Papel das proteínas Yops de Yersinia pseudotuberculosis na ativação dos linfócitos B

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo in vitro da efetividade do hidróxido de cálcio e da polimixina B na eliminação das endotoxinas presentes nos canais radiculares

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização imunofenotípica e avaliação quantitativa do infiltrado linfocítário junto às áreas de displasia epitelial da próstata de cães adultos sexualmente intactos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y enterocolitica 0:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y enterocolitica 0:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.

T lymphocyte subsets of the umbilical cord blood of dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Old beagle dogs have lower faecal concentrations of some fermentation products and lower peripheral lymphocyte counts than young adult beagles

The effects of age on microbiota composition, gut fermentation end-product formation and peripheral lymphocyte numbers were compared between old and young adult Beagle dogs fed four kibble diets differing in yeast cell wall contents. The experiment had a double 4 x 4 Latin square design, one with four mature dogs (4 years old) and the other with four old dogs (10 years old), with four replicates (diets) per dog. In each period a 15d adaptation period preceded a 5d total collection of faeces for the digestibility trial. on day 21, fresh faecal samples were collected for the determination of bacterial enumeration, pH, biogenic amine and short-chain fatty acid. Flow cytometry was used for immunophenotypic evaluation. Dogs were fed four kibble diets with similar composition with 0, 0.15, 0.30 and 0.45% of yeast cell wall (as-fed), respectively. Data were evaluated using general linear models of Statistical Analysis Systems statistical software (P<0.05). No evidence of a difference in faecal bacteria counts between ages was found (total aerobes, total anaerobes, Bifidobacterium, Lactobacillus, Clostridium and Escherichia coli: P. 0.15). Faecal concentrations of butyrate, histamine, agmatine and spermine were lower (P <= 0.05) and faecal pH was higher (P=0.03) in older dogs than in mature adult dogs, suggesting an alteration in bacterial metabolic activity, or in the rate of intestinal absorption of these compounds. Concentrations of T-lymphocytes, T-cytotoxic lymphocytes and B-lymphocytes were also lower (P <= 0.01) in older dogs than in mature adult dogs. The study confirmed alterations in peripheral lymphocytes and revealed a reduced concentration of some fermentation end products in the colon of old dogs.

Yersinia enterocolitica O : 3 isolated from patients with or without reactive arthritis induces polyclonal activation of B cells and autoantibody production in vivo

The mechanisms by which arthritis-provoking pathogens such as Yersinia enterocolitica interact with the human immune system to produce inflammatory synovitis are not well known. One of the immunomodulating mechanisms used against these pathogens is the polyclonal activation of lymphocytes. In this study, we investigated the extent of the B-lymphocyte activation induced in mice by a strain of Y. enterocolitica O:3 (FCF 526) isolated from a patient with arthritis, and compared it with two other strains, a virulent one (FCF 397[+]) isolated from a patient without arthritis and its plasmidless isogenic pair (FCF397[-]). Also we investigated the production of autoantibodies in mice infected with these different strains. SPF Swiss mice were infected intravenously with a suspension of Y. enterocolitica . Spleen cells were taken on days 7, 14, 21 and 28 after infection and the number of cells secreting nonspecific and specific antibodies of IgG 1 , IgG 2a , IgG 2b , IgG 3 , IgM and IgA isotypes were determined by the ELISPOT technique. The presence of autoantibodies in mouse serum was investigated by the dot-blot assay. The pattern of infection of the three bacterial strains were almost the same. We observed a general increase in the number of nonspecific Ig-secreting cells with all three strains, and the greatest increases observed were in the IgG 2a and IgG 3 isotypes. Only a small fraction of the immunoglobulins detected were antibacterial, suggesting that the rest resulted from polyclonal B cell activation. The strain isolated from the patient with arthritis (FCF526) induced the greatest production of autoantibodies, coinciding with the period in which the greatest activation of nonspecific B lymphocytes was seen. There were no signs of arthritis or inflammation in the joints of the infected animals. Based on our results, we were unable to determine whether there is an association between the arthritogenic capability of Y. enterocolitica and polyclonal activation of B cells.

Lymphoproliferative response and T lymphocyte subsets in a medium-term multi-organ bioassay for carcinogenesis in Wistar rats

The lymphoproliferative response and T lymphocyte subsets were evaluated at different stages of carcinogenesis in male Wistar, rats sequentially initiated with N-diethylnitrosamine (DEN), N-butyl-N-4(hydroxybutyl)nitrosamine (BBN), N-methyl-N-nitrosourea (MNU), dihydroxy-di-N-propylnitrosamine (DHPN) and N,N'-dimethylhydrazine (DMH) (DMBDD initiation). One group was evaluated at the 4th week and other initiated group at the 30th week. Two initiated groups were also exposed through diet to 7-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week. Two groups received only 2-AAF or PB until the 30th week. Five groups were studied to evaluate the effects of each initiator. The lymphoproliferative response was induced in vitro by concanavalin A and the percentage of T lymphocyte subsets was determined by flow cytometry, All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic: lesions than the untreated control group. The main target organs for tumor development were the liver, colon, urinary bladder, kidneys and Zymbal glands, mainly in the group treated with DMBDD + 2-AAF, There were no alterations of the lymphoproliferative response and of the T lymphocyte subsets percentage in the DMBDD-treated group at the 4th and 30th weeks. At the 30th week, the T lymphocyte subsets percentage was also not affected in the initiated groups after treatments with 2-AAF or PB. The lymphoproliferative response, however, was decreased in the DMBDD + 2-AAF group and in the groups treated only with 2-AAF or PB, the present results indicate that the initiating chemicals used in the DMBDD initiation protocol do not exert any influence on the immune system. The alteration of lymphoproliferative response induced at the advanced stage of carcinogenesis without alteration of T lymphocyte subsets may indicate that the influence of 2-AAF and PB on the immune system is functional and not toxic. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Propolis: lymphocyte proliferation and IFN-gamma production

We evaluated propolis influence on polyclonal activation of lymphocytes by concanavalin A (Con A). The in vitro experiments showed that propolis decreases splenocyte proliferation both in the absence or presence of Con A. The supression in mitogen-induced splenocyte proliferation also occurred when mice were treated intraperitoneally with propolis for 3 days. An increased of IFN-gamma production in the culture supernatants of the same cells was, observed. A dual action of propolis on lymphocyte activation was proposed: it decreases splenocyte proliferation in the presence or absence of Con A and stimulates IFN-gamma production by spleen cells. These results are important to understand the immunomodulatory action of propolis on the host's specific and non-specific immunity. (C) 2003 Elsevier B.V. Ireland Ltd. All rights reserved.

B-cell lymphoblastic lymphoma in the maxilla of a child: A rare case report

Lymphoblastic lymphoma is a malignant neoplasia that originates from B or T lymphocyte precursors and rarely occurs in the mouth. The authors report a rare case of B-cell lymphoblastic lymphoma in the maxilla of a child. Clinical examination revealed facial asymmetry with a swelling of the right maxilla, covered by healthy mucosa and painful to palpation. Radiographic examination revealed a poorly defined radiolucent lesion. Based on the hypothesis of malignant neoplasia of hematopoietic origin, an incisional biopsy was performed. Histological examination revealed malignant neoplasia with proliferation of monomorphic, lymphoid cells. Immunohistochemical staining was positive for leucocyte common antigen (LCA), CD 10, CD20, CD79, and terminal deoxynucleotidyl transferase (TdT). After the diagnosis of B-cell lymphoblastic lymphoma, the patient underwent chemotherapy, but died of leukoencephalopathy and demyelinization caused by high doses of methotrexate.

Interleukin-10 secreted by B-1 cells modulates the phagocytic activity of murine macrophages in vitro

As demonstrated previously in our laboratory, B-1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage-like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B-1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B-1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild-type mice. Moreover, macrophages from wild-type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1 cells down-regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B-1 cells are one of the main sources of interleukin (IL)-10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL-10 production by cells from wild-type mice. When B-1 cells from IL-10 knock-out mice and macrophages from wild-type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B-1 cells can influence the effector functions of macrophages in vitro via IL-10 secretion.

Association between polyclonal B cell activation and the presence of autoantibodies in mice infected with Yersinia enterocolitica O:3

Eight-week old conventional female Swiss mice were inoculated intravenously with Yersinia enterocolitica O:3. A second group of normal mice was used as control. Five mice from each group were bled by heart puncture and their spleens were removed for spleen cell collection on the 3rd, 5th, 7th, 10th, 14th and 21st day after infection. Immunoglobulin-secreting spleen cells were detected by the isotype-specific protein A plaque assay. Total immunoglobulin levels were determined in mouse serum by single radial immunodiffusion and the presence of autoantibodies was determined by ELISA. We observed a marked increase in the total number of cells secreting immunoglobulins of all isotypes as early as on the 3rd day post-infection and the peak of secretion occurred on the 7th day. At the peak of the immunoglobulin response, the total number of secreting cells was 19 times higher than that of control mice and most immunoglobulin-secreting cells were of the IgG2a isotype. On the 10th day post-infection, total serum immunoglobul in values were 2 times higher in infected animals when compared to the control group, and continued at this level up to the 21st day post-infection. Serum absorption with viable Y. enterocolitica cells had little effect on antibody levels detected by single radial immunodiffusion. Analysis of serum autoantibody levels revealed that Y. enterocolitica infection induced an increase of anti-myosin and anti-myelin immunoglobulins. The sera did not react with collagen. The present study demonstrates that Y. enterocolitica O:3 infection induces polyclonal activation of murine B cells which is correlated with the activation of some autoreactive lymphocyte clones.

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.