RESUMO
BACKGROUND: Roux-en-Y gastric bypass surgery provides a novel human model to investigate small bowel mucosal innate immunity, in which there is loss of gastric acid-mediated protection against orally-acquired microorganisms. AIM: To study changes in jejunal mucosal immunoreactivity of human defensin (HD)-5, an antimicrobial peptide normally produced by Paneth cells. METHODS: Mucosal samples were obtained from 18 female patients (24-54 years), from the same segment of jejunum during and after gastric bypass surgery. Samples were used for bacterial culture and immunohistochemistry using anti-HD-5 antibody. The number of immunoreactive cells per crypt and villus were determined and expressed as mean (SD). RESULTS: No bacteria were cultured from any of the perioperative jejunal samples but colonies of bacteria normally present in the pharynx were identified during culture of all postoperative jejunal biopsy specimens (1->100 colonies). Paneth cell numbers per crypt were unchanged after gastric bypass (4.16 (0.71) vs 4.24 (0.78)). However, following surgery, there was an increase in HD-5-positive intermediate cells per crypt (0.25 (0.41) vs 1.12 (0.66), p<0.01), HD-5 staining enterocytes per crypt (0.03 (0.09) vs 1.38 (1.10), p<0.01), HD-5 staining material in the crypt lumen (crypt lumens: 5.0% (10.9%) vs 68.1% (27.9%), p<0.01) and HD-5 immunoreactivity coating the luminal surface of villus enterocytes (villi sampled: 15.0% (31.0%) vs 67.5% (42.0%), p<0.01). CONCLUSIONS: Bacteria normally resident in the pharynx were present in the proximal jejunal mucosa following Roux-en-Y gastric bypass surgery. After gastric bypass, there was increased secretion of HD-5 and an increase in HD-5 expressing intermediate cells and enterocytes in the crypt. The increase in HD-5 expression in the jejunal mucosa following gastric bypass surgery is likely to be secondary to exposure to orally-acquired microorganisms.
Assuntos
Derivação Gástrica , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , alfa-Defensinas/metabolismo , Adulto , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Contagem de Células , Feminino , Humanos , Técnicas Imunoenzimáticas , Mucosa Intestinal/microbiologia , Jejuno/microbiologia , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Muramidase/metabolismo , Celulas de Paneth/patologia , Faringe/microbiologia , Período Pós-OperatórioRESUMO
The gastrointestinal (GI) tract is exposed to a wide range of microorganisms. The expression of antimicrobial peptides has been demonstrated in different regions of the GI tract, predominantly in epithelial cells, which represent the first host cells with which the microorganisms have to interact for invasion. The intestinal epithelial monolayer is complex, consisting of different cell types, and most have a limited lifespan. Of the GI antimicrobial peptides, alpha- and beta-defensins have been studied the most and are expressed by distinct types of epithelial cells. Enteric alpha-defensin expression is normally restricted to Paneth and intermediate cells in the small intestine. However, there are important differences between mice and humans in the processing of the precursor forms of enteric alpha-defensins. Parasite infection induces an increase in the number of enteric alpha-defensin-expressing Paneth and intermediate cells in the murine small intestine. In the chronically inflamed colonic mucosa, metaplastic Paneth cells (which are absent in the normal colon) also express enteric alpha-defensins. Epithelial expression of beta-defensins may be constitutive or inducible by infectious and inflammatory stimuli. The production of some members of the beta-defensin family appears to be restricted to distinct parts of the GI tract. Recent studies using genetically manipulated rodents have demonstrated the likely in vivo importance of enteric antimicrobial peptides in innate host defense against microorganisms. The ability of these peptides to act as chemoattractants for cells of the innate- and adaptive-immune system may also play an important role in perpetuating chronic inflammation in the GI tract.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Fenômenos Fisiológicos do Sistema Digestório , Sequência de Aminoácidos , Animais , Mucosa Gástrica/fisiologia , Regulação da Expressão Gênica , Humanos , Mucosa Intestinal/fisiologia , Dados de Sequência Molecular , Peptídeos/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , alfa-Defensinas/genéticaRESUMO
The human intestinal tract is constantly exposed to an enormous indigenous bacterial flora. It has recently been recognised that antimicrobial peptides of the defensin family likely play a role in protection against microbial invasion at a variety of mucosal epithelial surfaces, including that of the intestinal tract. To date, six alpha-defensins have been identified in humans. Four of these, designated Human Neutrophil Peptides (HNP) 1,2,3 and 4, form part of the armoury of neutrophils, where they participate in systemic innate immunity. The remaining two, Human Defensin (HD) 5 and 6, are expressed in intestinal Paneth cells, and probably contribute to innate defense of the GI mucosal surface. Murine intestinal alpha-defensins (the 'cryptdins') have been extensively studied, but less is known about their human counterparts. The putative mature HD-5 was chemically synthesised and used to raise polyclonal antiserum. Using this anti-HD-5 antiserum, the expression of HD-5 in normal and inflamed intestinal mucosal samples was studied using immunohistochemistry. HD-5 is expressed in Paneth cells and also in some villous epithelial cells in normal duodenum, jejunum and ileum. HD-5 is not expressed in the normal stomach or colon. In cases of gastritis, colonic Crohn's disease and ulcerative colitis, HD-5 is expressed in metaplastic Paneth cells. Utilizing the anti-HD-5 antiserum, native HD-5 was isolated and purified from acid extracts of normal terminal ileal mucosal epithelial cells using cation exchange and reverse phase high pressure liquid chromatography. The purified peptide was characterised using N-terminal amino acid sequence and mass spectral analysis. Antimicrobial activity of the peptide was assessed using a sensitive colony forming unit antimicrobial assay. HD-5 is stored in the predicted precursor form in Paneth cells, and this form does not have antimicrobial activity against a defensin sensitive Salmonella. Potential processing of the precursor form of the HD-5 peptide into a mature active form, was studied by stimulating Paneth cell granule secretion in freshly isolated, cultured ileal crypts. A truncated form of the precursor form of HD-5, but not the predicted mature form, was present in the culture supernatant. Recently published studies suggest that further processing of the molecule occurs in vivo. The expression of HNP 1-3 in the normal intestinal mucosa and in cases of inflammatory bowel disease was studied. In the normal intestinal mucosa, HNP are expressed only in sparse lamina propria neutrophils, and not in Paneth cells. In cases of active ulcerative colitis and Crohn's disease, scattered surface epithelial cells, as well as numerous lamina propria neutrophils, were seen to express HNP. In conclusion, HD-5 is stored only in its precursor form in normal ileal Paneth cells, and partial processing of the peptide to a mature form occurs during and/or after secretion. In inflammatory bowel disease, HD-5 is expressed in metaplastic Paneth cells in the colon, and HNP is expressed by some surface epithelial cells. These studies suggest that antimicrobial defensin peptides may be important in protection of the host against microbial invasion in states of intestinal inflammation.
Assuntos
Trato Gastrointestinal/imunologia , alfa-Defensinas/imunologia , Sequência de Aminoácidos , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Celulas de Paneth/imunologiaRESUMO
The gastrointestinal tract is constantly exposed to microorganisms, mainly a large and complex population of bacteria resident in the colon and distal small intestine. Although the normal host relationship with the resident luminal bacteria is often mutually beneficial, the host also requires protection against these microorganisms. Epithelial cells play a critical role in mediating these protective responses and there is increasing appreciation of the likely importance of antimicrobial peptides of the defensin family that they express. The enteric alpha-defensins (human defensins [HD]5 and 6) are expressed by Paneth cells, which are normally confined to the small intestine, but are also seen in the colon in patients with inflammatory bowel disease. Studies have shown that HD5 is stored in Paneth cell granules in precursor form and requires processing to the mature peptide. Human beta-defensin (HBD)1 is constitutively expressed in intestinal epithelial cells, whereas the expression of HBD2 is induced in inflammatory bowel disease. HBDs have also been shown to be chemotactic for immature dendritic cells and memory T cells. Thus, they may not only mediate innate immunity, but also regulate adaptive immune responses in inflammatory bowel disease.
Assuntos
Defensinas/fisiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Inflamação , Peptídeos/química , alfa-Defensinas/biossíntese , beta-Defensinas/biossínteseRESUMO
The side-effects suitable for monitoring in patients with inflammatory bowel disease being treated with the four main groups of drugs (5-aminosalicylic acid preparations, azathioprine and 6-mercaptopurine, methotrexate, and corticosteroids) are reviewed. On the basis of the reported frequency, severity and timing of side-effects, a practical scheme of monitoring is recommended. This includes a baseline measurement of full blood count, creatinine and liver function tests in all patients. In the absence of worrying symptoms, we recommend the following: (i) no monitoring for sulfasalazine; (ii) for other 5-aminosalicylic acid preparations, the measurement of creatinine at 6 and 12 months and then annually; (iii) for azathioprine/6-mercaptopurine, thiopurine methyltransferase genotype/phenotype determination has no role in treatment monitoring, but a full blood count at 2 weeks, 1 month, 3 months and then every 3 months should be performed; (iv) for methotrexate, a full blood count and liver function tests should be performed every 3 months; (v) for steroids, dual energy X-ray absorptiometry bone scanning should be performed at the start of therapy, every year in which steroids are used if the T score is < 0, and every 3-5 years if the T score is > 0.
Assuntos
Monitoramento de Medicamentos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Corticosteroides/efeitos adversos , Ácidos Aminossalicílicos/efeitos adversos , Azatioprina/efeitos adversos , Contagem de Células Sanguíneas , Creatinina/análise , Testes de Função Hepática , Mercaptopurina/efeitos adversos , Metotrexato/efeitos adversosRESUMO
BACKGROUND/AIMS: The normal intestinal epithelium is increasingly being recognised as an important component of the mucosal innate protection against microorganisms. Human neutrophil defensins 1-3 (HNP 1-3) and lysozyme are components of the systemic innate immunity. The aim of this study was to investigate the expression of HNP 1-3 and lysozyme in normal and active inflammatory bowel disease (IBD) mucosa. METHODS: Mucosal tissue sections were studied by immunohistochemistry using antibodies to neutrophil defensins 1-3 and lysozyme. Extracts of purified intestinal epithelial cells were used for immunoblotting studies and antimicrobial activity against the phoP negative strain of Salmonella typhimurium. RESULTS: Surface epithelial cells strongly immunoreactive for neutrophil defensins and lysozyme were seen in active ulcerative colitis and Crohn's disease (but not normal or inactive IBD) mucosal samples. Many of these cells coexpressed both of the antimicrobial proteins. Immunoblotting studies confirmed the expression of neutrophil defensins in extracts of purified ulcerative colitis epithelial cells, which also demonstrated antimicrobial activity. CONCLUSION: HNP 1-3 and lysozyme are expressed in surface enterocytes of mucosa with active IBD and they may play an important role in intestinal host defence against luminal microorganisms.
Assuntos
Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , alfa-Defensinas/metabolismo , Extratos Celulares/imunologia , Colite Ulcerativa/imunologia , Contagem de Colônia Microbiana , Doença de Crohn/imunologia , Enterócitos/imunologia , Células Epiteliais/imunologia , Humanos , Técnicas Imunoenzimáticas , Muramidase/metabolismo , Neutrófilos/imunologia , Salmonella typhimurium/imunologiaRESUMO
A comparison was made between membrane filtration and centrifugation for the isolation of Legionella pneumophila from seeded water samples. Using samples of varying concentration, the optimum speed and time of centrifugation were determined and the relationship between the number of organisms present in the water and the proportion recovered was examined. Following this, sequential routine environmental waters were filtered and centrifuged in parallel. Centrifugation and filtration using nitrocellulose filters were found to be comparable. The optimum speed and time of centrifugation was approximately 6000 g for 10 min. There was a constant proportion of viable organisms recovered irrespective of the concentration in the unspun samples.
Assuntos
Legionella/isolamento & purificação , Microbiologia da Água , Centrifugação , FiltraçãoRESUMO
BACKGROUND AND AIMS: Intestinal epithelial cell derived antimicrobial peptides of the defensin family may play a major role in host defence against microorganisms. Our aims were to (i) isolate, characterise, and investigate the processing of human defensin 5 (HD-5) in normal Paneth cells and (ii) investigate expression of HD-5 in active inflammatory bowel disease (IBD). METHODS: Antiserum raised against chemically synthesised putative mature HD-5 was used for immunohistochemistry and purification of HD-5 from extracts of normal terminal ileal crypts. RESULTS: In normal and Crohn's disease terminal ileum, HD-5 immunoreactivity was seen in Paneth cells and in some villous epithelial cells. Normal colonic mucosa did not express HD-5 but HD-5 immunoreactivity was seen in cells in the colonic crypt region of many IBD samples. N-terminal amino acid sequence analysis of HD-5 purified from normal terminal ileal Paneth cells consistently showed the predicted sequence of the precursor form of the peptide. Following stimulation of isolated intact normal terminal ileal crypts, a truncated form of HD-5, with the N-terminal sequence GEDNQLAIS, was detected in the supernatant. CONCLUSIONS: (i) HD-5 is present only in the precursor form in normal terminal ileal Paneth cells and is processed to the mature form during and/or after secretion, (ii) some villous epithelial cells express HD-5, and (iii) HD-5 is expressed by metaplastic Paneth cells in the colon in IBD.