Experimental models of acute and chronic liver failure in nude mice to study hepatocyte transplantation.

Although hepatocyte transplantation is a promising therapy for acute liver failure in human, there is still a lack of animal models suffering from hepatic injury in which the benefits of hepatocyte transplantation could be evaluated solely, without the bias caused by immunosuppression. As a consequence, the aim of the study was first to develop reproducible models of partial hepatectomy and of thioacetamide (TA)- or Jo2-induced acute liver failure in nude mice. Chronic liver disease was also investigated by repeated injections of sublethal doses of thioacetamide. Survival rates, routine histologic observations, alanin aminotransferase sera content, Ki67, and caspase 3 immunodetection were investigated both after 40% partial hepatectomy and after toxic-induced damages. Liver injuries were more severe and/or precocious in nude mice than in Balb/c mice for a given treatment with a maximum of acute injury obtained 24 h after single toxic injection, and were found to be transitory and reversible within 10 days. Toxics induced apoptosis followed by necrosis, confirming recent published data. Onset of fibrosis leading to reproducible chronic cirrhosis in nude mice correlated with increasing number of Ki67-positive cells, indicating that high levels of cell proliferation occurred. Chronic cirrhosis progressively reversed to fibrosis when the treatment ceased. Preliminary results demonstrated that engrafted xenogeneic hepatocytes could be detected in the host liver by anti-MHC class I immunohistochemistry. Fractions enriched in 2n or 4n hepatocytes by cell sorting using a flow cytometer were equivalent to the unpurified fraction in terms of engraftment in control nude mice or in nude mice subjected to PH. However, in mice suffering from liver injury 24 h after Jo2 or TA treatment, the engraftment of 2n hepatocytes was about twice that of an unpurified hepatocyte population or of a population enriched in 4n hepatocytes.

Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells.

Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins.

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells.

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.

Resveratrol analog (Z)-3,5,4'-trimethoxystilbene is a potent anti-mitotic drug inhibiting tubulin polymerization.

Resveratrol (3,5,4'-trihydroxystilbene) a natural polyphenol present in medicinal plants, grapes and wines, has potent chemopreventive properties on intestinal carcinogenesis. A methylated derivative (Z-3,5,4'-trimethoxystilbene: R3) was synthesized. R3 at 0.3 microM exerted a 80% growth inhibition of human colon cancer Caco-2 cells and arrested growth completely at 0.4 microM (R3 was 100-fold more active than resveratrol). The cis conformation of R3 was also 100-fold more potent than the trans isomer. R3 (0.3 microM) caused cell cycle arrest at the G2/M phase transition. The drug inhibited tubulin polymerization in a dose-dependent manner (IC50=4 microM), and it reduced also by 2-fold ornithine decarboxylase and s-adenosylmethionine decarboxylase activities. This caused the depletion of the polyamines, putrescine and spermidine, which are growth factors for cancer cells. R3 inhibited partially colchicine binding to its binding site on tubulin, indicating that R3 either partially overlaps with colchicine binding or that R3 binds to a specific site of tubulin that is not identical with the colchicine binding site modifying colchicine binding by allosteric influences. The resveratrol derivative (Z)-3,5,4'-trimethoxystilbene (R3) is an interesting anti-mitotic drug that exerts cytotoxic effects by depleting the intracellular pool of polyamines and by altering microtubule polymerization. Such a drug may be useful for the treatment of neoplastic diseases.

Expression and distribution of distinct variants of E-MAP-115 during proliferation and differentiation of human intestinal epithelial cells.

Epithelial cell proliferation and differentiation occur concomitant with striking remodeling of the cytoskeleton. Microtubules (MTs) play important roles in these processes, during which the MTs themselves are reorganized and stabilized by microtubule-associated proteins (MAPs). Among the proteins classified as structural MAPs, E-MAP-115 (also named ensconsin) is preferentially expressed in cells of epithelial origin. The aims of this study were, first, to determine if E-MAP-115, like other MAPs, is expressed as different isoforms during differentiation and, second, to perform a detailed analysis of the expression and distribution of any E-MAP-115 variants detected in intestinal epithelial cells during their polarization/differentiation. It was our expectation that these data would help us to develop hypotheses concerning the role of this MAP in epithelial development. We report the expression of three E-MAP-115 transcripts encoding isoforms of 115, 105, and 95 kDa; two display an expression gradient inverse to the third one as Caco-2 cells progress from proliferation through the stages of differentiation. To monitor the proteins produced from each transcript, we used purified polyclonal antibodies against synthetic peptides contained within the 115, 105, and 95 kDa isoforms to assay proliferating and differentiating CaCo-2 cells. Our results indicate that the expression and MT-binding capacity of the 115, 105, and 95 kDa isoforms vary upon proliferation/differentiation of the cells. E-MAP-115 proteins colocalize with MTs in proliferative and differentiated Caco-2 cells; in vivo, they are expressed in both crypt and villus epithelial cells where they are mainly concentrated at the apical pole of the cells.

The imidazoline receptors and the central regulation of the arterial blood pressure: a minireview

Recently, we proposed the hypothesis according to wich the central hypotensive effect of clonidine and related substances could be related to an action upon specific receptors, requiring the imidazoline or imidazoline-like structures, rather than alpha2-adrenoceptors. Since then, direct evidences have been accumulated to confirm the existence of a population of imidazoline specific binding sites in the brainstem of animals and man, more precisely in the Nucleus Reticularis Lateralis (NRL) region of the ventrolateral medulla (VLM), site of the antihypertensive action of clonidine. The purification of the putative endogenous ligand of the imidazoline receptors - named endazoline - is currently being attempted from human brain extracts. This new concept might at last lead to the expected dissociation of the pharmacological mechanisms involved, on the one hand, in the therapeutic antihypertensive effect, and on the other, in their major side-effect, which is sedation. In fact, it has been recently confirmed that hypotension is mediated by the activation of imidazoline preferring receptors (IPR) within the NRL region, while sedation is attributed to the inhibition of alpha2-adrenergic mechanisms in the locus coeruleus, which is involved in the control of the sleep-waking cycle. The IPRmay constitute on interesting target for new drugs in the treatment of arterial hypertension. Finally, dysfunctions of this modulatory system which could be involved in the pathophysiologyof some forms of the hypertensive disease are under investigation

Aspectos referentes ao melhor momento para o tratamento ortodôntico / Timing aspects of orthodontic treatment

Gens. interações e retroações cibernéticamente organizadas, conjuntamente formam o tamanho da mandíbula. Evidências indicam que, biológicamente, cefalométricamente e terapeuticamente, a face das crianças corresponde a 6 categorias auxológicas de crescimento, 11 tipos rotacionais e 33 grupos rotacionais. Quanto maior a categoria auxológica, um aparelho ortodôntico é mais efetivo, a nível tecidual. O tratamento funcional de uma má-oclusão de Classe II é insuperávelmente melhor corrigido quando executado durante a fase ascendente da curva de crescimento pubertal. O início da aceleração de crescimento, em um dado menino, pode ser constatado pela medida da estatura corporal após os 7 ou 8 anos de idade, em intervalos de 3 meses

Variaciones en la proporción de cambio del hueso alveolar del premolar humano durante el tratamiento, tanto con el aparato de Herbst como con el activador LSU (trad) / Variations in the change proportion of the alveolar bone of human premolar during treatment with Herbst appliance and LSU activator

1) El movimiento inducido ortodóncicamente es dependiente de la densidad ósea, del número de células involucradas respectivamente en la formación ósea y en la reabsorción ósea; en la velocidad de cambio o reversión del hueso alveolar y en la magnitud y tiempo de la fuerza aplicada. 2) El apropoado análisis cuantitiativo de parámetros bioquímicos de las proporciones de cambio alveolar, en el lado distal y en el lado mesial, da credibilidad para la realización, la dirección y la magnitud de un movimiento dentario espontáneo o inducido ortodóncicamente. 3) El aumento de la magnitud (del diente inducido ortodóncicamente) de la velocidad del cambio (reabsorción y neoformación ósea) es mayor cuando las fuerzas aplicadas (elásticos de clase II o clase III) son suaves e intermitentes que cuando son pesadas y continuas. El origen extrínseco de los encargados de la reabsorción ósea (monocitos circulantes y células similares) y, probablemente, de los de la neoformación, reconocen la gran efectividad de fuerzas livianas e intermitentes sobre fuerzas pesadas y continuas; las primeras tienen un menor efecto adverso de los tejidos locales y en las condiciones circulatorias. Fuerzas fuertes provocan un enorme aumento en la síntesis del colágeno II, que caracteriza la reparación del tejido conectivo. 4) La respuesta a las fuerzas ortodóncicas del hueso alveolar humano (de su proporción o cantidad de cambio) se hace mayor cuando el nivel tisular de crecimiento de la mandíbula aumenta; ejemplificando, cuando en nuestra categorización biológica uno progresa de 1 a 6. En forma no tan exacta, uno podría decir que la respuesta en la proporción de vuelco o cambios del hueso alveolar es mayor en la mandíbula con crecimiento rotacional anterior que con la de rotación posterior. 5) Los aparatos de Herbst actúan principalmente por inducción de un movimiento hacia adelante de los dientes laterales. De cualquier modo, de acuerdo con nuestro análisis de variaciones en la proporción de cambio del hueso alveolar en el lado mesial y en el distal del primer premolar, el movimiento hacia adelante es relativamente menos pronunciado en la categoría de crecimiento No.5 que en la categoría de crecimiento No.2. Como la efectividad del tratamiento es mayor en la categoría de crecimiento No.5, uno podría concluir que en un niño con un mayor potencial de crecimiento a nivel tisular mandibular el aparato podría también inducir un aumento, significativo clínicamente, en el cartílago condilar y en la c

Efeito dos aparelhos funcionais sobre a cartilagem do côndilo mandibular / Effect of functional appliances on the mandibular condylar cartilage

Neste artigo, os autores demonstram a importância dos hormônios do crescimento (STH, somatomedina) e os hormônios sexuais (testosterona, estrógenos, etc...) no controle do crescimento crânio-facial. Analisam também a integraçäo biológica entre estes hormônios e o efeito terapêutico dos aparelhos ortopédicos e ortodônticos. Ao mesmo tempo, elucidam o mecanismo biológico de certos aparelhos ortopédicos, sobre o crescimento mandibular, vistos sob o prisma da teoria do servo-sistema