Cristina Sancricca

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.

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.

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.

Isolation and characterization of mesoangioblasts from facioscapulohumeral muscular dystrophy muscle biopsies.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most frequent inherited muscle disease. Because in FSHD patients the coexistence of affected and unaffected muscles is common, myoblasts expanded from unaffected FSHD muscles have been proposed as suitable tools for autologous cell transplantation. Mesoangioblasts are a new class of adult stem cells of mesodermal origin, potentially useful for the treatment of primitive myopathies of different etiology. Here, we report the isolation and characterization of mesoangioblasts from FSHD muscle biopsies and describe morphology, proliferation, and differentiation abilities of both mesoangioblasts and myoblasts derived from various affected and unaffected muscles of nine representative FSHD patients. We demonstrate that mesoangioblasts can be efficiently isolated from FSHD muscle biopsies and expanded to an amount of cells necessary to transplant into an adult patient. Proliferating mesoangioblasts from all muscles examined did not differ from controls in terms of morphology, phenotype, proliferation rate, or clonogenicity. However, their differentiation ability into skeletal muscle was variably impaired, and this defect correlated with the overall disease severity and the degree of histopathologic abnormalities of the muscle of origin. A remarkable differentiation defect was observed in mesoangioblasts from all mildly to severely affected FSHD muscles, whereas mesoangioblasts from morphologically normal muscles showed no myogenic differentiation block. Our study could open the way to cell therapy for FSHD patients to limit muscle damage in vivo through the use of autologous mesoangioblasts capable of reaching damaged muscles and engrafting into them, without requiring immune suppression or genetic correction in vitro.

TWEAK in inclusion-body myositis muscle: possible pathogenic role of a cytokine inhibiting myogenesis

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 exert pleiotropic effects, including regulation of myogenesis. Sporadic inclusion-body myositis (IBM) is the most common muscle disease of the elderly population and leads to severe disability. IBM mesoangioblasts, different from mesoangioblasts in other inflammatory myopathies, display a myogenic differentiation defect. The objective of the present study was to investigate TWEAK-Fn14 expression in IBM and other inflammatory myopathies and explore whether TWEAK modulation affects myogenesis in IBM mesoangioblasts. TWEAK, Fn14, and NF-κB expression was assessed by immunohistochemistry and Western blot in cell samples from both muscle biopsies and primary cultures. Mesoangioblasts isolated from samples of IBM, dermatomyositis, polymyositis, and control muscles were treated with recombinant human TWEAK, Fn14-Fc chimera, and anti-TWEAK antibody. TWEAK-RNA interference was performed in IBM and dermatomyositis mesoangioblasts. TWEAK levels in culture media were determined by enzyme-linked immunosorbent assay. In IBM muscle, we found increased TWEAK-Fn14 expression. Increased levels of TWEAK were found in differentiation medium from IBM mesoangioblasts. Moreover, TWEAK inhibited myogenic differentiation of mesoangioblasts. Consistent with this evidence, TWEAK inhibition by Fn14-Fc chimera or short interfering RNA induced myogenic differentiation of IBM mesoangioblasts. We provide evidence that TWEAK is a negative regulator of human mesoangioblast differentiation. Dysregulation of the TWEAK-Fn14 axis in IBM muscle may induce progressive muscle atrophy and reduce activation and differentiation of muscle precursor cells.

Increased aging in primary muscle cultures of sporadic inclusion-body myositis.

Ageing is thought to participate to the pathogenesis of sporadic inclusion-body myositis (s-IBM). Although the regenerative potential of s-IBM muscle is reduced in vivo, age-related abnormalities of satellite cells possibly accounting for the decline of muscle repair have not been demonstrated. Here we show that proliferation rate and clonogenicity of s-IBM myoblasts are significantly lower and doubling time is longer than normal age-matched controls, indicating that proliferative capacity of s-IBM muscles becomes exhausted earlier. Telomere shortening is detected in s-IBM cells suggesting premature senescence. Differently from controls, s-IBM myoblasts show increased active beta-catenin mainly localized within myonuclei, indicating active Wnt stimulation. After many rounds of muscle growth, only s-IBM myoblasts accumulate congophilic inclusions and immunoreactive Abeta(1-40) deposits. Therefore, s-IBM myoblasts seem to have a constitutively impaired regenerative capacity and the intrinsic property, upon sufficient aging in vitro, to accumulate Abeta. Our results might be valuable in understanding molecular mechanisms associated with muscle aging underlying the defective regeneration of s-IBM muscle and provide new clues for future therapeutic strategies.

Mesoangioblasts from facioscapulohumeral muscular dystrophy display in vivo a variable myogenic ability predictable by their in vitro behavior.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most frequent inherited myopathy. We previously demonstrated that mesoangioblasts can be efficiently isolated from FSHD muscles, although their differentiation ability into skeletal muscle was variably impaired. This correlates with overall disease severity and degree of histopathologic abnormalities, since mesoangioblasts from morphologically normal muscles did not show any myogenic differentiation block. The aim of our present study was to verify whether mesoangioblasts from differentially affected FSHD muscles reproduce in vivo the same differentiation ability shown in vitro by studying their capability to form new muscle fibers during muscle regeneration of experimentally damaged muscles. We show that a diverse ability of FSHD mesoangioblasts to engraft and differentiate into skeletal muscle of SCID mice is strictly related to the characteristics of the muscle of origin, closely replicating in vivo what was previously observed in vitro. Moreover, we demonstrate that mesoangioblasts obtained from severely affected muscles scarcely integrate into muscle fibers, remaining mainly localized in the connective tissue. This suggests a defective migration in response to chemoattractants released by damaged fibers, as indicated by cell migration assays in response to HMGB1 and very low levels of RAGE expression, along with a decreased ability to fuse or to appropriately trigger the myogenic program. Our study indicates that FSHD mesoangioblasts from unaffected muscles can be used as selective treatment to halt muscle degeneration in severely affected muscles, and suggests that pharmacological and molecular interventions aimed to ameliorate homing and engraftment of transplanted autologous mesoangioblasts may open the way to cell therapy for FSHD patients, without requiring immunosuppression or genetic correction in vitro.

Clinical characteristics, course and prognosis of spinal multiple sclerosis.

Study design:Retrospective examination.Objective:To define the clinical characteristics and response to therapy of spinal multiple sclerosis (MS).Setting:Italy.Methods:Retrospective review was performed on 563 patients with clinical definite MS. Selection criteria were two or more spinal cord lesions in the presence of normal magnetic resonance imaging of the brain.Results:Spinal MS was diagnosed in 13 patients (2.3%) out of 563 with clinical definite MS. There were seven female and six male patients; nine had a relapsing–remitting (RR) and four, a primary progressive (PP) course. All patients were treated with immunosoppressive or immunomodulatory therapy. Mean disease duration in patients with RR–MS was 13.1±10.1 years with a mean age at onset of 29.5±14.3 years; the mean Expanded Disability Status Scale (EDSS) at the time of the study was 3.5±2.5 with a progression index of 0.28. Mean disease duration in patients with PP course was 7±6.2 years with a mean age at onset of 56.7±10.4 years; the mean EDSS at the time of the study was 6.2±2.0 with a progression index of 1.48.Conclusions:Patients with spinal RR–MS are characterised by an early disease onset with minimal or moderate disability progression; patients with spinal PP–MS show a late disease onset and more rapid disability progression. In our series of spinal MS patients, disability progression seems to be mainly due to the disease course and age at onset rather than to the site of lesion.

Evidence of involvement of leptin and IL-6 peptides in the action of interferon-beta in secondary progressive multiple sclerosis

Leptin is a peptide hormone which acts on cells of immune system by influencing the production of cytokines. Serum leptin levels and cytokine production by peripheral blood mononuclear cells (PBMC) were measured in 18 secondary progressive multiple sclerosis (SPMS) patients under IFN-beta-1b treatment. There were no overall effects on leptin, interleukin-6 (IL-6), IL-10 and IL-12 p40 after 2, 6 and 12 months of treatment. However, leptin and IL-6 decreased after 6 and 12 months of treatment in 12 patients who did not show progression of disability. Thus, our pilot data show that the beneficial effect of IFN-beta on some SPMS patients might be associated with the reduced levels of leptin and reduced IL-6 production by PBMC.

Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy

Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivostudies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders.