Chromobacterium violaceum genome: molecular mechanisms associated with pathogenicity

Chromobacterium violaceum is a versatile, Gram-negative beta-protebacterium that grows in a variety of ecosystems in tropical and subtropical areas, such as the water and borders of the Negro River, in the Amazon region of Brazil. Although it is a saprophyte and is generally considered non-pathogenic, sporadic cases of human infection have been described, mainly in young children and in immunodeficient individuals. Although rare, infections with C. violaceum are characterized by rapid dissemination and high mortality. With the complete genome sequence of C. violaceum now available, a detailed description of the molecular arsenal required for this bacterium's remarkable versatility has been revealed. Most importantly, a more detailed picture of its biotechnological properties, including the characteristic violacein pigment, has emerged. The complete genome sequence also enabled us to make a thorough examination of the repertoire of genes encoding probable virulence factors, which determine the potential for pathogenesis. We described a number of genes involved in infectious processes, such as host cell adhesion, [quot ]contact-dependent secretion[quot ] of factors that promote cell invasion, as well as other virulence factors, such as cytolytic proteins. We also described genes involved with the synthesis of lipopolysaccharides and proteoglycan, known to elicit the synthesis of pro-inflammatory cytokines and involved in the detoxification process, which may contribute to the evasion of the bacteria from the host immune response

Butyrate induces apoptosis in murine macrophages via caspase-3, but independent of autocrine synthesis of tumor necrosis factor and nitric oxide

We demonstrated that 4 mM butyrate induces apoptosis in murine peritoneal macrophages in a dose- and time-dependent manner as indicated by studies of cell viability, flow cytometric analysis of annexin-V binding, DNA ladder pattern and the determination of hypodiploid DNA content. The activity of caspase-3 was enhanced during macrophage apoptosis induced by butyrate and the caspase inhibitor z-VAD-FMK (100 æM) inhibited the butyrate effect, indicating the major role of the caspase cascade in the process. The levels of butyrate-induced apoptosis in macrophages were enhanced by co-treatment with 1 æg/ml bacterial lipopolysaccharide (LPS). However, our data indicate that apoptosis induced by butyrate and LPS involves different mechanisms. Thus, LPS-induced apoptosis was only observed when macrophages were primed with IFN-gamma and was partially dependent on iNOS, TNFR1 and IRF-1 functions as determined in experiments employing macrophages from various knockout mice. In contrast, butyrate-induced macrophage apoptosis was highly independent of IFN-gamma priming and of iNOS, TNFR1 and IRF-1 functions

Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes

The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31 percent) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38 percent) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs

Cytokines as determinants of disease and disease interactions

The immune response to pathogens results in both host resistance and immunopathology. Cytokines and in particular those lymphokines produced by Th1 and Th2 cells play a key role in determining the balance between these two immunologic outcomes. Recent data suggest that interleukin-10, a product of both Th2 cells and macrophages, protects the host against excessive immunopathology. The cytokine environment generated by different pathogens may also influence the course and outcome of infections with unrelated organisms. This relationship may be particularly important in the case of HIV-1 where prior Th1 or Th2 biases established by helminth or intracellular infections may influence either initial viral susceptibility or drive progression to AIDS through immune activation.

Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

Toxoplasma gandii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-gamma. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.

Effect of high sucrose diets on carcass composition in conventional and germ-free mice

1. O efeito da sacarose na dieta sobre a composiçäo da carcassa foi determinado em camundongos convencionais (CV) e isentos de germes (GF) alimentados com raçäo controle e com raçöes contendo 20 a 40% de sacarose. 2. Os camundongos GF na raçäo controle foram mais pesados do que os CV. Contudo, os camundongos CV alimentados com dieta com sacarose foram mais pesados que os GF. 3. Camundongos isentos de germes (GF) alimentados com dieta rica em sacarose mostraram um conteúdo lipídico menor na carcassa em relaçäo aos GF alimentados na raçäo controle e os outros CV. 4. Camundongos convencionais (CV) na dieta rica em sacarose foram mais pesados do que os animais CV na dieta controle. 5. O conteúdo aquoso na carcassa dos camundongos GF na dieta rica em sacarose foi maior do que nos controles GF e nos CV. 6. Esses resultados sugerem um efeito lipidogênico diferente da sacarose alimentar nos camundongos GF e CV

Immunocytochemical identification of Leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

O método da peroxidase-antiperoxidase foi utilizado para estudar as propriedades imunocitoquímicas de Leishmanias e de amastigotas do Trypanosoma cruzi, in situ, após os tecidos terem sido submetidos a diferentes tipos de fixaçäo. Anti-soros foram obtidos de coelhos cronicamente infectados com três cepas de T. cruzi ou imunizados com L. mexicana amazonensis e L. braziliensis guyanensis e aplicados nos cortes histológicos de 5*m de espessura. Os antígenos de T. cruzi foram coroados muito bem pelos três soros anti-T. cruzi e pelos dois soros anti-Leishmania com diluiçöes entre 1:1.000 e 1:2. Diferentemente, os antígenos de Leishmania foram revelados pelos soros anti-Leishmania somente em baixas diluiçöes, ou seja, entre 1:60 e 1:160 enquanto que os soros anti-T. cruzi, mesmo nestas diluiçöes baixas quando usados para revelar Leishmania. Embora näo haja explicaçäo clara para esta reaçäo imunocitoquímicacruzada "reversa-monodirecional" entre Leishmania e amastigotas de T. cruzi os resultados do presente trabalho mostram que anticorpos policlonais contra diferentes espécies de Leishmania, quando usados para detecçäo imunocitoquímica de Leishmania e T. cruzi in situ, reagem mais fortemente com amastigotas de T. cruzi do que com espécies homólogas