RESUMEN
Impaired E-cadherin (Cdh1) functions are closely associated with cellular dedifferentiation, infiltrative tumor growth and metastasis, particularly in gastric cancer. The class-I carcinogen Helicobacter pylori (H. pylori) colonizes gastric epithelial cells and induces Cdh1 shedding, which is primarily mediated by the secreted bacterial protease high temperature requirement A (HtrA). In this study, we used human primary epithelial cell lines derived from gastroids and mucosoids from different healthy donors to investigate HtrA-mediated Cdh1 cleavage and the subsequent impact on bacterial pathogenesis in a non-neoplastic context. We found a severe impairment of Cdh1 functions by HtrA-induced ectodomain cleavage in 2D primary cells and mucosoids. Since mucosoids exhibit an intact apico-basal polarity, we investigated bacterial transmigration across the monolayer, which was partially depolarized by HtrA, as indicated by microscopy, the analyses of the transepithelial electrical resistance (TEER) and colony forming unit (cfu) assays. Finally, we investigated CagA injection and observed efficient CagA translocation and tyrosine phosphorylation in 2D primary cells and, to a lesser extent, similar effects in mucosoids. In summary, HtrA is a crucially important factor promoting the multistep pathogenesis of H. pylori in non-transformed primary gastric epithelial cells and organoid-based epithelial models.
Asunto(s)
Proteínas Bacterianas , Cadherinas , Células Epiteliales , Mucosa Gástrica , Helicobacter pylori , Organoides , Humanos , Cadherinas/metabolismo , Organoides/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Antígenos Bacterianos/metabolismo , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Antígenos CD/metabolismo , Estómago/microbiología , Estómago/patología , Línea Celular , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/microbiología , Serina ProteasasRESUMEN
BACKGROUND: Gastric intestinal metaplasia (GIM) is an essential precancerous lesion. Although the reversal of GIM is challenging, it potentially brings a state-to-art strategy for gastric cancer therapeutics (GC). The lack of the appropriate in vitro model limits studies of GIM pathogenesis, which is the issue this work aims to address for further studies. METHOD: The air-liquid interface (ALI) model was adopted for the long-term culture of GIM cells in the present work. This study conducted Immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR), transcriptomic sequencing, and mucoproteomic sequencing (MS) techniques to identify the pathways for differential expressed genes (DEGs) enrichment among different groups, furthermore, to verify novel biomarkers of GIM cells. RESULT: Our study suggests that GIM-ALI model is analog to the innate GIM cells, which thus can be used for mucus collection and drug screening. We found genes MUC17, CDA, TRIM15, TBX3, FLVCR2, ONECUT2, ACY3, NMUR2, and MAL2 were highly expressed in GIM cells, while GLDN, SLC5A5, MAL, and MALAT1 showed down-regulated, which can be used as potential biomarkers for GIM cells. In parallel, these genes that highly expressed in GIM samples were mainly involved in cancer-related pathways, such as the MAPK signal pathway and oxidative phosphorylation signal pathway. CONCLUSION: The ALI model is validated for the first time for the in vitro study of GIM. GIM-ALI model is a novel in vitro model that can mimic the tissue micro-environment in GIM patients and further provide an avenue for studying the characteristics of GIM mucus. Our study identified new markers of GIM as well as pathways associated with GIM, which provides outstanding insight for exploring GIM pathogenesis and potentially other related conditions.
Asunto(s)
Metaplasia , Humanos , Aire , Modelos Biológicos , Mucosa Gástrica/patología , Mucosa Gástrica/metabolismo , Estómago/patología , Organoides/patología , Neoplasias Gástricas/patología , Neoplasias Gástricas/genética , Regulación Neoplásica de la Expresión Génica , Transcriptoma/genética , Intestinos/patologíaRESUMEN
BACKGROUND: Epigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail. RESULTS: Here, we establish gastric mucosa derived primary cell cultures as a reliable source of native human epithelium. We describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach, i.e., antrum, corpus, fundus together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1 and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach. By extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. We present insights into the dynamics of DNA methylation across anatomical regions of the healthy stomach and patterns of its change in disease. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Animales , Humanos , Metilación de ADN , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Epigénesis Genética , Infecciones por Helicobacter/genética , Células Epiteliales/patología , MamíferosRESUMEN
Crosstalk within the gastric epithelium, which is closely in contact with stromal fibroblasts in the gastric mucosa, has a pivotal impact in proliferation, differentiation and transformation of the gastric epithelium. The human pathogen Helicobacter pylori colonises the gastric epithelium and represents a risk factor for gastric pathophysiology. Infection of H. pylori induces the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is involved in the pro-inflammatory response but also in cell survival. In co-cultures with human gastric fibroblasts (HGF), we found that apoptotic cell death is reduced in the polarised human gastric cancer cell line NCI-N87 or in gastric mucosoids during H. pylori infection. Interestingly, suppression of apoptotic cell death in NCI-N87 cells involved an enhanced A20 expression regulated by NF-κB activity in response to H. pylori infection. Moreover, A20 acts as an important negative regulator of caspase-8 activity, which was suppressed in NCI-N87 cells during co-culture with gastric fibroblasts. Our results provide evidence for NF-κB-dependent regulation of apoptotic cell death in cellular crosstalk and highlight the protective role of gastric fibroblasts in gastric epithelial cell death during H. pylori infection.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Caspasa 8/metabolismo , Supervivencia Celular , Técnicas de Cocultivo , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Humanos , FN-kappa B/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The adult gastric mucosa is characterised by deep invaginations of the epithelium called glands. These tissue architectural elements are maintained with the contribution of morphogen signals. Morphogens are expressed in specific areas of the tissue, and their diffusion generates gradients in the microenvironment. Cells at different positions in the gland sense a specific combination of signals that instruct them to differentiate, proliferate, regenerate, or migrate. Differentiated cells perform specific functions involved in digestion, such as the production of protective mucus and the secretion of digestive enzymes or gastric acid. Biopsies from gastric precancerous conditions usually display tissue aberrations and change the shape of the glands. Alteration of the morphogen signalling microenvironment is likely to underlie those conditions. Furthermore, genes involved in morphogen signalling pathways are found to be frequently mutated in gastric cancer. We summarise the most recent findings regarding alterations of morphogen signalling during gastric carcinogenesis, and we highlight the new stem cell technologies that are improving our understanding of the regulation of human tissue shape.
Asunto(s)
Gastritis Atrófica , Infecciones por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Adulto , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/patología , Humanos , Neoplasias Gástricas/patología , Microambiente TumoralRESUMEN
NF-κB signaling pathways, induced by a variety of triggers, play a key role in regulating the expression of genes involved in the immune response and cellular responses to stress. The human pathogen Helicobacter pylori induces classical and alternative NF-κB signaling pathways via its effector ADP-L-glycero-ß-D-manno-heptose (ADP-heptose). We review H. pylori- and NF-κB-dependent alterations in cellular processes and associated maladaptation leading to deleterious gastric pathophysiology that have implications for the diagnosis and treatment of gastric diseases. Therapeutic options for gastric cancer (GC) include clinically relevant small molecule inhibitors of NF-κB and epigenetic therapy approaches. In this context, gastric organoid biobanks originated from patient material, represent a valuable platform for translational applications to predict patient responses to chemotherapy, with a view to personalized medicine.
Asunto(s)
Infecciones por Helicobacter , FN-kappa B , Neoplasias Gástricas , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/genética , Helicobacter pylori , Humanos , FN-kappa B/genética , Transducción de Señal , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/microbiologíaRESUMEN
The lining of the stomach is a tight monolayer of epithelial cells performing functions in digestion and a protective barrier against gastric acid, toxic metabolites and infectious agents, including Helicobacter pylori. The response of the epithelial barrier to infections underlies gastric pathologies, including gastric cancer. H. pylori has the unique capacity to colonise the gastric mucosa while evading the immune system. The colonised mucosa initiates an inflammatory response to fight the infection and a strong regenerative program to avoid barrier failure and ulceration. This response changes the morphology and cell composition of the gastric epithelium and in parallel it might contribute to the accumulation of somatic mutations leading to cellular transformation. Genetically modified mice, cell lines and human-derived organoids are the main biological models to study the gastric epithelial barrier. With these models it is possible to dissect the stepwise process of tissue adaptation to infection that places the epithelium at risk of malignant transformation.
Asunto(s)
Transformación Celular Neoplásica/genética , Infecciones por Helicobacter/fisiopatología , Neoplasias Gástricas/genética , Animales , Gerbillinae , Humanos , Modelos Moleculares , Neoplasias Gástricas/patologíaRESUMEN
BACKGROUND & AIMS: The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organized inside glands and crypts. Regeneration depends on Wnt/ß-catenin pathway activation, but to understand homeostasis and its dysregulation in disease, we need to identify the signaling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen-, and gastric acid-producing cells. METHODS: We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief, and parietal cells. We localized the source of the growth factors in the tissue of origin. RESULTS: We show that epidermal growth factor is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with bone morphogenetic factor/Noggin signals, epidermal growth factor controls the differentiation of foveolar cells vs parietal or chief cells. We also show that epidermal growth factor is likely to underlie alteration of the gastric mucosa in the precancerous condition atrophic gastritis. CONCLUSIONS: Use of our recently established mucosoid cultures in combination with analysis of the tissue of origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signaling microenvironment in the human gastric glands.
Asunto(s)
Tipificación del Cuerpo/efectos de los fármacos , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Mucosa Gástrica/efectos de los fármacos , Proteínas Portadoras/farmacología , Linaje de la Célula , Células Cultivadas , Microambiente Celular , Células Principales Gástricas/efectos de los fármacos , Células Principales Gástricas/metabolismo , Células Principales Gástricas/ultraestructura , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestructura , Gastritis Atrófica/metabolismo , Gastritis Atrófica/patología , Regulación del Desarrollo de la Expresión Génica , Humanos , Organoides , Células Parietales Gástricas/efectos de los fármacos , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/ultraestructura , Vía de Señalización WntRESUMEN
Genotoxic colibactin-producing pks+ Escherichia coli induce DNA double-strand breaks, mutations, and promote tumor development in mouse models of colorectal cancer (CRC). Colibactin's distinct mutational signature is reflected in human CRC, suggesting a causal link. Here, we investigate its transformation potential using organoids from primary murine colon epithelial cells. Organoids recovered from short-term infection with pks+ E. coli show characteristics of CRC cells, e.g., enhanced proliferation, Wnt-independence, and impaired differentiation. Sequence analysis of Wnt-independent organoids reveals an enhanced mutational burden, including chromosomal aberrations typical of genomic instability. Although we do not find classic Wnt-signaling mutations, we identify several mutations in genes related to p53-signaling, including miR-34a. Knockout of Trp53 or miR-34 in organoids results in Wnt-independence, corroborating a functional interplay between the p53 and Wnt pathways. We propose larger chromosomal alterations and aneuploidy as the basis of transformation in these organoids, consistent with the early appearance of chromosomal instability in CRC.
Asunto(s)
Células Epiteliales/metabolismo , Escherichia coli/metabolismo , Genómica , Péptidos/metabolismo , Policétidos/metabolismo , Animales , Aberraciones Cromosómicas , Colon/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/psicología , Daño del ADN , Células Epiteliales/patología , Escherichia coli/genética , Masculino , Ratones , Ratones Noqueados , Mutación , Organoides , Péptidos/genéticaRESUMEN
Carcinoma of the gallbladder (GBC) is the most frequent tumor of the biliary tract. Despite epidemiological studies showing a correlation between chronic infection with Salmonella enterica Typhi/Paratyphi A and GBC, the underlying molecular mechanisms of this fatal connection are still uncertain. The murine serovar Salmonella Typhimurium has been shown to promote transformation of genetically predisposed cells by driving mitogenic signaling. However, insights from this strain remain limited as it lacks the typhoid toxin produced by the human serovars Typhi and Paratyphi A. In particular, the CdtB subunit of the typhoid toxin directly induces DNA breaks in host cells, likely promoting transformation. To assess the underlying principles of transformation, we used gallbladder organoids as an infection model for Salmonella Paratyphi A. In this model, bacteria can invade epithelial cells, and we observed host cell DNA damage. The induction of DNA double-strand breaks after infection depended on the typhoid toxin CdtB subunit and extended to neighboring, non-infected cells. By cultivating the organoid derived cells into polarized monolayers in air-liquid interphase, we could extend the duration of the infection, and we observed an initial arrest of the cell cycle that does not depend on the typhoid toxin. Non-infected intoxicated cells instead continued to proliferate despite the DNA damage. Our study highlights the importance of the typhoid toxin in causing genomic instability and corroborates the epidemiological link between Salmonella infection and GBC.IMPORTANCE Bacterial infections are increasingly being recognized as risk factors for the development of adenocarcinomas. The strong epidemiological evidence linking Helicobacter pylori infection to stomach cancer has paved the way to the demonstration that bacterial infections cause DNA damage in the host cells, initiating transformation. In this regard, the role of bacterial genotoxins has become more relevant. Salmonella enterica serovars Typhi and Paratyphi A have been clinically associated with gallbladder cancer. By harnessing the stem cell potential of cells from healthy human gallbladder explant, we regenerated and propagated the epithelium of this organ in vitro and used these cultures to model S. Paratyphi A infection. This study demonstrates the importance of the typhoid toxin, encoded only by these specific serovars, in causing genomic instability in healthy gallbladder cells, posing intoxicated cells at risk of malignant transformation.
Asunto(s)
Daño del ADN , Células Epiteliales/microbiología , Células Epiteliales/patología , Vesícula Biliar/citología , Salmonella paratyphi A/patogenicidad , Adulto , Anciano , Animales , Células Cultivadas , Femenino , Vesícula Biliar/microbiología , Interacciones Huésped-Patógeno , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Serogrupo , Virulencia/genéticaRESUMEN
BACKGROUND: Deregulated c-Abl activity has been intensively studied in a variety of solid tumors and leukemia. The class-I carcinogen Helicobacter pylori (Hp) activates the non-receptor tyrosine kinase c-Abl to phosphorylate the oncoprotein cytotoxin-associated gene A (CagA). The role of c-Abl in CagA-dependent pathways is well established; however, the knowledge of CagA-independent c-Abl processes is scarce. METHODS: c-Abl phosphorylation and localization were analyzed by immunostaining and immunofluorescence. Interaction partners were identified by tandem-affinity purification. Cell elongation and migration were analyzed in transwell-filter experiments. Apoptosis and cell survival were examined by FACS analyses and MTT assays. In mice experiments and human biopsies, the involvement of c-Abl in Hp pathogenesis was investigated. RESULTS: Here, we investigated the activity and subcellular localization of c-Abl in vitro and in vivo and unraveled the contribution of c-Abl in CagA-dependent and -independent pathways to gastric Hp pathogenesis. We report a novel mechanism and identified strong c-Abl threonine 735 phosphorylation (pAblT735) mediated by the type-IV secretion system (T4SS) effector D-glycero-ß-D-manno-heptose-1,7-bisphosphate (ßHBP) and protein kinase C (PKC) as a new c-Abl kinase. pAblT735 interacted with 14-3-3 proteins, which caused cytoplasmic retention of c-Abl, where it potentiated Hp-mediated cell elongation and migration. Further, the nuclear exclusion of pAblT735 attenuated caspase-8 and caspase-9-dependent apoptosis. Importantly, in human patients suffering from Hp-mediated gastritis c-Abl expression and pAblT735 phosphorylation were drastically enhanced as compared to type C gastritis patients or healthy individuals. Pharmacological inhibition using the selective c-Abl kinase inhibitor Gleevec confirmed that c-Abl plays an important role in Hp pathogenesis in a murine in vivo model. CONCLUSIONS: In this study, we identified a novel regulatory mechanism in Hp-infected gastric epithelial cells by which Hp determines the subcellular localization of activated c-Abl to control Hp-mediated EMT-like processes while decreasing cell death.
Asunto(s)
Apoptosis , Movimiento Celular , Helicobacter pylori/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Línea Celular Tumoral , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Humanos , Modelos Biológicos , Fosforilación , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Proteína Quinasa C/metabolismo , Transporte de ProteínasRESUMEN
OBJECTIVE: Helicobacter pylori causes life-long colonisation of the gastric mucosa, leading to chronic inflammation with increased risk of gastric cancer. Research on the pathogenesis of this infection would strongly benefit from an authentic human in vitro model. DESIGN: Antrum-derived gastric glands from surgery specimens served to establish polarised epithelial monolayers via a transient air-liquid interface culture stage to study cross-talk with H. pylori and the adjacent stroma. RESULTS: The resulting 'mucosoid cultures', so named because they recapitulate key characteristics of the gastric mucosa, represent normal stem cell-driven cultures that can be passaged for months. These highly polarised columnar epithelial layers encompass the various gastric antral cell types and secrete mucus at the apical surface. By default, they differentiate towards a foveolar, MUC5AC-producing phenotype, whereas Wnt signalling stimulates proliferation of MUC6-producing cells and preserves stemness-reminiscent of the gland base. Stromal cells from the lamina propria secrete Wnt inhibitors, antagonising stem-cell niche signalling and inducing differentiation. On infection with H. pylori, a strong inflammatory response is induced preferentially in the undifferentiated basal cell phenotype. Infection of cultures for several weeks produces foci of viable bacteria and a persistent inflammatory condition, while the secreted mucus establishes a barrier that only few bacteria manage to overcome. CONCLUSION: Gastric mucosoid cultures faithfully reproduce the features of normal human gastric epithelium, enabling new approaches for investigating the interaction of H. pylori with the epithelial surface and the cross-talk with the basolateral stromal compartment. Our observations provide striking insights in the regulatory circuits of inflammation and defence.
Asunto(s)
Mucosa Gástrica/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/fisiología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/patología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Infecciones por Helicobacter/metabolismo , Homeostasis/fisiología , Interacciones Microbiota-Huesped/fisiología , Humanos , Moco/metabolismo , Antro Pilórico/metabolismo , Antro Pilórico/microbiología , Antro Pilórico/patología , Nicho de Células Madre , Células del Estroma/fisiología , Técnicas de Cultivo de Tejidos/métodosRESUMEN
BACKGROUND & AIMS: Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosa for decades. H pylori expression of cholesterol-α-glucosyltransferase (encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response. METHODS: MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patients undergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-ß-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed by immunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Δcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1-/- mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Δcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy. RESULTS: In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human ß-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNG-response gene IRF1 was substantially higher in PMSS1Δcgt-infected mice than PMSS1-infected mice. Ifngr1-/- mice were colonized by PMSS1 to a greater extent than control mice. CONCLUSIONS: H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which H pylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell-based vaccines against H pylori.
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Colesterol/metabolismo , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Gastritis/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Interferón gamma/metabolismo , Transducción de Señal , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Microambiente Celular , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Gastritis/genética , Gastritis/inmunología , Gastritis/microbiología , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/inmunología , Helicobacter pylori/patogenicidad , Interacciones Huésped-Patógeno , Humanos , Interferón gamma/inmunología , Interleucina-6/metabolismo , Interleucinas/metabolismo , Quinasas Janus/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Viabilidad Microbiana , Mutación , Cultivo Primario de Células , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Factor de Transcripción STAT1/metabolismo , Factores de Tiempo , Receptor de Interferón gamma , Interleucina-22RESUMEN
BACKGROUND: Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). METHODS: Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (ß2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-ß2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human ß2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human ß2GPI or after CL-micelle absorption. RESULTS: Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized ß2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-ß2GPI humoAbs. CONCLUSIONS: The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.
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Anticuerpos Antifosfolípidos/análisis , Síndrome Antifosfolípido/diagnóstico , Inmunoensayo/métodos , Adulto , Anciano , Síndrome Antifosfolípido/inmunología , Diagnóstico Diferencial , Femenino , Humanos , Infecciones/diagnóstico , Infecciones/inmunología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Although bacteria have long been associated with human cancer, drawing causal relationships has been difficult. In this issue of Cell Host & Microbe, Scanu et al. (2015) provide evidence for a transforming activity of Salmonella Typhimurium on predisposed host cells, which can subsequently form tumors in a xenograft model.
Asunto(s)
Neoplasias de la Vesícula Biliar/patología , Interacciones Huésped-Patógeno , Infecciones por Salmonella/patología , Salmonella enterica/patogenicidad , Animales , HumanosRESUMEN
The B cell lymphomas associated with Epstein-Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus-tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.
Asunto(s)
Linfocitos B/virología , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/fisiología , Mieloma Múltiple/patología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Linfocitos B/patología , Diferenciación Celular , Genes Virales , Herpesvirus Humano 4/genética , Humanos , Cadenas lambda de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/genética , Factores Reguladores del Interferón/biosíntesis , Factores Reguladores del Interferón/genética , MicroARNs/biosíntesis , MicroARNs/genética , Proteínas de Mieloma/biosíntesis , Proteínas de Mieloma/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Factor de Transcripción PAX5/biosíntesis , Factor de Transcripción PAX5/genética , Fenotipo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Sindecano-1/biosíntesis , Sindecano-1/genética , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/virología , Latencia del VirusRESUMEN
Epstein-Barr virus (EBV)-negative diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma-derived cell lines infected in vitro with a recombinant EBV expressed type II/III latency. High expression of EBNA2 inversely correlated with expression of germinal center (GC)-associated genes, BCL6 and TCL1. The decreased expression of BCL6 appeared to be dose dependent, with almost complete abrogation in highly EBNA2-expressing clones. The role of EBNA2 in negative regulation of these genes was confirmed by transfection and in a hormone-inducible EBNA2 cell system. LMP1 transfection reduced expression of TCL1, but not of BCL6, in DLBCLs. The GC-associated gene repression was at the transcriptional level and CBF1 independent. A decrease in HLA-DR, surface immunoglobulin M, and class II transactivator expression and an increase in CCL3, a BCL6 repression target, was observed in EBNA2-expressing clones. Since BCL6 is indispensable for GC formation and somatic hypermutations (SHM), we suggest that the previously reported lack of SHM seen in EBNA2-expressing GC cells from infectious mononucleosis tonsils could be due to negative regulation of BCL6 by EBNA2. These findings suggest that EBNA2 interferes with the GC phenotype.