RESUMEN
The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barrett's oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET-1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α3 , α4, α5 , α6 and αν ). Experimental findings were validated in human explant oesophageal biopsies, a rat model of gastroesophageal reflux disease (GORD) and in patient tissue microarrays. The bile acid deoxycholic acid (DCA) specifically reduced adhesion of HET-1A cells to vitronectin and reduced cell-surface expression of integrin-αν via effects on endocytic recycling processes. Increased expression of integrin-αv was observed in ulcerated tissue in a rat model of GORD and in oesophagitis and Barrett's intestinal metaplasia patient tissue compared to normal squamous epithelium. Increased expression of integrin-αν was observed in QH BO cells compared to HET-1A cells. QH cells were resistant to DCA-mediated loss of adhesion and reduction in cell-surface expression of integrin-αν . We demonstrated that a specific component of the gastric refluxate, DCA, affects the epithelial barrier through modulation of integrin αν expression, providing a novel mechanism for bile acid-mediated erosion of oesophageal squamous epithelium and promotion of BO. Strategies aimed at preventing bile acid-mediated erosion should be considered in the clinical management of patients with GORD.
Asunto(s)
Esófago de Barrett/metabolismo , Ácido Desoxicólico/farmacología , Células Epiteliales/efectos de los fármacos , Esofagitis/metabolismo , Reflujo Gastroesofágico/metabolismo , Integrina alfaV/genética , Animales , Esófago de Barrett/genética , Esófago de Barrett/patología , Adhesión Celular , Línea Celular , Colágeno/química , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Esofagitis/genética , Esofagitis/patología , Fibronectinas/química , Reflujo Gastroesofágico/genética , Reflujo Gastroesofágico/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Integrina alfaV/metabolismo , Integrinas/genética , Integrinas/metabolismo , Laminina/química , Permeabilidad/efectos de los fármacos , Transporte de Proteínas , Ratas , Análisis de Matrices Tisulares , Vitronectina/químicaRESUMEN
BACKGROUND: Eradication rates for current H. pylori therapies have fallen in recent years, in line with the emergence of antibiotic resistant infections. The development of therapeutic alternatives to antibiotics, such as immunomodulatory therapy and vaccines, requires a more lucid understanding of host-pathogen interactions, including the relationships between the organism and the innate immune response. Pellino proteins are emerging as key regulators of immune signaling, including the Toll-like receptor pathways known to be regulated by H. pylori. The aim of this study was to characterize the role of Pellino proteins in the innate immune response to H. pylori lipopolysaccharide. MATERIALS AND METHODS: Gain-of-function and loss-of-function approaches were utilized to elucidate the role of individual Pellino proteins in the Toll-like receptor 2-mediated response to H. pylori LPS by monitoring NF-ĸB activation and the induction of proinflammatory chemokines. Expression of Pellino family members was investigated in gastric epithelial cells and gastric tissue biopsy material. RESULTS: Pellino1 and Pellino2 positively regulated Toll-like receptor 2-driven responses to H. pylori LPS, whereas Pellino3 exerted a negative modulatory role. Expression of Pellino1 was significantly higher than Pellino3 in gastric epithelial cells and gastric tissue. Furthermore, Pellino1 expression was further augmented in gastric epithelial cells in response to infection with H. pylori or stimulation with H. pylori LPS. CONCLUSIONS: The combination of low Pellino3 levels together with high and inducible Pellino1 expression may be an important determinant of the degree of inflammation triggered upon Toll-like receptor 2 engagement by H. pylori and/or its components, contributing to H. pylori-associated pathogenesis by directing the incoming signal toward an NF-kB-mediated proinflammatory response.
Asunto(s)
Inmunidad Innata , Lipopolisacáridos/inmunología , Proteínas Nucleares/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Epiteliales/inmunología , Mucosa Gástrica/inmunología , Humanos , Proteínas Nucleares/genética , Técnicas de Cultivo de Órganos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Celecoxib is a COX-2 inhibitor drug that can be used to reduce the risk of colorectal adenocarcinoma. Glucocorticoids are used in the treatment of inflammatory bowel disease. A limitation to the use of both drug types is that they undergo absorption from the intestinal tract with serious side effects. The prodrug systems introduced here involve forming a nitro-substituted acylsulfonamide group in the case of celecoxib and a nitro-substituted 21-ester for the glucocorticoids. Drug release is triggered by the nitro reductase action of the colonic microflora, liberating a cyclization competent species. The release of the active parent drugs was evaluated in vitro using Clostridium perfringens and epithelial transport through Caco-2 monolayer evaluation was carried out to estimate the absorption properties of the prodrugs compared to the parental drugs.
Asunto(s)
Antineoplásicos/química , Budesonida/química , Nitrobencenos/química , Prednisolona/química , Profármacos/química , Pirazoles/química , Sulfonamidas/química , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Budesonida/uso terapéutico , Budesonida/toxicidad , Células CACO-2 , Celecoxib , Permeabilidad de la Membrana Celular/efectos de los fármacos , Clostridium perfringens/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Inhibidores de la Ciclooxigenasa 2/toxicidad , Humanos , Lactonas/química , Nitrorreductasas/metabolismo , Prednisolona/uso terapéutico , Prednisolona/toxicidad , Profármacos/uso terapéutico , Profármacos/toxicidad , Pirazoles/uso terapéutico , Pirazoles/toxicidad , Sulfonamidas/uso terapéutico , Sulfonamidas/toxicidadRESUMEN
Helicobacter pylori causes chronic gastritis, peptic ulcers, and gastric carcinoma. Gastric epithelial cells provide the first point of contact between H. pylori and the host. TLRs present on these cells recognize various microbial products, resulting in the initiation of innate immunity. Although previous reports investigated TLR signaling in response to intact H. pylori, the specific contribution of H. pylori LPS with regard to functional genomics and cell-signaling events has not been defined. This study set out to define downstream signaling components and altered gene expression triggered by H. pylori LPS and to investigate the role of the signaling protein tribbles 3 (TRIB3) during the TLR-mediated response to H. pylori LPS. Cotransfections using small interfering RNA and dominant-negative constructs demonstrated that H. pylori LPS functions as a classic TLR2 ligand by signaling through pathways involving the key TLR signaling components MyD88 adaptor-like, MyD88, IRAK1, IRAK4, TNFR-associated factor 6, IκB kinase ß, and IκBα. Microarray analysis, real-time PCR, and ELISA revealed the induction of a discrete pattern of chemokines as a direct effect of LPS:TLR2 signaling. H. pylori infection was associated with decreased expression of TRIB3 in human gastric epithelial cell lines and tissue samples. Additionally, H. pylori decreased expression of C/EBP homologous protein and activating transcription factor 4, the transcription factors involved in the induction of TRIB3 expression. Furthermore, knockdown of TRIB3 and C/EBP homologous protein enhanced TLR2-mediated NF-κB activation and chemokine induction in response to H. pylori LPS. Thus, modulation of TRIB3 by H. pylori and/or its products may be an important mechanism during H. pylori-associated pathogenesis.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Helicobacter pylori/inmunología , Lipopolisacáridos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Represoras/fisiología , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/biosíntesis , Línea Celular , Línea Celular Tumoral , Quimiocinas/biosíntesis , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células HEK293 , Humanos , Inmunidad Innata/genética , Interleucina-8/biosíntesis , Interleucina-8/genética , Lipopolisacáridos/aislamiento & purificación , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/inmunología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/biosíntesis , Transducción de Señal/genética , Receptor Toll-Like 2/fisiología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
This Letter generalizes the metabolism of the azo class of compounds by Clostridium perfringens, an anaerobe found in the human colon. A recently reported 5-aminosalicylic acid-based prednisolone prodrug was shown to release the drug when incubated with the bacteria, while the para-aminobenzoic acid (PABA) based analogue did not. Instead, it showed a new HPLC peak with a relatively close retention time to the parent which was identified by LCMS as the partially reduced hydrazine product. This Letter investigates azoreduction across a panel of substrates with varying degrees of electronic and steric similarity to the PABA-based compound. Azo compounds with an electron donating group on the azo-containing aromatic ring showed immediate disproportionation to their parent amines without any detection of hydrazine intermediates by HPLC. Compounds containing only electron withdrawing groups are partially and reversibly reduced to produce a stable detectable hydrazine. They do not disproportionate to their parent amines, but regenerate the parent azo compound. This incomplete reduction is relevant to the design of azo-based prodrugs and the toxicology of azo-based dyes.
Asunto(s)
Compuestos Azo/metabolismo , Clostridium perfringens/química , Diseño de Fármacos , Profármacos/síntesis química , Anaerobiosis , Compuestos Azo/química , Clostridium perfringens/metabolismo , Humanos , Estructura Molecular , Profármacos/química , Profármacos/metabolismoRESUMEN
Budesodine is a synthetic glurocorticoid that undergoes substantial first pass metabolism, limiting systemic exposure. Its use in treatment of inflammatory bowel disease would benefit from a targeting strategy that could lead to a local topical effect, improving safety and increasing anti-inflammatory efficacy. A two-step prodrug strategy involving azoreduction/cyclization that we developed previously for prednisolone is here applied with some variations to budesonide. The budesodine prodrugs were tested using an in vitro azoreductase assay simulating human colonic microflora. The kinetics of amino steroid ester cyclization and its pH dependence was also evaluated. The stability of the prodrugs systems in simulated human duodenal and gastric fluid was evaluated to determine the likelihood of intact intestinal transit. The propionic acid derived prodrug 3 undergoes rapid activation by Clostridium perfingens and its putative reduction product cyclizes with acceptable rapidity when synthesized independently. These properties of 3 suggest that it has potential in management of ulcerative colitis.
Asunto(s)
Budesonida/análogos & derivados , Budesonida/metabolismo , Colon/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Profármacos/metabolismo , Profármacos/farmacocinética , Budesonida/química , Clostridium perfringens , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Colon/microbiología , Ciclización , Sistemas de Liberación de Medicamentos , Humanos , Estructura Molecular , Nitrorreductasas , Especificidad de Órganos , Profármacos/químicaRESUMEN
BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) results from mutations in the SERPINA1 gene and classically presents with early-onset emphysema and liver disease. The most common mutation presenting with clinical evidence is the Z mutation, while the S mutation is associated with a milder plasma deficiency. AATD is an under-diagnosed condition and the World Health Organisation recommends targeted detection programmes for AATD in patients with chronic obstructive pulmonary disease (COPD), non-responsive asthma, cryptogenic liver disease and first degree relatives of known AATD patients. METHODS: We present data from the first 3,000 individuals screened following ATS/ERS guidelines as part of the Irish National Targeted Detection Programme (INTDP). We also investigated a DNA collection of 1,100 individuals randomly sampled from the general population. Serum and DNA was collected from both groups and mutations in the SERPINA1 gene detected by phenotyping or genotyping. RESULTS: The Irish National Targeted Detection Programme identified 42 ZZ, 44 SZ, 14 SS, 430 MZ, 263 MS, 20 IX and 2 rare mutations. Analysis of 1,100 randomly selected individuals identified 113 MS, 46 MZ, 2 SS and 2 SZ genotypes. CONCLUSION: Our findings demonstrate that AATD in Ireland is more prevalent than previously estimated with Z and S allele frequencies among the highest in the world. Furthermore, our targeted detection programme enriched the population of those carrying the Z but not the S allele, suggesting the Z allele is more important in the pathogenesis of those conditions targeted by the detection programme.
Asunto(s)
Deficiencia de alfa 1-Antitripsina/epidemiología , Distribución de Chi-Cuadrado , Análisis Mutacional de ADN , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Encuestas Epidemiológicas , Humanos , Irlanda/epidemiología , Tamizaje Masivo/métodos , Mutación , Oportunidad Relativa , Fenotipo , Prevalencia , Medición de Riesgo , Factores de Riesgo , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/diagnóstico , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
The design, synthesis and delivery potential of a new type of benzenesulfonamide cyclo-oxygenase-2 (COX-2) inhibitor prodrug is investigated using celecoxib. The approach involves a double prodrug that is activated first by azoreductases and then by cyclization triggering drug release. We studied the intramolecular aminolysis of the acylsulfonamide. The cyclization was surprisingly rapid at physiological pH and very fast at pH 5. The prodrug is activated specifically under conditions found in the colon but highly stable in the presence of human and rodent intestinal extracts. Finally, the prototype with celecoxib was transported much more slowly in the Caco-2 transepithelial model than the parent. The design therefore shows significant promise for the site specific delivery of benzenesulfonamide COX-2 inhibitors to the colon.
Asunto(s)
Colon/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacocinética , Diseño de Fármacos , Profármacos/farmacocinética , Pirazoles/farmacocinética , Sulfonamidas/farmacocinética , Animales , Células CACO-2 , Celecoxib , Clostridium perfringens/enzimología , Colon/microbiología , Neoplasias del Colon/tratamiento farmacológico , Ciclización , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/metabolismo , Humanos , NADH NADPH Oxidorreductasas/metabolismo , Nitrorreductasas , Profármacos/química , Profármacos/metabolismo , Pirazoles/química , Pirazoles/metabolismo , Ratas , Sulfonamidas/química , Sulfonamidas/metabolismo , BencenosulfonamidasRESUMEN
INTRODUCTION: Lymphocyte homeostasis is dependent on the γc cytokines. We hypothesised that sepsis in humans is associated with differential gene expression of the γc cytokines and their associated apoptosis mediators. METHODS: The study population consisted of a total of 60 patients with severe sepsis, 15 with gram negative bacteraemia, 10 healthy controls and 60 patients undergoing elective lung resection surgery. Pneumonia was diagnosed by CDC NNIC criteria. Gene expression in peripheral blood leukocytes (PBLs) of interleukin (IL)-2, 7, 15 and interferon (IFN)-γ, Bax, Bim, Bcl-2 was determined by qRT-PCR and IL-2 and IL-7 serum protein levels by ELISA. Gene expression of IL-2, 7 and IFN-γ was measured in peripheral blood leukocytes (PBL), cultured in the presence of lipopolysaccharide (LPS) and CD3 binding antibody (CD3ab) RESULTS: IL-2 gene expression was lower in the bacteraemia group compared with controls, and lower still in the sepsis group (P < 0.0001). IL-7 gene expression was similar in controls and bacteraemia, but lower in sepsis (P < 0.0001). IL-15 gene expression was similar in the three groups. Bcl-2 gene expression was less (P < 0.0001) and Bim gene expression was greater (P = 0.0003) in severe sepsis compared to bacteraemic and healthy controls. Bax gene expression was similar in the three groups.In lung resection surgery patients, post-operative pneumonia was associated with a perioperative decrease in IL-2 mRNA (P < 0.0001) and IL-7 mRNA (P = 0.003). IL-2 protein levels were reduced in sepsis and bacteraemia compared to controls (P = 0.02) but similar in pneumonia and non-pneumonia groups. IL-7 protein levels were similar in all groups.In cultured PBLs, IFN-γ gene expression was decreased in response to LPS and increased in response to CD3ab with sepsis: IL-7 gene expression increased in response to LPS in controls and to CD3ab with sepsis; Bcl-2 gene expression decreased in response to combined CD3ab and IL-2 with sepsis. CONCLUSIONS: Patients with infection and sepsis have deficient IL-2 and IL-7 gene expression in PBLs. Aberrant cytokine gene expression may precede the onset of infection.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/deficiencia , Quimiocinas C/deficiencia , Complicaciones Posoperatorias/genética , Sepsis/metabolismo , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis/genética , Bacteriemia/genética , Bacteriemia/metabolismo , Complejo CD3/inmunología , Células Cultivadas , Quimiocinas C/genética , Estudios de Cohortes , Femenino , Regulación Bacteriana de la Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/deficiencia , Interleucina-2/deficiencia , Interleucina-2/genética , Interleucina-7/deficiencia , Interleucina-7/genética , Lipopolisacáridos/farmacología , Masculino , Complicaciones Posoperatorias/microbiología , Estudios Prospectivos , Sepsis/genéticaRESUMEN
OBJECTIVE: The development and progression of severe sepsis is related to a deficiency in pro-inflammatory cytokine production, characterised by lesser IFNgamma levels, which are not explained by variations in levels of the main putative regulator of IFNgamma, namely IL-12. As alternative regulators of IFNgamma may be of greater importance in human sepsis, we investigated the hypothesis that the development of severe sepsis is related to variations in IL-18, IL-23 and IL-27 gene expression. DESIGN AND SETTING: A prospective observational trial in a mixed intensive care unit (ICU) and hospital wards in a university teaching hospital. PATIENTS AND PARTICIPANTS: Sixty-two ICU patients with severe sepsis, 13 bacteraemic patients with no acute critical illness, and 10 healthy controls. MEASUREMENTS AND RESULTS: All subjects were assayed for IL-18, IL-23 and IL-27 mRNA levels in peripheral blood. IL-27 mRNA levels distinguished between the three groups, with levels highest in the ICU group, intermediate in the bacteraemic group and lowest in the control group. IL-23 distinguished between the groups, with levels lowest in the ICU group. In late sepsis IL-23 and TNFalpha mRNA levels were directly related. IL-18 mRNA levels did not distinguish between the patient groups. CONCLUSIONS: We conclude that the deficient pro-inflammatory response in patients with sepsis is expansive and includes deficient IL-23 and excessive IL-27 gene expression. This provides further evidence that upregulation of a cytokine-based immune response is beneficial in sepsis.
Asunto(s)
Interferón gamma/deficiencia , Interleucina-17/sangre , Interleucina-18/sangre , Interleucina-23/sangre , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Bacteriemia/inmunología , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-17/genética , Interleucina-18/genética , Interleucina-23/genética , Masculino , Persona de Mediana Edad , Estudios Prospectivos , ARN Mensajero/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de SupervivenciaRESUMEN
Clostridium difficile is the leading cause of infectious antibiotic-associated diarrhoea, particularly among the elderly. Its surface-layer protein (SLP) was tested as a vaccine component in a series of immunization and challenge experiments with Golden Syrian hamsters, combined with different systemic and mucosal adjuvants. Some regimens were also tested in a nonchallenge BALB/c mouse model, enabling closer monitoring of the immune response. None of the regimens conferred complete protection in the hamster model, and antibody stimulation was variable within regimens, and generally modest or poor. Mice displayed stronger antibody responses to SLP compared with hamsters. Two hamsters of five given SLP with Ribi (monophosphoryl lipid A and synthetic trehalose dicorynomycolate) survived the challenge, as did two of three given SLP with Ribi and cholera toxin. This modest trend to protection is interpreted with caution, because the survivors had low anti-SLP serum antibody titres. The hamsters were an outbred line, and subject to more genetic variability than inbred animals; however, BALB/c mice also showed strongly variable antibody responses. There is a clear need for better adjuvants for single-component vaccines, particularly for mucosal delivery. The hamster challenge model may need to be modified to be useful in active immunization experiments with SLP.
Asunto(s)
Vacunas Bacterianas/inmunología , Clostridioides difficile/inmunología , Enterocolitis Seudomembranosa/prevención & control , Glicoproteínas de Membrana/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Esqueleto de la Pared Celular/administración & dosificación , Toxina del Cólera/administración & dosificación , Toxina del Cólera/inmunología , Factores Cordón/administración & dosificación , Cricetinae , Enterocolitis Seudomembranosa/inmunología , Femenino , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Lípido A/administración & dosificación , Lípido A/análogos & derivados , Mesocricetus , Ratones , Ratones Endogámicos BALB C , Análisis de SupervivenciaRESUMEN
Patient response to acute bacterial infection is highly variable. Differing outcomes in this setting may be related to variations in the immune response to an infectious insult. Using quantitative real-time polymerase chain reaction, we quantified gene expression of the tumor necrosis factor alpha(TNFalpha), interferon gamma (IFNgamma), and interleukin 10 (IL10), IL12p35, and IL4 genes in 3 patient groups. These groups consisted of an intensive care unit (ICU) cohort who presented with severe sepsis or septic shock, a group of noncritically ill ward patients with documented Gram-negative bacteremia, and a group of healthy controls. Greater interleukin 10 messenger RNA (mRNA) levels were detected in the ICU group in comparison with both the bacteremic and control groups (P < 0.0001). More TNF-alpha mRNA was detected in the ICU group when compared with the control group (P < 0.0001). However, TNF-alpha mRNA was most abundant in the bacteremic group (P = 0.0007). Lesser IFN-gamma mRNA levels were detected in the ICU group when compared with both the bacteremic and control groups (P < 0.0003). Cytokine mRNA levels were not associated with the occurrence of shock upon admission to ICU. On the seventh day of ICU stay, the presence of shock was associated with lesser IFN-gamma mRNA (P = 0.0004) and lesser TNF-alpha mRNA (P = 0.001). Survivors had greater TNF-alpha mRNA copy numbers on day 7 of ICU stay than nonsurvivors (P = 0.002). We conclude that a proinflammatory response is the appropriate response in the setting of infection and is associated with lesser requirements for inotropes and lesser mortality. Quantitative real-time polymerase chain reaction can be used to predict infection outcome in clinically relevant situations where enzyme-linked immunosorbent assay testing has proved disappointing.
Asunto(s)
Regulación de la Expresión Génica , Sepsis/diagnóstico , Sepsis/metabolismo , Choque/diagnóstico , Choque/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Humanos , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
INTRODUCTION: Asymmetrical dimethyl arginine (ADMA) is an endogenous non-selective inhibitor of nitric oxide synthase that may influence the severity of organ failure and the occurrence of shock secondary to an infectious insult. Levels may be genetically determined by a promoter polymorphism in a regulatory gene encoding dimethylarginine dimethylaminohydrolase II (DDAH II), which functions by metabolising ADMA to citrulline. The aim of this study was to examine the association between ADMA levels and the severity of organ failure and shock in severe sepsis and also to assess the influence of a promoter polymorphism in DDAH II on ADMA levels. METHODS: A prospective observational study was designed, and 47 intensive care unit (ICU) patients with severe sepsis and 10 healthy controls were enrolled. Serum ADMA and IL-6 were assayed on admission to the ICU and seven days later. Allelic variation for a polymorphism at position -449 in the DDAH II gene was assessed in each patient. Clinical and demographic details were also collected. RESULTS: On day 1 more ADMA was detectable in the ICU group than in the control group (p = 0.005). Levels subsequently increased during the first week in ICU (p = 0.001). ADMA levels were associated with vasopressor requirements on day one (p = 0.001). ADMA levels and Sequential Organ Failure Assessment scores were directly associated on day one (p = 0.0001) and day seven (p = 0.002). The degree of acidaemia and lactaemia was directly correlated with ADMA levels at both time points (p < 0.01). On day seven, IL-6 was directly correlated with ADMA levels (p = 0.006). The variant allele with G at position -449 in the DDAH II gene was associated with increased ADMA concentrations at both time points (p < 0.05). CONCLUSION: Severity of organ failure, inflammation and presence of early shock in severe sepsis are associated with increased ADMA levels. ADMA concentrations may be influenced by a polymorphism in the DDAH II gene.
Asunto(s)
Amidohidrolasas/sangre , Amidohidrolasas/genética , Arginina/análogos & derivados , Polimorfismo Genético/genética , Choque Séptico/genética , Alelos , Arginina/sangre , Arginina/genética , Femenino , Variación Genética , Humanos , Masculino , Estudios Prospectivos , Choque Séptico/sangreRESUMEN
Clostridium difficile is a major cause of antibiotic-associated diarrhoea and the primary cause of pseudomembraneous colitis in hospitalised patients. We assessed the protective effect of anti-surface layer protein (SLP) antibodies on C. difficile infection in a lethal hamster challenge model. Post-challenge survival was significantly prolonged in the anti-SLP treated group compared with control groups (P=0.0281 and P=0.0283). The potential mechanism of action of the antiserum was shown to be through enhancement of C. difficile phagocytosis. This report indicates that anti-SLP antibodies can modulate the course of C. difficile infection and may therefore merit closer investigation for use as constituents of multi-component vaccines against C. difficile associated diarrhoea.
Asunto(s)
Anticuerpos Antibacterianos/uso terapéutico , Proteínas Bacterianas/inmunología , Clostridioides difficile/inmunología , Enterocolitis Seudomembranosa/prevención & control , Inmunización Pasiva , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Línea Celular , Cricetinae , Modelos Animales de Enfermedad , Femenino , Humanos , Mesocricetus , Monocitos , FagocitosisRESUMEN
OBJECTIVES: The need for full colonoscopies in average-risk patients with non-specific colonic symptoms is controversial. We aimed to evaluate: (1) the yield of full colonoscopy; (2) the prevalence of proximal neoplasia in these patients; (3) the yield if any of doing full colonoscopies to diagnose proximal lesions in patients in whom the distal colon was clear; (4) the significance of this yield with respect to age. DESIGN: This is a retrospective analysis to assess the value of open access colonoscopy. PATIENTS AND METHODS: All patients who underwent a colonoscopy in our Endoscopy Unit during January 1996 to December 1999 were assessed (n = 3357). RESULTS: We analysed 945 patients with average risk and non-specific colonic symptoms (significant risk factors excluded). The overall yield of adenomas was 5.8%. The yield of distal adenomas in patients > or= 50 years of age was 8.2% (37 out of 450) versus 0.2% in the 50 years group (one out of 495; = 0.0001). The proximal adenoma yield in > or= 50 year olds was 3.8% (17 out of 495) versus 0.2% in < 50 year olds (one out of 495) (P = 0.0001). CONCLUSIONS: In a cohort of average-risk patients with non-specific colonic symptoms attending an "open access" colonoscopy clinic, the yield for proximal adenomas is small in the < 50 years group. In patients aged < 50 years, distal colonic examination is all that is required, whereas a full colonoscopy may be justified in patients > or = 50 years old.
Asunto(s)
Adenoma/diagnóstico , Neoplasias del Colon/diagnóstico , Colonoscopía/métodos , Adulto , Factores de Edad , Anciano , Neoplasias del Colon/patología , Pólipos del Colon/diagnóstico , Pólipos del Colon/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Helicobacter pylori (H. pylori) is the most important etiological agent of chronic active gastritis, peptic ulcer disease and gastric cancer. The aim of this study was to evaluate the efficacy of alkyl hydroperoxide reductase (AhpC) and mannosylated AhpC (mAhpC) as candidate vaccines in the C57BL/6J mouse model of H. pylori infection. Recombinant AhpC was cloned, over-expressed and purified in an unmodified form and was also engineered to incorporate N and C-terminal mannose residues when expressed in the yeast Pichia pastoris. Mice were immunized systemically and mucosally with AhpC and systemically with mAhpC prior to challenge with H. pylori. Serum IgG responses to AhpC were determined and quantitative culture was used to determine the efficacy of vaccination strategies. Systemic prophylactic immunization with AhpC/alum and mAhpC/alum conferred protection against infection in 55% and 77.3% of mice, respectively. Mucosal immunization with AhpC/cholera toxin did not protect against infection and elicited low levels of serum IgG in comparison with systemic immunization. These data support the use of AhpC as a potential vaccine candidate against H. pylori infection.
Asunto(s)
Vacunas Bacterianas/inmunología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/enzimología , Helicobacter pylori/inmunología , Peroxirredoxinas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Glicoproteínas/inmunología , Infecciones por Helicobacter/inmunología , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos C57BL , Pichia/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Barrett's esophagus is an increasingly common disease that is strongly associated with reflux of stomach acid and usually a hiatus hernia, and it strongly predisposes to esophageal adenocarcinoma (EAC), a tumor with a very poor prognosis. We report the first genome-wide association study on Barrett's esophagus, comprising 1,852 UK cases and 5,172 UK controls in the discovery stage and 5,986 cases and 12,825 controls in the replication stage. Variants at two loci were associated with disease risk: chromosome 6p21, rs9257809 (Pcombined=4.09×10(-9); odds ratio (OR)=1.21, 95% confidence interval (CI)=1.13-1.28), within the major histocompatibility complex locus, and chromosome 16q24, rs9936833 (Pcombined=2.74×10(-10); OR=1.14, 95% CI=1.10-1.19), for which the closest protein-coding gene is FOXF1, which is implicated in esophageal development and structure. We found evidence that many common variants of small effect contribute to genetic susceptibility to Barrett's esophagus and that SNP alleles predisposing to obesity also increase risk for Barrett's esophagus.
Asunto(s)
Esófago de Barrett/genética , Cromosomas Humanos Par 16 , Predisposición Genética a la Enfermedad , Complejo Mayor de Histocompatibilidad , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Modelos GenéticosRESUMEN
Photodynamic therapy (PDT) has developed over last century and is now becoming a more widely used medical tool having gained regulatory approval for the treatment of various diseases such as cancer and macular degeneration. It is a two-step technique in which the delivery of a photosensitizing drug is followed by the irradiation of light. Activated photosensitizers transfer energy to molecular oxygen which results in the generation of reactive oxygen species which in turn cause cells apoptosis or necrosis. Although this modality has significantly improved the quality of life and survival time for many cancer patients it still offers significant potential for further improvement. In addition to the development of new PDT drugs, the use of nanosized carriers for photosensitizers is a promising approach which might improve the efficiency of photodynamic activity and which can overcome many side effects associated with classic photodynamic therapy. This review aims at highlighting the different types of nanomedical approaches currently used in PDT and outlines future trends and limitations of nanodelivery of photosensitizers.
Asunto(s)
Composición de Medicamentos/tendencias , Nanocápsulas/química , Nanocápsulas/uso terapéutico , Nanomedicina/tendencias , Fotoquimioterapia/tendencias , Animales , HumanosRESUMEN
OBJECTIVES: The aim of this study was to investigate drug release from a double steroid prodrug, OPN501, which incorporates a phenylpropionate linker, and its phenylacetate analogue. The prodrugs, which were designed to deliver prednisolone to the colon for the treatment of inflammatory bowel disease, are based on a novel design that requires sequential azoreductase activity and cyclization of an amino ester to trigger drug release. We sought to explain the divergent effects of the two compounds in anti-inflammatory models and to justify the selection of OPN-501 for clinical development. METHODS: The compounds were incubated in mouse colonic contents (10%) fermented in brain heart infusion under anaerobic conditions. The disappearance of the prodrugs and release of prednisolone was monitored by HPLC. We then developed a method for assessment of prodrug activation using suspensions of Clostridium perfringens, an anaerobe from the human colon. The cyclization of the compounds was studied in various media, assessing the influence of pH and bulk solvent polarity on cyclization rate using HPLC and NMR. KEY FINDINGS: The prodrugs were activated via multiple pathways releasing prednisolone in mouse colonic ferment. The compounds released prednisolone by reduction-cyclization in C perfringens suspension. The active OPN-501 generated a stoichiometric amount of prednisolone following azoreductase activation, whereas its analogue did not. The pH rate profile for the cyclization of the amino intermediates of the two compounds revealed significant differences in rate at pH values relevant to the inflamed colon, which explain in part the different amounts of drug produced. CONCLUSIONS: The steroid prodrug OPN-501 has optimal drug release characteristics for colon targeting because of a kinetic advantage of a six-membered ring formation in the aminolysis reactions of anilides. The results are relevant to the development of OPN-501 but also to cyclization strategies in prodrug design especially for colon targeting.
Asunto(s)
Colon/metabolismo , Sistemas de Liberación de Medicamentos/métodos , NADH NADPH Oxidorreductasas/metabolismo , Prednisolona/administración & dosificación , Profármacos/administración & dosificación , Aminoácidos/química , Animales , Humanos , Concentración de Iones de Hidrógeno , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , NitrorreductasasRESUMEN
Identification of the molecular mechanisms of host-pathogen interaction is becoming a key focus of proteomics. Analysis of these interactions holds promise for significant developments in the identification of new therapeutic strategies to combat infectious diseases, a process that will also benefit parallel improvements in molecular diagnostics, biomarker identification and drug discovery. This review highlights recent advances in functional proteomics initiatives in infectious disease with emphasis on studies undertaken within physiologically relevant parameters that enable identification of the infectious proteome rather than that of the vegetative state. Deciphering the molecular details of what constitutes physiologically relevant host-pathogen interactions remains an underdeveloped aspect of research into infectious disease. The magnitude of this deficit will be largely influenced by the ease with which model systems can be established to investigate such interactions. As the selective pressures exerted by the host on an infecting pathogen are numerous, the adequacy of certain model systems should be considered carefully.