RESUMO
The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal infection with pathogens increase the expression of Reg3g and Reg3b in the murine ileum. Reg3g is directly bactericidal for gram-positive bacteria, but the exact role of Reg3b in bacterial infections is unknown. To investigate the possible protective role of Reg3b in intestinal infection, Reg3b knockout (Reg3b(-/-)) mice and wild-type (WT) mice were orally infected with gram-negative Salmonella enteritidis or gram-positive Listeria monocytogenes. At day 2 after oral Listeria infection and at day 4 after oral Salmonella infection, mice were sacrificed to collect intestinal and other tissues for pathogen quantification. Protein expression of Reg3b and Reg3g was determined in intestinal mucosal scrapings of infected and noninfected mice. In addition, ex vivo binding of ileal mucosal Reg3b to Listeria and Salmonella was investigated. Whereas recovery of Salmonella or Listeria from feces of Reg3b(-/-) mice did not differ from that from feces of WT mice, significantly higher numbers of viable Salmonella, but not Listeria, bacteria were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the Reg3b(-/-) mice than from those of WT mice. Mucosal Reg3b binds to both bacterial pathogens and may interfere with their mode of action. Reg3b plays a protective role against intestinal translocation of the gram-negative bacterium S. enteritidis in mice but not against the gram-positive bacterium L. monocytogenes.
Assuntos
Trato Gastrointestinal/imunologia , Listeriose/imunologia , Proteínas/imunologia , Proteínas/metabolismo , Salmonelose Animal/imunologia , Animais , Feminino , Trato Gastrointestinal/microbiologia , Deleção de Genes , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Fígado/microbiologia , Linfonodos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas a Pancreatite , Proteínas/genética , Salmonella enteritidis/imunologia , Salmonella enteritidis/patogenicidade , Baço/microbiologiaRESUMO
Perturbation of the intestinal microbiota by antibiotics predisposes the host to food-borne pathogens like Salmonella. The effects of antibiotic treatment on intestinal permeability during infection and the efficacy of dietary components to improve resistance to infection have not been studied. Therefore, we investigated the effect of clindamycin on intestinal barrier function in Salmonella-infected rats. We also studied the ability of dietary calcium and tannic acid to protect against infection and concomitant diarrhea and we assessed intestinal barrier function. Rats were fed a purified control diet including the permeability marker chromium EDTA (CrEDTA) (2 g/kg) or the same diet supplemented with calcium (4.8 g/kg) or tannic acid (3.75 g/kg). After adaptation, rats were orally treated with clindamycin for 4 d followed by oral infection with Salmonella enteritidis. Two additional control groups were not treated with antibiotics and received either saline or Salmonella. Urine and feces were collected to quantify intestinal permeability, diarrhea, cytotoxicity of fecal water, and Salmonella excretion. In addition, Salmonella translocation was determined. Diarrhea, CrEDTA excretion, and cytotoxicity of fecal water were higher in the clindamycin-treated infected rats than in the non-clindamycin-treated infected control group. Intestinal barrier function was less in the Salmonella-infected rats pretreated with antibiotics compared with the non-clindamycin- treated rats. Both calcium and tannic acid reduced infection-associated diarrhea and inhibited the adverse intestinal permeability changes but did not decrease Salmonella colonization and translocation. Our results indicate that calcium protects against intestinal changes due to Salmonella infection by reducing luminal cytotoxicity, whereas tannic acid offers protection by improving the mucosal resistance.
Assuntos
Antibacterianos/farmacologia , Cálcio da Dieta/administração & dosagem , Mucosa Intestinal/efeitos dos fármacos , Infecções por Salmonella/patologia , Taninos/administração & dosagem , Animais , Mucosa Intestinal/microbiologia , Masculino , Ratos , Ratos WistarRESUMO
An increased intestinal permeability is associated with several diseases. Nutrition can influence gut permeability. Previously, we showed that dietary Ca decreases whereas dietary short-chain fructo-oligosaccharides (scFOS) increase intestinal permeability in rats. However, it is unknown how and where in the gastrointestinal tract Ca and scFOS exert their effects. Rats were fed a Western low-Ca control diet, or a similar diet supplemented with either Ca or scFOS. Lactulose plus mannitol and Cr-EDTA were added to the diets to quantify small and total gastrointestinal permeability, respectively. Additionally, colonic tissue was mounted in Ussing chambers and exposed to faecal water of these rats. Dietary Ca immediately decreased urinary Cr-EDTA excretion by 24 % in Ca-fed rats compared with control rats. Dietary scFOS increased total Cr-EDTA permeability gradually with time, likely reflecting relatively slow gut microbiota adaptations, which finally resulted in a 30 % increase. The lactulose:mannitol ratio was 15 % higher for Ca-fed rats and 16 % lower for scFOS-fed rats compared with control rats. However, no dietary effect was present on individual urinary lactulose and mannitol excretion. The faecal waters did not influence colonic permeability in Ussing chambers. In conclusion, despite effects on the lactulose:mannitol ratio, individual lactulose values did not alter, indicating that diet did not influence small-intestinal permeability. Therefore, both nutrients affect permeability only in the colon: Ca decreases, while scFOS increase colonic permeability. As faecal water did not influence permeability in Ussing chambers, probably modulation of mucins and/or microbiota is important for the in vivo effects of dietary Ca and scFOS.
Assuntos
Cálcio da Dieta/farmacologia , Colo/efeitos dos fármacos , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Animais , Colo/metabolismo , Dieta , Fezes/química , Masculino , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Wistar , Água/análiseRESUMO
BACKGROUND: Dietary polyunsaturated fatty acids (PUFA), in particular the long chain marine fatty acids docosahexaenoic (DHA) and eicosapentaenoic (EPA), are linked to many health benefits in humans and in animal models. Little is known of the molecular response to DHA and EPA of the small intestine, and the potential contribution of this organ to the beneficial effects of these fatty acids. Here, we assessed gene expression changes induced by DHA and EPA in the wildtype C57BL/6J murine small intestine using whole genome microarrays and functionally characterized the most prominent biological process. RESULTS: The main biological process affected based on gene expression analysis was lipid metabolism. Fatty acid uptake, peroxisomal and mitochondrial beta-oxidation, and omega-oxidation of fatty acids were all increased. Quantitative real time PCR, and -- in a second animal experiment -- intestinal fatty acid oxidation measurements confirmed significant gene expression differences and showed in a dose-dependent manner significant changes at biological functional level. Furthermore, no major changes in the expression of lipid metabolism genes were observed in the colon. CONCLUSION: We show that marine n-3 fatty acids regulate small intestinal gene expression and increase fatty acid oxidation. Since this organ contributes significantly to whole organism energy use, this effect on the small intestine may well contribute to the beneficial physiological effects of marine PUFAs under conditions that will normally lead to development of obesity, insulin resistance and diabetes.
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Intestino Delgado/metabolismo , Metabolismo dos Lipídeos , Animais , Colo/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Fenótipo , Regiões Promotoras Genéticas , RNA/análise , Fatores de Transcrição/metabolismoRESUMO
We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1beta. In addition, colonic mucosal gene expression of individual rats was analyzed using whole-genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1beta levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD.
Assuntos
Cálcio da Dieta/uso terapêutico , Cálcio/uso terapêutico , Colite/tratamento farmacológico , Diarreia/tratamento farmacológico , Matriz Extracelular/efeitos dos fármacos , Absorção Intestinal/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Cálcio/farmacologia , Cálcio da Dieta/farmacologia , Colite/genética , Colite/metabolismo , Colo/efeitos dos fármacos , Colo/imunologia , Colo/metabolismo , Diarreia/metabolismo , Suplementos Nutricionais , Modelos Animais de Doenças , Ácido Edético/administração & dosagem , Ácido Edético/urina , Fezes , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Antígeno HLA-B27/genética , Interleucina-1beta/metabolismo , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Permeabilidade/efeitos dos fármacos , Pró-Colágeno/genética , Pró-Colágeno/metabolismo , Ratos , Ratos TransgênicosRESUMO
BACKGROUND: Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.Rats were fed a control diet or the same diet supplemented with buthionine sulfoximine (BSO; glutathione depletion) or cystine (glutathione maintenance). Inert chromium ethylenediamine-tetraacetic acid (CrEDTA) was added to the diets to quantify intestinal permeability. At day 4 after oral gavage with Salmonella enteritidis (or saline for non-infected controls), Salmonella translocation was determined by culturing extra-intestinal organs. Liver and ileal mucosa were collected for analyses of glutathione, inflammation markers and oxidative damage. Faeces was collected to quantify diarrhoea. RESULTS: Glutathione depletion aggravated ileal inflammation after infection as indicated by increased levels of mucosal myeloperoxidase and interleukin-1beta. Remarkably, intestinal permeability and Salmonella translocation were not increased. Cystine supplementation maintained glutathione in the intestinal mucosa but inflammation and oxidative damage were not diminished. Nevertheless, cystine reduced intestinal permeability and Salmonella translocation. CONCLUSION: Despite increased infection-induced mucosal inflammation upon glutathione depletion, this tripeptide does not play a role in intestinal permeability, bacterial translocation and diarrhoea. On the other hand, cystine enhances gut barrier function by a mechanism unlikely to be related to glutathione.
Assuntos
Translocação Bacteriana/fisiologia , Glutationa/fisiologia , Mucosa Intestinal/fisiologia , Salmonelose Animal/fisiopatologia , Animais , Translocação Bacteriana/efeitos dos fármacos , Butionina Sulfoximina/farmacologia , Cistina/administração & dosagem , Cistina/farmacologia , Diarreia/etiologia , Diarreia/fisiopatologia , Suscetibilidade a Doenças , Glutationa/antagonistas & inibidores , Ileíte/fisiopatologia , Interleucina-1beta/análise , Lipopolissacarídeos/toxicidade , Fígado/metabolismo , Masculino , Óxido Nítrico/metabolismo , Estresse Oxidativo , Peroxidase/análise , Ratos , Ratos Wistar , Salmonelose Animal/complicações , Salmonelose Animal/microbiologia , Salmonella enteritidis/fisiologia , Organismos Livres de Patógenos EspecíficosRESUMO
BACKGROUND: Microbial infections induce ileal pancreatitis-associated protein/regenerating gene III (PAP/RegIII) mRNA expression. Despite increasing interest, little is known about the PAP/RegIII protein. Therefore, ileal mucosal PAP/RegIII protein expression, localization, and fecal excretion were studied in rats upon Salmonella infection. RESULTS: Salmonella infection increased ileal mucosal PAP/RegIII protein levels in enterocytes located at the crypt-villus junction. Increased colonization and translocation of Salmonella was associated with higher ileal mucosal PAP/RegIII levels and secretion of this protein in feces. CONCLUSIONS: PAP/RegIII protein is increased in enterocytes of the ileal mucosa during Salmonella infection and is associated with infection severity. PAP/RegIII is excreted in feces and might be used as a new and non-invasive infection marker.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Fezes/química , Íleo/metabolismo , Lectinas Tipo C/metabolismo , Salmonelose Animal/metabolismo , Salmonella enteritidis/patogenicidade , Animais , Antígenos de Neoplasias/genética , Translocação Bacteriana , Biomarcadores/metabolismo , Biomarcadores Tumorais/genética , Cálcio da Dieta/metabolismo , Modelos Animais de Doenças , Ingestão de Alimentos , Enterócitos/metabolismo , Enterócitos/microbiologia , Fezes/microbiologia , Ileíte/metabolismo , Ileíte/microbiologia , Íleo/microbiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Lectinas Tipo C/genética , Masculino , Proteínas Associadas a Pancreatite , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Salmonelose Animal/microbiologia , Índice de Gravidade de Doença , Fatores de TempoRESUMO
BACKGROUND: Dietary non-digestible carbohydrates stimulate the gut microflora and are therefore presumed to improve host resistance to intestinal infections. However, several strictly controlled rat infection studies showed that non-digestible fructo-oligosaccharides (FOS) increase, rather than decrease, translocation of Salmonella towards extra-intestinal sites. In addition, it was shown that FOS increases intestinal permeability already before infection. The mechanism responsible for this adverse effect of FOS is unclear. Possible explanations are altered mucosal integrity due to changes in tight junctions or changes in expression of defense molecules such as antimicrobials and mucins. To examine the mechanisms underlying weakening of the intestinal barrier by FOS, a controlled dietary intervention study was performed. Two groups of 12 rats were adapted to a diet with or without FOS. mRNA was collected from colonic mucosa and changes in gene expression were assessed for each individual rat using Agilent rat whole genome microarrays. RESULTS: Among the 997 FOS induced genes we observed less mucosal integrity related genes than expected with the clear permeability changes. FOS did not induce changes in tight junction genes and only 8 genes related to mucosal defense were induced by FOS. These small effects are unlikely the cause for the clear increase in intestinal permeability that is observed. FOS significantly increased expression of 177 mitochondria-related genes. More specifically, induced expression of genes involved in all five OXPHOS complexes and the TCA cycle was observed. These results indicate that dietary FOS influences intestinal mucosal energy metabolism. Furthermore, increased expression of 113 genes related to protein turnover, including proteasome genes, ribosomal genes and protein maturation related genes, was seen. FOS upregulated expression of the peptide hormone proglucagon gene, in agreement with previous studies, as well as three other peptide hormone genes; peptide YY, pancreatic polypeptide and cholecystokinin. CONCLUSION: We conclude that altered energy metabolism may underly colonic barrier function disruption due to FOS feeding in rats.
Assuntos
Colo/metabolismo , Carboidratos da Dieta/farmacologia , Genes Mitocondriais , Oligossacarídeos/farmacologia , Animais , Peso Corporal , Carboidratos da Dieta/administração & dosagem , Ingestão de Alimentos/genética , Expressão Gênica , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Masculino , Oligossacarídeos/administração & dosagem , Permeabilidade , Ratos , Ratos WistarRESUMO
BACKGROUND: Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point). As fructo-oligosaccharides (FOS) affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. RESULTS: Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase), antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2), inflammation (e.g. calprotectin), oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2) and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9). Furthermore, Salmonella translocation increased serum IFN gamma and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap), showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. CONCLUSION: We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in mucosal gene expression.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Expressão Gênica , Intestino Delgado/metabolismo , Lectinas Tipo C/metabolismo , Salmonella enteritidis/fisiologia , Administração Oral , Animais , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Intestino Delgado/microbiologia , Lectinas Tipo C/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Associadas a Pancreatite , Ratos , Salmonelose Animal/microbiologia , Salmonella enteritidis/química , Salmonella enteritidis/genética , Salmonella enteritidis/imunologiaRESUMO
PURPOSE: To conduct clinical and genetic studies in a European family with autosomal dominant Stargardt-like macular dystrophy (adSTGD-like MD) and to investigate the functional consequences of a novel ELOVL4 mutation. METHODS: Ophthalmic examination and mutation screening by direct sequencing of the ELOVL4 gene was performed in two affected individuals. Wild-type and mutant ELOVL4 genes were expressed as enhanced green fluorescent protein (EGFP) fusion proteins in transient transfection in NIH-3T3 and HEK293 cells. To determine the subcellular localization of ELOVL4, an endoplasmic-reticulum (ER)-specific marker for pDsRed2-ER was cotransfected with ELOVL4 constructs. Transfected cells were viewed by confocal microscopy. Western blot analysis was performed to assess protein expression using an anti-GFP antibody. RESULTS: Affected patients exhibited macular atrophy with surrounding flecks characteristic of adSTGD-like MD. A novel ELOVL4 p.Tyr270X mutation was detected in affected individuals. In cell-transfection studies, wild-type ELOVL4 localized preferentially to the ER. In contrast, the mutant protein appeared to be mislocalized within transfected cells. CONCLUSIONS: In a European family with adSTGD-like MD, a novel ELOVL4 mutation was found to underlie the disorder. Transfection studies indicated that, unlike wild-type ELOVL4, the mutant protein does not localize to the ER but rather appears to be sequestered elsewhere in an aggregated pattern in the cytoplasm. Further analysis of the function of normal and mutant ELOVL4 will provide insight into the mechanism of macular degeneration.
Assuntos
Proteínas do Olho/genética , Genes Dominantes , Degeneração Macular/genética , Proteínas de Membrana/genética , Mutação , Adolescente , Adulto , Atrofia , Western Blotting , Linhagem Celular , Códon de Terminação , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas do Olho/metabolismo , Feminino , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes , Macula Lutea/patologia , Degeneração Macular/patologia , Degeneração Macular/fisiopatologia , Proteínas de Membrana/metabolismo , Microscopia Confocal , Linhagem , Distribuição Tecidual , Transfecção , Tirosina , Acuidade VisualRESUMO
OBJECTIVE: Research on dietary modulation of inflammatory bowel disease is in its infancy. Dietary heme, mimicking red meat, is cytotoxic to colonic epithelium and thus may aggravate colitis. Alternatively, heme-induced colonic stress might also result in potential protective heat-shock proteins (HSPs). Therefore, we investigated the effect of dietary heme on trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. METHODS: Rats were fed a high-fat control diet or a similar diet supplemented with heme. After dietary adaptation, rats were rectally infused with TNBS for colitis induction or saline for sham treatment. Colitis severity was evaluated and several markers were quantified in colonic mucosa isolated 1 wk after colitis induction. Furthermore, cytotoxicity of fecal water and serum α-1-acid glycoprotein were measured. RESULTS: Dietary heme increased cytotoxicity of the fecal water. Heme-fed sham-treated rats had higher colonic HSP-25 and heme-oxygenase-1 mRNA levels, which was confirmed by immunohistochemistry. HSP induction by heme was associated with decreased protein levels of myeloperoxidase and interleukin-1ß after subsequent TNBS infusion. However, no dietary effects were observed on histologic colitis score. Furthermore, body weight gain, colon length, and food intake were lower and α-1-acid glycoprotein concentrations were higher in heme-fed colitic rats. In addition, somatostatin, involved in mucosal repair, was not changed with TNBS infusion in heme-fed rats. CONCLUSION: Dietary heme adversely affects colitis, despite HSP induction. We speculate that the irritating influence of dietary heme, being continuously present in the colon, impairs recovery after colitis induction. A diet high in red meat might be a risk factor for inflammatory bowel disease development.
Assuntos
Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Proteínas de Choque Térmico/metabolismo , Heme/efeitos adversos , Inflamação/induzido quimicamente , Animais , Biomarcadores , Colo/metabolismo , Colo/patologia , Enterobacter/metabolismo , Heme Oxigenase-1/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Interleucina-1beta/análise , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Lactobacillaceae/metabolismo , Masculino , Peroxidase/análise , Ratos , Ratos Wistar , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
BACKGROUND: Oxidative stress is presumed to play an important role in inflammatory bowel disease (IBD). Accordingly, antioxidant supplementation might be protective. Dietary calcium inhibited colitis development in HLA-B27 transgenic rats, an animal model mimicking IBD. As antioxidants might act at mucosa level and calcium predominantly in the gut lumen, we hypothesize that the combination has additive protective effects on colitis development. METHODS: HLA-B27 rats were fed a control diet or the same diet supplemented with the antioxidants glutathione, vitamin C, and vitamin E, or supplemented with both antioxidants and calcium. Oxidative stress in colonic mucosa, colonic inflammation, intestinal permeability, and diarrhea were quantified. RESULTS: Intestinal permeability, diarrhea, myeloperoxidase, and interleukin-1ß levels were significantly lower in rats fed both antioxidants and calcium compared to rats supplemented with antioxidants only. No beneficial effects were observed in rats fed the diet supplemented with antioxidants only. Strikingly, despite extremely low colonic mucosal glutathione levels in HLA-B27 rats, there was no oxidative stress-related damage. Subsequent analyses showed no defect in expression of glutathione synthesis genes. Additional experiments, comparing young and older HLA-B27 rats, showed that glutathione levels and also reactive oxygen species production decreased with progression of intestinal inflammation. CONCLUSIONS: Antioxidant supplementation was ineffective in HLA-B27 rats despite low mucosal glutathione levels, because colitis development did not coincide with oxidative stress in this model. This indicates that the neutrophilic respiratory burst, and thus innate immune defense, is compromised in HLA-B27 rats. As supplementation with both calcium and antioxidants attenuated colitis development, we speculate that this protective effect is attributed to calcium only.
Assuntos
Antioxidantes/administração & dosagem , Cálcio da Dieta/administração & dosagem , Colite/patologia , Suplementos Nutricionais , Glutationa/metabolismo , Antígeno HLA-B27/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colite/tratamento farmacológico , Colite/metabolismo , Modelos Animais de Doenças , Feminino , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: To investigate the clinical spectrum and molecular causes of retinal dystrophies in 3 families. DESIGN: Family molecular genetics study. PARTICIPANTS: Sixteen patients and 15 relatives in 3 families. METHODS: Members of 3 families with multiple ABCA4-associated retinal disorders were clinically evaluated. Deoxyribonucleic acid samples of all affected individuals and their family members were analyzed for variants in all 50 exons of the ABCA4 gene. MAIN OUTCOME MEASURES: ABCA4-associated retinal phenotypes and mutations in the ABCA4 gene. RESULTS: In family A, 2 sisters were diagnosed with Stargardt's disease (STGD); the eldest sister was compound heterozygous for the mild 2588G-->C and the severe 768G-->T mutation. Another patient in this family with a severe type of retinitis pigmentosa (RP) carried the 768G-->T mutation homozygously. In family B, 2 siblings presented with an RP of severity similar to that encountered in family A. Both were homozygous for the severe IVS33+1G-->A mutation. Two other family members with STGD were compound heterozygous for the 2588G-->C and IVS33+1G-->A mutations. In family C, all 5 siblings of generation II demonstrated age-related macular degeneration (AMD). In generations III and IV, 2 STGD patients and 1 cone-rod dystrophy (CRD) patient were present. In 1 STGD patient we identified a heterozygous 768G-->T mutation. Sequence analysis of the entire ABCA4 gene did not reveal the remaining 2 mutations. Nevertheless, the 2 patients with STGD, the patient with CRD, and 2 of the AMD patients shared a common haplotype spanning the ABCA4 gene. CONCLUSIONS: Different mutations in the ABCA4 gene are the cause of STGD and RP or CRD in at least 2 and, possibly, 3 families. Patients with RP caused by ABCA4 mutations are characterized by an early onset and rapid progression of their retinal dystrophy, with extensive chorioretinal atrophy resulting in a very low visual acuity. Various combinations of relatively rare retinal disorders such as STGD, CRD, and RP in one family may not be as uncommon as once believed, in view of the relatively high carrier frequency of ABCA4 mutations (about 5%) in the general population.