RESUMO
BACKGROUND: We found a high incidence of thrombotic deaths in COX-1(+/-)COX-2(-/-) mice and sought to define the mechanism of these events. The cyclooxygenase products thromboxane A(2) and prostacyclin are important in the regulation of coagulation but their role in fibrinolysis is largely unexplored. PAI-1 blocks fibrinolysis by inhibiting plasminogen activator. AIM: Our objective was to explain the mechanism of increased thrombosis associated with the COX-1(+/-)COX-2(-/-) genotype. METHODS: Carotid artery occlusion times were measured after photochemical injury. PAI-1 levels were measured in the plasma by ELISA. PAI-1 levels in the aorta were measured by RT-PCR and Western blotting. Urinary metabolites of Thromboxane A(2) and prostacyclin were measured by ELISA. RESULTS: The COX-1(+/-)COX-2(-/-) genotype is associated with a decreased time to occlusion in the carotid artery thrombosis model (30 ± 5 minutes vs 60 ± minutes in wild type, p<.001). The COX-1(-/-)COX-2(+/+), COX-1(+/-)COX-2(+/-) and COX-1(+/-)COX-2(+/+) all had occlusion times similar to wild type. COX-1(+/+)COX-2(-/-) had a prolonged occlusion time. COX-1(+/-)COX-2(-/-) had increased PAI-1 levels in the plasma and aorta and with a prolonged euglobulin lysis time (37.4 ± 10.2 hours vs 15.6 ± 9.8 hours in wild type, p<.004). The decreased time to occlusion in the COX-1(+/-)COX2(-/-) mice was normalized by an inhibitory antibody to PAI-1 whereas the antibody had no effect on the time to occlusion in wild type mice. CONCLUSION: The COX-1(+/-)COX-2(-/-) genotype is associated with a shortened time to occlusion in the carotid thrombosis model and the shortened time to occlusion is mediated through increased PAI-1 levels resulting in decreased fibrinolysis.
Assuntos
Estenose das Carótidas/genética , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Animais , Estenose das Carótidas/metabolismo , Primers do DNA , Modelos Animais de Doenças , Feminino , Fibrinólise , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: In a predefined analysis, data were pooled from eight blinded, randomized, controlled trials, and separately from three long-term, open-label trials to determine the rate of upper gastrointestinal ulcer complications with the cyclo-oxygenase-2 selective inhibitor, valdecoxib, vs. non-selective non-steroidal anti-inflammatory drugs. METHODS: In randomized, controlled trials, 7434 osteoarthritis and rheumatoid arthritis patients received placebo (n = 973), valdecoxib 5-80 mg daily (n = 4362), or a non-selective non-steroidal anti-inflammatory drug (naproxen, ibuprofen or diclofenac; n = 2099) for 12-26 weeks. In long-term, open-label trials, 2871 patients received valdecoxib 10-80 mg daily for up to 1 year. All potential events were reviewed by a blinded, independent review committee based on a priori definitions of ulcer complications (perforations, obstructions, bleeds). RESULTS: In randomized, controlled trials, 19 of 955 potential events were adjudicated to be ulcer complications. Valdecoxib was associated with a significantly lower ulcer complication rate than non-selective non-steroidal anti-inflammatory drugs (0.68% vs. 1.96%, all patients; 0.29% vs. 2.08%, non-aspirin users; P < 0.05). In long-term, open-label trials, seven of 310 potential events were adjudicated to be ulcer complications; the annualized incidence for valdecoxib was 0.39% (seven of 1791 patient-years) for all patients and 0.2% (three of 1472 patient-years) for non-aspirin users. CONCLUSIONS: Valdecoxib, including above recommended doses, is associated with a significantly lower rate of upper gastrointestinal ulcer complications than therapeutic doses of non-selective non-steroidal anti-inflammatory drugs.
Assuntos
Inibidores de Ciclo-Oxigenase/efeitos adversos , Isoenzimas/antagonistas & inibidores , Isoxazóis/efeitos adversos , Úlcera Péptica/induzido quimicamente , Sulfonamidas/efeitos adversos , Adulto , Idoso , Anti-Inflamatórios não Esteroides/efeitos adversos , Artrite Reumatoide/tratamento farmacológico , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Feminino , Humanos , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Osteoartrite/tratamento farmacológico , Prostaglandina-Endoperóxido Sintases , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Cyclooxygenase (Cox)-2 expression and inhibition were investigated in a rabbit ileal loop model of Clostridium difficile colitis and diarrhea. Intestinal tissue stimulated with C. difficile toxin A showed up-regulation of Cox-2 expression in lamina propria macrophages and elevated prostaglandin levels. Toxin A-stimulated loops exhibited severe inflammation and increased secretory volume. Celecoxib, a specific Cox-2 inhibitor, significantly reduced toxin A-induced prostaglandin production. Furthermore, celecoxib (> or =0.02 mg/mL) blocked both histologic damage (mean histologic grade, 1.25 vs. 3.44 in rabbits receiving toxin A alone; P<.0005) and secretion (volume:length ratio, 0.18 vs. 0.72 in those receiving toxin A alone; P=.002) in toxin A-stimulated loops in a dose-related manner. Thus, toxin A induced expression of Cox-2 in the host, and prostaglandins produced through Cox-2 were involved in the mediation of the increased secretion of electrolytes and water and the inflammatory response induced by toxin A.
Assuntos
Toxinas Bacterianas/toxicidade , Clostridioides difficile/metabolismo , Colite/metabolismo , Dinoprostona/metabolismo , Enterotoxinas/toxicidade , Íleo/metabolismo , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Clostridioides difficile/patogenicidade , Colite/microbiologia , Colite/patologia , Ciclo-Oxigenase 2 , Diarreia/microbiologia , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Íleo/enzimologia , Íleo/patologia , Proteínas de Membrana , CoelhosRESUMO
We sought to determine if infection of the colon with Entamoeba histolytica induces the expression of cyclooxygenase-2 and, if it does, to determine the contribution of prostaglandins produced through cyclooxygenase-2 to the host response to amebic infection. Human fetal intestinal xenografts were implanted subcutaneously in mice with severe combined immunodeficiency and allowed to grow; the xenografts were then infected with E. histolytica trophozoites. Infection with E. histolytica resulted in the expression of cyclooxygenase-2 in epithelial cells and lamina propria macrophages. Infection with E. histolytica increased prostaglandin E(2) (PGE2) levels 10-fold in the xenografts and resulted in neutrophil infiltration, as manifested by an 18-fold increase in myeloperoxidase activity. Amebic infection also induced an 18-fold increase in interleukin 8 (IL-8) production and a >100-fold increase in epithelial permeability. Treatment of the host mouse with indomethacin, an inhibitor of cyclooxygenase-1 and cyclooxygenase-2, or with NS-398, a selective inhibitor of cyclooxygenase-2, resulted in (i) decreased PGE(2) levels, (ii) a decrease in neutrophil infiltration, (iii) a decrease in IL-8 production, and (iv) a decrease in the enhanced epithelial permeability seen with amebic infection. These results indicate that amebic infection in the colon induces the expression of cyclooxygenase-2 in epithelial cells and macrophages. Moreover, prostaglandins produced through cyclooxygenase-2 participate in the mediation of the neutrophil response to infection and enhance epithelial permeability.
Assuntos
Colo/enzimologia , Doenças do Colo/enzimologia , Entamebíase/enzimologia , Isoenzimas/biossíntese , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Colo/parasitologia , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Indução Enzimática , Humanos , Interleucina-1/biossíntese , Interleucina-8/biossíntese , Proteínas de Membrana , Camundongos , Camundongos SCID , NF-kappa B/fisiologia , Peroxidase/metabolismoRESUMO
The mechanisms allowing the gastrointestinal immune system to avoid an inappropriate inflammatory response to nonpathogenic luminal Ags are poorly understood. We have previously described a role for cyclooxygenase (COX)-2-dependent arachidonic acid metabolites produced by the murine small intestine lamina propria in controlling the immune response to a dietary Ag. To better understand the role of COX-2-dependent arachidonic acid metabolites produced by the lamina propria, we examined the pattern of expression and the cellular source of COX-2 and COX-2-dependent PGE(2). We now demonstrate that non-bone marrow-derived lamina propria stromal cells have basal COX-2 expression and that COX-2-dependent PGE(2) production by these cells is spontaneous and continuous. The other mucosal and nonmucosal lymphoid compartments examined do not share this phenotype. In contrast to the majority of descriptions of COX-2 expression, COX-2 expression by lamina propria stromal cells is not dependent upon exogenous stimuli, including adhesion, LPS signaling via Toll-like receptor 4, or the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-1 beta. These findings, in conjunction with the known immunomodulatory capacities of PGs, suggest that COX-2 expression by the small intestine lamina propria is a basal state contributing to the hyporesponsiveness of the intestinal immune response.
Assuntos
Dinoprostona/biossíntese , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Intestino Delgado/enzimologia , Intestino Delgado/imunologia , Isoenzimas/fisiologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Animais , Células da Medula Óssea/química , Células da Medula Óssea/imunologia , Adesão Celular/imunologia , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Ciclo-Oxigenase 2 , Vida Livre de Germes , Imunofenotipagem , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Isoenzimas/biossíntese , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prostaglandina-Endoperóxido Sintases/biossíntese , Solubilidade , Células Estromais/enzimologia , Células Estromais/metabolismoRESUMO
Investigators have increasingly appreciated the importance of the breakdown of the epithelial barrier in the pathogenesis of inflammatory bowel disease. Epithelial cells secrete trefoil factors, heat shock proteins, and secretory leukocyte proteinase inhibitors that protect the integrity of the epithelial barrier. Other agents, including growth factors, regulate the repair of the epithelium after injury has occurred. Drugs that would either enhance protection of barrier function from injury or accelerate repair after injury would have therapeutic potential in inflammatory bowel disease.
RESUMO
Eicosanoids are arachidonic acid metabolites formed through the cyclooxygenase or one of the lipoxygenase metabolic pathways. They include the prostaglandins (PG), leukotrienes (LT), thromboxanes (Tx), peptidoleukotrienes, and 5-hydroxyeicosatetraenoic acid (5-HETE). This unit describes procedures for measuring individual eicosanoids using several commercial kits. These include kits for an enzyme immunoasssay (ELISA) and a radioimmunoassay (RIA). A method is also described that can be used to analyze the full range of eicosanoids produced in a biological system, and a support protocol presents an acetone/chloroform extraction method for purifying eicosanoids prior to assay. Another support protocol describes a different extraction protocol in which samples are run over an octadecylsilyl silica column.
Assuntos
Eicosanoides/análise , Ensaio de Imunoadsorção Enzimática/métodos , Prostaglandinas/análise , Radioimunoensaio/métodos , Kit de Reagentes para Diagnóstico , Animais , HumanosRESUMO
DNA arrays capable of simultaneously measuring expression of thousands of genes in clinical specimens from affected and normal individuals have the potential to provide information about disease pathogenesis not previously possible. Few studies have applied mRNA profiling to diseases involving complex tissues like the intestinal mucosa, reflecting the unique challenges inherent to this type of analysis. We report the analysis of mucosal gene expression in ulcerative colitis (UC) patients and inflamed and noninflamed control specimens. Genes can be used as markers for cell recruitment, activation, and mucosal synthesis of immunoregulatory molecules. Self-organizing maps were applied to cluster and analyze gene expression patterns and were paired with histopathological scores to identify genes associated with increased disease activity. Clustering was achieved on the basis of differences in expression levels across individual specimens. Several inflammatory mediators were identified as likely determinants of characteristic histological features of active UC. These results provide proof of principle for application of functional genomics to larger inflammatory bowel disease populations for gene discovery, to facilitate identification of disease subgroups on the basis of gene expression signatures, and for prediction of disease behavior or optimal therapeutic intervention.
Assuntos
Perfilação da Expressão Gênica , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/química , Análise de Sequência com Séries de Oligonucleotídeos , Adolescente , Adulto , Criança , Mapeamento Cromossômico , Colite Ulcerativa/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , RNA Mensageiro/análiseRESUMO
Prostaglandins may play an important role in regulating normal renewal of gastrointestinal epithelium, epithelial injury repair, and initiation or progression of intestinal neoplasia. Synthesis of prostaglandins is catalyzed by either of two cyclooxygenase isoforms, Cox-1 and Cox-2. Cox-1 is the predominant cyclooxygenase isoform found in the normal intestine. In contrast, Cox-2 is present at low levels in normal intestine but is elevated at sites of inflammation and in adenomas and carcinomas. To determine directly whether prostaglandins synthesized by Cox-1 or Cox-2 regulate crypt epithelial cell fate after genotoxic or cytotoxic injury, we examined apoptosis, prostaglandin synthesis, and crypt stem cell survival after gamma-irradiation in Cox-1(-/-) and Cox-2(-/-) mice. Cox-1(-/-) mice had increased crypt epithelial cell apoptosis and decreased clonogenic stem cell survival compared with wild-type littermates. PGE(2) synthesis was also diminished in Cox-1(-/-) mice compared with wild-type controls in unstressed intestine and after radiation injury. In contrast, apoptosis, stem cell survival, and intestinal PGE(2) synthesis in Cox-2(-/-) mice after irradiation were the same as in wild-type littermates. Crypt stem cell survival after irradiation was inhibited by a highly specific neutralizing antibody to PGE(2), suggesting that this prostaglandin mediates stem cell fate in vivo. These data suggest that prostaglandins synthesized by Cox-1 regulate multiple steps that determine the fate of crypt epithelial cell after genotoxic or cytotoxic injury.
Assuntos
Mucosa Intestinal/enzimologia , Mucosa Intestinal/efeitos da radiação , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Lesões Experimentais por Radiação/enzimologia , Animais , Anticorpos/farmacologia , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Dinoprostona/imunologia , Feminino , Mucosa Intestinal/citologia , Masculino , Proteínas de Membrana , Camundongos , Camundongos Knockout , Testes de Neutralização , Lesões Experimentais por Radiação/genética , Células-Tronco/citologia , Células-Tronco/enzimologia , Células-Tronco/efeitos da radiaçãoRESUMO
CONTEXT: Conventional nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with a spectrum of toxic effects, notably gastrointestinal (GI) effects, because of inhibition of cyclooxygenase (COX)-1. Whether COX-2-specific inhibitors are associated with fewer clinical GI toxic effects is unknown. OBJECTIVE: To determine whether celecoxib, a COX-2-specific inhibitor, is associated with a lower incidence of significant upper GI toxic effects and other adverse effects compared with conventional NSAIDs. DESIGN: The Celecoxib Long-term Arthritis Safety Study (CLASS), a double-blind, randomized controlled trial conducted from September 1998 to March 2000. SETTING: Three hundred eighty-six clinical sites in the United States and Canada. PARTICIPANTS: A total of 8059 patients (>/=18 years old) with osteoarthritis (OA) or rheumatoid arthritis (RA) were enrolled in the study, and 7968 received at least 1 dose of study drug. A total of 4573 patients (57%) received treatment for 6 months. INTERVENTIONS: Patients were randomly assigned to receive celecoxib, 400 mg twice per day (2 and 4 times the maximum RA and OA dosages, respectively; n = 3987); ibuprofen, 800 mg 3 times per day (n = 1985); or diclofenac, 75 mg twice per day (n = 1996). Aspirin use for cardiovascular prophylaxis (=325 mg/d) was permitted. MAIN OUTCOME MEASURES: Incidence of prospectively defined symptomatic upper GI ulcers and ulcer complications (bleeding, perforation, and obstruction) and other adverse effects during the 6-month treatment period. RESULTS: For all patients, the annualized incidence rates of upper GI ulcer complications alone and combined with symptomatic ulcers for celecoxib vs NSAIDs were 0.76% vs 1.45% (P =.09) and 2. 08% vs 3.54% (P =.02), respectively. For patients not taking aspirin, the annualized incidence rates of upper GI ulcer complications alone and combined with symptomatic ulcers for celecoxib vs NSAIDs were 0.44% vs 1.27% (P =.04) and 1.40% vs 2.91% (P =.02). For patients taking aspirin, the annualized incidence rates of upper GI ulcer complications alone and combined with symptomatic ulcers for celecoxib vs NSAIDs were 2.01% vs 2.12% (P =.92) and 4.70% vs 6.00% (P =.49). Fewer celecoxib-treated patients than NSAID-treated patients experienced chronic GI blood loss, GI intolerance, hepatotoxicity, or renal toxicity. No difference was noted in the incidence of cardiovascular events between celecoxib and NSAIDs, irrespective of aspirin use. CONCLUSIONS: In this study, celecoxib, at dosages greater than those indicated clinically, was associated with a lower incidence of symptomatic ulcers and ulcer complications combined, as well as other clinically important toxic effects, compared with NSAIDs at standard dosages. The decrease in upper GI toxicity was strongest among patients not taking aspirin concomitantly. JAMA. 2000;284:1247-1255
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Inibidores de Ciclo-Oxigenase/efeitos adversos , Gastroenteropatias/induzido quimicamente , Isoenzimas/antagonistas & inibidores , Isoenzimas/farmacologia , Prostaglandina-Endoperóxido Sintases/farmacologia , Sulfonamidas/efeitos adversos , Idoso , Análise de Variância , Artrite Reumatoide/tratamento farmacológico , Aspirina/efeitos adversos , Celecoxib , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Diclofenaco/efeitos adversos , Método Duplo-Cego , Feminino , Humanos , Ibuprofeno/efeitos adversos , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Osteoartrite/tratamento farmacológico , Úlcera Péptica/induzido quimicamente , Modelos de Riscos Proporcionais , Estudos Prospectivos , PirazóisRESUMO
BACKGROUND & AIMS: The bone marrow and the intestine are the major sites of radiation-induced injury. The cellular response to radiation injury in the intestine or bone marrow can be modulated by agents given before irradiation. Lipopolysaccharide is known to be radioprotective in the bone marrow, but its effect on the intestine is not known. We sought to determine if lipopolysaccharide is radioprotective in the intestine and, if so, to determine the mechanism of its radioprotective effects. METHODS: Mice were treated with parenteral lipopolysaccharide or vehicle and then irradiated (14 Gy total body irradiation in a cesium irradiator). The number of surviving intestinal crypts was assessed 3.5 days after irradiation using a clonogenic assay. RESULTS: Parenteral administration of lipopolysaccharide 2-24 hours before irradiation resulted in a 2-fold increase in the number of surviving crypts 3.5 days after irradiation. The radioprotective effects of lipopolysaccharide could be eliminated by coadministration of a selective inhibitor of cyclooxygenase 2. Lipopolysaccharide was radioprotective in wild-type mice but not in mice with a disrupted cyclooxygenase 2. Parenteral administration of lipopolysaccharide resulted in increased production of prostaglandins in the intestine and in the induction of cyclooxygenase 2 expression in subepithelial fibroblasts and in villous, but not crypt, epithelial cells. CONCLUSIONS: Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-dependent pathway.
Assuntos
Dinoprostona/metabolismo , Intestino Delgado/enzimologia , Lipopolissacarídeos/farmacologia , Lesões Experimentais por Radiação/tratamento farmacológico , Lesões Experimentais por Radiação/metabolismo , Protetores contra Radiação/farmacologia , Animais , Western Blotting , Temperatura Corporal , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/efeitos da radiação , Feminino , Genótipo , Indometacina/farmacologia , Intestino Delgado/patologia , Isoenzimas/análise , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrobenzenos/farmacologia , Prostaglandina-Endoperóxido Sintases/análise , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Lesões Experimentais por Radiação/patologia , Sulfonamidas/farmacologiaRESUMO
The multiple drug resistance protein, MDR1, is highly expressed on the apical surface of intestinal epithelial cells. The physiologic substrate of this protein remains unclear. Several studies using compounds known to act as MDR1 inhibitors have suggested that MDR1 may be involved in the transport of cholesterol from the plasma membrane to the endoplasmic reticulum where it is esterified. To examine the role of MDR1 in cholesterol uptake by intestinal cells, the rat intestinal epithelial cell line IEC-18, was stably transfected with human MDR1. MDR1-transfected cells exhibited increased expression of MDR1 protein, reduced accumulation of vinblastine and increased uptake of [(3)H]cholesterol from cholesterol/monolein/taurocholate micelles. These studies provide the first direct evidence that the level of MDR1 expression in intestinal cells can influence the amount of cholesterol taken up by those cells. This is also the first demonstration that a multiple drug resistance protein can function in the net uptake, rather than efflux, of a substrate.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Anticorpos/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Expressão Gênica , Genes MDR , Humanos , Micelas , Ratos , Transfecção , Verapamil/farmacologia , Vimblastina/metabolismoRESUMO
BACKGROUND: Phytosterol feeding in human clinical trials has had generally small and inconsistent effects on serum cholesterol concentrations, raising doubts about the importance of phytosterols in natural diets and supplements. OBJECTIVE: The hypothesis tested was that the low intestinal bioavailability of purified phytosterols can be increased by formulation with lecithin. DESIGN: The ability of sitostanol to reduce cholesterol absorption was measured directly by including hexadeuterated cholesterol tracer in a standard test breakfast and measuring plasma tracer concentration 4 and 5 d later by gas chromatography-negative ion mass spectrometry. The tracer amount after a test meal containing sitostanol was compared with that after an identical meal containing placebo. Each subject served as his or her own control and the order of testing was random. Sitostanol was formulated either as a powder or as a sonicated micellar solution with lecithin. A total of 38 single-meal tests were performed in 6 healthy subjects. RESULTS: Sitostanol powder (1 g) reduced cholesterol absorption by only 11.3 +/- 7.4% (P = 0.2), confirming in vitro data showing poor solubility of sitostanol powder in artificial bile. In contrast, sitostanol in lecithin micelles reduced cholesterol absorption by 36.7 +/- 4.2% (P = 0.003) at a dose of 700 mg and by 34.4 +/- 5.8% (P = 0.01) at a dose of 300 mg. CONCLUSIONS: Sitostanol reduced cholesterol absorption at doses lower than reported previously, but only if presented in lecithin micelles. Properly formulated sitostanol as well as naturally occurring complexes of phytosterol and phospholipid might be therapeutically useful for cholesterol lowering.
Assuntos
Anticolesterolemiantes/administração & dosagem , Colesterol/sangue , Fosfatidilcolinas , Sitosteroides/administração & dosagem , Adulto , Anticolesterolemiantes/farmacologia , Disponibilidade Biológica , Colesterol/farmacocinética , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Absorção Intestinal/efeitos dos fármacos , Masculino , Micelas , Pós , Sitosteroides/farmacologiaRESUMO
While unphysiologically large cholesterol doses are known to reduce percent cholesterol absorption, smaller amounts are reported to have no effect in human subjects. To determine the dose;-response relation between dietary cholesterol consumed and the efficiency of intestinal cholesterol absorption, we fed 18 normal subjects two test meals containing different amounts of natural cholesterol. In each test pentadeuterated cholesterol tracer was given orally, hexadeuterated cholesterol tracer was given intravenously, and the tracer ratio was measured in plasma 4 days later by gas chromatography/negative ion mass spectrometry. Baseline cholesterol absorption in the presence of 26 mg cholesterol tracer was 40.7 +/- 2.3%. This decreased by 4.9 percentage points (P = 0.05) when a total of 188 mg cholesterol was included in the meal and by 15.6 percentage points (P = 0.006) when 421 mg cholesterol was given, showing that the efficiency of cholesterol absorption declines appreciably even with modest increases in cholesterol dose. Considerable variation was noted in the response of different subjects and, on the higher cholesterol dose, dietary cholesterol absorption varied 5-fold from 40 mg to 212 mg. Fasting plasma insulin was correlated with the ability to absorb higher cholesterol doses without loss of efficiency (r(s) = 0.700, P = 0.036). Percent cholesterol absorption in a single meal is significantly influenced by the amount of cholesterol in that meal, suggesting that acute dietary factors influencing cholesterol absorption need further study.
Assuntos
Colesterol na Dieta/farmacocinética , Absorção Intestinal , Administração Oral , Adulto , Índice de Massa Corporal , Colesterol/sangue , Deutério , Relação Dose-Resposta a Droga , Feminino , Humanos , Injeções Intravenosas , Insulina/sangue , Lipoproteínas/sangue , Masculino , Pessoa de Meia-Idade , Estatísticas não ParamétricasRESUMO
Intestinal inflammatory diseases are mediated by dysregulated immune responses to undefined luminal antigens. Feeding hen egg-white lysozyme to mice expressing a transgenic T-cell receptor that recognizes hen egg-white lysozyme peptide 46-61 resulted in no intestinal pathology; however, simultaneous administration of cyclooxygenase-2 inhibitors and dietary hen egg-white lysozyme resulted in increased proliferation of lamina propria mononuclear cells and crypt epithelial cells, crypt expansion and villus blunting. Lamina propria mononuclear cells produce high levels of cyclooxygenase-2-dependent arachidonic acid metabolites, which act as immunomodulators in the immune response to dietary antigen. These findings establish that cyclooxygenase-2-dependent arachidonic acid metabolites are essential in the development and maintenance of intestinal immune homeostasis.
Assuntos
Ácido Araquidônico/metabolismo , Isoenzimas/metabolismo , Muramidase/imunologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/biossíntese , Ativação Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Nitrobenzenos/farmacologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Sulfonamidas/farmacologiaRESUMO
Percent cholesterol absorption was measured in 94 normal subjects aged 17- 80 years while consuming diets generally low in cholesterol (mean intake = 226 +/- 126 mg/day). A new dual stable isotope method was used where a cholesterol tracer containing 6 extra mass units was given intravenously and another tracer with 5 extra mass units was given orally during a standard test meal. The ratio of tracers in plasma was determined by negative ion mass spectrometry of pentafluorobenzoyl sterol esters. Absorption values ranged widely from 29.0% to 80.1% with mean 56.2 +/- 12.1 (SD) %. Cholesterol absorption was significantly increased in African-Americans (63.4 +/- 11.8% vs. 55.1 +/- 11.9%, P = 0.027) but was similar for women (53.3 +/- 11.9%) and men (57.6 +/- 12.1%). It was not related to plasma lipoproteins, age, apoE3/E3 or E3/E4 genotype, or chronic dietary intake of energy, fat, or cholesterol quantitated from 7- day food records. However, dietary cholesterol intake was positively related to plasma cholesterol (P = 0.036) and triglycerides (P = 0.026). The milligram amount of dietary cholesterol absorbed (but not percent absorption) was positively correlated with fasting plasma insulin (r = 0.525, P < 0.0001), C-peptide (r = 0.367, P = 0.0003) and glucagon (r = 0.421, P < 0.0001) independent of gender, body fat percent and age.The efficiency of intestinal cholesterol absorption and the milligram amount of dietary cholesterol absorbed were not related to plasma cholesterol or LDL cholesterol in individuals consuming a low-cholesterol low-fat diet. The dominant factor determining dietary cholesterol absorption was intake rather than absorption efficiency. Dietary cholesterol and fat were strongly and independently related to hormonal measures of insulin resistance.-Bosner, M. S., L. G. Lange, W. F. Stenson, and R. E. Ostlund, Jr. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.
Assuntos
Colesterol na Dieta/farmacocinética , Colesterol/sangue , Adulto , Composição Corporal , Radioisótopos de Carbono , Deutério , Feminino , Humanos , Insulina/sangue , Absorção Intestinal/fisiologia , Lipídeos/sangue , Lipoproteínas/sangue , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Grupos Raciais , Distribuições Estatísticas , Estatística como AssuntoRESUMO
BACKGROUND & AIMS: Although dextran sodium sulfate (DSS)-induced colitis is a commonly used model of colonic injury, the mechanism of this model is not understood. The aim of this study was to determine the contribution of prostaglandins to the mechanism of DSS-induced epithelial injury. METHODS: Mice were treated with 3% DSS in the drinking water for 5 days followed by water only (recovery). Tissue prostaglandin E2 (PGE2) levels were measured, proliferating cells per cecal crypt were determined by bromodeoxyuridine labeling, and the cellular localization of cyclooxygenase (COX)-1 and COX-2 was determined by immunohistochemistry. RESULTS: DSS decreased the number of proliferating epithelial cells per crypt by approximately 90% and decreased the height of cecal crypts by 40%. Administration of dimethyl PGE2 with DSS reversed the effect of DSS on proliferation but not its effect on crypt shortening. COX-1 was expressed in the crypt epithelium and lamina propria mononuclear cells; DSS treatment down-regulated COX-1 expression only in the epithelium. Dimethyl PGE2 reversed the effect of DSS on COX-1 expression. Recovery was associated with a return to normal COX-1 expression in the epithelium. COX-2 was expressed in lamina propria mononuclear cells. CONCLUSIONS: Epithelial cell proliferation in the presence of DSS contains a PGE2-sensitive component.
Assuntos
16,16-Dimetilprostaglandina E2/farmacologia , Ceco/efeitos dos fármacos , Sulfato de Dextrana/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Animais , Bromodesoxiuridina/metabolismo , Ceco/metabolismo , Ceco/patologia , Divisão Celular/efeitos dos fármacos , Ciclo-Oxigenase 1 , Sulfato de Dextrana/antagonistas & inibidores , Feminino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Isoenzimas/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C3H , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
BACKGROUND & AIMS: Prostaglandins are synthesized by cyclooxygenases (COX)-1 and -2. The expression and cellular localization of COX-1 and COX-2 in normal human colon and inflammatory bowel disease (IBD) surgical resections were studied. METHODS: COX-1 and COX-2 protein expression and cellular localization were assessed by Western blotting and immunohistochemistry. RESULTS: COX-1 protein was expressed at equal levels in normal, Crohn's disease, and ulcerative colitis colonic epithelial cells. COX-2 protein was not detected in normal epithelial cells but was detected in Crohn's disease and ulcerative colitis epithelial cells. Immunohistochemistry of normal, Crohn's colitis, and ulcerative colitis tissue showed equivalent COX-1 expression in epithelial cells in the lower half of the colonic crypts. COX-2 expression was absent from normal colon, whereas in Crohn's colitis and ulcerative colitis, COX-2 was observed in apical epithelial cells and in lamina propria mononuclear cells. In Crohn's ileitis, COX-2 was present in the villus epithelial cells. In ulcerative colitis, colonic epithelial cells expressing COX-2 also expressed inducible nitric oxide synthase. CONCLUSIONS: COX-1 was localized in the crypt epithelium of the normal ileum and colon, and its expression was unchanged in IBD. COX-2 was undetectable in normal ileum or colon, but it was induced in apical epithelial cells of inflamed foci in IBD.
Assuntos
Colo/enzimologia , Doenças Inflamatórias Intestinais/enzimologia , Mucosa Intestinal/enzimologia , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Colite/enzimologia , Colite Ulcerativa/enzimologia , Doença de Crohn/enzimologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Indução Enzimática/fisiologia , Feminino , Humanos , Ileíte/enzimologia , Doenças Inflamatórias Intestinais/cirurgia , Isoenzimas/genética , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/metabolismo , Valores de ReferênciaRESUMO
Increased intestinal fluid secretion is a protective host response after enteric infection with invasive bacteria that is initiated within hours after infection, and is mediated by prostaglandin H synthase (PGHS) products in animal models of infection. Intestinal epithelial cells are the first host cells to become infected with invasive bacteria, which enter and pass through these cells to initiate mucosal, and ultimately systemic, infection. The present studies characterized the role of intestinal epithelial cells in the host secretory response after infection with invasive bacteria. Infection of cultured human intestinal epithelial cell lines with invasive bacteria, but not noninvasive bacteria, is shown to induce the expression of one of the rate-limiting enzymes for prostaglandin formation, PGHS-2, and the production of PGE2 and PGF2alpha. Furthermore, increased PGHS-2 expression was observed in intestinal epithelial cells in vivo after infection with invasive bacteria, using a human intestinal xenograft model in SCID mice. In support of the physiologic importance of epithelial PGHS-2 expression, supernatants from bacteria-infected intestinal epithelial cells were shown to increase chloride secretion in an in vitro model using polarized epithelial cells, and this activity was accounted for by PGE2. These studies define a novel autocrine/paracrine function of mediators produced by intestinal epithelial cells in the rapid induction of increased fluid secretion in response to intestinal infection with invasive bacteria.