Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant genetic disease mainly characterized by ptosis and dysphagia. We conducted a phase I/IIa clinical study (ClinicalTrials.gov NCT00773227) using autologous myoblast transplantation following myotomy in adult OPMD patients. This study included 12 patients with clinical diagnosis of OPMD, indication for cricopharyngeal myotomy, and confirmed genetic diagnosis. The feasibility and safety end points of both autologous myoblast transplantation and the surgical procedure were assessed by videoendoscopy in addition to physical examinations. Potential therapeutic benefit was also assessed through videoendoscopy and videofluoroscopy of swallowing, quality of life score, dysphagia grade, and a drink test. Patients were injected with a median of 178 million myoblasts following myotomy. Short and long-term (2 years) safety and tolerability were observed in all the patients, with no adverse effects. There was an improvement in the quality of life score for all 12 patients, and no functional degradation in swallowing was observed for 10 patients. A cell dose-dependant improvement in swallowing was even observed in this study. This trial supports the hypothesis that a local injection of autologous myoblasts in the pharyngeal muscles is a safe and efficient procedure for OPMD patients.

Corticosteroid insensitivity (CSI) represents a profound challenge in managing patients with asthma. We recently demonstrated that short exposure of airway smooth muscle cells (ASMCs) to proasthmatic cytokines drastically reduced their responsiveness to glucocorticoids (GCs), an effect that was partially mediated via interferon regulatory factor-1, suggesting the involvement of additional mechanisms (Am J Respir Cell Mol Biol 2008;38:463-472). Although GC receptor (GR) can be phosphorylated at multiple serines in the Nterminal region, the major phosphorylation sites critical for GR transcriptional activity are serines 211 (Ser211) and 226 (Ser226). We tested the novel hypothesis that cytokine-induced CSI in ASMCs is due to an impaired GR phosphorylation. Cells were treated with TNF-a (10 ng/ml) and IFN-g (500 UI/ml) for 6 hours and/or fluticasone (100 nm) added 2 hours before. GR was constitutively phosphorylated at Ser226 but not at Ser211 residues. Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211 but not on Ser226 residues while increasing the expression of Ser/Thr protein phosphatase (PP)5 but not that of PP1 or PP2A. Transfection studies using a reporter construct containing GC responsive elements showed that the specific small interfering RNAinduced mRNA knockdown of PP5, but not that of PP1 or PP2A, partially prevented the cytokine suppressive effects on GR-meditated transactivation activity. Similarly, cytokines failed to inhibit GC-induced GR-Ser211 phosphorylation when expression of PP5 was suppressed. We propose that the novel mechanism that proasthmatic cytokine-induced CSI in ASMCs is due, in part, to PP5-mediated impairment of GR-Ser211 phosphorylation.Keywords: serine/threonine protein phosphatase; airway smooth muscle; asthma; corticosteroid insensitivity; airway remodeling Although in the majority of patients with asthma symptoms are well controlled by inhaled glucocorticoids (GCs), a subgroup of patients suffering from severe asthma respond poorly to GC therapy (1, 2). This group is responsible for a disproportionate share of health care costs and morbidity associated with the disease; as a result, corticosteroid insensitivity (CSI) in patients with severe asthma represents a significant unmet clinical need (3). Recent studies performed on bronchial biopsies from patients with asthma showed a persistent expression of eotaxin (4), CCL19 (5), ADAM33, and ADAM8 (6, 7) in airway smooth muscle (ASM) bundles despite patients being treated with inhaled or oral GCs. The expression of these GC-insensitive proasthmatic proteins by ASM correlated with the severity of asthma. We and others replicated this finding in vitro using cultured human ASM cells (ASMCs) exposed to a combination of TNF-a and IFN-g (8-10). We showed that the induction of a variety of proasthmatic genes, namely CD38, CXCL10, CX3CL1, and CCL5, by TNF-a and IFN-g was surprisingly insensitive to the inhibition by GC (9, 11). This "corticosteroidinsensitive" state induced by TNF-a and IFN-g was associate...

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease characterized by a typical regional distribution, featuring composed patterns of clinically affected and unaffected muscles. No treatment is available for this condition, in which the pathophysiological mechanism is still unknown. Autologous transfer of myoblasts from unaffected to affected territories could be considered as a potential strategy to delay or stop muscle degeneration. To evaluate the feasibility of this concept, we explored and compared the growth and differentiation characteristics of myoblasts prepared from phenotypically unaffected muscles of five FSHD patients and 10 control donors. According to a clinically approved procedure, 10 9 cells of a high degree of purity were obtained within 16-23 days. More than 80% of these cells were myoblasts, as demonstrated by labeling of the muscle markers CD56 and desmin. FSHD myoblasts presented a doubling time equivalent to that of control cells; they kept high proliferation ability and did not show early telomere shortening. In vitro, these cells were able to differentiate and to express muscle-specific antigens. In vivo, they participated to muscle structures when injected into immunodeficient mice. These data suggest that myoblasts expanded from unaffected FSHD muscles may be suitable tools in view of autologous cell transplantation clinical trials.

Like many steroid receptors, the glucocorticoid (GC) receptor (GR) is a phosphoprotein. Although there are multiple phosphorylation sites critical for GR transcriptional activity (i.e., serine [S]203, S211, and S226), their respective role in driving GR functions is highly cell specific. We have recently identified protein phosphatase 5 as an essential Ser/Thr phosphatase responsible for impairing GR function via S211 dephosphorylation in airway smooth muscle (ASM) cells. Because p38 mitogen-activated protein kinase (MAPK) directly phosphorylates GR in different cell types in a stimulus-and celldependent manner, we investigated the role of p38 MAPK on GR phosphorylation and function in ASM cells. Cells were transfected with 100 nM p38 MAPK small interfering RNA or 2 mg MAPK kinase 3 expression vector (a specific kinase that directly activates p38 MAPK) in the presence or absence of fluticasone (100 nM) and/or p38 MAPK pharmacological inhibitor SB203580. We found that p38 MAPK blockade positively regulates GR nuclear translocation and GR-dependent induction of the steroid-target gene GC-induced leucine zipper in a hormone-independent manner. We also found that p38 MAPK-dependent regulation of GR functions was associated with a differential action on GR phosphorylation at S203 and S211 residues. This study demonstrated that the inactive state of GR in resting conditions is not only ensured by the absence of the GC ligand but also by p38 MAPK-dependent phosphorylation of unliganded GR at specific residues, which appears to be important in determining the overall GC responsiveness of ASM cells.

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Although the majority of patients with asthma are well controlled by inhaled glucocorticoids (GCs), patients with severe asthma are poorly responsive to GCs. This latter group is responsible for a disproportionate share of health care costs associated with asthma. Recent studies in immune cells have incriminated interferon-γ (IFN-γ) as a possible trigger of GC insensitivity in severe asthma; however, little is known about the role of IFN-γ in modulating GC effects in other clinically relevant nonimmune cells, such as airway epithelial cells. We hypothesized that IFN-γ-induced JAK/STAT-associated signaling pathways in airway epithelial cells are insensitive to GCs and that strategies aimed at inhibiting JAK/STAT pathways can restore steroid responsiveness. Using Western blot analysis we found that all steps of the IFN-γ-induced JAK/STAT signaling pathway were indeed GC insensitive. Transfection of cells with reporter plasmid showed IFN-γ-induced STAT1-dependent gene transcription to be also GC insensitive. Interestingly, real-time PCR analysis showed that IFN-γ-inducible genes (IIGs) were differentially affected by GC, with CXCL10 being GC sensitive and CXCL11 and IFIT2 being GC insensitive. Further investigation showed that the differential sensitivity of IIGs to GC was due to their variable dependency to JAK/STAT vs. NF-κB signaling pathways with GC-sensitive IIGs being more NF-κB dependent and GC-insensitive IIGs being more JAK/STAT dependent. Importantly, transfection of cells with siRNA-STAT1 was able to restore steroid responsiveness of GC-insensitive IIGs. Taken together, our results show the insensitivity of IFN-γ-induced JAK/STAT signaling pathways to GC effects in epithelial cells and also suggest that targeting STAT1 could restore GC responsiveness in patients with severe asthma.

In the present study, modifications in cytosolic expressed proteins during human myoblast differentiation were studied by dialysis-assisted 2-DE (DAGE, [1]). About 1000 spots were analysed on the 5th and 13th day of differentiation with a dynamic range of protein expression exceeding 1000-fold. During myogenic differentiation, the number of nonmatching spots as well as the extent of quantitative differences between matched spots significantly increased. Over one hundred differentially expressed spots were excised and identified by MALDI-TOF MS. The differentiation-associated expression pattern of eight proteins was validated by Western blot analysis. Differential expression of several proteins was demonstrated for the first time in human myotubes. Interestingly, Ingenuity pathway analysis grouped 30 of these proteins into two overlapping networks containing as principal nodes IGF-1 and tumour necrosis factor, two proteins known to play a crucial role in cytogenesis. Our results illustrate the large rearrangement of the proteome during the differentiation of human myoblasts and provide evidence for new partners involved in this complex process.

The coronavirus pandemic, known as coronavirus disease 2019 (COVID-19), is an infectious respiratory disease caused by SARS-CoV-2, a novel coronavirus first identified in patients from Wuhan, China. Since December 2019, SARS-CoV-2 spread swiftly around world and infected more than 25 million people and caused more than 800,000 deaths in 188 countries. Chronic respiratory diseases such as asthma and COPD appear to be a risk factor for COVID-19; however, their prevalence remains controversial. In fact, studies in China reported lower rates of chronic respiratory conditions in patients with COVID-19 than in the general population, while the trend is reversed in the United States and Europe. Although the underlying molecular mechanisms of a possible interaction between COVID-19 and chronic respiratory diseases remain unknown, some observations can help to resolve them. Indeed, physiological changes, immune response, or medication used against SARS-CoV-2 may have greater impact on patients with chronic respiratory conditions, already debilitated by chronic inflammation, dyspnea, and the use of immunosuppressant drugs like corticosteroids. In this review, we discuss the importance and the impact of COVID-19 on asthmatics and COPD patients, the possible available treatments, and patient management during the pandemic.