Marcella Caggiula

pSTAT1, pSTAT3, and T-bet expression in peripheral blood mononuclear cells from relapsing-remitting multiple sclerosis patients correlates with disease activity.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system, and it is considered to be a T helper 1 (Th1) cell‐mediated autoimmune disease. T‐bet has been identified as a key transcription factor for the development of Th1 cells and the induction of interferon (IFN)‐γ production. T‐bet is induced during T‐cell activation by the IFN‐γ signal transducer and activator of transcription (STAT)‐1 signalling pathway. In this study we found an up‐regulation of T‐bet and pSTAT1 in peripheral blood CD4+ and CD8+ T cells and monocytes from relapsing‐remitting MS patients in relapse compared with patients in remission and with healthy subjects. The increased expression of pSTAT1 strongly correlated with T‐bet expression in CD4+ and CD8+ cells and monocytes from patients in relapse and was associated with an increased production of IFN‐γ by peripheral blood mononuclear cells (PBMCs). pSTAT3 was also up‐regulated in CD4+ and CD8+ cells and monocytes from patients in relapse and was associated with an increased production of interleukin (IL)‐10 but not of IL‐6. pSTAT1, pSTAT3, and T‐bet expression strongly correlated with Gd‐DTPA‐enhanced lesions on brain and spinal cord magnetic resonance imaging. Our data show for the first time that there is an up‐regulation of type 1 immunity‐correlated transcription factors such as STAT1 and T‐bet in peripheral blood subpopulations of MS patients in the active phase of disease. The evaluation of T‐bet and pSTAT1 expression in peripheral blood CD4+, CD8+ T cells and monocytes could be used as a marker of disease activity in relapsing‐remitting MS.

Leptin as a marker of multiple sclerosis activity in patients treated with interferon-beta

The role of leptin was investigated in relapsing-remitting multiple sclerosis (MS). Control and MS patients showed comparable baseline serum leptin levels. During the first year of IFNbeta-1a treatment, leptin significantly decreased since 2 months after starting therapy in 11 patients who had no relapses. A significant decrease in IL12/IL10 ratio was observed in this group of patients only after 1 year of treatment. An increase of leptin was observed before the first clinical exacerbation in 13 relapsing patients. Leptin may play a pathogenic role in MS and can be a useful marker of disease activity and response to therapy.

Regulatory T cells fail to suppress CD4+T-bet+ T cells in relapsing multiple sclerosis patients

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system and a defect in the regulatory T‐cell subset seems to be involved in the pathogenesis of the disease. Foxp3 is a transcription factor that is selectively expressed in CD4+ CD25+ regulatory T cells and is required for their development and function. T‐bet is a key transcription factor for the development of T helper 1 (Th1) cells. We found that both the percentage of circulating CD4+ CD25+ Foxp3+ cells and Foxp3 expression were lower in relapsing‐remitting (RR) MS patients during relapses than during remission. Otherwise, the percentage of CD4+ T‐bet+ T cells and T‐bet expression in CD4+ T cells were higher in relapsing than in remitting RRMS patients. CD4+ CD25+ T cells both from relapsing and from remitting RRMS patients showed significantly less capacity than corresponding cells from healthy subjects to suppress autologous CD4+ CD25− T‐cell proliferation, despite a similar Foxp3 expression level. CD4+ CD25+ T cells from healthy subjects and patients in remission clearly reduced T‐bet mean fluorescence intensity (MFI) in CD4+ CD25− T cells up to a ratio of 1:10, whereas CD4+ CD25+ T cells from patients in relapse were able to reduce T‐bet expression only at a high ratio. Our data indicate that the increased number of regulatory T (T‐reg) cells and the increased Foxp3 expression in circulating CD4+ CD25+ T cells may contribute to the maintenance of tolerance in the remission phase of MS. Moreover, the inhibitory capacity of CD4+ CD25+ T cells seems to be impaired in relapsing patients under inflammatory conditions, as shown by the high levels of T‐bet expression in CD4+ T cells.

IL17 and IFNgamma production by peripheral blood mononuclear cells from clinically isolated syndrome to secondary progressive multiple sclerosis.

We evaluated the spontaneous IL17, IFNgamma and IL10 production by peripheral blood mononuclear cells from patients affected by clinically isolated syndromes (CIS) suggestive of multiple sclerosis (MS) both in acute phase and in remission, relapsing remitting MS (RRMS) both in relapse and in remission, not-relapsing secondary progressive MS (SPMS) and controls. We observed higher IL17 levels in CIS patients both in acute phase and in remission than in SPMS patients and controls. On the contrary no difference in IL17 production was observed among RRMS patients and CIS, SPMS patients and controls. IFNgamma levels were significantly higher in CIS patients in acute phase than in CIS and RRMS patients in remission, SPMS patients and controls. Moreover, we observed higher IFNgamma spontaneous production in relapsing RRMS patients than in remitting RRMS and SPMS patients and controls. IL10 levels were significantly higher in remitting CIS and in relapsing RRMS patients than in SPMS patients and controls. There was no difference in IFNgamma, IL10 and IL17 levels between SPMS patients and controls. Our data suggest that IL17 might play a crucial role mainly in the early phase of MS, while IFNgamma seems to be involved both in the early phase and in the following relapses of the disease.

Neurotrophic factors and clinical recovery in relapsing-remitting multiple sclerosis.

Pathogenic autoimmune cells are demonstrated to be able to produce neurotrophic factors during acute phase of multiple sclerosis (MS). In this study, we determined the production of various neurotrophins [brain‐derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell line‐derived neurotrophic factor (GDNF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4)] and some pro‐inflammatory cytokines [tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ)] by unstimulated peripheral blood mononuclear cells (PBMC) in 21 relapsing‐remitting MS patients during different phases of disease (stable, relapse and post‐relapse). During acute phase of disease, we detected a considerable increase of BDNF, TNF‐α and IFN‐γ production, while significantly higher levels of GDNF, NGF, NT3 and NT4 were found in post‐relapse phase. When neurotrophin production was correlated with clinical outcome (complete or partial recovery from new symptoms), we found a significantly higher BDNF production in relapse phase followed by increased GDNF, NGF, NT3 and NT4 levels during post‐relapse phase in subjects with complete remission only. During relapse phase, we detected a significant increase of pro‐inflammatory cytokines, that was more evident in patients with partial recovery. The neuroprotective potential of immune cells seems to be inversely correlated with disease duration and with the age of patients.

Leptin Enhances the Release of Cytokines by Peripheral Blood Mononuclear Cells from Relapsing Multiple Sclerosis Patients

Leptin, a hormone synthesized mainly by adipocytes, can modulate the immune response and seems to be involved in the induction of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS). However, the possible role of leptin in MS pathogenesis has not yet been elucidated. In this study we investigated the effect of leptin on cytokine production by peripheral blood mononuclear cells (PBMCs) of MS patients (either in the acute or in the stable phase of the disease) and healthy controls. We also analyzed leptin effects on cytokine production by monocytes in relapsing MS patients. Our data showed that leptin induced tumor necrosis factor-alpha, interleukin-6, and interleukin-10 production by PBMCs of patients in an acute phase of disease but not in patients in a stable phase or in healthy controls. Moreover, we found no effect of leptin in monocytes from relapsing MS patients. Therefore we conclude that leptin may modulate the MS inflammatory process during relapses.

Serum Levels of Anti-Myelin Antibodies in Relapsing-Remitting Multiple Sclerosis Patients during Different Phases of Disease Activity and Immunomodulatory Therapy

Antibodies against myelin oligodendrocyte antigens have been found in the immunoreactive brain lesions of Multiple Sclerosis (MS) patients. Recently it has been proposed that these antibodies can be used as a prognostic marker in the course of disease. However, the serum levels of these autoantibodies during different phases of disease activity or after an immunomodulatory therapy have been poorly investigated. In this study the serum levels of anti-myelin oligodendrocyte glycoprotein (MOG) (directed against the epitopes 1–26 and 15–40) and anti-myelin basic protein (MBP) antibodies were sequentially measured in the same MS patient either in relapse or remission phases. We found that MS patients in the relapse phase had higher serum anti-MOG (peptides 1–26 and 15–40) and anti-MBP antibody levels than controls. In addition, the levels of anti-MOG 1–26 were also elevated during the relapse as compared with the remission phase but no significant changes were found in the levels of anti-MOG 15–40 of anti-MBP antibodies. We also evaluated the effect of interferon-beta (β) therapy on anti-myelin antibodies. 1-year of interferon-β treatment did not induce any changes in the levels of anti-MOG and anti-MBP antibodies. In conclusion, these data indicate that the use of peripheral levels of autoantibodies against MOG and MBP as marker of multiple sclerosis might be complicated by the phase of disease activity and by the epitope of the MOG protein used.

Severe disability in patients with relapsing-remitting multiple sclerosis is associated with profound changes in the regulation of leptin secretion.

Objectives: Experimental evidences indicate that leptin is involved in the neuroinflammatory process sustaining multiple sclerosis (MS). However, the relationship between leptin and body fat, as assessed by body mass index (BMI), in MS was not previously evaluated. It was the aim of this study to compare serum leptin levels between patients with MS and healthy controls and to evaluate the possible relationship between circulating leptin levels and disease severity. Patients and Methods: Eighty-four MS patients and 57 sex-matched healthy volunteers were enrolled. Serum leptin levels were measured in all patients and controls. MS patients were stratified in 3 groups according to their degree of disability as assessed by the Expanded Disability Status Scale (EDSS). Patients were classified as having low (33 patients with an EDSS score <1.5), intermediate (28 patients with an EDSS score from 2 to 3) and high disability (23 patients with an EDSS score ≥3.5). Results: No significant differences in serum leptin levels and BMI were observed between patients and controls. In patients with MS, serum leptin levels were significantly correlated with BMI in those patients with low (R2 = 0.363; p < 0.001) and intermediate disability (R2 = 0.408; p < 0.001), but not in patients with a higher disability score (R2 = 0.064; p = 0.256). Conclusion: BMI, the major determinant of leptin level in physiological conditions, has a minor role in determining the serum levels of leptin in MS patients with a high EDSS score. Future longitudinal studies will be required in order to provide further insights into the regulation of leptin secretion in patients with MS.

A human anti-neuronal autoantibody against GABAB receptor induces experimental autoimmune agrypnia

In the serum and cerebrospinal fluid of a patient with recurrent acute episodes of respiratory crises, autonomic symptoms and total insomnia (agrypnia), we identified a novel anti-neural complement fixing antibody directed against GABA(B) receptor (GABA(B)R). Patient purified IgG recognized a band of approximately 110 kDa on protein extracts of mouse cerebellum, cortex and brainstem and immunolabelled cultured Chinese hamster ovary (CHO) cells, transfected with human GABA(B)R1 and rat GABA(B)R2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both patient purified IgG and anti-GABA(B)R1 antibody. In order to verify the pathogenic role of these purified antibodies, we injected patient IgG intrathecally into cisterna magna of C57BL/6 mice pre-implanted with EEG electrodes and we observed severe ataxia followed by breathing depression and total suppression of slow wave sleep, as evidenced by EEG recording, in a dose-dependent manner. Immunohistochemistry on brain sections of mice injected with patient IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on Purkinje cells and cerebellar granular layer. After incubation with anti-GABA(B)R antibody, a marked reduction of receptor immunostaining was found with relative sparing of neuronal architecture. In conclusion we recognized an anti-neuronal autoantibody directed against GABA(B)R that is associated with autoimmune agrypnia and we showed that our patient purified IgG was able to induce in mice experimental autoimmune agrypnia characterized by a complex neurological syndrome affecting several CNS functions.

Relapsing-remitting autoimmune agrypnia

A woman affected by multiple cranial nerve palsy developed several episodes of total insomnia and respiratory crises resulting from central breathing depression associated with dysautonomic symptoms. Oligoclonal IgG bands were present in her cerebrospinal fluid, and immunohistochemistry showed increased binding of serum and cerebrospinal fluid on γ‐aminobutyric acid‐ergic, synapse‐rich neuronal cells. Immunosuppressive treatment and plasma exchange were followed by clinical improvement, with restoration of sleep architecture and disappearance of respiratory crises, suggesting autoimmune pathogenesis of the syndrome.