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Liver transplantation is an established treatment for acute and chronic liver disease. However, because of the shortage of donor organs, it does not fulfill the needs of all patients. Hepatocyte transplantation is promising as an alternative method for the treatment of end-stage liver disease and as bridging therapy until liver transplantation. Our group has been working on the optimization of matrix-based hepatocyte transplantation. In order to increase cell survival after transplantation, freshly isolated human hepatocytes were seeded onto biodegradable poly(l-lactic acid) (PLLA) polymer scaffolds and were cultured in a flow bioreactor. PLLA discs were seeded with human hepatocytes and exposed to a recirculated medium flow for 6 days. Human hepatocytes formed spheroidal aggregates with a liver-like morphology and active metabolic function. Phase contrast microscopy showed increasing numbers of spheroids of increasing diameter during the culture period. Hematoxylin and eosin histology showed viable and intact hepatocytes inside the spheroids. Immunohistochemistry confirmed sustained hepatocyte function and a preserved hepatocyte-specific cytoskeleton. Albumin, alpha-1-antitrypsin, and urea assays showed continued production during the culture period. Northern blot analysis demonstrated increasing albumin signals. Scanning electron micrographs showed hepatocyte spheroids with relatively smooth undulating surfaces and numerous microvilli. Transmission electron micrographs revealed intact hepatocytes and junctional complexes with coated pits and vesicles inside the spheroids. Therefore, we conclude that primary human hepatocytes, precultured in a flow bioreactor on a PLLA scaffold, reorganize to form morphologically intact liver neotissue, and this might offer an optimized method for hepatocyte transplantation because of the expected reduction of the initial cell loss, the high regenerative potential in vivo, and the preformed functional integrity.

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Liver neo-tissue suitable for transplantation has not been established. Primary rat hepatocytes were cultured on three-dimensional biodegradable polymer matrices in a pulsatile flow bioreactor with the intention of inducing tissue formation and improving cell survival. Functional and structural analysis of the hepatocytes forming liver neo-tissue was performed. Biodegradable poly(L-lactic acid) (PLLA) polymer discs were seeded with 4 x 10(6) primary rat hepatocytes each, were exposed to a pulsatile medium flow of 24 mL/min for 1, 2, 4, or 6 days and were investigated for monoethylglycinexylidine (MEGX) formation, ammonia detoxification, Cytokeratin 18 (CK18) expression, and preserved glycogen storage. Fine structural details were obtained using scanning and transmission electron microscopy. Spheroids of viable hepatocytes were formed. MEGX-specific production was maintained and ammonia removal capacity remained high during the entire flow-culture period of 6 days. CK18 distribution was normal. Periodic-acid- Schiff reaction demonstrated homogenous glycogen storage. The hepatocytes reassembled to form intercellular junctions and bile canaliculi. Functional and morphological analysis of rat hepatocytes forming spheroids in a pulsatile flow bioreactor indicated preserved and intact hepatocyte morphology and specific function. Pulsatile flow culture on PLLA scaffolds is a promising new method of hepatic tissue engineering leading to liver neo-tissue formation.

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Nuclear deposits of stainable iron in hepatocytes are a sign of liver iron overload in mice. Animals with no, partial or total knock-out of the HFE alleles, the deletion of which is responsible for hereditary haemochromatosis, were given different forms of dietary iron to measure nuclear iron deposits which were then related to cytoplasmic iron load. Wild type and heterozygous HFE-knock-out mice kept for 52 weeks on a standard diet showed no such deposits. These were, however, demonstrated in low numbers and with small diameters in homozygous HFE-knock-out mice kept on this diet. Nuclear iron deposits were most abundant in all type of mice fed carbonyl iron (2.5% w/w) for 52 weeks almost irrespective of their genetic background. The diameter of these deposits increased with the genetically conditioned extent of hepatocellular iron overload. Mice that were fed a diet containing TMH-ferrocene for 4 weeks showed amounts of hepatic iron that were comparable to those in the carbonyl iron-fed group but nuclear deposits were small and present in only 0.3% of the hepatocytes. While surrounding karyoplasm was immunostained for H- and L-ferritin, the nuclear iron deposits were not. As the nuclear iron deposits corresponded electron microscopically to aggregated ferritin molecules, they represent a non-immunoreactive form of presumably denatured ferritin.

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Gene marking with replication-defective retroviral vectors has been used for more than 20 years to track the in vivo fate of cell clones. We demonstrate that retroviral integrations themselves may trigger nonmalignant clonal expansion in murine long-term hematopoiesis. All 29 insertions recovered from clones dominating in serially transplanted recipients affected loci with an established or potential role in the self-renewal or survival of hematopoietic stem cells. Transcriptional dysregulation occurred in all 12 insertion sites analyzed. These findings have major implications for diagnostic gene marking and the discovery of genes regulating stem cell turnover.

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Recent conceptual and technical improvements have resulted in clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects, based on preclinical and clinical data. To assess the therapeutic potential of genetic interventions in hematopoietic cells, it will be important to derive a classification of side effects, to obtain insights into their underlying mechanisms, and to use rigorous statistical approaches in comparing data. We here review side effects related to target cell manipulation; vector production; transgene insertion and expression; selection procedures for transgenic cells; and immune surveillance. We also address some inherent differences between hematopoiesis in the most commonly used animal model, the laboratory mouse, and in humans. It is our intention to emphasize the need for a critical and hypothesis-driven analysis of "transgene toxicology," in order to improve safety, efficiency, and prognosis for the yet small but expanding group of patients that could benefit from gene therapy.

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The lectin Chelidonium majus agglutinin (CMA) was previously shown to visualise endothelia of all blood vessels and those lining sinuses of red pulp, stromal reticular meshwok (RM) and dendritic cells of lymphatic follicles in white pulp of the spleen in rats. The aim of the present study was the analysis of CMA and some other lectins in labelling RM, vascular structures and macrophages in lymph nodes of rats. It appeared that CMA stained the entire RM, dendritic cells, lining cells of sinuses and all types of blood vessels. Sinus-lining cells of lymph nodes were labelled with CMA and mannose-, GalNac-, and sialic acid-specific lectins. Moreover, lymph node macrophages were labelled above all by mannose specific lectins. The broad lectin-binding pattern of sinuses--not observed in rat spleen- and CMA-reactivity of both sinus-lining and dendritic cells corroborates the hypothesis that lymph node sinus-lining endothelia are precursors or a special type of immune accessory cells.

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In the HFE-gene era, precise diagnostic parameters remain important to characterize individual iron stores, because the indication for therapy and prognosis are mainly related to the extent of iron loading. The frequently used serum ferritin interferes with non-iron related factors such as inflammation and may produce falsely positive values. We used a SQUID-biosusceptometer in a large series of patients (n = 679) to measure liver iron concentration in the differential diagnosis and therapy control of hereditary hemochromatosis (SQUID = superconducting quantum interference device). This truly non-invasive technique is sensitive, reliable, fast (online results), and also cost-effective when compared to invasive liver biopsy. Recently, ferritin iron content was propagated as a better parameter than ferritin protein. However, we found a poor correlation between ferritin iron and individual liver iron concentrations in patients with iron overload. Ferritin iron saturation varied in a range between 3 and 10%, independent from liver iron concentration. No differences were found between patients with hemochromatosis and secondary iron overload disease. Only patients with liver cell damage had increased ferritin iron saturations. In conclusion the diagnostic values of serum ferritin protein and iron to assess iron overload are limited.

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