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Background: Lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma, are one of the differentials for peripheral lymphadenopathy and are difficult to diagnose clinically. Biopsy is essential for diagnosing lymphoma, although it is invasive. Non-invasive methods are required to identify patients with suspected lymphoma who should undergo a biopsy. The relevance of the monocyte-to-lymphocyte ratio has recently been reported to be a useful diagnostic marker in children with lymphoma and a prognostic marker of various other diseases. This study aimed to determine the relevance of the monocyte-to-lymphocyte ratio in the diagnosis of lymphoma in adults. Methods: The study included 246 adult outpatients (median age of 49.0 years) presenting with peripheral lymphadenopathy. The final diagnosis was determined by reviewing the medical records. We categorized all patients into either the lymphoma group or the non-lymphoma group. The lymphoma group included patients who underwent biopsy and were diagnosed with lymphoma by histopathology, while the non-lymphoma group included those diagnosed with disease excluding lymphoma. The monocyte-to-lymphocyte ratios were compared between the two groups. Results: Of the participants, 33 (13.4%) were assigned to the lymphoma group. The median age of the lymphoma and non-lymphoma groups were 67.0 years (interquartile range [IQR] 55.5-75.5 years) and 46.0 years (IQR 36.0-61.0 years), respectively. The lymphocyte and monocyte levels showed no significant differences between the two groups individually. Nonetheless, the monocyte-to-lymphocyte ratio was significantly higher in the lymphoma group (median, 0.36; IQR, 0.24-0.73) than in the non-lymphoma group (median, 0.29; IQR, 0.21-0.43; P = 0.022), independent of lymph node diameter ≥ 1 cm and C-reactive protein levels. Conclusion: This study suggests that the monocyte-to-lymphocyte ratio can be a helpful diagnostic marker for lymphoma in adults with peripheral lymphadenopathy when the etiology is unclear even after a medical interview and physical examination.
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Infection with cagA-positive Helicobacter pylori strains plays an etiological role in the development of gastric cancer. The CagA protein is injected into gastric epithelial cells through a bacterial Type IV secretion system. Inside the host cells, CagA promiscuously associates with multiple host cell proteins including the prooncogenic phosphatase SHP2 that is required for full activation of the Ras-ERK pathway. CagA-SHP2 interaction aberrantly activates SHP2 and thereby deregulates Ras-ERK signaling. Cancer is regarded as a disease of the genome, indicating that H. pylori-mediated gastric carcinogenesis is also associated with genomic alterations in the host cell. Indeed, accumulating evidence has indicated that H. pylori infection provokes DNA double-stranded breaks (DSBs) by both CagA-dependent and CagA-independent mechanisms. DSBs are repaired by either error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ). Infection with cagA-positive H. pylori inhibits RAD51 expression while dampening cytoplasmic-to-nuclear translocalization of BRCA1, causing replication fork instability and HR defects (known as "BRCAness"), which collectively provoke genomic hypermutation via non-HR-mediated DSB repair. H. pylori also subverts multiple DNA damage responses including DNA repair systems. Infection with H. pylori additionally inhibits the function of the p53 tumor suppressor, thereby dampening DNA damage-induced apoptosis, while promoting proliferation of CagA-delivered cells. Therefore, H. pylori cagA-positive strains promote abnormal expansion of cells with BRCAness, which dramatically increases the chance of generating driver gene mutations in the host cells. Once such driver mutations are acquired, H. pylori CagA is no longer required for subsequent gastric carcinogenesis (Hit-and-Run carcinogenesis).
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Infecciones por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carcinogénesis/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Humanos , Neoplasias Gástricas/metabolismoRESUMEN
The initial step in bacterial infection is adherence of the bacterium to the target cell surface. Helicobacter pylori exploits the interaction of bacterial adhesin protein HopQ with human epithelial CEACAMs (CEACAM1, 5, and 6) to stably adhere to gastric epithelial cells, which is necessary for delivery of the H. pylori CagA oncoprotein into the epithelial cells via a type IV secretion system. In contrast to human CEACAMs, however, HopQ does not interact with Ceacam1 (mouse CEACAM1) in vitro or in CHO cells ectopically expressing Ceacam1. Since the mouse genome lacks Ceacam5 and Ceacam6, no significant HopQ-Ceacam interaction may occur in mouse gastric epithelial cells. Here, we found that the mouse stomach has a much lower expression level of Ceacam1 than the expression level of CEACAM1 in the human stomach. Consistently, mouse gastric epithelial cells resist CagA delivery by cagA-positive H. pylori, and the delivery is restored by ectopic expression of human CEACAM1 or CEACAM5 in mouse gastric epithelial cells. Thus, despite the fact that mice are routinely used for H. pylori infection studies, a low expression level of Ceacam1 in the mouse stomach together with the loss or greatly reduced interaction of HopQ with Ceacams make the mouse an inappropriate model for studying the role of H. pylori-delivered CagA in gastric pathogenesis, including the development of gastric cancer.
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Infecciones por Helicobacter , Helicobacter pylori , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cricetinae , Cricetulus , Células Epiteliales/metabolismo , Helicobacter pylori/metabolismo , Ratones , Transporte de Proteínas , Estómago , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismoRESUMEN
The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.
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Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Roturas del ADN de Doble Cadena , Replicación del ADN , Regulación hacia Abajo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Proteínas Oncogénicas/metabolismo , Transducción de Señal , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Línea Celular , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Humanos , Proteínas Oncogénicas/genéticaRESUMEN
Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible "hit-and-run mechanism" of H. pylori CagA for gastric carcinogenesis.
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Antígenos Bacterianos/metabolismo , Proteína BRCA1/metabolismo , Proteínas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Inestabilidad Genómica , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Neoplasias Gástricas/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinogénesis/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , Células Epiteliales/microbiología , Femenino , Regulación Neoplásica de la Expresión Génica , Helicobacter pylori/patogenicidad , Interacciones Huésped-Patógeno , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Serina-Treonina Quinasa 3 , Transducción de Señal , Estómago/microbiología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Cervical lymphadenopathy is commonly seen in general practice, and its etiology is diverse. Establishing the diagnostic strategy for lymphadenopathy would be desirable to avoid overlooking neoplasms or other critical conditions. This study aims to identify the useful laboratory parameters for cervical lymphadenopathy that require clinical observation or intervention. METHODS: The participants were outpatients presenting cervical swelling or cervical lymph node (LN) pain who consulted the General Internal Medicine department from 2010 to 2016. We evaluated the characteristics, physical findings, and laboratory parameters with final diagnoses by multivariate logistic regression analysis. We categorized the final diagnoses as "Clinical Intervention Required Group (CIRG)" including necrotizing lymphadenitis, hematologic neoplasms, metastatic lymphadenopathy, tuberculous lymphadenitis, bacterial infectious diseases, infectious mononucleosis, autoimmune diseases, and other abnormal conditions or "No-CIRG" not requiring further clinical observation or intervention. RESULTS: We evaluated 409 participants, with 130 (31.8%) diagnosed as belonging to the CIRG. There was an association between CIRG and various parameters: age ≥60 years old (adjusted odds ratio [AOR], 2.70; 95% confidence interval [CI], 1.48-4.90), having a referral (AOR, 1.83; 95% CI, 1.12-3.00), diameter of LN ≥ 2 cm (AOR, 1.91; 95% CI, 1.05-3.48), fixed LNs (AOR, 2.74; 95% CI, 1.02-7.37), and lactate dehydrogenase (LD) ≥400 U/L (AOR, 3.78; 95% CI, 1.46-9.77). Eighty-two percent of LD ≥ 400 cases in the CIRG were infectious mononucleosis or necrotizing lymphadenitis. CONCLUSIONS: Besides the clinical indicators reported previously, we may apply an elevated LD level as a useful indicator of cervical lymphadenopathy that requires further clinical observation or intervention.
RESUMEN
Chronic infection with Helicobacter pylori cagA-positive strains is causally associated with the development of gastric diseases, most notably gastric cancer. The cagA-encoded CagA protein, which is injected into gastric epithelial cells by bacterial type IV secretion, undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) segments (EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D), which are present in various numbers and combinations in its C-terminal polymorphic region, thereby enabling CagA to promiscuously interact with SH2 domain-containing host cell proteins, including the prooncogenic SH2 domain-containing protein tyrosine phosphatase 2 (SHP2). Perturbation of host protein functions by aberrant complex formation with CagA has been considered to contribute to the development of gastric cancer. Here we show that SHIP2, an SH2 domain-containing phosphatidylinositol 5'-phosphatase, is a hitherto undiscovered CagA-binding host protein. Similar to SHP2, SHIP2 binds to the Western CagA-specific EPIYA-C segment or East Asian CagA-specific EPIYA-D segment through the SH2 domain in a tyrosine phosphorylation-dependent manner. In contrast to the case of SHP2, however, SHIP2 binds more strongly to EPIYA-C than to EPIYA-D. Interaction with CagA tethers SHIP2 to the plasma membrane, where it mediates production of phosphatidylinositol 3,4-diphosphate [PI(3,4)P2 ]. The CagA-SHIP2 interaction also potentiates the morphogenetic activity of CagA, which is caused by CagA-deregulated SHP2. This study indicates that initially delivered CagA interacts with SHIP2 and thereby strengthens H. pylori-host cell attachment by altering membrane phosphatidylinositol compositions, which potentiates subsequent delivery of CagA that binds to and thereby deregulates the prooncogenic phosphatase SHP2.
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Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Secuencias de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular , Membrana Celular/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Dominios Homologos srcRESUMEN
High-molecular-weight hyaluronan, a major component of the extracellular matrix, is anti-oncogenic, whereas low-molecular-weight hyaluronan is pro-oncogenic, though the mechanisms underlying the size-dependent opposite bioactivities of hyaluronan remain uncertain. We show here that treatment with high-molecular-weight hyaluronan stimulates tumor-suppressive Hippo signaling in breast epithelial cells. Mechanistically, clustering of the CD44 extracellular domain by high-molecular-weight hyaluronan leads to recruitment of the polarity-regulating kinase PAR1b by the CD44 intracellular domain, which results in disruption of the Hippo signaling-inhibitory PAR1b-MST complex. Once liberated from PAR1b, MST activates Hippo signaling. Conversely, low-molecular-weight hyaluronan, which is produced by hyaluronidase-mediated degradation of high-molecular-weight hyaluronan, inhibits Hippo signaling by competing with high-molecular-weight hyaluronan for CD44 binding. Triple-negative breast cancers with higher hyaluronidase-2 expression show poorer prognosis than those with lower hyaluronidase-2 expression. Consistently, decreased hyaluronidase-2 is associated with reduced tumorigenicity in a tumor xenograft model. Hence, perturbation of high-molecular-weight hyaluronan-mediated Hippo signaling activation contributes to cancer aggressiveness.
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Receptores de Hialuranos/metabolismo , Ácido Hialurónico/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Recuento de Células , Línea Celular Tumoral , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Femenino , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Xenoinjertos , Vía de Señalización Hippo , Humanos , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Vertebral osteomyelitis can result in serious complications if diagnosis is delayed. Magnetic resonance imaging (MRI) is considered the most useful modality for the early diagnosis of vertebral osteomyelitis. We examined three patients with vertebral osteomyelitis whose initial MRI (obtained <2 weeks after the onset of symptoms) did not reveal obvious findings of pyogenic vertebral osteomyelitis. However, follow-up MRI clearly demonstrated typical findings of the disease. This case series illustrates that a repeat MRI must be performed 2-4 weeks after the onset of symptoms in patients presenting with clinical manifestations and microbiological findings suggestive of vertebral osteomyelitis.
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Recent studies have indicated that increased expression of the M2 isoform of pyruvate kinase (PKM2) is involved in glycolysis and tumor development. However, little is known about the role of PKM2 in gastric cancer (GC). Therefore, we examined the expression and function of PKM2 in human GC. We evaluated PKM1 and PKM2 expression by quantitative RT-PCR in gastric tissues from 10 patients who underwent gastric endoscopic submucosal dissection, 80 patients who underwent gastrectomy, and seven healthy volunteers, and analyzed the correlation with clinicopathological variables. To assess the function of PKM2, we generated PKM2-knockdown GC cells, and investigated the phenotypic changes. Furthermore, we examined the induction of PKM2 expression by cytotoxin-associated gene A (CagA), a pathogenic factor of Helicobacter pylori, using CagA-inducible GC cells. We found that PKM2 was predominantly expressed not only in GC lesions but also in the normal gastric regions of GC patients and in the gastric mucosa of healthy volunteers. The PKM2 expression was significantly higher in carcinoma compared to non-cancerous tissue and was associated with venous invasion. Knockdown of PKM2 in GC cells caused significant decreases in cellular proliferation, migration, anchorage-independent growth, and sphere formation in vitro, and in tumor growth and liver metastasis in vivo. The serine concentration-dependent cell proliferation was also inhibited by PKM2 silencing. Furthermore, we found that PKM2 expression was upregulated by CagA by way of the Erk pathway. These results suggested that enhanced PKM2 expression plays a pivotal role in the carcinogenesis and development of GC in part by regulating cancer-specific metabolism.
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Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Isoformas de Proteínas/genética , Neoplasias Gástricas/genética , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Anciano , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glucólisis/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Sistema de Señalización de MAP Quinasas/genética , Masculino , Isoformas de Proteínas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Regulación hacia Arriba/genética , Proteínas de Unión a Hormona TiroideRESUMEN
Most if not all gastric cancers are associated with chronic infection of the stomach mucosa with Helicobacter pylori cagA-positive strains(1-4). Approximately 10% of gastric cancers also harbour Epstein-Barr virus (EBV) in the cancer cells(5,6). Following delivery into gastric epithelial cells via type IV secretion(7,8), the cagA-encoded CagA protein undergoes tyrosine phosphorylation on the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs initially by Src family kinases (SFKs) and then by c-Abl(9,10). Tyrosine-phosphorylated CagA binds to the pro-oncogenic protein tyrosine phosphatase SHP2 and thereby deregulates the phosphatase activity(11,12), which has been considered to play an important role in gastric carcinogenesis(13). Here we show that the SHP2 homologue SHP1 interacts with CagA independently of the EPIYA motif. The interaction potentiates the phosphatase activity of SHP1 that dampens the oncogenic action of CagA by dephosphorylating the CagA EPIYA motifs. In vitro infection of gastric epithelial cells with EBV induces SHP1 promoter hypermethylation, which strengthens phosphorylation-dependent CagA action via epigenetic downregulation of SHP1 expression. Clinical specimens of EBV-positive gastric cancers also exhibit SHP1 hypermethylation with reduced SHP1 expression. The results reveal that SHP1 is the long-sought phosphatase that can antagonize CagA. Augmented H. pylori CagA activity, via SHP1 inhibition, might also contribute to the development of EBV-positive gastric cancer.
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Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Helicobacter pylori/patogenicidad , Herpesvirus Humano 4/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Proteína Tirosina Fosfatasa no Receptora Tipo 6/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Carcinogénesis , Línea Celular Tumoral , Metilación de ADN , Células Epiteliales/microbiología , Células Epiteliales/virología , Humanos , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
Early detection of gastric cancers saves lives, but remains a diagnostic challenge. In this study, we aimed to identify cell-surface biomarkers of early gastric cancer. We hypothesized that a subset of plasma membrane proteins induced by the Helicobacter pylori oncoprotein CagA will be retained in early gastric cancers through non-oncogene addiction. An inducible system for expression of CagA was used to identify differentially upregulated membrane protein transcripts in vitro. The top hits were then analyzed in gene expression datasets comparing transcriptome of gastric cancer with normal tissue, to focus on markers retained in cancer. Among the transcripts enriched upon CagA induction in vitro, a significant elevation of CEACAM6 was noted in gene expression datasets of gastric cancer. We used quantitative digital immunohistochemistry to measure CEACAM6 protein levels in tissue microarrays of gastric cancer. We demonstrate an increase in CEACAM6 in early gastric cancers, when compared to matched normal tissue, with an AUC of 0.83 for diagnostic validity. Finally, we show that a fluorescently conjugated CEACAM6 antibody binds avidly to freshly resected gastric cancer xenograft samples and can be detected by endoscopy in real time. Together, these results suggest that CEACAM6 upregulation is a cell surface response to H. pylori CagA, and is retained in early gastric cancers. They highlight a novel link between CEACAM6 expression and CagA in gastric cancer, and suggest CEACAM6 to be a promising biomarker to aid with the fluorescent endoscopic diagnosis of early neoplastic lesions in the stomach.
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Antígenos Bacterianos/fisiología , Antígenos CD/análisis , Proteínas Bacterianas/fisiología , Biomarcadores de Tumor/análisis , Moléculas de Adhesión Celular/análisis , Neoplasias Gástricas/diagnóstico , Animales , Técnica del Anticuerpo Fluorescente , Proteínas Ligadas a GPI/análisis , Infecciones por Helicobacter/metabolismo , Humanos , Ratones , Regulación hacia ArribaRESUMEN
Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis.
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Proteínas Portadoras/metabolismo , Movimiento Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Familia-src Quinasas/metabolismo , Secuencias de Aminoácidos , Animales , Biocatálisis , Proteína Tirosina Quinasa CSK , Proteínas Portadoras/química , Forma de la Célula , Células Cultivadas , Activación Enzimática , Retroalimentación Fisiológica , Adhesiones Focales , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Invasividad Neoplásica , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Especificidad por SustratoRESUMEN
More than 50% of people in the world are infected with Helicobacter pylori (H. pylori), which induces various gastric diseases. Especially, epidemiological studies have shown that H. pylori infection is a major risk factor for gastric cancer. It has been reported that the levels of interleukin (IL)-1ß are upregulated in gastric tissues of patients with H. pylori infection. In this study, we investigated the induction mechanism of IL-1ß during H. pylori infection. We found that IL-1ßmRNA and protein were induced in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells after H. pylori infection. This IL-1ß production was inhibited by a caspase-1 inhibitor and a ROS inhibitor. Furthermore, K(+) efflux and Ca(2+) signaling were also involved in this process. These data suggest that NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) and its complex, known as NLRP3 inflammasome, are involved in IL-1ß production during H. pylori infection because it is reported that NLRP3 inflammasome is activated by ROS, K(+) efflux and/or Ca(2+) signaling. These findings may provide therapeutic strategy for the control of gastric cancer in H. pylori-infected patients.
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Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Adenosina Trifosfato/metabolismo , Señalización del Calcio , Caspasa 1/metabolismo , Línea Celular , Espacio Extracelular/metabolismo , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Humanos , Interleucina-1beta/genética , Espacio Intracelular/metabolismo , Macrófagos/inmunología , Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
CagA, encoded by cytotoxin-associated gene A (cagA), is a major virulence factor of Helicobacter pylori, a gastric pathogen involved in the development of upper gastrointestinal diseases. Infection with cagA-positive H. pylori may also be associated with diseases outside the stomach, although the mechanisms through which H. pylori infection promotes extragastric diseases remain unknown. Here, we report that CagA is present in serum-derived extracellular vesicles, known as exosomes, in patients infected with cagA-positive H. pylori (n = 4). We also found that gastric epithelial cells inducibly expressing CagA secrete exosomes containing CagA. Addition of purified CagA-containing exosomes to gastric epithelial cells induced an elongated cell shape, indicating that the exosomes deliver functional CagA into cells. These findings indicated that exosomes secreted from CagA-expressing gastric epithelial cells may enter into circulation, delivering CagA to distant organs and tissues. Thus, CagA-containing exosomes may be involved in the development of extragastric disorders associated with cagA-positive H. pylori infection.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Exosomas/metabolismo , Helicobacter pylori/fisiología , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Transporte Biológico , Biomarcadores , Línea Celular , Cromatografía Liquida , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Humanos , Transporte de Proteínas , Neoplasias Gástricas/etiología , Espectrometría de Masas en Tándem , Factores de VirulenciaRESUMEN
Helicobacter pylori cagA-positive strain delivers the CagA oncoprotein into gastric epithelial cells and at the same time elicits stomach inflammation. To experimentally investigate the pathophysiological interplay between CagA and inflammation, transgenic mice systemically expressing the bacterial cagA gene were treated with a colitis inducer, dextran sulfate sodium (DSS). Compared with control mice, DSS-induced colitis was markedly deteriorated in cagA-transgenic mice. In the colonic epithelia of cagA-transgenic mice, there was a substantial decrease in the level of IκB, which binds and sequesters NF-κB in the cytoplasm. This IκB reduction was due to CagA-mediated inhibition of PAR1, which may stimulate IκB degradation by perturbing microtubule stability. Whereas the CagA-mediated IκB reduction did not automatically activate NF-κB, it lowered the threshold of NF-κB activation by inflammogenic insults, thereby contributing to colitis exacerbation in cagA-transgenic mice. CagA also activates inflammasomes independently of NF-κB signaling, which further potentiates inflammation. The incidence of colonic dysplasia was elevated in DSS-treated cagA-transgenic mice due to a robust increase in the number of pre-cancerous flat-type dysplasias. Thus, CagA deteriorated inflammation, whereas inflammation strengthened the oncogenic potential of CagA. This work revealed that H. pylori CagA and inflammation reinforce each other in creating a downward spiral that instigates neoplastic transformation.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Carcinogénesis/inmunología , Colitis/inmunología , Neoplasias Gastrointestinales/inmunología , Inflamasomas/inmunología , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Carcinogénesis/efectos de los fármacos , Colitis/inducido químicamente , Sulfato de Dextran , Femenino , Neoplasias Gastrointestinales/inducido químicamente , Masculino , Ratones , Ratones TransgénicosRESUMEN
Helicobacter pylori strains carrying the cagA gene are associated with severe disease outcomes, most notably gastric cancer. CagA protein is delivered into gastric epithelial cells by a type IV secretion system. The translocated CagA undergoes tyrosine phosphorylation at the C-terminal EPIYA motifs by host cell kinases. Tyrosine-phosphorylated CagA acquires the ability to interact with and activate SHP2, thereby activating mitogenic signaling and inducing cell morphological transformation (hummingbird phenotype). CagA also interacts with PAR1b via the CM sequence, resulting in induction of junctional and polarity defects. Furthermore, CagA-PAR1b interaction stabilizes the CagA-SHP2 complex. Because transgenic mice systemically expressing CagA develop gastrointestinal and hematological malignancies, CagA is recognized as a bacterium-derived oncoprotein. Interestingly, the C-terminal region of CagA displays a large diversity among H. pylori strains, which influences the ability of CagA to bind to SHP2 and PAR1b. In the present study, we investigated the biological activity of v225d CagA, an Amerindian CagA of H. pylori isolated from a Venezuelan Piaroa Amerindian subject, because the variant CagA does not possess a canonical CM sequence. We found that v225d CagA interacts with SHP2 but not PAR1b. Furthermore, SHP2-binding activity of v225d CagA was much lower than that of CagA of H. pylori isolated from Western countries (Western CagA). v225d CagA also displayed a reduced ability to induce the hummingbird phenotype than that of Western CagA. Given that perturbation of PAR1b and SHP2 by CagA underlies the oncogenic potential of CagA, the v225d strain is considered to be less oncogenic than other well-studied cagA-positive H. pylori strains.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Células COS , Chlorocebus aethiops , Perros , Células Epiteliales/microbiología , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Humanos , Células de Riñón Canino Madin Darby , Datos de Secuencia Molecular , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismoRESUMEN
Escherichia coli Orf135 hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-oxo-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP. Nucleotide pool sanitization by Orf135 is important since nucleotides are continually subjected to potential damage by reactive oxygen species produced during respiration. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, even though they recognize various substrates and possess a variety of substrate binding pockets. We investigated the tertiary structure of Orf135 and its interaction with a 2-hydroxy-dATP analog using NMR. We report on the solution structure of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrates.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Escherichia coli/enzimología , Pirofosfatasas/química , Adenosina Trifosfato/química , Secuencias de Aminoácidos , Sitios de Unión , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Especificidad por SustratoRESUMEN
BACKGROUND: Most of what is known about the Helicobacter pylori (H. pylori) cytotoxin, CagA, pertains to a much-vaunted role as a determinant of gastric inflammation and cancer. Little attention has been devoted to potential roles of CagA in the majority of H. pylori infected individuals not showing oncogenic progression, particularly in relation to host tolerance. Regenerating islet-derived (REG)3γ encodes a secreted C-type lectin that exerts direct bactericidal activity against Gram-positive bacteria in the intestine. Here, we extend this paradigm of lectin-mediated innate immunity, showing that REG3γ expression is triggered by CagA in the H. pylori-infected stomach. METHODOLOGY/PRINCIPAL FINDINGS: In human gastric mucosal tissues, REG3γ expression was significantly increased in CagA-positive, compared to CagA-negative H. pylori infected individuals. Using transfected CagA-inducible gastric MKN28 cells, we recapitulated REG3γ induction in vitro, also showing that tyrosine phosphorylated, not unphosphorylated CagA triggers REG3γ transcription. In concert with induced REG3γ, pro-inflammatory signalling downstream of the gp130 cytokine co-receptor via the signal transducer and activator of transcription (STAT)3 and transcription of two cognate ligands, interleukin(IL)-11 and IL-6, were significantly increased. Exogenous IL-11, but not IL-6, directly stimulated STAT3 activation and REG3γ transcription. STAT3 siRNA knockdown or IL-11 receptor blockade respectively abrogated or subdued CagA-dependent REG3γ mRNA induction, thus demonstrating a requirement for uncompromised signalling via the IL-11/STAT3 pathway. Inhibition of the gp130-related SHP2-(Ras)-ERK pathway did not affect CagA-dependent REG3γ induction, but strengthened STAT3 activation as well as augmenting transcription of mucosal innate immune regulators, IL-6, IL-8 and interferon-response factor (IRF)1. CONCLUSIONS/SIGNIFICANCE: Our results support a model of CagA-directed REG3γ expression in gastric epithelial cells via activation of the IL-11/gp130/STAT3 pathway. This response might allow Gram-negative H. pylori to manipulate host immunity to favour its own survival, by reducing the fitness of co-habiting Gram-positive bacteria with which it competes for resources in the gastric mucosal niche.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Regulación de la Expresión Génica , Helicobacter pylori/fisiología , Proteínas/genética , Factor de Transcripción STAT3/metabolismo , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Línea Celular , Receptor gp130 de Citocinas/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Mucosa Gástrica/citología , Mucosa Gástrica/inmunología , Helicobacter pylori/metabolismo , Humanos , Inmunidad Innata , Interleucina-11/metabolismo , Quinasas Janus/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Asociadas a Pancreatitis , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Activación Transcripcional , Tirosina/metabolismoRESUMEN
Helicobacter pylori CagA is delivered into gastric epithelial cells, where undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif to interact with Src homology 2-containing protein tyrosine phosphatase-2 (SHP2) oncoprotein. CagA also binds to partitioning-defective 1 (PAR1) polarity-regulating kinase via the CagA multimerization (CM) sequence. To investigate pathophysiological role of CagA-SHP2 and/or CagA-PAR1 interaction in H. pylori infection, we generated H. pylori isogenic strains producing a phosphorylation-resistant CagA and a CagA without CM sequence. Infection studies revealed that deregulation of epithelial cell motility was more prominent in the wild-type strain than in the mutant strains. Thus, both CagA-SHP2 and CagA-PAR1 interactions are involved in the pathogenicity of cagA-positive H. pylori.