Even though anti‐cancer chemotherapy has been continuously improved during the last decades. problems with adverse effects and drug resistance still constitutes a considerable obstacle and sets a demand for new effective treatment options. Tissue homeostasis in multi‐cellular organisms is maintained through intrinsic cell death, apoptosis, which removes unwanted or damaged cells. Disrupted apoptosis is an important factor in tumorgenesis and drug resistance, therefore induction or restoration of apoptotic pathways is also important for the treatment of cancer. Several naturally occurring bacterial toxins have the ability to induce apoptosis and could thus be candidates to complement or improve the therapeutic effect of other anticancer drugs. The bacterial toxins, adenylate cyclase (AC) toxin from Bordetella pertussis, α‐toxin from Staphylococcus aureus and verotoxin‐1 (VT‐1) from Escherichia coli were investigated for their ability to induce apoptosis in different tumor cell lines. Toxin induction of cell death was investigated by cell viability assays, end‐stage apoptosis induction by DNA‐fregmentation (TUNEL) assay. Toxin receptor expression and signal transduction pathways to apoptosis were investigated by flow cytometry, caspase enzyme activity assays and western blot. Immunohistochemistry was used for identification of toxin receptor expression in tumor tissue samples. AC‐toxin was cytotoxic and induced apoptosis in cultured malignant plural mesothelioma (MPM) and small‐cell lung cancer (SCLC) cells. Low‐toxic concentrations of AC‐toxin enhanced cisplatin cytotoxicity and apoptosis in both cell lines. MPM‐cells with acquired cisplatin resistance were more sensitive to α‐toxin than the less resistant parental MPM cell line. A low‐toxic concentration of α‐toxin re‐sensitized resistant MPM cells to cisplatin cytotoxicity by apoptosis induced through the mitochondrial pathway without detectable activation of common up‐stream apoptosis signalling proteins. VT‐1 was highly cytotoxic and induced apoptosis in globotriosylceramide (Gb3) ‐expressing glioma, breast cancer and non‐small‐cell lung cancer (NSCLC) cells but was not cytotoxic to non‐Gb3‐expressing cells. PPMP, an inhibitor of glucosylceramide synthesis which makes exposed cells unable to synthesize Gb3 rendered Gb3‐expressing cells resistant to VT‐1. MPM cells with acquired‐cisplatin resistance expressed Gb3 in contrast to the absent of expression in the less resistant parental cell line. Gb3, could however be up‐regulated by cisplatin in Gb3‐negative MPM‐cells. Presence of a low‐toxic concentration of VT‐1 potentiated cisplatin‐induced cytotoxicity and apoptosis in the cisplatin‐resistance MPM cell line. VT‐1 was a potent inducer of apoptosis, probably via stress‐induced Mitogen‐activated protein kinase (MAPK)‐signaling involving c‐Jun N‐terminal kinase (JNK) and p38, leading to disruption of the mitochondrial membrane integrety, activation of caspase‐9 and ‐3, and ultimately DNA fragmentation and cell death. Gb3 expression was demonstrated in clinical specimens of glioblastoma and breast cancer making these tumor types interesting for further VT‐1 studies. We conclude that bacterial toxins may be used to induce apoptosis in several types of cancer cells. Low concentrations of verotoxin‐1 and α‐toxin may potentially be used to overcome acquired cisplatin‐resistance in cancer patients.

O Clostridium perfringens é um microrganismo anaeróbio que está presente no solo e no trato intestinal dos mamíferos. Provoca intoxicação alimentar nos seres humanos, doenças enterotoxêmicas nos animais domésticos e gangrena gasosa em ambos os grupos. O C. perfringens é classificado em cinco tipos (A, B, C, D e E) mediante a produção de quatro toxinas principais (alfa, beta, épsilon e iota). Neste trabalho foi possível padronizar a técnica de PCR para detectar a presença dos genes cpa, cpb e etx a partir de culturas de C. perfringens. A sensibilidade analítica da técnica de PCR a partir de culturas de C. perfringens foi de 2,27 ng/µL para o gene cpa, 22,7 pg/µL para o gene cpb e 22,7 pg/µL para o gene etx. A pesquisa dos genes cpa, cpb e etx partir de 35 amostras de C. perfringens isoladas de bovinos revelou que 16 (45,7%) eram do tipo A; 18 (51,4%) eram do tipo C e 1 (2,9%) era do tipo B. Não foi observada nenhuma amostra do tipo D. A metodologia de PCR revelou-se útil na tipificação de amostras de C. perfringens isoladas de bovinos, contribuindo para o diagnóstico dessa bacteriose neste país, eliminando as dificuldades de tipificação oriundas do alto custo e da indisponibilidade de anti-soros para a tipificação pela reação de soroneutralização e evitando a utilização de animais de laboratório. / Clostridium perfringens is an anaerobic micro-organism that is present in the soil and gastrointestinal tract of mammals. It causes food poisoning in humans, enterotoxemic diseases in domestic animals and gas gangrene in both. C. perfringens is classified into five types (A, B, C, D and E) according to the production of four major toxins (alpha, beta, epsilon and iota). In this trial was possible to standardize the PCR?s technique to detect cpa, cpb and etx genes from cultures of C. perfringens. PCR?s analythical sensibility was 2.27 ng/µL for cpa gene, 22.7 pg/µL for cpb gene and 22.7 pg/µL for etx gene. The research of cpa, cpb and etx genes from 35 samples of C. perfringens isolated from cattle reveals that 16 (45.7%) were classified as type A, 18 (51.4%) as type C and 1 (2.9%) as type B. No sample of type D was observed. PCR?s technique reveals to be usefull to typify samples of C. perfringens isolated from cattle, contributing to diagnose of this bacterial disease in this country and solving typifing problems represented by the high costs of the process and by the lack of antiserum that is required to typify the micro-organism by seroneutralization. PCR?s technique avoid the use of laboratory animals, too.

No description available.

A bovinocultura brasileira possui grande ênfase no mercado nacional. Doenças que acometem rebanhos comprometem o mercado além de gerarem grandes prejuízos econômicos. O Clostridium novyi tipo B provoca necrose hepática em bovinos através da produção da alfa toxina, uma potente exotoxina que reduz a produtividade através de alterações como hemoglobinúria, redução do apetite, febre, letargia, diminuição da produção de leite e sangue nas fezes. Conter o microrganismo causador torna-se uma busca necessária tanto do ponto de vista econômico quanto social. Entretanto, o controle da doença ainda é realizado por vacinas formuladas com múltiplos antígenos. A emulsão pode ser uma alternativa promissora para a melhoria da adsorção de antígenos nas formulações vacinais. Camundongos da linhagem Swiss foram utilizados a fim de se avaliar aspectos clínicos e validar resultados referentes a composição de uma nova formulação vacinal contendo adjuvante Montanide ISA 61 VG e antígeno inativado de C. novyi. Os testes de caracterização e antigenicidade indicaram a presença da proteína alfa toxina na composição avaliada. A imunogenicidade do antígeno inativado em emulsão W/O (água/óleo) foi verificada e a proporção empregada (40/60) mostrou ser ideal no uso de múltiplos antígenos, apresentando inocuidade, estabilidade do produto, liberação controlada e estímulo da resposta imune. A determinação da concentração de antígeno foi averiguada pela relação antígeno ativo e inativado com soros de animais doentes, visto a eficácia vacinal de 40%. A adequação da concentração de alfa toxina inativada na emulsão mostrou ser necessária para atingir melhores valores de proteção animal. Análises de hemograma, bioquímicas e morfologia de fígado, baço e coxa contribuíram para elucidar os efeitos da emulsão e comprovar necrose hepática nos grupos não imunizados, além de sugerir avanços na adsorção de vacinas. Os resultados possibilitaram o estabelecimento de um modelo murino de infecção de C. novyi com futuras aplicações relacionadas à produção vacinal com múltiplos antígenos emulsificados para controle das clostridioses. / Brazilian cattle breeding has great emphasis on the national market. Diseases that affect herds compromise the market as well as generate great economic losses. Clostridium novyi type B causes hepatic necrosis in cattle through the production of alpha toxin, a potent exotoxin that reduces productivity through changes such as hemoglobinuria, reduced appetite, fever, lethargy, decreased milk and stool production. Containing the causative micro-organism becomes a necessary quest both economically and socially. However, control of the disease is still performed by vaccines formulated with multiple antigens. The emulsion may be a promising alternative for the improvement of antigen adsorption in vaccine formulations. Swiss strain mice were used to evaluate clinical aspects and validate results regarding the composition of a new vaccine formulation containing Montanide ISA 61 VG adjuvant and C. novyi inactivated antigen. Characterization and antigenicity tests indicated the presence of the alpha toxin protein in the evaluated composition. The immunogenicity of antigen inactivated in W / O emulsion (water / oil) was verified and the ratio employed (40/60) showed to be ideal in the use of multiple antigens, presenting innocuousness, product stability, controlled release and stimulation of the immune response. The determination of the antigen concentration was investigated by the active antigen ratio and inactivated with sera from sick animals, since the vaccine efficacy was 40%. The suitability of the inactivated alpha toxin concentration in the emulsion was shown to be necessary to achieve better animal protection values. Hemogram, biochemical and liver, spleen and thigh morphology contributed to elucidate the effects of the emulsion and to verify hepatic necrosis in the nonimmunized groups, in addition to suggesting advances in the adsorption of vaccines. The results allowed the establishment of a murine model of C. novyi infection with future applications related to the vaccine production with multiple emulsified antigens to control clostridia.