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INTRODUCTION: As required by standards organizations, Héma-Québec Cord Blood Bank performs enumeration of nucleated red blood cells (NRBCs) in cord blood units (CBUs). This study presents the validation and implementation approaches developed to transfer the routine NRBC enumeration from the manual blood film method to a flow cytometric assay. METHODS: The flow cytometry method was adapted from Tsuji (Cytometry, 37, 1999, 291). This assay was validated to assess the specificity, detection limit, repeatability, and reproducibility of the method, including interoperator and interlaboratory testing. Finally, postimplementation follow-up and adjustments were performed for CBU over a 7-month period. RESULTS: Blood film and flow cytometry NRBC enumerations showed a strong correlation (n = 40; Pearson's r correlation = 0.90). Validation was successful as exemplified by the correlation in interlaboratory testing (n = 30; r = 0.98). During implementation, our routine laboratory analyses revealed that CBU with low NRBC content (≤2%), representing 26% of all CBU tested, resulted in 15% of repeated reading and/or staining and was the principal source of nonconformity. Small adjustments in the standard operating procedures (SOPs), including a fixed 200-event setting in the NRBC gate for the second reading of the replicates, have completely solved this issue. CONCLUSION: Flow cytometric NRBC enumerations, now implemented in Héma-Québec Public Cord Blood Bank, is an improvement in the efficiency of our operations by integrating the count for NRBC into our flow cytometry platform.
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Eritroblastos/citología , Sangre Fetal/citología , Citometría de Flujo/métodos , Citometría de Flujo/normas , Bancos de Sangre/normas , Recuento de Eritrocitos/métodos , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Almacenamiento de Sangre/métodosRESUMEN
Trillions of bacteria inhabit our intestine, forming a community called the microbiota, whose contributions are essential to maintain host homeostasis. Disruption of this normal microbial-host communication network has deleterious consequences for the host and is associated with intestinal pathologies such as inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Here we present key concepts and mechanisms by which bacteria may participate in intestinal pathology, and discuss possible means to therapeutically target the microbiome.
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Bacterias/metabolismo , Carcinogénesis , Neoplasias Colorrectales/microbiología , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino/microbiología , Intestinos/microbiología , Neoplasias Colorrectales/etiología , Humanos , Enfermedades Inflamatorias del Intestino/etiología , MicrobiotaRESUMEN
Prebiotics are selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health. The aim of this study was to evaluate the influence of a ß(1-4)galacto-oligosaccharides (GOS) formulation consisting of 90% pure GOS (GOS90), on the composition and activity of the mouse gut microbiota. Germ-free mice were colonised with microbiota from four pathogen-free wt 129 mice donors (SPF), and stools were collected during a feeding trial in which GOS90 was delivered orally for 14 days. Pyrosequencing of 16S rDNA amplicons showed that Bifidobacterium and specific Lactobacillus, Bacteroides and Clostridiales were more prevalent in GOS90-fed mice after 14 days, although the prebiotic impact on Bifidobacterium varied among individual mice. Prebiotic feeding also resulted in decreased abundance of Bacteroidales, Helicobacter and Clostridium. High-throughput quantitative PCR showed an increased abundance of Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis and Bifidobacterium gallicum in the prebiotic-fed mice. Control female mice showed a higher diversity (phylogenetic diversity (PD) = 15.1 ± 3.4 in stools and PD = 13.0 ± 0.6 in intestinal contents) than control males (PD = 7.8 ± 1.6 in stool samples and PD = 9.5 ± 1.0 in intestinal contents). GOS90 did not modify inflammatory biomarkers (interleukin (IL)-6, IL-12, IL-1ß, interferon gamma and tumour necrosis factor alpha). Decreased butyrate, acetate and lactate concentrations in stools of prebiotic fed mice suggested an increase in colonic absorption and reduced excretion. Overall, our results demonstrate that GOS90 is capable of modulating the intestinal microbiome resulting in expansion of the probiome (autochtonous commensal intestinal bacteria considered to have a beneficial influence on health).
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Bifidobacterium/fisiología , Microbioma Gastrointestinal , Oligosacáridos/metabolismo , Prebióticos/administración & dosificación , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Bifidobacterium/genética , Bifidobacterium/aislamiento & purificación , Femenino , Fermentación , Galactosa/metabolismo , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Interleucina-12/genética , Interleucina-12/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Ratones , Ratones de la Cepa 129 , Oligosacáridos/administración & dosificación , Oligosacáridos/análisis , Prebióticos/análisisRESUMEN
Parkinson's disease (PD) patients often suffer from gastrointestinal (GI) impairments that are associated with the alteration of dopaminergic (DAergic) neurons in the myenteric nervous system. Growing evidence suggests that inflammation originating from the gut may have a major impact in both the initiation and progression of PD. Here, we investigated the role of the innate immune response in neurodegeneration occurring in central nervous system (CNS) and enteric nervous system (ENS) in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism in both humans and animal models. We found a strong immune response in the gut of mice treated with MPTP, as demonstrated by the prominent presence of macrophages derived from CD115(+) CD11b(+) Ly6C(Hi) monocytes, known as M1 monocytes, and increased production of IL-1ß and IL-6. Partial depletion of proinflammatory M1 monocytes through intravenous injections of clodronate-encapsulated liposome protects against MPTP-induced reduction of tyrosine hydroxylase (TH) expression in the ENS. In contrast, loss of striatal TH expression in the CNS after MPTP intoxication occurs regardless of partial monocyte depletion. Examination of brain tissue revealed that microglial activation, comprising the majority of the immune response in the CNS after MPTP injections is unaffected by M1 depletion. In vitro experiments revealed that MPTP and MPP(+) act directly on monocytes to elicit a proinflammatory response that is, in part, dependent on the MyD88/NF-κB signaling pathway resulting in nitrite and proinflammatory cytokine production. Taken together, our results demonstrate a critical role for proinflammatory M1 monocytes/macrophages in DAergic alterations occurring in the GI, but not in the brain, in the MPTP model of PD.
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Ganglios Basales/metabolismo , Intoxicación por MPTP/metabolismo , Monocitos/metabolismo , Plexo Mientérico/metabolismo , Animales , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , RatonesRESUMEN
BACKGROUND: In a recent double-blinded clinical trial, the probiotic combination of Lactobacillus acidophilus NCFM (L-NCFM) and B-LBi07 reduced bloating symptoms in patients with functional bowel disorders; an effect more evident in those who reported abdominal pain. In mice, L-NCFM but not B-LBi07 induced colonic mu-opioid receptor (MOR) and cannabinoid receptor 2 (CB2) expression, and reduced visceral sensitivity. AIMS: To determine if L-NCFM was the active component in the clinical trial and to investigate the mechanism of action in humans with mild to moderate abdominal pain. METHODS: Caucasian women (n = 20) 18-70 years with mild to moderate abdominal pain were enrolled in a double-blind, two-armed, single-centre study. Patients were given either L-NCFM alone or in combination with B-LBi07 for 21 days at a total dose of 2 × 10(10) CFU b.d. Colonic biopsies were collected during unsedated, unprepped flexible sigmoidoscopy before and at the end of probiotic consumption. mRNA and immunostaining were then performed on these biopsies. Patients kept symptom diaries for the 7 days prior to starting probiotic therapy and for the last 7 days of therapy. RESULTS: L-NCFM alone, but not with B-LBi07, induced colonic MOR mRNA and protein expression, as well as downstream signalling, as measured by enterocyte STAT3-phosphorylation. In contrast, CB2 expression was decreased. Both treatment groups trended towards improvement in symptoms, but the study was insufficiently powered to draw meaningful conclusions. CONCLUSIONS: Lactobacillus acidophilus NCFM modulates mu-opioid receptor expression and activity, while the combination of L-NCFM and B-LBi07 does not. This study provides a possible mechanism for action by which probiotics modulates pain sensation in humans (Clinical Trial Number: NCT01064661).
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Dolor Abdominal/tratamiento farmacológico , Mucosa Intestinal/metabolismo , Lactobacillus acidophilus , Probióticos/uso terapéutico , Receptores Opioides mu/genética , Dolor Abdominal/metabolismo , Dolor Abdominal/patología , Adolescente , Adulto , Anciano , Colon/metabolismo , Colon/patología , Método Doble Ciego , Enterocitos/metabolismo , Femenino , Humanos , Mucosa Intestinal/patología , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptor Cannabinoide CB2/genética , Factor de Transcripción STAT3/metabolismo , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Chronic ethanol abuse and haemorrhagic shock are major causes of global mortality and, separately, induce profound hepato- and immune-toxic effects via activation of NF-κB. Here, we assessed the effects of chronic ethanol intake upon the pathophysiological derangements after haemorrhagic shock with subsequent resuscitation (H/R), with particular attention to the contribution of NF-κB. EXPERIMENTAL APPROACH: Transgenic NF-κB(EGFP) mice, expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-κB cis-elements were fed a Lieber-DeCarli diet containing ethanol (EtOH-diet) or an isocaloric control diet for 4 weeks and were then pairwise subjected to H/R. Liver tissues and peripheral blood were sampled at 2 or 24 h after H/R. Cytokines in blood and tissue and leukocyte activation (as CD11b expression) were measured, along with EGFP as a marker of NF-κB activation. KEY RESULTS: The EtOH-diet increased mortality at 24 h after H/R and elevated liver injury, associated with an up-regulation of NF-κB-dependent genes and IL-6 release; it also increased production of NF-κB-driven intercellular adhesion molecule 1 (ICAM-1) and EGFP in liver tissue. At 2h after the H/R procedure in ethanol-fed mice we observed the highest proportion of NF-κB activated non-parenchymal cells and an NF-κB-dependent increase in polymorphonuclear leukocyte CD11b expression. CONCLUSIONS AND IMPLICATIONS: The EtOH-diet exacerbated liver injury after H/R, accompanying an overwhelming hepatic and systemic immune response. Our findings contribute to evidence implicating NF-κB as a key player in the orchestration of the immune response in haemorrhagic shock patients with a history of chronic ethanol abuse.
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Consumo de Bebidas Alcohólicas/metabolismo , Hígado/metabolismo , FN-kappa B/metabolismo , Resucitación , Choque Hemorrágico/metabolismo , Choque Hemorrágico/terapia , Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/inmunología , Animales , Antígeno CD11b/metabolismo , Modelos Animales de Enfermedad , Hígado Graso Alcohólico/inmunología , Hígado Graso Alcohólico/metabolismo , Hígado Graso Alcohólico/patología , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hemodinámica , Hepatomegalia/inmunología , Hepatomegalia/metabolismo , Hepatomegalia/patología , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Hígado/inmunología , Hígado/patología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/genética , Necrosis , Regiones Promotoras Genéticas , Choque Hemorrágico/genética , Choque Hemorrágico/inmunología , Choque Hemorrágico/patología , Choque Hemorrágico/fisiopatología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
OBJECTIVE: Despite the evidence of cognitive deficits in Multiple Sclerosis (MS) patients, evaluation of their cognitive integrity is often limited to the use of clinical interviews and questionnaires. However, the consensus in the literature is that these patients under- or overestimate their deficits and repercussions. The objective of this study was to clarify why some patients overestimate while others underestimate their memory deficits. METHOD: Fifty-four participants (30 MS, 24 controls) completed the Prospective and Retrospective Memory Questionnaire (PRMQ) and were tested on a battery of neuropsychological tests. Based on the test results, MS patients were categorized as having either mild or moderate/severe cognitive deficits. RESULTS: The moderate/severe MS group differed from the two other groups on the Rey Auditory Verbal Learning Test (RAVLT) but did not differ from the control group on the PRMQ. Conversely, the mild MS group did not differ from the control group on the RAVLT but did report significantly more problems than this group on the PRMQ. There was no difference between the two clinical groups on the Depression Index (Beck) but there was a significant correlation (r=.409) between the depression scores and the overestimation of prospective memory problems (PRMQ). CONCLUSION: The results explain the contradiction in the literature. It is the mild group who overestimates, maybe because they are overly concerned by their deficits, whereas the cognitive impairments of the moderate/severe group lead them to underestimate and may make their self-assessment unreliable. Formal testing or information from a significant other would be advisable.
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Trastornos del Conocimiento/etiología , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/etiología , Esclerosis Múltiple/complicaciones , Adulto , Análisis de Varianza , Estudios de Casos y Controles , Evaluación de la Discapacidad , Femenino , Humanos , Masculino , Recuerdo Mental/fisiología , Persona de Mediana Edad , Pruebas Neuropsicológicas , Encuestas y Cuestionarios , Aprendizaje Verbal/fisiologíaRESUMEN
The gastrointestinal epithelium functions as an important physical barrier that separates the rich, diverse, and potentially immunogenic luminal content from the underlying mucosal immune system. In pathological situations such as inflammatory bowel disease, ischemic/hypoxic episodes and bacterial infection, insults to the intestinal epithelium threaten the integrity of the mucosal barrier and represent a huge challenge for the host. During episodes of epithelial injury and barrier breakdown, the host initiates a rapid wound healing response aimed at resealing the gap region and reestablishing homeostasis. This response named "restitution" involves migration of epithelial cells toward the injured regions, as well as epithelial cell proliferation until the gap is closed and the barrier function is reestablished. These biological processes are influenced by a variety of factors derived from the gastrointestinal microenvironment, including host epithelial and lamina propria cells, as well as the microbiota, and the dietary and non-dietary components present in the gastrointestinal lumen. In this manuscript, we will review both host signaling events and luminal factors that influence the wound healing response and have an impact on host homeostasis.
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Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/patología , Inmunidad Innata/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Cicatrización de Heridas/inmunología , Animales , Humanos , Modelos InmunológicosRESUMEN
Curcumin (diferulolylmethane) demonstrates profound anti-inflammatory effects in intestinal epithelial cells (IEC) and in immune cells in vitro and exhibits a protective role in rodent models of chemically induced colitis, with its presumed primary mechanism of action via inhibition of NF-kappaB. Although it has been demonstrated effective in reducing relapse rate in ulcerative colitis patients, curcumin's effectiveness in Crohn's disease (CD) or in Th-1/Th-17 mediated immune models of CD has not been evaluated. Therefore, we investigated the effects of dietary curcumin (0.1-1%) on the development of colitis, immune activation, and in vivo NF-kappaB activity in germ-free IL-10(-/-) or IL-10(-/-);NF-kappaB(EGFP) mice colonized with specific pathogen-free microflora. Proximal and distal colon morphology showed a mild protective effect of curcumin only at 0.1%. Colonic IFN-gamma and IL-12/23p40 mRNA expression followed similar pattern ( approximately 50% inhibition at 0.1%). Secretion of IL-12/23p40 and IFN-gamma by colonic explants and mesenteric lymph node cells was elevated in IL-10(-/-) mice and was not decreased by dietary curcumin. Surprisingly, activation of NF-kappaB in IL-10(-/-) mice (phospho-NF-kappaBp65) or in IL-10(-/-);NF-kappaB(EGFP) mice (whole organ or confocal imaging) was not noticeably inhibited by curcumin. Furthermore, we demonstrate that IL-10 and curcumin act synergistically to downregulate NF-kappaB activity in IEC and IL-12/23p40 production by splenocytes and dendritic cells. In conclusion, curcumin demonstrates limited effectiveness on Th-1 mediated colitis in IL-10(-/-) mice, with moderately improved colonic morphology, but with no significant effect on pathogenic T cell responses and in situ NF-kappaB activity. In vitro studies suggest that the protective effects of curcumin are IL-10 dependent.
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Colitis/tratamiento farmacológico , Curcumina/farmacología , Dieta , Interleucina-10/genética , Células TH1/fisiología , Animales , Colitis/microbiología , Colitis/patología , Colon/patología , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Inmunohistoquímica , Interleucina-10/metabolismo , Mucosa Intestinal/citología , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Organismos Libres de Patógenos Específicos , Bazo/citología , Células TH1/efectos de los fármacosRESUMEN
Transfectants of human CM and NES2Y beta cell lines and primary islets transfected by FADD-DN (dominant-negative form of Fas-associated death domain), a mutant of FADD and/or a superrepressor of nuclear factor kappaB (NF-kappaB) (AdIkappaB(SA)2), were examined for their susceptibility to the TRAIL (TNF-related apoptosis-inducing ligand)-induced death signal pathway, compared with controls, wild-type cells, and vector transfectants in caspase fluorescence, Western blot, electrophoretic mobility shift, apoptosis, and cytotoxicity assays. FADD-DN inhibited caspase-8 activation induced by TRAIL in the transfectants of CM and NES2Y cells. TRAIL-induced apoptosis and cytotoxicity to the FADD-DN transfectants were decreased in comparison to those responses in controls (CM, p < 0.01 and p < 0.01; NES2Y, p < 0.05, and p < 0.02, respectively). When CM, NES2Y, and primary islet cells were transfected by AdIkappaB(SA)2, TRAIL-induced IkappaB degradation and nuclear translocation of NF-kappaB p50/p65 were blocked. TRAIL-induced apoptosis and cytotoxicity to AdIkappaB(SA)2 transfectants of these cells were also reduced (CM, p < 0.02 and p < 0.02; NES2Y, p < 0.01 and p < 0.01, respectively, and islet p < 0.01 for cytotoxicity). Finally, cytotoxicity induced by TRAIL in CM and NES2Y cells transfected with both FADD-DN and AdIkappaB(SA)2 was reduced, compared with that observed in these cells transfected with either FADD-DN alone or AdIkappaB(SA)2 alone, suggesting that FADD and NF-kappaB have synergistic proapoptotic regulatory effects on the susceptibility of beta cell lines and islet cells to TRAIL-induced destruction.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Reguladoras de la Apoptosis/farmacología , Apoptosis/fisiología , Células Secretoras de Insulina/fisiología , Glicoproteínas de Membrana/farmacología , FN-kappa B/fisiología , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Apoptosis/efectos de los fármacos , Caspasa 8 , Caspasas/metabolismo , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Mutación/genética , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sulfasalazina/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Transcripción ReIA/metabolismo , TransfecciónRESUMEN
Herbal medicine has become an increasing popular therapeutic alternative among patients suffering from various inflammatory disorders. The Salvia miltiorrhizae water-soluble extract (SME) have been shown to possess antioxidant and anti-inflammatory properties in vitro. However, the mechanism of action and impact of SME on LPS-induced gene expression is still unknown. We report that SME significantly abrogated LPS-induced IkappaB phosphorylation/degradation, NF-kappaB transcriptional activity and ICAM-1 gene expression in rat IEC-18 cells. Chromatin immunoprecipitation assay demonstrated that LPS-induced RelA recruitment to the ICAM-1 gene promoter was inhibited by SME. Moreover, in vitro kinase assay showed that SME directly inhibits LPS induced IkappaB kinase (IKK) activity in IEC-18 cells. To investigate the physiological relevance of SME inhibitory activity on NF-kappaB signalling, we used small intestinal explants and primary intestinal epithelial cells derived from a transgenic mouse expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-kappaB cis-elements (cis-NF-kappaB(EGFP)). SME significantly blocked LPS-induced EGFP expression and IkappaBalpha phosphorylation in intestinal explants and primary IECs, respectively. However, salvianolic acid B, an activate component of SME did not inhibit NF-kappaB transcriptional activity and IkappaB phosphorylation/degradation in IEC-18 cells. These results indicate that SME blocks LPS-induced NF-kappaB signalling pathway by targeting the IKK complex in intestinal epithelial cells. Modulation of bacterial product-mediated NF-kappaB signalling by natural plant extracts may represent an attractive strategy towards the prevention and treatment of intestinal inflammation.
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Medicamentos Herbarios Chinos/farmacología , Mucosa Intestinal/efectos de los fármacos , Lipopolisacáridos/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , Salvia miltiorrhiza , Animales , Benzofuranos/farmacología , Células Cultivadas , Inmunoprecipitación de Cromatina/métodos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas I-kappa B/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Mucosa Intestinal/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , ARN Mensajero/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transducción de Señal/efectos de los fármacos , Solubilidad , Translocación Genética , AguaRESUMEN
Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression. Here, we show that loss of E-cadherin leads to induction of nuclear factor kappa B (NFkappaB) activity in melanoma cell lines. Melanoma cells show constitutively active NFkappaB, whereas no activity is found in primary melanocytes. After re-expression of E-cadherin in melanoma cells, strong downregulation of NFkappaB activity was found. Consistently, NFkappaB activity was induced in primary human melanocytes after inhibition of E-cadherin activity by functionally blocking anti-E-cadherin antibodies. Interestingly, re-expression of E-cadherin-blocked p38 MAPK activity and the p38 MAPK inhibitors SB203580 and SB202190 almost completely prevented NFkappaB activation in melanoma cells. Furthermore, cytoplasmatic beta-catenin induced p38 and NFkappaB activation in malignant melanoma. To our knowledge, this is the first report suggesting a correlation between E-cadherin and NFkappaB activity in melanocytes and melanoma cells. In summary, we conclude that loss of E-cadherin and cytoplasmatic beta-catenin induces p38-mediated NFkappaB activation, potentially revealing an important mechanism of tumorigenesis in malignant melanomas.
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Cadherinas/metabolismo , Melanoma/metabolismo , FN-kappa B/metabolismo , Western Blotting , ADN sin Sentido/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Humanos , Reacción en Cadena de la Polimerasa , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
Molecular and biochemical analysis indicates that nuclear transcription factor kappaB (NF-kappaB)-inducing kinase (NIK) mediates IKK activation and NF-kappaB transcriptional activity. However, gene deletion studies suggest that NIK triggers gene expression without affecting IkappaBalpha degradation and NF-kappaB DNA binding activity. In order to investigate the role of NIK in NF-kappaB transcriptional activity, we used mouse embryonic fibroblasts (MEF) derived from wild-type (wt) and IkappaB kinase gamma (IKKgamma) gene deficient (IKKgamma(-/-)) mice. We report that although TNF-induced NF-kappaB transcriptional activity is abolished in IKKgamma(-/-) cells, adenoviral gene delivery of NIK (Ad5NIK) still enhanced transcriptional activity and IL-6 mRNA accumulation. Moreover, NIK targets the transactivation function of NF-kappaB through stimulation of the transactivation domain (TAD) of RelA (S536) in IKKgamma(-/-) cells. Interestingly, Ad5NIK, but not TNF, induces RelA S536 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in IKKgamma(-/-) cells. Functional analysis demonstrated that Ad5NIK-induced NF-kappaB transcriptional activity, IL-6 mRNA expression and RelA phosphorylation are inhibited by the p38 inhibitor SB203580, suggesting a role for this MAPK in NIK signaling to NF-kappaB. These data demonstrate for the first time the presence of an IKKgamma-independent NIK/p38 MAPK-dependent signaling pathway that activates NF-kappaB and induces pro-inflammatory gene expression through RelA phosphorylation.
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FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Activación Transcripcional , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Eliminación de Gen , Quinasa I-kappa B , Interleucina-6/genética , Ratones , Ratones Noqueados/genética , Mutagénesis , FN-kappa B/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , ARN Mensajero/metabolismo , Factor de Transcripción ReIA , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Quinasa de Factor Nuclear kappa BRESUMEN
We have previously shown that non-pathogenic Gram negative bacteria induce RelA phosphorylation, nuclear factor (NF)-kappaB transcriptional activity and pro-inflammatory gene expression in intestinal epithelial cells (IEC) in vivo and in vitro. In this study, we investigated the molecular mechanism of immune-epithelial cell cross-talk on Gram-negative enteric bacteria-induced NF-kappaB signalling and pro-inflammatory gene expression in IEC using HT-29/MTX as well as CaCO-2 transwell cultures Interestingly, while differentiated HT-29/MTX cells are unresponsive to non-pathogenic Gram negative bacterial stimulation, interleukin-8 (IL-8) mRNA accumulation is strongly induced in Escherichia coli- but not Bacteroides vulgatus-stimulated IEC cocultured with peripheral blood (PBMC) and lamina propria mononuclear cells (LPMC). The presence of PBMC triggered both E. coli- and B. vulgatus-induced mRNA expression of the Toll-like receptor-4 accessory protein MD-2 as well as endogenous IkappaBalpha phosphorylation, demonstrating similar capabilities of these bacteria to induce proximal NF-kappaB signalling. However, B. vulgatus failed to trigger IkappaBalpha degradation and NF-kappaB transcriptional activity in the presence of PBMC. Interestingly, B. vulgatus- and E. coli-derived lipopolysaccharide-induced similar IL-8 mRNA expression in epithelial cells after basolateral stimulation of HT-29/PBMC cocultures. Although luminal enteric bacteria have adjuvant and antigenic properties in chronic intestinal inflammation, PBMC from patients with active ulcerative colitis and Crohn's disease differentially trigger epithelial cell activation in response to E. coli and E. coli-derived LPS. In conclusion, this study provides evidence for a differential regulation of non-pathogenic Gram-negative bacteria-induced NF-kappaB signalling and IL-8 gene expression in IEC cocultured with immune cells and suggests the presence of mechanisms that assure hyporesponsiveness of the intestinal epithelium to certain commensally enteric bacteria.
Asunto(s)
Bacterias Gramnegativas/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , FN-kappa B/metabolismo , Animales , Antígenos Bacterianos/inmunología , Bacteroides/inmunología , Células CACO-2 , Células Cultivadas , Técnicas de Cocultivo , Células Epiteliales/inmunología , Escherichia coli/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Inmunidad Mucosa , Enfermedades Inflamatorias del Intestino/microbiología , Interleucina-8/biosíntesis , Interleucina-8/genética , Leucocitos Mononucleares/inmunología , Fosforilación , ARN Mensajero/genética , Transducción de Señal/inmunologíaRESUMEN
Several effects of bile acids (BAs) on colonic epithelial cells (CECs) have been described, including induction of proliferation and apoptosis. Some of these effects are mediated through activation of the NF-kappa B transcriptional system. In this study, we investigated the molecular mechanisms underlying the BA-induced gene expression in CECs. The human CEC line HT-29 and primary human CECs were treated with dilutions of salts of deoxycholic acid (DCA) and taurodeoxycholic acid (TDCA). NF-kappa B binding activity was analyzed with EMSA, RelA translocation with immunofluorescence, and I kappa B alpha- and RelA-phosphorylation with Western blot analysis. IL-8 mRNA and protein expression were assessed by quantitative PCR and ELISA. Functional impact of NF-kappa B activation was determined by blocking the proteasome activity with MG132 or by preventing IKK activity with a dominant-negative IKK beta delivered by adenoviral dominant-negative (dn) IKK beta (Ad5dnIKK beta). DCA and TDCA induced IL-8 expression in a dose- and time-dependent manner. It is interesting that DCA but not TDCA induced I kappa B alpha-phosphorylation, RelA translocation, and NF-kappa B binding activity. Accordingly, the proteasome inhibitor MG132 blocked DCA- but not TDCA-induced IL-8 gene expression. In contrast, TDCA-induced IL-8 gene expression correlated with enhanced RelA phosphorylation, which was blocked by Ad5dnIKK beta. Our data suggest that DCA-induced signal transduction mainly utilized the I kappa B degradation and RelA nuclear translocation pathway, whereas TDCA primarily induced IL-8 gene expression through RelA phosphorylation. These differences may have implications for the understanding of the pathophysiology of inflammation and carcinogenesis in the gut.
Asunto(s)
Colon/metabolismo , Ácido Desoxicólico/farmacología , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido Taurodesoxicólico/farmacología , Ácidos y Sales Biliares/farmacología , Células Cultivadas , Colon/citología , Colon/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-8/genética , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Butyrate, a short-chain fatty acid released by colonic bacteria and administered therapeutically in inflammatory bowel diseases, exerts immunomodulatory properties. The aim of the study was to determine the functional consequences of butyrate exposure on the proinflammatory responsiveness of human intestinal epithelial cells (IEC). IL-8 promoter activity in IEC pretreated with butyrate then exposed to proinflammatory stimuli was assayed by transfection of luciferase constructs. IL-8 secretion was determined by ELISA and neutrophil migration by flow cytometry. Receptor mRNA was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Butyrate modulated proinflammatory IL-8 secretion differentially in Caco-2 and HT-29 cells on the transcriptional level. Pointing to the potentially underlying mechanism of increased IL-1 beta-stimulated IL-8 secretion in HT-29 cells, butyrate up-regulated IL-1RI mRNA but not IL-1RII. Butyrate pretreatment of IEC lines stimulated by IL-1 beta modulated neutrophil migration significantly: reduction towards Caco-2 and enhancement towards HT-29/p cells. Pharmacological inhibition of protein tyrosine phosphatases or treatment with mesalamine or sulphasalazine diminished IL-1 beta-stimulated IL-8 secretion by butyrate-exposed HT-29 cells substantially. Immunomodulatory effects of butyrate on IEC are functionally relevant for neutrophil migration. Pharmacological inhibition of enhanced IL-1 beta-mediated IL-8 secretion in a subpopulation of IEC may improve the clinical efficacy of butyrate.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Butiratos/uso terapéutico , Interleucina-1/inmunología , Interleucina-8/metabolismo , Mucosa Intestinal/inmunología , Antiinflamatorios no Esteroideos/uso terapéutico , Células CACO-2 , Quimiotaxis de Leucocito/efectos de los fármacos , Células HT29 , Humanos , Interleucina-8/genética , Mucosa Intestinal/efectos de los fármacos , Mesalamina/uso terapéutico , ARN Mensajero/análisis , Receptores de Interleucina-1/genética , Sulfasalazina/uso terapéutico , Transcripción GenéticaRESUMEN
Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.
Asunto(s)
Apoptosis/fisiología , Interferón gamma/farmacología , Interleucina-1/farmacología , Islotes Pancreáticos/fisiología , FN-kappa B/antagonistas & inhibidores , Adenoviridae/genética , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Proteínas I-kappa B/genética , Proteínas I-kappa B/farmacología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratas , Ratas WistarRESUMEN
BACKGROUND & AIMS: Our previous studies have shown that Cryptosporidium parvum induces biliary epithelial cell apoptosis in vivo and causes apoptosis in bystander uninfected biliary epithelia in vitro. We analyzed C. parvum-induced nuclear factor kappa B (NF-kappaB) activation in human biliary epithelial cells and assessed its relevance to epithelial cell apoptosis. METHODS: In vitro models of cryptosporidial infection using a human biliary epithelial cell line were used to assay C. parvum- induced NF-kappaB activation and associated apoptosis. RESULTS: Degradation of I(kappa)B and nuclear translocation of the NF-kappaB family of proteins (p65 and p50) were observed in the biliary epithelial cell cultures directly exposed to the parasite. Activation of NF-kappaB was found only in directly infected cells (but not in bystander uninfected cells). A time-dependent secretion of a known NF-kappaB gene product, interleukin 8, from infected cell cultures was detected. C. parvum-induced biliary epithelial cell apoptosis was limited to bystander uninfected cells. In contrast, inhibition of NF-kappaB activation resulted in apoptosis in directly infected cells and significantly enhanced C. parvum-induced apoptosis in bystander uninfected cells. CONCLUSIONS: These observations support the concept that, while C. parvum triggers host cell apoptosis in bystander uninfected biliary epithelial cells, which may limit spread of the infection, it directly activates the NF-kappaB/I(kappa)B system in infected biliary epithelia thus protecting infected cells from death and facilitating parasite survival and propagation.
Asunto(s)
Apoptosis , Conductos Biliares/parasitología , Cryptosporidium parvum/fisiología , FN-kappa B/metabolismo , Animales , Conductos Biliares/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Humanos , Interleucina-8/biosíntesis , Microscopía InmunoelectrónicaRESUMEN
BACKGROUND AND AIMS: Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells. METHODS: In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography. RESULTS: The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly. CONCLUSIONS: COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.