RESUMO
Bacteroides fragilis is a common colonic symbiote of which one subtype, enterotoxigenic Bacteroides fragilis (ETBF), causes inflammatory diarrhea. However, asymptomatic ETBF colonization is common. Through its primary virulence factor, B. fragilis toxin (BFT), ETBF causes asymptomatic, chronic colitis in C57BL/6 mice and increased colon tumorigenesis in multiple intestinal neoplasia mice. Human studies suggest an association between ETBF infection, inflammatory bowel disease, and colon cancer. Additional studies on ETBF epidemiology are, therefore, crucial. The goal of this study is to develop a reliable fecal diagnostic for ETBF. To develop a sensitive assay for ETBF, we tested multiple protocols on mouse stools spiked with serially diluted ETBF. Each assay was based on either touchdown or quantitative polymerase chain reaction (qPCR) and used primers targeted to bft to detect ETBF. Using touchdown PCR or qPCR, the mean ETBF detection limit was 1.55 × 10(6) colony-forming units (CFU)/g stool and 1.33 × 10(4) CFU/g stool, respectively. Augmentation of Bacteroides spp. growth in fecal samples using PYGB (Peptone Yeast Glucose with Bile) broth enhanced ETBF detection to 2.93 × 10(2) CFU/g stool using the touchdown PCR method and 2.63 × 10(2) CFU/g stool using the qPCR method. Fecal testing using combined culture-based amplification and bft touchdown PCR is a sensitive assay for the detection of ETBF colonization and should be useful in studying the role of ETBF colonization in intestinal diseases, such as inflammatory bowel disease and colon cancer. We conclude that touchdown PCR with culture-based amplification may be the optimal ETBF detection strategy, as it performs as well as qPCR with culture-based amplification, but is a less expensive technique.
Assuntos
Toxinas Bacterianas/metabolismo , Infecções por Bacteroides/diagnóstico , Bacteroides fragilis/isolamento & purificação , Enterotoxinas/metabolismo , Fezes/microbiologia , Animais , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/patogenicidade , Colite/microbiologia , Enterocolite/microbiologia , Neoplasias Gastrointestinais/microbiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mature B cells can alter their antibody repertoires by several mechanisms, including immunoglobulin heavy chain variable region (V(H)) replacement. This process changes the antigen combining site by replacing a portion of the original V(H)/diversity/heavy chain joining region (V(H)DJ(H)) rearrangement with a corresponding portion of a new V(H) segment. This exchange can involve cryptic heptamer-like sequences embedded in the coding regions of V(H) genes. While studying the B lymphocytes that expand in the synovial tissues of patients with rheumatoid arthritis (RA), clones with V(H)DJ(H) variants that were apparently generated by V(H) replacement were identified with surprising frequency (approximately 8%). Examples of multiple independent V(H) replacement events occurring in distinct progeny clones were also identified. These secondary V(H) rearrangements were documented at both the cDNA and genomic DNA levels and involved several heptamer-like sequences at four distinct locations within V(H) (three sites in framework region 3 and one in complementarity determining region 2). The identification of blunt-ended double-stranded DNA breaks at the embedded heptamers and the demonstration of recombinase activating gene (RAG) expression suggested that these rearrangements could occur in the synovial tissues, presumably in pseudo-germinal centers, and that they could be mediated by RAG in a recognition signal sequence-specific manner. The presence of V(H) mutations in the clones that had undergone replacement indicated that these B cells were immunocompetent and could receive and respond to diversification signals. A relationship between these secondary V(H) gene rearrangements and the autoimmunity characteristic of RA should be considered.
Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/genética , Região Variável de Imunoglobulina/genética , Membrana Sinovial/imunologia , Adulto , Sequência de Bases , Feminino , Biblioteca Gênica , Variação Genética , Articulação do Quadril , Humanos , Isotipos de Imunoglobulinas/genética , Articulação do Joelho , Masculino , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Alinhamento de SequênciaAssuntos
Subpopulações de Linfócitos B/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Células-Tronco Neoplásicas/patologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antígenos CD/análise , Antígenos de Diferenciação/análise , Antígenos de Neoplasias/análise , Antígenos CD5/análise , Diferenciação Celular , Divisão Celular , Transformação Celular Neoplásica/patologia , Replicação do DNA , DNA de Neoplasias/genética , Células-Tronco de Carcinoma Embrionário , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas , Centro Germinativo/patologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana , NAD+ Nucleosidase/análise , Telômero/ultraestruturaRESUMO
Human C5a is a potent chemoattractant for granulocytes, monocytes, and dendritic cells. In mice C5a has been shown to be chemotactic for germinal center (GC) B cells. To date, no information is available on the effects of C5a on human B cell locomotion. Here we demonstrate that rC5a increases polarization and migration of human tonsillar B cells. The locomotory response was due to both chemokinetic and chemotactic activities of rC5a. Moreover, memory and, at a lesser extent, naive B cell fractions from purified tonsillar populations displayed rC5a-enhanced migratory properties, whereas GC cells did not. Flow cytometry revealed C5aR (CD88) on approximately 40% memory and 10% naive cells, respectively, whereas GC cells were negative. Immunohistochemistry showed that a few CD88+ cells were of the B cell lineage and localized in tonsillar subepithelial areas, where the majority of memory B cells settle. Pretreatment of memory B cells with the CD88 mAb abolished their migratory responsiveness to rC5a. Finally, the C5 gene was found to be expressed in naive, GC, and memory B lymphocytes at both the mRNA and the protein level. This study delineates a novel role for C5a as a regulator of the trafficking of human memory and naive B lymphocytes and supports the hypothesis that the B cells themselves may serve as source of C5 in secondary lymphoid tissues.