Cell Transplantation

The increased survival of aplastic anemia patients treated by human leukocyte antigens (HLA)-identical marrow transplants is due in part to a decrease in the incidence of graft rejection. The decrease in rejection, in turn, results from the more judicious use of transfusions before transplant, the removal of sensitizing white blood cells from transfusion products, and improvements in the immunosuppressive conditioning programs used to prepare patients for transplant. In regards to the latter, radiation-based programs have been effective, although a cydophosphamide/antithymocyte globulin program begins to look impressive with only one rejection among 33 patients transplanted. In regards to transfusions before transplant, in vitro radiation of all blood products may further reduce the risk of sensitization to minor histocompatibility antigens in the future. The incidence and severity of acute graft-versus-host disease (GvHD) have declined with the use of the methotrexate/cyclosporine regimen, and this has also contributed to improved survival. Chronic GvHD is difficult to treat and future emphasis should be on prevention rather than on treatment. Whether an extended course of cyclosporine beyond 6 mo after transplant will reduce the risk of chronic GvHD is under study. As more and more patients become long-term survivors, problems from long-term sequelae from the conditioning programs and from postgrafting immunosuppression must be considered, in particular secondary malignancies. Perhaps less toxic conditioning programs can be designed. Because of the higher likelihood of causing secondary cancer, possible deleterious effects on growth and development for pediatric patients, and the problem of sterility, radiation-based regimens should not be used in HLA-identical recipients.

Somatic cell gene therapy is a new field of biomedical research that encompasses a variety of traditional basic research and clinical disciplines. This new approach to therapeutics has the potential to prevent, treat, or cure a variety of inherited and acquired diseases. Two divergent strategies of hepatocyte transplantation are being employed in animal models and clinical trials in an attempt to correct genetic deficiencies. Allogeneic hepatocyte transplantation has two main advantages over autologous cell transplantation. First, invasive surgical procedures are not required in the recipient. Second, allogeneic cells can be administered repetitively, so that multiple harvests are not necessary. The major drawbacks to allogeneic hepatocyte transplants are rejection and the risks of immunosuppression. Although there is no clinical experience with the treatment of genetic disease by allogeneic hepatocyte transplantation, a variety of animal models have been characterized, including the Gunn rat (UDP-glucuronosyl transferase deficient), the Nagase analbuminemic rat, and the Watanabe heritable hyperlipidemic rabbit (LDL receptor deficient). The use of genetically corrected autologous cells represents a different and more elegant approach to the correction of inherited disease. A segment of liver is harvested from the affected individual. Recombinant retroviruses are used to transduce normal genes— with a variety of promoter/enhancer constructs —into the patients own hepatocytes. The genetically corrected hepatocytes are then transplanted back into the patient. This approach, known as ex vivo gene therapy, eliminates the risk of rejection and the need for immunosuppression. The safety and efficacy of this approach has been proven in a variety of preclinical animals models, including Watanabe rabbits, dogs, and Papio spp. A clinical trial for the treatment of familial hypercholesterolemia is currently in progress. A number of approaches for the reintroduction of hepatocytes into the recipient have been proposed, including cathetermediated delivery into the inferior mesenteric vein, the umbilical vein, or into the spleen. Candidate diseases, which are likely to result in the first clinical trials include familial hypercholesterolemia, ornithine transcarbamylase deficiency, Crigler-Najjar syndrome, alpha]-antitrypsin deficiency, and phenylketonuria.

We have investigated the cellular organization in two different types of retinal transplants using cell type-specific monoclonal antibodies. Both fragments and cell suspensions of E17-E19 Sprague–Dawley rat retina were transplanted to a subretinal site in congenic adult rat hosts. After a survival time of 28 days, the transplants were stained by immunocytochemistry with antibodies against rhodopsin, which stained rods; with antibodies against HPC-1, which stained amacrine cells and outer and inner plexiform layers; and with antibodies against vimentin, which stained Müller cell fibers and horizontal cells. In the host retina, the distribution of immunocytochemical staining was similar, irrespective of transplantation technique. In the transplants, the antirhodopsin staining showed that fragment transplants developed photoreceptors in rosettes, whereas in cell suspension transplants, this staining showed a scattered distribution of photoreceptors. The HPC-1 staining showed that regions corresponding to the inner nuclear layer surrounded both types of transplants and made large invaginations into them. In one case, using the cell suspension technique, fibres were found to run from the inner plexiform layer of the transplant to the outer plexiform layer of the host. The vimentin staining revealed a disorganized array of Müller cell fibres in both types of transplants, but with some concentration to the regions corresponding to the inner plexiform layer.

Nineteen different antisera raised against mammalian hormones were used to identify the occurrence and distribution of endocrine cells in the gut of grass carp (Ctenopharyngodon idellus). Positive reactions were obtained in gut epithelium with antisera gastrin, glucagon, gastric inhibitory peptide, leucine enkephalin, substance P, and bovine pancreatic polypeptide. No immunoreactive product was formed using antisera against somatostatin, 5-hydroxy-tryptamine, insulin, avian pancreatic polypeptide, motilin, cholecystokinin, secretin, neurotensin, vasoactive intestinal polypeptide, bombesin, neuron-specific enolase, prochymosin, and pepsinogen. The exact distribution mapping of six kinds of immunoreactive endocrine cells throughout the gut of grass carp (C. idellus) is presented. The morphological characteristics of immunoreactive endocrine cells is described. Their distribution characteristics and possible modes of secretion and function are discussed. Finally, the possible relationship between the transplantation of these cells in the gastro-entero-pancreatic endocrine system is discussed.

Alginate is a key reagent in the preparation of microcapsules for cell transplantation. To address the question of the intracapsular alginate concentration, a sensitive assay has been developed to quantify the alginate content of microcapsules. The method is based on the metachromatic change induced by alginate binding to the dye, 1,9-dimethyl methylene blue (DMMB). The assay has a high sensitivity and precision. It covers a wide concentration range enabling the measurement of alginate in dilute supernatants as well as in microcapsules. For the latter, the membrane is initially dissolved by incubating the microcapsules in an alkaline medium. The effect of potentially interfering substances (poly-l-lysine (PLL), citrate, chloride, sodium) and of pH has been studied. Poly-l-lysine interfered with the assay at pH 6.5 but not at pH 13. Interference by sodium augmented with increasing sodium concentration and reached a plateau at 200 mM. This problem was overcome by routinely adjusting all samples to 500 mM sodium. The other substances tested had a negligible effect on the assay. The reliable measurement of alginate with this new assay will allow the optimization of the intracapsular alginate concentration.