Disseminated sparganosis in a cynomolgus macaque (Macaca fascicularis).

An adult male cynomolgus macaque (Macaca fascicularis) from Mauritius arrived at our facility in France after a 1-year period of quarantine in Spain. Clinical examination soon after arrival revealed the presence of numerous firm cutaneous and subcutaneous nodules (0.1-0.5 cm diameter) in the scrotal and inguinal areas, and persistent mild eosinophilia. On necropsy examination additional similar nodules were found in the peritoneum and abdominal wall, omentum and mesentery. Microscopical examination revealed disseminated eosinophilic granulomas containing tapeworm larvae identified as Spirometra erinaceieuropaei by direct sequencing of the cox1 gene.

Absence of an intrathecal immune reaction to a helper-dependent adenoviral vector delivered into the cerebrospinal fluid of non-human primates.

Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration.

[Promoting myelin repair in disorders such as multiple sclerosis and some types of leukodystrophy: current studies]. / Approche expérimentale des stratégies réparatrices des maladies demyélinisantes du système nerveux central.

Several ways of promoting myelin repair in myelin disorders such as multiple sclerosis and certain types of leukodystrophies are currently being investigated. Numerous studies suggest that it is possible to repair the central nervous system (CNS) by cell transplantation or by enhancing endogenous remyelination. Investigations in animal models indicate that cell therapy results in robust anatomical and functional recovery of acute myelin lesions. These models are also used to explore and validate the role of candidate molecules to stimulate endogenous remyelination by activating the myelin competent population or providing neuroprotection. However, in view of the heterogeneity of the lesion environment in MS, it seems more likely that cell therapy alone will not be able to contribute efficiently to the repair of the lesion. Further developments should indicate whether combining multiple approaches will be more powerful to achieve global myelin repair in the CNS than applying these strategies alone.

Migration and multipotentiality of PSA-NCAM+ neural precursors transplanted in the developing brain.

By optimizing the previously described strategy for obtention of spheres enriched in PSA-NCAM+ precursors, we prepared PSA-NCAM-immunoselected cell populations from cerebral hemispheres of neonatal MBP-LacZ transgenic mice. These cells expressed Nestin, exhibited clonal expansion potential and formed spheres, which were initially enriched in PSA-NCAM+ cells but became enriched in GD3+ oligodendrocyte progenitors after 1 week in B104 contionned medium. One month after their periventricular transplantation into the brain of wild-type and/or shiverer newborn mice, cells from PSA-NCAM+ spheres exhibited a higher rostral migration potential than cells from GD3+ spheres, and clearly contributed to myelination in the olfactory bulb. In shiverer hosts, both sphere populations generated oligodendrocytes with similar myelination potential. In addition PSA-NCAM+ sphere cells generated GFAP+ astrocytes and NeuN+ neurons, depending on their site of insertion. These results evidence the high plasticity of newborn PSA-NCAM+ neural precursors and suggest that they are promising tools for cell therapy of CNS diseases, including myelin disorders.

Developmental pattern of expression of the alpha chemokine stromal cell-derived factor 1 in the rat central nervous system.

Stromal cell-derived factor 1 (SDF-1) is an alpha-chemokine that stimulates migration of haematopoietic progenitor cells and development of the immune system. SDF-1 is also abundantly and selectively expressed in the developing and mature CNS, as we show here. At embryonic day 15, SDF-1 transcripts were detected in the germinal periventricular zone and in the deep layer of the forming cerebral cortex. At birth, granule cells in the cerebellum and glial cells of the olfactory bulb outer layer showed an SDF-1 in situ hybridization signal that decreased progressively within the next 2 weeks. In other regions such as cortex, thalamus and hippocampus, SDF-1 transcripts detected at birth progressively increased in abundance during the postnatal period. SDF-1 protein was identified by immunoblot and/or immunocytochemistry in most brain regions where these transcripts were detected. SDF-1 was selectively localized in some thalamic nuclei and neurons of the fifth cortical layer as well as in pontine and brainstem nuclei which relay the nociceptive response. The presence of SDF-1 transcripts in cerebellar granule cells was correlated with their migration from the external to the inner granular layers with disappearance of the signal when migration was completed. In contrast, SDF1 mRNA signal increased during formation of the hippocampal dentate gyrus and stayed high in this region throughout life. The selective and regulated expression of SDF-1 in these regions suggests a role in precursor migration, neurogenesis and, possibly, synaptogenesis. Thus this alpha chemokine may be as essential to nervous system function as it is to the immune system.

Do central nervous system axons remyelinate?

In multiple sclerosis (MS), one of the most frequent demyelinating diseases in man, remyelination of demyelinating lesions exists but is often incomplete. Also reported in experimental models of demyelination, this phenomenom confirms the regenerating potential of the demyelinated central nervous system (CNS) and, in particular, the existence of an endogenous mechanism of oligodendrocyte renewal. Failure in efficient remyelination could result from exhaustion of the pool of remyelinating cells, loss of axons and absence of a permissive environment for remyelination. Identifying the nature and the origin of the cells capable of generating new oligodendrocytes for remyelination could contribute to strategies to activate these cells, and thereby enhance their potential for myelin repair. Within the adult CNS, several cell types are capable of generating new oligodendrocytes following myelin damage: post-mitotic oligodendrocytes frequently found at the lesion site, oligodendrocyte progenitors whose existence has been confirmed both in vitro and in vivo, and multipotent cells localized in the germinative areas of the brain and the spinal cord. Although restricted to particular sites of the CNS, these multipotent cells, which maintain the capacity to self-renew and to migrate throughout adulthood, could constitute a powerful source of remyelinating cells. The study of the mechanisms of proliferation, migration and differentiation of these cells in response to demyelination should allow the definition of new strategies to promote endogenous remyelination and develop therapeutic approaches for demyelinating diseases such as MS. This goal is an appealing alternative to the transplantation of myelin-forming cells and should efficiently complement strategies aimed at reducing neuronal loss and inflammation.

Neurosteroid progesterone is up-regulated in the brain of jimpy and shiverer mice.

Concentrations of neurosteroids have been measured in the brains of postnatal myelin mutants jimpy (jp) and shiverer (shi) mice and of their normal controls. Progesterone (PROG) concentrations were increased more than threefold in the brains of mutant mice. Marked astroglial reaction occurs in the brains of jp mice and to a much smaller extent in shi ones. Whereas the mitochondrial benzodiazepine/diazepam binding inhibitor (DBI) receptor (MBR) was below the immunohistochemical detection limit in normal mice (except in the choroid plexus and ependyma cells), it was significantly expressed in many reactive astrocytes of jp and shi mice brains. DBI-like peptides, investigated either by immunohistochemistry or by radioimmunoassay, were expressed to similar extents in mutant and control mice. Reversed-phase HPLC indicated that DBI-like peptides were almost exclusively of the triakontatetraneuropeptide size. It was concluded that the increased expression of MBR (involved in the intramitochondrial delivery of cholesterol to P450scc) likely accounts for the large PROG content in mutant mice brain. The role of PROG in myelin repair is discussed.

Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination.

Identifying a source of cells with the capacity to generate oligodendrocytes in the adult CNS would help in the development of strategies to promote remyelination. In the present study, we examined the ability of the precursor cells of the adult mouse subventricular zone (SVZ) to differentiate into remyelinating oligodendrocytes. After lysolecithin-induced demyelination of the corpus callosum, progenitors of the rostral SVZ (SVZa) and the rostral migratory pathway (RMS), expressing the embryonic polysialylated form of the neural cell adhesion molecule (PSA-NCAM), increased progressively with a maximal expansion occurring after 2 weeks. This observation correlated with an increase in the proliferation activity of the neural progenitors located in the SVZa and RMS. Moreover, polysialic acid (PSA)-NCAM-immunoreactive cells arizing from the SVZa were detected in the lesioned corpus callosum and within the lesion. Tracing of the constitutively cycling cells of the adult SVZ and RMS with 3H-thymidine labelling showed their migration toward the lesion and their differentiation into oligodendrocytes and astrocytes but not neurons. These data indicate that, in addition to the resident population of quiescent oligodendrocyte progenitors of the adult CNS, neural precursors from the adult SVZ constitute a source of oligodendrocytes for myelin repair.

Mouse oligospheres: from pre-progenitors to functional oligodendrocytes.

To study the biology and repair capacities of mouse oligodendroglial cells, we established cultures of cells purified from neonatal wild-type and 9.6-kb MBP-LacZ transgenic newborn mice cerebral hemispheres as free-floating aggregates in the continuous presence of neuroblastoma conditioned medium (N1-B104). In vitro analysis indicated that the initial cell preparations were enriched in oligodendrocyte pre-progenitors that expressed PSA-NCAM and GAP-43 but not GD3, O4, NF68 or glial fibrillary acidic protein (GFAP) markers. These pre-progenitors required increased concentrations of insulin and progesterone to allow their survival in vitro. With time in culture, spheres composed of oligodendrocyte pre-progenitors became oligospheres enriched in oligodendrocyte progenitors expressing GAP-43 and GD3. As well as conserving bipotentiality in vitro, these spheres were able to form myelin in vivo after transplantation into the neonatal shiverer mouse brain. Thus, the oligosphere strategy is a powerful method for generating large populations of mouse oligodendrocyte pre-progenitors and progenitors. The ability to generate oligospheres from transgenic mice will be instrumental in the further dissection of the molecular and cellular mechanisms of myelination and remyelination of the central nervous system.

In vitro and in vivo behaviour of NDF-expanded monkey Schwann cells.

Schwann cells, the myelin-forming cells of the peripheral nervous system may play a major role in the regeneration and remyelination not only of the peripheral but also of the central nervous system. The discovery of the mitogenicity of human recombinant forms of neuregulins (glial growth factors) on primate Schwann cells allows us to envisage a considerable expansion of these cells in culture with a view to autologous transplantation in the central nervous system. To assay this possibility, we used human recombinant neu-differentiation factor beta (NDFbeta) to expand monkey Schwann cells derived from perinatal and adult nerve biopsies. We report that NDFbeta containing the epidermal growth factor (EGF)-like domain (residues 177-228) is a potent mitogen for monkey Schwann cells but is more effective on perinatal than adult Schwann cells. Moreover, continuous treatment with NDFbeta, does not seem to prevent Schwann cells differentiation into myelin-forming cells after their transplantation into the demyelinated mouse spinal cord. These observations, in addition to the close similarities of in vitro behaviour which exist between human and monkey Schwann cells, indicate that monkey Schwann cells could be an ideal tool to study the potential and limits of autologous transplantation in a non-human primate model of central nervous system demyelination.

Dendritic cells route human immunodeficiency virus to lymph nodes after vaginal or intravenous administration to mice.

We have developed a murine model to study the involvement of dendritic cells (DC) in human immunodeficiency virus (HIV) routing from an inoculation site to the lymph nodes (LN). Murine bone marrow-derived DC migrate to the draining LN within 24 h after subcutaneous injection. After incubation of these cells with heat-inactivated (Hi) HIV type 1 (HIV-1), HIV RNA sequences were detected in the draining LN only. Upon injection of DC pulsed with infectious HIV, the virus recovered in the draining LN was still able to productively infect human T cells. After a vaginal challenge with Hi HIV-1, the virus could be detected in the iliac and sacral draining LN at 24 h after injection. After an intravenous challenge, the virus could be detected in peripheral LN as soon as 30 min after injection. The specific depletion of a myeloid-related LN DC population, previously shown to take up blood macromolecules and to translocate them into the LN, prevented HIV transport to LN. Together, our data demonstrate the critical role of DC for HIV routing to LN after either a vaginal or an intravenous challenge, which does not require their infection. Therefore, despite the fact that the mouse is not infectable by HIV, this small animal model might be useful to test preventive strategies against HIV.

Schwann cell transplantation and myelin repair of the CNS.

Studies with experimental models of dysmyelination and demyelination have shown that rodent Schwann cells including a Schwann cell line, transplanted in the central nervous system compete with host oligodendrocytes to remyelinate denuded central axons of the spinal cord. The myelin produced by transplanted SC around these central nervous system axons is structurally normal and restores, secure nerve conduction. In the presence of a favorable substrate, transplanted Schwann cells migrate over considerable distances (several mm) and are recruited by a demyelinated lesion which they will partially repair Thus Schwann cells, which can also support axonal growth, may be instrumental in central nervous system repair. In addition, the possibility of obtaining large quantities of human and non-human primate Schwann cells, makes it possible to consider autologous Schwann cell transplantation as a potential therapy for demyelinating or traumatic diseases. The various differences which may exist between rodents and humans, however, require further investigation of this possibility in a non-human primate model of demyelination. These experiments should provide not only insights on the potential of autologous transplantation in primates but also a better understanding of the process of central remyelination.

Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential.

The limited availability of enriched populations of oligodendroglial progenitors has impeded the exploration of the complex spatio-temporal mechanisms which dictate the chemical "language" of their biology. We have developed a technique to prepare homotypic aggregates of oligodendrocyte progenitors called "oligospheres." These were obtained using various approaches (sieving, Percoll gradient separation and differential adhesion) to purify oligodendroglial progenitors from newborn rat brain. Culturing cells in a mixture of N1 defined medium and conditioned medium from the B104 neuronal cell line in the absence of adhesive substrate allowed to expand routinely and extensively for several months, the oligodendrocyte progenitor population. Under these conditions, the resulting population consisted of 98% GD3-positive/GFAP-negative cells. After dissociation and plating on polyornithine coated substrates, in the presence of low (2%) or high (20%) serum, oligosphere-derived oligodendrocyte progenitors were induced to differentiate into GalC-positive oligodendrocytes or GFAP-positive astrocytes, respectively. When transplanted into the newborn shiverer mouse brain, oligospheres were able to provide a focal reservoir of migrating and myelinating cells. Oligospheres are thus ideal tools for exploring the biological and molecular events of the oligodendrocyte lineage both in vitro and in vivo.

Order-disorder phenomena in myelinated nerve sheaths. VI. The effects of quaking, jimpy and shiverer mutations: an X-ray scattering study of mouse sciatic and optic nerves.

We report the X-ray scattering study of sciatic and optic nerve myelin from shiverer, jimpy and quaking mice mutants and from the corresponding controls. These three mutations are known to affect dramatically central nervous system (CNS) myelin and to induce comparatively minor alterations in peripheral nervous system (PNS) myelin. Scattering experiments and data reduction were carried out using the techniques and algorithms developed in our laboratory and previously applied to several problems involving the structure of myelin. In sciatic nerve the fraction of myelin elementary pairs of membranes (total myelin) decreases in shiverer and quaking nerves (by approximately 30%) but not in jimpy nerves; in all three mutants the fraction of myelin membrane pairs that are not regularly stacked in the sheaths (loose myelin), the average number of membranes per sheath and the packing disorder are the same as in the control nerves; the repeat distance D and the membrane distance Dcyt across the cytoplasmic space increase in shiverer and decrease in jimpy; in quaking, D also decreases and the decrease is smaller than in jimpy and is not specific for Dcyt; small changes are also observed in the electron density profiles. As for the optic nerve the myelin content decreases dramatically in the three mutants; the very weak signal attests to a tiny amount of pairs of membranes structurally similar to normal CNS myelin. It is surprising that the structure of CNS myelin should be almost normal in the absence of the major structural components, namely myelin basic protein (MBP) for shiverer of proteolipid protein (PLP) for jimpy. The question arises whether the composition of the residual pairs of membranes, operationally identified as myelin in the X-ray scattering experiments, mirrors the composition determined by chemical means on the fraction of nerve tissue histologically identified as myelin, or whether in all circumstances it remains approximately the same.

Myelination by transplanted human and mouse central nervous system tissue after long-term cryopreservation.

Human and mouse oligodendrocytes were transplanted, after a long period of cryostorage, into newborn mouse brain. Tissue fragments were obtained from brain and spinal cord of 10-week-old human fetuses and from the periventricular zone of embryonic and newborn mouse brains. Samples were stored at -180 degrees C for periods of 3 days to over 5 years. Frozen or fresh fragments were transplanted into the brains of newborn shiverer mutant mice, which are deficient in myelin basic protein (MBP). Normal myelin, produced by grafted oligodendrocytes, was detected by immunohistochemistry with an anti-MBP antiserum. The best results were obtained with isospecific grafts. The timing of myelin appearance did not depend significantly on the species or age of the donor. Myelination obtained with mouse grafts was more profuse when the donor was younger (embryonic versus newborn). Cryopreservation over 5 years did not impede the graft's ability to produce myelin and can be considered for long-term storage of oligodendrocytes in view of cell therapy.

Glial transplants: an in vivo analysis of extrinsic and intrinsic determinants of dysmyelination in genetic variants.

Myelination in the CNS depends on the ability of oligodendrocytes (Ols) to efficiently colonize the brain, differentiate, and express a precise balance of specific genes necessary for myelin synthesis. Mutations in these genes produce different types of dysmyelination in animal as in human. Defects in the synthesis of myelin constituents usually lead to mild dysmyelinations. IN contrast, mutations affecting the gene encoding the proteolipid, another major protein of myelin, produce various perturbations of Ols biology suggesting a pleiotropic effect of the gene in the development of the CNS. Studies on expansion of cell population and survival have provided contradictory information on the extrinsic and intrinsic action of the gene on Ols biology. On one hand, in vitro studies using conditioned media as in vivo studies on heterozygotes, and transplantations experiments suggest that excess of programmed cell death in these mutants is ruled out by intrinsic factors which could act during embryonic life. On the other hand, attempts to compensate the gene defect by transgenic correction demonstrate a dominant negative effect of the jp mutation on both survival and functional potential of Ols. Finally, total suppression of PLP gene expression has a restricted effect on myelin structure without excess of cell death. These contradictory results are discussed in the perspective of regulation of cell death by competition for growth factors in limiting amount. The proposed model suggests that this contradiction is only apparent, and that excess of cell death in PLP/DM20 mutant is intrinsically determined by diminished competitivity of the mutant Ols for limited amounts of environmental factors.