Polyautoimmunity in a Greek cohort of multiple sclerosis.

OBJECTIVES: The aim of this study was to assess the existence of polyautoimmunity in a Greek cohort of multiple sclerosis (MS), particularly multiple autoimmune syndrome (MAS), i.e., the presence of three or more distinct autoimmune disorders (ADs) in the same individual. METHODS: Cross-sectional control study. RESULTS: The overall prevalence of polyautoimmunity in 2140 MS patients (female to male ratio: 2.1:1) was 8.3% (vs 6.07% in 1580 matched control participants, P = 0.008) mainly due to differences in autoimmune thyroid disorders (AITD) and vitiligo. The prevalence of MAS was 1.0%. The most frequent diseases encountered in MS were organ-specific ADs. There was no statistical difference in the total rates of ADs between female and male MS patients. There were higher rates of AITD in women (P = 0.004) and higher rates of iritis (P = 0.039) and ankylosing spondylitis (P = 0.003) in men. MS was diagnosed in the same year with AD in 7.4% of patients with additional ADs, earlier than AD in 42.0% and later than AD in 50.6%. CONCLUSION: Polyautoimmunity and particularly MAS occur more frequently in MS patients than in control participants indicating that MS may be part of a generalized susceptibility to autoimmunity. Therefore, polyautoimmunity may be implicated in the etiopathogenesis of MS-related ADs, with a potential impact on relative therapeutic strategies.

Capturing the elasticity and morphology of live fibroblast cell cultures during degradation with atomic force microscopy.

Atomic force microscopy, in a liquid environment, was used to capture in vitro the morphological and mechanical changes that cultured fibroblasts undergo as time elapses from the completion of the cell culture. Topography images illustrated that initially, the nucleus had a height of 1.18 ± 0.2 µm, and after 48 h it had decreased to 550 ± 60 nm; similarly, the cell membrane exhibited significant shrinkage from 34 ± 4 to 23 ± 2 µm. After each image scan, atomic force microscopy indentation was performed on the centre of the nucleus, to measure the changes in the cell elasticity. Examination of the force-distance curves indicated that the membrane elastic modulus at the nucleus remained the same within the time frame of 48 h, even though the cell morphology had significantly changed.

Perimetric and peri-papillary nerve fibre layer thickness findings in multiple sclerosis.

BACKGROUND AND PURPOSE: Several previous studies have employed optical coherence tomography (OCT) of the optic disc and 'white-on-white' automated perimetry to evaluate optic neuritis (ON) associated with multiple sclerosis (MS). This study employed OCT, white-on-white automated perimetry as well as 'blue-on-yellow' automated perimetry to evaluate MS patients with or without episodes of ON. METHODS: The MS group consisted of 56 patients with MS (27 patients with no history of ON in both eyes and 29 patients with at least one ON attack in one or both eyes), whereas the control group consisted of 56 age- and sex-matched healthy subjects. All patients underwent a complete neurological and ophthalmological examination. Peri-papillary retinal nerve fibre layer thickness (RNFLT) was evaluated using OCT. The mean defect and pattern standard deviation for both white-on-white and blue-on-yellow perimetry were also recorded. RESULTS: RNFLT and perimetric scores were significantly lower in MS group without a history of ON and in the unaffected eyes of MS group with unilateral ON, compared with controls. MS group with more than one ON episodes had significantly compromised blue-on-yellow perimetric indices, compared with patients with one ON episode, whereas respective differences for white-on-white perimetry were not statistically significant. CONCLUSIONS: The significantly lower RNFLT and perimetric scores in MS group patients without ON, compared with control group, may possibly be attributed to sub-clinical episodes of ON or to retrograde degeneration of nerve cells from sub-clinical post-chiasmal lesions. Blue-on-yellow perimetry may be advantageous over white-on-white perimetry in evaluating MS-associated functional defects.

P0 glycoprotein peptides 56-71 and 180-199 dose-dependently induce acute and chronic experimental autoimmune neuritis in Lewis rats associated with epitope spreading.

Two synthetic peripheral nerve myelin P0 protein peptides, an immunodominant (amino acids 180-199) and a cryptic (amino acids 56-71) one, induced an acute or chronic course of experimental autoimmune neuritis (EAN) in Lewis rats, when given at low dose (50-100 microg/rat) or high dose (250 microg/rat), respectively. Corresponding to the different clinical course, pathological changes and immune responses were found: (1) Onset of clinical signs of P0 peptide 56-71 (P0 56-71) induced EAN was 1-3 days later than in P0 peptide 180-199 (P0 180-199) induced EAN at all immunizing doses, whereas the peak of the disease occurred at a similar time point post immunization (p.i.), i.e. at days 14-16 p.i. in P0 56-71 induced EAN and at day 16 p.i. in P0 180-199 induced EAN. (2) Intramolecular epitope spreading as assessed by delayed type hypersensitivity response occurred in P0 56-71 induced EAN at both low and high antigen doses and in P0 180-199 induced EAN at high antigen dose (250 microg/rat) only. (3) P0 180-199 stimulated higher levels of interferon-gamma production in P0 180-199 induced EAN than in P0 56-71 induced EAN and vice versa. (4) Histopathologic evaluation revealed a similar grade of mononuclear cell infiltration in the sciatic nerves of both types of EAN, but more severe demyelination was found in P0 180-199 induced EAN compared to P0 56-71 induced EAN. The results support the hypothesis that high dose autoantigen immunization induces extensive determinant spreading and chronic course of autoimmune diseases.

Protective effect of Rolipram in experimental autoimmune neuritis: protection is associated with down-regulation of IFN-gamma and inflammatory chemokines as well as up-regulation of IL-4 in peripheral nervous system.

Rolipram, a phosphodiesterase type 4 inhibitor, is reported to have anti-inflammatory effects. It can markedly downregulate antigen-driven T cell proliferation and suppress TNF-(alpha and TNF-beta production in vitro and in vivo, which have led to its use in the treatment of a number of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN). EAN is a CD4+ T cell-mediated demyelinating autoimmune disease of peripheral nervous system (PNS) that represents an animal model for the study of the immunopathogenesis and immunotherapy of Guillain-Barré syndrome (GBS) in human. In the previous study, we reported that suppression of EAN by Rolipram was associated with down-regulated myelin antigen-induced T cell responses as well as downregulated IFN-gamma and TNF-alpha production. Here we report that EAN induced in Lewis rats by inoculation with the PNS P2 protein peptide 57-81 and Freund's complete adjuvant (FCA), was strongly suppressed by Rolipram administered twice daily intraperitoneally from day 9 post immunization (p.i.), i.e. after onset of clinical EAN to day 18 p.i. This clinical effect was associated with dose-dependent down-regulated production of IFN-gamma and the chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha, MIP-2 and monocyte chemotactic protein-1(MCP-1) as well as up-regulated IL-4 production in sciatic nerve sections from Rolipram-treated EAN rats at maximum of clinical EAN, i.e. on day 14 p.i.. These findings suggest that Rolipram may be useful in certain T cell-dependent autoimmune diseases and inflammatory neuropathies. These observations call for further studies on the potential role of Rolipram in the treatment of autoimmune diseases.

High inflammation and mild demyelination in the peripheral nervous system induced by an intraneural injection of RR interleukin-4.

To examine the direct effects of IL-4 on peripheral nervous system (PNS) we injected recombinant rat IL-4 (rrIL-4) into the sciatic nerve of normal adult Lewis rats. Histopathological and immunohistochemical observations revealed that 1 day after injection, a large number of macrophages and MHC class II-positive cells appeared within both the perineurium and endoneurium. Only few CD4(+)and CD8(+)T cells existed in the endoneurium. From day 4 to day 7, we observed a gradual decline of inflammation, but the number of infiltrates in rrIL-4 injected nerves was significantly higher compared with sterile PBS-injected control group. On the contrary, demyelination affected significantly fewer nerve fibres in the rrIL-4-injected nerves compared with control group on day 7. Intraneural injection of rrIL-4 results in high grade inflammation and mild demyelination in the PNS.

Enhancement of acute phase and inhibition of chronic phase of experimental autoimmune neuritis in Lewis rats by intranasal administration of recombinant mouse interleukin 17: potential immunoregulatory role.

Experimental autoimmune neuritis (EAN) is a CD4(+) T-cell-mediated demyelinating disease of the peripheral nervous system (PNS). We examined the effect of recombinant mouse interleukin 17 (rmIL-17) on chronic EAN induced in Lewis rats by inoculation of P2 57-81 peptide in Freund's complete adjuvant. Animals were treated nasally for 6 days with either 0.1 or 0.9 microg/rat/day rmIL-17 from the onset of neurological signs, i.e., days 9 to 14 postimmunization (p.i.). Prolonged follow-up demonstrated a chronic course in control and rmIL-17-treated rats. Treated rats had more severe disease initially (days 18-36 p.i.) with a stronger enhancing effect observed with the higher rmIL-17 dose. At day 19 rmIL-17-treated rats showed increased infiltration of inflammatory cells into the sciatic nerve, more severe demyelination, augmented proliferation of regional lymph node cells, and increased serum levels of tumor necrosis factor-alpha. After the initial phase of disease enhancement the IL-17-treated EAN rats improved gradually and ultimately recovered completely, whereas the control EAN rats remained affected until the end of the observation (day 120 p.i.). The lower dose of rmIL-17 induced an earlier recovery from clinical deficits than the higher one. The results indicate that IL-17 plays an immunoregulatory role in chronic EAN which could have implications for immunomodulatory treatments of chronic autoimmune disease of the PNS.

Rolipram suppresses experimental autoimmune neuritis and prevents relapses in Lewis rats.

Rolipram, a phosphodiesterase type 4 inhibitor, can markedly down-regulate antigen-driven T cell proliferation and suppress TNF-alpha production in vitro and in vivo. Here we report the effects of Rolipram on experimental autoimmune neuritis (EAN), which can be induced by immunization with myelin components of the peripheral nervous system (PNS) combined with Freund's complete adjuvant (FCA), and which represents a CD4+ T cell-mediated animal model for human Guillain-Barré syndrome. EAN induced in Lewis rats by inoculation with the PNS P2 protein peptide 57-81 and FCA was strongly suppressed by Rolipram administered twice daily intraperitoneally from day 9 post immunization (p.i.), i.e. after onset of clinical EAN. Suppression of EAN was associated with down-regulated myelin antigen-induced T cell responses as well as down-regulated IFN-gamma and TNF-alpha production. A relapse of clinical EAN occurred upon treatment of a short duration (7 days), while prolongation of treatment resulted in the prevention of clinical EAN relapse. There was no relationship between clinical EAN relapse and high levels of TNF-alpha. The immunomodulatory effects of Rolipram call for further research into the potential role of drugs acting on the immune system in the treatment of autoimmune diseases.

Intranasal administration of recombinant mouse interleukin-12 increases inflammation and demyelination in chronic experimental autoimmune neuritis in Lewis rats.

To examine whether interleukin (IL)-12 modulates ongoing chronic experimental autoimmune neuritis (EAN), we evaluated the effects of recombinant mouse IL-12 (rmIL-12) in Lewis rats with chronic EAN, induced by immunization with P0 peptide (180-199) plus complete Freund's adjuvant. Rats were treated intranasally with either 0.1 or 1 microg/rat/day rmIL-12 for 6 days from the onset of clinical chronic EAN, on days 5-10 postimmunization (p.i.). Only high-dose rmIL-12 exacerbated chronic EAN. This clinical effect was associated with higher numbers of inflammatory cells and more severe demyelination in sciatic nerve sections on days 15 and 80 p.i. compared with low-dose rmIL-12-treated rats and phosphate-buffered saline (PBS)-treated control rats. High-dose rmIL-12 increased significantly the lymph node mononuclear cell proliferation in response to P0 peptide 180-199 and IFN-gamma production in the sciatic nerves. These data indicate that intranasally administered IL-12 acts as a proinflammatory cytokine in chronic EAN. Effective inhibition of IL-12 in vivo could be considered for therapeutic use in chronic inflammatory demyelinating polyradiculoneuropathy.

Increased IL-1beta, IL-8, and IL-17 mRNA expression in blood mononuclear cells observed in a prospective ischemic stroke study.

BACKGROUND AND PURPOSE: Ischemic brain injury secondary to arterial occlusion is characterized by acute local inflammation, which involves accumulation of polymorphonuclear neutrophils (PMN). Factors that influence the recruitment of PMN could represent new therapeutic targets in acute stroke. In this prospective study we evaluated numbers of peripheral blood mononuclear cells (PBMC) expressing mRNA for interleukin (IL)-1beta, IL-8, and IL-17 and macrophage inflammatory protein-1alpha (MIP-1alpha) after ischemic stroke. METHODS: Peripheral blood was obtained on days 1 to 3, 4 to 10, and 20 to 31 after onset of symptoms. In situ hybridization with radiolabeled synthetic oligonucleotide probes was adopted to measure cytokine mRNA expression in PBMC. Plasma and cerebrospinal fluid levels of IL-8 were measured by an enzyme-linked immunosorbent assay. RESULTS: Most patients with ischemic stroke had clearly elevated numbers of IL-1beta, IL-8, and IL-17 mRNA expressing PBMC 1 to 3 days after onset of symptoms compared with healthy individuals (P<0. 0001 for all comparisons). At follow-up after 20 to 31 days, numbers of IL-8 mRNA expressing PBMC were lower than during the acute stage (P<0.001), but only IL-1beta and IL-17 mRNA expression had returned to the level of the healthy individuals. Numbers of MIP-1alpha mRNA expressing PBMC did not differ between patients with ischemic stroke and healthy individuals at any time point. A correlation was observed between numbers of IL-1beta, IL-8, and IL-17 mRNA expressing PBMC and the degree of neurological impairment as measured by the Scandinavian Stroke Scale 1 to 3 days after onset of symptoms (r=0.5; P<0.01 for all correlations). CONCLUSIONS: A longitudinal study of patients with ischemic stroke revealed systemic increases of levels of IL-1beta, IL-8, and IL-17 that correlated with Scandinavian Stroke Scale scores. IL-8 levels were further increased in cerebrospinal fluid.

Dynamics of production of MIP-1alpha, MCP-1 and MIP-2 and potential role of neutralization of these chemokines in the regulation of immune responses during experimental autoimmune neuritis in Lewis rats.

Experimental autoimmune neuritis (EAN) is an inflammatory autoimmune demyelinating disease of the peripheral nervous system (PNS) and represents an animal model of Guillain-Barré syndrome (GBS), which is a major inflammatory demyelinating disease of the PNS in humans. In the present study, the dynamics of the expression of the chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-2 and monocyte chemotactic protein-1 (MCP-1) were determined in the sciatic nerves of EAN rats. Additionally, the effect of neutralizing antibodies against MIP-1alpha, MIP-2 and MCP-1 on the clinical course of EAN and the chemokine expression was investigated. The maximum of MIP-1alpha positive cells in the sciatic nerves was seen on day 14 post immunization (p.i.) correlating with the development of severe clinical signs. Administration of an anti-MIP-1alpha antibody suppressed the clinical signs of EAN and inhibited inflammation and demyelination in the sciatic nerve. Peak numbers of MCP-1 positive cells in the sciatic nerves were detected on day 7 p.i. Administration of an anti-MCP-1 antibody caused a delay of onset of EAN. However, 4 of the 6 EAN rats receiving the anti-MCP-antibody showed the same degree of inflammatory cell infiltration and demyelination in the sciatic nerves as sham-treated EAN rats, whereas only 2 EAN rats had less inflammation and demyelination. The numbers of MIP-2 positive cells reached a maximum on day 21 p.i. Anti-MIP-2 antibody failed to suppress the clinical signs of EAN and the inflammation and demyelination in the sciatic nerves. Only administration of the anti-MIP-1alpha antibody resulted in a significant reduction in the number of chemokine (MIP-1alpha)-positive cells and ED1-positive macrophages in the sciatic nerves. The present results demonstrate that MIP-1alpha and MCP-1 may play a role in the immunopathogenesis of EAN, and that MIP-1alpha induced trafficking of inflammatory cells can be inhibited by immunoneutralization. Further elucidation of the regulation and coordination of MIP-1alpha and MCP-1 production may lead to new therapeutic approaches to GBS in humans.

Local effects of recombinant rat interleukin-6 on the peripheral nervous system.

Interleukin-6 (IL-6) is a multifunctional cytokine with a broad range of activities and can affect a variety of target cells or systems in multiple ways. However, there is currently no consensus on how IL-6 directly affects the peripheral nervous tissue. We performed histopathological and immunohistochemical analyses to investigate the direct effects of recombinant rat IL-6 (rrIL-6) following its intraneural injection into the sciatic nerve of adult Lewis rats. One day after injection, a large number of macrophages, major histocompatibility complex (MHC) class II positive cells, and CD4+ and CD8+ T cells appeared within the perineurium and endoneurium. From day 4 to day 7 after injection, we observed a gradual increase of inflammation and demyelination. On day 7, demyelination affected more than 80% of nerve fibres. In contrast, in the sterile phosphate-buffered saline (PBS)-injected control group, lower inflammation and fewer demyelinating nerve fibres were observed on days 4 and 7. Thus, intraneural injection of rrIL-6 into the sciatic nerve induces high inflammation and severe demyelination. This study improves our understanding of the effector mechanisms underlying inflammation and demyelination and identifies IL-6 as an essential mediator of inflammation and demyelination in the peripheral nervous system after local administration.

Inflammation and severe demyelination in the peripheral nervous system induced by the intraneural injection of recombinant mouse interleukin-12.

Inflammatory cytokines appear to be involved in damage to Schwann cells, myelin and axons, although the exact role of the different cytokines is uncertain. The direct injection model offers a new and simplified way of examining the mechanisms of early inflammation in the peripheral nervous system. The present study was performed to assess the direct effects of interleukin (IL)-12 on rat sciatic nerves injected with recombinant mouse IL-12. Histological and immunohistochemical examination 24 h after injection showed early inflammation as well as demyelination within the injected nerve fibres. By 4 days the inflammatory and demyelinating changes were significantly increased. Seven days after injection, the endoneurium still contained significant numbers of inflammatory cells and the demyelination was even more severe. Control rats injected with sterile phosphate-buffered saline exhibited no such inflammatory and demyelinating response. These changes are similar to those seen in inflammatory and demyelinating disorders of the peripheral nervous system and suggest that IL-12 could be relevant to the pathogenesis of demyelinating diseases such as Guillain-Barré syndrome.

High levels of IL-10 secreting cells are present in blood in cerebrovascular diseases.

Ischemic stroke is associated with altered systemic immune responses both early after the onset and in the recovery phase. Interleukin (IL)-10, a Th2 related cytokine, has multiple effects on different cell types, including T and B lymphocytes, monocytes, neutrophils and mast cells. IL-4 is another Th2 cytokine that inhibits the synthesis of pro-inflammatory cytokines by Th1 clones. We used enzyme-linked immunospot assays to detect and enumerate blood mononuclear cells (MNC) secreting IL-10 and IL-4 spontaneously as well as after stimulation with myelin basic protein (MBP), considered to be an autoantigen of possible pathogenic importance in, for example, multiple sclerosis, to evaluate the involvement of anti-inflammatory cytokines in ischemic stroke. All patients with ischemic stroke and cerebral hemorrhage had strongly elevated numbers of IL-10 secreting blood MNC compared with healthy individuals. Numbers of MBP-reactive IL-10 secreting blood MNC were also elevated in a proportion of the patients with stroke and hemorrhage. Levels of IL-4 secreting blood MNC did not differ in ischemic stroke versus healthy individuals. The anti-inflammatory IL-10 could play a pivotal role in ischemic stroke as well as cerebral hemorrhage.

Nasal administration of recombinant rat IL-4 ameliorates ongoing experimental autoimmune neuritis and inhibits demyelination.

Experimental autoimmune neuritis (EAN) is a CD4(+)T cell-mediated demyelinating disease of the peripheral nervous system (PNS) and serves as an experimental model for human immune-demyelinating neuropathies. In this study, we examined the effect of recombinant rat interleukin-4 (rrIL-4) on chronic EAN in Lewis rats induced by immunization with P0 peptide 180-199 and complete Freund's adjuvant (CFA). We estimated that nasal administration of rrIL-4, in dose ranges of 0.1-1 microg/rat/day in the initial phase of EAN, decreased the severity and the duration of clinical EAN. Hyporesponsiveness of T cells, downregulation of Th1 cell responses (INF-gamma), but increased levels of specific IgG1 isotypes document that nasal administration of rrIL-4 was systemically immune effective. Low grade inflammation and complete lack of regional demyelination within the sciatic nerves were seen in rrIL-4 treated rats. Based on these observations we suggest that nasal administration of IL-4 could be further evaluated, considering its possible use in human immune-demyelinating neuropathies.