Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression.

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (gamma-irradiation, depletion of the peripheral immune system) or administration of "mega" doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-gamma-producing T cells re-emerged and skin grafts were rejected at approximately 100 days. When applied to a murine beta-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.

Noscapine inhibits tumor growth with little toxicity to normal tissues or inhibition of immune responses.

Noscapine, a phthalideisoquinoline alkaloid derived from opium, has been used as an oral anti-tussive agent and has shown very few toxic effects in animals or humans. Recently, we reported that noscapine binds stoichiometrically to tubulin and promotes microtubule polymerization. Noscapine causes growth arrest of tumor cells in mitosis and induces apoptosis of tumor cells in vitro. Previous experiments also showed that noscapine has potent antitumor activity in mice when administered parenterally or by gastric lavage. Here, we report that the anti-mitotic effect was specific to noscapine since closely related compounds did not inhibit the growth of a lymphoma cell line. In addition, noscapine was shown to be effective in reducing the growth of the lymphoma and increasing the survival of tumor-bearing mice when administered in the drinking water. It is noteworthy that, noscapine showed little or no toxicity to kidney, liver, heart, bone marrow, spleen or small intestine at tumor-suppressive doses. Furthermore, oral noscapine did not inhibit primary immune responses, which are critically dependent upon proliferation of lymphoid cells. Thus, our results indicate that noscapine has the potential to be an effective chemotherapeutic agent for the treatment of human cancer.

Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection.

The double-stranded (ds) RNA-dependent protein kinase PKR is considered to play an important role in interferon's (IFN's) response to viral infection. Here, we demonstrate that mice lacking PKR are predisposed to lethal intranasal infection by the usually innocuous vesicular stomatitis virus, and also display increased susceptibility to influenza virus infection. Our data indicate that in normal cells, PKR primarily prevents virus replication by inhibiting the translation of viral mRNAs through phosphorylation of eIF2alpha, while concomitantly assisting in the production of autocrine IFN and the establishment of an antiviral state. These results show that PKR is an essential component of innate immunity that acts early in host defense prior to the onset of IFN counteraction and the acquired immune response.

Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaling pathway.

Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-alpha/beta greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.

In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice.

We have previously demonstrated that high levels of allogeneic, donor-derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34+ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45+ human cells. CD45+ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3+ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16-fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.

Sustained multilineage engraftment of allogeneic hematopoietic stem cells in NOD/SCID mice after in utero transplantation.

Substantial barriers exist to the engraftment of hematopoietic cells in mice after in utero transplantation. Although high levels of donor-derived hematopoiesis have been reported in SCID mice, the majority of chimeric recipients exhibit decreasing levels of donor cells over time. To directly test whether the natural killer cell and macrophage activity of the recipients represents a barrier to sustained engraftment, fetal NOD/SCID mice were injected on day 13.5 of gestation with an enriched congenic hematopoietic progenitor cell population. Forty-four percent of pups showed the presence of Ly5.1+ donor cells 4 weeks after transplantation. The mean number of donor-derived nucleated cells in the peripheral blood (PB) was 30%. Although the majority of circulating donor cells were lymphocytes, up to 15% expressed myelomonocytic markers. Serial PB samples from individual mice indicated that the percentage of circulating donor cells increased from 17% to 55% between 4 and 24 weeks. At 6 months posttransplantation, an increased frequency of multilineage, donor-derived cells was also observed in the bone marrow (BM) and the spleen of chimeric recipients. The engraftment of pluripotent hematopoietic stem cells was evaluated by transplanting BM from chimeric mice into irradiated congenic recipients. Irradiated secondary recipients also exhibited multilineage donor-derived hematopoiesis in the PB, BM, and spleen for up to 6 months. These results show that the in utero transplantation of lineage-depleted BM cells into NOD/SCID recipients produces a high frequency of sustained engraftment of allogeneic hematopoietic stem cells.

Myelination following transplantation of EGF-responsive neural stem cells into a myelin-deficient environment.

Epidermal growth factor (EGF)-responsive stem cells have been identified in the murine central nervous system. These cells can be isolated from the brain and maintained in an undifferentiated state in vitro in the presence of EGF. After removing EGF, the cells cease mitosis and can be induced to differentiate into neurons, astrocytes, and oligodendrocytes. We demonstrate that when the undifferentiated stem cells (nestin-positive) are injected into the myelin-deficient rat spinal cord, they respond to cues within the mutant CNS and differentiate into myelinating oligodendrocytes, in contrast to their behavior in vitro, where they mainly form astrocytes. The cells provide a valuable model system for the study of the development of early oligodendrocytes from multipotent neural stem cells. Because these cells are influenced to divide using growth factors, rather than oncogenes, and because they appear to make appropriate lineage decisions when transplanted into a mutant environment, they may provide an excellent source of cells for a variety of future therapies using cellular transplantation.

Myelination of the canine central nervous system by glial cell transplantation: a model for repair of human myelin disease.

There is a lack of effective means of promoting remyelination of the central nervous system (CNS) in humans with chronic demyelinating disease. We have investigated the ability of transplanted glia to myelinate areas of the CNS equivalent to focal demyelinated lesions in multiple sclerosis (MS). In these studies we show that transplantation of oligodendrocytes or their progenitors into the CNS of a neonatal or adult canine myelin mutant results in repair of large areas similar in size to many MS plaques. Progenitor or pre-progenitor cells of the oligodendrocyte lineage have the greatest capacity for myelination following grafting, although cells of neonatal origin may also be used. Such an approach may therefore have therapeutic value in the repair of focal lesions in human myelin disease.

Acute dispersion of glial cells following transplantation into the myelin-deficient rat spinal cord.

Evaluation of glial cell migration following transplantation can be difficult as the force of the injection itself may cause the cells to become immediately dispersed. In this study we evaluated the extent of spread of cells after injection of 1 microliter of a dissociated cell suspension (50,000 cells/microliter) into the dorsal columns of the thoracolumbar spinal cord in the neonatal myelin-deficient (md) rat. Spinal cords were examined at 0, 4, and 24 h after injection to determine the dispersion of cells away from the initial site of deposition. Examination of skip-serial sections collected at 50-microns intervals rostral and caudal to the site of transplantation showed that the injection could result in a spread of transplanted cells up to 1,600 microns. Migration should therefore be defined as the detection of cells beyond the rostral-caudal boundaries defined by the injection deposition. Cell dispersion should be taken into account when evaluating the results of migration in previous and future experiments concerning glial cell transplantation.

Transplantation of an oligodendrocyte cell line leading to extensive myelination.

Oligodendrocytes, the myelin-forming cells of the central nervous system, can be generated from progenitor cell lines and assayed for their myelinating properties after transplantation. A growth-factor-dependent cell line of rat oligodendrocyte progenitors (CG4) was carried through 31-48 passages before being transplanted into normal newborn rat brain or the spinal cord of newborn myelin-deficient (md) rats. In md rat spinal cord, CG4 oligodendrocyte progenitors migrated up to 7 mm along the dorsal columns, where they divided and myelinated numerous axons 2 weeks after grafting. CG4 cells were transfected with the bacterial lacZ gene and selected for high beta-galactosidase expression. The cell migration and fate of these LacZ+ cells were analyzed after transplantation. In normal newborn brain, LacZ+ oligodendrocyte progenitors migrated along axonal tracts from the site of injection and integrated in the forming white matter. In md rats, extensive migration (up to 12 mm) was revealed by staining for beta-galactosidase activity of the intact spinal cord where many grafted cells had moved into the posterior columns. Similar migration and integration of grafted cells occurred in the spinal cord of normal myelinated rats and after a noninvasive grafting procedure. Thus, oligodendrocyte progenitors can maintain their ability to migrate and myelinate axons in vivo after multiple passages in vitro. Such progenitor cell lines can be used to study the molecular mechanisms underlying oligodendrocyte development and the repair of myelin in dysmyelinating diseases.

Phosphorylation-dependent immunoreactivity of neurofilaments and the rate of slow axonal transport in the central and peripheral axons of the rat dorsal root ganglion.

The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [35S]Methionine was injected into the DRG, and the "front" of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were approximately 1.5-fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, approximately 1.7-fold; tubulin/actin, 2.1-fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation-dependent immunoreactivity of the high-molecular-weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation-related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.

Myelination by cryopreserved xenografts and allografts in the myelin-deficient rat.

This study examined the ability of freshly prepared and cryopreserved canine oligodendrocytes to myelinate axons following transplantation into the myelin deficient (md) rat. The effects of immunosuppression, and the age of the donor tissue, were also examined. Canine glial cells, dissociated from the spinal cords at E50, P2, P20, P28, and P50, were transplanted into the spinal cords of myelin-deficient rats as single cell suspensions. Both cryopreserved (E50 and P28) and freshly dissociated tissue (P2, P20, and P50) were able to form myelin within 13 days of transplantation. Cells from younger donors (< P20) myelinated more md axons than those from older donors. In those rats which received xenografts and which were treated with cyclosporin A there was markedly less cellular infiltration than in untreated animals. For comparison with these xenografts, fresh and cryopreserved adult rat glia were also transplanted. Eight days after transplantation, myelination by allografts of cryopreserved rat glia was qualitatively similar to that produced by freshly prepared cells. These results show that oligodendrocytes transplanted as xenografts are capable of myelinating rat axons, and that cryopreserved glia retain their capacity to myelinate in vivo.

Changes in slow axonal transport of tubulin induced by local application of colchicine to rabbit vagus nerve.

The biochemical and morphological responses of the rabbit vagus nerve to local application of colchicine and to nerve crush were investigated. Fourteen days after the cervical vagus nerve had been crushed or subjected to local application of colchicine for 2 h, nodose ganglia of anaesthetized rabbits were either injected with [35S]methionine or [3H]leucine for studies of slow and fast axonal transport, respectively, or prepared for light microscopical examination. The radio-labelled proteins of the faster of the two slow transport groups (SCb; 25-30 mm day-1) were separated by one- or two-dimensional polyacrylamide gel electrophoresis and both radio-labelled tubulin and actin were quantified by densitometry from resulting fluorographs of gels. A relative increase in radio-labelled tubulin was found in SCb in the crushed and colchicine-treated nerves; this increase persisted for up to 50 days after nerve crush. Morphological changes in nerve cell bodies induced by colchicine were similar, but smaller in magnitude than those in crushed nerves. It is concluded that a temporary arrest of axonal transport produced by colchicine can lead to a redistribution of tubulin transport comparable with that found in regenerating nerve.

Transplantation of glial cells enhances action potential conduction of amyelinated spinal cord axons in the myelin-deficient rat.

A central issue in transplantation research is to determine how and when transplantation of neural tissue can influence the development and function of the mammalian central nervous system. Of particular interest is whether electrophysiological function in the traumatized or diseased mammalian central nervous system can be improved by the replacement of cellular elements that are missing or damaged. Although it is known that transplantation of neural tissue can lead to functional improvement in models of neurological disease characterized by neuronal loss, less is known about results of transplantation in disorders of myelin. We report here that transplantation of glial cells into the dorsal columns of neonatal myelin-deficient rat spinal cords leads to myelination and a 3-fold increase in conduction velocity. We also show that impulses can propagate into and out of the transplant region and that axons myelinated by transplanted cells do not have impaired frequency-response properties. These results demonstrate that myelination following central nervous system glial cell transplantation enhances action potential conduction in myelin-deficient axons, with conduction velocity approaching normal values.

Axonal transport and morphological changes following nerve compression. An experimental study in the rabbit vagus nerve.

Axonal transport and morphological changes were studied in the rabbit vagus nerve after the nerves had been subjected to compression at either 0, 50 or 200 mmHg for two hours. Slow axonally transported proteins, tubulin and actin, were radiolabelled with 35S-methionine two, seven or 14 days after the injury and the distribution of radiolabelled tubulin and actin within component b of slow transport was measured three days later by densitometric analysis of fluorographs of polyacrylamide gel. No significant differences were found in the distribution of tubulin two (50 and 200 mmHg) or seven (200 mmHg) days after injury, but at 14 days (200 mmHg) there was significantly increased radiolabelling of tubulin relative to actin in the nerve 60 to 70 mm from the nodose ganglion. Morphometric measurements of the nerve cell bodies two days after the compression injury at 200 mmHg revealed no significant changes. Previous work has shown that morphological changes, similar to those found after axotomy, were present in nerve cell bodies seven days after a compression injury. This, taken together with the present results, indicates that compression can induce both morphological and biochemical changes in the neurone. The altered axonal transport of tubulin associated with nerve injury follows a slower time course and does not precede the morphological changes. The findings may be of relevance when discussing the double crush syndrome.

Functional capacities of transplanted cell-sorted adult oligodendrocytes.

Previous work has shown that transplanted dissociated glial cells derived from neonatal rats can myelinate areas of the myelin deficient (md) rat spinal cord. In the present studies we have examined the ability of adult glial cells, either as suspension of mixed glial cells or as 01 positive oligodendrocytes purified by cell sorting, to myelinate md rat axons following transplantation. Mixed glial cell preparations from adult rats myelinated large areas of either, dorsal, lateral or ventral columns. These areas contained clumped, transplanted oligodendrocytes. In addition, preparations containing over 90% 01 positive oligodendrocytes were also capable of myelinating large numbers of axons upon transplantation. These results suggest that adult oligodendrocytes may play an important role in remyelination of the adult CNS.

Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport.

In the brains of aged humans and cases of Alzheimer disease, deposits of amyloid in senile plaques are located in proximity to nerve processes. The principal component of this extracellular amyloid is beta/A4, a peptide derived from a larger amyloid precursor protein (APP), which is actively expressed in brain and systemic organs. Mechanisms that result in the proteolysis of APP to form beta/A4, previously termed beta-amyloid protein, and the subsequent deposition of the peptide in brain are unknown. If beta/A4 in senile plaques is derived from neuronally synthesized APP and deposited at locations remote from sites of synthesis, then APP must be transported from neuronal cell bodies to distal nerve processes in proximity to deposits of amyloid. In this study, using several immunodetection methods, we demonstrate that APP is transported axonally in neurons of the rat peripheral nervous system. Moreover, our investigations show that APP is transported by means of the fast anterograde component. These findings are consistent with the hypothesis of a neuronal origin of beta/A4, in which amyloid is deposited in the brain parenchyma of aged individuals and cases of Alzheimer disease. In this setting, we suggest that APP is synthesized in neurons and delivered to dystrophic nerve endings, where subsequent alterations of local processing of APP result in deposits of brain amyloid.

Changes in fast axonally transported proteins in the regenerating rabbit vagus nerve.

Regeneration was induced in rabbit vagus nerves by a crush injury. Fourteen and 50 days later [35S]methionine-radiolabelled, fast axonally transported proteins were separated by one- and two-dimensional electrophoresis. Quantitative densitometric analysis of fluorographs from one-dimensional separations showed increases in the radiolabelling of fast transported proteins of 45 and 20 kDa, and a decrease in the radiolabelling of a 26 kDa protein 14 days after crush injury, which were confirmed by two-dimensional separations. These increases were maintained at 50 days post-crush. The proteins have similar molecular weights and isoelectric points to two growth-associated proteins known to have important roles in growth and regeneration.

Treatment with an aldose reductase inhibitor can reduce the susceptibility of fast axonal transport following nerve compression in the streptozotocin-diabetic rat.

The effect of treatment with an aldose reductase inhibitor on the susceptibility of peripheral nerves to compression was studied in rats made diabetic by the injection of streptozotocin (50 mg.kg-1). The response to nerve compression was determined in untreated diabetic rats after 22 days of diabetes and compared with the response in two similar groups of diabetic rats which had been treated with the aldose reductase inhibitor 'Statil' (ICI 128436; 25 mg.kg-1.day-1 orally) either from the induction of diabetes or for 7 days prior to nerve compression. Two groups of non-diabetic rats were treated with 'Statil' for either 22 days or 7 days to act as controls. Inhibition of fast axonally transported proteins was induced by local compression of the sciatic nerves 4 h after application of 3H-leucine to the motor neurone cell bodies in the spinal cord. The inhibition of fast axonal transport was quantified by calculation of a transport block ratio. Compression at 30 mmHg for 3 h induced a significantly greater (p less than 0.05) inhibition of axonal transport at the site of compression in nerves of untreated diabetic rats (transport block ratio 0.96 +/- 0.24, n = 8) than in nerves of control rats treated with the aldose reductase inhibitor for either the shorter time of 7 days (0.71 +/- 0.17, n = 10) or the longer time of 22 days (0.69 +/- 0.08, n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Monoamine oxidase activities in dissociated cell fractions from rat skeletal muscle.

Collagenase was used to dissociate rat skeletal muscle (gastrocnemius) into its constituent cells, from which a myocyte fraction enriched in striated muscle cells, and a non-myocyte fraction containing cells of connective tissue and vascular origin, were prepared. The activities of amine oxidase enzymes and alkaline phosphatase (AP) were then assayed in these fractions, and also in homogenates prepared from corresponding samples of non-dissociated tissue. The specific activities of the semicarbazide-sensitive amine oxidase (SSAO) and AP were considerably higher (30 to 35-fold) in non-myocyte than in myocyte fractions. AP is generally considered to be present predominantly in vascular cells of skeletal muscle, with little, if any, in skeletal muscle cells themselves. Thus, the results obtained may indicate a similar localization for SSAO activity. Support for this came from histochemical studies, which showed staining for SSAO primarily over the walls of larger blood vessels in the muscle. Unlike SSAO and AP, were marked differences in MAO-A activity between myocyte and non-myocyte fractions were not found, suggesting that MAO-A is more probably a constituent of cells within both fractions.