RESUMEN
In this work, we present DrugSolver CavitomiX, a novel computational pipeline for drug repurposing and identifying ligands and inhibitors of target enzymes. The pipeline is based on cavity point clouds representing physico-chemical properties of the cavity induced solely by the protein. To test the pipeline's ability to identify inhibitors, we chose enzymes essential for SARS-CoV-2 replication as a test system. The active-site cavities of the viral enzymes main protease (Mpro) and papain-like protease (Plpro), as well as of the human transmembrane serine protease 2 (TMPRSS2), were selected as target cavities. Using active-site point-cloud comparisons, it was possible to identify two compounds-flufenamic acid and fusidic acid-which show strong inhibition of viral replication. The complexes from which fusidic acid and flufenamic acid were derived would not have been identified using classical sequence- and structure-based methods as they show very little structural (TM-score: 0.1 and 0.09, respectively) and very low sequence (~ 5%) identity to Mpro and TMPRSS2, respectively. Furthermore, a cavity-based off-target screening was performed using acetylcholinesterase (AChE) as an example. Using cavity comparisons, the human carboxylesterase was successfully identified, which is a described off-target for AChE inhibitors.
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COVID-19 , Ácido Fusídico , Humanos , Ácido Fusídico/farmacología , Acetilcolinesterasa , Ácido Flufenámico/farmacología , SARS-CoV-2 , Péptido Hidrolasas , PapaínaRESUMEN
The current COVID19 pandemic is caused by a positive-sense single-stranded RNA virus, which presents high mutational rates. The development of effective therapeutics and mitigation strategies using vaccination or therapeutic antibodies faces serious challenges because of the regular emergence of immune escape variants of the virus. An efficient approach would involve the use of an agent to non-specifically limit or block viruses contacting the mucosae and therefore entering the body. Here, we investigated the ability of a micronized purified clinoptilolite-tuff to bind and neutralize different viruses from the Coronaviridae family. Using plaque assay, RT-qPCR and immunostaining, the adsorption and inactivation of the seasonal human coronavirus HCoV-229E and of 2 SARS-CoV-2 variants were demonstrated. The resulting data suggest that purified clinoptilolite-tuff could be used as an ingredient in new medical devices and/or pharmaceuticals to prevent or mitigate SARS-CoV-2 dissemination.
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COVID-19 , Coronaviridae , Humanos , SARS-CoV-2RESUMEN
Here, we report on the outcome of the 2nd International Danube Symposium on advanced biomarker development that was held in Vienna, Austria, in early 2018. During the meeting, cross-speciality participants assessed critical aspects of non-invasive, quantitative biomarker development in view of the need to expand our understanding of disease mechanisms and the definition of appropriate strategies both for molecular diagnostics and personalised therapies. More specifically, panelists addressed the main topics, including the current status of disease characterisation by means of non-invasive imaging, histopathology and liquid biopsies as well as strategies of gaining new understanding of disease formation, modulation and plasticity to large-scale molecular imaging as well as integrative multi-platform approaches. Highlights of the 2018 meeting included dedicated sessions on non-invasive disease characterisation, development of disease and therapeutic tailored biomarkers, standardisation and quality measures in biospecimens, new therapeutic approaches and socio-economic challenges of biomarker developments. The scientific programme was accompanied by a roundtable discussion on identification and implementation of sustainable strategies to address the educational needs in the rapidly evolving field of molecular diagnostics. The central theme that emanated from the 2nd Donau Symposium was the importance of the conceptualisation and implementation of a convergent approach towards a disease characterisation beyond lesion-counting "lumpology" for a cost-effective and patient-centric diagnosis, therapy planning, guidance and monitoring. This involves a judicious choice of diagnostic means, the adoption of clinical decision support systems and, above all, a new way of communication involving all stakeholders across modalities and specialities. Moreover, complex diseases require a comprehensive diagnosis by converging parameters from different disciplines, which will finally yield to a precise therapeutic guidance and outcome prediction. While it is attractive to focus on technical advances alone, it is important to develop a patient-centric approach, thus asking "What can we do with our expertise to help patients?"
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Biomarcadores/metabolismo , Congresos como Asunto/organización & administración , Imagen Molecular/métodos , Neoplasias/patología , Informe de Investigación , Austria , Biomarcadores/análisis , Humanos , Agencias Internacionales , Imagen Molecular/instrumentación , Imagen Molecular/tendencias , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Neoplasias/terapiaRESUMEN
Comparison of published biomedical studies shows that a large proportion are irreproducible, causing severe damage to society and creating an image of wasted investments. These observations are of course damaging to the biomedical research field, which is currently full of future promise. Precision medicine and disease prevention are successful, but are progressing slowly due to irreproducible study results. Although standardization is mentioned as a possible solution, it is not always clear how this could decrease or prevent irreproducible results in biomedical studies. In this article more insight is given into what quality, norms, standardization, certification, accreditation and optimized infrastructure can accomplish to reveal causes of irreproducibility and increase reproducibility when collecting biomaterials. CEN and ISO standards for the sample pre-analytical phase are currently being developed with the support of the SPIDIA4P project, and their role in increasing reproducibility in both biomedical research and diagnostics is demonstrated. In particular, it is described how standardized methods and quality assurance documentation can be exploited as tools for: 1) recognition and rejection of 'not fit for purpose' samples on the basis of detailed sample metadata, and 2) identification of methods that contribute to irreproducibility which can be adapted or replaced.
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Materiales Biocompatibles/análisis , Investigación Biomédica/normas , Fase Preanalítica/normas , Humanos , Reproducibilidad de los ResultadosRESUMEN
Hepatocellular carcinoma (HCC) develops as a consequence of chronic inflammatory liver diseases such as chronic hepatitis B virus (HBV) infection. The transcription factor c-Jun/activator protein 1 (AP-1) is strongly expressed in response to inflammatory stimuli, promotes hepatocyte survival during acute hepatitis and acts as an oncogene during chemically induced liver carcinogenesis in mice. Here, we therefore aimed to characterize the functions of c-Jun during HBV-related liver tumorigenesis. To this end, transgenic mice expressing all HBV envelope proteins (HBV(+)), an established model of HBV-related HCC, were crossed with knockout mice lacking c-Jun specifically in hepatocytes and tumorigenesis was analyzed. Hepatic expression of c-Jun was strongly induced at several time points during tumorigenesis in HBV(+) mice, whereas expression of other AP-1 components remained unchanged. Importantly, formation of premalignant foci and tumors was strongly reduced in HBV(+) mice lacking c-Jun. This phenotype correlated with impaired hepatocyte proliferation and increased expression of the cell cycle inhibitor p21, whereas hepatocyte survival was not affected. Progression and prognosis of HBV-related HCC correlates with the expression of the cytokine osteopontin (Opn), an established AP-1 target gene. Opn expression was strongly reduced in HBV(+) livers and primary mouse hepatocytes lacking c-Jun, demonstrating that c-Jun regulates hepatic Opn expression in a cell-autonomous manner. These findings indicate that c-Jun has important functions during HBV-associated tumorigenesis by promoting hepatocyte proliferation as well as progression of dysplasia. Therefore, targeting c-Jun may be a useful strategy to prevent hepatitis-associated tumorigenesis.
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Carcinoma Hepatocelular/metabolismo , Transformación Celular Viral , Virus de la Hepatitis B/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Proliferación Celular/genética , Virus de la Hepatitis B/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Hígado/patología , Hígado/virología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción AP-1/genéticaRESUMEN
Biobanks containing human biological samples and associated data are key resources for the advancement of medical research. Efficient access to samples and data increases competitiveness in medical research, reduces effort and time for achieving scientific results and promotes scientific progress. In order to address upcoming health challenges, there is increasing need for transnational collaboration. This requires innovative solutions improving interoperability of biobanks in fields such as sample and data management as well as governance including ethical and legal frameworks. In this context, rights and expectations of donors to determine the usage of their biological material and data and to ensure their privacy have to be observed. We discuss the benefits of biobanks, the needs to support medical research and the societal demands and regulations, in particular, securing the rights of donors and present IT solutions that allow both to maintain the security of personal data and to increase the efficiency of access to data in biobanks. Disclosure filters are discussed as a strategy to combine European public expectations concerning informed consent with the requirements of biobank research.
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Bancos de Muestras Biológicas/legislación & jurisprudencia , Investigación Biomédica/legislación & jurisprudencia , Seguridad Computacional/legislación & jurisprudencia , Difusión de la Información/legislación & jurisprudencia , Almacenamiento y Recuperación de la Información/métodos , Privacidad/legislación & jurisprudencia , Bancos de Muestras Biológicas/ética , Investigación Biomédica/ética , HumanosRESUMEN
In recent years a number of new therapeutics has been developed that were not general toxins and inhibitors of cell division like classical chemotherapeutics, but were designed to target a specific pathway. A prerequisite for this development was the comprehensive characterization of molecular alterations occurring in human hepatocellular carcinoma (HCC). However, while much knowledge of the molecular pathogenesis of human HCC has been gained, the model systems used to test the functional relevance of these alterations and applied for preclinical evaluation of drug candidates are still poorly characterized. In this paper, we reviewed the literature about several commonly used HCC cell lines and xenotransplantation models and present our own data on the molecular characterization of these. Results obtained demonstrate that it is important to have a sound knowledge of the specific molecular constitution of the experimental model and to carefully evaluate the functional status of the pathway of interest. For this reason, we make the gene expression profiles publicly available to help researchers making an informed decision about which model to use.
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Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Modelos Biológicos , Trasplante Heterólogo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/cirugía , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/cirugía , Ratones , Ratones DesnudosRESUMEN
Endometrial stromal sarcomas are rare and molecular mechanisms involved in their pathogenesis are poorly understood. Covalent modifications of histone proteins, in particular de/acetylation of lysine residues, play an important role in the regulation of gene transcription in normal and neoplastic cells, but there are only limited data about these processes in solid mesenchymal tumours. We treated endometrial stromal sarcoma cells (ESS-1) and non-malignant human endometrial stromal cells (HESCs) with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor. SAHA was able to mediate the cell cycle and expression of genes related to the malignant phenotype of endometrial stromal tumours, eg p21(WAF1) and HDAC7. SAHA led to dose-dependent differentiation and death of ESS-1 cells but not of HESCs. Exposure of HESCs to SAHA resulted only in slightly decreased cell proliferation. SAHA also increased the p21(WAF1) expression and caused significant changes in the cell cycle by inhibiting the G1/S transition in ESS-1 cells. Recovery experiments indicated that these changes became irreversible when the tumour cells were treated with SAHA for longer than 24 h. In our experimental system, not apoptotic but autophagic processes were responsible for the cell death. Monodansyl cadaverine accumulation in treated ESS-1 cells and decreased expression of the mTOR and phospho-S6 ribosomal protein (S6rp) additionally supported this observation. Taken together, our study indicates that HDACs might be considered as potential drug targets in the therapy of stromal sarcomas and that SAHA might be a promising therapeutic agent for endometrial stromal sarcoma.
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Neoplasias Endometriales/tratamiento farmacológico , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/uso terapéutico , Proteínas Quinasas/metabolismo , Sarcoma Estromático Endometrial/tratamiento farmacológico , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias Endometriales/patología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Histona Desacetilasas/metabolismo , Humanos , Immunoblotting/métodos , Microscopía Electrónica , Sarcoma Estromático Endometrial/patología , Serina-Treonina Quinasas TOR , VorinostatRESUMEN
High quality human biological samples (e.g. blood, tissue or DNA) with associated, well documented clinical and research data are key resources for advancement of life sciences, biotechnology, clinical medicine, drug development and also molecular pathology. Millions of samples of diseased tissues have been collected in the context of routine histopathological diagnosis and are stored in the archives of hospitals and institutes of pathology. A concerted effort is necessary to overcome the current fragmentation of the European biobanking community in order to tap the full research potential of existing biobanks. A pan-European research infrastructure for biobanking and biomolecular resources (BBMRI) is currently in its planning phase. The mission is to link and provide access to local biobanks of different formats, including tissue collections, harmonize standards, establish operational procedures which properly consider ethical, legal, societal aspects, and to secure sustainable funding. Pathology plays a key role in development and administration of tissue banks and is, thus, a major partner for collaboration, expertise and construction of this pan-European research infrastructure.
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Bancos de Muestras Biológicas/organización & administración , Biotecnología/organización & administración , Patología Clínica , Investigación/organización & administración , Bancos de Tejidos/organización & administración , Biología Computacional , Europa (Continente) , Humanos , Cooperación InternacionalRESUMEN
Inclusion bodies are characteristic morphological features of various neuronal, muscular and other human disorders. They share common molecular constituents such as p62, chaperones and proteasome subunits. The proteins within aggregates are misfolded with increased beta-sheet structure, they are heavily phosphorylated, ubiquitinylated and partially degraded. Furthermore, involvement of proteasomal system represents a common feature of virtually all inclusions. Multiple aggregates contain intermediate filament proteins as their major constituents. Among them, Mallory-Denk bodies (MDBs) are the best studied. MDBs represent hepatic inclusions observed in diverse chronic liver diseases such as alcoholic and non-alcoholic steatohepatitis, chronic cholestasis, metabolic disorders and hepatocellular neoplasms. MDBs are induced in mice fed griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine and resolve after discontinuation of toxin administration. The availability of a drug-induced model makes MDBs a unique tool for studying inclusion formation. Our review summarizes the recent advances gained from this model and shows how they relate to observations in other aggregates. The MDB formation-underlying mechanisms include protein misfolding, chaperone alterations, disproportional protein expression with keratin 8>keratin 18 levels and subsequent keratin 8 crosslinking via transglutaminase. p62 presence is crucial for MDB formation. Proteasome inhibitors precipitate MDB formation, whereas stimulation of autophagy with rapamycin attenuates their formation.
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Cuerpos de Inclusión/metabolismo , Queratinas/metabolismo , Hepatopatías/metabolismo , Animales , Humanos , Cuerpos de Inclusión/patología , Hepatopatías/patología , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFbeta-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.
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Carcinoma Hepatocelular/fisiopatología , Comunicación Celular , Neoplasias Hepáticas/fisiopatología , Animales , Línea Celular Tumoral , Células Epiteliales , Humanos , Ratones , RatasRESUMEN
Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising approximately 70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.
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Filamentos Intermedios/metabolismo , Queratinas/metabolismo , Hepatopatías/metabolismo , Hígado/metabolismo , Humanos , Cuerpos de Inclusión/patología , Filamentos Intermedios/patología , Hígado/patología , Hepatopatías/patología , Orgánulos/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
Vacuole membrane protein 1 (Vmp1) is described as a cancer-relevant cell cycle modulator, but the function of this protein and its mode of action in tumor progression are still unknown. In this study, we show that the VMP1 mRNA level is significantly reduced in kidney cancer metastases as compared to primary tumors. Further, VMP1 expression is also decreased in the invasive breast cancer cell lines HCC1954 and MDA-MB-231 as compared to the non-invasive cell lines MCF-12A, T-47D and MCF-7. We show for the first time that Vmp1 is a plasma membrane protein and an essential component of initial cell-cell contacts and tight junction formation. It interacts with the tight junction protein Zonula Occludens-1 and colocalizes in spots between neighboring HEK293 cells. Downregulation of VMP1 by RNAi results in loss of cell adherence, and increases the invasion capacity of the non-invasive kidney cancer cell line Caki-2. In conclusion, our findings establish Vmp1 to be a novel cell-cell adhesion protein and that its expression level determines the invasion and metastatic potential of cancer cells.
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Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Neoplasias Renales/patología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Vacuolas/química , Animales , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Células COS , Carcinoma Ductal de Mama/química , Carcinoma Ductal de Mama/genética , Adhesión Celular/genética , Comunicación Celular/genética , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Humanos , Neoplasias Renales/química , Neoplasias Renales/genética , Proteínas de la Membrana/biosíntesis , Invasividad Neoplásica , Uniones Estrechas/química , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Células Tumorales Cultivadas , Vacuolas/patologíaRESUMEN
Biobanks are a challenge and topic for governance. Today, biobanks are identified as a biomedical scientific/infrastructural development that warrants a political/legal/ethical reaction with the goal to integrate biobanks into the preexisting fabric of regulation, medicine, law and society. Biobank governance is always a response to sociocultural challenges and requires the building of trust, acceptance, and careful political negotiation. Biobanks are regulated in networks of governance in which the state is one actor next to others, and the ordering and structuring of the interaction between biobanks, society, and politics operates through a variety of actors, on different levels and along particular rationalities. Such networks of governance reflect, to some extent, a postregulatory state in which governance has become a complicated architecture and field of action involving a multitude of forces and rationalities. Biobank governance is still a relatively new field of political-legal intervention and it will be crucial for the future of biobanks to establish governance regimes that appropriately link research with society and politics.
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Discusiones Bioéticas , Política de Salud/tendencias , Autonomía Personal , Salud Pública , Bancos de Tejidos , Política de Salud/legislación & jurisprudencia , Humanos , Bancos de Tejidos/ética , Bancos de Tejidos/legislación & jurisprudencia , Bancos de Tejidos/organización & administraciónRESUMEN
In the context of the Austrian Genome Program, a tissue bank is being established (Genome Austria Tissue Bank, GATiB) which is based on a collection of diseased and corresponding normal tissues representing a great variety of diseases at their natural frequency of occurrence from a non-selected Central European population of more than 700,000 patients. Major emphasis is put on annotation of archival tissue with comprehensive clinical data, including follow-up data. A specific IT infrastructure supports sample annotation, tracking of sample usage as well as sample and data storage. Innovative data protection tools were developed which prevent sample donor re-identification, particularly if detailed medical and genetic data are combined. For quality control of old archival tissues, new techniques were established to check RNA quality and antigen stability. Since 2003, GATiB has changed from a population-based tissue bank to a disease-focused biobank comprising major cancers such as colon, breast, liver, as well as metabolic liver diseases and organs affected by the metabolic syndrome. Prospectively collected tissues are associated with blood samples and detailed data on the sample donor's disease, lifestyle and environmental exposure, following standard operating procedures. Major emphasis is also placed on ethical, legal and social issues (ELSI) related to biobanks. A specific research project and an international advisory board ensure the proper embedding of GATiB in society and facilitate international networking.
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Genoma , Bancos de Tejidos/organización & administración , Austria , Bases de Datos Factuales , Humanos , Cooperación Internacional , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/patología , Neoplasias/genética , Neoplasias/patología , Control de Calidad , Bancos de Tejidos/normas , Bancos de Tejidos/tendenciasRESUMEN
Defective protein folding is responsible for many diseases. Although these diseases seem to be quite diverse at the first glance, there is evidence for common pathogenetic principles. The basis of the pathological changes is the cell's inability to prevent protein misfolding, to revert misfolded proteins to normal or to eliminate misfolded proteins by degradation. This could result in deposition of potentially cytotoxic protein aggregates (protein aggregation diseases). Chronic degenerative diseases of the central nervous system (e.g. Alzheimer's and Parkinson's disease), the amyloidoses, but also chronic liver diseases, for example alcoholic and nonalcoholic steatohepatitis, belong to this category of disorders. This review highlights general pathogenic principles of protein aggregation diseases based on immunohistochemical and biochemical studies as well as observations in a mouse model for protein aggregation in the context of alcoholic and nonalcoholic steatohepatitis. The cellular defense mechanisms involved in protein quality control as well as the pathogenesis of protein aggregation diseases will be discussed.
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Chaperonas Moleculares/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Pliegue de Proteína , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Amiloidosis/metabolismo , Amiloidosis/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Enfermedad Crónica , Modelos Animales de Enfermedad , Hígado Graso Alcohólico/metabolismo , Hígado Graso Alcohólico/patología , Humanos , Ratones , Chaperonas Moleculares/química , Enfermedades Neurodegenerativas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Conformación ProteicaRESUMEN
Mallory bodies (MBs) and intracellular hyaline bodies (IHBs) are cytoplasmic hepatocellular inclusions that consist of aggregated proteins. MBs are characteristically associated with alcoholic and non-alcoholic steatohepatitis, but may also be found in chronic cholestatic and metabolic (eg copper intoxication) diseases and hepatocellular neoplasms, particularly hepatocellular carcinomas. IHBs have hitherto only been described in hepatocellular carcinoma cells. In the present study hepatocellular carcinomas (HCCs) and a case of idiopathic copper toxicosis were evaluated with respect to the presence and mutual relationship of MBs and IHBs. IHBs alone were present in 8.6%, MBs alone in 16.1% and both types of inclusion in 7.5% of HCCs. It is shown that IHBs may also occur in non-neoplastic hepatocytes in association with idiopathic copper toxicosis, together with MBs. In HCCs and idiopathic copper toxicosis, MBs and IHBs may be present within the same cell. Moreover, hybrid inclusions holding an intermediate position between MBs and IHBs regarding light microscopy, ultrastructure and composition exist. MBs and IHBs contain p62, a stress-inducible adapter protein, as the major constituent. In MBs p62 is associated with keratins, whereas classical IHBs lack keratins. Light microscopic, electron microscopic and immunohistochemical data suggest a close pathogenetic relationship between MBs and IHBs. Both types of inclusion are the result of over-expression and accumulation of the stress protein p62. If p62 is induced alone, or at least prevails, IHBs may arise by aggregation. However, if abnormal keratins are present in addition to p62, p62 associates and co-aggregates with keratins, finally leading to classical MBs.
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Carcinoma Hepatocelular/ultraestructura , Hepatocitos/ultraestructura , Cuerpos de Inclusión/ultraestructura , Neoplasias Hepáticas/ultraestructura , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma Hepatocelular/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas , Cobre/envenenamiento , Hepatocitos/metabolismo , Humanos , Hialina , Cuerpos de Inclusión/metabolismo , Queratinas/metabolismo , Hepatopatías/patología , Neoplasias Hepáticas/metabolismo , Microscopía Electrónica , Microscopía Inmunoelectrónica , Proteínas de Neoplasias/metabolismo , Proteína Sequestosoma-1RESUMEN
Morphologic criteria of steatohepatitis are steatosis, ballooning of hepatocytes, often but not constantly associated with Mallory bodies, pericellular fibrosis and inflammation. Liver cirrhosis follows in about 20-50%. With respect to etiology an alcoholic and non-alcoholic type can be distinguished, the latter being a characteristic hepatic lesion associated with the metabolic syndrome (type II diabetes, insulin resistance, obesity, dyslipidemia). Ballooning of hepatocytes as well as Mallory body formation are associated with a disturbance of the keratin intermediate filament cytoskeleton. Mallory bodies are protein aggregates consisting of keratin (particularly keratin 8), p62, a stress-induced adapter protein involved in signal transduction pathways, heat shock proteins, and ubiquitin. Oxidative stress is involved in Mallory body formation. Major sources of oxidative stress in alcoholic and non-alcoholic steatohepatitis are the microsomal biotransformation system (cytochrome P-450) and the mitochondria, together with an impaired antioxidant defense system. Oxidative stress leads to misfolding/unfolding, abnormal phosphorylation of keratins and disturbance of keratin 8: keratin 18 ratio, and thus interferes with intermediate filament assembly. Moreover, impairment of cellular defense against abnormal proteins, i. e. chaperone action and proteasomal degradation, leads to the accumulation of abnormal aggregation--prone keratins (particularly keratin 8) which after ubiquitination associate with the stress-induced ubiquitin-binding protein p62 to form Mallory bodies. Thus, Mallory body formation resembles an "off-folding" protein response of the amyloid type. These pathogenetic principles of the human disease are supported by immunohistochemical and gene expression studies in experimental animals and by transfection experiments in tissue culture cells.
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Hígado Graso Alcohólico/patología , Hígado Graso/patología , Diagnóstico Diferencial , Hígado Graso/complicaciones , Hígado Graso/fisiopatología , Hígado Graso Alcohólico/complicaciones , Hígado Graso Alcohólico/fisiopatología , Humanos , Cuerpos de Inclusión/patología , Queratinas/análisis , Queratinas/aislamiento & purificaciónRESUMEN
Tumour-associated macrophages (TAM) are involved in tumour angiogenesis and anti-tumour immune response. In colorectal cancer (CRC), an association of high microvascular density (MVD) and unfavourable prognosis has been reported by some investigators. However, heterogeneous patient groups were studied. We, therefore, analysed the correlation between TAM and MVD and the prognostic relevance of MVD, TAM and T lymphocyte infiltration for long-term survival in a homogeneous group of 70 patients with moderately differentiated cancers of the International Union Against Cancer (UICC) stages II and III, who did not receive chemotherapy. MVD was evaluated using immunohistochemistry with antibodies against CD34 and von Willebrand factor (vWF). TAM and T lymphocytes were visualised with antibodies against CD68 and CD3, respectively. Statistical analysis did not reveal a significant correlation between TAM and T lymphocyte numbers and MVD. Multivariate analysis of immunohistochemical data from all CRC patients and the subgroup of patients with UICC stage-II CRC identified TAM- and vWF-positive microvessel numbers as prognostically relevant markers. Low numbers of TAM- and high numbers of vWF-positive microvessels were associated with an unfavourable prognosis. In conclusion, TAM- and vWF-positive microvessel numbers may serve as independent prognostic markers for patients with UICC stage-II and -III CRC and may help to identify patients with an unfavourable prognosis.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Macrófagos/inmunología , Factor de von Willebrand/metabolismo , Anciano , Capilares/metabolismo , Neoplasias Colorrectales/irrigación sanguínea , Femenino , Humanos , Inmunohistoquímica , Masculino , Estadificación de Neoplasias , Neovascularización Patológica , Pronóstico , Análisis de Supervivencia , Linfocitos T/inmunologíaRESUMEN
We investigated the efficacy and safety of the cationic polymer polyethylenimine (PEI) as a potential tool for intrauterine gene delivery into livers of fetal mice in the last trimester of pregnancy (E17.5). Using luciferase as a reporter gene, transferrin-conjugated and ligand-free PEI/DNA complexes (containing 3 microg DNA) with varying PEI-nitrogen/DNA-phosphate (N/P) ratios and different PEI forms, branched (800, 25 kDa) and linear (22 kDa), were compared with naked DNA. Transgene expression was measured 48 h after administration of PEI/DNA complexes or naked DNA. Highest luciferase activity (9.8 x 10(3) relative light units (RLU)/mg of tissue protein) was observed with ligand-free PEI22/DNA mixtures at N/P 6.0. In addition, this formulation was associated with very low toxicity as compared to the other PEI/DNA-injected groups. Using beta-galactosidase as a reporter gene, transfection of single, but also small, clusters of cells was demonstrated throughout the liver. Injection of 3 microg naked DNA resulted in an 11-fold lower transgene expression value (0.9 x 10(3) RLU/mg of tissue protein) as compared to PEI22/DNA complexes. However, the administration of higher concentrated naked DNA (9 microg) into fetal livers yielded expression levels of 3.2 x 10(4) RLU/mg of tissue protein, a more than three-fold increase compared to PEI22/DNA complexes. Furthermore, the gene transfer efficacy of concentrated naked DNA was approximately 40 times higher in fetuses than in adults (0.8 x 10(3) RLU/mg of tissue protein), indicating that fetal tissue is especially amenable to the uptake and expression of naked DNA.