RESUMO
A novel rapid ex vivo assay was developed as part of a concept to determine potential tailor-made combinations of pre- and probiotics for individual farms. Sow faecal slurries from 20 German pig farms were anaerobically incubated with pre- and probiotics or their combinations together with pathogenic strains that are of interest in pig production. Aliquots of these slurries were then incubated with media containing antibiotic mixtures allowing only growth of the specific pathogen. Growth was monitored and lag time was used to determine the residual fitness of the pathogenic strains. The background growth could be inhibited for an Escherichia coli- and a Clostridium difficile- but not for a Clostridium perfringens strain. The prebiotic fructo-oligosaccharides (FOS) and its combination with probiotics reduced the residual fitness of the E. coli strain in some farms. However, notable exceptions occurred in other farms where FOS increased the fitness of the E. coli strain. Generally, combinations of pre- and probiotics did not show additive effects on fitness for E. coli but displayed farm dependent differences. The effects of pre- and probiotics on the residual fitness of the C. difficile strain were less pronounced, but distinct differences between single application of prebiotics and their combination with probiotics were observed. It was concluded that the initial composition of the microbiota in the samples was more determinative for incubations with the C. difficile strain than for incubations with the E. coli strain, as the presumed fermentation of prebiotic products showed less influence on the fitness of the C. difficile strain. Farm dependent differences were pronounced for both pathogenic strains and therefore, this novel screening method offers a promising approach for pre-selecting pre- and probiotics for individual farms. However, evaluation of farm metadata (husbandry, feed, management) will be crucial in future studies to determine a tailor-made solution for combinations of pre- and probiotics for individual farms. Also, refinement of the ex vivo assay in terms of on-farm processing of samples and validation of unambiguous growth for pathogenic strains from individual farms should be addressed.
Assuntos
Clostridioides difficile , Escherichia coli , Prebióticos , Probióticos , Animais , Clostridioides difficile/patogenicidade , Escherichia coli/patogenicidade , Fazendas , Feminino , Alemanha , Oligossacarídeos , SuínosRESUMO
The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-ß), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for in vitro research using 3D high-density culture systems.
RESUMO
Primary human hepatocytes (PHH) are the "gold standard" for in vitro toxicity tests. However, 2D PHH cultures have limitations that are due to a time-dependent dedifferentiation process visible by morphological changes closely connected to a decline of albumin production and CYP450 activity. The 3D in vitro culture corresponds to in vivo-like tissue architecture, which preserves functional characteristics of hepatocytes, and therefore can at least partially overcome the restrictions of 2D cultures. Consequently, several drug toxicities observed in vivo cannot be reproduced in 2D in vitro models, for example, the toxic effects of acetaminophen. The objective of this study was to identify molecular differences between 2D and 3D cultivation which explain the observed toxicity response. Our data demonstrated an increase in cell death after treatment with acetaminophen in 3D, but not in 2D cultures. Additionally, an acetaminophen concentration-dependent increase in the CYP2E1 expression level in 3D cultures was detected. However, during the treatment with 10 mM acetaminophen, the expression level of SOD gradually decreased in 3D cultures and was undetectable after 24 h. In line with these findings, we observed higher import/export rates in the membrane transport protein, multidrug resistance-associated protein-1, which is known to be specific for acetaminophen transport. The presented data demonstrate that PHH cultured in 3D preserve certain metabolic functions. Therefore, they have closer resemblance to the in vivo situation than PHH in 2D cultures. In consequence, 3D cultures will allow for a more accurate hepatotoxicity prediction in in vitro models in the future.
Assuntos
Acetaminofen/toxicidade , Técnicas de Cultura de Células/métodos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Acetaminofen/metabolismo , Acetaminofen/farmacocinética , Morte Celular/efeitos dos fármacos , Citocromo P-450 CYP2E1/metabolismo , Relação Dose-Resposta a Droga , Humanos , Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Cultura Primária de Células/métodos , Superóxido Dismutase/metabolismoRESUMO
Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura/métodos , Corpos Embrioides/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Teratoma/patologia , Animais , Diferenciação Celular , Desenho de Equipamento , Perfilação da Expressão Gênica , Camadas Germinativas/metabolismo , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Perfusão , Células-Tronco Pluripotentes/citologiaRESUMO
Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.
Assuntos
Citocinas/metabolismo , Hepatócitos/metabolismo , Modelos Animais , Modelos Biológicos , Complexos Multienzimáticos/metabolismo , Transdução de Sinais/fisiologia , Biologia de Sistemas/normas , Animais , Simulação por Computador , CamundongosRESUMO
The kinetics of 18 amino acids, ammonia (NH3) and urea (UREA) in 18 liver cell bioreactor runs were analyzed and simulated by a two-compartment model consisting of a system of 42 differential equations. The model parameters, most of them representing enzymatic activities, were identified and their values discussed with respect to the different liver cell bioreactor performance levels. The nitrogen balance based model was used as a tool to quantify the variability of runs and to describe different kinetic patterns of the amino acid metabolism, in particular with respect to glutamate (GLU) and aspartate (ASP).
Assuntos
Aminoácidos/metabolismo , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Hepatócitos/metabolismo , Fígado Artificial , Modelos Biológicos , Engenharia Tecidual/métodos , Células Cultivadas , Simulação por Computador , Humanos , Cinética , Taxa de Depuração MetabólicaRESUMO
Cell-based extracorporeal liver support is an option to assist or replace the failing organ until regeneration or until transplantation can be performed. The use of porcine cells or tumor cell lines is controversial. Primary human liver cells, obtained from explanted organs found to be unsuitable for transplantation, are a desirable cell source as they perform human metabolism and regulation. The Modular Extracorporeal Liver Support (MELS) concept combines different extracorporeal therapy units, tailored to suit the individual and intra-individual clinical needs of the patient. A multi-compartment bioreactor (CellModule) is loaded with human liver cells obtained by 5-step collagenase liver perfusion. A cell mass of 400 g - 600 g enables the clinical application of a liver lobe equivalent hybrid organ. A detoxification module enables single pass albumin-dialysis via a standard high-flux dialysis filter, and continuous veno-venuous hemodiafiltration may be included if required. Cells from 54 human livers have been isolated (donor age: 56 +/- 13 years, liver weight: 1862 +/- 556 g resulting in a viability of 55.0 +/- 15.9%). These grafts were not suitable for LTx, due to steatosis (54%), cirrhosis (15%), fibrosis (9%), and other reasons (22%). Out of 36 prepared bioreactors, 10 were clinically used to treat 8 patients with liver failure. The overall treatment time was 7-144 hours. No adverse events were observed. Initial clinical applications of the bioreactor evidenced the technical feasibility and safety of the system.
Assuntos
Circulação Extracorpórea , Hepatócitos , Falência Hepática Aguda/terapia , Fígado Artificial , Reatores Biológicos , Estudos de Viabilidade , Hepatócitos/metabolismo , Humanos , Pessoa de Meia-Idade , Doadores de TecidosRESUMO
Initial results of the clinical use of primary porcine liver cells for extracorporeal liver support are being reviewed as the cell source is controversial. According to Eurotransplant data 20-25% of explanted donor livers are not transplanted, due to factors such as steatosis or cirrhosis. This number corresponds to the number of patients with acute liver failure who require bridging therapy to transplantation. Primary human liver cells from transplant discards can be isolated, purified and maintained in bioreactors and provide an alternative for cell-based extracorporeal liver support therapy. A four-compartment bioreactor enables recovery from preservation and isolation injury in a three-dimensional network of interwoven capillary membranes with integrated oxygenation, rendering the liver cells from these discarded donor organs viable for clinical utilization. Patient contact with additional animal-derived biomatrix and fetal calf serum can be avoided. The initiation of an in vitro cultivation phase allows cell stabilization, quality control, and immediate availability of a characterized system without cryopreservation. The hypothesis of this paper is that with appropriate logistics and four-compartment bioreactor technology, cells from human liver transplant discards can serve the demand for cell-based therapy, including extracorporeal liver support.
Assuntos
Circulação Extracorpórea , Hepatócitos , Falência Hepática Aguda/terapia , Fígado Artificial , Animais , Reatores Biológicos , Células Cultivadas , Humanos , SuínosRESUMO
OBJECTIVE: Hepatic in vitro studies, like those on hypoxia/reperfusion injury in liver transplants, demand large numbers of cultivated sinusoidal endothelial cells (SECs). In this article, we present and evaluate a new method for the isolation of SECs from porcine and human livers. METHODS: SECs were isolated employing a four-step collagenase perfusion. The sinusoidal character of the cells was validated by transmission and scanning electron microscopy, exclusion of Weibel-Palade bodies and factor VIII-related antigen, expression of scavenger receptor, and incorporation of latex beads. RESULTS: In 23 pigs, an average of 9 x 10(4) SECs were harvested from each liver. Cells were cultivated under standard conditions, as well as in multilayer cocultures of isolated SECs and hepatocytes in a "sandwich" configuration. Standard cultures showed an average of 90% SECs in primary cultures and 100% SECs after the first passage. The possibility of isolation of SECs from human livers was demonstrated in eight cases. CONCLUSION: With the four-step collagenase perfusion it is possible to easily isolate large numbers of viable and pure SECs from one organ. A further advantage is the possibility of isolating hepatocytes from the same organ.
Assuntos
Separação Celular/métodos , Hepatócitos/ultraestrutura , Fígado/citologia , Proteínas de Membrana , Receptores de Lipoproteínas , Animais , Materiais Biocompatíveis , Separação Celular/instrumentação , Células Cultivadas , Técnicas de Cocultura , Colágeno , Colagenases , Combinação de Medicamentos , Endotélio/citologia , Hepatócitos/química , Humanos , Laminina , Masculino , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Microscopia Imunoeletrônica , Perfusão , Proteoglicanas , Receptores Imunológicos/análise , Receptores Depuradores , Receptores Depuradores Classe B , SuínosRESUMO
Since the liver is the main organ involved in the metabolism and the toxicity of xenobiotics, isolated rat hepatocytes have been increasingly used in recent years as a model to identify pharmacological and toxicological responses of drugs. However, it is generally recognised that isolated hepatocytes retain most of their functions only for a short period. For this reason, numerous models and techniques have been developed to study and improve the metabolic capacity of hepatocytes in vitro over an extended time period and in application for drug metabolism studies. In the present study, we compared four different cell culture models to fulfill these requirements and have therefore harvested hepatocytes and cultured them in different culture systems over two weeks. In order to prove certain advantages or disadvantages of each model, we compared the metabolic capacity, albumin secretion, the release of cytosolic and mitochondrial enzymes, as well as the capacity to metabolise diclofenac (DF). We found that rat hepatocytes in all studied culture models (except the Unisyn Bioreactor) were able to metabolise DF to the same extent as found in vivo. However, the concentration of metabolites was found to decrease with culture time using the monolayer although the DF metabolite level in the collagen Sandwich culture was higher than that of the monolayer culture. The 3D-membrane bioreactor preserved the metabolic capacity for a prolonged period of time. The concentrations of DF metabolites in the Unisyn hollow fiber bioreactor were below the detection limit, which corresponded to other parameters such as albumin secretion and cytochrome P450 activity, disqualifying this culture system clearly for the use of in vitro primary hepatocyte cultures. The other three systems all have their place in drug metabolism with different advantages. However, our studies clearly showed that hepatocytes cultured within a collagen sandwich or in the 3D-membrane bioreactor qualify to study various aspects of drug metabolisms over a long time period. Further studies are needed to prove if the later two culture models may really help to reduce animal testing.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Diclofenaco/farmacocinética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Modelos Biológicos , Alanina Transaminase/análise , Animais , Aspartato Aminotransferases/análise , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Citosol/enzimologia , Hepatócitos/citologia , Cinética , L-Lactato Desidrogenase/análise , Masculino , Metilcolantreno/farmacologia , Mitocôndrias Hepáticas/enzimologia , Omeprazol/farmacologia , Ratos , Ratos Sprague-Dawley , Albumina Sérica/biossínteseRESUMO
An important consideration for the utilisation of in vitro culture models for studies on drug metabolism as an alternative to animal testing is the maintenance of a defined degree of cell differentiation. Thus, in vitro conditions reflecting as near as possible the in vivo situation of the cells within the whole organ are required. A bioreactor was developed for the cultivation of liver cells which allows the reorganisation of hepatocytes and non-parenchymal cells of the liver in coculture to form three-dimensional, tissue-like structures including extracellular matrix components produced by the cells. In this study, the vitality and metabolic activity of isolated rat hepatocytes was investigated over a two week culture period in bioreactors. The results show that after a reorganisation phase, the cells preserve specific functions, such as protein and urea synthesis capacity and specific cytochrome P450 activities during the culture period, with maximal values during the first week. Possible applications of the model in pharmaceutical industry are studies on metabolite patterns, enzyme induction, drug-drug-interactions, first pass effects and long-term toxicity of drugs.
Assuntos
Alternativas aos Testes com Animais , Reatores Biológicos , Fígado/citologia , Fígado/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Cumarínicos/farmacocinética , Hepatócitos/citologia , Masculino , Oxazinas/farmacocinética , Ratos , Ratos WistarRESUMO
In order to investigate locally produced mediators during the process of organ storage in liver transplantation, we collected the liver preservation solution effluent of 15 transplanted livers and compared it with serum samples taken preoperatively from donor and recipient, as well as 60 min after reperfusion. The mean ischemia time +/- SEM was 10 h 10 min +/- 53 min. Mean concentrations in University of Wisconsin preservation solution effluent were: interleukin-(IL-)1beta 154 +/- 77 pg/ml; IL-1 receptor antagonist (IL-1 ra) 1281 +/- 309 pg/ml; IL-6 412 +/- 90 pg/ml; and for tumor necrosis factor-(TNF-)alpha 74 +/- 21 pg/ml. Cytokine levels in the donors were lower than those detected in the effluent. All measured cytokines showed higher concentrations in the effluent compared to those of the recipient prior to the operation. With respect to a comparison of donor and recipient values, no correlation is evident. Likewise, the ischemic time does not correlate with effluent values. Further development of liver preservation concepts requires information about the state of the graft before reperfusion. Data on cytokine liberation may serve as a helpful tool for the further development of preservation concepts because they enable an estimation of cell activation during preservation.
Assuntos
Citocinas/metabolismo , Transplante de Fígado/fisiologia , Fígado , Preservação de Órgãos , Adenosina , Adulto , Alopurinol , Feminino , Glutationa , Humanos , Insulina , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Isquemia , Fígado/imunologia , Transplante de Fígado/imunologia , Masculino , Pessoa de Meia-Idade , Soluções para Preservação de Órgãos , Rafinose , Sialoglicoproteínas/metabolismo , Doadores de Tecidos , Fator de Necrose Tumoral alfa/metabolismoAssuntos
Rejeição de Enxerto/fisiopatologia , Transplante de Rim/fisiologia , Animais , Doença Crônica , Ciclosporina/farmacologia , Rejeição de Enxerto/patologia , Imunossupressores/farmacologia , Transplante de Rim/imunologia , Transplante de Rim/patologia , Proteinúria , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Reoperação , Doadores de Tecidos , Transplante HomólogoRESUMO
BACKGROUND: Sinusoidal endothelial cells (SEC) are significantly more vulnerable to cold storage and reperfusion than hepatocytes. Swelling and disruption of the sinusoidal lining induce the microcirculatory disturbances seen after reperfusion. In this article, the investigation of a method to assess the adhesion and morphology of SEC in vitro during reperfusion after preservation is described. METHODS: Time-lapse video microscopy analysis was performed and cell detachment rates and cell lengths were determined. Preservation intervals between 6 and 24 hr and flow rates ranging from 3 L/min to 9 L/min (resulting in shear stresses between 5.1 and 15.3 dynes/cm2 on the monolayer surface) during reperfusion period were compared. SEC that were stored for 6 hr in University of Wisconsin solution and nonpreserved control cultures were compared. RESULTS: Varying the preservation intervals from 6 hr to 24 hr during reperfusion at a flow rate of 3 L/min led to increased cell erosion rates (6 hr, 35.5+/-15.2%; 12 hr, 38.0+/-7.6%; 18 hr, 54.3+/-5.7%; 24 hr, 76.7+/-6.7%; nonpreserved cells, 3.4+/-3.4%). Storage periods from 12 hr to 24 hr led to significantly higher cell detachment rates than occurred in nonpreserved cells. CONCLUSIONS: This method allows the investigation of the adhesion capability and morphology of individual cells in vitro. Indications of the kind of preservation/reperfusion injury that occurs after treatment with several preservation solutions and the resultant repair behavior can be obtained.
Assuntos
Endotélio Vascular/citologia , Fígado , Soluções para Preservação de Órgãos , Preservação de Órgãos/métodos , Reperfusão , Adenosina , Alopurinol , Animais , Adesão Celular , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Endotélio Vascular/fisiologia , Glutationa , Insulina , Fígado/citologia , Fígado/fisiologia , Circulação Hepática , Masculino , Microcirculação , Microscopia de Vídeo , Rafinose , Suínos , Fatores de TempoRESUMO
Previous studies on pathophysiological mechanisms of chronic graft rejection demonstrated the impact of both alloresponsiveness and nonspecific immunological events on the process. To study the role of alloantigen-specific factors further, we hypothesized an acceleration of chronic graft rejection after presensitization. Chronically rejected renal allografts in the established Fischer 344 --> Lewis rat model were replaced sequentially by native allografts of donor origin. Grafting of second allografts was performed 2, 4, 8, and 12 weeks after the original transplantation and followed long term. Second allografts demonstrated significantly ameliorated functional and structural alterations with few cellular infiltrates. These changes were independent from the time interval between first and second engraftment (2-12 weeks); immunosuppressive treatment after the second engraftment was not influential. The nonresponsiveness was not restricted to the second kidney allografts, as heart allografts of donor origin in these recipients also functioned indefinitely, whereas third-party grafts (Lewis x Brown Norway F1) and Fischer 344 heart grafts in untreated Lewis control rats were acutely rejected. Thus, donor-specific and tissue-nonspecific graft acceptance is achieved by second engraftment of donor-specific allografts in a model of chronic graft rejection. Those observations demonstrate the synergistic effects of alloresponsiveness and of the injured graft itself for the development of chronic graft failure.
Assuntos
Transplante de Rim/imunologia , Animais , Doença Crônica , Ciclosporina/uso terapêutico , Rejeição de Enxerto/fisiopatologia , Rejeição de Enxerto/prevenção & controle , Tolerância Imunológica , Molécula 1 de Adesão Intercelular/análise , Transplante de Rim/patologia , Masculino , Proteinúria/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Reoperação , Fatores de Tempo , Transplante Homólogo/patologiaRESUMO
Sinusoidal endothelial cells are significantly more vulnerable to cold storage and reperfusion than hepatocytes. In this study, a method for assessing the repair behavior of sinusoidal endothelial cells in vitro, after preservation, was investigated. Time-lapse video microscopy analysis was performed and migration rates, division rates, and cell detachment rates were determined. Preservation intervals between 3 and 24 hr and reoxygenation times between 4 and 24 hr were compared. A comparison between sinusoidal endothelial cultures that were stored for 6 hr in University of Wisconsin solution and nonpreserved control cultures was performed. This method allows the investigation of the repair capability of individual cells in vitro. Indications of the kind of preservation/reoxygenation injury that occurs after treatment with several preservation solutions and the resultant repair behavior can be obtained.
Assuntos
Endotélio Vascular/citologia , Transplante de Fígado/patologia , Microscopia de Vídeo , Preservação de Órgãos , Traumatismo por Reperfusão/patologia , Animais , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/efeitos adversos , Movimento Celular/efeitos dos fármacos , Endotélio Vascular/ultraestrutura , Masculino , Mitose/efeitos dos fármacos , Soluções para Preservação de Órgãos/farmacologia , Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , SuínosAssuntos
Aminoácidos/farmacologia , Endotélio/citologia , Frutose/farmacologia , Glucose/farmacologia , Fígado/citologia , Soluções para Preservação de Órgãos , Traumatismo por Reperfusão/prevenção & controle , Preservação de Tecido , Adenosina , Aerobiose , Alopurinol , Animais , Hipóxia Celular , Separação Celular/métodos , Células Cultivadas , Endotélio/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Glutationa , Glicina/farmacologia , Insulina , L-Lactato Desidrogenase , Fígado/efeitos dos fármacos , Masculino , Rafinose , Serina/farmacologia , SuínosRESUMO
A remarkable property of the integral glycoprotein dipeptidyl peptidase IV (DPP IV, CD 26) is its affinity to proteins of the extracellular matrix (ECM). By in vitro binding assays we have shown that DPP IV binds to collagens; preferentially to the collagens I and III, which are both characterized by the formation of large triplehelical domains. No binding of DPP IV to laminin or fibronectin could be observed. Within collagen I, the alpha 1(I) chain was found to be the most prominent binding ligand of DPP IV. A monoclonal anti DPP IV antibody (13.4) specifically inhibited the interaction of DPP IV with collagen I. Peptide mapping and N-terminal sequencing revealed that the corresponding epitope of mAb 13.4 is located in the cysteine-rich domain of DPP IV. We therefore conclude that the putative collagen binding site of DPP IV is different from the region of the catalytic site containing the exopeptidase activity, which is located at the C-terminal portion of the molecule.