RESUMEN
AIMS: Somatic mutations in genes encoding chromatin remodellers have been reported recently in several cancer types, including approximately half of cholangiocarcinomas. One of the most commonly mutated chromatin remodellers in cholangiocarcinoma is the Polybromo-1 (PBRM1) gene located on chromosome 3p21, which encodes a subunit of the SWI/SNF complex. The aim of this study was to determine the timing of PBRM1 mutations in biliary carcinogenesis. METHODS AND RESULTS: In order to accomplish this goal, we used immunohistochemistry to assess PBRM1 protein expression in a series of precursor lesions and invasive biliary carcinomas. Previous studies have correlated loss of protein expression on immunohistochemistry with inactivating mutations in this tumour suppressor gene. We found that PBRM1 loss occurred in approximately 26% of invasive cancers, but PBRM1 expression was retained in all biliary intra-epithelial neoplasia (BilIN) specimens, including 25 intrahepatic BilINs and 19 gallbladder BilINs. CONCLUSIONS: These findings indicate that PBRM1 mutation (and resultant loss of expression) is a late event during biliary carcinogenesis. In addition, we confirm a lack of prognostic significance of PBRM1 status in invasive intrahepatic cholangiocarcinoma. This study provides important insights into the basic mechanisms of chromatin remodelling genes in carcinogenesis.
Asunto(s)
Neoplasias de los Conductos Biliares/genética , Transformación Celular Neoplásica/genética , Colangiocarcinoma/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Neoplasias de los Conductos Biliares/mortalidad , Colangiocarcinoma/mortalidad , Proteínas de Unión al ADN , Humanos , Estimación de Kaplan-Meier , Mutación , Pronóstico , Modelos de Riesgos ProporcionalesAsunto(s)
Brotes de Enfermedades , Microbiología Ambiental , Vivienda para Animales , Infecciones por Salmonella/epidemiología , Salmonella enterica/aislamiento & purificación , Animales , Humanos , Michigan/epidemiología , Servicios Postales , Aves de Corral , Infecciones por Salmonella/microbiología , Salmonella enterica/genética , Serogrupo , Estados Unidos/epidemiologíaRESUMEN
Centers for Disease Control and Prevention (CDC), health departments, and other state and federal partners have linked contact with live poultry to 70 human Salmonella outbreaks in the United States from 2000 to 2017, which resulted in a total of 4,794 illnesses, 894 hospitalizations, and 7 deaths. During human salmonellosis outbreaks environmental sampling is rarely conducted as part of the outbreak investigation. CDC was contacted by state health officials on June 12, 2018, to provide support during an investigation of risk factors for Salmonella infections linked to live poultry originating at a mail-order hatchery. From January 1, 2018, to June 15, 2018, 13 human Salmonella infections in multiple states were attributed to exposure to live poultry from a single hatchery. Two serotypes of Salmonella were associated with these infections, Salmonella Enteritidis and Salmonella Litchfield. Molecular subtyping of the S. Enteritidis clinical isolates revealed they were closely related genetically (within 0 to 9 alleles) by core genome multi-locus sequence typing (cgMLST) to isolates obtained from environmental samples taken from hatchery shipping containers received at retail outlets. Environmental sampling and onsite investigation of practices was conducted at the mail-order hatchery during an investigation on June 19, 2018. A total of 45 environmental samples were collected, and 4 (9%) grew Salmonella. A chick box liner from a box in the pre-shipping area yielded an isolate closely related to the S. Enteritidis outbreak strain (within 1 to 9 alleles by cgMLST). The onsite investigation revealed lapses in biosecurity, sanitation, quality assurance, and education of consumers. Review of Salmonella serotype testing performed by the hatchery revealed that the number of samples and type of samples collected monthly varied. Also, S. Enteritidis was identified at the hatchery every year since testing began in 2016. Recommendations to the hatchery for biosecurity, testing, and sanitation measures were made to help reduce burden of Salmonella in the hatchery and breeding flocks, thereby reducing the occurrence of human illness.
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Brotes de Enfermedades , Enfermedades de las Aves de Corral/microbiología , Salmonelosis Animal/microbiología , Infecciones por Salmonella/microbiología , Salmonella/aislamiento & purificación , Adolescente , Adulto , Anciano , Crianza de Animales Domésticos , Animales , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Vigilancia de la Población , Aves de Corral , Salmonella/clasificación , Infecciones por Salmonella/epidemiología , Salmonelosis Animal/epidemiología , Transportes , Estados Unidos/epidemiología , Adulto JovenRESUMEN
The adipose tissue-derived hormone, leptin, acts via its receptor (LepRb) in the brain to regulate energy balance and neuroendocrine function. Parsing the biology of leptin requires understanding LepRb signaling and the roles for specific signaling pathways in neural and physiological leptin action. Since the leptin acts via a broadly distributed network of LepRb-expressing neurons, understanding the function of each of these LepRb neural populations will also be crucial. Here, we review the status of knowledge regarding the molecular mediators of leptin action and the neural substrate via which leptin acts to regulate physiologic processes.
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Hipotálamo/fisiología , Leptina/fisiología , Neuronas/fisiología , Receptores de Leptina/fisiología , Transducción de Señal/fisiología , Animales , Glucemia/metabolismo , Humanos , Hipotálamo/citología , Janus Quinasa 2/metabolismoRESUMEN
Semiquantitative immunohistochemistry (IHC) is commonly used in combination with fluorescence in situ hybridization (FISH) to detect HER2 amplification in gastroesophageal adenocarcinomas. Most laboratories apply these tests in a sequential algorithm, using IHC as a frontline test and reserving FISH for IHC-equivocal cases. To gain a better understanding of the concordance of IHC and FISH results at our institution, we identified all gastroesophageal adenocarcinomas at our institution tested for HER2 (n=125). Matched IHC and FISH were available for 116 cases (94%). Cases consisted of adenocarcinoma of the distal esophagus (22%), gastroesophageal junction (24%), stomach (43%), and metastatic sites (12%). A total of 88 cases (70%) were biopsies, whereas 37 cases (30%) were resections. Overall, 15 cases (13%) were HER2 positive (IHC 3+ and/or FISH amplified). A total of 60 cases (52%) were IHC score 0; none of these were HER2 amplified by FISH. A total of 30 cases (26%) were IHC 1+; 5 (17%) of these cases were HER2 amplified by FISH. A total of 20 cases (17%) were IHC 2+; 4 (20%) of these cases were HER2 amplified by FISH. A total of 6 cases were IHC score 3+; all of these were HER2 amplified by FISH. Although there was a high overall concordance between IHC and FISH results (96%), a subset (17%) of IHC-negative cases (score 1+) were HER2 amplified as evaluated by FISH, representing 33% of all HER2 amplified cases. This suggests that the common practice of limited FISH testing to IHC 2+ cases will miss a significant number of HER2 amplified cases.
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Adenocarcinoma/diagnóstico , Inmunohistoquímica/normas , Hibridación Fluorescente in Situ/normas , Receptor ErbB-2/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Errores Diagnósticos , Unión Esofagogástrica/patología , Femenino , Amplificación de Genes , Humanos , Receptor ErbB-2/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The Ink4a/Arf locus is functionally linked to the Rb and p53 pathways through the action of its two gene products. Mouse models null for this locus show rapid onset of cancer with a preponderance of lymphomas and sarcomas. We report on a study of cell lines derived from sarcomas arising in Ink4a/Arf null mice. The cytogenetics of these lines was monitored over the course of serial passage. Results indicate that early passage cells are relatively normal. However, after multiple passages chromosomal instability becomes apparent as evidenced by increasing tetraploidy and aneuploidy, and the concomitant loss of clonality. To further evaluate the effect of Ink4a/Arf-deficiency on chromosomal stability in vitro, we isolated Ink4a/Arf deficient primary murine embryonic fibroblasts (MEFs), serially passaged them, and analyzed their chromosomal stability by spectral karyotyping (a 24-color chromosome paint-FISH technique). We found that chromosomal instability in Ink4a/Arf deficient MEFs developed with the same timing as seen in cell lines derived from Ink4a/Arf deficient sarcomas. Thus, chromosomal instability seen in Ink4a/Arf deficient tumors in vitro may be unrelated to the original phenotype of the tumor in vivo. Therefore, interpretation of cytogenetic data from cell lines derived from Ink4a/Arf deficient tumors should be done on early passage cells.
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Inestabilidad Cromosómica , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Sarcoma/genética , Animales , Fibroblastos , Cariotipificación , Ratones , Fenotipo , Reproducibilidad de los Resultados , Sarcoma/patología , Células Tumorales CultivadasRESUMEN
Autoimmune metaplastic atrophic gastritis (AMAG) is a significant risk factor for pernicious anemia and gastric neoplasia. Still, the histologic features of AMAG are frequently overlooked, especially in the early stages of the disease. The purpose of our study, therefore, was to catalogue the progression of histologic changes that precede the development of AMAG in affected individuals. Over a 2-year period (2012 to 2014), the diagnosis of AMAG was rendered on material from 113 patients seen at Johns Hopkins Hospital (â¼1.8% of "in house" gastric biopsies). Prior gastric body biopsies had been performed on 54 (48%) patients in the cohort, and the majority of these specimens had also shown AMAG. Eighteen of the previous biopsies, however, carried a diagnosis other than AMAG: 13 inactive chronic gastritis, 2 acute Helicobacter pylori gastritis, and 1 each of eosinophilic gastritis, iron pill gastritis, and proton-pump inhibitor-like effect. Upon review of these 18 biopsies, the most common histologic findings were heavy full-thickness or deep lamina propria chronic inflammation (12), inflammatory destruction of oxyntic glands (12), metaplasia (intestinal, pyloric, or pancreatic acinar) (10), prominent lamina propria eosinophils (8), and parietal cell pseudohypertrophy (4). At least 2 of these features were present in the majority (13, 72%) of the biopsies. In addition, 7 (58%) of these patients were also found to have another autoimmune or inflammatory disorder before the diagnosis of AMAG. Although subtle, histologic features of developing AMAG are identifiable in routine gastric body biopsies. When metaplasia, full-thickness chronic inflammation, and/or oxyntic destruction are seen, a note suggesting laboratory testing and/or close clinical follow-up in this subset of patients may be warranted.
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Enfermedades Autoinmunes/patología , Gastritis Atrófica/patología , Estómago/patología , Adulto , Anciano , Anciano de 80 o más Años , Enfermedades Autoinmunes/metabolismo , Baltimore , Biomarcadores/análisis , Biopsia , Estudios de Casos y Controles , Errores Diagnósticos , Progresión de la Enfermedad , Registros Electrónicos de Salud , Femenino , Gastritis Atrófica/metabolismo , Humanos , Inmunohistoquímica , Masculino , Metaplasia , Persona de Mediana Edad , Células Parietales Gástricas/química , Células Parietales Gástricas/patología , Valor Predictivo de las Pruebas , Pronóstico , Estómago/química , Factores de TiempoRESUMEN
Tactile corpuscle-like bodies (TCLB) are microscopic Schwannian structures that simulate the superficial mechanoreceptors of the peripheral nervous system (Wagner-Meissner corpuscles). They have been described nearly exclusively in peripheral nerve sheath tumors, namely diffuse neurofibromas, and schwannomas but also in cellular nevi. There are rare reports of these structures in the gastrointestinal tract (predominantly the lower tract), with the presumption that they are incidental reactive neural proliferations. We compiled 9 cases showing this rare phenomenon in gastrointestinal-type mucosa in nonsyndromic patients to further characterize its features. There were 6 men and 3 women (age range, 39 to 79 y, mean 56 y) with lesions involving esophagus/gastro-esophageal junction (n=7), sigmoid colon (n=1), and gastric heterotopia of the cricopharynx (n=1). Endoscopic examination was abnormal in 6 of the 7 cases (including changes consistent with Barrett esophagus and polypoid/nodular mucosa) and normal in 1 of 7 cases for which this information was available. The histologic features were similar in all cases, with unencapsulated clusters of lamellated and concentrically arranged spindle cells in the lamina propria. The foci of TCLB ranged in size from <0.1 to 1.5 mm in the greatest dimension. Abnormal histopathologic findings were identified in the background mucosa in 6 of 9 cases (including Barrett esophagus, active and inactive chronic gastritis, enterochromaffin-like cell hyperplasia, and gastric intestinal metaplasia). None of the patients showed signs of neurofibromatosis type 1, multiple endocrine neoplasia type 2B, Cowden syndrome, or other inherited syndrome. No morbidity related to TCLB was reported for the patients with available follow-up.
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Tracto Gastrointestinal/patología , Mecanorreceptores/patología , Células de Schwann/patología , Adulto , Anciano , Biomarcadores/análisis , Biopsia , Linaje de la Célula , Diagnóstico Diferencial , Endoscopía Gastrointestinal , Femenino , Tracto Gastrointestinal/química , Humanos , Inmunohistoquímica , Hallazgos Incidentales , Masculino , Mecanorreceptores/química , Persona de Mediana Edad , Membrana Mucosa/química , Membrana Mucosa/patología , Valor Predictivo de las Pruebas , Regeneración , Células de Schwann/química , Estados UnidosRESUMEN
Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry analysis of JAK2 activated by an erythropoietin-bound chimeric erythropoietin receptor/leptin receptor. Phosphospecific antibodies suggest that the phosphorylation of all three tyrosines increases in response to GH. Compared with wild-type JAK2, which is constitutively active when overexpressed, JAK2 lacking tyrosine 868, 966, or 972 has substantially reduced activity. Coexpression with GH receptor and protein tyrosine phosphatase1B allowed us to investigate GH-dependent activation of these mutated JAK2s in human embryonic kidney 293T cells. All three mutated JAK2s are activated by GH, although to a lesser extent than wild-type JAK2. The three mutated JAK2s also mediate GH activation of signal transducer and activator of transcription 3 (Stat3), signal transducer and activator of transcription 5b (Stat5b) and ERK1, but at reduced levels. Coexpression with Src-homology 2B1beta (SH2B1beta), like coexpression with GH-bound GH receptor, partially restores the activity of all three JAK2 mutants. Based on these results and the crystal structure of the JAK2 kinase domain, we hypothesize that small changes in the conformation of the regions of JAK2 surrounding tyrosines 868, 966, and 972 due to e.g. phosphorylation, binding to a ligand-bound cytokine receptor, and/or binding to Src-homology 2B1, may be essential for JAK2 to assume a maximally active conformation.
Asunto(s)
Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Tirosina/metabolismo , Animales , Western Blotting , Línea Celular , Hormona del Crecimiento/metabolismo , Humanos , Inmunoprecipitación , Janus Quinasa 2/genética , Ratones , Mutagénesis Sitio-Dirigida , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica/genética , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Ratas , Transducción de Señal/genética , Transducción de Señal/fisiología , Relación Estructura-Actividad , Espectrometría de Masas en Tándem , Tirosina/químicaRESUMEN
Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr(317) in the FERM domain and Tyr(637) in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr(317) promotes increased Jak2 activity, and the phosphorylation of Tyr(317) during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr(317) in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr(317) in the attenuation of Jak2 signaling. In contrast, mutation of Tyr(637) decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr(637) phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr(317) and Tyr(637) act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.
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Citocinas/metabolismo , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Retroalimentación Fisiológica , Humanos , Janus Quinasa 2/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Espectrometría de Masas en Tándem , Tirosina/químicaRESUMEN
Perturbations in the functional integrity of the leptin axis are obvious candidates for mediation of altered adiposity. In a large number of genetic association studies in humans, the nonconservative LEPR Q223R allele has been inconsistently associated with adiposity. Subtle, long-term effects of such genetic variants can be obscured by effects of the environment and other confounders that render definitive inferences difficult to reach. We directly assessed the biological effects of this variant in 129P3/J mice segregating for the humanized Lepr allele at codon 223. No effects of this allele were detected on body weight, composition, or energy expenditure in animals fed diets of varying fat content over periods as long as 235 days. In vitro, Q223R did not affect leptin signaling as reflected by activation of STAT3. We conclude that Q223R is unlikely to play a significant role in regulation of human adiposity. This approach to vetting of human allelic variation might be more widely used.
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Tejido Adiposo/fisiología , Composición Corporal/genética , Obesidad/genética , Receptores de Leptina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Cartilla de ADN , Dieta con Restricción de Grasas , Células Madre Embrionarias/fisiología , Exones , Femenino , Regulación de la Expresión Génica , Variación Genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido SimpleRESUMEN
An XWnt8-Fz5 fusion protein synergizes with LRP6 to potently activate beta-catenin-dependent signaling. Here, we generated a fusion in which XWnt8 was fused to the N-terminus of LRP6 and show it synergizes with both Fz4 and Fz5 to potently transactivate beta-catenin-dependent Wnt signaling. Based on this, we hypothesized that the main function of Wnt is to nucleate the formation of a physical complex between LRP6 and a Frizzled. Dkk1, but not the related Dkk3, binds LRP6 and inhibits canonical Wnt signaling by blocking the interaction of Wnt and LRP6. Therefore, we reasoned that a covalent fusion of Dkk1 to Fz5 (Dkk1-Fz5) would mimic Wnt ligand by nucleating the formation of a complex containing Fz5 and LRP6, while Dkk3 (Dkk3-Fz5) would not. We found that Dkk1-Fz5, but not Dkk3-Fz5, potently synergized with LRP6 to activate signaling in a dishevelled-dependent manner.