RESUMEN
BACKGROUND: Studies of the effects of antiretroviral drugs on lipid metabolism are limited by the availability of suitable models. We have thus developed an animal model utilising Göttingen mini-pigs. The normal lipid metabolism of mini-pigs closely reflects that of humans and they are expected to have similar reactions to antiretroviral drugs. METHODS: The pigs were treated orally with high doses of the protease inhibitors ritonavir and saquinavir for 4 weeks. The model allows repeated concomitant biopsies from liver, muscle, adipose tissue and plasma samples. RESULTS: The study showed a general decrease in polyunsaturated fatty acids; changes in both saturated and monounsaturated fatty acids were also apparent after antiretroviral treatment. The changes were observed after 4 weeks of treatment. At 4 weeks post-treatment, the levels of all fatty acids were lower compared with pretreatment levels, suggesting a prolonged effect of the antiretroviral drug treatment lasting beyond the 4 week post-treatment observation period. CONCLUSIONS: The Göttingen mini-pig model is a promising animal model for rapid screening of the metabolic effects induced by antiretroviral drugs.
Asunto(s)
Fármacos Anti-VIH , Metabolismo de los Lípidos/efectos de los fármacos , Modelos Animales , Ritonavir , Saquinavir , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacología , Esquema de Medicación , Ácidos Grasos/metabolismo , Humanos , Ritonavir/administración & dosificación , Ritonavir/farmacología , Saquinavir/administración & dosificación , Saquinavir/farmacología , Porcinos , Porcinos Enanos , Factores de TiempoRESUMEN
Monocytes/macrophages are known to represent a potential reservoir of human immunodeficiency virus type 1 (HIV-1), which ensures continuous replication of the virus in patients on highly active antiretroviral therapy (HAART). Infected macrophages are a highly productive source of HIV-1 during infections with common opportunistic pathogens. Previous studies report that toll like receptors (TLR)s play a role in HIV-1 replication in macrophages. Here, we investigate the three main pathways activated through TLR4 and the interactions with the HIV-1 long terminal repeat (LTR), using human embryonic kidney (HEK) 293 cells expressing TLR4 and transfected with a luciferase reporter under the control of the HIV-1 LTR. Here, we demonstrate, that TLR4-mediated activation of HIV-LTR is largely governed by the nuclear factor-kappaB pathway. Neither of the mitogen-activated protein kinases ERK1/2, JNK, or p38 nor the transcription factor interferon regulatory factor 3 were involved in the direct transactivation of HIV-LTR through stimulation of TLR4.
Asunto(s)
Duplicado del Terminal Largo de VIH , Factor 3 Regulador del Interferón/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , FN-kappa B/fisiología , Receptor Toll-Like 4/fisiología , Línea Celular , Humanos , Lipopolisacáridos/farmacología , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Transepithelial migration of polymorphonuclear neutrophils (PMN) plays a crucial role in inflammatory conditions of the intestine, such as inflammatory bowel diseases. Hypertonic saline (HS) exerts various inhibitory effects on PMN function. We hypothesized that HS could inhibit transepithelial migration of PMN and thereby prevent inflammatory events in experimental colitis. Isolated human PMN were treated with HS (40 mM), and their transmigration across a monolayer of T84 epithelial cells was induced by N-formyl-methionyl-leucyl-phenylalanine. Monolayer disruption was assessed by monitoring changes in transepithelial conductance in an Ussing chamber. Colitis in mice was induced by oral administration of dextran sulfate sodium (DSS). Animals were treated with 4 or 8 ml/kg of 7.5% saline intraperitoneally two times daily for 7 days. Controls received equivalent volumes of normal saline (NS, n = 6) or no intraperitoneal treatment (DSS, n = 12). The severity of inflammation was evaluated based on disease activity index and histology score. HS treatment of PMN in vitro significantly reduced cell migration and the disruption of T84 monolayers compared with untreated control cells (n = 5, P < 0.05). This effect of HS was dose dependent. HS treatment in vivo also reduced colitis-induced gut tissue damage, as indicated by an improved histology score compared with the NS and DSS groups. We conclude that HS inhibits transepithelial migration of PMN in vitro and gut tissue damage in vivo in a mouse model of colitis. Thus HS may have clinical value to reduce PMN-mediated intestinal damage.
Asunto(s)
Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Colitis/prevención & control , Mucosa Intestinal/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Solución Salina Hipertónica/farmacología , Animales , Antiinflamatorios/administración & dosificación , Antígeno CD11b/metabolismo , Células Cultivadas , Colitis/inducido químicamente , Colitis/inmunología , Colitis/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Humanos , Inyecciones Intraperitoneales , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Neutrófilos/patología , Permeabilidad , Solución Salina Hipertónica/administración & dosificación , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
Bile acid epimers and side-chain homologues are present in the human colon. To test whether such bile acids possess secretory activity, cultured T84 colonic epithelial cells were used to quantify the secretory properties of synthetic epimers and homologues of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). In our study, chloride secretion was measured as changes in short-circuit current (DeltaI(sc), in microA/cm2) with the use of voltage-clamped monolayers of T84 cells mounted in Ussing chambers. Bile acids were added at 0.5 mM, a concentration that did not alter transepithelial resistance. Data were expressed as peak DeltaI(sc) (means +/- SD). When added bilaterally, DCA stimulated a DeltaI(sc) response of 15.7 +/- 12.5 microA/cm2. The 12beta-OH epimer of DCA was less potent (DeltaI(sc) = 8.0 +/- 1.7 microA/cm2), whereas its 3beta-OH epimer had no effect. CDCA stimulated secretion (DeltaI(sc) = 8.2 +/- 5.5 microA/cm2), whereas both its 7beta-OH and 3beta-OH epimers were inactive, as was lithocholic acid. HomoDCA (1 additional side-chain carbon) was active (DeltaI(sc) = 7.8 +/- 4.8 microA/cm2), whereas norDCA (1 fewer carbon) and dinorDCA (2 fewer carbons) were not. Taurine conjugates of DCA and CDCA stimulated secretion (DeltaI(sc) = 12.3 +/- 7.5 and 8.8 +/- 4.8 microA/cm2, respectively) from the basolateral side but not the apical side. Uptake of taurine conjugates from the basolateral but not the apical side was shown by mass spectrometry. These studies indicate marked structural specificity for bile acid-induced chloride secretion and show that modification of bile acid structure by colonic bacteria modulates the secretory properties of these endogenous secretagogues.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacología , Colon/fisiología , Ácido Desoxicólico/análogos & derivados , Ácido Desoxicólico/farmacología , Mucosa Intestinal/fisiología , Línea Celular , Ácido Quenodesoxicólico/farmacocinética , Ácido Desoxicólico/farmacocinética , Electrofisiología , Humanos , Mucosa Intestinal/efectos de los fármacos , Espectrometría de Masas , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiologíaRESUMEN
The Salmonella effector protein SigD is an inositol phosphate phosphatase that inhibits phosphatidylinositol 3-kinase-dependent signaling. Because epidermal growth factor (EGF) inhibits chloride secretion via phosphatidylinositol 3-kinase, we explored whether Salmonella infection might modify the inhibitory effect of EGF. As expected, EGF inhibited chloride secretion induced by carbachol in T(84) epithelial cells. Infection with wild-type (WT) but not sigD(-) mutant S. typhimurium SL1344 decreased CCh-stimulated chloride secretion. Moreover, WT but not sigD(-) Salmonella reduced the inhibitory effect of EGF on carbachol-stimulated chloride secretion. Complementation of sigD restored the ability of mutant Salmonella to reverse the inhibitory effect of EGF. EGF-induced EGF receptor phosphorylation was similar in cells infected with either WT or mutant Salmonella, and neither WT nor sigD(-) Salmonella altered recruitment of the p85 subunit of phosphatidylinositol 3-kinase to EGF receptor, implying that SigD acts downstream of these signaling events. Furthermore, transepithelial resistance fell more rapidly in cells infected with WT vs. sigD(-) Salmonella, indicating an early role for SigD in reducing barrier function, perhaps via activation of protein kinase C. We conclude that the Salmonella bacterial effector protein SigD may play critical roles in the pathogenesis of disease caused by this microorganism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cloruros/metabolismo , Células Epiteliales/microbiología , Mucosa Intestinal/microbiología , Infecciones por Salmonella/fisiopatología , Animales , Línea Celular , Factor de Crecimiento Epidérmico/fisiología , Células Epiteliales/fisiología , Receptores ErbB/metabolismo , Humanos , Inmunohistoquímica , Mucosa Intestinal/fisiología , Salmonella typhimurium/patogenicidad , Transducción de Señal/fisiología , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiologíaRESUMEN
IFN-gamma is elevated in intestinal inflammation and alters barrier and transport functions in human colonic epithelial cell lines, but its effects on normal human small intestinal epithelium in vivo are poorly defined. We investigated effects of prolonged IFN-gamma exposure on ion transport and expression of transporters by using human fetal small intestinal xenografts. Xenograft-bearing mice were injected with IFN-gamma, and 24 h later xenografts were harvested and mounted in Ussing chambers. Baseline potential difference (PD) was not affected by IFN-gamma treatment. However, conductance was enhanced and agonist-stimulated ion transport was decreased. IFN-gamma also decreased expression of the Na+-K+-2Cl- cotransporter and the alpha-subunit of Na+-K+-ATPase compared with controls, whereas levels of the calcium-activated Cl- channel and CFTR were unaltered. Thus prolonged exposure to IFN-gamma leads to decreased ion secretion due, in part, to decreased ion transporter levels. These findings demonstrate the implications of elevated IFN-gamma levels in human small intestine and validate the human intestinal xenograft as a model to study chronic effects of physiologically relevant stimuli.
Asunto(s)
Interferón gamma/farmacología , Intestinos/enzimología , Simportadores de Cloruro de Sodio-Potasio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Transporte Biológico Activo/efectos de los fármacos , Western Blotting , Calcio/fisiología , Carbacol/farmacología , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/metabolismo , Cloruros/metabolismo , AMP Cíclico/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Dinoprostona/farmacología , Regulación hacia Abajo/efectos de los fármacos , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Intestinos/trasplante , Ratones , Pruebas de Precipitina , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Miembro 2 de la Familia de Transportadores de Soluto 12 , Estimulación Química , Trasplante HeterólogoRESUMEN
We have previously shown that Gq protein-coupled receptor (GqPCR) agonists stimulate epidermal growth factor receptor (EGFr) transactivation and activation of mitogen-activated protein kinases (MAPK) in colonic epithelial cells. This constitutes a mechanism by which Cl- secretory responses to GqPCR agonists are limited. In the present study we examined a possible role for the EGFr in regulating Cl- secretion stimulated by agonists that act through GsPCRs. All experiments were performed using monolayers of T84 colonic epithelial cells grown on permeable supports. Protein phosphorylation and protein-protein interactions were analyzed by immunoprecipitation and Western blotting. Cl- secretion was measured as changes in short-circuit current (DeltaIsc) across voltage-clamped T84 cells. The GsPCR agonist, vasoactive intestinal polypeptide (VIP; 100 nM), rapidly stimulated EGFr phosphorylation in T84 cells. This effect was mimicked by a cell-permeant analog of cAMP, Bt2cAMP/AM (3 microM), and was attenuated by the protein kinase A (PKA) inhibitor, H-89 (20 microM). The EGFr inhibitor, tyrphostin AG1478 (1 microM), inhibited both Bt2cAMP/AM-stimulated EGFr phosphorylation and Isc responses. VIP and Bt2cAMP/AM both stimulated ERK MAPK phosphorylation and recruitment of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to the EGFr in a tyrphostin AG1478-sensitive manner. The PI3K inhibitor, wortmannin (50 nM), but not the ERK inhibitor, PD 98059 (20 microM), attenuated Bt2cAMP/AM-stimulated secretory responses. We conclude that GsPCR agonists rapidly transactivate the EGFr in T84 cells by a signaling pathway involving cAMP and PKA. Through a mechanism that likely involves PI3K, transactivation of the EGFr is required for the full expression of cAMP-dependent Cl- secretory responses.
Asunto(s)
Receptores ErbB/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Sulfonamidas , Activación Transcripcional , Androstadienos/farmacología , Western Blotting , Línea Celular , Cloruros/metabolismo , Cloro/metabolismo , Colon/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Flavonoides/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Humanos , Iones , Isoquinolinas/farmacología , Sistema de Señalización de MAP Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Quinazolinas , Factores de Tiempo , Tirosina/metabolismo , Tirfostinos/farmacología , Péptido Intestinal Vasoactivo/química , WortmaninaRESUMEN
Specific effects of the cytotoxic secondary lipid oxidation product, 4-hydroxynonenal (10(-8)-10(-4) M), on intact sheets of rat jejunum were measured as changes in short circuit current (delta(I)sc) following cumulative addition to either the mucosal or serosal side, using the analogous aldehyde, nonenal, as reference. 4-Hydroxynonenal stimulated I(sc) from the serosal side (maximal delta(I)sc = 27.2 +/- 3.5 microA/cm2, mean +/- SEM, N = 32) while nonenal stimulated I(sc) primarily from the mucosal side (maximal delta(I)sc = 16.2 +/- 3.4 microA/cm2, N = 20). Inhibition by 100 microM bumetanide (4-hydroxynonenal: 88.9 +/- 3.0%, N = 6, p < 0.05, nonenal: 69.3 +/- 2.9%, N = 6, P < 0.05) indicated chloride secretion. Nonenal-induced delta(I)sc was inhibited (72.5 +/- 1.2%, N = 8, P < 0.05) by a combination of nordihydroguaiaretic acid (100 microM) and piroxicam (10 microM), while 4-hydroxynonenal-induced delta(I)sc, was abolished by piroxicam (N = 8, P < 0.001) and inhibited by 1 microM tetrodotoxin (69.8 +/- 9.7%, N = 6, P < 0.001). These data indicate that 4-hydroxynonenal stimulates chloride secretion mediated by prostaglandins and the enteric nervous system. The site of action (serosal) being opposite to the reference aldehyde.
Asunto(s)
Cloruros/metabolismo , Eicosanoides/farmacología , Mucosa Intestinal/fisiología , Secreciones Intestinales/efectos de los fármacos , Yeyuno/fisiología , Aldehídos/farmacología , Análisis de Varianza , Animales , Mucosa Intestinal/química , Transporte Iónico/fisiología , Yeyuno/química , Peroxidación de Lípido/fisiología , Masculino , Masoprocol/farmacología , Oxidantes/farmacología , Piroxicam/farmacología , RatasRESUMEN
Enteric Salmonella infection is accompanied by inflammation and diarrhea, and yet little is known about its effects on intestinal epithelial physiology. Since species differences limit the utility of animal tissues and cell lines lack relevant cell-cell interactions, we have used a human model of fetal intestine grown as xenografts in SCID mice. We investigated here the effects of Salmonella enterica serovar Typhimurium SL1344 on xenograft ion transport. Harvested xenografts were stripped of seromuscular layers by blunt dissection, infected with Salmonella, and mounted in Ussing chambers. Salmonella infection for 1 h increased baseline ion transport without altering tissue conductance or morphology. The increased transport was blocked by the cyclooxygenase inhibitor, indomethacin, or the specific Cox-2 inhibitor, NS-398. Further, xenografts infected for 2 h showed increased secretory responses to the calcium-dependent agonist, carbachol, and the cyclic AMP-dependent agonists prostaglandin E(2) (PGE(2)) and forskolin, which were blocked by indomethacin. Western blot experiments revealed that infection was accompanied by increased cyclooxygenase 2 (Cox-2) expression, with no change in Cox-1 levels. Immunoassay demonstrated basolateral PGE(2) release, which was inhibited by indomethacin. Histological examination of infected xenografts illustrated that upregulated Cox-2 expression was restricted to the epithelium and that little or no invasion of the tissue by Salmonella occurred for up to 2 h. In summary, Salmonella infection rapidly increases Cox-2 expression in human intestinal tissue, accounting for increased epithelial ion transport characteristic of infectious diarrhea.