V. Navikas

Tumor necrosis factor α-308 alleles in multiple sclerosis and optic neuritis

Abstract Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is believed to play an important role in multiple sclerosis (MS) pathogenesis. A bi-allelic polymorphism in the TNF-α promoter region (TNFα-308), has been reported to influence levels of TNF-α production. In the present study, we investigated the TNFα-308 polymorphism in 93 patients with MS, 17 patients with optic neuritis (ON) and 95 healthy individuals using an allele-specific PCR technique. Allelic genotype was compared with TNF-α mRNA expression levels and HLA class II phenotypes. No significant difference regarding the TNFα-308 polymorphism was observed between MS patients and controls. Specifically, the less common allele, TNF2, which is associated with higher expression levels of TNF-α, was somewhat less frequent among MS patients. In fact, analysis of 19 patients homozygous for the MS associated HLA-DR-DQ haplotype HLA-Dw2 showed that this haplotype does not carry the TNF2 allele. In addition, in 47 patients, the TNF-α alleles did not correlate with expression levels measured as numbers of TNF-α expressing cells. Thus, we found no evidence for an important role of TNFα-308 polymorphism for genetic susceptibility to MS.

Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Abstract Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.

Tumor necrosis factor-α, lymphotoxin, interleukin (IL)-6, IL-10, IL-12 and perforin mRNA expression in mononuclear cells in response to acetylcholine receptor is augmented in myasthenia gravis

Myasthenia gravis (MG) is a neuromuscular disorder mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) on the postsynaptic membrane of the neuromuscular junction. The production of antibodies is regulated by T cells by means of immunoregulatory cytokines. To investigate the involvement of TNF-alpha, lymphotoxin (LT), IL-6, IL-10, IL-12 and perforin in MG, numbers of cytokine mRNA expressing blood mononuclear cells (MNC) were examined in patients with MG and controls. LT belongs to the Th1 cell related cytokines, IL-6 and IL-10 to the Th2 type, TNF-alpha is produced by both Th1 and Th2 cells, IL-12 induces T cell switch towards the Th1 type and perforin is an effector molecule inducing cell lysis. Short term culture of MNC with AChR revealed augmented levels of AChR-reactive TNF-alpha, LT, IL-6, IL-10, IL-12 and perforin mRNA expressing cells in MG compared to levels obtained without AChR or in presence of control antigen. AChR-reactive TNF-alpha, IL-6, IL-10, IL-12 and perforin mRNA expressing cells were higher in MG than controls. These findings suggest that the cytokines TNF-alpha, LT, IL-6, IL-10 and IL-12, and the cytolytic effector molecule perforin are also involved in MG.

Linomide (roquinimex) affects the balance between pro‐ and anti‐inflammatory cytokines in vitro in multiple sclerosis

Objectives ‐ Multiple sclerosis (MS) is characterized by high levels of circulating mononuclear cells (MNC) that respond to myelin proteins like myelin basic protein (MBP) in vitro by expressing mRNA of both pro‐inflammatory cytokines, e.g. interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α) and lymphotoxin (LT) that may make MS worse, and anti‐inflammatory cytokines like IL‐4, IL‐10 and transforming growth factor‐β (TGF‐β) that may act beneficially. Substances that down‐regulate cytokines such as TNF‐α or promote IL‐10 or TGF‐β can be anticipated to affect MS beneficially. Material and methods ‐ In situ hybridization to detect and enumerate IFN‐γ, TNF‐α, LT, IL‐4, IL‐10 and TGF‐β mRNA expressing blood MNC after stimulation with myelin basic protein (MBP), control antigens and without antigen in presence and absence of Linomide (roquinimex, LS‐2616) was employed. In parallel, ELISPOT assay to detect MBP‐ and PHA‐reactive IFN‐γ secreting blood MNCLinomide was used. Results ‐ Here we report that Linomide, a synthetic immunomodulator, at concentrations effective in vivo reduces the number of MBP‐reactive TNF‐a and increases MBP‐reactive IL‐10 and TGF‐β mRNA expressing MNC from MS patients’blood when analysed in vitro. Compared to dexamethasone, Linomide up‐regulated levels of blood MNC expressing mRNA of TGF‐β after culture in presence of MBP. Conclusions ‐ Changes of cytokine balance towards a production of anti‐inflammatory cytokines could be a desirable effect to be evaluated in future drug studies of Linomide‐like substances. At present, Linomide is not evaluable in MS clinical trials due to side‐effects.

Autoantigen-induced IL-13 mRNA expression is increased in blood mononuclear cells in myasthenia gravis and multiple sclerosis

Evidence has been presented for the involvement of immune mechanisms in the pathogenesis of myasthenia gravis (MG) and multiple sclerosis (MS). The production of autoantibodies in both diseases is regulated by T‐cells by means of cytokines. Interleukin‐13 (IL‐13) is mainly produced by T‐helper type 2 cells and induces B‐cell proliferation and antibody class switch. The role of IL‐13 in MG and MS is not known. We employed in situ hybridization with synthetic radiolabelled oligonucleotide probes to detect and enumerate blood and cerebrospinal fluid (CSF) mononuclear cells (MNC) expressing IL‐13 mRNA from patients with MG, MS, optic neuritis (ON), other inflammatory neurological diseases (OIND) and healthy controls. MG is associated with elevated levels of acetylcholine receptor (AChR) reactive IL‐13 mRNA expressing blood MNC compared to control patients. In MS, numbers of MBP‐reactive IL‐13 mRNA expressing MNC were higher compared to cultures without antigen stimulation. The levels of MBP‐reactive IL‐13 mRNA positive MNC were higher in MS compared to MG, but not other controls. There were no differences in spontaneous IL‐13 mRNA expressing blood MNC numbers between MG, MS, ON and control patients. The data suggest the involvement of IL‐13 in both MG and MS.

Soluble CD30 levels in plasma and cerebrospinal fluid in multiple sclerosis, HIV infection and other nervous system diseases

The CD30 molecule, a member of tumor necrosis factor superfamily, has been suggested to be preferentially expressed and released in soluble form by activated T cells that produce T helper 2 type (Th2) cytokines. To evaluate whether determination of soluble CD30 (sCD30) levels could have a diagnostic value in diseases associated with Th1 and Th2 cytokine involvement, we investigated sCD30 in plasma and cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS), HIV infection and other nervous system diseases. There was no statistically significant difference for plasma sCD30 levels in these clinical groups. However, patients with HIV infection had higher levels of sCD30 in CSF than MS patients. The mean sCD30 values were 3 to 6 folds higher in plasma than in CSF in all patient groups. No relationships were found between sCD30 levels and different clinical variables of MS and HIV infection, except that higher plasma sCD30 levels in HIV‐infected patients were found in those with higher CD4+ T cell counts (> 200 ± 106) compared to the group with lower cell counts. The findings indicate that determinations of plasma and CSF sCD30 levels in MS and HIV infection have limited or no value as diagnostic or prognostic indicator.

Cytokines in multiple sclerosis (MS) and HIV infection

HIV-I INFECTION OF HUMAN EMBRYONIC ASTROCYTES. A.M. Di Ricnzo, A.C. Sanlarcanrclo. C. Palladino. E. Olivctta, F. Aloisi, G.B. Rossi. P. Vcrani and G. Levi. Islih!to Supcriorc di Sanit% Rome. Italy. Cuhurcs highly cnrichcd in GFAP+ awoqtcs. and virluallv devoid of macrophagcs/rnicroglia. csublishcd from the human embryonic brain. wcrc cxposcd lo ccl1 free vims of cithcr monoc)~olropic. (HIV-Ba-L) or Iympholropic (HIV-1118) strain and csamincd for ~hc prcscncc of ~21, RT actiwly and c~~opalhic clTccts :II dilTcrcn! limes allcr infection. We found Ihat both HIV-I strains can infccl human embryonic aslrocytes. Except for an early transient peak ofp21 occurring at day 2 after infection with HIVBa-L alone or wilh HIV-IIIB in the presence of TNFa and II-lp, the infection of astrocytes appears to bc nonpermissive. The possibility that undclecuble Icvels of virus are produced by astrocytes was made unlikely by he finding Ihal p2&ncgalive astrocyle supcrnatants were not able to inrcc! pcrmissivc cells (CEM-SS or PBMC). On the other hand, viral production was consis(cnlly obscrvcd when infected astrocytes were coculmrcd with pcrmissivz cells Inrcclion of aslroqlcs by HIV-I was hlrthcr supporlcd by the fmdmg that prowrus pcrsisls in lhcsc cells. By performing a acslcd PCR. HIV-I DNA could bc dclcctcd in astroqles cuhurcd in ihc abscocc of pcrmissi\c cells for as long as one month postin&lion. Morcovcr. IO vcrib if proviral transcrip0on occurs, we are prcscmly performing RT-PCR Tar gag. lal and ncf’genes. Our obscnations suppon the concept that aswocgcs may play a role in Ihc spread of HIV-l in&lion wilhin ~hc brain and in lhc palhogcncsis of ncoro-AIDS.

Increased levels of perforin and IL-6 in multiple sclerosis (MS)

Gender and associated factors have an impact on multiple sclerosis (MS) disease. Epidemiological studies demonstrate a distinct and worldwide preponderance of female patients. The average female to male ratio in MS is 2: 1. Other correlations with gender have been reported as a preferential transmission for females, a significant paucity of father-son pairs, an excess of female offspring in male MS patients and a specific clinical course or reponse to treatments. HormonaI changes associated with menopause, may also have an influence on disease evolution. Sex steroids can act on the immune system via alteration of T and B cell function and phenotype, via influence on the kinetics of immunoglobulines synthesis or via responsiveness to sex hormones of various cytokines, namely tumor necrosis factors and interferons. Another aspect concerns the influence of pregnancy and postpartum on MS disease evolution. Up to 1950, it was considered that pregnancy adversely affected MS. Since then, ideas have radically changed and the most recent studies conclude that pregnancy is associated with a lower risk of onset and a better prognosis. Since a few years, we have a better understanding of the mechanisms underlying the physiological immunosuppression associated with pregnancy and influencing MS evolution.