Extended interval dosing of natalizumab in multiple sclerosis.

BACKGROUND: Natalizumab (NTZ), a monoclonal antibody to human α4ß1/ß7 integrin, is an effective therapy for multiple sclerosis (MS), albeit associated with progressive multifocal leukoencephalopathy (PML). Clinicians have been extending the dose of infusions with a hypothesis of reducing PML risk. The aim of the study is to evaluate the clinical consequences of reducing NTZ frequency of infusion up to 8 weeks 5 days. METHODS: A retrospective chart review in 9 MS centres was performed in order to identify patients treated with extended interval dosing (EID) regimens of NTZ. Patients were stratified into 3 groups based on EID NTZ treatment schedule in individual centres: early extended dosing (EED; n=249) every 4 weeks 3 days to 6 weeks 6 days; late extended dosing (LED; n=274) every 7 weeks to 8 weeks 5 days; variable extended dosing (n=382) alternating between EED and LED. These groups were compared with patients on standard interval dosing (SID; n=1093) every 4 weeks. RESULTS: 17% of patients on SID had new T2 lesions compared with 14% in EID (p=0.02); 7% of patients had enhancing T1 lesions in SID compared with 9% in EID (p=0.08); annualised relapse rate was 0.14 in the SID group, and 0.09 in the EID group. No evidence of clinical or radiographic disease activity was observed in 62% of SID and 61% of EID patients (p=0.83). No cases of PML were observed in EID group compared with 4 cases in SID cohort. CONCLUSIONS: Dosing intervals up to 8 weeks 5 days did not diminish effectiveness of NTZ therapy. Further monitoring is ongoing to evaluate if the risk of PML is reduced in patients on EID.

Identifying and treating patients with suboptimal responses.

Multiple sclerosis (MS) is an immune-mediated neurologic disease in which acute inflammatory events early in the disease course contribute to subsequent neurologic disability. The early relapsing inflammatory phase is followed by a progressive degenerative phase in which the frequency of acute inflammatory attacks diminishes but progressive loss of neurologic function continues. Current immune therapies are most effective in suppressing the acute inflammatory events that characterize the earlier stages of disease. Optimal suppression of these inflammatory events is likely to have the best potential for delaying or preventing loss of axons and decline in neurologic function. In view of these considerations, and because MS is a heterogeneous disease and response to disease-modifying agents (DMA) varies across individuals, it is important to identify suboptimal responders as early as possible to allow therapeutic modification while the opportunity to avert future loss of function remains. At present, no criteria for identifying suboptimal responders have been validated. In January 2004, a group of neurologists from 16 MS centers in the United States met to develop a consensus on criteria for defining suboptimal response for use in compelling clinical situations and to prompt clinical studies to validate the efficacy of these criteria. Consensus criteria included relapse rates of either 1/year or unchanged from pretreatment rates, incomplete recovery from multiple attacks, evolution of polyregional neurologic involvement, recurrent brainstem or spinal cord lesions, and cumulative loss of neurologic function sufficient to disrupt daily activities. The panel then considered the use of mitoxantrone for patients with worsening MS and a suboptimal response to DMA therapy.

TrkA induces differentiation but not apoptosis in C6-2B glioma cells.

Nerve growth factor (NGF) binds to the TrkA tyrosine kinase and the p75 neurotrophin receptors. Depending upon which receptor is activated, NGF can induce differentiation or apoptosis. C6-2B glioma cells express the p75 receptor, but NGF decreases their growth only when TrkA is introduced (C6trk). It is unclear, however, whether TrkA reduces C6-2B cell growth by apoptosis or differentiation. To examine which mechanisms account for the anti-proliferative effect of NGF in these cells, we first analyzed whether NGF causes apoptosis by flow cytometry, two-site immunoassay and in situ TUNEL. None of these methods indicated that C6trk undergo apoptosis. Additional apoptotic markers, such as Bcl-2, Bax, Bad, p53, caspase 3, and NF-kappaB were also used. C6trk cells exhibited lower levels of Bcl-2 compared with the parental C6 mock cells, but no changes in the levels of other apoptotic proteins. Moreover, NGF increased AP-1 binding activity in C6trk cells, suggesting that NGF may induce differentiation. We then examined whether TrkA changes the glioma phenotype. In C6trk cells, but not in C6mock cells, NGF enhanced the levels of neuron-specific enolase as well as the levels of A2B5 and 2', 3'-cyclic nucleotide 3'-phosphodiesterase, markers for oligodendrocytes, without affecting the expression of other neuronal markers. Our data suggest that the antiproliferative properties of TrkA may rely on its ability to induce differentiation of C6 cells from undifferentiated glioma to oligodendrocytes.

Reversible tacrolimus-induced neurotoxicity isolated to the brain stem.

Diplopia, nystagmus, visual hallucinations, and internuclear ophthalmoplegia developed in a 30-year-old woman 84 days after she received a matched, unrelated bone marrow transplant for chronic myeloid leukemia. A regimen of tacrolimus had been administered since the transplantation was performed. MR imaging revealed bilaterally symmetric regions of signal abnormality with abnormal contrast enhancement in the brain stem. No supratentorial abnormality was present. Tacrolimus therapy was discontinued, and the symptoms resolved. MR imaging that was performed 10 days after tacrolimus was discontinued showed resolution of the abnormalities.

Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.

G207 is a conditionally replicating derivative of herpes simplex virus (HSV) type-1 strain F engineered with deletions of both gamma(1)34.5 loci and a lacZ insertion disabling the UL39 gene. We have demonstrated the efficacy of G207 in treating malignant glial tumors in athymic mice, as well as the safety of intracerebral G207 inoculation in mice and in Aotus nancymai. We sought to determine the safety of G207 inoculation into cerebral malignant glial tumors in humans. Criteria for inclusion into this dose-escalation study were the diagnosis of histologically proven malignant glioma, Karnofsky score > or = 70, recurrence despite surgery and radiation therapy, and an enhancing lesion greater than 1 cm in diameter. Serial magnetic resonance images were obtained for volumetric analysis. The trial commenced at a dose of 10(6) plaque forming units (p.f.u.) inoculated at a single enhancing site and was completed when the 21st patient was inoculated with 3x10(9) p.f.u. at five sites. While adverse events were noted in some patients, no toxicity or serious adverse events could unequivocally be ascribed to G207. No patient developed HSV encephalitis. We found radiographic and neuropathologic evidence suggestive of anti-tumor activity and long-term presence of viral DNA in some cases.

Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content.

The M213-20 and M213-1L cell lines were immortalized from rat striatum using the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano et al., 1994, Exp. Neurol. 124:395-400). Cell lines that produce large amounts of GABA may be useful for transplantation into the brain in conditions such as Huntington's disease or epilepsy, where localized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, to increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD(67) cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed increased GAD(67) expression compared to the parent M213-20 and M213-1L cell lines. Staining for the EBNA antigen and Southern blots demonstrated that the pREP10 plasmid was stably maintained in the cells for at least 12-15 months in culture. Several clones were isolated in which GABA concentrations were increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared to the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678 +/- 0.4 micromol/g prot). The ability of these cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be used with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes.

Neurochemical alterations in the cerebellum of a murine model of Niemann-Pick type C disease.

Niemann-Pick disease Type C (NPC) is a progressive neurovisceral metabolic disorder that is caused in most patients by a defect in a recently found gene, NPC-1. Neurological damage includes visual disorders such as vertical supranuclear gaze palsy, movement disorders such as dystonia and ataxia, dementia, and seizures. So far the biochemical deficit, most likely manifested by delayed intracellular cholesterol transport, has not been correlated with the progressive neurological damage. A mutant Balb/C mouse with a defect in the same gene is used as a model to study NPC. Pathological examination of brain tissue obtained by autopsy from NPC patients or brains of affected NPC mice of different ages, revealed signs of extensive damage throughout the brain, including neurofibrillary tangles and intracellular storage of various compounds. Loss of cerebellar Purkinje cells was the most significant specific damage. The present study examined whether the neurochemical changes present in the NPC mouse brain were related to the pathological changes. The results show major alterations in the levels of serotonin and its main metabolite, 5-hydroxyindoleacetic acid, in the cerebellum and cortex of NPC mice. The levels of the inhibitory amino acid glycine were threefold higher in the cerebellum of NPC mice and those of glutamate and GABA decreased in the cortex. Tyrosine hydroxylase immunoreactivity was present in Purkinje cells, and the levels of L-DOPA increased specifically in the vermis of the cerebellum. These results are the first to indicate changes in neurotransmitters in NPC and that these could be correlated with some of the neuropathology of this disease.

Dopamine turnover and metabolism in the striatum of parkinsonian rats grafted with genetically-modified human astrocytes.

The potential of a novel therapeutic approach for treating Parkinson's disease, which involves the transplantation of a transfected human astrocyte cell line SVG-TH, that stably expresses the rate-limiting enzyme for dopamine production, tyrosine hydroxylase, was examined. SVG-TH and untransfected parent cells were grafted into the diseased striatum of rats in which Parkinson's disease had been induced by the administration of 6-hydroxydopamine. The in situ production and spillover of 3,4-dihydroxyphenylalanine (the precursor of dopamine), dopamine and their metabolites in the striatal extracellular fluid of the grafted rats was determined in conscious animals using the microdialysis technique and a high pressure liquid chromatography apparatus. Alleviation of symptoms of Parkinson's disease (abnormal movements) was evaluated by rotation tests. Upon transplantation of the SVG-TH cells into the striatum of the parkinsonian rats, the levels of dopamine in extracellular fluid of the striatum reached those of the normal rats, and correlated well with the improvement (74%) in their rotating behaviour (behavioural deficit). The levels of the two main dopamine metabolites, dihydroxyphenylacetic acid and homovanillic acid, were low in the lesioned rats, even after SVG-TH transplantation. An alternative route of metabolism of dopamine may occur in the transplanted striatum, since the dopamine metabolite, 3-O-methoxy-4-hydroxy-phenylethylamine, appeared, which indicates activity of catechol-O-methyl transferase. Upon blockade of L-aromatic-amino acid decarboxylase, 3,4-dihydroxyphenylalanine accumulated in extracellular fluid of the 6-hydroxydopamine-lesioned and SVG-TH-grafted rats, which indicated that these cells produced active tyrosine hydroxylase in vivo. These findings indicate the potential of treating Parkinson's disease by the intrabrain grafting of human astrocyte cells transfected with the rate limiting enzyme for dopamine production.

TrkA receptors delay C6-2B glioma cell growth in rat striatum.

Nerve growth factor (NGF) acts as an anti-mitogenic factor in C6-2B glioma cells stably expressing TrkA (C6trk+). To study the effect of TrkA on cell growth in vivo, we grafted mock and C6trk+ cells into the striatum of ACI nude rats. Thy 1.1 and p75NTR immunohistochemistry revealed that wild type C6-2B cells formed a tumor mass in the striatum by 14 days. In contrast, C6trk+ transplanted rats did not show the presence of a significant tumor mass until 71 days. Analysis of this tumor showed that expression of TrkA was retained, but the synthesis of NGF was abolished. Our data encourage the speculation that expression of TrkA in glioblastoma in vivo will attenuate tumor progression.

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells.

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.

N18-RE-105 cells: differentiation and activation of p53 in response to glutamate and adriamycin is blocked by SV40 large T antigen tsA58.

Process extension was induced in cells of the N18-RE-105 neuroblastoma-retinal hybrid line by toxic agents, including glutamate and the p53-inducing anticancer agents adriamycin and etoposide. Both adriamycin and glutamate activated p53 as measured by a plasmid transfection assay. It was therefore hypothesized that SV40 large T antigen, which binds p53, would interfere with cellular differentiation. To test this hypothesis, the temperature-sensitive form of SV40 large T was transduced into N18-RE-105 cells by retroviral infection. SV40 large T-infected cells became de-differentiated, grew in tightly-packed colonies, lost expression of neurofilament, and lost the ability to differentiate in response to glutamate and adriamycin. The de-differentiating effect of SV40 large T antigen may be due to binding and inactivation of cellular proteins, such as p53, p107, p130, p300, and retinoblastoma protein, which are important in cellular growth and differentiation. It is suggested that p53 may play a role in cellular differentiation, perhaps under unusual circumstances involving stress or cytotoxicity.

Growth properties of neural cell lines immortalized with the tsA58 allele of SV40 large T antigen.

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33 degrees C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5 degrees C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5 degrees C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5 degrees C for all three cell lines. After 3 wk at 39.5 degrees C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

Engraftment of C6-2B cells into the striatum of ACI nude rats as a tool for comparison of the in vitro and in vivo phenotype of a glioma cell line.

The C6-2B is a well-characterized glioma cell line used extensively in the study of malignant glial biology. While the C6-2B cell line has traditionally been thought of as a homogenous cell line, the in vitro phenotype of the C6-2B cell line can vary considerably depending on the culture technique used and the stratum on which the cells are grown. Thus, we asked whether the in vitro phenotype of the C6-2B cell line was significantly different than the in vivo phenotype of the cell line once it was engrafted into the striatum of nude rats. Under culture conditions used in our laboratory, 100% of the C6 cells were found to express p75, the low-affinity nerve growth factor (NGF) receptor, and Major Histocompatability Class I (MHC Class I), while only 10-15% demonstrated vimentin reactivity. Immunohistochemistry was consistently negative for GFAP, trkA (the high-affinity receptor for NGF), CD4, CD8, and a macrophage specific marker (Ox-41). Once engrafted into the striatum of nude rats, the cells remained 100% p75 and MHC Class I positive, and again, only 15% of the cells demonstrated vimentin reactivity. The grafted cells retained this characteristic for 28 days in vivo. Although an immunoincompetent host was selected to minimize the effects an inflammatory response would have on the graft, a transient inflammatory response was detected. During the first week of engraftment, numerous MHC class II cells, some of which were macrophages, were seen infiltrating the graft. However, by 4 weeks postengraftment, no inflammatory cells were appreciated in the graft and surprisingly little reactive gliosis was seen in the penumbra of the tumor mass. Thus, the limited number of in vitro phenotypic characteristics we examined in the C6-2B cell line remained constant once the cells were engrafted into the striatum of athymic nude rats.

Metabolite abnormalities in progressive multifocal leukoencephalopathy by proton magnetic resonance spectroscopy.

OBJECTIVE: To evaluate progressive multifocal leukoencephalopathy (PML) lesions using proton magnetic resonance spectroscopy (1H MRS). DESIGN: CSF polymerase chain reaction (PCR) detection for JC viral (JCV) DNA; MRI and localized 1H MRS in the PML lesions, normal-appearing contralateral brain regions (CONTRA), and in matched brain regions of normal subjects. SETTING: University-affiliated medical center. PATIENTS OR PARTICIPANTS: 20 AIDS patients with clinical diagnosis of PML, 16 had tissue and/or CSF evidence of JCV infection; 20 age-matched normal subjects. MAIN OUTCOME MEASURES: Metabolites from 1H MRS: N-acetyl aspartate (NA), creatine (CR), choline-containing compounds (CHO), myoinositol (MI), glutamine/glutamate (GLX), lactate, and lipids. RESULTS: CSF PCR for JCV DNA showed 86% sensitivity. MRI showed characteristic demyelinating lesions; commonest locations were frontal lobe and cerebellum. 1H MRS in the lesions showed decreased NA (-35%; p < 0.0001) and CR (-18%; p = 0.003), increased CHO (+28%; p = 0.0005), occasional increased MI, and excess lactate (15/20 lesions) and lipids (18/20). In the CONTRA, MRS showed trends for increased CR (+15%), CHO (+15%), MI (+13%), and lower GLX (-9%; p = 0.02). Six patients, studied longitudinally (4-18 months), showed progressive spectroscopic changes; two patients with longest survival showed the highest MI. CONCLUSIONS: These MRS findings are consistent with neuropathologic observations of neuronal loss, cell membrane and myelin breakdown, and increased glial activity in PML lesions. The CONTRA abnormalities may be due to remote effects of PML or direct HIV-1 infection. 1H MRS may be useful for characterization and follow-up evaluation of PML lesions.

Depletion of T lymphocytes with immunotoxin retards the progress of experimental allergic encephalomyelitis in rhesus monkeys.

FN18-CRM9 is an anti-rhesus anti-CD3 immunotoxin that can transiently deplete T cells to 1% of initial values in both the blood and lymph node compartments and can induce long-term tolerance to mismatched renal allografts. We have investigated the ability of this immunotoxin to interdict the course of an experimental rhesus T-cell-driven autoimmune disease, experimental allergic encephalomyelitis (EAE) induced by myelin basic protein. Monkeys showing CSF pleocytosis were then treated with FN18-CRM9 alone or in combination with cranial irradiation (325 or 650 cGy). EAE in nontreated control monkeys progressed rapidly. Paralysis occurred 4-6 days after CSF pleocytosis. Paralysis was either delayed or never occurred in treated monkeys, and histopathology revealed few inflammatory plaques that were notable for their low or absent T cell content. While T cells repopulate in the periphery posttreatment, they do not return to the CNS in large numbers, suggesting that the newly repopulated T cells have lost their previously acquired CNS homing capability. Anti-CD3 immunotoxin may be useful in treating clinical T-cell-driven autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Efficient gene transfer into primary and immortalized human fetal glial cells using adeno-associated virus vectors: establishment of a glial cell line with a functional CD4 receptor.

Adeno associated virus (AAV) is a non-pathogenic dependent parvovirus with a broad host range, capable of high levels of transduction and stable integration into the host cell genome. We have investigated the potential for using AAV as a vector for gene transfer into glial cells of the human fetal nervous system. Recombinant AAV vectors expression either the reporter gene beta-galactosidase or a human CD4 receptor were able to transduce both primary glial cells of the human fetal nervous system and an SV40 immortalized human fetal glial cell line (SVG). No difference in transduction efficiency was observed between the primary cells and the cell line which in both cases was as high as 95%. Stable transfectants of the glial cell line expressing the CD4 receptor were selected. An SVG/CD4 expressing line was then established. The presence of the CD4 receptor was confirmed by immunohistochemistry, Westerm immuno-blotting and flow cytometric analysis. The CD4 receptor was shown to be functional by infection of the SVG/CD4 cell line with the human immunodeficiency virus (HIV). Upon infection, the SVG/CD4 cells produced 20-fold higher levels of the HIV intracellular core antigen P24 than the CD4 negative parental cells and in addition formed syncytia. The use of AAV vectors should prove useful in biological investigations of human glial cells and offers promise as a means of ex vivo and in vivo gene delivery.

JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: implications for viral latency.

The human polyomavirus JC virus (JCV) infects myelin-producing cells in the central nervous system, resulting in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV-induced PML occurs most frequently in immunosuppressed individuals, with the highest incidence in human immunodeficiency type 1-infected patients, ranging between 4 and 6% of all AIDS cases. Although JCV targets a highly specialized cell in the central nervous system, infection is widespread, with more than 80% of the human population worldwide demonstrating serum antibodies. A number of clinical and laboratory studies have now linked the pathogenesis of PML with JCV infection in lymphoid cells. For example, JCV-infected lymphocytes have been suggested as possible carriers of virus to the brain following reactivation of a latent infection in lymphoid tissues. To further define the cellular tropism associated with JCV, we have attempted to infect immune system cells, including CD34+ hematopoietic progenitor cells derived from human fetal liver, primary human B lymphocytes, and human tonsillar stromal cells. Our results demonstrate that these cell types as well as a CD34+ human cell line, KG-1a, are susceptible to JCV infection. JCV cannot, however, infect KG-1, a CD34+ cell line which differentiates into a macrophage-like cell when treated with phorbol esters. In addition, peripheral blood B lymphocytes isolated by flow cytometry from a PML patient demonstrate JCV infection. These results provide direct evidence that JCV is not strictly neurotropic but can infect CD34+ hematopoietic progenitor cells and those cells which have differentiated into a lymphocytic, but not monocytic, lineage.

In vitro properties of a newly established medulloblastoma cell line, MCD-1.

Medulloblastomas are poorly differentiated brain tumors believed to arise from primitive pleuripotential stem cells, and tend to express mixed neuronal and glial properties. In the present study, we examined immunohistochemical and neurotransmitter phenotypic properties in a newly established medulloblastoma cell line, MCD-1. MCD-1 cells were immortal, not contact-inhibited, but did not grow in soft agar. Immunohistochemical studies showed positive staining for neurofilament protein (NF), neuron-specific enolase (NSE), synaptophysin, MAP 2, tau, NCAM 180, vimentin, and S-100 protein. The cells expressed specific uptake of glutamate, serotonin, and choline, but not GABA or dopamine. A significant increase in process extension was seen in response to agents that enhance intracellular cyclic AMP, especially 3-isobutyl-1-methylxanthine (IBMX). Process formation induced by IBMX was associated with a decrease in cell proliferation as evidenced by a reduction in numbers of cells incorporating 5-bromo-2-deoxyuridine (BrdU). No increase in process extension was observed following exposure to NGF or retinoic acid. MCD-1 cells were shown to produce transforming growth factor beta (TGF beta), and were immunopositive for mutant p53. Transfection assays with the PG13-Luc reporter plasmid, which contains a p53-responsive enhancer element and a luciferase reporter gene, suggested MCD-1 cells are deficient in wild-type p53 and do not activate p53 on treatment with the anticancer agent adriamycin. The MCD-1 cell line is suggested to represent an abnormally differentiated cell type, which has some properties consistent with a multipotent neuronal phenotype while retaining some properties of immature cells of a glial lineage. The MCD-1 cell line can be used to provide a model of a medulloblastoma cell line that is resistant to growth-controlling and anticancer agents.

Immunohistochemical staining of fixed tissue using antigen retrieval and a thermal cycler.

The use of monoclonal antibodies to identify antigens in perfused fixed tissue is currently very challenging. Recently, many antigen retrieval methods have been described that require heating the tissue on slides using a microwave oven. Here, we describe a protocol for antigen retrieval that uses a commercially available solution called Citra and a DNA thermal cycler to heat the tissue sections. This method (i) allows for precise control of the temperature and time (unlike the microwave oven), (ii) uses very small quantities of the antigen retrieval solution, (iii) stains either free-floating sections or sections mounted on slides and (iv) achieves uniform staining throughout the tissue. In the paraformaldehyde-perfused fixed rat cerebellum, the monoclonal antibody glial fibrillary acidic protein conveys the clarity of the Bergmann and stellate glia in the strata. Scanning confocal images through the center of the tissue demonstrates the depth of antibody penetration achieved using this method. Finally, using a monoclonal antibody against the simian virus 40 (SV40) T antigen allowed positive, unequivocal identification of a grafted cell line, immortalized using SV40 T protein, thus demonstrating the true value of this method in an experimental paradigm.