Identification of a set of genes potentially responsible for resistance to ferroptosis in lung adenocarcinoma cancer stem cells.

Scientific literature supports the evidence that cancer stem cells (CSCs) retain inside low reactive oxygen species (ROS) levels and are, therefore, less susceptible to cell death, including ferroptosis, a type of cell death dependent on iron-driven lipid peroxidation. A collection of lung adenocarcinoma (LUAD) primary cell lines derived from malignant pleural effusions (MPEs) of patients was used to obtain 3D spheroids enriched for stem-like properties. We observed that the ferroptosis inducer RSL3 triggered lipid peroxidation and cell death in LUAD cells when grown in 2D conditions; however, when grown in 3D conditions, all cell lines underwent a phenotypic switch, exhibiting substantial resistance to RSL3 and, therefore, protection against ferroptotic cell death. Interestingly, this phenomenon was reversed by disrupting 3D cells and growing them back in adherence, supporting the idea of CSCs plasticity, which holds that cancer cells have the dynamic ability to transition between a CSC state and a non-CSC state. Molecular analyses showed that ferroptosis resistance in 3D spheroids correlated with an increased expression of antioxidant genes and high levels of proteins involved in iron storage and export, indicating protection against oxidative stress and low availability of iron for the initiation of ferroptosis. Moreover, transcriptomic analyses highlighted a novel subset of genes commonly modulated in 3D spheroids and potentially capable of driving ferroptosis protection in LUAD-CSCs, thus allowing to better understand the mechanisms of CSC-mediated drug resistance in tumors.

Multiple Sclerosis Onset before and after COVID-19 Vaccination: Can HLA Haplotype Be Determinant?

A few cases of multiple sclerosis (MS) onset after COVID-19 vaccination have been reported, although the evidence is insufficient to establish causality. The aim of this study is to compare cases of newly diagnosed relapsing-remitting MS before and after the outbreak of the COVID-19 pandemic and the impact of COVID-19 vaccination. Potential environmental and genetic predisposing factors were also investigated, as well as clinical patterns. This is a single-centre retrospective cohort study including all patients who presented with relapsing-remitting MS onset between January 2018 and July 2022. Data on COVID-19 vaccination administration, dose, and type were collected. HLA-DRB1 genotyping was performed in three subgroups. A total of 266 patients received a new diagnosis of relapsing-remitting MS in our centre, 143 before the COVID-19 pandemic (until and including March 2020), and 123 during the COVID-19 era (from April 2020). The mean number of new MS onset cases per year was not different before and during the COVID-19 era and neither were baseline patients' characteristics, type of onset, clinical recovery, or radiological patterns. Fourteen (11.4%) patients who subsequently received a new diagnosis of MS had a history of COVID-19 vaccination within one month before symptoms onset. Patients' characteristics, type of onset, clinical recovery, and radiological patterns did not differ from those of patients with non-vaccine-related new diagnoses of MS. The allele frequencies of HLA-DRB1*15 were 17.6% and 22.2% in patients with non-vaccine-related disease onset before and during the COVID-19 era, respectively, while no case of HLA-DRB1*15 was identified among patients with a new diagnosis of MS post-COVID-19 vaccine. In contrast, HLA-DRB1*08+ or HLA-DRB1*10+ MS patients were present only in this subgroup. Although a causal link between COVID-19 vaccination and relapsing-remitting MS cannot be detected, it is interesting to note and speculate about the peculiarities and heterogeneities underlying disease mechanisms of MS, where the interactions of genetics and the environment could be crucial also for the follow-up and the evaluation of therapeutic options.

Serum-Circulating microRNAs in Sporadic Inclusion Body Myositis.

BACKGROUND: Sporadic inclusion body myositis (s-IBM) represents a unique disease within idiopathic inflammatory myopathies with a dual myodegenerative-autoimmune physiopathology and a lack of an efficacious treatment. Circulating miRNA expression could expand our knowledge of s-IBM patho-mechanisms and provide new potential disease biomarkers. To evaluate the expression of selected pre-amplified miRNAs in the serum of s-IBM patients compared to those of a sex- and age-matched healthy control group, we enrolled 14 consecutive s-IBM patients and 8 sex- and age-matched healthy controls. By using two different normalization approaches, we found one downregulated and three upregulated miRNAs. hsa-miR-192-5p was significantly downregulated, while hsa-miR-372-3p was found to be upregulated more in the s-IBM patients compared to the level of the controls. The other two miRNAs had a very low expression levels (raw Ct data > 29). hsa-miR-192-5p and hsa-miR-372-3p were found to be significantly dysregulated in the serum of s-IBM patients. These miRNAs are involved in differentiation and regeneration processes, thus possibly reflecting pathological mechanisms in s-IBM muscles and potentially representing disease biomarkers.

Liposome-based nanoparticles impact on regulatory and effector phenotypes of macrophages and T cells in multiple Sclerosis patients.

Current available treatments of Multiple Sclerosis (MS) reduce neuroinflammation acting on different targets on the immune system, but potentially lead to severe side effects and have a limited efficacy in slowing the progression of the disease. Here, we evaluated in vitro the immunomodulatory potential of a new class of nanoparticles - liposomes, constituted by a double-layer of phosphatidylserine (PSCho/PS), and double-faced, with an outer layer of phosphatidylserine and an inner layer of phosphatidic acid (PSCho/PA), either alone or in the presence of the myelin basic protein (MBP) peptide (residues 85-99) (PSCho/PS-MBP and PSCho/PA-MBP). Results showed that PSCho/PS are equally and efficiently internalized by pro- and anti-inflammatory macrophages (M1 and M2 respectively), while PSCho/PA were internalized better by M2 than M1. PSCho/PS liposomes were able to inhibit the secretion of innate pro-inflammatory cytokine IL-1ß. PSCho/PS liposomes expanded Tregs, reducing Th1 and Th17 cells, while PSCho/PA liposomes were unable to dampen pro-inflammatory T cells and to promote immune-regulatory phenotype (Treg). The ability of PSCho/PS liposomes to up-regulate Treg cells was more pronounced in MS patients with high basal expression of M2 markers. PSCho/PS liposomes were more effective in decreasing Th1 (but not Th17) cells in MS patients with a disease duration >3 months. On the other hand, down-modulation of Th17 cells was evident in MS patients with active, Gadolinium enhancing lesions at MRI and in MS patients with a high basal expression of M1-associated markers in the monocytes. The same findings were observed for the modulation of MBP-driven Th1/Th17/Treg responses. These observations suggest that early MS associate to a hard-wired pro-Th1 phenotype of M1 that is lost later during disease course. On the other hand, acute inflammatory events reflect a temporary decrease of M2 phenotype that however is amenable to restauration upon treatment with PSCho/PS liposomes. Thus, together these data indicate that monocytes/macrophages may play an important regulatory function during MS course and suggest a role for PSCho/PS and PSCho/PS-MBP as new therapeutic tools to dampen the pro-inflammatory immune responses and to promote its regulatory branch.

CSF CXCL13 and Chitinase 3-like-1 Levels Predict Disease Course in Relapsing Multiple Sclerosis.

Several biomarkers from multiple sclerosis (MS) patients' biological fluids have been considered to support diagnosis, predict disease course, and evaluate treatment response. In this study, we assessed the CSF concentration of selected molecules implicated in the MS pathological process. To investigate the diagnostic and prognostic significance of CSF concentration of target candidate biomarkers in both relapsing (RMS, n = 107) and progressive (PMS, n = 18) MS patients and in other inflammatory (OIND, n = 10) and non-inflammatory (ONIND, n = 15) neurological disorders. We measured the CSF concentration of APRIL, BAFF, CHI3L1, CCL-2, CXCL-8, CXCL-10, CXCL-12, CXCL-13 through a Luminex Assay. MS patients were prospectively evaluated, and clinical and radiological activity were recorded. CHI3L1 and CXCL13 CSF levels were significantly higher in both MS groups compared to control groups, while CCL2, BAFF, and APRIL concentrations were lower in RMS patients compared to PMS and OIND. Considering RMS patients with a single demyelinating event, higher concentrations of CHI3L1, CXCL10, CXCL12, and CXCL13 were recorded in patients who converted to clinically defined MS(CDMS). RMS patients in the CXCL13 and CHI3L1 high concentration group had a significantly higher risk of relapse (HR 12.61 and 4.57), MRI activity (HR 7.04 and 2.46), and of any evidence of disease activity (HR 12.13 and 2.90) during follow-up. CSF CXCL13 and CHI3L1 levels represent very good prognostic biomarkers in RMS patients, and therefore can be helpful in the treatment choice. Higher CSF concentrations of neuro-inflammatory biomarkers were associated with a higher risk of conversion to CDMS in patients with a first clinical demyelinating event. Differential CSF BAFF and APRIL levels between RMS and PMS suggest a different modulation of B-cells pathways in the different phases of the disease.

The Expanding Role of the Infectious Disease Expert in the Context of the MS Centre.

INTRODUCTION: The complexity of the MS patient's management is constantly growing. Consequently, the MS care unit requires a multidisciplinary approach, including an infectious disease specialist to minimise the risk of infectious complications related both to the disease and DMTs. MATERIALS AND METHODS: We retrospectively evaluated the infectious disease consultations performed from 2015 to 2019 in our MS centre. RESULTS: We identified 107 patients with at least one infectious disease consultation out of 1088 patients. We found a progressive increase in the number of consultations from 2015 to 2019. Nearly half of the consultations were requested at the time of starting MS treatment. The most frequent requests were represented by chronic or acute infections. The most prevalent infectious agents were Herpesviridae and Mycobacterium tuberculosis. Antibiotic or antiviral treatment and prophylactic treatment or vaccination represented together the most frequent outcomes of the consultations. Finally, a treatment delay was significantly associated with the advice of a prophylactic treatment or of a vaccination. CONCLUSION: There is an increasing awareness of the potential infectious complications of MS and of exposure to DMTs. The interaction between the MS neurologist and infectious disease specialist is fundamental to minimise the infectious risk related to the disease and to the DMTs, with a progressive shift from complication management to a broader prevention workup at the time of MS diagnosis, including both vaccination and prophylactic treatments.

Technology outcome measures in neuromuscular disorders: A systematic review.

BACKGROUND AND PURPOSE: Portable and wearable devices can monitor a number of physical performances and lately have been applied to patients with neuromuscular disorders (NMDs). METHODS: We performed a systematic search of literature databases following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) principles, including all studies reporting the use of technological devices for motor function assessment in NMDs from 2000 to 2021. We also summarized the evidence on measurement properties (validity, reliability, responsiveness) of the analyzed technological outcome measures. RESULTS: One hundred studies fulfilled the selection criteria, most of them published in the past 10 years. We defined four categories that gathered similar technologies: gait analysis tools, for clinical assessment of pace and posture; continuous monitoring of physical activity with inertial sensors, which allow "unsupervised" activity assessment; upper limb evaluation tools, including Kinect-based outcome measures to assess the reachable workspace; and new muscle strength assessment tools, such as Myotools. Inertial sensors have the evident advantage of being applied in the "in-home" setting, which has become especially appealing during the COVID-19 pandemic, although poor evidence from psychometric property assessment and results of the analyzed studies may limit their research application. Both Kinect-based outcome measures and Myotools have already been validated in multicenter studies and different NMDs, showing excellent characteristics for application in clinical trials. CONCLUSIONS: This overview is intended to raise awareness on the potential of the different technology outcome measures in the neuromuscular field and to be an informative source for the design of future clinical trials, particularly in the era of telemedicine.

Immunogenicity and safety of mRNA COVID-19 vaccines in people with multiple sclerosis treated with different disease-modifying therapies.

The potential impact of disease-modifying therapies (DMTs) for multiple sclerosis (MS) on COVID-19 vaccination is poorly understood. According to recent observations, the humoral immune response could be impaired in patients treated with ocrelizumab or fingolimod. Our study evaluated the immunogenicity and safety of mRNA COVID-19 vaccines in a convenience sample of 140 MS patients treated with different DMTs, undergoing vaccination between April and June 2021. Humoral immune response was tested 1 month after the second dose, using a chemiluminescent microparticle immunoassay to detect IgG against SARS-CoV-2 nucleoprotein. We explored the potential correlation between the IgG titer and DMTs. All patients in treatment with first-line DMTs, natalizumab, cladribine, and alemtuzumab, developed a measurable humoral response. In patients treated with ocrelizumab and fingolimod, the IgG level was significantly lower, but only some patients (22.2% for fingolimod and 66% for ocrelizumab) failed to develop a measurable humoral response. In the ocrelizumab group, the IgG level was positively correlated with the time from last infusion. No SARS-CoV-2 infections were reported after vaccination. The most reported side effects were pain at the injection site (57.1%) and fatigue (37.9%). No patient experienced severe side effects requiring hospitalization. Our study confirms that COVID-19 vaccination is safe and well-tolerated in MS patients and should be recommended to all patients regardless of their current DMTs. Since fingolimod and ocrelizumab could reduce the humoral immune response, in patients treated with these drugs, detecting SARS-CoV-2 antibodies could be helpful to monitor the immune response after vaccination.

Phosphodiesterase 5a Signalling in Skeletal Muscle Pathophysiology.

Phosphodiesterase 5A (PDE5A) is involved in cGMP hydrolysis, regulating many physiological processes. Increased activity of PDE5A has been found in several pathological conditions, and the pharmacological inhibition of PDE5 has been demonstrated to have several therapeutic applications. We have identified the presence of three different Pde5a isoforms in cardiomyocytes, and we have found that the expression of specific Pde5a isoforms may have a causal role in the onset of pathological responses in these cells. In our previous study, we demonstrated that PDE5A inhibition could ameliorate muscular dystrophy by acting at different levels, as assessed by the altered genomic response of muscular cells following treatment with the PDE5A inhibitor tadalafil. Thus, considering the importance of PDE5A in various pathophysiological conditions, we further investigated the regulation of this enzyme. Here, we analysed the expression of Pde5a isoforms in the pathophysiology of skeletal muscle. We found that skeletal muscle tissues and myogenic cells express Pde5a1 and Pde5a2 isoforms, and we observed an increased expression of Pde5a1 in damaged skeletal muscles, while Pde5a2 levels remained unchanged. We also cloned and characterized the promoters that control the transcription of Pde5a isoforms, investigating which of the transcription factors predicted by bioinformatics analysis could be involved in their modulation. In conclusion, we found an overexpression of Pde5a1 in compromised muscle and identified an involvement of MyoD and Runx1 in Pde5a1 transcriptional activity.

Disease Reactivation after Fingolimod Discontinuation in Pregnant Multiple Sclerosis Patients.

Recent studies estimated an incidence of 4-25% of disease rebound after withdrawal of fingolimod (FTY) for any reason, but specific data on disease reactivation after FTY withdrawal due to pregnancy are limited. The aim of the study was to evaluate the frequency and predictors of disease reactivation in patients who stopped FTY for pregnancy. A multicentre retrospective cohort study was conducted in four Italian MS centres in 2013-2019. Both planned and unplanned pregnancies were included. The annualized relapse rate (ARR) was calculated before FTY treatment, during FTY treatment, during pregnancy and during the year after delivery. In total, 27 patients (mean age 29 years) were included. The ARR 1 year before FTY treatment was 1.3. Patients were exposed to FTY for a median of 2.9 years. The ARR was 0.04 during the last year before conception (p < 0.001 compared with the ARR before FTY treatment). Eleven patients became pregnant after a mean of 88 days following FTY discontinuation, whereas 16 patients stopped FTY after pregnancy confirmation. Relapses were observed in 22% of patients during pregnancy and in 44% in the postpartum period. ARR increased both during pregnancy (0.49; p = 0.027) and in the first year after delivery (0.67; p < 0.001) compared to the last year before pregnancy. Compared with radiological assessment before pregnancy, more patients showed new or enlarging T2 lesions (63% vs 30%; p = 0.02) and gadolinium-enhancing lesions (44% vs 0; p = 0.0001) on brain Magnetic Resonance Imaging. Relapses during pregnancy were the only significant predictor for postpartum relapses (OR 1.9, 95% CI 1.11-3.1). One case of spontaneous abortion and no cases of abnormal foetal development were observed. Despite adequate and prolonged control of disease activity, women who discontinue FTY because of pregnancy are at risk for disease reactivation. In patients who relapsed during pregnancy, the initiation of high-efficacy disease modifying drugs (DMDs) soon after delivery is advisable to prevent postpartum relapses.

Anti-cN1A Antibodies Are Associated with More Severe Dysphagia in Sporadic Inclusion Body Myositis.

In recent years, an autoantibody directed against the 5'-citosolic nucleotidase1A (cN1A) was identified in the sera of sporadic inclusion body myositis (s-IBM) patients with widely variable sensitivity (33%-76%) and specificity (87%-100%). We assessed the sensitivity/specificity of anti-cN1A antibodies in an Italian cohort of s-IBM patients, searching for a potential correlation with clinical data. We collected clinical data and sera from 62 consecutive s-IBM patients and 62 other inflammatory myopathies patients. Testing for anti-cN1A antibodies was performed using a commercial ELISA. Anti-cN1A antibodies were detected in 23 s-IBM patients, resulting in a sensitivity of 37.1% with a specificity of 96.8%. Positive and negative predictive values were 92.0% and 60.6%, respectively. We did not find significant difference regarding demographic variables, nor quadriceps or finger flexor weakness. Nevertheless, we found that anti-cN1A-positive patients presented significantly lower scores in IBMFRS item 1 (swallowing, p = 0.045) and more frequently reported more severe swallowing problems, expressed as an IBMFRS item 1 score ≤ 2 (p < 0.001). We confirmed the low sensitivity and high specificity of anti-cN1A Ab in s-IBM patients with a high positive predictive value. The presence of anti-CN1A antibodies identified patients with a greater risk of more severe dysphagia.

Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line.

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy.

Pathways Implicated in Tadalafil Amelioration of Duchenne Muscular Dystrophy.

Numerous therapeutic approaches for Duchenne and Becker Muscular Dystrophy (DMD and BMD), the most common X-linked muscle degenerative disease, have been proposed. So far, the only one showing a clear beneficial effect is the use of corticosteroids. Recent evidence indicates an improvement of dystrophic cardiac and skeletal muscles in the presence of sustained cGMP levels secondary to a blocking of their degradation by phosphodiesterase five (PDE5). Due to these data, we performed a study to investigate the effect of the specific PDE5 inhibitor, tadalafil, on dystrophic skeletal muscle function. Chronic pharmacological treatment with tadalafil has been carried out in mdx mice. Behavioral and physiological tests, as well as histological and biochemical analyses, confirmed the efficacy of the therapy. We then performed a microarray-based genomic analysis to assess the pattern of gene expression in muscle samples obtained from the different cohorts of animals treated with tadalafil. This scrutiny allowed us to identify several classes of modulated genes. Our results show that PDE5 inhibition can ameliorate dystrophy by acting at different levels. Tadalafil can lead to (1) increased lipid metabolism; (2) a switch towards slow oxidative fibers driven by the up-regulation of PGC-1α; (3) an increased protein synthesis efficiency; (4) a better actin network organization at Z-disk.

Inflammation in muscular dystrophy and the beneficial effects of non-steroidal anti-inflammatory drugs.

INTRODUCTION: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular dystrophy (DMD), but it is unclear whether their efficacy is dependent on their anti-inflammatory activity. METHODS: To address this issue, mdx mice were treated daily with methylprednisolone and non-steroidal anti-inflammatory drugs (NSAIDs: aspirin, ibuprofen, parecoxib). RESULTS: NSAID treatment was effective in ameliorating muscle morphology and reducing macrophage infiltration and necrosis. The percentage of regenerating myofibers was not modified by the treatments. The drugs were effective in reducing COX-2 expression and inflammatory cytokines, but they did not affect utrophin levels. The effects of the treatments on contractile performance were analyzed. Isometric tension did not differ in treated and untreated muscle, but the resistance to fatigue was decreased by treatment with methylprednisolone and aspirin. CONCLUSIONS: NSAIDs have a beneficial effect on mdx muscle morphology, pointing to a crucial role of inflammation in the progression of DMD.

ß1-syntrophin modulation by miR-222 in mdx mice.

BACKGROUND: In mdx mice, the absence of dystrophin leads to the deficiency of other components of the dystrophin-glycoprotein complex (DAPC), making skeletal muscle fibers more susceptible to necrosis. The mechanisms involved in the disappearance of the DAPC are not completely understood. The muscles of mdx mice express normal amounts of mRNA for the DAPC components, thus suggesting post-transcriptional regulation. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the hypothesis that DAPC reduction could be associated with the microRNA system. Among the possible microRNAs (miRs) found to be upregulated in the skeletal muscle tissue of mdx compared to wt mice, we demonstrated that miR-222 specifically binds to the 3'-UTR of ß1-syntrophin and participates in the downregulation of ß1-syntrophin. In addition, we documented an altered regulation of the 3'-UTR of ß1-syntrophin in muscle tissue from dystrophic mice. CONCLUSION/SIGNIFICANCE: These results show the importance of the microRNA system in the regulation of DAPC components in dystrophic muscle, and suggest a potential role of miRs in the pathophysiology of dystrophy.

Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference.

RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression-based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression-based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.

V1a vasopressin receptor expression is modulated during myogenic differentiation.

Neurohypophyseal peptides potently stimulate myogenic differentiation by acting through different receptors of the same family. Here, we show that L6C5 myogenic cells express, at a high density, a single class of V1a Arg8-vasopressin (AVP) receptor. The expression of the vasopressin receptor of type 1a (V1aR) is significantly higher in proliferating myoblasts than in differentiated myotubes. The differentiation-related decrease of V1aR expression was evident both at the mRNA and at the protein level as shown by the reduction of [(3)H]-AVP binding. However, in L6C5 cells transfected with a synthetic construct containing the luciferase gene driven by the 2 kb upstream region of V1aR, we observed a stimulation of the activity of the promoter when the cells were cultured in differentiative medium. The down-regulation of the V1aR correlated with a decreased half-life of its mRNA (half-life 5.86+/-0.74 hr in 10% fetal bovine serum [FBS] versus 3.53+/-0.72 hr in 1% FBS). Cyclosporine A and dexamethasone, but not 5'-azacytidine, treatments of cells in differentiation medium restored the V1aR level to that measured in proliferating L6C5 cells, thus confirming the role of post-transcriptional mechanisms in the modulation of V1aR expression. Taken together, these data show that mRNA stability plays a role in modulating protein expression during the myogenic differentiation process.