RESUMEN
BACKGROUND: Antimicrobial peptides are key players of initial innate immune responses to human pathogens. Two major representatives, the human beta defensin 2 and 3 (hBD2 and hBD3), are both known to be regulated by, and to affect viability of, Helicobacter pylori. Previously, it was demonstrated in vitro that H. pylori actively abrogates hBD3 expression during prolonged infections. Here, we comprehensively assessed hBD2 and hBD3 expression ex vivo in the gastric mucosa of healthy individuals. MATERIALS AND METHODS: Twenty volunteers (H. pylori positive and H. pylori negative: n = 10) were enrolled. Helicobacter pylori-positive subjects underwent eradication therapy and repeated the protocol. Expression of both defensins was assessed by quantitative RT-PCR and ELISA, and correlated with histopathologic degree of gastritis. RESULTS: hBD2 and hBD3 were found to be ubiquitously expressed in all three groups. In general, hBD2 levels were elevated in relation to H. pylori infection (up to 40-fold). This upregulation correlated with degree of gastritis in corpus and antrum. In contrast, hBD3 protein levels were significantly decreased, while corresponding mRNA amounts remained unchanged. Eradication therapy led to normalization of mucosal hBD2 expression, while hBD3 expression demonstrated high interindividual variations among individuals. CONCLUSIONS: Both defensins are ubiquitously but differentially expressed in gastric mucosa in relation to H. pylori infection. Ex vivo data support the notion that H. pylori infection is associated with reduced hBD3 expression in chronic active gastritis.
Asunto(s)
Mucosa Gástrica/patología , Perfilación de la Expresión Génica , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , beta-Defensinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Mucosa Gástrica/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Histocitoquímica , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Here we demonstrate basophile structures located in the arteriolar wall and being associated with a plasma-protein-leakage. We assume, that the structures indicate blood-brain-barrier-disturbances and degenerative small vessel wall alterations.
Asunto(s)
Endotelio Vascular/metabolismo , Agaricus/metabolismo , Factores de Edad , Animales , Arteriolas/metabolismo , Barrera Hematoencefálica , Encéfalo/metabolismo , Antígeno CD11c/biosíntesis , Capilares/metabolismo , Citocinas/metabolismo , Células Dendríticas/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interferón gamma/metabolismo , Lectinas Tipo C/biosíntesis , Masculino , Ratones , Microcirculación , Ratas , Bazo/citología , beta-Glucanos/metabolismoRESUMEN
AIMS: Gastro-oesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. This study was aimed at investigating the role of desmosomal proteins in relation to GERD. METHODS AND RESULTS: Ninety-five patients with GERD-related symptoms (erosive, n = 51; non-erosive, n = 44) and 27 patients lacking those symptoms were included. Endoscopic and histological characterization of oesophagitis was performed according to the Los Angeles and Ismeil-Beigi criteria, respectively. Multiple biopsies were taken from the oesophageal mucosa of each patient. Gene expression analysis of plakoglobin, desmoglein-1, desmoglein-2 and desmoglein-3 was performed by quantitative real time (RT)-polymerase chain reaction and immunohistochemistry in the oesophageal mucosa. Routine histology revealed specific GERD-related alterations, such as dilatation of intercellular spaces (DIS), basal cell hyperplasia (BCH), and elongation of the papillae, in the oesophageal mucosa of patients with GERD, as compared with controls (all parameters: P < 0.05). All four genes and corresponding proteins were found to be up-regulated by between 1.7 and 8.1-fold (transcript level, P < 0.05; protein level, P < 0.05). Induced gene expression levels of plakoglobin, desmoglein-1 and desmoglein-2 correlated significantly with DIS and BCH. CONCLUSIONS: Taken together, the uniform up-regulation of desmosomal genes/proteins in the oesophageal mucosa of patients with GERD supports the concept of architectural and molecular changes in the desmosomal compartment in the pathogenesis of GERD.
Asunto(s)
Desmogleínas/genética , Desmosomas/patología , Esófago/patología , Reflujo Gastroesofágico/patología , Membrana Mucosa/patología , gamma Catenina/genética , Adolescente , Adulto , Anciano , Biomarcadores/metabolismo , Biopsia , Desmogleínas/metabolismo , Desmosomas/genética , Desmosomas/metabolismo , Endoscopía Gastrointestinal/métodos , Esofagitis/genética , Esofagitis/metabolismo , Esofagitis/patología , Esófago/metabolismo , Femenino , Reflujo Gastroesofágico/genética , Reflujo Gastroesofágico/metabolismo , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Membrana Mucosa/metabolismo , Estudios Prospectivos , Regulación hacia Arriba , Adulto Joven , gamma Catenina/metabolismoRESUMEN
BACKGROUND: Non-invasive tools for gastric cancer screening and diagnosis are lacking. Serological testing with the detection of pepsinogen 1 (PG1), pepsinogen 2 (PG2) and gastrin 17 (G17) offers the possibility to detect preneoplastic gastric mucosal conditions. Aim of this study was to assess the performance of these serological tests in the presence of gastric neoplasia. METHODS: Histological and serological samples of 118 patients with gastric cancer have been assessed for tumor specific characteristics (Laurén type, localisation), degree of mucosal abnormalities (intestinal metaplasia, atrophy) and serological parameters (PG1, PG2, PG1/2-ratio, G17, H. pylori IgG, CagA status). Association of the general factors to the different serological values have been statistically analyzed. RESULTS: Patients with intestinal type gastric cancer had lower PG1 levels and a lower PG1/2-ratio compared to those with diffuse type cancer (p = 0.003). The serum levels of PG2 itself and G17 were not significantly altered. H. pylori infection in general had no influence on the levels of PG1, PG2 and G17 in the serum of gastric cancer patients. There was a trend towards lower PG1 levels in case of positive CagA-status (p = 0.058). The degree of both intestinal metaplasia and atrophy correlated inversely with serum levels for PG1 and the PG1/2-ratio (p < 0.01). Laurén-specific analysis revealed that this is only true for intestinal type tumors. Univariate ANOVA revealed atrophy and CagA-status as the only independent factors for low PG1 and a low PG1/2-ratio. CONCLUSIONS: Glandular atrophy and a positive CagA status are determinant factors for decreased pepsinogen 1 levels in the serum of patients with gastric cancer. The serological assessment of gastric atrophy by analysis of serum pepsinogen is only adequate for patients with intestinal type cancer.
Asunto(s)
Adenocarcinoma/sangre , Adenocarcinoma/patología , Biomarcadores de Tumor/sangre , Mucosa Gástrica/patología , Neoplasias Gástricas/sangre , Neoplasias Gástricas/patología , Anciano , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/sangre , Atrofia , Proteínas Bacterianas/sangre , Femenino , Gastrinas/sangre , Infecciones por Helicobacter/sangre , Helicobacter pylori/inmunología , Humanos , Masculino , Persona de Mediana Edad , Pepsinógeno A/sangre , Lesiones Precancerosas/sangre , Estudios RetrospectivosRESUMEN
BACKGROUND: Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD. METHODS: Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)]. RESULTS: Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD. CONCLUSIONS: Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD.
Asunto(s)
Reflujo Gastroesofágico/fisiopatología , Proteínas de Uniones Estrechas/fisiología , Adulto , Anciano , Esofagitis/patología , Esofagitis/fisiopatología , Esofagoscopía , Femenino , Reflujo Gastroesofágico/patología , Gastroscopía , Regulación de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas de Uniones Estrechas/genética , Adulto JovenRESUMEN
Our previous studies have shown an association between Helicobacter pylori infection, the strong up-regulation of cathepsin X (CTSX, also called cathepsin Z/P), and the development of gastric cancer. In the present study, we analyzed primary and conventional gastric epithelial cell lines to establish an optimal in vitro mouse model system for the examination of H. pylori-induced overexpression of Ctsx in a functional way. Gastric epithelial cells were isolated from stomachs of wild-type C57BL6/N and Ctsx(-/-) mice and compared with the gastric cancer cell line CLS103. Indirect co-cultures of epithelial cells and macrophages were infected with H. pylori strain SS1 and analyzed for the expression of cathepsins, cytokines, and adhesion factors. Cellular interactions, migration capability, and adherence of H. pylori were assessed using time-lapse video microscopy and colony-forming assays. Isolated primary cells from wild-type and transgenic mice revealed qualities and expression profiles similar to those of corresponding tissue samples. Adherence of H. pylori was significantly higher in primary compared with commercially cells. Thus, induction of cathepsins, cytokines, and adhesion proteins was detected solely in primary cells and co-cultured macrophages. Microarray and migration experiments indicated that Ctsx is involved in B/T-cell proliferation/migration and adhesion of macrophages. Primary epithelial cells from stomach of Ctsx(-/-) mice represent an excellent model of H. pylori gastritis to elaborate the special functions of Ctsx in regulating the immune response to H. pylori.
Asunto(s)
Catepsina Z/metabolismo , Citocinas/metabolismo , Células Epiteliales/citología , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Macrófagos/citología , Animales , Movimiento Celular , Técnicas de Cocultivo , Células Epiteliales/microbiología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Estómago/microbiologíaRESUMEN
BACKGROUND & AIMS: Although the "submucosal cushion" technique or injection-assisted polypectomy (IAP) is often used to resect colon polyps, little is known on the influence of this technique on histologic interpretation. We aimed to evaluate whether the use of a submucosal cushion improves the histologic and margin evaluation of colon polyps. METHODS: Consecutive patients undergoing polypectomy with and without IAP were included. An experienced blinded gastrointestinal pathologist evaluated the specimens using standardized criteria. RESULTS: One hundred eleven sessile colon adenomas were analyzed (IAP, n = 65, standard, n = 46). Two-thirds of polyps ranged in size from 10 to 20 mm; the average polyp size was 13.2 mm for IAP and 9.9 mm for standard snare polypectomy (P = .001). The cautery degree, cautery amount, and margin evaluability, did not differ substantially with regard to the resection technique. For polyps ≥10-20 mm, the overall architecture quality was better in polyps resected with standard technique as compared with IAP. CONCLUSIONS: The utilization of IAP did not result in a better margin evaluability of the resected polyp. Overall, IAP does not result in a better histologic polyp evaluability.
Asunto(s)
Neoplasias del Colon/diagnóstico , Neoplasias del Colon/patología , Mucosa Intestinal/patología , Pólipos/diagnóstico , Pólipos/patología , Procedimientos Quirúrgicos Operativos/métodos , Anciano , Neoplasias del Colon/cirugía , Femenino , Histocitoquímica/métodos , Humanos , Mucosa Intestinal/cirugía , Masculino , Microscopía/métodos , Persona de Mediana Edad , Pólipos/cirugíaRESUMEN
INTRODUCTION: Protease-activated receptors (PAR) are seven transmembrane receptors that are expressed throughout the gastrointestinal tract. In vitro experiments using gastric tumor cell lines, murine models and one clinical study provided evidence for a potential role of PAR2 in Helicobacter pylori-induced gastritis. AIM: To investigate PAR2 expression in H. pylori-infected patients and correlation with proinflammatory IL-8, IL-1ß as well as histologic changes of the mucosa. Furthermore, PAR2 expression was studied in context to mucosal amounts of secretory leukocyte protease inhibitor (SLPI), a putative regulator of PAR2. METHODS: Twenty-two H. pylori-infected patients and 72 H. pylori-negative subjects underwent upper GI endoscopy. In antrum-derived mucosal biopsies, PAR2, IL-1ß, IL-8, and SLPI expression was analyzed by quantitative RT-PCR, and in part by ELISA and immunohistochemistry. Histopathologic evaluation of gastritis was performed according to the updated Sydney classification. RESULTS: IL-8 gene expression was 5-fold increased in the mucosa of H. pylori-infected patients compared with non-infected (p < .0001), whereas no differences for PAR2 and IL-1ß mRNA amounts were observed between both groups. PAR2 gene expression correlated positively with transcript levels of IL-8, IL-1ß as well mucosal SLPI levels in H. pylori-infected patients (r: 0.47-0.84; p < .0001), whereas no correlation was found with the degree of gastritis. CONCLUSIONS: PAR2 represents an additive pathway of IL-8 secretion and proinflammatory effects in H. pylori-induced gastritis. Reduced SLPI levels leading to higher serine protease activities in the mucosa of infected subjects might regulate PAR2 activation.
Asunto(s)
Mucosa Gástrica/patología , Gastritis/patología , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , Interacciones Huésped-Patógeno , Receptor PAR-2/metabolismo , Adulto , Animales , Femenino , Perfilación de la Expresión Génica , Histocitoquímica , Humanos , Inmunohistoquímica , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Masculino , Ratones , Persona de Mediana EdadRESUMEN
BACKGROUND: Mucosal levels of Secretory Leukocyte Protease Inhibitor (SLPI) are specifically reduced in relation to H. pylori-induced gastritis. Progranulin is an epithelial growth factor that is proteolytically degraded into fragments by elastase (the main target of SLPI). Considering the role of SLPI for regulating the activity of elastase, we studied whether the H. pylori-induced reduction of SLPI and the resulting increase of elastase-derived activity would reduce the Progranulin protein levels both ex vivo and in vitro. METHODS: The expression of Progranulin was studied in biopsies of H. pylori-positive, -negative and -eradicated subjects as well as in the gastric tumor cell line AGS by ELISA, immunohistochemistry and real-time RT-PCR. RESULTS: H. pylori-infected subjects had about 2-fold increased antral Progranulin expression compared to H. pylori-negative and -eradicated subjects (P < 0.05). Overall, no correlations between mucosal Progranulin and SLPI levels were identified. Immunohistochemical analysis confirmed the upregulation of Progranulin in relation to H. pylori infection; both epithelial and infiltrating immune cells contributed to the higher Progranulin expression levels. The H. pylori-induced upregulation of Progranulin was verified in AGS cells infected by H. pylori. The down-regulation of endogenous SLPI expression in AGS cells by siRNA methodology did not affect the Progranulin expression independent of the infection by H. pylori. CONCLUSIONS: Taken together, Progranulin was identified as novel molecule that is upregulated in context to H. pylori infection. In contrast to other diseases, SLPI seems not to have a regulatory role for Progranulin in H. pylori-mediated gastritis.
Asunto(s)
Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Péptidos y Proteínas de Señalización Intercelular/metabolismo , ARN Mensajero/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Línea Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Expresión Génica , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Progranulinas , Estómago/citología , Estómago/microbiología , Transcripción GenéticaRESUMEN
BACKGROUND/AIMS: At present, the dilation of esophageal intercellular spaces (ICS) is considered an early morphologic marker of acid damage in patients with GERD. Nevertheless, previous electron microscopic (EM) studies had focused only on the suprabasal layer of squamous epithelium or did not nearly specify which layer of squamous epithelium was studied. Therefore, we aimed to assess the volumetric amount of the ICS in all layers of SE in patients with GERD. METHODS: In this study, 48 patients were prospectively included (NERD = 18, ERD = 17; Barrett's esophagus = 5, controls = 8). All patients with ERD and NERD had typical reflux symptoms, as assessed by a valid GERD questionnaire. ICS volume was assessed by electron microscopy in the superficial, prickle cell, and basal layers of esophageal squamous epithelium using the method of Weibel. RESULTS: ERD was associated with increased ICS volume in the basal layer (LA-A, p = 0.038; LA-B, p = 0.005) and prickle cell layer (LA-A, p = 0.006; LA-B, p = 0.007) as compared to controls. Comparisons between NERD and ERD patients revealed more dilated ICS in the basal layer (LA-B, p = 0.007), prickle cell layer (LA-A, p = 0.008; LA-B, p = 0.001) and superficial layer (LA-B, p = 0.018) in patients with ERD. CONCLUSIONS: Not only the diameter but also the volume of the ICS is increased in patients with GERD. Furthermore, the dilation of ICS is present in all three layers of the SE, being more pronounced in the basal layer. These findings support the concept that the impairment of the esophagus begins in the deeper parts of the esophageal epithelium.
Asunto(s)
Células Epiteliales/ultraestructura , Esófago/patología , Esófago/ultraestructura , Espacio Extracelular , Reflujo Gastroesofágico/patología , Esófago de Barrett/patología , Estudios de Casos y Controles , Epitelio/patología , Epitelio/ultraestructura , HumanosRESUMEN
The aim of the current study was to investigate the role of promoter methylation of adenomatous polyposis coli (APC) and epithelial cadherin (E-cadherin) genes in endometrial tumorigenesis. The methylation status of both genes was investigated in 43 cases of normal endometrium, 21 simple hyperplasia, 17 atypical hyperplasia, and 86 endometrial carcinoma (EC). Additionally, the methylation pattern of both genes was analyzed in 24 primary ECs and their corresponding metastases. DNA methylation of the APC gene increased from atypical hyperplasia (23.5%) to endometrial carcinoma, reaching its highest level of 77.4% in early stage cancer (FIGO I and II) and decreasing stepwise to 24.2% in advanced stage carcinomas (FIGO III and IV). No methylation of APC was found in normal endometrium or simple hyperplasia. Methylation of E-cadherin was found only in EC (22.1%). The mean age of the patients with aberrant APC methylation was 68.8 years and was significantly higher compared to the mean age (60.9 years) of the patients without methylation of APC promoter (P = 0.02). APC promoter methylation significantly correlated with decreased protein expression of APC (P = 0.039), with increased expression of the Ki-67 proliferative marker (P = 0.006) and decreased metastatic potential (P = 0.002). There was no correlation between APC and E-cadherin methylation patterns and the other clinicopathologic features, nor with patient outcome. Our results suggest that hypermethylation of APC promoter region is an early event in endometrial tumorigenesis.
Asunto(s)
Metilación de ADN , Neoplasias Endometriales/genética , Genes APC , Regiones Promotoras Genéticas , Adulto , Anciano , Anciano de 80 o más Años , Cadherinas/genética , Neoplasias Endometriales/etiología , Neoplasias Endometriales/patología , Femenino , Humanos , Antígeno Ki-67/análisis , Persona de Mediana EdadRESUMEN
OBJECTIVES: The proteinase-activated receptor-2 (PAR-2) is activated by serine proteases and has been demonstrated to induce proinflammatory and neuroinflammatory effects. It is considered to alter transepithelial resistance and mediates visceral hypersensitivity. This study aimed to evaluate the expression of PAR-2 in human esophageal mucosa of patients with gastroesophageal reflux disease (GERD) in relation to mucosal alterations. METHODS: The study included 123 patients with GERD stratified to erosive reflux disease (n=50), non-erosive reflux disease (n=46), and reflux-negative patients as controls (n=27). Endoscopic and histopathological characterization was performed according to the Los Angeles classification and modified Ismail-Beigi criteria, respectively. PAR-2 expression was analyzed by quantitative reverse transcription (RT)-PCR and immunohistochemistry. The gene expression levels of interleukin (IL)-8 were determined by quantitative RT-PCR and correlated to PAR-2 expression in each patient. Performing in vitro studies, esophageal squamous cell lines (KYSE 150, KYSE 450) were incubated, adjusted to different pH (7.0, 6.0, and 5.0), and exposed to bile acids and PAR-2-activation peptide (SLIGKV-NH(2)). RESULTS: PAR-2 gene expression was 7- to 10-fold upregulated (P<0.0001) in the mucosa of patients with GERD and correlated positively with IL-8 expression and with histomorphological alterations (dilated intercellular spaces, papillary elongation, basal cell hyperplasia (BCH); P<0.01). Immunohistochemistry showed an intense staining of PAR-2 throughout all epithelial layers in patients with GERD compared with controls (P=0.0005). In vitro studies revealed a 1.5- to 20-fold induction of PAR-2 gene expression in esophageal squamous cells by acidified medium (P<0.01), but not by additional bile acids. The activation of PAR-2 leads to expression and secretion of IL-8. CONCLUSIONS: This study provides evidence of the functional importance of PAR-2-mediated pathways in the pathogenesis of GERD and GERD-associated mucosal alterations and inflammatory changes.
Asunto(s)
Esófago/metabolismo , Mucosa Gástrica/metabolismo , Reflujo Gastroesofágico/metabolismo , Receptor PAR-2/metabolismo , Adolescente , Adulto , Anciano , Línea Celular , Endoscopía Gastrointestinal , Esófago/patología , Femenino , Mucosa Gástrica/patología , Reflujo Gastroesofágico/genética , Reflujo Gastroesofágico/patología , Humanos , Inmunohistoquímica , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor PAR-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Regulación hacia ArribaRESUMEN
Death-associated protein kinase (DAPK) is a serine/threonine kinase that contributes to pro-apoptotic signaling on cytokine exposure. The role of DAPK in macrophage-associated tumor cell death is currently unknown. Recently, we suggested a new function for DAPK in the induction of apoptosis during the interaction between colorectal tumor cells and tumor-associated macrophages. Using a cell-culture model with conditioned supernatants of differentiated/activated macrophages (U937) and human HCT116 colorectal tumor cells, we replicated DAPK-associated tumor cell death; this model likely reflects the in vivo tumor setting. In this study, we show that tumor necrosis factor-alpha exposure under conditions of macrophage activation induced DAPK-dependent apoptosis in the colorectal tumor cell line HCT116. Simultaneously, early phosphorylation of p38 mitogen-activated protein kinase (phospho-p38) was observed. We identified the phospho-p38 mitogen-activated protein kinase as a novel interacting protein of DAPK in tumor necrosis factor-alpha-induced apoptosis. The general relevance of this interaction was verified in two colorectal cell lines without functional p53 (ie, HCT116 p53(-/-) and HT29 mutant) and in human colon cancer and ulcerative colitis tissues. Supernatants of freshly isolated human macrophages were also able to induce DAPK and phospho-p38. Our findings highlight the mechanisms that underlie DAPK regulation in tumor cell death evoked by immune cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Neoplasias Colorrectales/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Proteínas Quinasas Asociadas a Muerte Celular , Humanos , Inmunoprecipitación , Interferón gamma/inmunología , Interferón gamma/farmacología , Activación de Macrófagos , Macrófagos/enzimología , Fosforilación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: The presence of enterohepatic Helicobacter species (EHS) is commonly noted in mouse colonies. These infections often remain unrecognized but can cause severe health complications or more subtle host immune perturbations and therefore can confound the results of animal experiments. The aim of this study was to isolate and characterize a putative novel EHS that has previously been detected by PCR screening of specific-pathogen-free mice. MATERIALS AND METHODS: Biochemical analysis of enzyme activities (API campy), morphologic investigation (Gram-staining and electron microscopy) and genetic analyses (16SrRNA and 23SrRNA analyses, DNA fingerprinting, restriction fragment polymorphisms, and pulsed-field gel electrophoresis) were used to characterize isolated EHS. Genomic DNA fragments were sequenced to develop a species-specific PCR detection assay. RESULTS: Scanning electron microscopy revealed the presence of spiral-shaped EHS, which varied in length (2.5-6 µm) and contained single monopolar or single bipolar sheathed flagella. The bacteria were grown under anaerobic conditions, preferably on agar plates containing serum or blood. The 16SrRNA, genetic, and biochemical analyses indicated the identification of a novel EHS species, named Helicobacter magdeburgensis. We also examined the genome content using pulsed-field gel electrophoresis. Based on the pattern produced by two restriction enzymes, BamIII and KspI, the genome size was determined to be about 1.7-1.8 Mbp. CONCLUSION: We isolated and characterized a novel EHS species, H. magdeburgensis, morphologically, biochemically, and genetically. These results are important for future studies on the prevalence and pathophysiologic relevance of such infections. Our PCR assay can be used to detect and discriminate H. magdeburgensis from other Helicobacter species.
Asunto(s)
Infecciones por Helicobacter/veterinaria , Helicobacter/clasificación , Helicobacter/aislamiento & purificación , Intestinos/microbiología , Animales , Técnicas de Tipificación Bacteriana , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Electroforesis en Gel de Campo Pulsado , Genotipo , Helicobacter/citología , Helicobacter/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Análisis de Secuencia de ADNRESUMEN
Cathepsin X (CTSX) is strongly up-regulated in Helicobacter pylori-infected gastric mucosa and intestinal-type gastric cancer. The overexpression of CTSX is mediated predominantly by associated macrophages; depends on a functional type IV-secretion system; and leads to increased migration of gastric epithelial cells. In the present study, we analysed the role of CagA in CTSX overexpression and identified H. pylori-induced inflammatory factors and signalling pathways required for stimulating CTSX expression by H. pylori. Gastric epithelial cells were co-cultured with macrophages in Transwell chambers of 0.4 microm pore size, enabling exchange of fluids but retracting H. pylori. N87 gastric epithelial cells were infected with H. pylori P1 wild-type strain in the presence of inhibitors for p38, JNK, and ERK1/2 signal transduction pathways. Furthermore, cytokines and growth factors were tested for their regulatory function using inhibitory antibodies, and their gene expression was studied by quantitative RT-PCRs and western blots. CTSX is strongly up-regulated at both the mRNA and the protein levels by TNF-alpha, IL-1beta, IL-6, and IL-8, depending on cell type. All these cytokines were found to be increased by five- to ten-fold in macrophages by H. pylori infection of co-cultured N87 gastric epithelial cells. In macrophages, H. pylori up-regulated CTSX via ERK1/2 signalling pathways, and in N87 cells via JNK irrespective of p38 signalling. Our results suggest that H. pylori induced overexpression of CTSX in macrophages and epithelium through specific cytokines that are initiated by CagA-dependent pathways in a cell type-dependent manner.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Catepsinas/metabolismo , Gastritis/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Regulación hacia Arriba , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Western Blotting/métodos , Estudios de Casos y Controles , Catepsina K , Catepsinas/genética , Línea Celular , Citocinas/metabolismo , Células Epiteliales/metabolismo , Femenino , Mucosa Gástrica/metabolismo , Gastroscopía , Humanos , Inmunohistoquímica , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
BACKGROUND: Despite evidence for the association of distal gastric cancer (GC) with the H. pylori infection, relevance of the infection for proximal GC is uncertain. AIMS: We analysed the prevalence of H.pylori in proximal and distal GC and its association with premalignant mucosal alterations in different gastric locations. METHODS: We performed a retrospective analysis on 152 patients with GC, stratified according to the location of the main tumor mass into proximal (n = 73) and distal (n = 79) GC. H.pylori prevalence and CagA-status were determined by serology. Intestinal metaplasia (IM), glandular atrophy and mucosal inflammation were diagnosed from histological specimens and graded according to the updated Sydney-classification. RESULTS: H.pylori prevalence (78.1 vs. 82.3%) and CagA-status (77.2 vs. 84.6%) were similar in proximal and distal GC as well as in intestinal and diffuse GC. IM (79.8 vs. 60.3%; P = 0.012) and atrophy (50.0 vs. 19.1%; P < 0.001) were more frequent in the mucosa surrounding intestinal tumors. There was a higher degree of surrounding IM in case of distally located compared to proximal tumors (P = 0.001). Overall, IM was more severe in the antrum than the corpus. In contrast, there was more severe active inflammation in the corpus than the antrum (P = 0.017). CONCLUSION: The prevalence of H.pylori is similar in proximal and distal GC if precise allocation of the primary tumor has been performed, especially at the esophagogastric junction. Distal tumors of the intestinal type are more often associated with local IM than proximal and diffuse type carcinomas. This suggests a distinct pathophysiological relevance of these mucosal alterations.
Asunto(s)
Infecciones por Helicobacter/epidemiología , Neoplasias Gástricas/epidemiología , Anciano , Atrofia/patología , Comorbilidad , Femenino , Mucosa Gástrica/patología , Helicobacter pylori , Humanos , Masculino , Metaplasia/patología , Persona de Mediana Edad , Prevalencia , Estudios Retrospectivos , Factores de RiesgoRESUMEN
AIMS: In skull base chordoma, c-MET expression has been reported to correlate with younger patient age and favourable prognosis; however, it also contributes to tumour invasiveness, especially in recurrent lesions, suggesting variable roles for c-MET according to clinical status. The aim of this study was to investigate the significance of c-MET expression in spinal chordoma, which affects patients who are 10-20 years older than those with skull base chordoma. METHODS AND RESULTS: Using immunohistochemical techniques, the expression of c-MET and its ligand, hepatocyte growth factor (HGF) was investigated in 34 primary spinal chordomas and compared with other clinicopathological parameters. Expression of c-MET and HGF was observed in 85.3 and 21.7% of lesions, respectively. c-MET expression correlated with the expression of an epithelial marker, low-molecular-weight cytokeratin (CAM5.2). Lesions with higher c-MET expression showed significantly stronger expression of proteinases, including matrix metalloproteinase (MMP)-1 and MMP-2. However, c-MET expression was not associated with patient age, proliferative ability estimated by MIB-1 labelling index, or prognosis. CONCLUSIONS: c-MET expression was observed in most spinal chordomas and correlated with the expression of CAM5.2, suggesting a relationship to an epithelial phenotype.
Asunto(s)
Cordoma/metabolismo , Queratinas/biosíntesis , Metaloproteinasa 1 de la Matriz/biosíntesis , Metaloproteinasa 2 de la Matriz/biosíntesis , Proteínas Proto-Oncogénicas c-met/biosíntesis , Neoplasias de la Médula Espinal/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Biomarcadores de Tumor/análisis , Cordoma/mortalidad , Cordoma/patología , Femenino , Factor de Crecimiento de Hepatocito/biosíntesis , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Neoplasias de la Médula Espinal/mortalidad , Neoplasias de la Médula Espinal/patologíaRESUMEN
BACKGROUND: Gastroesophageal reflux disease (GERD) leads to endoscopic and histomorphological changes in the gastroesophageal (GE) mucosa. AIMS: To evaluate the expression of the cytokines interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in the GE mucosa in GERD patients and controls and to correlate the cytokine expression with the histomorphological parameters. METHODS: One hundred and fifteen patients, 48 with erosive reflux disease (ERD) and 41 with non-erosive reflux disease (NERD) with typical GERD-related symptoms, and 26 controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and Ismeil-Beigi/Vieth criteria, respectively. Mucosal gene expression levels of IL-1beta and IL-8 were analyzed by real-time RT-PCR. RESULTS: ERD and NERD patients revealed significant higher levels of IL-1beta and IL-8 transcript levels in the cardia and esophageal mucosa than controls. The esophageal mucosa revealed elevated IL-8 (2.5- and 8.7-fold) and IL-1beta (4.1- and 7.8-fold) transcript levels in NERD and ERD, respectively. Histological analysis demonstrated a stepwise increase of dilatation of intercellular spaces and the degree of basal cell hyperplasia from controls, NERD towards ERD. Gene expression levels of both cytokines correlated with histology. CONCLUSIONS: ERD and NERD are associated with an induction of the proinflammatory cytokines IL-1beta and IL-8 that correlates with histomorphological changes in esophageal mucosa.
Asunto(s)
Reflujo Gastroesofágico/patología , Interleucina-1beta/genética , Interleucina-8/genética , Mucosa Intestinal/patología , Adolescente , Adulto , Anciano , Femenino , Reflujo Gastroesofágico/genética , Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
INTRODUCTION: Chronic inflammation at the cardia occurs in gastroesophageal reflux disease (GERD), as well as in the presence of Helicobacter pylori. Regulatory T cells have been demonstrated for H. pylori-induced gastritis, whereas their role has not been studied in GERD. METHODS: We prospectively analyzed the expression of FOXP3, a marker of various regulatory T cells, as well as the mucosal transcript levels of TGF-beta1 and IL-10. RNA and protein levels have been determined in cardiac biopsies of 70 patients stratified according to GERD (n = 22), controls (n = 17), and H. pylori (n = 31). RESULTS: GERD presented with chronic inflammation and reduced FOXP3-mRNA in the cardiac mucosa (-84%), whereas H. pylori-positive patients revealed a 25.1-fold increase of FOXP3 gene expression. These results were verified by the regulatory cytokines IL-10 and TGF-beta1, and by the immunohistochemical detection of intramucosal FOXP3-expressing T cells. CONCLUSION: Chronic inflammation at the cardia associated with either GERD or H. pylori differs concerning the presence of FOXP3-expressing T cells. In contrast to H. pylori, FOXP3-expressing T cells are not associated with GERD-associated carditis.