Extensive remyelination of the CNS leads to functional recovery.

Remyelination of the CNS in multiple sclerosis is thought to be important to restore conduction and protect axons against degeneration. Yet the role that remyelination plays in clinical recovery of function remains unproven. Here, we show that cats fed an irradiated diet during gestation developed a severe neurologic disease resulting from extensive myelin vacuolation and subsequent demyelination. Despite the severe myelin degeneration, axons remained essentially intact. There was a prompt endogenous response by cells of the oligodendrocyte lineage to the demyelination, with remyelination occurring simultaneously. Cats that were returned to a normal diet recovered slowly so that by 3-4 months they were neurologically normal. Histological examination of the CNS at this point showed extensive remyelination that was especially notable in the optic nerve where almost the entire nerve was remyelinated. Biochemical analysis of the diet and tissues from affected cats showed no dietary deficiencies or toxic accumulations. Thus, although the etiology of this remarkable disease remains unknown, it shows unequivocally that where axons are preserved remyelination is the default pathway in the CNS in nonimmune-mediated demyelinating disease. Most importantly, it confirms the clinical relevance of remyelination and its ability to restore function.

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.

Embryonic stem cell-derived glial precursors: a source of myelinating transplants.

Self-renewing, totipotent embryonic stem (ES) cells may provide a virtually unlimited donor source for transplantation. A protocol that permits the in vitro generation of precursors for oligodendrocytes and astrocytes from ES cells was devised. Transplantation in a rat model of a human myelin disease shows that these ES cell-derived precursors interact with host neurons and efficiently myelinate axons in brain and spinal cord. Thus, ES cells can serve as a valuable source of cell type-specific somatic precursors for neural transplantation.

In vitro differentiation of transplantable neural precursors from human embryonic stem cells.

The remarkable developmental potential and replicative capacity of human embryonic stem (ES) cells promise an almost unlimited supply of specific cell types for transplantation therapies. Here we describe the in vitro differentiation, enrichment, and transplantation of neural precursor cells from human ES cells. Upon aggregation to embryoid bodies, differentiating ES cells formed large numbers of neural tube-like structures in the presence of fibroblast growth factor 2 (FGF-2). Neural precursors within these formations were isolated by selective enzymatic digestion and further purified on the basis of differential adhesion. Following withdrawal of FGF-2, they differentiated into neurons, astrocytes, and oligodendrocytes. After transplantation into the neonatal mouse brain, human ES cell-derived neural precursors were incorporated into a variety of brain regions, where they differentiated into both neurons and astrocytes. No teratoma formation was observed in the transplant recipients. These results depict human ES cells as a source of transplantable neural precursors for possible nervous system repair.

Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells.

Magnetic resonance (MR) tracking of magnetically labeled stem and progenitor cells is an emerging technology, leading to an urgent need for magnetic probes that can make cells highly magnetic during their normal expansion in culture. We have developed magnetodendrimers as a versatile class of magnetic tags that can efficiently label mammalian cells, including human neural stem cells (NSCs) and mesenchymal stem cells (MSCs), through a nonspecific membrane adsorption process with subsequent intracellular (non-nuclear) localization in endosomes. The superparamagnetic iron oxide nanocomposites have been optimized to exhibit superior magnetic properties and to induce sufficient MR cell contrast at incubated doses as low as 1 microg iron/ml culture medium. When containing between 9 and 14 pg iron/cell, labeled cells exhibit an ex vivo nuclear magnetic resonance (NMR) relaxation rate (1/T2) as high as 24-39 s-1/mM iron. Labeled cells are unaffected in their viability and proliferating capacity, and labeled human NSCs differentiate normally into neurons. Furthermore, we show here that NSC-derived (and LacZ-transfected), magnetically labeled oligodendroglial progenitors can be readily detected in vivo at least as long as six weeks after transplantation, with an excellent correlation between the obtained MR contrast and staining for beta-galactosidase expression. The availability of magnetodendrimers opens up the possibility of MR tracking of a wide variety of (stem) cell transplants.

Mature results of a randomized trial of two doses of cisplatin for the treatment of ovarian cancer. Scottish Gynecology Cancer Trials Group.

PURPOSE: In 1992, we reported the first results of a randomized study in ovarian cancer, comprising two doses of cisplatin and indicated a significant difference (P = .0008) in median survival. Four years later, we now describe the results of this trial. PATIENTS AND METHODS: After a median follow-up of 4 years and 9 months, 115 of 159 cases of advanced ovarian cancer, originally randomized to receive six cycles of cyclophosphamide 750 mg/m2 and either a high dose (HD) of 100 mg/m2 cisplatin or a low dose (LD) of 50 mg/m2 (LD) cisplatin, have now died. RESULTS: The overall survival for HD and LD patients is 32.4% and 26.6%, respectively, and the overall relative death rate is 0.68 (P = .043). This represents a reduction in overall benefit with longer follow-up compared with the first 2 years (relative death rate of 0.52). Toxicity, particularly neurotoxicity, is still evident in the fourth year (10/31 on HD compared with 1/24 on LD). CONCLUSION: Our recommended dose of cisplatin in combination schedule is therefore 75 mg/m2, representing the optimal balance between efficacy and toxicity.

Glial cell transplants: experimental therapies of myelin diseases.

Transplantation of cells into the CNS of human patients with neurodegenerative disorders offers a radical new approach to the treatment of previously incurable diseases. Considerable success has been achieved in Parkinson's disease following transplantation of human fetal dopaminergic neurons. Disorders of myelination of the brain, of either inherited or acquired origin, might also be treated by glial cell transplantation although there are additional challenges. Cells of the oligodendrocyte lineage have been found to be capable of myelinating axons on transplantation into numerous experimental pathological environments, including the CNS of myelin mutants and focal areas of demyelination in normal animals made by injection of myelinotoxic chemicals. In general, primary cells and progenitors are likely to have the greatest myelinating capacity. Cell lines can also be used, but those driven by oncogenes may produce little myelin, and tumor formation is likely. Schwann cells are also a potential source of cells, possibly as a homograft, and may be primed by treatment ex vivo with glial growth factors. The variable CNS milieu seen in human myelin disease will mean that transplanted cells must be able to migrate appropriately and myelinate axons in an adult, pathological environment, and this awaits experimental confirmation. Physiological analysis of transplants in such situations in adult animals will provide the functional data which may expedite clinical trials.

Early postnatal development of glial cells in the canine cervical spinal cord.

To study qualitative and quantitative changes in the glial cell population of young postnatal dogs, the cervical spinal cords of 20 beagle pups, ranging in age from 1 to 28 days, were prepared for light and electron microscopy. Glial cells in the lateral corticospinal tract were classified and quantified directly on the electron microscope. Quantification was performed by means of a stereological method designed to correct for sampling bias, and glia were classified according to morphological criteria as immature glial cell precursors, light and dark oligodendrocytes, astrocytes, and microglia. Glial cell precursors, which include undifferentiated glioblasts, oligodendroblasts, and astroblasts, predominated in the first few days after birth, constituting 43% of the glial cell population, and then declined to less than 5% by 28 days. Light and dark oligodendrocytes differed morphologically in their electron density and the appearance of their organelles. Light oligodendrocytes increased slightly prior to myelination, and then declined, whereas dark oligodendrocytes continued to increase throughout the 4-week period and became the predominant cell type at 28 days (66%). In contrast to the oligodendroglial population, the sizes of the astroglial and microglial cell populations were relatively stable. This study shows that the population of immature glial cell precursors, abundant at birth in the lateral corticospinal tract, appear to be differentiating primarily into oligodendroglia, because this population exhibits a rapid increase in size, and relatively little change occurs in the astrocyte population. The trends in glial cell development in the dog are similar to those reported for rodents, although there may be some variation in the maturation and activity of oligodendrocytes.

Oligodendrocytes are not inherently programmed to myelinate a specific size of axon.

Current studies support the morphological classification of oligodendrocytes proposed by Del Rio Hortega ([1922] Bol. R. Soc. Esp. Hist. Nat. 10:25-29; [1924] C.R. Soc. Biol. 91:818-820), in which cells either myelinate multiple internodes that are associated with small axons, or they myelinate restricted/single internodes of large-diameter axons. The reasons why an oligodendrocyte myelinates a particular calibre of axon are unknown. Because progenitors are generated in restricted, subventricular zones, an intrinsic program would imply that germinal centres contain a mixture of cells, each committed to myelinate axons of a particular size. Conversely, each cell could have the potential ability to myelinate any size axon. We tested this latter hypothesis that oligodendrocyte progenitors are uncommitted in their ability to myelinate a particular axon size. We introduced oligodendrocyte lineage cells from the optic nerve, which normally encounter only small-diameter axons, to a myelin-deficient environment containing a large range of axon sizes. Dissociated, mixed glial cells from the optic nerve were characterised immunocytochemically and were grafted into the spinal cord ventral column of neonatal, myelin-deficient rat mutants. Examination of the patches of myelin produced by these cells at different times after transplantation revealed that optic nerve oligodendrocytes were capable of producing a widespread, nonselective myelination of axons that were destined to have both small or large calibres. Thus, an axonal or local signal, and not an intrinsic program, is probably responsible for the previously described oligodendrocyte diversity.

The immune status of the myelin deficient rat and its immune responses to transplanted allogeneic glial cells.

This study examined the immunological responsiveness of the myelin deficient (md) rat, and its immune response to transplanted allogeneic glial cells, with and without immunosuppression therapy. Skin grafts from an ACI strain of rat were found to be acutely rejected by Wistar md rats. Anti-donor cytotoxic antibody was produced and alloreactive T helper cells were expanded in these mutants after skin sensitization. Equivalent high frequencies of precursor cytotoxic T lymphocytes (pCTLs) specific to the ACi MHC antigens were observed in both normal controls and mutants. An immune response was noted when allogeneic glial cells were transplanted into the spinal cords of md rats, which was effectively suppressed for 2 weeks post transplantation by treatment with either cyclosporin A (CsA) or a monoclonal antibody to the interleukin-2 receptor (IL-2R). These results demonstrate that md rats are immunocompetent, but that CNS allograft rejection can be prevented by immunosuppressive agents.

Isolation of actinomycetes from cervical specimens.

Cervical smears from 30 women were examined. Fifteen of these had actinomycetes visible by direct smear examination and the organisms were isolated in 13. Of the remainder, all had negative smears and culture was similarly negative in 12, whilst in 3, the cultures were either positive (1) or suspicious (2).

Myelin mutants: new models and new observations.

The myelin mutants have been extensively used as tools to study the complex process of myelination in the central and peripheral nervous system. A multidisciplinary approach to the study of these models ultimately allows a correlation to be made between phenotype and genotype. This correlation may then lead to the formation of new hypotheses about the functions of the products of genes involved in myelination. This review presents a number of new myelin mutants which have recently been described. The species involved include mouse, rat, rabbit, hamster, and dog models. The genetic defect has not been elucidated in all of these animals, but most have been characterized clinically and pathologically, and, in some cases, biochemically. In addition, a better known myelin mutant, the trembler mouse, is discussed. Recent molecular findings have brought this fascinating mutant to the forefront of the field of peripheral nervous system research. The range of abnormalities in the mutants described in this review includes defects in specific myelin proteins, suspected abnormalities in membrane formation, and apparent defects of the oligodendrocyte cytoskeleton. These findings underscore the complexity of the myelination process and highlight the numerous ways in which it can be disrupted.

Abnormal expression of SNS/PN3 sodium channel in cerebellar Purkinje cells following loss of myelin in the taiep rat.

Using in situ hybridization and immunochemical methods, we have observed an increase in the expression of SNS/PN3 sodium channel mRNA and protein in cerebellar Purkinje cells of the taiep rat. These changes are present in taiep rats at 12 months of age, following loss of myelin, but not at one month, prior to loss of myelin. Increased SNS/PN3 expression is not associated with aging per se, because it was not observed in control rats at 12 months of age. These results suggest that altered sodium channel expression in Purkinje cells may contribute to the ataxia that occurs in taiep rats.

Fertility after cryosurgery of the cervix.

Fertility in patients following cryosurgery of the cervix was evaluated in 412 women. Cryosurgery does not appear to have any effect on subsequent fertility when compared with the general population.

Myelin proteins in the CNS of 'shaking pups'.

Myelin proteins were quantitated in whole tissue and isolated 'myelin fractions' from spinal cord, brainstem and hemispheres of 'shaking pups', a mutation in Springer-Spaniel dogs characterized by hypomyelination of the CNS. The amount of myelin basic protein (MBP) in the brainstem of affected 4-week-old pups was 2.6% of that in age-matched controls, while the levels of 2'3'-cyclic nucleotide phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG) were 10% and 15% of the control levels, respectively. Similar results were obtained in the spinal cord and hemispheres, and the amounts of these proteins in the mutant pups did not change substantially between 4 and 16 weeks of age. The amount of the 21 kDa MBP compared to the 18 kDa MBP was relatively increased in the shaking pups, suggesting that the small amount of myelin formed was immature. The yields of myelin fractions from the mutant pups were very low; e.g., the yield from the brainstems of 4-week-old mutants was only 2.4% of that from age-matched controls and the yield did not increase by 16 weeks. The isolated myelin fractions contained very little MBP (less than 0.5% of total protein) or proteolipid protein, indicating that they were a very immature form of myelin or consisted largely of non-myelin contaminants. MAG in the 'myelin fractions' from the mutant brainstems were 9-15 fold higher and CNP levels were 2-3 fold higher than those in whole homogenates, suggesting that the isolated fractions were enriched in oligodendroglia-derived membranes. Overall, the biochemical results are consistent with a severe hypomyelination of the CNS in which a small amount of immature myelin is formed.

Node-like axonal undercoating in the optic nerve of heterozygous myelin-deficient rats.

Focal regions of node-like axonal undercoating occur occasionally in the myelin-deficient rat spinal cord. In the optic nerve of heterozygous myelin-deficient rats, areas of amyelination are present, within which many axons show regions of node-like undercoating. These structures were most easily seen when fixation resulted in round axonal profiles. In one-year-old rats, these structures were common. The ubiquitous association of patches of axonal undercoating with regions of chronic amyelination and demyelination indicates that chronically demyelinated axons differ structurally from recently demyelinated axons. It is possible that such axons may have lost their ability to recruit myelin-forming cells.

Myelin mosaicism in female heterozygotes of the canine shaking pup and myelin-deficient rat mutants.

Female heterozygotes of the shaking pup and myelin-deficient rat sex-linked recessive traits, show myelin mosaicism of the optic nerve and spinal cord. This is most marked in the optic nerve especially in the rat where mosaic patches persist with aging. In both the rat and dog, abnormal oligodendrocytes with distended rough endoplasmic reticulum are found in the abnormal patches and are a marker of the trait. Female heterozygote dogs can develop a marked tremor which disappears with age.

Shaking pups: a disorder of central myelination in the Spaniel dog. Part 1. Clinical, genetic and light-microscopical observations.

A new disorder of central myelination has been recognised in male Springer Spaniel pups which is probably inherited in a sex-linked recessive mode. The affected animals were much reduced in weight an size and showed gross generalised tremor, particularly when aroused, at about 10-12 days of age. Affected pups were studied between 1 and 3 months of age. There was severe hypomyelination throughout the CNS which was more marked in the cerebrum and optic nerves than in the spinal cord. The amount of myelin at each location increased with age. Axonal calibre also increased and there was no difference between the axonal diameters of affected and age-matched normal pups. Axons were either naked or surrounded by a disproportionately thin layer of myelin. Myelinated internodes tended to be short and heminodes were frequent. Vacuoles were present adjacent to axons or within glia but there was no evidence of demyelination. Total glial numbers were not reduced and numerous oligodendroglial and astrocytic nuclei identified. Peripheral, cranial and autonomic nerves were myelinated normally. It is suggested that there is an abnormality of oligodendroglial metabolism such that they cannot form and maintain normal myelin. Consequently the radial and longitudinal extensions of their plasma membranes are reduced. The vacuoles may represent a breakdown of defective myelin lipids as suggested in certain murine mutants. This defect of myelination provides a further model in which normal and disordered myelinogenesis can be studied.

Transplantation of rat Schwann cells grown in tissue culture into the mouse spinal cord.

Injections of lysolecithin were used to produce acute focal demyelination in the dorsal columns of 2 strains of mice, the myelin mutant quaking and the normal C57BL/6J. A small collection of rat Schwann cells grown in tissue culture was transplanted with their collagen substrate into this demyelinated area. The host mice were immune-suppressed to prevent graft rejection. Evidence of remyelination by Schwann cells was seen in the dorsal columns from 2-18 weeks after implantation. Proof that these Schwann cells were foreign to the host was derived from their rejection after the recipient mice were allowed to recover immunological competence by discontinuation of the immune suppression and by transferring immune cells sensitized against the donor tissue. It was concluded that Schwann cells grown in vitro retain their potential to produce myelin when returned to an in vivo situation and can myelinate central axons of a xenogenic host.

Identification of octadeca-9,11-dienoic acid and its significance in the diagnosis of cervical intraepithelial neoplasia.

In 1987 it was claimed using high performance liquid chromatography (HPLC) that the molar ratio of octadeca-9,11-dienoic acid to its parent linoleic acid (18:2 (9,12)) was raised in malignant and premalignant conditions of the cervix. In this study based on a gas chromatographic procedure which prevents autoxidation and avoids the detection limitations of HPLC, we have shown that assay of these fatty acids has no role to play in the assessment of cervical neoplasia.