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2.
Ann Surg ; 259(4): 715-22, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23598380

RESUMEN

OBJECTIVE: To determine the efficacy of oral supplementation of the gut enzyme intestinal alkaline phosphatase (IAP) in preventing antibiotic-associated infections from Salmonella enterica serovar Typhimurium (S. Typhimurium) and Clostridium difficile. BACKGROUND: The intestinal microbiota plays a pivotal role in human health and well-being. Antibiotics inherently cause dysbiosis, an imbalance in the number and composition of intestinal commensal bacteria, which leads to susceptibility to opportunistic bacterial infections. Previously, we have shown that IAP preserves the normal homeostasis of intestinal microbiota and that oral supplementation with calf IAP (cIAP) rapidly restores the normal gut flora. We hypothesized that oral IAP supplementation would protect against antibiotic-associated bacterial infections. METHODS: C57BL/6 mice were treated with antibiotic(s) ± cIAP in the drinking water, followed by oral gavage of S. Typhimurium or C. difficile. Mice were observed for clinical conditions and mortality. After a defined period of time, mice were killed and investigated for hematological, inflammatory, and histological changes. RESULTS: We observed that oral supplementation with cIAP during antibiotic treatment protects mice from infections with S. Typhimurium as well as with C. difficile. Animals given IAP maintained their weight, had reduced clinical severity and gut inflammation, and showed improved survival. CONCLUSIONS: Oral IAP supplementation protected mice from antibiotic-associated bacterial infections. We postulate that oral IAP supplementation could represent a novel therapy to protect against antibiotic-associated diarrhea (AAD), C. difficile-associated disease (CDAD), and other enteric infections in humans.


Asunto(s)
Fosfatasa Alcalina/uso terapéutico , Antibacterianos/efectos adversos , Clostridioides difficile , Infecciones por Clostridium/prevención & control , Fármacos Gastrointestinales/uso terapéutico , Infecciones por Salmonella/prevención & control , Salmonella typhimurium , Administración Oral , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/farmacología , Animales , Antibacterianos/administración & dosificación , Biomarcadores/metabolismo , Infecciones por Clostridium/etiología , Colon/efectos de los fármacos , Colon/metabolismo , Colon/microbiología , Diarrea/etiología , Diarrea/prevención & control , Femenino , Fármacos Gastrointestinales/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Endogámicos C57BL , Infecciones por Salmonella/etiología , Estreptomicina/administración & dosificación , Estreptomicina/efectos adversos , Resultado del Tratamiento
3.
Proc Natl Acad Sci U S A ; 110(17): 7003-8, 2013 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-23569246

RESUMEN

Metabolic syndrome comprises a cluster of related disorders that includes obesity, glucose intolerance, insulin resistance, dyslipidemia, and fatty liver. Recently, gut-derived chronic endotoxemia has been identified as a primary mediator for triggering the low-grade inflammation responsible for the development of metabolic syndrome. In the present study we examined the role of the small intestinal brush-border enzyme, intestinal alkaline phosphatase (IAP), in preventing a high-fat-diet-induced metabolic syndrome in mice. We found that both endogenous and orally supplemented IAP inhibits absorption of endotoxin (lipopolysaccharides) that occurs with dietary fat, and oral IAP supplementation prevents as well as reverses metabolic syndrome. Furthermore, IAP supplementation improves the lipid profile in mice fed a standard, low-fat chow diet. These results point to a potentially unique therapy against metabolic syndrome in at-risk humans.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/farmacología , Síndrome Metabólico/tratamiento farmacológico , Absorción/efectos de los fármacos , Administración Oral , Fosfatasa Alcalina/administración & dosificación , Fosfatasa Alcalina/genética , Animales , Compuestos Azo , Línea Celular , Cartilla de ADN/genética , Lipopolisacáridos , Hígado/metabolismo , Síndrome Metabólico/etiología , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Triglicéridos/metabolismo
4.
Am J Physiol Gastrointest Liver Physiol ; 304(6): G597-604, 2013 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23306083

RESUMEN

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación , Enfermedades Inflamatorias del Intestino , Intestino Delgado/metabolismo , Uridina Difosfato/metabolismo , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Células Cultivadas , Humanos , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Interleucina-8/análisis , Interleucina-8/metabolismo , Mucosa Intestinal/inmunología , Lipopolisacáridos/metabolismo , Ratones , Ratones Noqueados , Receptores Purinérgicos P2/metabolismo
5.
J Gastrointest Surg ; 15(5): 860-9, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21360208

RESUMEN

BACKGROUND: The brush-border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and detoxifies different toll-like receptor ligands. This study aimed to determine the therapeutic effects of locally administered calf IAP (cIAP) in a cecal ligation and puncture (CLP) model of polymicrobial sepsis. METHODS: C57BL/6 mice underwent CLP followed by intraperitoneal injection of cIAP or normal saline. Blood leukocyte counts, levels of cytokines and liver enzymes, and lung myeloperoxidase activity were determined. Peritoneal lavage fluid (PLF) was assayed for neutrophil infiltration and both aerobic and anaerobic bacterial counts. RESULTS: After intraperitoneal injection, cIAP activity in PLF decreased 50% within 15 min with minimal activity evident at 4 h. Compared with irrigation with normal saline, cIAP irrigation increased the 7-day survival rate in mice undergoing CLP, with maximal effects seen at 25 units of cIAP (0% vs. 46% survival rate, respectively; p < 0.001). cIAP treatment reduced lung inflammation, liver damage and levels of tumor necrosis factor alpha and interleukin-6. CONCLUSIONS: Peritoneal irrigation with cIAP significantly enhances survival in a mouse model of peritonitis, likely through reduction of local inflammation and remote organ damage. We suggest that intraperitoneal cIAP irrigation could be a novel therapy for intra-abdominal sepsis.


Asunto(s)
Fosfatasa Alcalina/administración & dosificación , Lavado Peritoneal/métodos , Peritonitis/prevención & control , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Resultado del Tratamiento
6.
Inflamm Bowel Dis ; 17(2): 532-42, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20645323

RESUMEN

BACKGROUND: The brush border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and is protective against dextran sulfate sodium (DSS)-induced acute injury in rats. The present study evaluated the potential therapeutic role for orally administered calf IAP (cIAP) in two independent mouse models of chronic colitis: 1) DSS-induced chronic colitis, and 2) chronic spontaneous colitis in Wiskott-Aldrich Syndrome protein (WASP)-deficient (knockout) mice that is accelerated by irradiation. METHODS: The wildtype (WT) and IAP knockout (IAP-KO) mice received four cycles of 2% DSS ad libitum for 7 days. Each cycle was followed by a 7-day DSS-free interval during which mice received either cIAP or vehicle in the drinking water. The WASP-KO mice received either vehicle or cIAP for 6 weeks beginning on the day of irradiation. RESULTS: Microscopic colitis scores of DSS-treated IAP-KO mice were higher than DSS-treated WT mice (52±3.8 versus 28.8±6.6, respectively, P<0.0001). cIAP treatment attenuated the disease in both groups (KO=30.7±6.01, WT=18.7±5.0, P<0.05). In irradiated WASP-KO mice cIAP also attenuated colitis compared to control groups (3.3±0.52 versus 6.2±0.34, respectively, P<0.001). Tissue myeloperoxidase activity and proinflammatory cytokines were significantly decreased by cIAP treatment. CONCLUSIONS: Endogenous IAP appears to play a role in protecting the host against chronic colitis. Orally administered cIAP exerts a protective effect in two independent mouse models of chronic colitis and may represent a novel therapy for human IBD.


Asunto(s)
Fosfatasa Alcalina/administración & dosificación , Colitis/prevención & control , Modelos Animales de Enfermedad , Animales , Enfermedad Crónica , Colitis/inducido químicamente , Colitis/enzimología , Citocinas/metabolismo , Sulfato de Dextran/toxicidad , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peroxidasa/metabolismo , Síndrome de Wiskott-Aldrich , Proteína del Síndrome de Wiskott-Aldrich/fisiología
7.
Am J Physiol Gastrointest Liver Physiol ; 299(2): G467-75, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20489044

RESUMEN

Intestinal alkaline phosphatase (IAP) is a small intestinal brush border enzyme that has been shown to function as a gut mucosal defense factor, but its precise mechanism of action remains unclear. We investigated the effects of IAP on specific bacteria and bacterial components to determine its molecular targets. Purulent fluid from a cecal ligation and puncture model, specific live and heat-killed bacteria (Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes), and a variety of proinflammatory ligands (LPS, CpG DNA, Pam-3-Cys, flagellin, and TNF) were incubated with or without calf IAP (cIAP). Phosphate release was determined by using a malachite green assay. The various fluids were applied to target cells (THP-1, parent HT-29, and IAP-expressing HT-29 cells) and IL-8 secretion measured by ELISA. cIAP inhibited IL-8 induction by purulent fluid in THP-1 cells by >35% (P < 0.005). HT29-IAP cells had a reduced IL-8 response specifically to gram-negative bacteria; >90% reduction compared with parent cells (P < 0.005). cIAP had no effect on live bacteria but attenuated IL-8 induction by heat-killed bacteria by >40% (P < 0.005). cIAP exposure to LPS and CpG DNA caused phosphate release and reduced IL-8 in cell culture by >50% (P < 0.005). Flagellin exposure to cIAP also resulted in reduced IL-8 secretion by >40% (P < 0.005). In contrast, cIAP had no effect on TNF or Pam-3-Cys. The mechanism of IAP action appears to be through dephosphorylation of specific bacterial components, including LPS, CpG DNA, and flagellin, and not on live bacteria themselves. IAP likely targets these bacterially derived molecules in its role as a gut mucosal defense factor.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Fenómenos Fisiológicos Bacterianos , ADN Bacteriano/metabolismo , Flagelina/metabolismo , Mucosa Intestinal/metabolismo , Lipopolisacáridos/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Abdomen/microbiología , Absceso/metabolismo , Fosfatasa Alcalina/farmacología , Animales , Bacterias/efectos de los fármacos , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/prevención & control , Bovinos , Línea Celular , Escherichia coli/fisiología , Células HT29 , Humanos , Interleucina-8/antagonistas & inhibidores , Interleucina-8/biosíntesis , Mucosa Intestinal/microbiología , Listeria monocytogenes/fisiología , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Salmonella typhimurium/fisiología , Receptores Toll-Like/metabolismo
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