assess changes in DNA and/or intestinal mucosa that correlate to cancer development. Tests of phagocytosis in the spleen and differential count in blood cells allow the inference of modulation of the immune system as well as to propose a way of eliminating cells with DNA damage. Supplementation with the mushroom was carried out under pre-treatment, simultaneous treatment, post-treatment and pre-treatment. +. continuous conditions. Statistical analysis demonstrated that the mushroom did not have genotoxic activity but showed antigenotoxic activity. Supplementation caused an increase in the number of monocytes and in phagocytic activity, suggesting that supplementation increases a proliferation of monocytes, consequently increasing phagocytic capacity especially in the groups pre-treatment, simultaneous and pre-treatment. +. continuous. The data suggest that A. blazei could act as a functional food capable of promoting immunomodulation which can account for the destruction of cells with DNA alterations that correlate with the development of cancer, since this mushroom was demonstrated to have a preventive effect against pre-neoplastic colorectal lesions evaluated by the aberrant crypt foci assay. According to these results and the literature, it is believed that supplementation with A. blazei can be an efficient method for the prevention of cancer as well as possibly being an important coadjuvant treatment in chemotherapy. © 2011 Elsevier Inc.

Pathological and molecular analysis of Turkey coronavirus replication in captive wild Turkey embryos (broad-breasted bronze breeder)

The degree of genetic and pathologic variation exhibited by a turkey Coronavirus (TCoV) strain was investigated after nine serial passages in 25-day-old turkey embryos obtained from wild broad-breasted bronze breeders. In spite of spleen, liver, kidneys, cloacal bursa and thymus have been collected and analysed, the main histopathological changes were only documented in the intestine sections. Microscopic lesions were characterized as mild enteritis, low degree of enterocyte vacuolization and detachment of the intestinal villous after five consecutive passages and were considered absent in the last passages. Genealogic analysis based on S1 and S2 DNA sequences suggested that Brazilian isolate might be considered as originated from TCoV strains circulating in the United States, as 100% identity with TCoV-Gl strain. Although S1 S2 sequences from each passage revealed no significant point mutations, and no correlation could be speculate between S2 nucleotide changes and pathologic features in infected embryos. This is the first demonstration of wild turkey embryos as a model for TCoV isolation and propagation.

Antitumor properties of Ganoderma lucidum polysaccharides and terpenoids

Ganoderma lucidum is an edible medicinal mushroom with immunomodulatory and antitumor properties, which are mainly attributed to polysaccharides and triterpenes that can be isolated from mycelia, fruiting bodies and spores. G. lucidum has been us d in a powdered form, as a medicinal beverage and a nutraceutical food (usually dried). In the present review we report some historical facts and the experimental evidence that polysaccharides and triterpenes obtained from this mushroom present potential antitumor activity. Direct effects on tumor cells include induction of apoptosis and interference in the cell cycle, whereas indirect effects are based on the modulation of immune response, usually impaired by cancer cells. Data indicate that G. lucidum can be used as a complementary tool for treatment of cancer patients. © by São Paulo State University.