Supervivenica de hepatocitos alogénicos trasplantados en timo, en ausencia de terapia inmunosupresora / Survival o fallogeneic hepatocytes transplanted into thymus, without immunossuprresive therapy

El trasplante hepático constituye actualmente, una realidad clínica en situaciones de fallo hepático. Por otro lado, en el caso de diversos errores congénitos del metabolismo, en los cuales un solo fallo en una reacción de una ruta metabólica, conducea un fallo multiorgánico, la sustitución de un hígado morfológicamente normal parece una medida demasiado agresiva. Por ello, el uso de terapias celulares para el tratamiento de estas entidades está cada vez mas presente. Sin embargo, ambas estrategias terapéuticas se asocian a los efectos derivados de la inmunosupresión. La búsqueda de métodos que eliminen la necesidad de inmunosupresión es uno de los principales objetivos dentro del campode los trasplantes. La inyección intratímica de aloantígenos ha demostrado, en ciertos modelos experimentales, inducir tolerancia inmunológica. Sin embargo, los resultados con el trasplante intratímico de hepatocitos son contradictorios. Por este motivo, el objetivo de nuestro estudio ha sido estudiar el trasplante de hepatocitos en un órgano inmunológicamente privilegiado, como es el timo, donde se puede estudiar la funcionalidad y supervivencia de las células trasplantadas, así como estudiar la hipótesis de que el trasplante intratímico podría inducir un cierto grado de inmunomodulación en las células del sistemainmune. Se realizó trasplante de hepatocitos procedentes de ratas Fisher 344, en timo de ratas Gunn que habían recibido una dosis previa única de ciclosporina A. Después del trasplante, las ratas no recibieron nuevas dosis de ciclosporina, y fueron sacrificadas a diferentes tiempos. Se realizó microscopia óptica y se determinaron los niveles séricos y biliares de bilirrubina. También se estudió la presencia de mRNAs específicos y las poblaciones linfocitarias CD4 y CD8 en timo y sangre. Los datos obtenidos demuestran que los hepatocitos trasplantados sobreviven al menos 6 semanas, en los animales que habían recibido una dosis de ciclosporina A, antes del trasplante (AU)

Liver transplantation is currently a clinical reality, and it is well established as a treatment for acute organ failure. On the other hand, in the case of inborn errors of metabolism, in which a single step in a metabolic pathway leads to multiple organ failure, the replacement of a morphologically normal liver would be an aggressive measure. Thus, the use of cell therapy in these diseases is being considered. However, both strategies are associated with the effects derived from immunosuppression. The search for methods resulting in the avoidance of immunosupression is nowadays one of the main objectives within the field of transplantation. Intrathymic alloantigen injection has proved to induce immunological tolerance in certain experimental models. However, the results of intrathymic hepatocyte transplantation are contradictory. For this reason our objective was to study the transplantation of hepatocytes into an immunologically privileged organ such as the thymus, where we could assess the functionality and survival of the transplanted cells, as well as test the hypothesis that the intrathymic transplant could induce a certain immunomodulation in the cells of the immune system. Gunn rats, half of which received a single pre-transplant dose of cyclosporine A, were transplanted into the thymus with hepatocytes from Fisher 344 rats. After the transplant, rats did not receive any further dose of cyclosporine A, and they were sacrificed at different times. Light microscopy, bilirubin levels in both serum and bile, and presence of specific mRNAs were determined. Also, CD4 and CD8 lymphocyte subsets were studied in both thymus and blood. The results demonstrated thattransplanted hepatocytes survive for six weeks in animals which had been given cyclosporine A prior to the transplant (AU)

Evolution of the activity of UGT1A1 throughout the development and adult life in a rat.

Biliary excretion is the main route of disposal of bilirubin and impaired excretion results in jaundice, a well recognisable symptom of liver disease. Conjugation of bilirubin in the liver is essential for its clearance. The glucuronidation of bilirubin is catalysed by the microsomal UDP-glucuronosyltransferase UGT1A1. Patients with Crigler-Najjar syndrome type 1 and Gunn rats, mutant strain of the Wistar rats, bear an autosomal recessive disorder resulting in hyperbilirubinemia. The aim of this work is to add new data about activity of UGT1A1 during the perinatal period and adult life. The results showed that activity of UGT1A1 is detectable from day 22 of the gestation. After birth, activity of UGT1A1 gradually increases and reaches the levels of adult life. Furthermore, bilirubin azopigments have been separated and characterized by thin layer chromatography. We have found that concentration of samples by evaporation and ulterior storing at -20 degrees C seemed to be suitable for the maintenance of samples.

Hepatic proliferation in Gunn rats transplanted with hepatocytes: effect of retrorsine and tri-iodothyronine.

Hepatocyte transplantation would offer an attractive alternative to liver transplantation in the treatment of inborn errors of liver metabolism. However, a major problem in most transplantation studies to date has been the limited growth of transplanted cells in the recipient organ. We performed a strategy for selective proliferation of transplanted cells by interfering with the proliferative capacity of resident hepatocytes, using the pyrrolizidine alkaloid retrorsine and then transplanting liver cells in conjunction with repeated administration of triiodothyronine, an inducer of hepatocyte proliferation in rats. In the present study, foetal and adult syngeneic hepatocyte transplantation into spleen was performed in retrorsine-treated hyperbilirubinemic Gunn rats. In parallel, repeated injections of triiodothyronine were given to recipients. Rats were sacrificed at 1, 7, 30 and 90 days after transplantation and blood and bile samples were taken to assess the functionality of transplanted cells. The proliferative activity of transplanted hepatocytes was evaluated using proliferating cell nuclear antigen labelling index. In summary, both adult and foetal hepatocyte transplantation were effective in correcting a metabolic abnormality in Gunn rats for as long as 3 months. The RS/T3 model, as a measure to increase graft function, could represent an important advance to future clinical application of hepatocyte transplantation.

A compared study on the purification of fetal and adult rat hepatocyte suspensions by biomagnetic isolation techniques and flow cytometry analysis.

An analysis of liver cell populations from both adult and 21 day pregnancy rat fetuses (E21) was carried out. The results show that E21 hepatocytes express OX-43, as do endothelial cells but not adult hepatocytes. OX-43 could be used in future as a cell marker for the hepatocyte maturation.

Effects of endogenous and exogenous glucocorticoids on liver differentiation.

The effects of maternal bilateral adrenalectomy on day 1 of gestation and betamethasone treatment on fetal liver development were compared, in terms of biochemical and morphological parameters. For fetuses 20 days old (E20), absence of maternal glucocorticoids during gestation caused an increase in the number of nuclei in whole livers, and a significantly decrease of both body weight and protein content per nucleus, in comparison with the control group (C). Betamethasone injection on days 15, 16 and 17 of gestation into adrenalectomized pregnant rats (ADX + BET) did not completely prevent these effects. The electron microscopic analysis of the ADX fetal liver (E20) showed some hepatocyte lesions such as loss of cytoplasmic organelles, increase in hematopoietic cell number as well as a lower cellular maturation in comparison with the control group. The fetal liver from ADX + BET mothers 20 days after gestation displayed a noticeable involution of the hematopoietic component in spite of its relatively immature stage. However, there was no significant change in the degree of fetal hepatocyte lesions. Therefore, supply of maternal glucocorticoids from the beginning of gestation is essential for maintenance of the integral structure of the rat fetal hepatic parenchyma, for the correct maturation of the blood strains and for the beginning of involution of the hematopoietic tissue at the end of gestation.

Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation.

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Nagase analbuminemic rats (NAR), a strain which bears a mutation that determines the impossibility of the normal splicing of the albumin mRNA were used as recipients. The transplanted FH immediately migrated to the liver via portal vein, and anchored there. To assess the functional state of the transplanted cells, one month after transplantation, the expression of the albumin gene was studied in the liver of the recipients.

Expression of bilirubin UDP-glucuronosyltransferase (bUGT) throughout fetal development: intrasplenic transplantation into Gunn rats to correct enzymatic deficiency.

The aim of this work was to determine the pattern of expression of hepatic bilirubin UDP-glucuronosyltransferase throughout fetal development in rats, with the purpose of using fetal hepatocytes at the most appropiate stage of development for transplantation into Gunn rats lacking bilirubin UDP-glucuronosyltransferase activity and then assessing the therapeutic capacity of the implants. The results show that at day 13 of gestational life there is already bilirubin UDP-glucuronosyltransferase gene expression. Twenty-one-day fetal hepatocyte transplantation was also performed into the spleens of hyperbilirubinemic Gunn rats, when alpha-fetoprotein mRNA is still detectable. At 15, 30, and 90 days after transplantation, a mild decrease in total bilirubin serum levels was observed. An increase in bile conjugated bilirubin also was observed at 30 and 90 days. These data suggest the favorable evolution of transplanted cells and show its feasibility for therapy.

Liver gene expression and increase in albumin synthesis by fetal hepatocytes transplanted into analbuminemic rats.

This report describes the evolution of hepatocytes isolated from 21-day fetuses and transplanted into spleens of Nagase analbuminemic rats which have negligible serum albumin levels due to a mutation affecting albumin mRNA processing. Albumin and alpha-fetoprotein expression, in addition to other parameters related to cellular proliferation status (thymidine kinase and proliferating cell nuclear antigen expression) were studied as indicative of the behavior and evolution of the cells. In recipient rats, only a few clusters of hepatocytes could be observed in the red pulp of the spleen 24 h after transplantation. The fetal hepatocytes migrated to the liver and could be seen in portal branches immediately after transplantation. Fifteen days later, albumin mRNA was detected in recipient livers and was expressed throughout the entire 3-month study. Alpha-fetoprotein was not detected. Cell proliferation was not relevant, although 3 months after transplantation, the proliferation rates appeared to show a tendency to increase. These data demonstrate that fetal hepatocytes transplanted into spleen migrate to liver, settle there and acquire an adult phenotype free of malignant transformation. Our study is a first step towards the thorough understanding of fetal hepatocyte transplantation. The next steps will involve in-depth studies of the possibilities of genetic manipulation to achieve a high degree of repopulation/expression, employing the least possible number of donor cells, and of how the cells reach the liver parenchyma, overcoming the endothelial barrier.

Effect of metyrapone administration in pregnant rats on monoamine concentration in fetal brain.

Studies performed in our laboratory indicate that the adrenal deprivation during gestation can greatly influence the fetal catecholamines development in several cerebral areas. The present study was undertaken to determine whether the administration of metyrapone to pregnant rats affects the content of monoamines in fetal brain at term. To test wether the content of monoamines in fetal brain is regulated, at least in part, by endogenous glucocorticoids, pregnant rats were injected for 5 days prior to delivery with metyrapone, an adrenal 11-beta-steroid hidroxylase inhibitor which crosses the placenta and blocks endogenous glucocorticoid synthesis, or saline. On day 21 of gestation, delivery of all animals was accomplished by cesarean section. The encephalons were extracted and immediately dissected in metencephalon, mesencephalon, diencephalon and telencephalon. Monoamine determination was carried out using HPLC-ED. The results obtained indicate that the metyrapone treatment increases both DA and 5-HT and their metabolites in the brain studied areas.

A histopathological study of anoxic-resuscitated liver allografts.

The possibility of resuscitating livers after warm ischaemia has been recently suggested. The aim of the present investigation was to analyse the effects of anoxia on the morphology of hepatic cells, to determine whether these effects are reversible after providing a resuscitation period between warm ischaemia (WI) and cooling, and to study the behaviour of the resuscitated liver in the recipient organism. Ten female, Large-White pigs acted as donors for 10 recipient animals of the same kind who received an orthotopic liver graft. Recipients were divided into two groups depending on whether the livers they received had undergone a resuscitation period (Group I (n=5) where animal livers were subjected to 5 min warm ischaemia (WI) without resuscitation, and Group II (n=5) where the livers were subjected to 5 min WI followed by 5 min resuscitation). Morphological and ultrastructural studies of liver cells were performed using light and electron microscopy. ATP, ADP and AMP levels were determined in liver biopsies by high performance liquid chromatography (HPLC). Plasma AST and bilirubin levels in the two groups were compared 24 h after transplantation. After 5 min of anoxia, hepatocytes showed two morphological patterns in response to WI. Some were appreciably condensed with dark mitochondria, peroxisomes and some cytoplasmic vacuoles. Others showed electronlucent organelles, inflamed mitochondria with broken cristae and disorganized endoplasmic reticulum. Hepatocytes showed globular microvilli and bleb formation with migration towards the sinusoids. One hour after the revascularisation of the resuscitated livers, the hepatocytes showed nearly normal morphological characteristics. However, the hepatocytes of non-resuscitated organs continued to show alterations. Kupffer cells were activated in the livers of both experimental groups. Ultrastructural changes and total tissue adenine nucleotide (TAN) levels recovered completely in resuscitated livers soon after transplant. These results suggest that when short WI periods are followed by equivalent periods of resuscitation, the hepatocytes of transplanted livers recover from the effects of anoxia.

Effects of refeeding of undernourished and insulin treatment of diabetic neonatal rats on IGF and IGFBP.

The effect of refeeding and insulin treatment of undernourished and diabetic neonatal rats, respectively, on the regulation of insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP) was investigated. The changes in body weight, insulinemia, glycemia, serum IGF-I, and growth hormone (GH) as well as the increase of the 30-kDa IGFBP in undernourished and diabetic neonatal rats previously shown elsewhere were reversed by refeeding and insulin treatment, respectively. Also, changes in liver mRNA expression of IGF-I and-II and IGFBP-1 and -2 were restored in refed undernourished and IGF-I and IGFBP-1 levels recovered in insulin-treated diabetic rats. However, serum GH was still below normal after rehabilitation in both situations. Thus the present results support the idea of a GH-independent IGF/ IGFBP regulation mediated by a balance of insulin and nutrients as has already been suggested in previous neonatal studies.

Comparative in vivo and in vitro models to approach the cellular basis of endotoxic shock. The role of sinusoidal liver cells.

During endotoxic shock, the liver exerts a lipopolysaccharide (LPS) clearance function with the participation of both parenchymal and sinusoidal cells. Liver damage could be caused by LPS direct action, hypoxia and/or inflammatory mediators released by Kupffer cells. The aim of this study is to establish an experimental model that could allow us to understand the direct E. coli 011:B4 LPS action on sinusoidal cells. A comparative study was carried out, in vivo and in vitro, using either a rat reversible endotoxic shock model or sinusoidal cell cultures. The LPS was found to induce important and similar morphological alterations both in vivo and in vitro, specially in Kupffer cells. These cells present mitochondrial damage, nuclear membrane swelling, and increased number of phagosomes, including lamellar bodies. An immunocolloidal gold technique shows, in vitro, the LPS mainly located on Kupffer cell membrane and in phagosomes. The LPS binding to membrane, as a primary step of Kupffer cell activation, increases the phagocytosis. This effect could be related to a decrease of fluidity on the external membrane portion.

Hepatic resuscitation after warm anoxia: one approach for increasing the donor pool for liver transplantation.

The deleterious effects of warm anoxia on the liver are seen to be irreversible if cooling and transplantation (LT) follow immediately after. The aim of our study is to demonstrate that livers subjected to anoxia may be suitable for LT if a period of resuscitation is interposed before the cooling process. Forty female Large White pigs were used. Preservation (Euro-Collins solution) and LT technique were the same in all 20 procedures. All donors underwent clamping of the porta hepatis at the end of harvesting dissection. In the so-called "resuscitated" groups (AR and BR), the clamp was released for a period of time before the liver was cooled. Then, all livers underwent 2 h of cold ischemia followed by LT. Ultrastructural study showed better maintenance of mitochondria and sinusoidal cell integrity in resuscitated livers after LT. Liver synthesis of total adenine nucleotides, graft function and recipient survival were found to be better in the "resuscitated" groups. In conclusion, anoxic livers may be retrieved for LT if a resuscitation period (i.e. aerobic perfusion) is allowed prior to cold preservation. Longer periods of warm anoxia are needed to further support these preliminary results.

Influence of maternal adrenalectomy and glucocorticoid administration on the development of rat cerebral cortex.

In order to determine the incidence of maternal glucocorticoids on morphological parameters in fetal development, we performed optic and electron microscopic analysis of the cerebral cortex of fetuses of 16 and 20 days of gestation, from control (C) and pregnant rats bilaterally adrenalectomized on day 1 of gestation (ADX). We also studied fetuses 20 days old from pregnant rats betamethasone-injected on days 15, 16 and 17 (BET), and adrenalectomized on day 1 and betamethasone-injected on days 15, 16 and 17 (ADX+BET). Absence of maternal glucocorticoids during gestation caused, in fetuses 16 and 20 days old, a marked increase of cellular density, laxity of tissue and lower cellular maturation in comparison with the control group. Beta-methasone injected into sham-operated animals (BET) caused a slight advance in relation to controls in developmental parameters such as cellular density, maturation and synapse formation. Betamethasone injection into adrenalectomized animals prevented the lower degree of maturation characteristic of the adrenalectomized group, although an increase of cellular density could be detected. The cerebral cortex from fetuses of 16 days of gestation from adrenalectomized mothers also showed an increase of cellular density as compared with the control group. These results show that glucocorticoids participate in prenatal rat brain in control mechanisms of cellular division and maturation.

Effects of maternal adrenalectomy and glucocorticoid administration on the development of rat hippocampus.

Optic and electron microscopic analysis were performed on the hippocampus of fetuses of 20 days gestation, from pregnant rats bilaterally adrenalectomized on day 1 of gestation (ADX) and control (C), in an attempt to determine the incidence of maternal glucocorticoids on morphological parameters in fetal development. In addition, we studied 20 day old fetuses from pregnant rats betamethasone-injected on days 15, 16 and 17 (BET), and adrenalectomized on day 1 and betamethasone-injected on days 15, 16 and 17 (ADX + BET). The adrenalectomy led to a decreased total cell number and a marked depletion of the dentate gyrus of the hippocampus, and also to widespread indifferentiation, both in the pyramidal cell layer of the Ammon's horn and the dentate gyrus, as well as a decreased cell number in CA3 stratum pyramidale. No differences in cell number were found in CA1. So, the ADX effect is in relation to the neurogenic gradient. The main effect of the exogenous glucocorticoid treatment was increased maturation in relation to the control group. Betamethasone injection in adrenalectomized animals prevented the lower degree of maturation of the adrenalectomized group but not the impaired cell number. These results show that glucocorticoids participate in prenatal hippocampus in control mechanisms of cellular division and in maturation.

Physiological changes in antioxidant defences in fetal and neonatal rat liver.

This study describes the physiological changes in the activities of the hepatic antioxidant enzymes superoxide dismutase isoenzymes (Cu/Zn-and Mn-superoxide dismutase) and catalase, in the glutathione content and in the lipid peroxidation levels in fetal (20 and 21 days of gestation) and neonatal rat liver (Days 1, 8, 15, and 22 post partum). The catalase and superoxide dismutase activities decreased before birth and increased after birth. The oxidized:reduced glutathione (GSSG:GSH) ratio declined before birth, but it increased between Days 1 and 15 post partum and then remained stable. Finally, newborn rat liver from the 1st day of life shows the highest susceptibility to lipid peroxidation. These results suggest that the changes in antioxidant defences could be related mainly to the beginning of diet intake after birth, which entails a higher hepatic metabolism rate, as well as a higher oxygen consumption.

Intracellular calcium alterations and free radical formation evaluated by flow cytometry in endotoxin-treated rat liver Kupffer and endothelial cells.

During endotoxic shock, the liver exerts an endotoxin (lipopolysaccharide, LPS) clearance function with the participation of both sinusoidal (mainly Kupffer and endothelial cells) and parenchymal cells. In order to determine the specificity and diversity of response of each liver cell type, the effect of Escherichia coli 0111:B4 endotoxin (LPS) on intracellular Ca2+ content and reactive oxygen metabolite production in rat liver Kupffer, endothelial and parenchymal cells, was evaluated by flow cytometry during short treatment times (from 0-2 min) with a low dose of LPS (10 micrograms/ml). Concerning sinusoidal cells, LPS produced a significant increase of intracellular Ca2+ in both endothelial and Kupffer cells. The LPS effect on Kupffer cells was higher than on endothelial cells. When intracellular reactive oxygen metabolite production was evaluated in LPS-treated sinusoidal cells, a fast and significant increase of Kupffer cells in activated state (cells with a high reactive oxygen intermediate production) was observed. However, endothelial cells did not show LPS-induced changes in their intracellular reactive oxygen metabolite content. All these results support a rapid activation of liver Kupffer cells by endotoxin consistent with the major role of this cellular type as active first line of defense during endotoxic shock. The liver endothelial cells are also involved in the first steps of the cell damage showing intracellular Ca2+ alterations. Liver parenchymal cells did not show any response at these experimental conditions (short treatment time and low LPS dose) indicating that longer treatment times are needed for LPS binding and action in agreement with previous studies.

Effect of early maternal adrenalectomy on antioxidant enzymes, GSH, ascorbate, and uric acid in the rat fetal lung at term.

Previous studies have shown that the increase of the enzymatic antioxidant defense that takes place in the fetal rat lung at the end of gestation can be accelerated by the synthetic glucocorticoid dexamethasone and diminished by metyrapone, a blocker of glucocorticoid synthesis. Since it is known that the fetal adrenal does not start to synthesize corticosterone until the last 20% of gestation, pregnant rats were bilaterally adrenalectomized on the first day of gestation in order to clarify the role of the endogenous maternal hormone on the development of the enzymatic and nonenzymatic antioxidant systems of fetal lung. This early adrenalectomy did not change fetal lung catalase, glutathione peroxidase, glutathione reductase, cytochrome oxidase, GSH, ascorbate, and uric acid at term. The presence of the maternal glands is not essential for lung antioxidant development in the fetus and that the stimulus of fetal corticosterone during the last 20% of gestation is enough to achieve a normal maturation of the fetal lung enzymatic and nonenzymatic antioxidant systems.