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1.
Nature ; 592(7854): 450-456, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33762733

RESUMEN

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.


Asunto(s)
Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Inmunoterapia , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/terapia , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/inmunología , Animales , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinogénesis/inmunología , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/inmunología , Progresión de la Enfermedad , Humanos , Hígado/inmunología , Hígado/patología , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/patología , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Factor de Necrosis Tumoral alfa/inmunología
2.
J Lipid Res ; 60(7): 1270-1283, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31113816

RESUMEN

Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2-/- mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.


Asunto(s)
Hígado/metabolismo , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Oxiesteroles/metabolismo , Adulto , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Colesterol/sangre , Cromatografía Liquida , Citometría de Flujo , Humanos , Hidroxicolesteroles/sangre , Hidroxicolesteroles/metabolismo , Masculino , Espectrometría de Masas , Ratones , Ratones Noqueados , Persona de Mediana Edad , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Enfermedad del Hígado Graso no Alcohólico/sangre , Oxiesteroles/sangre , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo
3.
J Hepatol ; 70(4): 700-709, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30553841

RESUMEN

BACKGROUND & AIMS: In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma. METHODS: Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo. RESULTS: In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRß inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases. CONCLUSION: PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis. LAY SUMMARY: Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.


Asunto(s)
Neoplasias de los Conductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Hígado/patología , Linfangiogénesis/efectos de los fármacos , Linfocinas/metabolismo , Linfocinas/farmacología , Miofibroblastos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Neoplasias de los Conductos Biliares/patología , Fibroblastos Asociados al Cáncer/metabolismo , Línea Celular Tumoral , Colangiocarcinoma/patología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Xenoinjertos , Humanos , Mesilato de Imatinib/farmacología , Masculino , Ratones , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Endogámicas F344 , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor C de Crecimiento Endotelial Vascular/biosíntesis
4.
Hepatology ; 67(1): 247-259, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-28802066

RESUMEN

Cholangiocyte senescence has been linked to primary sclerosing cholangitis (PSC). Persistent secretion of growth factors by senescent cholangiocytes leads to the activation of stromal fibroblasts (ASFs), which are drivers of fibrosis. The activated phenotype of ASFs is characterized by an increased sensitivity to apoptotic stimuli. Here, we examined the mechanisms of apoptotic priming in ASFs and explored a combined targeting strategy to deplete senescent cholangiocytes and ASFs from fibrotic tissue to ameliorate liver fibrosis. Using a coculture system, we determined that senescent cholangiocytes promoted quiescent mesenchymal cell activation in a platelet-derived growth factor (PDGF)-dependent manner. We also identified B-cell lymphoma-extra large (Bcl-xL) as a key survival factor in PDGF-activated human and mouse fibroblasts. Bcl-xL was also up-regulated in senescent cholangiocytes. In vitro, inhibition of Bcl-xL by the small molecule Bcl-2 homology domain 3 mimetic, A-1331852, or Bcl-xL-specific small interfering RNA induced apoptosis in PDGF-activated fibroblasts, but not in quiescent fibroblasts. Likewise, inhibition of Bcl-xL reduced the survival and increased apoptosis of senescent cholangiocytes, compared to nonsenescent cells. Treatment of multidrug resistance 2 gene knockout (Mdr2-/- ) mice with A-1331852 resulted in an 80% decrease in senescent cholangiocytes, a reduction of fibrosis-inducing growth factors and cytokines, decrease of α-smooth muscle actin-positive ASFs, and finally in a significant reduction of liver fibrosis. CONCLUSION: Bcl-xL is a key survival factor in ASFs as well as in senescent cholangiocytes. Treatment with the Bcl-xL-specific inhibitor, A-1331852, reduces liver fibrosis, possibly by a dual effect on activated fibroblasts and senescent cholangiocytes. This mechanism represents an attractive therapeutic strategy in biliary fibrosis. (Hepatology 2018;67:247-259).


Asunto(s)
Benzotiazoles/farmacología , Conductos Biliares/citología , Colangitis Esclerosante/patología , Fibroblastos/efectos de los fármacos , Isoquinolinas/farmacología , Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Animales , Biopsia con Aguja , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Colangitis Esclerosante/tratamiento farmacológico , Modelos Animales de Enfermedad , Resistencia a Múltiples Medicamentos , Fibroblastos/metabolismo , Fibroblastos/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Terapia Molecular Dirigida , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Distribución Aleatoria , Valores de Referencia
5.
Liver Int ; 39 Suppl 1: 63-78, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30907492

RESUMEN

Tumour microenvironment is a complex, multicellular functional compartment that, particularly when assembled as an abundant desmoplastic reaction, may profoundly affect the proliferative and invasive abilities of epithelial cancer cells. Tumour microenvironment comprises not only stromal cells, mainly cancer-associated fibroblasts, but also immune cells of both the innate and adaptive system (tumour-associated macrophages, neutrophils, natural killer cells, and T and B lymphocytes), and endothelial cells. This results in an intricate web of mutual communications regulated by an extensively remodelled extracellular matrix, where the tumour cells are centrally engaged. In this regard, cholangiocarcinoma, in particular the intrahepatic variant, has become the focus of mounting interest in the last years, largely because of the lack of effective therapies despite its rising incidence and high mortality rates worldwide. On the other hand, recent studies in pancreatic cancer, which similarly to cholangiocarcinoma, is highly desmoplastic, have argued against a tumour-promoting function of the tumour microenvironment. In this review, we will discuss recent developments concerning the role of each cellular population and their multifaceted interplay with the malignant biliary epithelial counterpart. We ultimately hope to provide the working knowledge on how their manipulation may lead to a therapeutic gain in cholangiocarcinoma.


Asunto(s)
Neoplasias de los Conductos Biliares/inmunología , Colangiocarcinoma/inmunología , Células Endoteliales/inmunología , Fibroblastos/inmunología , Microambiente Tumoral/inmunología , Inmunidad Adaptativa , Animales , Neoplasias de los Conductos Biliares/fisiopatología , Colangiocarcinoma/fisiopatología , Modelos Animales de Enfermedad , Fibroblastos/patología , Humanos , Inmunidad Innata
6.
Liver Int ; 37(3): 369-376, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-27678216

RESUMEN

BACKGROUND: Novel direct antiviral agents (DAA) targeting hepatitis C virus (HCV) have revolutionized the treatment of chronic hepatitis C infection (CHC). Rates of sustained virological response (SVR) to treatment have drastically improved since introduction of DAA. Transient Elastography (TE) is an ultrasound based, non-invasive technique to assess liver stiffness (LS). We examined the changes in TE values and fibrosis scores FIB-4 and APRI after DAA treatment of CHC. METHODS: 549 patients who received a DAA based treatment for CHC were screened and 392 were included. TE values recorded prior to therapy and within 18 months after therapy were evaluated. In addition, FIB-4 and APRI scores were calculated and histopathological results were recorded if available. RESULTS: Median TE prior to DAA treatment was 12.65 kPa (IQR 9.45-19.2 kPa) and decreased to 8.55 kPa (IQR 5.93-15.25) post-treatment. This finding is statistically significant (P<.001) and equals a TE regression of 32.4% after DAA treatment. Median FIB-4 and APRI values significantly decreased from 2.54 (IQR 1.65-4.43) and 1.10 (IQR 0.65-2.43) to 1.80 (IQR 1.23-2.84, P<.001) and 0.43 (IQR 0.3-0.79, P<.001) respectively. CONCLUSION: Patients with SVR after DAA therapy showed significant regression of TE values. Rapid decrease in TE was in concordance with regression of validated fibrosis scores FIB-4 and APRI. It remains to be examined whether this indicates a true regression of fibrosis or merely resolution of chronic liver inflammation with subsequent improvement of TE values and laboratory parameters.


Asunto(s)
Antivirales/uso terapéutico , Aspartato Aminotransferasas/sangre , Plaquetas/citología , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Cirrosis Hepática/diagnóstico por imagen , Biomarcadores/sangre , Progresión de la Enfermedad , Quimioterapia Combinada , Diagnóstico por Imagen de Elasticidad , Femenino , Hepacivirus , Humanos , Hígado/diagnóstico por imagen , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Respuesta Virológica Sostenida , Suiza
7.
Apoptosis ; 20(9): 1211-28, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26201457

RESUMEN

Proteasome activator PA28γ (REGγ, Ki antigen) has recently been demonstrated to display anti-apoptotic properties via enhancing Mdm2-p53 interaction, thereby facilitating ubiquitination and down-regulation of the tumor suppressor p53. In this study we demonstrate a correlation between cellular PA28γ levels and the sensitivity of cells towards apoptosis in different cellular contexts thereby confirming a role of proteasome activator PA28γ as an anti-apoptotic regulator. We investigated the anti-apoptotic role of PA28γ upon UV-C stimulation in B8 mouse fibroblasts stably overexpressing the PA28γ-encoding PSME3 gene and upon butyrate-induced apoptosis in human HT29 adenocarcinoma cells with silenced PSME3 gene. Interestingly, our results demonstrate that PA28γ has a strong influence on different apoptotic hallmarks, especially p53 phosphorylation and caspase activation. In detail, PA28γ and effector caspases mutually restrict each other. PA28γ is a caspase substrate, if PA28γ levels are low. In contrast, PA28γ overexpression reduces caspase activities, including the caspase-dependent processing of PA28γ. Furthermore, overexpression of PA28γ resulted in a nuclear accumulation of transcriptional active p53. In summary, our findings indicate that even in a p53-dominated cellular context, pro-apoptotic signaling might be overcome by PA28γ-mediated caspase inhibition.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autoantígenos/metabolismo , Caspasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Apoptosis/efectos de la radiación , Butiratos/farmacología , Citocromos c/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de la radiación , Células HT29/efectos de la radiación , Humanos , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/metabolismo , Rayos Ultravioleta/efectos adversos
8.
BMC Musculoskelet Disord ; 15: 414, 2014 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-25482151

RESUMEN

BACKGROUND: PA28γ (also known as Ki, REG gamma, PMSE3), a member of the ubiquitin-and ATP-independent proteasome activator family 11S, has been proved to show proteasome-dependent and -independent effects on several proteins including tumor suppressor p53, cyclin-dependent kinase inhibitor p21 and steroid receptor co-activator 3 (SCR-3). Interestingly, PA28γ is overexpressed in pathological tissue of various cancers affecting e. g. breast, bowl and thyroids. Furthermore, anti-PA28γ autoantibodies have been linked to several autoimmune disorders. The aim of this study was to develop and evaluate a novel and sensitive PA28γ sandwich ELISA for the quantification of PA28γ serum levels in patients with cancer and autoimmune diseases for diagnostic and prognostic purposes. METHODS: PA28γ-specific polyclonal antibodies and recombinant His-tagged PA28γ were purified and used to develop a sandwich ELISA for the detection of circulating PA28γ. With this new assay, PA28γ serum levels of patients with various cancers, rheumatoid arthritis (RA), Sjögren's syndrome (SS), adult-onset Still's disease (AOSD) and different connective-tissue diseases (CTD) were compared with healthy control subjects. Anti-PA28γ autoantibodies were additionally confirmed using a newly developed microbead assay. RESULTS: The developed PA28γ sandwich ELISA showed a high specificity with a detection limit of 3 ng/ml. A significant up-regulation of circulating PA28γ was detected in the sera of patients with cancer, RA, SS and CTD. A significant correlation was observed dependent on age as well as anti-PA28γ autoantibody levels with circulating PA28γ protein levels. Furthermore, PA28γ serum levels showed a correlation with disease activity in patients with RA under treatment with the T-cell directed biological compound abatacept according to disease activity score 28 (DAS28) and erythrocyte sedimentation rate (ESR). CONCLUSION: The application of PA28γ as a novel biomarker for diagnostic purposes of a specific disease is limited, since elevated levels were observed in different disorders. However, the correlation with disease activity in patients with RA suggests a prognostic value, which needs to be addressed by further studies. Therefore our results show that PA28γ is a useful marker which should be included in studies related to novel treatments, e.g. abatacept.


Asunto(s)
Artritis Reumatoide/sangre , Artritis Reumatoide/diagnóstico , Autoantígenos/sangre , Complejo de la Endopetidasa Proteasomal/sangre , Autoantígenos/biosíntesis , Biomarcadores/sangre , Estudios de Seguimiento , Humanos , Complejo de la Endopetidasa Proteasomal/biosíntesis , Regulación hacia Arriba/fisiología
9.
Nat Rev Gastroenterol Hepatol ; 17(9): 557-588, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32606456

RESUMEN

Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted.


Asunto(s)
Neoplasias de los Conductos Biliares/diagnóstico , Neoplasias de los Conductos Biliares/terapia , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/terapia , Neoplasias de los Conductos Biliares/etiología , Colangiocarcinoma/etiología , Humanos
10.
Mucosal Immunol ; 12(3): 733-745, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30742043

RESUMEN

The gene encoding for Epstein-Barr virus-induced G-protein-coupled receptor 2 (EBI2) is a risk gene for inflammatory bowel disease (IBD). Together with its oxysterol ligand 7α,25-dihydroxycholesterol, EBI2 mediates migration and differentiation of immune cells. However, the role of EBI2 in the colonic immune system remains insufficiently studied. We found increased mRNA expression of EBI2 and oxysterol-synthesizing enzymes (CH25H, CYP7B1) in the inflamed colon of patients with ulcerative colitis and mice with acute or chronic dextran sulfate sodium (DSS) colitis. Accordingly, we detected elevated levels of 25-hydroxylated oxysterols, including 7α,25-dihydroxycholesterol in mice with acute colonic inflammation. Knockout of EBI2 or CH25H did not affect severity of DSS colitis; however, inflammation was decreased in male EBI2-/- mice in the IL-10 colitis model. The colonic immune system comprises mucosal lymphoid structures, which accumulate upon chronic inflammation in IL-10-deficient mice and in chronic DSS colitis. However, EBI2-/- mice formed significantly less colonic lymphoid structures at baseline and showed defects in inflammation-induced accumulation of lymphoid structures. In summary, we report induction of the EBI2-7α,25-dihydroxycholesterol axis in colitis and a role of EBI2 for the accumulation of lymphoid tissue during homeostasis and inflammation. These data implicate the EBI2-7α,25-dihydroxycholesterol axis in IBD pathogenesis.


Asunto(s)
Colitis/metabolismo , Colon/patología , Receptores Acoplados a Proteínas G/metabolismo , Estructuras Linfoides Terciarias/patología , Animales , Movimiento Celular , Células Cultivadas , Colitis/inducido químicamente , Colitis/inmunología , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxiesteroles/metabolismo , Receptores Acoplados a Proteínas G/genética , Factores Sexuales , Transducción de Señal
11.
Swiss Med Wkly ; 148: w14560, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29376557

RESUMEN

BACKGROUND: The introduction of direct acting antivirals (DAAs) for the therapy of chronic hepatitis C (CHC) has revolutionised treatment and marks a paradigm shift in the approach to this disease, rendering interferon-based therapies obsolete. AIMS OF THE STUDY: We retrospectively and prospectively evaluated treatment results after the introduction of DAA in Switzerland in a cohort of patients with CHC. METHODS: We examined 565 patients who received DAA treatment for CHC between November 2013 and June 2016 with regard to HCV genotype, fibrosis stadium, treatment and outcome. In addition, outcome of re-treatment and resistance-associated substitutions (RAS) in patients that did not achieve sustained virological response (SVR) were evaluated. The majority of patients participate in the Swiss Hepatitis C Cohort Study. Data were evaluated in an intention-to-treat and a modified intention-to-treat analysis. RESULTS: Overall SVR rate for all patients was 94% (530 of 565, 95% CI 92-96%). Of 350 patients with HCV genotype 1 CHC, 335 achieved SVR, resulting in an SVR rate of 96% (335 of 350, 95% CI 94-98%). Patients with HCV genotype 2 achieved SVR in 94% (48 of 51, 95% CI 87-100%). Patients with HCV genotype 3 showed SVR of 92% (98 of 107, 95% CI 87-97%). In patients with HCV genotype 4, the SVR rate was substantially lower at 85% (49 of 57, 95% CI 76-94%). The rate of advanced liver fibrosis (Metavir F3/F4) assessed by means of liver biopsy or Fibroscan® in the entire patient population was 71% (404 of 565). Out of 35 patients that did not achieve SVR after DAA treatment, 32 had a relapse and 3 patients showed viral breakthrough. In 17 of 35 cases (49%) patients were treatment naïve and 21 of 35 patients (60%) were cirrhotic. RAS genotyping of HCV was performed in 14 patients. Nine of these 14 patients (60%) carried mutations in the NS5A region of the virus genome. Twenty-seven percent of patients who experienced treatment failure were not treated with recommended regimens as a result of drug availability and reimbursement limitations. CONCLUSION: In Switzerland, novel DAA treatments for CHC reflect the positive results from registration trials. Genotypes 2 and 4 remained more difficult to treat between 2014 and 2016. Patients who experienced a relapse after DAA treatment in Switzerland predominantly showed mutations in the NS5A region of the virus genome. DAA treatment limitations in Switzerland did prevent optimal treatment regimens in some patients.


Asunto(s)
Antivirales/uso terapéutico , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Quimioterapia Combinada , Femenino , Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/genética , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Estudios Retrospectivos , Suiza , Resultado del Tratamiento
12.
Anal Chim Acta ; 908: 150-60, 2016 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-26826697

RESUMEN

Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.


Asunto(s)
Epítopos/química , Dispositivos Laboratorio en un Chip , Péptidos/química , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Datos de Secuencia Molecular , Oligonucleótidos
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