RESUMO
A clinical isolate of Campylobacter jejuni, previously found to produce a toxin active in cell culture assays, was used for identification and characterisation of a cytotoxic porin-lipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by high-performance liquid chromatography of crude concentrated culture supernate and DEAE-anion exchange chromatography. The complex had a toxic activity of 20.1 tissue culture dose50 (TCD50)/microg of protein for HEp-2 cells, 7.49 TCD50/microg of protein for HeLa cells and 1.87 TCD50/microg of protein for Chinese hamster ovary cells. Analysis by SDS-PAGE revealed a single protein band of 45 kDa and a high mol. wt carbohydrate moiety. The complex gave a positive result in the Limulus amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins Galanthus nivalis agglutinin, Maackia amurensis agglutinin and Datura stramonium agglutinin. The cytotoxic activity associated with the complex was heat-labile at 70 degrees C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperiodate and the glycosidases neuraminidase and N-glycosidase F. Sequencing of the N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C. jejuni. The cytotoxic activity of the complex was neutralised by a polyclonal, homologous antiserum, which reacted on Western blot with the 45-kDa protein, but not by polyclonal antisera raised against a number of other bacterial toxins.
Assuntos
Campylobacter jejuni/metabolismo , Citotoxinas/química , Lipopolissacarídeos/química , Porinas/química , Sequência de Aminoácidos , Animais , Western Blotting , Células CHO , Campylobacter jejuni/genética , Sequência de Carboidratos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cricetinae , Citotoxinas/isolamento & purificação , Citotoxinas/toxicidade , DNA Bacteriano/análise , Relação Dose-Resposta a Droga , Galanthus , Células HeLa , Humanos , Teste do Limulus , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/toxicidade , Dados de Sequência Molecular , Testes de Neutralização , Reação em Cadeia da Polimerase , Porinas/isolamento & purificação , Porinas/toxicidade , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Samples of human plasma from anthrax vaccine adsorbed (AVA, BioThrax)-vaccinated individuals were used to demonstrate passive protection of A/J mice from a lethal challenge with the Sterne strain of anthrax bacteria. The maximum concentration of human anti-protective antigen IgG in mouse sera 24 h after injection of 260 microg of anti-PA IgG was 134 microg/ml, declining to 91 microg/ml at 72 h (half-life=101.7 h). Mice showed significant survival (pAssuntos
Vacinas contra Antraz/imunologia
, Antraz/prevenção & controle
, Soros Imunes/administração & dosagem
, Soros Imunes/imunologia
, Animais
, Anticorpos Antibacterianos/sangue
, Feminino
, Humanos
, Imunização Passiva
, Camundongos
, Testes de Neutralização
, Estatística como Assunto
, Análise de Sobrevida