Epstein-Barr virus and multiple sclerosis: interaction with HLA.

Epstein-Barr virus (EBV) infection, history of infectious mononucleosis (IM) and HLA-A and DRB1 have all been proposed as risk factors for multiple sclerosis (MS). Our aim was to analyse possible interactions between antibodies against Epstein-Barr virus nuclear antigen 1 (EBNA1) or EBNA1 fragments, presence of DRB1*15 and absence of A*02. The study population includes newly diagnosed cases and matched controls. Interaction on the additive scale was calculated using attributable proportion due to interaction (AP), which is the proportion of the incidence among individuals exposed to two interacting factors that is attributable to the interaction per se. IM showed association with MS, odds ratio (OR)=1.89 (1.45-2.48% confidence interval (CI)), as did raised EBNA1 IgG OR=1.74 (1.38-2.18 95%CI). All EBNA1 fragment IgGs were associated with MS risk. However, EBNA1 fragment 385-420 IgG levels were more strongly associated to MS than total EBNA1 IgG, OR=3.60 (2.75-4.72 95%CI), and also interacted with both DRB1*15 and absence of A*02, AP 0.60 (0.45-0.76 95%CI) and AP 0.39 (0.18-0.61 95%CI), respectively. The observed interaction between HLA class I and II genotype and reactivity to EBV-related epitopes suggest that the mechanism through which HLA genes influence the risk of MS may, at least in part, involve the immune control of EBV infection.

Epstein-Barr virus nuclear antigen-1 B-cell epitopes in multiple sclerosis twins.

BACKGROUND: Compared with quantitative observations, the search for qualitative changes that may characterize the immune response to Epstein-Barr virus (EBV) in multiple sclerosis (MS) has been less intense. OBJECTIVE: To examine the B-cell epitopes of antibodies against the Epstein-Barr nuclear antigen-1 (EBNA-1) and their relevance for MS, through a study in disease-discordant identical twins. METHODS: We evaluated the antibodies to all unique, maximally overlapping octapeptides of EBNA-1 in 12 pairs of monozygotic (MZ) twins (9 MS-discordant, 3 healthy), 3 non-twin patients and 2 healthy subjects. All except one of the patients were untreated. The EBV serology of these individuals had been assessed in advance using commercially available and in-house enzyme-linked immunosorbent assay (ELISA) kits, including assays for antibodies against select peptides of EBNA-1: EBNA-72 (GAGGGAGAGG) and EBNA-206 (EADYFEYHQEGGPDGE). RESULTS: The glycine-alanine rich domain of EBNA-1 was immunodominant in all subjects. Compared with healthy individuals, and similarly to what has been described in infectious mononucleosis (IM) patients, affected co-twins and non-twin patients had a significantly increased response to another EBNA-1 epitope (aa. 401-411). CONCLUSION: In a study that controls for confounders, our data focus an EBNA-1 specificity that may be associated with MS pathogenesis.

The costimulatory molecule B7 is expressed on human microglia in culture and in multiple sclerosis acute lesions.

B7 is a costimulatory molecule which is expressed on antigen-presenting cells and which plays a pivotal role in T cell activation and proliferation. To elucidate mechanisms regulating intracerebral immune responses, expression of B7 was examined in cultured microglial cells and in brain tissue from control and multiple sclerosis patients. Using immunocytochemical and polymerase chain reaction techniques, we show that B7 was expressed in cultured microglial cells from the human embryonic brain. Microglia also bound the soluble form of the B7 receptor CTLA-4 (CTLA-4-Ig). B7 gene expression and binding of anti-B7 antibodies and CTLA-4-Ig increased after treatment with interferon-gamma. B7 was not inducible in human astrocytes. Human microglia expressed other costimulatory molecules, such as intercellular adhesion molecule-1, LFA-1 and LFA-3. In sections of multiple sclerosis brains, B7 immunoreactivity was detected on activated microglia and infiltrating macrophages within active lesions. In chronic lesions, only perivascular cells were stained. B7 immunoreactivity was undetectable in sections from Alzheimer's disease or normal brain tissue. These data suggest that B7 may be involved in T cell activation and lesion development in multiple sclerosis and that the regulated expression of B7 on microglia may contribute to the local stimulation of T cell proliferation and effector functions.

Regulation of prostanoid synthesis in microglial cells and effects of prostaglandin E2 on microglial functions.

Brain prostanoid levels are normally low but can increase after ischemia and during inflammatory and infectious diseases. High prostanoid levels can affect brain function in several ways. In particular, prostaglandin E2 (PGE2) might exert both immunodepressive and proinflammatory actions. The present short review focuses on the regulation of prostanoid synthesis in microglial cultures and on the possible role of PGE2 in the down-regulation of microglial activation induced by lipopolysaccharide (LPS). Our studies were carried out using purified mouse or rat microglial cultures. LPS induced a dose-dependent expression of the inducible isoform of cyclooxygenase (COX-2), both in neonatal and adult microglial cultures. In the latter, the inducibility of COX-2 increased with time in culture, paralleling the acquisition of a more 'activated' microglial phenotype, and appeared to account for the time-dependent increase in the PGE2/TXB2 production ratio. The LPS-induced COX-2 expression and prostanoid production were down-regulated by potentially neurotoxic agents, such as nitric oxide (NO), the proinflammatory cytokine IFN-gamma (which acted both directly and indirectly, through its NO-inducing activity) and the HIV regulatory protein tat. On the other hand, COX-2 expression was up-regulated by the macrophage-deactivating cytokine TGF-beta 1, by exogenous PGE2 itself, which acted through EP2 receptors linked to cyclic AMP generation, and by non steroidal anti-inflammatory drugs. Interestingly, PGE2 utilized the same EP2 receptor-mediated signal transduction mechanism to down-regulate the expression of the inducible NO synthase and the production of NO. Largely, but not exclusively, through its effect on cyclic AMP, PGE2 can also: i) depress the expression of major histocompatibility complex class II antigens and of the costimulatory molecule B7-2; ii) down-regulate TNF and up-regulate IL-10 microglial production; iii) inhibit microglial IL-12 secretion. These observations, together with literature data on in vivo models of central nervous system (CNS) diseases, suggest a neuroprotective role of PGE2 in pathological conditions.

Glia-T cell dialogue.

Interactions of CD4(+) T helper (Th) cells with microglia and astrocytes are likely to play an important role in regulating immune responses as well as tissue damage and repair during infectious and autoimmune central nervous system (CNS) diseases. T cells secreting Th1-type cytokines provide inducing signals for microglia to mature into functional antigen presenting cells (APC). The ability of microglia to act as efficient APC for the restimulation of Th1 cells suggests a role for these cells in the local amplification of pro-inflammatory immune responses. Conversely, the Th2-inducing capacity of microglia and astrocytes together with their ability to produce anti-inflammatory mediators could play a role in providing counter-regulatory signals limiting CNS inflammation. In this article, we review recent studies addressing the functional significance of T cell-CNS glia interactions and present new data on the expression of cyclooxygenase-2, the inducible enzyme involved in prostanoid biosynthesis, in microglia and astrocytes during the course of experimental allergic encephalomyelitis.

Human myelin basic protein specific T cell lines display differential cytotoxicity against astrocytes, but are consistently cytotoxic against monocytes.

In experimental allergic encephalomyelitis (EAE) myelin basic protein (MBP) specific T cells differ in their encephalitogenic potential. To investigate the functional diversity of human MBP specific T cell lines, we analysed their cytotoxic activity against human astrocytes and monocytes. Five out of 14 MBP specific T cell lines killed astrocytes in the presence of MBP. Nevertheless, all lines lysed blood derived monocytes. T cell lines that lysed astrocytes efficiently in the presence of MBP, recognized peptide aa 80-99/86-105 in context with HLA-DRB5 * 0101, peptide aa 50-69/61-83 in context with HLA-DRB1 * 1501 and peptides aa 139-153, and aa 148-162 in context with HLA-DRB1 * 0101. There was no correlation of MBP-mediated lysis of astrocytes with TCR-Vbeta usage, HLA-restriction and the production of tumor-necrosis-factor-alpha (TNF-alpha), lymphotoxin (LT) and interferon-gamma (IFN-gamma). Different lysis of astrocytes, however, revealed a functional heterogeneity of MBP specific T cells, which was not observed by using monocytes as targets.

Analysis of B7-1 and B7-2 costimulatory ligands in cultured mouse microglia: upregulation by interferon-gamma and lipopolysaccharide and downregulation by interleukin-10, prostaglandin E2 and cyclic AMP-elevating agents.

Recent evidence indicates that membrane-bound costimulatory molecules of the B7 family are important for T-cell activation and are upregulated in IFN gamma-stimulated human microglia and in multiple sclerosis active lesions. In this study we have performed a detailed analysis of B7-1 and B7-2 expression and regulation in cultured mouse glial cells using immunocytochemical and semi-quantitative reverse transcriptase-polymerase chain reaction techniques. In an immortalized mouse microglial cell line (BV-2), expression of B7-1 and B7-2 was enhanced by interferon-gamma (IFN gamma). IFN gamma was a weak inducer of B7-2 mRNA and immunoreactivity in microglia primary cultures obtained from the neonatal mouse brain, whereas lipopolysaccharide, tumour necrosis factor-alpha, colony-stimulating factors and interleukin-1 beta did not affect microglial B7-2 expression. Combined IFN gamma and lipopolysaccharide treatment very effectively upregulated the B7-2 gene expression and immunoreactivity in microglia, but not in astrocytes. In both glial cell types, expression of B7-1 was not induced by any of the above agents. Among known microglia/macrophage deactivators, interleukin-10, prostaglandin E2 and cAMP-elevating agents, but not transforming growth factor-beta 1 and interleukin-4, inhibited B7-2 transcripts and immunoreactivity in IFN gamma/LPS-stimulated microglia, thus suggesting possible paracrine and autocrine mechanisms for regulating the expression of this important T-cell costimulatory signal in the brain.

Norepinephrine and vasoactive intestinal peptide induce IL-6 secretion by astrocytes: synergism with IL-1 beta and TNF alpha.

Resident glial cells and invading inflammatory cells are responsible for cytokine production within the brain. Astrocytes are known to secrete a variety of cytokines upon stimulation with cytokines themselves, protein kinase C activators, bacterial or viral constituents. Astrocytes also have surface receptors for a wide number of neurotransmitters and neuropeptides and some of these substances affect astrocyte immune functions, such as major histocompatibility complex (MHC) class II antigen expression. To elucidate the activity of neuromediators on cytokine secretion by glial cells, we studied the secretion of interleukin-6 (IL-6) by cultured rat astrocytes after incubation with various neurotransmitters and neuropeptides. Norepinephrine (NE) and the beta-adrenergic agonist isoproterenol (IPT) induced IL-6 secretion in a dose-dependent fashion. NE effect was predominantly mediated by beta 2-adrenergic receptors with a minor contribution of alpha 1-adrenergic receptors. The induction of IL-6 release by dibutyryl-cAMP indicated that IL-6 secretion secondary to beta 2-adrenergic receptor activation probably occurs through cAMP signalling pathways. Vasoactive intestinal peptide (VIP) was the sole neuropeptide able to induce IL-6 secretion. NE and VIP promoted IL-6 mRNA synthesis and both substances synergized with interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) in inducing IL-6 release. Our findings provide further evidence that neurons modulate astrocyte cytokine production and thereby regulate central nervous system immune functions.

Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures.

Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum, for 2,5,8 or 12 days in vitro (DIV), in conditions giving a purity greater than 90%. The results obtained can be summarized as follows: (1) Light microscopic autoradiography showed that cultured granule cells and their processes can accumulate the glutamate analog [3H]D-aspartate once they have reached an advanced degree of morphological differentiation (8 and 12 DIV), but, even then, only a limited number of cells was heavily labeled. In contrast, astrocytes were heavily labeled at all stages. (2) Calcium-dependent, high [K+]-induced release, or tetrodotoxin-sensitive, veratridine-induced release of [3H]D-aspartate from granule cell-enriched cultures was detectable only in cultures of 8 or 12 DIV. (3) When subject to 3 consecutive depolarizations, cultured granule cells maintained their ability to release [3H]D-aspartate and endogenous glutamate almost unchanged. (4) Newly synthesized [3H]glutamate was autoradiographically localized in both neurons and astrocytes (the latter, however, were not preferentially labeled as with [3H]D-aspartate), but was specifically released from neuronal structures (perikarya and processes) by depolarizing stimuli.

Cytokine regulation of astrocyte function: in-vitro studies using cells from the human brain.

Participation of astrocytes in central nervous system pathophysiology is likely to involve cytokines, both as stimulators and mediators of astrocyte function. We have used highly enriched human astrocyte cultures as an experimental tool to investigate the influence of cytokines on adhesion molecule expression and synthesis of mediators that are probably important in immune and inflammatory reactions involving the nervous system and in cerebral tissue repair. The response of astrocytes to interferon-gamma mainly resulted in increased expression of major histocompatibility complex antigens and co-stimulatory molecules (intercellular adhesion molecule-1, LFA-1 alpha) which mediate astrocyte-T-cell interactions. Another co-stimulatory molecule, B7, was neither expressed nor inducible by IFN-gamma and other cytokines. TNF-alpha and IL-1 beta were more efficient in stimulating synthesis of immunoregulatory and proinflammatory cytokines (IL-6, IL-8 and colony-stimulating factors), cytokine antagonists (TNF-alpha soluble receptors), or cytokines with a possible neuroprotective role (leukemia inhibitory factor); they also increased expression of some co-stimulatory molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1). Transforming growth factor-beta 1 was a strong inducer of leukemia inhibitory factor, but did not affect either major histocompatibility complex/co-stimulatory molecule expression or cytokine synthesis. Thus, different cytokines activate distinct functional programs in astrocytes, which may play a specific role in different brain diseases or at different stages of the same disease. It was additionally observed that the response of human astrocytes to cytokines (in particular the inducible synthesis of certain cytokines) varied greatly depending on the presence or absence of neurons in the culture system. This finding suggests that neuronal-glial interactions may be implicated in determining the activation threshold of astrocytes to inflammatory cytokines.

Is the oligodendroglial differentiation of bipotential oligodendrocyte-type 2 astrocyte progenitors promoted by autocrine factors?

When immature O-2A (oligodendrocyte-type 2 astrocyte) lineage cells purified from rat mixed cortical glial cultures were subcultured at low density in 10% fetal calf serum (FCS)-containing medium they largely differentiated into type-2 astrocytes. When the same number of cells was subcultured at high density in the same volume of medium the proportion of O-2A progenitors differentiating into oligodendrocytes was substantially increased. The possibility that oligodendrocyte differentiation in high-density cultures is facilitated by autocrine factors is supported by the observation that a medium conditioned by high-density subcultures of purified O-2A cells contains high molecular weight (greater than 30 kDa), non-mitogenic factor(s) capable of inducing a rapid differentiation of immature 0-2A cells into oligodendrocytes even in low-density cultures.

Anterior tension-free repair under local anesthesia of abdominal wall hernias in continuous ambulatory peritoneal dialysis patients.

BACKGROUND: A 10-year experience of abdominal wall hernia repair performed with anterior tension-free mesh or plug technique under local anesthesia in end-stage renal failure patients submitted to continuous ambulatory peritoneal dialysis (CAPD) is described in order to assess the safety and effectiveness of this approach. METHODS: Between January 1993 and December 2002, 18 hernia repairs were performed under local anesthesia in 16 patients (14 males and two females) with a mean age of 70 years (48-78). One umbilical and three unilateral inguinal hernias were observed and repaired before starting peritoneal dialysis (PD), while two umbilical, eight unilateral, and two bilateral groin hernias developed and were then treated during PD. Repairs were performed electively in all but one case, which was an emergency operation for strangulation. An ipsilateral scrotal swelling was also present in two indirect unilateral inguinal hernias. In these cases, the hernia sac was ligated before entering, while in the others it was simply dissected and inverted. RESULTS: Patients were discharged the same day or the day after surgery. No local or general immediate or late complications occurred. CAPD in subjects operated on during PD treatment was resumed the same day of surgery. In no instance was hernia recurrence or leak of dialysis solution observed at follow-up examinations. CONCLUSIONS: The absence of surgical and general complications and the nearly immediate resumption of PD indicate the anterior tension-free repair under local anesthesia as a safe and effective technique for CAPD patients even in an ambulatory or day-surgery setting.

Peripheral tissue release of interleukin-6 in patients with chronic kidney diseases: effects of end-stage renal disease and microinflammatory state.

To examine if uremia influences muscle interleukin-6 (IL-6) metabolism we studied the exchange of IL-6 across the forearm in 16 patients with chronic kidney disease (CKD) (stages 3 and 4), in 15 hemodialysis (HD)-treated end-stage renal disease (ESRD) patients (n=15), and in six healthy controls. In addition, we performed an analysis of both IL-6 protein and IL-6 mRNA expression in muscle of CKD (stage 4) patients showing evidence of inflammation and in controls. A release of IL-6 from the forearm was observed in patients with elevated IL-6 plasma levels. Arterial IL-6 was directly related to released IL-6 (r=0.69; P<0.004) in HD patients. Both IL-6 protein and IL-6 mRNA expression were increased in muscle of inflamed CKD patients vs controls (P<0.05). Although muscle net protein balance was similar in all patients, it was significantly more negative in HD patients with high than in those with low IL-6 plasma levels (P<0.05). In addition, net protein balance was related to the forearm release of IL-6 in HD patients only (r=0.47; P<0.038). These data demonstrate that IL-6 expression is upregulated in muscle, and that muscle tissue, by releasing this cytokine, may contribute to the inflammatory response in HD patients. The release of IL-6 from peripheral tissues is associated with an increase in muscle protein loss in HD patients, suggesting that muscle release of IL-6 is linked to protein catabolism in these patients. The release of IL-6 from peripheral tissues may act as a signal for the inflammatory response and contribute to functional dysregulation in uremia.

ATP regulates oligodendrocyte progenitor migration, proliferation, and differentiation: involvement of metabotropic P2 receptors.

Extracellular nucleotides act as potent signaling molecules in the neuron-glia and glia-glia communication, via the activation of specific ligand-gated P2X and G-protein-coupled metabotropic P2Y receptors. Most of the data available about the effects of P2 receptor activation in the CNS concern astrocytes, microglia, and neurons. To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in rat oligodendrocyte progenitors (OPs) and investigated the effects of ATP and its breakdown products on their functions. We describe here that rat OPs express different types of P2 receptors and that nucleotide-induced Ca(2+) raises in these progenitor cells are mainly due to the activation of P2X(7) ionotropic and ADP-sensitive P2Y(1) metabotropic receptors. We also show that ATP and ADP stimulate OP migration, inhibit the mitogenic response of OPs to PDGF and promote oligodendrocyte differentiation. The pharmacological profile of the nucleotide-induced effects demonstrates the important regulatory role of P2Y(1) receptor signaling in OP functions. These findings suggest that ATP, which is released in high amounts under inflammatory conditions and following cell death, might regulate remyelination processes in inflammatory demyelinating diseases of the CNS, like multiple sclerosis.

Metabotropic P2 receptor activation regulates oligodendrocyte progenitor migration and development.

To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in cultured rat oligodendrocyte progenitors and investigated the effects of ATP and its breakdown products on the migration and proliferation of this immature glial cell population. Using Western blot analysis, we show that oligodendrocyte progenitors express several P2X (P2X(1,2,3,4,7)) and P2Y (P2Y(1,2,4)) receptors. Intracellular Ca(2+) recording by Fura-2 video imaging allowed to determine the rank potency order of the P2 agonists tested: ADPbetaS = ADP = Benzoyl ATP > ATP > ATPgammaS > UTP, alpha,beta-meATP ineffective. Based on the above findings, on pharmacological inhibition by the antagonists oxATP and MRS2179, and on the absence of alpha,betameATP-induced inward current in whole-cell recording, P2X(7) and P2Y(1) were identified as the main ionotropic and metabotropic P2 receptors active in OPs. As a functional correlate of these findings, we show that ATP and, among metabotropic agonists, ADP and the P2Y(1)-specific agonist ADPbetaS, but not UTP, induce oligodendrocyte progenitor migration. Moreover, ATP and ADP inhibited the proliferation of oligodendrocyte progenitors induced by platelet-derived growth factor, both in purified cultures and in cerebellar tissue slices. The effects of ATP and ADP on cell migration and proliferation were prevented by the P2Y(1) antagonist MRS2179. By confocal laser scanning microscopy, P2Y(1) receptors were localized in NG2-labeled oligodendrocyte progenitors in the developing rat brain. These data indicate that ATP and ADP may regulate oligodendrocyte progenitor functions by a mechanism that involves mainly activation of P2Y(1) receptors.