Sensitivity, specificity, and accuracy of molecular profiling on circulating cell-free DNA in refractory or relapsed multiple myeloma patients, results of MM-EP1 study.

As a promising alternative to bone marrow aspiration (BMA), mutational profiling on blood-derived circulating cell-free tumor DNA (cfDNA) is a harmless and simple technique to monitor molecular response and treatment resistance of patients with refractory/relapsed multiple myeloma (R/R MM). We evaluated the sensitivity and specificity of cfDNA compared to BMA CD138 positive myeloma plasma cells (PCs) in a series of 45 R/R MM patients using the 29-gene targeted panel (AmpliSeq) NGS. KRAS, NRAS, FAM46C, DIS3, and TP53 were the most frequently mutated genes. The average sensitivity and specificity of cfDNA detection were 65% and 97%, respectively. The concordance per gene between the two samples was good to excellent according to Cohen's κ coefficients interpretation. An increased number of mutations detected in cfDNA were associated with a decreased overall survival. In conclusion, we demonstrated cfDNA NGS analysis feasibility and accuracy in R/R MM patients who may benefit from early phase clinical trial.

Acute intermittent hypoxia enhances regeneration of surgically repaired peripheral nerves in a manner akin to electrical stimulation.

The intrinsic repair response of injured peripheral neurons is enhanced by brief electrical stimulation (ES) at time of surgical repair, resulting in improved regeneration in rodents and humans. However, ES is invasive. Acute intermittent hypoxia (AIH) - breathing alternate cycles of regular air and air with ~50% normal oxygen levels (11% O2), considered mild hypoxia, is an emerging, promising non-invasive therapy that promotes motor function in spinal cord injured rats and humans. AIH can increase neural activity and under moderately severe hypoxic conditions improves repair of peripherally crushed nerves in mice. Thus, we posited an AIH paradigm similar to that used clinically for spinal cord injury, will improve surgically repaired peripheral nerves akin to ES, including an impact on regeneration-associated gene (RAG) expression-a predictor of growth states. Alterations in early RAG expression were examined in adult male Lewis rats that underwent tibial nerve coaptation repair with either 2 days AIH or normoxia control treatment begun on day 2 post-repair, or 1 h ES treatment (20 Hz) at time of repair. Three days post-repair, AIH or ES treatments effected significant and parallel elevated RAG expression relative to normoxia control at the level of injured sensory and motor neuron cell bodies and proximal axon front. These parallel impacts on RAG expression were coupled with significant improvements in later indices of regeneration, namely enhanced myelination and increased numbers of newly myelinated fibers detected 20 mm distal to the tibial nerve repair site or sensory and motor neurons retrogradely labeled 28 mm distal to the repair site, both at 25 days post nerve repair; and improved return of toe spread function 5-10 weeks post-repair. Collectively, AIH mirrors many beneficial effects of ES on peripheral nerve repair outcomes. This highlights its potential for clinical translation as a non-invasive means to effect improved regeneration of injured peripheral nerves.

Electrical stimulation as a conditioning strategy for promoting and accelerating peripheral nerve regeneration.

The delivery of a nerve insult (a "conditioning lesion") prior to a subsequent test lesion increases the number of regenerating axons and accelerates the speed of regeneration from the test site. A major barrier to clinical translation is the lack of an ethically acceptable and clinically feasible method of conditioning that does not further damage the nerve. Conditioning electrical stimulation (CES), a non-injurious intervention, has previously been shown to improve neurite outgrowth in vitro. In this study, we examined whether CES upregulates regeneration-associated gene (RAG) expression and promotes nerve regeneration in vivo, similar to a traditional nerve crush conditioning lesion (CCL). Adult rats were divided into four cohorts based on conditioning treatment to the common peroneal (fibular) nerve: i) CES (1h, 20Hz); ii) CCL (10s crush); iii) sham CES (1h, 0Hz); or iv) naïve (unconditioned). Immunofluorescence and qRT-PCR revealed significant RAG upregulation in the dorsal root ganglia of both CES and CCL animals, evident at 3-14days post-conditioning. To mimic a clinical microsurgical nerve repair, all cohorts underwent a common peroneal nerve cut and coaptation one week following conditioning. Both CES and CCL animals increased the length of nerve regeneration (3.8-fold) as well as the total number of regenerating axons (2.2-fold), compared to the sham and naïve-conditioned animals (p<0.001). These data support CES as a non-injurious conditioning paradigm that is comparable to a traditional CCL and is therefore a novel means to potentially enhance peripheral nerve repair in the clinical setting.

Reaching training in rats with spinal cord injury promotes plasticity and task specific recovery.

In the current study we examined the effects of training in adult rats with a cervical spinal cord injury (SCI). One group of rats received 6 weeks of training in a single pellet reaching task immediately after injury, while a second group did not receive training. Following this period changes in cortical levels of BDNF and GAP-43 were analysed in trained and untrained animals and in a group with training but no injury. In another group of rats, functional recovery was analysed in the reaching task and when walking on a horizontal ladder. Thereupon, the cortical forelimb area was electrophysiologically examined using micro-stimulation followed by tracing of the lesioned corticospinal tract (CST). We found that trained rats improved substantially in the reaching task, when compared to their untrained counterparts. Trained rats however, performed significantly worse with their injured forelimb when walking on a horizontal ladder. In parallel to the improved recovery in the trained task, we found that the cortical area where wrist movements could be evoked by micro-stimulation expanded in trained rats in comparison to both untrained and uninjured rats. Furthermore, collateral sprouting of lesioned CST fibres rostral to the injury was increased in trained rats. Post-injury training was also found to increase cortical levels of GAP-43 but not BDNF. In conclusion we show that training of a reaching task promotes recovery of the trained task following partial SCI by enhancing plasticity at various levels of the central nervous system (CNS), but may come at the cost of an untrained task.

Neurotrophin-3 attenuates galanin expression in the chronic constriction injury model of neuropathic pain.

We have recently shown that exogenous neurotrophin-3 (NT-3) acts antagonistically to nerve growth factor (NGF) in regulation of nociceptor phenotype in intact neurons and suppresses thermal hyperalgesia and expression of molecules complicit in this behavioral response induced by chronic constriction injury (CCI) of the sciatic nerve. The present study examines whether there is a global influence of NT-3 in mitigating alterations in peptide and NGF receptor expression; molecules believed to also contribute to CCI-associated pain. Thus, the influence of NT-3 on phenotypic changes in dorsal root ganglion (DRG) neurons in rats coincident with CCI was examined using in situ hybridization. Seven days following injury, the incidence of expression of the neuropeptides galanin and pituitary adenylate cyclase-activating polypeptide (PACAP) was increased in L5 sensory neurons ipsilateral to the injury from 12% to 60% and 16% to 37% respectively, in addition to an increased level of expression. In contrast, there was no consistent significant change in tropomyosin-related kinase A (trkA) expression following CCI. Intrathecal infusion of NT-3 globally mitigated both the increased incidence and elevated levels of galanin messenger RNA (mRNA) expression observed following CCI, reducing the former from 60% to 39%. NT-3 infusion resulted in a limited reduction in the incidence and level of neuronal PACAP in medium to large size, but not small size, DRG neurons. NT-3 had no significant net effect on CCI-induced alterations in trkA mRNA expression.

Effect of anti-nerve growth factor treatment on pituitary adenylate cyclase activating polypeptide expression in adult sensory neurons exposed to adjuvant induced inflammation.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) is increased in sensory neurons exposed to adjuvant induced peripheral inflammation. Local elevation in expression of the neurotrophin nerve growth factor (NGF) is a main factor contributing to the neuronal response to inflammation. This study examines the role of endogenous NGF in inflammation-associated increases in PACAP expression using the adjuvant-induced peripheral inflammation model with or without systemic administration of antibodies against NGF. Quantitative in situ hybridization was used to detect changes in neuronal PACAP mRNA expression and to correlate this expression with neuronal mRNA expression of the NGF receptor tyrosine kinase (trk) A. The results from this study show that inflammation triggered increases in PACAP expression occurs in small- to medium-sized dorsal root ganglion (DRG) neurons that also express trkA, and that this elevation in PACAP expression is prevented by systemic injection of anti-NGF. This supports a role for NGF as a positive regulator of PACAP expression during inflammation.

Dynamic patterns of BDNF expression in injured sensory neurons: differential modulation by NGF and NT-3.

It has been suggested that altered retrograde neurotrophin support contributes to the phenotypic switch observed in BDNF expression in injured sensory neurons. Thus, modulatory influences of NGF and NT-3 on BDNF expression in injured adult rat DRG neurons were examined using in situ hybridization and immunohistochemical approaches. Quantitative analysis reveals a biphasic response to sciatic nerve injury, whereby in the first day following injury, BDNF expression is up-regulated in approximately 83% of injured neurons including all small neurons, and a larger pool of trkB expressing neurons than in intact. By 1 week and up to 3 weeks later expression is still seen in approximately 66% of injured neurons, but the characteristic phenotypic switch in the subpopulations expressing BDNF occurs, whereby expression in the trkA population is reduced and expression in trkB- and in trkC-positive neurons is elevated. NGF infusion results in elevated levels of BDNF expression in both intact and injured trkA-positive neurons, accompanied by reduced trkB expression. NT-3 acts in an opposite fashion effecting a down-regulation in BDNF expression in intact neurons and preventing/reducing the injury-associated increases in BDNF expression in both trkC- and nontrkC-expressing subpopulations of injured neurons. These effects suggest NGF can regulate BDNF expression in trkA-expressing neurons regardless of the axonal state and that elevated levels of BDNF may contribute to the down-regulation in trkB expression associated with these states. Furthermore, the findings demonstrate that NT-3 can act in an antagonistic fashion to NGF in the regulation of BDNF expression in intact neurons, and mitigate BDNF's expression in injured neurons.

Does diabetes target ganglion neurones? Progressive sensory neurone involvement in long-term experimental diabetes.

Targeting of dorsal root ganglia by diabetes could account for the selective sensory abnormalities that patients with early diabetic polyneuropathy develop. In this work, we addressed survival, phenotype and gene expression in sensory neurones in lumbar dorsal root ganglia in a long-term model of experimental streptozotocin-induced diabetes in rats, designed to reflect human disease. Motor and sensory conduction slowing developed early, by the 2-month time point. At 2 months, sensory neurones had no detectable alterations in their calibre or gene expression, assessed using quantitative in situ hybridization studies for mRNA markers that included alpha CGRP, beta CGRP, NFM, t alpha 1-tubulin, SP, VIP, B50 (GAP43), galanin, somatostatin, PACAP, HSP27, c-jun, SNAP 25, p75, TrkA, TrkB and TrkC. By 12 months, however, diabetics had developed neurone perikaryal and distal axon atrophy, accompanied by generalized downregulation of mRNA expression, particularly of CGRP transcripts, PACAP, SP, NFM, p75, trkA and trkC. With the exception of HSP-27, no elevation in mRNAs that increase after injury, such as VIP, galanin, CCK, PACAP, B50 and t alpha 1-tubulin, was observed and constitutive levels, when detectable, trended towards lower rather than increased levels. There was relative preservation of neurone numbers at 12 months; only a non-significant trend towards fewer diabetic neurones was detected using a rigorous and systematic physical dissector counting approach through the entire L5 ganglia. There was no change in the relative populations of CGRP- and SP-immunoreactive neurones. Our findings indicate that even long-term experimental diabetes is associated with relative preservation of sensory neurone populations, but the neurones are atrophic and their gene expression is altered. This pattern of change differs from that following axotomy, implies a degenerative rather than an injury phenotype and has important implications for how such neurones might be rescued.

Exogenous NT-3 and NGF differentially modulate PACAP expression in adult sensory neurons, suggesting distinct roles in injury and inflammation.

Expression of pituitary adenylate cyclase-activating polypeptide in sensory neurons varies with injury or inflammation. The neurotrophins NGF and NT-3 are profound regulators of neuronal peptidergic phenotype in intact and injured sensory neurons. This study examined their potential for modulation of PACAP expression in adult rat with intact and injured L4-L6 spinal nerves with or without immediate or delayed intrathecal infusion of NT-3 or NGF. Results indicate that in L5 DRG, few trkC neurons express high levels of PACAP mRNA in the intact state, but many do following injury. The elevated expression in injured neurons is mitigated by NT-3 infusion, suggesting a role for NT-3 in returning the 'injured phenotype' back towards an 'intact phenotype'. NGF dramatically up-regulated PACAP expression in trkA-positive neurons in both intact and injured DRGs, implicating NGF as a positive regulator of PACAP expression in nociceptive neurons. Surprisingly, NT-3 modulates PACAP expression in an antagonistic fashion to NGF in intact neurons, an effect most evident in the trkA neurons not expressing trkC. Both NT-3 and NGF infusion results in decreased detection of PACAP protein in the region of the gracile nuclei, where central axons of the peripherally axotomized large sensory fibers terminate. NGF infusion also greatly increased the amount of PACAP protein detected in the portion of the dorsal horn innervated by small-medium size DRG neurons, while both neurotrophins appear able to prevent the decrease in PACAP expression observed in these afferents with injury. These results provide the first insights into the potential molecules implicated in the complex regulation of PACAP expression in sensory neurons.

In vitro production of dendritic cells from human blood monocytes for therapeutic use.

Dendritic cells (DC) are professional antigen-presenting cells that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34(+) pluripotent hematopoietic progenitor cells have been now developed. For this purpose, their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. In the present review, we give our experience of such a procedure: it includes collection of mononuclear cells by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + human serum (autologous patient's serum or AB serum) or in a serum-free medium (AIM V). The characteristics of monocyte-derived DC grown in these various conditions varied mainly regarding their phenotype and their morphology in confocal microscopy, whereas no significant differences were found in their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The DC were also cryopreserved in bags (either by putting the bags directly in a -80 degrees C mechanical freezer or using a classical liquid nitrogen controlled-rate freezer at -1 degrees C/min) in a solution containing 10% dimethyl sulfoxide (Me(2)SO) and 2% human albumin in doses of DC available for several infusions. The mean recoveries after freezing and thawing were not statistically different (around 70%). The immunophenotype of DC, as well as the T lymphocyte-stimulating capacity, were not modified by the freezing--thawing procedure. The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined DC. Clinical trials using DC already published will be discussed.

Nitric oxide synthase activity and expression in experimental diabetic neuropathy.

The changes of nitric oxide synthase (NOS) activity and expression in experimental diabetic neuropathy have not been examined. Increases in ganglia NOS might be similar to those that follow axotomy, whereas declines in endothelial NOS (eNOS) and immunological NOS (iNOS) might explain dysfunction of microvessels or macrophages. In this work, we studied NOS activity in lumbar dorsal root ganglia (DRG) of rats with both short- and long-term experimental streptozotocin-induced diabetes and correlated it with expression of each of the 3 NOS isoforms. NOS enzymatic activity in DRG increased after 12 months of diabetes. This increase, however, was not accompanied by an increase in neuronal NOS immunohistochemistry or mRNA. Immunohistochemical and RT-PCR studies did not identify changes of eNOS expression in 12-month sciatic nerves or DRG from diabetics. Two-month diabetic DRG had increased eNOS mRNA and there was novel eNOS labeling of capsular DRG and perineurial cells. iNOS mRNA levels were lower in diabetics at both time points in peripheral nerves but were unchanged in DRG. Diabetic ganglia showed an increase in NOS activity not explained by novel NOS isoform synthesis. The increases may compensate for NO "quenching" by endproducts of glycosylation. Declines in iNOS may indicate impaired macrophage function.

Fas-mediated apoptosis of mouse embryo stem cells: its role during embryonic development.

PROBLEM: Fas antigen (APO-1/CD95) can regulate the activity of various cells during adulthood. This study aimed at determining whether Fas may also be involved in the regulation of very early events such as the embryo preimplantation stage. METHOD OF STUDY: We used mouse embryo stem (ES) cell line as a model for testing the effect of Fas crosslinking upon anti-Fas monoclonal antibody (MoAb) treatment. In addition, this treatment was also applied to in-vitro mouse-embryo culture. RESULTS: Flow-cytometry analysis of cultured ES cells demonstrated an increase in Fas expression. unchanged in the presence of mouse interleukin-2, while greatly upregulated in the presence of lipopolysaccharide (LPS). As determined by various means, ES cells may undergo a Fas-mediated apoptosis, slightly but significantly intensified by the addition of LPS to cell cultures. We also report that anti-Fas MoAb directly inhibited two-cell stage mouse-embryo (preimplantation) development in in-vitro culture conditions. CONCLUSION: These data suggest a novel mechanism controlling the regulation of physiological cell turnover as well as blastocyst implantation in early embryo development.

In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy.

DC are professional APC that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34+ pluripotent HPC have been developed recently. However, the various steps of their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. Our study focused on setting up such a full procedure, including collection of mononuclear cells (MNC) by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + autologous serum (SAuto) in sterile Teflon bags. The procedure was first developed with apheresis products from 7 healthy donors. Its clinical feasibility was then tested on 7 patients with breast cancer. The characteristics of monocyte-derived DC grown with SAuto (or in some instances with a pooled AB serum) were compared with those obtained in the presence of FBS by evaluation of their phenotype, their morphology in confocal microscopy, and their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined SAuto-derived DC as efficient as those derived with FBS.

Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures.

OBJECTIVE: In previous work, we showed that CD34+ bone marrow cells can be successfully expanded along the myeloid pathway in stroma- and serum-free conditions in the presence of SCF+IL-3+IL-6+Flt3-l+G-CSF+MGDF. Due to the lack of phenotypically detectable lymphoid cells, it was necessary to address the question of the lymphoid potential of the expanded populations under these conditions. MATERIALS AND METHODS: The present report describes a long-term culture system that supports human B- and NK-cell differentiation from the day 14 fraction without further selection of the more primitive cells. In NK proliferation assays, the cells were maintained over stroma cells in the presence of IL-2 for 4-5 weeks. NK initiating cells (NK-IC) were determined by a limiting dilution assay. In B-cell cultures, the expanded cells were maintained over MS5 in the presence of Flt3-l for 4-8 weeks. RESULTS: NK cells rose from 0.2%+/-0.04% at culture initiation to 71%+/-6% at week 5. These cells displayed cytolytic activity. NK-IC evaluation showed a mean 18-fold expansion in the day 14 expanded fraction as compared to the initial day 0 fraction. Similarly, CD19+ cells rose from 0.1% at culture initiation to 30%+/-1% at week 6. Cells produced under these B-LTC conditions were CD34-CD19+CD10+. We also demonstrated that the CD34+/Lin- sorted cells from the day 14 fraction gave rise to NK and B cells. CONCLUSION: This culture system permits the revelation of a population that, although poorly represented in terms of phenotypically detectable cells, nevertheless retains high levels of lymphoid NK and B potential after 14 days expansion. Such data suggest the persistence, or expansion, of lymphoid progenitors and, hence, the multipotentiality of the expanded progenitor/stem cells.

Anatomical evidence supporting the potential for modulation by multiple neurotrophins in the majority of adult lumbar sensory neurons.

Neurotrophins exert effects on sensory neurons through receptor tyrosine kinases (trks) and a common neurotrophin receptor (p75). Quantitative in situ hybridization studies were performed on serial sections to identify neurons expressing single or multiple neurotrophin trk receptor mRNA(s) in adult lumbar dorsal root ganglion (DRG) in order to examine the possibility of multi-neurotrophin modulation of phenotype via different trk receptors or various trk isoforms. Expression of mRNA encoding trkA, trkB, trkC, or p75 is restricted to select subpopulations representing approximately 41%, 33%, 43%, and 79% of DRG neurons, respectively. Colocalization studies reveal that approximately 10% of DRG neurons coexpress trkA and trkB mRNA; 19% coexpress trkA and trkC mRNA; and 18% coexpress trkB and trkC mRNA. Trilocalization of all three trk mRNAs is rare, with approximately 3-4% of neurons in this category. Overall incidence of expression of more than one full length trk mRNA occurs in approximately 40% of DRG neurons, whereas expression of individual trk mRNA is found in approximately 34%. Full length trk receptor mRNA is rarely detected without p75, implicating the latter in neuronal response to neurotrophins. Examination of two full-length isoforms of trkA reveal that they are coexpressed with relative levels of expression positively correlated. TrkC mRNAs corresponding to 14- or 39-amino acid insert isoforms colocalize with the non-insert trkC isoform, but the converse is not necessarily true. The data suggest that substantial subpopulations of adult sensory neurons may be modulated through interactions with multiple neurotrophins, the consequences of which are largely unknown.

MAP kinase inactivation is required only for G2-M phase transition in early embryogenesis cell cycles of the starfishes Marthasterias glacialis and Astropecten aranciacus.

Downregulation of MAP kinase is a universal consequence of fertilization in the animal kingdom. Here we show that oocytes of the starfishes Astropecten aranciacus and Marthasterias glacialis complete meiotic maturation and form a pronucleus when treated with 1-methyladenine and then complete DNA replication and arrest at G2 if not fertilized. Release of G2 by fertilization or a variety of parthenogenetic treatments is associated with inactivation of MAP kinase. Prevention of MAP kinase inactivation by microinjection of Ste11-DeltaN, a constitutively active budding yeast MAP kinase kinase kinase, arrests fertilized eggs at G2 in either the first or the second mitotic cell cycle, in a dose-dependent manner. G1 arrest is never observed. Conversely, inactivation of MAP kinase by microinjection of the MAP kinase-specific phosphatase Pyst-1 releases mature starfish oocytes from G2 arrest. The role of MAP kinase in arresting cell cycle at various stages in oocytes of different animal species is discussed.

Direct cloning of astrocytes from primary culture without previous immortalization.

In primary cultures, much evidence shows the existence of different subtypes of astrocytes that are not all identified. One methodology for studying these subtypes can be their cloning. The present investigation shows a method for a direct cloning of astrocytes without previous immortalization. Astrocytes from the cerebral cortex of newborn rats were cultured, purified by shaking, and harvested by trypsinization. One single astrocyte was plated in a small volume of a homemade cloning medium. After getting a colony, successive platings were made using larger and larger vessels, up to 60-mm-diameter petri dishes. Then, subcultures were made. The yield of the cloning was similar to that of common eukaryotic cell clonings. All along the cloning procedure, the cells were positively immunostained with anti-glial fibrillary acidic protein antibodies. Cloned cells from some batches were spindle-shaped, looking like fibroblasts. Nevertheless, they were immunostained with anti-glial fibrillary acidic protein antibodies, unlike true fibroblasts. These spindle-shaped astrocytes were compared to cells from an astrocytoma cell line that had the same shape. The growth pattern of the astrocytoma cells was different from that of the astrocytes cloned from the primary cultures. All the types of studied cells contained glycogen. On the basis of the criteria of morphology, of glial fibrillary acidic protein immunolabeling, and of glycogen synthesis, the cloned cells kept the characteristics of astrocytes. This study shows that it is perfectly possible to get clones of astrocytes from one astrocyte without previous immortalization, giving thus a convenient material for the study of astrocyte biology.

Collaborative and reciprocal effects of ciliary neurotrophic factor and nerve growth factor on the neuronal phenotype of human neuroblastoma cells.

We have probed the molecular basis of functional effects of ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) on aspects of the neuronal differentiation of LA-N-2 neuroblastoma cells. The influence of CNTF on the cholinergic phenotype can be accounted for by transcriptional/translational effects without implicating posttranslational mechanisms. Although both NGF receptors are expressed constitutively by LA-N-2 cells, CNTF has a marked stimulatory effect on trkA mRNA and protein. The NGF receptors are functional in serum-free conditions where they mitigate CNTF effects on cell adhesion but do not support process extension. Following priming by CNTF, NGF and CNTF have synergistic influences on process formation but not on choline acetyltransferase-specific activity.

Isolation of carbohydrate metabolic clones from cultured astrocytes.

Astrocytes are the principal sites of glycogen synthesis in the nervous tissue. Growing evidence shows that there are many types of astrocytes. The aim of the present investigation was to isolate different types of astrocytes that display different carbohydrate anabolism. Astrocytes from newborn rat brain were directly cloned from primary cultures without a previous transformation. Many clones were obtained, and they were termed CP clones. Another series of clones, termed SV clones, were obtained after the transfection of the primary cultures by the SV40 T antigen. The effectiveness of the transfection was verified by the rate of DNA synthesis using flow cytometry and by the presence of plasmid DNA in the genomic DNA of the astrocytes using the Southern blot method. After the transfection, the growth velocity increased greatly. The size and shape of the astrocytes were the same for each cell in a given clone, regardless of the cloning method utilized. However, these sizes and shapes could be different from one clone to another in CP clones, whereas all the astrocytes of all the SV clones looked like each other. All the clones obtained stained positively with anti-glial fibrillary acidic protein antibodies. Glycogen stained in the clones using concanavalin A-horseradish peroxidase. The glycogen content was also measured using biochemical analysis. Concordant results obtained using two methods showed that some clones contained an important quantity of glycogen while other clones contained a small amount, in the CP series as well as in the SV series. This property was the same for the intracellular glucose concentrations. The activity of the gluconeogenic enzyme fructose-1, 6-bisphosphatase was measured in each clone using spectrophotometry. This activity was also significantly different from one clone to another. The clones containing large amounts of glycogen had important fructose-1,6-bisphosphatase activity. The present results show that it is possible to clone astrocytes either directly from primary cultures without immortalization or after their transformation. When analyzing these clones, it appears that carbohydrate anabolism can be significantly different from one astrocyte to another. This difference may also exist in vivo.