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Biomedical subjects

M P Pender

Publications and source records attributed to M P Pender.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cytokine expression by inflammatory cells obtained from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis induced by inoculation with myelin basic protein and adjuvants.

Inflammatory cells were obtained from the spinal cords of rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of mRNA for interleukin-2 (IL-2), IL-4, IL-10 and interferon-gamma (IFN-gamma) by cells from groups of rats studied 10-21 days after inoculation. On all days of study, the inflammatory cells, which were predominantly lymphocytes, expressed mRNA for IL-2, IL-4, IL-10 and IFN-gamma. In the mRNA from normal rat spinal cord tissue, there was little expression of cytokine mRNA. Cells from a short-term MBP-reactive T cell line expressed all the cytokines. Densitometry was used to measure the products of PCR, to assess the expression of each cytokine relative to that of beta-actin. IL-2 mRNA was expressed throughout the course of disease and reached a peak on day 18, during late clinical recovery. IFN-gamma was expressed throughout the course of the disease and was also high during late recovery. IL-4 mRNA was present in the spinal cord throughout the course of the disease, with a slight rise during late recovery. Relative expression of IL-10 rose to a peak on days 17-19, during late recovery from clinical disease. This study indicates that IL-2, IL-4, IL-10 and IFN-gamma are expressed by inflammatory cells in the spinal cord in EAE, with the relative expression of all cytokines being high during late clinical recovery.

Adjuvants, Immunologic↗
PubMed1998

Genetically determined failure of activation-induced apoptosis of autoreactive T cells as a cause of multiple sclerosis.

I postulate that multiple sclerosis is an autoimmune disease that involves genetically determined failure of activation-induced apoptosis of autoreactive T cells in the central nervous system. Activation of central-nervous-system-reactive T cells in peripheral lymphoid organs by exposure to crossreacting antigens or superantigens derived from common infectious agents may trigger attacks of multiple sclerosis. In normal individuals these activated T cells are deleted by activation-induced apoptosis, but in individuals predisposed to multiple sclerosis they survive, proliferate, and damage the central nervous system. The clinical course of multiple sclerosis may vary according to the antigens in the central nervous system being targeted: targeting of myelin antigens leads to a relapsing-remitting course of clinical recovery due to remyelination or other mechanisms; targeting of axonal antigens leads to a progressive course from onset because axonal regeneration is limited in the central nervous system. This hypothesis can account for many characteristics of multiple sclerosis and has predictions that can be tested.

Animals↗
PubMed1998

Soluble antigen therapy induces apoptosis of autoreactive T cells preferentially in the target organ rather than in the peripheral lymphoid organs.

The administration of soluble myelin proteins is an effective way of down-regulating the inflammation in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. To shed more light on the mechanism of this antigen-specific therapy, we determined the effect of the intraperitoneal (i.p.) injection of soluble myelin basic protein (MBP) on Tcell apoptosis in the CNS and peripheral lymphoid organs of Lewis rats with EAE induced by inoculation with MBP and complete Freund's adjuvant. In particular we assessed the level of apoptosis of Vbeta8.2+ Tcells, which constitute the predominant encephalitogenic MBP-reactive T cell population in the Lewis rat. The daily i.p. injection of MBP for 3 days from the onset of neurological signs inhibited the further development of neurological signs of EAE. Using two-color flow cytometry we found that a single i.p. injection of MBP increased the level of apoptosis of the Vbeta8.2+ T cell population in the CNS to 26.2% compared to 7.4% in saline-treated rats and 7.6% in ovalbumin-treated rats. In contrast, treatment with MBP did not increase the level of apoptosis of the Vbeta8.2+ population in the popliteal lymph node draining the inoculation site (1.4%) or in the spleen (1.6%) above that occurring in saline-treated rats (1.6% and 1.1%, respectively). Limiting dilution analysis revealed that the frequency of T cells reactive to the major encephalitogenic epitope, MBP72-89, was decreased in the CNS but not in the popliteal lymph node by this treatment. Three-color flow cytometry in MBP-treated rats demonstrated that CNS Vbeta8.2+ T cells expressing Fas (CD95) and Fas ligand were highly vulnerable to apoptosis compared to Vbeta8.2+ Tcells not expressing these proteins. We conclude that the i.p. injection of MBP increases the spontaneously occurring Fas-mediated activation-induced apoptosis of autoreactive T cells in the CNS in EAE and that this contributes to the therapeutic effect of the injection.

Acute Disease↗
PubMed1998

Increased circulating antiganglioside antibodies in primary and secondary progressive multiple sclerosis.

Plasma samples from 70 patients with multiple sclerosis (MS), 41 patients with other neurological diseases (OND), and 38 healthy subjects were examined for antibodies against gangliosides GM1, GM3, GD1a, GD1b, and GD3 using enzyme-linked immunosorbent assays. The percentages of subjects with increased anti-GM3 responses were significantly higher in the primary progressive MS (56.3%) and secondary progressive MS (42.9%) groups than in the relapsing-remitting MS (2.9%), healthy subject (2.6%), and OND (14.6%) groups. Elevated antiganglioside antibodies may be secondary to axonal damage or may be a cause of axonal damage and accumulating disability in progressive MS. In either case, they may serve as a marker of axonal damage in MS.

Antibodies↗
PubMed1998

Phagocytosis of apoptotic lymphocytes by oligodendrocytes in experimental autoimmune encephalomyelitis.

The alterations in oligodendrocytes in myelin basic protein-induced acute experimental autoimmune encephalomyelitis (EAE) in the Lewis rat were studied using the technique of pre-embedding immunolabelling with the Rip monoclonal antibody, which specifically labels the cytoplasm of the cell bodies and processes of oligodendrocytes. In spinal cord sections of untreated and dexamethasone-treated rats with EAE, we found that apoptotic lymphocytes were phagocytosed by Rip-positive oligodendrocytes as well as by CD11b/c-positive macrophages/microglia and by astrocytes expressing glial fibrillary acidic protein. Morphologically normal lymphocytes were also internalised by these cell type, a phenomenon known as emperipolesis. We suggest that this phenomenon represents the phagocytosis of lymphocytes that have undergone the plasma membrane alterations of apoptosis without yet manifesting the nuclear condensation of apoptosis. We also found an increase in the number of clusters of two to four oligodendrocytes, mainly in the grey matter, suggesting proliferation of cells of the oligodendrocyte lineage.

Animals↗
PubMed1998

Microglia are more susceptible than macrophages to apoptosis in the central nervous system in experimental autoimmune encephalomyelitis through a mechanism not involving Fas (CD95).

Morphological studies have shown that macrophages and microglia undergo apoptosis in the central nervous system (CNS) in acute experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. To assess the relative levels of macrophage and microglial apoptosis, and the molecular mechanisms involved in this process, we used three-colour flow cytometry to identify CD45lowCD11b/c+ microglial cells and CD45highCD11b/c+ macrophages in the inflammatory cells isolated from the spinal cords of Lewis rats 13 days after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Simultaneously, we analyzed the DNA content of these cell populations to assess the proportions of cells undergoing apoptosis and in different stages of the cell cycle or examined their expression of three apoptosis-regulating proteins, i.e. Fas (CD95), Fas ligand (FasL) and Bcl-2. Microglia were highly vulnerable to apoptosis and were over-represented in the apoptotic population. Macrophages were less susceptible to apoptosis than microglia and underwent mitosis more frequently than microglia. The different susceptibilities of microglia and macrophages to apoptosis did not appear to be due to variations in Fas, FasL or Bcl-2 expression, as the proportions of microglia and macrophages expressing these proteins were similar, and were relatively high. Furthermore, in contrast to T cell apoptosis, apoptosis of microglia/macrophages did not occur more frequently in cells expressing Fas or FasL, or less frequently in cells expressing Bcl-2. These results indicate that the apoptosis of microglia and CNS macrophages in EAE is not mediated through the Fas pathway, and that Bcl-2 expression does not protect them from apoptosis. Expression of FasL by macrophages and microglia may contribute to the pathogenesis and immunoregulation of EAE through interactions with Fas+ oligodendrocytes and Fas+ T cells. The high level of microglial apoptosis in EAE indicates that microglial apoptosis may be an important homeostatic mechanism for controlling the number of microglia in the CNS following microglial activation and proliferation.

Animals↗
PubMed1998

Goodpasture's syndrome associated with multiple sclerosis.

Multiple sclerosis (MS) has been reported to occur in association with other autoimmune diseases. Here we report the case of a woman with primary progressive MS who developed and died of Goodpasture's syndrome associated with anti-glomerular basement membrane antibodies. Of interest she had the human leukocyte antigen haplotype (DRB1*1501-DQA1*0102-DQB1*0602) most frequently associated with these two diseases; however, it is likely that other genes, particularly those involved with immunoregulation, also contributed to her susceptibility to these diseases. The association of MS and autoimmune renal disease may be more common than currently recognized.

Adult↗
PubMed1997

Increased immunoreactivity to two overlapping peptides of myelin proteolipid protein in multiple sclerosis.

We tested the proliferative responses of peripheral blood mononuclear cells from 61 patients with multiple sclerosis, 56 healthy control subjects and 52 patients with other neurological diseases to seven synthetic peptides of myelin proteolipid protein (PLP) and 19 synthetic peptides of myelin basic protein (MBP). Increased proliferative responses to two overlapping PLP peptides, PLP184-199 and PLP190-209 were found significantly more frequently in blood from patients with relapsing-remitting or secondary progressive multiple sclerosis (52.3%), but not from those with primary progressive multiple sclerosis (18.2%), than in that from healthy control subjects (8.9%) and patients with other neurological diseases (20.8%). Reactivity to these PLP peptides was most frequently seen in blood from patients with multiple sclerosis of 6-15 years duration and with moderate to severe disability (Kurtzke's Expanded Disability Status Scale > 4.0); the blood from 15 of 19 patients in this group reacted to one or both of the peptides. Both peptides could be recognized by short-term T-cell lines specific for whole PLP, and lines specific for one or other of the two overlapping peptides were able to recognize whole PLP, indicating that these peptides can be processed naturally from the intact molecule. This region of PLP is encephalitogenic in a number of strains of mice. Samples from multiple sclerosis patients did not react more frequently to any of the MBP peptides than those from healthy control subjects. The proportions of patients with other neurological diseases whose blood responded to the MBP peptides that most frequently elicited responses in blood from multiple sclerosis patients were significantly lower than the proportions of multiple sclerosis patients and healthy control subjects whose blood responded to these peptides.

Adolescent↗
PubMed1997

Increased apoptosis of T lymphocytes and macrophages in the central and peripheral nervous systems of Lewis rats with experimental autoimmune encephalomyelitis treated with dexamethasone.

Using light and electron microscopic histological and immunocytochemical techniques, we investigated the effects of the glucocorticoid dexamethasone on T cell and macrophage apoptosis in the central nervous system (CNS) and peripheral nervous system (PNS) of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP). A single subcutaneous injection of dexamethasone markedly augmented T cell and macrophage apoptosis in the CNS and PNS and microglial apoptosis in the CNS within 6 hours (h). Pre-embedding immunolabeling revealed that dexamethasone increased the number of apoptotic CD5+ cells (T cells or activated B cells), alphabeta T cells, and CD11b+ cells (macrophages/microglia) in the meninges, perivascular spaces, and CNS parenchyma. The induction of increased apoptosis was dose-dependent. Daily dexamethasone treatment suppressed the neurological signs of EAE. However, the daily injection of a dose of dexamethasone (0.25 mg/kg), which, after a single dose, did not induce increased apoptosis in the CNS or PNS, was as effective in inhibiting the neurological signs of EAE as the high dose (4 mg/kg), which induced a marked increase in apoptosis. This indicates that the beneficial clinical effect of glucocorticoid therapy in EAE does not depend on the induction of increased apoptosis. The daily administration of dexamethasone for 5 days induced a relapse that commenced 5 days after cessation of treatment, with the severity of the relapse tending to increase with dexamethasone dosage.

Animals↗
PubMed1996

A simple technique for flat osmicating and flat embedding of immunolabelled vibratome sections of the rat spinal cord for light and electron microscopy.

We describe here a simple technique for flat-osmicating and flat-embedding of immunolabelled vibratome sections. The technique is particularly useful for large specimens such as whole cross-sections of rat spinal cord. After the vibratome section has been flat-osmicated on a flat surface under a glass coverslip, it is dehydrated and then embedded by placing it in a small drop of epoxy resin on a flat base of polypropylene plastic and overlaying a small square piece of cellulose acetate cut from heat-resistant overhead projector transparency film for photocopying. The thin layer of resin containing the flat-embedded vibratome section is then separated from the base and glued onto the flat end of a pre-polymerised blank block of resin. The method produces flat-embedded vibratome sections and thus allows serial large uniformly labelled semithin and ultrathin sections to be obtained of the whole cross-section of the rat spinal cord. This facilitates the observation and quantification of labelled cells in the specimen. Because of its simplicity the technique also allows one worker to process more than 100 vibratome sections at the one time.

Animals↗
PubMed1996

Corticosteroid treatment of experimental autoimmune encephalomyelitis in the Lewis rat results in loss of V beta 8.2+ and myelin basic protein-reactive cells from the spinal cord, with increased total T-cell apoptosis but reduced apoptosis of V beta 8.2+ cells.

We have studied the effects of corticosteroid treatment on the numbers of lymphocytes obtained from the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Flow cytometric studies showed that treatment with dexamethasone (4 mg/kg) 8-12 h prior to study on day 14 after inoculation resulted in a reduction in the numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells in the spinal cord. Limiting dilution analysis indicated that dexamethasone treatment 12 h prior to study on day 12 after inoculation reduced the frequencies of MBP-reactive and interleukin-2-responsive lymphocytes in the spinal cord to low levels, but reduced the frequency of concanavalin-A-responsive lymphocytes to a lesser extent. Using propidium iodide staining of nuclear chromatin we also studied lymphocyte apoptosis. Greater numbers of apoptotic cells were found in the cells extracted from the spinal cords of rats, examined on day 14, that had been treated 1-12 h previously with dexamethasone, than in saline-treated controls. This increased level of apoptosis was observed in the CD5+ and TCR alpha beta + cell populations. At 1-4 h after dexamethasone treatment there was a reduction in the selective apoptosis of V beta 8.2+ cells that normally occurs during spontaneous recovery from EAE. Therefore apoptosis of V beta 8.2+ cells cannot explain the reduction in the numbers of V beta 8.2+ cells and MBP-reactive cells in the CNS after dexamethasone treatment. By 8-12 h after dexamethasone treatment the selectivity of the apoptotic process was restored. These studies suggest that a reduction in the number of T-lymphocytes in the central nervous system contributes to the beneficial effects of corticosteroids in EAE.

Animals↗
PubMed1996

A study of human T-cell lines generated from multiple sclerosis patients and controls by stimulation with peptides of myelin basic protein.

We generated T-cell lines from the peripheral blood of controls and of patients with multiple sclerosis (MS) by stimulation with overlapping synthetic peptides representing the entire sequences of all four isoforms of human myelin basic protein (MBP). The T-cell lines reacted to a wide range of epitopes in the major isoforms of MBP and to epitopes that were present only in the minor isoforms. Many MS patients and controls had T-cells responding to one or more cryptic MBP epitopes, as indicated by the generation of a peptide-specific T-cell line(s) by stimulation with synthetic peptides but not by stimulation with whole MBP. About one-third of the peptide-generated lines were cytotoxic. Although we have shown that this technique of peptide stimulation is effective in generating human antiviral cytotoxic CD8+ T-cell lines, all the cytotoxic MBP-specific lines generated by this method were predominantly CD4+. Our study did not reveal any significant differences, between MS patients and controls, in reactivity to epitopes within any of the isoforms of MBP.

Adolescent↗
PubMed1996

Apoptosis of V beta 8.2+ T lymphocytes in the spinal cord during recovery from experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein.

To study T cell apoptosis during spontaneous recovery from experimental autoimmune encephalomyelitis (EAE), we extracted lymphocytes from the spinal cords of Lewis rats with EAE induced by inoculation with myelin basic protein (MBP) and adjuvants. Using flow cytometry we assessed the numbers of CD5+ and TCR alpha beta + lymphocytes, as well as V beta 8.2+ lymphocytes, which constitute the predominant encephalitogenic MBP-reactive cells in Lewis rats. Rats developed neurological signs of disease 10-12 days after inoculation. The peak of disease was on day 14 after inoculation and was followed by clinical recovery. The numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells obtained from the spinal cord were greatest on day 13. During spontaneous clinical recovery, there was a decline in the numbers of all the cells studied, with a selective loss of V beta 8.2+ cells from the CD5+ and TCR alpha beta + populations. To determine whether the decline in lymphocyte numbers was due to apoptosis, we used simultaneous surface labelling and propidium iodide staining of the DNA of the cells extracted from the spinal cord. From day 14 onwards, there was selective enrichment of V beta 8.2+ cells in the apoptotic population, and the percentage of V beta 8.2+ cells undergoing apoptosis was greater than the percentages of CD5+ and TCR alpha beta + cells undergoing apoptosis. These findings indicate that recovery from acute EAE is associated with the selective apoptosis, in the central nervous system, of these disease-relevant cells. The findings in this study of actively induced EAE are similar to those of our previous study of EAE induced by transfer of encephalitogenic MBP-specific T cells (Z. Tabi et al., Eur. J. Immunol. 24: 2609-2617, 1994) and further support the hypothesis that selective apoptosis of autoreactive T cells in the central nervous system is of primary importance in spontaneous recovery from EAE.

Animals↗
PubMed1995

Restoration of conduction in the spinal roots correlates with clinical recovery from experimental autoimmune encephalomyelitis.

In the Lewis rat, acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants is characterized by tail and hindlimb weakness that resolves spontaneously after several days. In rats with neurological signs of this form of EAE (MBP-EAE) we have previously demonstrated demyelination and nerve conduction block in the proximal peripheral nervous system (PNS) and in the central nervous system (CNS). The present study was performed to assess conduction in the PNS and CNS, after recovery from acute MBP-EAE, using direct recordings from surgically exposed spinal roots and spinal cord dorsal columns. The study revealed that 1-2 weeks after clinical recovery from tail paralysis there was almost complete restoration of conduction in the sacral spinal roots but persistent severe conduction abnormalities in the dorsal columns. Significant restoration of conduction through the dorsal columns occurred over the following 2 weeks. These findings indicate that PNS conduction block due to a demyelinating polyradiculitis is a major cause of the neurological signs of acute MBP-EAE in the Lewis rat.

Action Potentials↗