Gender differences in autoimmune disease.

Autoimmune diseases are a range of diseases in which the immune response to self-antigens results in damage or dysfunction of tissues. Autoimmune diseases can be systemic or can affect specific organs or body systems. For most autoimmune diseases there is a clear sex difference in prevalence, whereby females are generally more frequently affected than males. In this review, we consider gender differences in systemic and organ-specific autoimmune diseases, and we summarize human data that outlines the prevalence of common autoimmune diseases specific to adult males and females in countries commonly surveyed. We discuss possible mechanisms for sex specific differences including gender differences in immune response and organ vulnerability, reproductive capacity including pregnancy, sex hormones, genetic predisposition, parental inheritance, and epigenetics. Evidence demonstrates that gender has a significant influence on the development of autoimmune disease. Thus, considerations of gender should be at the forefront of all studies that attempt to define mechanisms that underpin autoimmune disease.

HLA and amyotrophic lateral sclerosis: a systematic review and meta-analysis.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with loss of upper and lower motor neurones. It leads to death by respiratory failure and has a typical prognosis of 2-3 years. The immune system has been shown to play a role in the pathophysiology of ALS. Some of the most important immune genes are within the human leukocyte antigen (HLA) region, and a recent genome-wide association study (GWAS) has identified a risk allele for ALS within the HLA region. Older studies have also suggested an HLA association with ALS, with certain HLA alleles showing differing expression between patients and controls. This systematic review and meta-analysis examines the previous studies performed in this field.Methods: We used established publication search engines. Findings were excluded if they did not meet the selection criteria. We then undertook statistical meta-analysis on the eligible papers, using a fixed effects model.Results: There were eight eligible papers. There were three statistically significant meta-analysis findings, although these would not be significant after correction for multiple comparisons. The frequencies of HLA-A9 and HLA-DR4 genotypes were lower in ALS subjects than controls, and HLA-B35 was higher in ALS subjects.Discussion: This systematic review and meta-analysis do not confirm all the previously reported associations of HLA with ALS, but shows three alleles of interest. However, there are limitations to the studies, which include the use of older serotyping methodology and the small numbers of subjects. Given the recent GWAS association with HLA, further modern HLA studies are warranted.

Cloning of a new form of EAAT2/GLT-1 from human and rodent brains.

Glutamate transporter 1 is the principal transporter that mediates glutamate clearance in the mammalian brain. In rodents, it is referred to as GLT-1, whereas in humans it is referred to as EAAT2. We have cloned a novel and abundantly expressed carboxyl-terminal splice variant of this transporter in both rodents and humans, which we denote as GLT-1d/EAAT2d. The novel splice variant results from usage of internal splice sites and the splicing event leads to novel extra sequence spliced in after exon 10. The open reading frames of GLT-1d and EAAT2d encode proteins of 572 and 566 amino acids respectively; both contain a C-terminal PDZ motif. When expressed in COS7 cells, the proteins function as glutamate transporters that are inhibited by dihydrokainate (a GLT-1/EAAT2 transporter inhibitor). RT-PCR amplification using GLT-1d specific primers confirmed expression of message in all brain regions examined (forebrain, midbrain, hindbrain and cerebellum) as well as spinal cord, astrocyte cultures, retina and peripheral tissues (liver, testis, small intestine and lung). Quantitative RT-PCR analysis showed that expression of GLT-1d is developmentally regulated. In adult human brain, EAAT2d message is âˆ¼ 30% of the level of EAAT2a message (the most abundant form), potentially making it the second most abundantly expressed form of EAAT2 in the brain. The amino terminal region of GLT-1d is also alternately spliced; the brain and testis forms contain a sequence corresponding to the amino acid sequence MASTEG whereas the corresponding liver sequence is MVS. In summary, we have cloned a novel EAAT2/GLT-1 splice variant from human and rodent brains. The splice variant is abundantly expressed in the brain, spinal cord, retina, liver and testis; it is a functional glutamate transporter; therefore, we conclude that it will likely have a functional role in glutamate homeostasis in the rodent and human nervous system, during development, adulthood, and plausibly in pathological states.

Monocyte CD14 and HLA-DR expression increases with disease duration and severity in amyotrophic lateral sclerosis.

Objective: To investigate changes in immune markers and frequencies throughout disease progression in patients with amyotrophic lateral sclerosis (ALS). Methods: In this longitudinal study, serial blood samples were collected from 21 patients with ALS over a time period of up to 16 months. Flow cytometry was used to quantitate CD14, HLA-DR, and CD16 marker expression on monocyte subpopulations and neutrophils, as well as their cell population frequencies. A Generalized Estimating Equation model was used to assess the association between changes in these immune parameters and disease duration and the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R). Results: CD14 expression on monocyte subpopulations increased with both disease duration and a decrease in ALSFRS-R score in patients with ALS. HLA-DR expression on monocyte subpopulations also increased with disease severity and/or duration. The expression of CD16 did not change relative to disease duration or ALSFRS-R. Finally, patients had a reduction in non-classical monocytes and an increase in the classical to non-classical monocyte ratio throughout disease duration. Conclusion: The progressive immunological changes observed in this study provide further support that monocytes are implicated in ALS pathology. Monocytic CD14 and HLA-DR surface proteins may serve as a therapeutic target or criteria for the recruitment of patients with ALS into clinical trials for immunomodulatory therapies.

Recombinant EPF/chaperonin 10 promotes the survival of O4-positive pro-oligodendrocytes prepared from neonatal rat brain.

Chaperonin 10 (cpn 10) is a small heat-shock protein that is usually intracellular. Early pregnancy factor (EPF), a biologically active protein that was first described in the serum of pregnant mammals, is homologous to cpn 10. EPF/cpn 10 has been reported to have effects on immunomodulation and cell survival and to inhibit activation of toll-like receptors by lipopolysaccharide. We found that recombinant EPF/cpn 10 was able to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, which is a disease causing inflammation and demyelination of the brain and spinal cord. This beneficial effect could be due to anti-inflammatory and/or cell survival properties of EPF/cpn 10. We aimed to assess the effects of cpn 10 on cells of the oligodendrocyte lineage because oligodendrocytes are the brain cells that produce myelin and that are depleted in multiple sclerosis. Two forms of recombinant EPF/cpn 10 were prepared in the pGEX expression system and in the baculovirus expression system. Purified O4(+) pro-oligodendrocytes were prepared from the brains of day-old Wistar rats and isolated by cell sorting with flow cytometry. Single cells were dispensed into micro-well plates and tested for survival in the presence of a range of concentrations of the two forms of cpn 10. We also studied the effects of bFGF, PDGF, IGF-1 and insulin as controls. With cpn 10 present, there was enhanced survival of O4(+) cells.

Increased levels of activated T-cells and reduced levels of CD4/CD25+ cells in peripheral blood of Guillain-Barré syndrome patients compared to controls.

Guillain-Barré syndrome (GBS) is a severe, self-limiting, autoimmune motor neuropathy. This study was performed to investigate the numbers of activated T-cells and regulatory T-cells, and CD95 and bcl-2 expression in GBS patients compared to controls. The percentage of cells expressing CD69 (activated T-cells) was increased in the blood of both patients with GBS and those with other neuropathies compared to healthy controls. GBS patients displayed significant decreases in the percentage of T-lymphocytes (CD3) and CD4/CD25+ cells (T regulatory cells) compared to patients with other neuropathies and a reduction in the percentage of cytotoxic/suppressor T-lymphocytes (CD8) compared to healthy controls. CD95 expression was reduced in GBS compared to patients with other neuropathies and expression of Bcl-2 was increased in GBS compared to healthy controls. We therefore suggest that in GBS there are increased activated T-cells and disturbances in regulatory T-cells and T-cell apoptosis.

Antibody responses to peptides of peripheral nerve myelin proteins P0 and P2 in patients with inflammatory demyelinating neuropathy.

BACKGROUND: Antibodies with reactivity to peripheral nerve myelin have previously been found in the serum, and bound to peripheral nerves of patients with Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). AIM: To investigate the presence of antibodies reactive to specific peptide sequences within the myelin proteins P0 and P2 in patients with GBS, in patients with CIDP, in healthy controls and in patients with other neuropathies (ON). METHODS: Blood was obtained from 48 patients with GBS, 36 with CIDP, 48 with ON and 38 controls. ELISA was used to detect antibody responses to peptides of the human peripheral myelin proteins P0 and P2. Blood samples were collected from patients with GBS in early, peak and recovery stages of GBS to analyse antibody levels throughout the course of the disease. RESULTS: Significantly increased total IgG levels were found in patients with GBS compared with other groups. A higher percentage of patients with GBS at the peak of disease had antibody reactivity to P2(14-25) compared with patients with CIDP and control groups. In patients with GBS and CIDP, the percentages of patients with antibody reactivity to P2(61-70), and peptides derived from P0, were comparable to the control groups. Although some individual patients with GBS had high titres of reactivity to the peptide antigens tested, most patients with GBS and CIDP had levels of antibody similar to controls. CONCLUSION: Our data suggest that increased IgG levels and increased antibody reactivity to P2(14-25) in patients with GBS at the peak of disease may play a contributory role in the disease process in some patients with demyelinating forms of GBS.

Postural orthostatic tachycardia syndrome: an underrecognized disorder.

BACKGROUND: Postural orthostatic tachycardia syndrome (POTS), a clinical syndrome of orthostatic intolerance characterized by excessive tachycardia and symptoms of cerebral hypoperfusion on standing, is not well recognized in Australia. The aim was to study the clinical symptomatology, results of autonomic testing and outcome in patients with POTS. METHODS: Sixteen subjects from a tertiary referral centre who met the criteria for POTS were studied between January 2003 and January 2006. Ten of these patients consented to be interviewed using a validated autonomic symptom questionnaire. Heart rate responses to deep breathing and the Valsalva manoeuvre were measured using Colin BP-508 machine (WR Medical Electronic Co., Stillwater, MN, USA). Tilt studies were carried out for 10 min to 80 degrees of head-up tilting. Patient outcome was assessed as functionally normal, able to stand 30 min without symptoms, able to work and carry out recreational activities or worse on follow up. RESULTS: The mean age of 10 subjects was 24.9 +/- 6.8 years, six being women. The mean duration of symptoms was 70.7 months (range 3-228 months). The common presenting orthostatic symptoms were light-headedness (100%), palpitations (90%), pallor (90%), weakness (80%) and clammy skin (80%). The mean heart rate increment during the tilt study was 51.7 +/- 14.3 b.p.m. The mean duration of follow up was 8.9 months (range 1-16 months). Only five patients were functioning normally at the follow-up visit. CONCLUSION: POTS is an underrecognized but persistent autonomic disorder in young patients with a variety of symptoms and variable outcome.

Extra-motor abnormalities in amyotrophic lateral sclerosis: another layer of heterogeneity.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease defined by the presence of muscle weakness. The motor features of disease are heterogeneous in site of onset and progression. There are also extra-motor features in some patients. The genetic basis for extra-motor features is uncertain. The heterogeneity of ALS is an issue for clinical trials. Areas covered: This paper reviews the range and prevalence of extra-motor features associated with ALS, and highlights the current information about genetic associations with extra-motor features. Expert commentary: There are extra-motor features of ALS, but these are not found in all patients. The most common is cognitive abnormality. More data is required to ascertain whether extra-motor features arise with progression of disease. Extra-motor features are reported in patients with a range of causative genetic mutations, but are not found in all patients with these mutations. Further studies are required of the heterogeneity of ALS, and genotype/phenotype correlations are required, taking note of extra-motor features.

Serial measurements of phosphorylated neurofilament-heavy in the serum of subjects with amyotrophic lateral sclerosis.

There is a need for a blood biomarker of disease activity in ALS. This marker needs to measure the loss of motor neurones. Phosphorylated neurofilament heavy chain (pNfH) in the serum is a biomarker of axonal injury. Previous studies have found that levels of pNfH are elevated in ALS. We have performed a serial study of pNfH levels in 98 subjects from our ALS clinic. There was significant elevation of levels of pNfH in subjects with ALS compared to controls, although there was considerable variability. In studies of individuals who had two or more serial samples, we found that the levels of pNfH increased over time in the early stage of disease. Levels were low in subjects with long survival. The rate of rise of pNfH was inversely correlated with survival. We suggest that the initial level of pNfH is a marker of disease severity and that changes in pNfH levels are markers of disease progression.

Altered expression of metabolic proteins and adipokines in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the loss of upper cortical and lower motor neurons. ALS causes death within 2-5years of diagnosis. Diet and body mass index influence the clinical course of disease, however there is limited information about the expression of metabolic proteins and fat-derived cytokines (adipokines) in ALS. In healthy controls and subjects with ALS, we have measured levels of proteins and adipokines that influence metabolism. We find altered levels of active ghrelin, gastric inhibitory peptide (GIP), pancreatic polypeptide (PP), lipocalin-2, plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6) and 8 (IL-8), and tumor necrosis factor alpha (TNFα) in the plasma of ALS patients relative to controls. We also observe a positive correlation between the expression of plasma nerve growth factor (NGF) relative to disease duration, and an inverse correlation between plasma glucagon and the ALS functional rating scale-revised (ALSFRS-R). Further studies are required to determine whether altered expression of metabolic proteins and adipokines contribute to motor neuron vulnerability and how these factors act to modify the course of disease.

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.

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.

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) alphabeta+ 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 alphabeta+ cells that were apoptotic. There was a decline in the percentage of apoptotic T cells that were Vbeta8.2+, compared to the percentage of non-apoptotic T cells that were Vbeta8.2+, 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 Vbeta8.2+ encephalitogenic T cells that normally occurs during spontaneous recovery from acute EAE.

The effects of prophylactic cyclosporin A on experimental allergic neuritis (EAN) in the Lewis rat. Induction of relapsing EAN using low dose cyclosporin A.

Experimental allergic neuritis (EAN) was induced in Lewis rats by inoculation with bovine intradural root myelin plus adjuvants. Animals treated with high dose (30 mg/kg) cyclosporin A (CsA) 3 times per week did not develop clinical EAN during the period of CsA treatment, but had an episode of EAN after cessation of CsA treatment. Animals treated with low dose (4 mg/kg) CsA 3 times per week developed EAN during the period of treatment, and after cessation of CsA treatment all of these animals developed relapsing EAN with disease continuing for up to four episodes. In contrast, 30-40% of untreated animals had a mild second episode of EAN but no further attacks. Histological studies performed in treated and untreated animals at the time of clinical episodes revealed inflammation and demyelination in the spinal roots and dorsal root ganglia. When animals were challenged with a second inoculation at age 7 months, one of 15 untreated control animals but none of the CsA treated animals developed an episode of EAN.

Inflammatory cells, microglia and MHC class II antigen-positive cells in the spinal cord of Lewis rats with acute and chronic relapsing experimental autoimmune encephalomyelitis.

Chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord and adjuvants and treatment with low dose cyclosporin A (CsA). Acute EAE was induced by the same method without CsA treatment. Immunocytochemistry and flow cytometry were used to assess inflammatory cells and MHC class II (Ia) antigen expression in the central nervous system of these rats. The inflammatory infiltrate was composed mainly of CD4+ T cells and macrophages, and alpha beta T cells constituted about 65% of the CD2+ T cells. After recovery from acute EAE and during the first remission of CR-EAE, the number of T cells was significantly less than in the preceding episodes. The number of T cells was higher in the second episode of CR-EAE than in the first remission. Throughout the course of CR-EAE, the majority of the CD2+ T cells were CD45RC-. The ratio of IL-2R+ cells to CD2+ cells ranged from 10.5 to 24.0%. The ratio of CD4+ T cells to B cells was lower in the later episodes of CR-EAE than in the first episode. Ia antigen was expressed on infiltrating round cells at all stages of CR-EAE and on microglial cells (identified by dendritic morphology) with increasing intensity throughout the course of CR-EAE. With flow cytometry, the number of Ia+ cells obtained from the spinal cord rose throughout the course of CR-EAE. The number of FSClowOX1low cells, which we consider represent microglia, also increased during the course of CR-EAE.

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.

Class II antigen expression and T lymphocyte subsets in chronic inflammatory demyelinating polyneuropathy.

The inflammatory infiltrate within human sural nerve, biopsied from six patients with active chronic inflammatory demyelinating neuropathy (CIDP) was studied for T lymphocyte subsets and Class II antigen (Ia)-expressing cells. Immunohistochemical staining with mouse monoclonal antibodies, acid phosphatase staining, and electron microscopy were used to provide an alternative assessment of macrophage and other mononuclear cell numbers. In normal control nerves Class II antigen was present upon endothelial cells, very occasional mononuclear cells and sparsely within the perineurium. In CIDP nerve dense Class II antigen staining was prominent within nerve fascicles, in capillary endothelial cells and within the perineurium. T lymphocytes of suppressor and helper type were present in small numbers only. Moderate numbers of macrophage-monocytes were found in the patients within nerve fascicles but these cells accounted for only part of the dense Ia staining. Since two nerves with hypertrophic changes, Schwann cells forming 'onion bulbs', were clearly Ia positive, the dense and widespread staining in all nerves studied is best explained by Ia antigen expression upon mononuclear and some Schwann cells.