Effects of Cyclosporin A Treatment on Clinical Course and Inflammatory Cell Apoptosis in Experimental Autoimmune Encephalomyelitis Induced in Lewis Rats by Inoculation with Myelin Basic Protein

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by inoculation with myelin basic protein (MBP) and adjuvants. Rats were treated with second daily injections of saline or cyclosporin A (CsA) from the day of inoculation. Saline-treated rats had an acute episode of disease followed by clinical recovery. Rats treated with CsA 16 or 32 mg/kg had minimal signs of EAE at the usual time after inoculation, but developed signs of disease after treatment was ceased. Rats treated with CsA 8 mg/kg had a delayed first episode of disease and then developed a relapsing or a chronic persistent course of disease. CsA 4 mg/kg delayed the onset of disease. To study the effects of CsA on the inflammatory infiltrate, cells were extracted from the spinal cords of rats with EAE, 16 h after a single injection of CsA or saline. Extracted cells were labelled with antibodies to T cells, CD11b/c (macrophages/microglia), CD95 (Fas) and Fas ligand. CsA 4 mg/kg did not alter the composition of the inflammatory infiltrate. Treatment with higher single doses of CsA caused a dose-dependent decline in the percentage of T cell receptor (TCR)alpha beta+ cells in the inflammatory infiltrate. All doses of CsA caused a significant increase in the number and percentage of cells that were apoptotic. CsA treatment caused an increase in the percentages of CD5+ and TCR alpha beta+ cells that were apoptotic. There was a decline in the percentage of apoptotic T cells that were V beta 8.2+, compared to the percentage of non-apoptotic T cells that were V beta 8.2+, in CsA treated rats compared to saline-treated controls. This suggests that, while CsA treatment caused a non-specific increase in the overall level of T cell apoptosis in the spinal cord, it abrogated the selective apoptosis of V beta 8.2+ encephalitogenic T cells that normally occurs during spontaneous recovery from acute EAE

Antigen-Specific Down-Regulation of Myelin Basic Protein-Reactive T Cells During Spontaneous Recovery From Experimental Autoimmune Encephalomyelitis: Further Evidence of Apoptotic Deletion of Autoreactive T Cells in the Central Nervous System

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by the i.v. injection of 107 cloned V beta 8.2+ T cells specific for the 72-89 peptide of guinea pig myelin basic protein (MBP). Some animals were injected simultaneously with 107 cloned T cells specific for ovalbumin (OVA). Lymphocytes were isolated from the spinal cord and from the peripheral lymphoid organs of these rats and the frequencies of MBP-peptide-specific or OVA-specific proliferating cells were estimated by limiting dilution analysis at different times after cell transfer. The frequencies of cells specific for MBP_72-89 or OVA in the spinal cord were highest 5 days after cell transfer (MBP_72-89, 1 in 1149; OVA, 1 in 1116). On day 7, when the rats were recovering, the frequency of cells specific for MBP_72-89 in the spinal cord fell dramatically t

Macrophage apoptosis in the central nervous system in experimental autoimmune encephalomyelitis

Using light and electron microscopy, we have demonstrated that macrophage apoptosis (programmed cell death) occurs in the central nervous system (CNS) in Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) and chronic relapsing EAE. Apoptotic macrophages were identified by the presence of an apoptotic nucleus in a cell with cytoplasm containing myelin debris but no intermediate filaments. They were found in the meninges, perivascular spaces and in the parenchyma of the white and grey matter of the spinal cord. In acute EAE the apoptotic macrophages were most frequently seen at the time of maximal neurological signs and during the early stages of clinical recovery. Several possible mechanisms may be responsible for the macrophage apoptosis: the release or withdrawal of cytokines; T-cell cytotoxicity; the effect of activated macrophage products, such as nitric oxide; and a direct effect of endogenous glucocorticoids. Macrophage apoptosis, together with the T-cell apoptosis we have previously described in the CNS in EAE, may contribute to the down-regulation of this autoimmune disease

Conduction abnormalities are restricted to the central nervous system in experimental autoimmune encephalomyelitis induced by inoculation with proteolipid protein but not with myelin basic protein

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) and can be induced by inoculation of animals with homogenized CNS tissue or highly purified myelin proteins such as myelin basic protein (MBP) or proteolipid protein (PLP). It is widely studied as a possible animal model of multiple sclerosis. We performed the present neurophysiological study to define the location of nerve conduction abnormalities in EAE induced by immunization with PLP (PLP-EAE) and in EAE induced by immunization with MBP (MBP-EAE) in the Lewis rat. In rats with tail weakness due to acute PLP-EAE, conduction was normal in the spinal nerve roots and peripheral nerves but there was evidence of conduction block in a high proportion of the fibres in the dorsal columns of the lumbosacral spinal cord. In contrast, in acute MBP-EAE, there was conduction block in a high proportion of fibres in the sacral dorsal and ventral roots of the peripheral nervous system (PNS) and in the dorsal columns of the lumbosacral spinal cord. The distribution of nerve conduction abnormalities is consistent with previous histological studies showing that inflammation and primary demyelination are restricted to the CNS in PLP-EAE, but are present in the CNS and in the spinal roots of the PNS in MBP-EAE. The restriction of functional abnormalities to the CNS in PLP-EAE but not in MBP-EAE may have implications for the human inflammatory demyelinating diseases, including multiple sclerosis

Apoptosis of αβ T lymphocytes in the nervous system in experimental autoimmune encephalomyelitis: Its possible implications for recovery and acquired tolerance

We have recently shown that apoptosis, an active process of cellular self-destruction, occurs in the central nervous system in Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Conventional light and electron microscopic studies suggested that some of the apoptotic cells were oligodendrocytes and that others were hematogenous mononuclear cells. To determine whether any of the apoptotic cells were T lymphocytes, we used the technique of pre-embedding immunolabelling which allows sufficient preservation of the ultrastructure to permit recognition of apoptotic changes while at the same time preserving surface antigens so that the identity of the apoptotic cells can be determined by immunocytochemistry. Light microscopic immunocytochemistry using the mono-clonal antibodies OX-34 (CD2) and R73 (alpha beta T-cell receptor) revealed that 10% of the CD2+ cells and 5% of the alpha beta T lymphocytes in the parenchyma of the spinal cord were dying by apoptosis. The presence of apoptotic alpha beta T cells was confirmed by electron microscopy. About half of all the apoptotic cells within the spinal cord were labelled by these antibodies. It is possible that some of the unlabelled apoptotic cells were also T lymphocytes but that others were glial cells such as oligodendrocytes. One possible interpretation of this T-cell apoptosis is that it represents activation-induced cell death, which has recently been shown to provide a mechanism of clonal elimination of mature as well as immature autoreactive T cells. Another possible interpretation is that it is a result of corticosterone released during the course of EAE. The apoptotic elimination of target-antigen-specific lymphocytes within the target organ in this autoimmune disease may contribute to the subsidence of inflammation and, if ongoing, to the development of tolerance

Cyclosporin A Treatment Modulates Cytokine mRNA Expression by Inflammatory Cells Extracted from the Spinal Cord of Rats with Experimental Autoimmune Encephalomyelitis Induced by Inoculation with Myelin Basic Protein

In Lewis rats, treatment with high doses of cyclosporin A (CsA) suppresses clinical signs of experimental autoimmune encephalomyelitis (EAE), although disease occurs when treatment is ceased. Treatment with low doses of CsA causes EAE to take a chronic relapsing course. We have previously shown that CsA treatment causes a decline in the number of T cells and increased inflammatory cell apoptosis in the spinal cord. The present study was undertaken to assess whether CsA therapy also modulates cytokine mRNA expression by inflammatory cells in the spinal cord of rats with EAE, looking for changes that might contribute to the observed effects of CsA on the course of EAE. EAE was induced in Lewis rats by inoculation with myelin basic protein and adjuvants. At the peak of neurological signs, on day 14 after inoculation, rats were given a single intraperitoneal injection of saline, or CsA at a dose of 8, 16, 32 or 64 mg/kg. The next day, rats were sacrificed, the spinal cords removed, inflammatory cells were extracted from the cords, and mRNA isolated from these cells. Expression of cytokine mRNA was assessed by semi-quantitative reverse transcription polymerase chain reaction (PCR) and by quantitative real-time PCR. With both techniques, we found that CsA suppressed the expression of interferon-gamma mRNA and interleukin-2 (IL-2) mRNA. With real-time PCR, we found that CsA caused significantly increased expression of transforming growth factor-beta mRNA. With the different techniques, we observed no consistent pattern of alteration of expression of interleukin-10 or interleukin-4 mRNA. It is possible that these changes in cytokine mRNA expression contribute to the modulation of the clinical course of EAE that is produced by CsA treatment

The Proximal Peripheral Nervous System Is A Major Site Of Demyelination In Experimental Autoimmune Encephalomyelitis Induced In The Lewis Rat By A Myelin Basic Protein-Specific T Cell

Experimental autoimmune encephalomyelitis (EAE) was induced in the Lewis rat by the passive transfer of a cytotoxic CD4 + T cell clone specific for the 72-89 peptide of guinea-pig myelin basic protein (MBP). Histological studies on rats with neurological signs showed that inflammation was present in the proximal peripheral nervous system (PNS), namely the spinal roots, as well as in the central nervous system (CNS). The main sites of demyelination were the spinal roots in the PNS, and the spinal cord root entry and exit zones in the CNS. The major involvement of the proximal PNS in autoimmune disease directed at MBP is in marked contrast to EAE induced by immunisation with myelin proteolipid protein, where the inflammation and demyelination are restricted to the CNS. These findings may have implications for the human inflammatory demyelinating diseases including multiple sclerosis, in which MBP is a putative target antigen

The effect of ageing on human lymphocyte subsets: comparison of males and females

There is reported to be a decline in immune function and an alteration in the frequency of circulating lymphocytes with advancing age. There are also differences in ageing and lifespan between males and females. We performed this study to see if there were differences between males and females in the frequency of the different lymphocyte subsets with age.Using flow cytometry we have examined different populations of peripheral blood leukocytes purified from healthy subjects with age ranging from the third to the tenth decade. We used linear regression analysis to determine if there is a linear relationship between age and cell frequencies. For the whole group, we find that with age there is a significant decline in the percentage of naïve T cells and CD8(+) T cells, and an increase in the percentage of effector memory cells, CD4(+)foxp3(+) T cells and NK cells. For all cells where there was an effect of ageing, the slope of the curve was greater for men than for women and this was statistically significant for CD8(+)alphabeta(+) T cells and CD3(+)CD45RA(-)CCR7(-) effector memory cells. There was also a difference for naïve cells but this was not significant.The cause of the change in percentage of lymphocyte subsets with age, and the different effects on males and females is not fully understood but warrants further study

Case report: Adult-onset limb girdle muscular dystrophy in sibling pair due to novel homozygous LAMA2 missense variant

Recessive pathogenic variants in the laminin subunit alpha 2 (LAMA2) gene cause a spectrum of disease ranging from severe congenital muscular dystrophy to later-onset limb girdle muscular dystrophy (LGMDR23). The phenotype of LGMDR23 is characterized by slowly progressive proximal limb weakness, contractures, raised creatine kinase, and sometimes distinctive cerebral white matter changes and/or epilepsy. We present two siblings, born to consanguineous parents, who developed adult-onset LGMDR23 associated with typical cerebral white matter changes and who both later developed dementia. The male proband also had epilepsy and upper motor neuron signs when he presented at age 72. Merosin immunohistochemistry and Western blot on muscle biopsies taken from both subjects was normal. Whole exome sequencing revealed a previously unreported homozygous missense variant in LAMA2 [Chr6(GRCh38):g.129297734G>A; NM_000426.3:c.2906G>A; p.(Cys969Tyr)] in the proband. The same homozygous LAMA2 variant was confirmed by Sanger sequencing in the proband's affected sister. These findings expand the genotypic and phenotypic spectrum of LGMDR23

Increased Circulating T Cell Reactivity to GM1 Ganglioside in Patients with Guillain-Barre Syndrome

This study was performed to determine whether increased ganglioside-specific T cell reactivity can be detected in the peripheral blood of patients with Guillain-Barre syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). T cell responsiveness to the gangliosides GM1, GM3, GD1a, GD1b, GD3, GT1b, GQ1b and sulphatide was assessed in peripheral blood mononuclear cells from untreated GBS patients (57), CIDP patients (43), patients with other peripheral neuropathies (55) and healthy control subjects (74) in a standard 6-day proliferation assay. Increased T cell reactivity to GM1 occurred in GBS patients compared to healthy controls and patients with other neuropathies. There was increased reactivity to GM3 in GBS patients compared to patients with other neuropathies but not compared to healthy controls. The frequencies of increased T cell reactivity to GM1 and GM3 in CIDP patients were intermediate between those of GBS patients and controls. We suggest that T cell reactivity to gangliosides might play a contributory role in the pathogenesis of GBS and perhaps CIDP