Agata Katia Patanella

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.

Correlations between peripheral blood mononuclear cell production of BDNF, TNF-alpha, IL-6, IL-10 and cognitive performances in multiple sclerosis patients.

The aim of this study was to investigate the role of Brain Derived Neurotrophic Factor (BDNF) and inflammatory factors in the development of cognitive dysfunctions in Multiple Sclerosis (MS). We correlated peripheral blood mononuclear cell (PBMC) production of BDNF, Tumor Necrosis Factor‐alpha (TNF‐α), Interleukin (IL)‐6 and IL‐10 with performances on specific neuropsychological tasks in a selected series of MS patients. We studied a sample of 30 patients with relapsing‐remitting (RR)MS, segregated by gender and matched for age, education, disease duration, type of immunomodulating therapy, degree of disability and overall cognitive status. We found that low BDNF levels were correlated with increased time of execution on a divided attention and visual scanning task whereas high levels of IL‐6 were correlated with low Mini Mental State Examination scores. We did not observe any significant correlations between IL‐10, TNF‐α levels and cognitive performances in our patients. In conclusion our study shows a correlation between low BDNF and high IL‐6 production by PBMCs and poorer performances in cognitive tasks in RRMS patients suggesting a possible role of these factors in cognitive impairment in MS.

CD8+ T Cells in Facioscapulohumeral Muscular Dystrophy Patients with Inflammatory Features at Muscle MRI

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited disease, and although strongly suggested, a contribution of inflammation to its pathogenesis has never been demonstrated. In FSHD patients, we found by immunohistochemistry inflammatory infiltrates mainly composed by CD8+ T cells in muscles showing hyperintensity features on T2-weighted short tau inversion recovery magnetic resonance imaging (T2-STIR-MRI) sequences. Therefore, we evaluated the presence of circulating activated immune cells and the production of cytokines in patients with or without muscles showing hyperintensity features on T2-STIR-MRI sequences and from controls. FSHD patients displaying hyperintensity features in one or more muscles showed higher CD8+pSTAT1+, CD8+T-bet+ T cells and CD14+pSTAT1+, CD14+T-bet+ cells percentages and IL12p40, IFNγ and TNFα levels than patients without muscles displaying hyperintense features and controls. Moreover, the percentages of CD8+pSTAT1+, CD8+T-bet+ and CD14+pSTAT1+ cells correlated with the proportion of muscles displaying hyperintensity features at T2-STIR sequences. These data indicate that circulating activated immune cells, mainly CD8+ T cells, may favour FSHD progression by promoting active phases of muscle inflammation.

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.

Increased CD4(+)CD25(+)Foxp3(+) T Cells in Peripheral Blood of Celiac Disease Patients: Correlation with Dietary Treatment.

Regulatory CD4+ CD25+Foxp3+ T cells are involved in the regulation of immune response and inhibit protective antitumor immunity. Celiac disease (CD), a food gluten-sensitive enteropathy, is considered a T-cell-mediated autoimmune disease and is generally associated with an overall increased risk of cancer in CD patients. We observed a higher percentage of circulating CD4+CD25+Foxp3+ T cells and an increased Foxp3 expression in CD4+CD25+ T cells from untreated than from treated CD patients. In co-culture, CD4+CD25+ T cells from both treated and untreated CD patients significantly suppressed the proliferation of autologous CD4+CD25(-) T cells similarly to values in healthy subjects. Our study suggests that Treg proportion and Foxp3 expression in circulating CD4+CD25+ T cells could justify the increased global risk of malignancy in CD population and support the efficacy of lifelong gluten-free diet in the reduction of the cancer risk.

pSTAT1, pSTAT3, and T-bet as markers of disease activity in chronic inflammatory demyelinating polyradiculoneuropathy.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is considered an auto‐immune disorder. We evaluated expression of pSTAT1, T‐bet, and pSTAT3 in circulating T‐cells, B‐cells, and monocytes and spontaneous production of interleukin‐17 (IL17), interferon‐gamma (IFNγ), and interleukin‐10 (IL10) by peripheral blood mononuclear cells (PBMCs) from 14 active CIDP patients compared with 6 patients with long‐lasting remission and 20 controls. Active disease patients showed higher pSTAT1, T‐bet, and pSTAT3 in CD4+ T‐cells than controls (p < 0.001, p = 0.0002, p = 0.0097, respectively) and remission patients (p < 0.001, p = 0.0036, p = 0.0008, respectively). pSTAT1, T‐bet, and pSTAT3 were also higher in monocytes from active CIDP patients than controls (p = 0.0011, p = 0.0041, p = 0.0413, respectively) and remission patients (p = 0.0073, p = 0.0274, p = 0.0251, respectively). Moreover in CD8+ T‐cells, pSTAT3 expression was higher in active CIDP patients than in remission patients (p = 0.0345) and in controls (p = 0.0023). IL17 and IFNγ production were significantly higher in active CIDP patients than in controls (p < 0.0395, p = 0.0010, respectively); IFNγ levels were higher also in remission CIDP patients (p = 0.0073). IL10 levels were higher in active phase patients than in controls (p = 0.0334). Our data suggest that pSTAT1, T‐bet, and pSTAT3 can be considered putative markers of disease activity and potential targets for specific therapies.

CD4+T-bet+, CD4+pSTAT3+ and CD8+T-bet+ T cells accumulate in peripheral blood during NZB treatment.

Circulating T cells and monocytes expressing T-bet, pSTAT1 and pSTAT3 increase in relapsing–remitting multiple sclerosis (RRMS) during relapse. Natalizumab (NZB) is an effective drug in RRMS, but exacerbation of the disease after its discontinuation has been described in some patients. The aim of this research was to study the effect of NZB treatment on circulating lymphomonocyte subpopulations expressing T-bet, pSTAT1, pSTAT3 and CD4+CD25+Foxp3+ regulatory T cells. Flow cytometry was used to evaluate the percentages of circulating CD4+ and CD8+ T cells, CD14+ monocytes and B cells expressing T-bet, pSTAT1, and pSTAT3, and CD4+CD25+Foxp3+ regulatory T cells from RRMS patients before and after 6–12 NZB infusions. In NZB-treated RRMS patients, the percentages of CD4+pSTAT1+ and CD8+pSTAT1+ T cells, CD14+pSTAT1+ monocytes, CD4+T-bet+, CD8+T-bet+ and CD4+pSTAT3+ T cells and CD14+pSTAT3+ monocytes increased after 12 drug infusions and were similar to those observed in untreated relapsing RRMS patients. Otherwise in vitro NZB exposure of peripheral blood mononuclear cells from untreated RRMS patients and controls had no effect. It was concluded that NZB treatment determines an accumulation of CD4+pSTAT1+, CD8+pSTAT1+, CD4+T-bet+, CD8+T-bet+ and CD4+STAT3+ T cells in peripheral blood that may account for the exacerbation of the disease observed in some patients after the discontinuation of the drug.

Refractory generalized seizures and cerebellar ataxia associated with anti-GAD antibodies responsive to immunosuppressive treatment

Anti-glutamic acid decarboxylase antibodies (anti-GAD-Ab) may play a pathogenic role in Stiff-man syndrome [1], cerebellar ataxia withmulti-endocrine auto-immune disease [2] and drug-resistant epilepsy [3]. We described a 42-year-old woman who developed type-1 diabetes, Hashimoto s thyroiditis, vitamin B12 deficiency, horizontal and upbeat nystagmus, cerebellar ataxia and pharmacoresistant generalized seizureswith thepresenceof anti-GAD-Ab. The patient was affected by psoriasis and vitiligo. In March 2004, she developed dizziness and diplopia. She was found to have hyperglycaemia and impaired insulin secretion. She was positive for antithyroglobulin and anti-thyroid peroxidase antibodies, and her TSH levels were elevated. A brain magnetic resonance imaging (MRI) showed mild cerebellar vermis atrophy. Type-1 diabetes and subclinical hypothyroidism because of Hashimoto s thyroiditis were diagnosed. In April 2004, the patient experienced tonic clonic generalized seizures. Neurological examination showed evidence of bilateral eyelid ptosis prevailingon left side, horizontal and upbeat nystagmus, and gait ataxia. Deep sensory function and deep tendon reflexes were normal. A new brain MRI confirmed the cerebellar vermis atrophy (Fig. 1).Cerebrospinal fluid showedan increased IgG index (> 1.7). She was found to have a deficit of vitamin B12. She was positive for anti-ANA (1:40) and antigastric parietal cell antibodies (70 U/ml). Molecular analysis for expanded CAG repeats in the gene for SCA 1-3, 6, 7, 12 and 17 was negative. The patient was well substituted for vitamin B12; however, there was no improvement of neurological symptoms after substitution. Additional therapy was started, initially consisting of oxcarmazepine (900 mg/day); then levetiracetam (3000 mg/day) and finally phenobarbital (150 mg/day) were added without benefit on seizures. In October 2004, the frequency of seizures increased (5–10 episodes/month), and she developed cerebellar ataxia. Serum anti-GAD-Ab level was 67.4 U/ml (n.v. < 1 U/ml). Therapy with prednisone 50 mg/die and azathioprine 100 mg/die resulted in a marked improvement of cerebellar ataxia and epilepsy (3–4 episodes/year). Immunosuppressive therapy was progressively tapered down to suspension in December 2006. At present, the number of seizure episodes is 3–4/year, and the patient is treated with levetiracetam (3000 mg/day) and pregabalin (450 mg/day). Neurological examination shows evidence of only nystagmus and left eyelid ptosis. Anti-GAD-Ab is negative. Cerebellar ataxia [2] as well as nystagmus [4,5] associated with anti-GAD-Ab have been previously described. High titres of anti-GAD-Ab have also been associated with some cases of epilepsy [3,6]. Immunosuppressive treatment has been performed in some of these patients, with somewhat varying success [4,6,7]. In our patient, prednisolone and azathioprine treatment markedly improved the cerebellar ataxia and reduced the frequency of seizures. These data support the autoimmune pathogenesis of our patient s complex clinical syndrome. In our opinion, it is important to search for anti-GAD-Ab in presence of neurological symptoms such as cerebellar ataxia, nystagmus and pharmacoresistant epilepsy in which a combination of immunosuppressive drugs may be very effective.