Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157: H7 / Resposta de anticorpos obtidas em camundongos imunizados com linhagens vacinais de Bacillus subtilis expressando a subunidade B da Stx2 de Escherichia coli O157: H7 enterohemorrágica

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Até o presente o momento, não há vacina ou imunoterapia disponível para pacientes com Síndrome Hemolítica Urêmica (SHU) induzida pela toxina Shiga-like (Stx) produzida por linhagens de Escherichia coli entero-hemorragica (EHEC), tais como as pertencentes ao sorotipo O157:H7. Neste trabalho, avaliamos a performance de Bacillus subtilis, uma espécie bacteriana gram-positiva não-patogênica formadora de esporos, como veículo vacinal para a expressão da subunidade B da Stx2B (Stx2B). Uma linhagem vacinal recombinante de B. subtilis expressando Stx2B, sob o controle de um promoter induzível por estresse, foi administrada a camundongos BALB/c por via oral, nasal ou subcutânea usando células vegetativas e esporos. Camundongos imunizados com células vegetativas e esporos pela via oral desenvolveram títulos anti-Stx2B baixos, mas específicos, de IgG sérico e IgA fecal, enquanto camundongos imunizados com esporos recombinates desenvolveram resposta anti-Stx2B apenas após a administração pela via parenteral. No entanto, anticorpos produzidos em camundongos imunizados com a linhagem recombinante de B. subtilis não inibiram os efeitos tóxicos da toxina nativa em condições in vitro e in vivo, sugerindo que a quantidade e/ou a qualidade da resposta imune gerada não suportam uma neutralização efetiva da Stx2 produzidas por linhagens de EHEC.

Activación de la respuesta inmune innata y específica en los pacientes con sindrome urémico hemolítico: papel dual a traves de la paticipacion en los mecanismos pagogenicos y protectores / Activation of innate and specific immune responses in hemolytic uremic syndrome (HUS)-patients

The central role of the immune system is the preservation of the health against several pathogenic microbes and injury agents. However, on special conditions defensive mechanisms triggered towards the foreign agent can damage the host. Clinical and experimental evidence indicate that inflammatory reaction triggered by the main components of Shiga toxin (Stx)-producing Escherichia coil (STEC), participate in the evolution to the complete form of HUS. When children are diagnosed of HUS, they present evidence that have suffered a very strong and early inflammatory response. These features include: the presence of a marked neutrophilia, the polymorfonuclear leucocytes (PMN) are "deactivated or exhausted" and the monocytes are differentiated towards an inflammatory phenotype (CD14-reduced and CD16-enhanced membrane expression). In addition, HUS-patients show a marked reduction in the absolute and relative number of leucocytes carrying the receptor (CX3CR1) for the chemokine "Fractalkine" (FKN, CX3CL1), which are the classic monocytes and Natural Killer cells (NK). All these cells express a high cytotoxic potencial. The chemokine FKN is expressed in endothelial and epithelial renal cells, and is involved in the pathogenic mechanism of different nephropathies. Noteworthy, we found a significant correlation between the severity of the renal damage (as days of anuria) and the alterations described above. Finally, the protective role of specific immune response, mainly through the antibody production with Stx-neutralizing capacity, is discussed.

Detección de la actividad de desoxiribonucleasa (DNasa) en cepas de Clostridium chauvoei / [Deoxyribonuclease activity detection in Clostridium chauvoei strains]

Beta toxin of C. chauvoei has desoxiribonuclease (DNase) activity which is regarded as one of its virulence factors. The production of DNase was detected in strains isolated from bovines, using as controls C. chauvoei ATCC 10092, and C. perfringens Type A and C. septicum, both laboratory isolates. The enzyme activity was made evident on a DNA substrate observing the macroscopic degradation. A simple methodology was developed using a commercial medium for DNase test, with the incorporation of sterile horse serum. Each strain was streaked on the surface of the medium, incubated in anaerobic atmosphere at 37 degrees C for 48 hours. The plates were revealed with HCI 1 N. The appearance of a clear and transparent zone around and under the microbial growing was considered a positive reaction. Enzyme activity was detected in 10 of 12 strains and also in the controls. The serum addition to the commercial basal medium allows the optimum development of the microorganism showing the enzymatic digestion zone.