[show abstract][hide abstract] ABSTRACT: Necrotizing autoimmune myopathies (NAM) have recently been defined as a distinct group of severe acquired myopathies, characterized by prominent myofiber necrosis without significant muscle inflammation. Because of the lack of appropriate biomarkers, these diseases have been long misdiagnosed as atypical forms of myositis. NAM may be associated to autoantibodies directed against signal recognition particle (SRP) or 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). The objective of this work was to quantify anti-HMGCR autoantibodies in patients with suspicion of NAM through the development of a new addressable laser bead immunoassay (ALBIA).
Recombinant HMGCR C-domain was bound to fluorescent beads. After incubation with serum, autoantibodies were revealed using class- or subclass-specific anti-human immunoglobulin G (IgG) antibodies. Anti-HMGCR levels were assayed in 150 patients with suspicion of NAM, 142 controls with different inflammatory/autoimmune diseases and 100 healthy donors. Inhibition with free recombinant HMGCR and immunoprecipitation experiments confirmed test specificity. Reproducibility and repeatability were determined from sera with various levels of anti-HMGCR autoantibodies. A multiplex assay (ALBIA-NAM) was also developed to permit the simultaneous quantification of anti-HMGCR and anti-signal recognition particle autoantibodies.
No controls scored positive. Of 150 patients with suspicion of NAM, 24% were positive for anti-HMGCR autoantibodies with levels ranging from 24 to 2,656 AU/mL. Anti-HMGCR positivity could be associated to a cytoplasmic pattern in immunofluorescence assay on HEp-2 cells. Anti-HMGCR-positive patients had high creatine kinase (CK) levels (mean 6,630 IU/L) and only 40% of them had been exposed to statins. Multiplex ALBIA-NAM was equally as effective as monoplex anti-HMGCR and anti-SRP ALBIA.
Both monoplex ALBIA-HMGCR and multiplex ALBIA-NAM reliably detect and quantify anti-HMGCR autoantibodies. A positive result allows ascribing patients with a necrotizing myopathy to an autoimmune form. Anti-HMGCR autoantibodies may be found in patients who have not taken statins.
Arthritis research & therapy 02/2014; 16(1):R39. · 4.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fecal incontinence (FI) remains a socially isolating condition with profound impact on quality of life for which autologous myoblast cell therapy represents an attractive treatment option. We developed an animal model of FI and investigated the possibility of improving sphincter function by intra-sphincteric injection of syngeneic myoblasts. Several types of anal cryoinjuries were evaluated on anesthetized Fischer rats receiving analgesics. The minimal lesion yielding sustainable anal sphincter deficiency was a 90° cryoinjury of the sphincter, repeated after a 24h interval. Anal sphincter pressure was evaluated longitudinally by anorectalmanometry under local electro-stimulation. Myoblasts were prepared using a protocol mimicking a clinical-grade process and further transduced with a GFP-encoding lentiviral vector before intra-sphincteric injection. Experimental groups were: uninjured controls; cryoinjured + PBS; and cryoinjured + myoblasts (different doses or injection site). Myoblast injection was well tolerated. Transferred myoblasts expressing GFP integrated into the sphincter and differentiated in situinto dystrophin-positive mature myofibers. Posttreatment sphincter pressures increased overtime.At D60, pressures in the treated group were significantly higher than those ofPBS-injected controls and notsignificantly different from those of normal rats. Longitudinal follow-up showed stability of the therapeutic effect on sphincter function over a period of 6 months. Intra-sphincteric myoblast injections at the lesion borders were equally as effective as intra-lesion administration, but an injection opposite to the lesion was not. These results provide proof of principle for myoblast cell therapy in fecal incontinence in a rat model. This strategy is currently being evaluated in humans in a randomized double-blind placebo-controlled clinical trial.
[show abstract][hide abstract] ABSTRACT: Immune-mediated necrotizing myopathy (IMNM) associated with statin use and anti 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibody is a new and emerging entity that supports a link between statin use and IMNM and raises the questions of distinct clinical phenotypes and treatment strategy. We describe the clinical and histopathological characteristics of a patient and discuss the spectrum of IMNM and statin-induced myopathies. A 65-year-old man was suffering from proximal muscle weakness and elevated CK levels, following exposure to statin therapy. The symptoms worsened despite discontinuation of the drug. At that point, no myositis-specific or -associated antibodies were detected. Malignancy screening did not reveal abnormalities. Muscle biopsy demonstrated a predominantly necrotizing myopathy with minimal lymphocytic infiltrates, MHC class I expression in necrotic muscle fibers, and complement deposition on scattered non-necrotic muscle fibers. Muscle protein analysis by western blot was normal. The patient did not improve with steroid and methotrexate and required monthly intravenous immunoglobulin (IVIG) therapy. Muscle strength gradually improved, CK levels normalized and IVIG were stopped 1year later. Screening for anti-HMGCR antibodies, not available at the time of presentation, was highly positive. Identification of anti-HMGCR antibodies in statin-exposed patients with myopathy appears to be helpful both for differential diagnosis and for treatment strategy. In patients who did not improve after discontinuation of the statin treatment, a muscle biopsy should be performed as well as screening for anti-HMGCR antibodies. Patients with this disorder require aggressive immunosuppressive treatment.
[show abstract][hide abstract] ABSTRACT: Genetic alterations have recently been described as emerging during the culture of embryonic stem cells or induced pluripotent stem cells, raising concerns about their safety in future clinical use. Myoblasts are adult stem cells with important therapeutic potential that have been used in clinical trials for almost twenty years but their genome integrity has not yet been established. Here, we produced 10 human myoblast preparations and investigated their genomic stability. At the third passage, half of the preparations had a normal karyotype and half showed 1-4 alterations/30 metaphases. Chromosome 2 trisomy was found in 1-2/30 metaphases and/or 2/100 nuclei by FISH in 3/10 samples, and there was no other recurrent anomaly. When prolonging cultures, these erratic abnormalities were never associated with a growth advantage. Cellular senescence was manifested in all samples by growth arrest before passage 15. Expression of hTERT was always negative. Molecular analysis of individual p53 transcripts did not reveal tumorigenic mutations. CGH array (10 samples) and exome sequencing (1 sample) failed to detect copy number variations or accumulation of mutations, respectively. Myoblasts did not grow either in soft agar or in vivo after injection in immuno-deficient mice. Hence, occasional genomic abnormalities may occur during myoblast culture but are not associated with risk of transformation.
[show abstract][hide abstract] ABSTRACT: Muscle fibers do not normally express major histocompatibility complex class I (MHC-I) molecules, and their reexpression is a hallmark of inflammatory myopathies. It has been shown in mice that overexpression of MHC-I induces a poorly inflammatory myositis accompanied by the unfolded protein response (UPR), but it is unclear whether it is attributable to T-cell-mediated MHC-I-dependent immune responses or to MHC-I forced expression per se. Indeed, besides presenting antigenic peptides to CD8(+) T cells, MHC-I may also possibly exert nonimmunologic, yet poorly understood pathogenic effects. Thus, we investigated the pathogenicity of MHC-I expression in muscle independently of its immune functions. HT transgenic mice that conditionally overexpress H-2K(b) in muscle were bred to an immunodeficient Rag2(-/-) background. The muscle proteome was analyzed by label-free high-resolution protein quantitation and Western blot. Despite the absence of adaptive immunity, HT Rag2(-/-) mice developed a very severe myopathy associated with the cytoplasmic accumulation of H-2K(b) molecules. The UPR was manifest by up-regulation of characteristic proteins. In humans, we found that HLA class I molecules not only were expressed at the sarcolemma but also could accumulate intracellularly in some patients with inclusion body myositis. Accordingly, the UPR was triggered as a function of the degree of HLA accumulation in myofibers. Hence, reexpression of MHC-I in normally negative myofibers exerts pathogenic effects independently of its immune function.
American Journal Of Pathology 07/2013; · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Spinal cord injury (SCI) induces a permanent disability in patients. To this day no curative treatment can be proposed to restore lost functions. Therefore, extensive experimental studies have been conducted to induce recovery after SCI. One of the most promising therapies is based on the use of olfactory ensheathing cells (OECs). OECs can be obtained from either the olfactory bulbs (OB-OECs) or from olfactory mucosa (OM-OECs), involving a less invasive approach for autotransplantation. However the vast majority of experimental transplantations have been focusing on OB-OECs although the OM represents a more accessible source of OECs. Importantly, the ability of OM-OECs in comparison to OB-OECs to induce spinal cord recovery in the same lesion paradigm has never been described. We here present data using a multiparametric approach, based on electrophysiological, behavioral, histological and magnetic resonance imaging experiments on the repair potential of OB-OECs and OM-OECs from either primary or purified cultures after a severe model of SCI. Our data demonstrate that transplantation of OECs obtained from OB or OM induces electrophysiological and functional recovery, reduces astrocyte reactivity and glial scar formation and improves axonal regrowth. We also show that the purification step is essential for OM-OECs while not required for OB-OECs. Altogether, our study strongly indicates that transplantation of OECs from OM represents the best benefit/risk ratio according to the safety of access of OM and the results induced by transplantations of OM-OECs. Indeed, purified OM-OECs in addition to induce recovery can integrate and survive up to 60 days into the spinal cord. Therefore, our results provide strong support for these cells as a viable therapy for SCI.
PLoS ONE 01/2013; 8(4):e62860. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: La myopathie nécrosante auto-immune (MNAI) associée à l’utilisation des statines et à la présence d’anticorps anti-3-hydroxy-3-méthylglutaryl-coenzyme A réductase (HMGCR) est une entité nouvelle et émergeante qui renforce le lien entre l’exposition aux statines et la survenue de MNAI et soulève la question d’éventuels phénotypes cliniques et de stratégies thérapeutiques distincts. Nous décrivons les caractéristiques cliniques et histologiques d’un patient et discutons le spectre des MNAI et des myopathies induites par les statines. Un homme âgé de 65 ans avait souffert d’une faiblesse musculaire proximale et d’un taux élevé de CPK, après avoir reçu un traitement par statines. Les symptômes se sont aggravés malgré l’arrêt de ces dernières. À ce moment, aucun anticorps spécifique ou associé aux myosites n’a été détecté. La recherche de néoplasies était négative. La biopsie musculaire avait montré un aspect de myopathie nécrosante avec un infiltrat lymphocytaire minime, une expression du CMH de classe I dans les fibres musculaires nécrosées et des dépôts de complément sur quelques fibres musculaires non nécrosées dispersées. L’analyse des protéines musculaires par Western Blot était normale. Le patient n’était pas amélioré par les corticoïdes et le méthotrexate et avait nécessité des perfusions d’immunoglobulines IV (IGIV). La force musculaire s’est améliorée progressivement, le taux des CPK s’est normalisé et les IGIV étaient arrêtés un an plus tard. La recherche d’anticorps anti-HMGCR, non disponible au moment de la présentation clinique, était fortement positive. L’identification des anticorps anti-HMGCR chez les patients sous statines présentant une myopathie semble intéressante afin d’aider au diagnostic différentiel et de guider le traitement. Chez les patients ne s’améliorant pas à l’arrêt du traitement par statines, une biopsie musculaire doit être réalisée ainsi que la recherche d’anticorps anti-HMGCR. Les patients présentant ces anomalies nécessitent des traitements immunosuppresseurs agressifs.
[show abstract][hide abstract] ABSTRACT: We describe the effectiveness of combinatorial therapy with plasma exchanges and methylprednisolone pulses followed by intravenous cyclophosphamide in a young girl with anti-signal recognition particle 54 (SRP54) antibody-associated myopathy. We also use a newly described quantitative assay to demonstrate the close association between the titers of anti-SRP54 antibodies and disease activity. This is the first report of a pediatric patient indicating that the serum levels of anti-SRP54 antibodies are also beneficial for monitoring the disease activity of progressive necrotizing myopathy.
[show abstract][hide abstract] ABSTRACT: To investigate the influence of myoinjury on antigen presentation to T cells in draining lymph nodes (LNs).
Muscle crush was performed in mice injected with exogenous ovalbumin (OVA) and in transgenic SM-OVA mice expressing OVA as a muscle-specific self antigen. Antigen exposure and the resulting stimulation of T cell proliferation in draining LNs was assessed by transferring carboxyfluorescein succinimidyl ester (CFSE)-labeled OVA-specific CD8+ and CD4+ T cells from OT-I and OT-II mice and by measuring the dilution of CFSE, which directly reflects their proliferation. The role of monocyte-derived dendritic cells (DCs) in T cell priming was assessed using pharmacologic blockade of DC migration. Immunofluorescence was used to detect CD8+ T cells, inflammatory monocyte-derived DCs, and type I major histocompatibility complex (MHC)-expressing myofibers in crushed muscle, and to assess expression of perforin, interferon-γ (IFNγ), interleukin-2 (IL-2), IL-10, and transforming growth factor β1 (TGFβ1).
OVA injection into intact muscle induced strong proliferation of CD4+ and CD8+ T cells, indicating efficient exposure of soluble antigens in draining LNs. OVA-specific CD8+ T cell proliferation in draining LNs of SM-OVA mice required myoinjury and was unaffected by pharmacologic inhibition of monocyte-derived DC migration. On day 7 postinjury, activated CD8+ T cells expressing perforin, IFNγ and IL-2 were transiently detected in crushed muscle, and these cells were in close contact with class I MHC-positive regenerating myofibers. Beginning on day 7, the immunosuppressive cytokines IL-10 and TGFβ1 were conspicuously expressed by CD11b+ cells, and CD8+ T cells rapidly disappeared from the healing muscle.
Myofiber damage induces an episode of muscle antigen-specific CD8+ T cell proliferation in draining LNs. Activated CD8+ T cells transiently infiltrate the injured muscle, with prompt control by immunosuppressive cues. Inadequate control might favor sustained autoimmune myositis.
[show abstract][hide abstract] ABSTRACT: Endogenous danger signals released during cell damage contribute to alert the immune system. Typically, their release results in the activation and maturation of innate immune cells, and the production of pro-inflammatory cytokines. In addition, extracellular NAD(+) stimulates immune responses by hindering regulatory T cells (Tregs), and could, therefore, represent the prototype of a new category of danger signals.
Microbes and Infection 05/2012; · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.
PLoS ONE 01/2012; 7(7):e41269. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Muscle potentially represents the most abundant source of autoantigens of the body and can be targeted by a variety of severe autoimmune diseases. Yet, the mechanisms of immunological tolerance toward muscle autoantigens remain mostly unknown. We investigated this issue in transgenic SM-Ova mice that express an ovalbumin (Ova) neo-autoantigen specifically in skeletal muscle. We previously reported that antigen specific CD4(+) T cell are immunologically ignorant to endogenous Ova in this model but can be stimulated upon immunization. In contrast, Ova-specific CD8(+) T cells were suspected to be either unresponsive to Ova challenge or functionally defective. We now extend our investigations on the mechanisms governing CD8(+) tolerance in SM-Ova mice. We show herein that Ova-specific CD8(+) T cells are not detected upon challenge with strongly immunogenic Ova vaccines even after depletion of regulatory T cells. Ova-specific CD8(+) T cells from OT-I mice adoptively transferred to SM-Ova mice started to proliferate in vivo, acquired CD69 and PD-1 but subsequently down-regulated Bcl-2 and disappeared from the periphery, suggesting a mechanism of peripheral deletion. Peripheral deletion of endogenous Ova-specific cells was formally demonstrated in chimeric SM-Ova mice engrafted with bone marrow cells containing T cell precursors from OT-I TCR-transgenic mice. Thus, the present findings demonstrate that immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8(+) T cells.
PLoS ONE 01/2012; 7(5):e36444. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vascular rejection after organ transplantation is characterized by an arterial occlusive lesion, resulting from intimal proliferation occurring in response to arterial wall immune aggression. Our hypothesis is that an early endothelial repair may prevent vascular graft rejection. The aim of the current study was to compare different pharmacologic progenitor cell mobilizing treatments for their protective effects against vascular rejection.
Aortic transplants were made from balb/c donor to C57Bl/6 recipient mice. Three different mobilizing pharmacologic agents were used: low molecular weight fucoidan (LMWF), simvastatin, and AMD3100. The circulating levels of progenitor cells were found to be increased by all three treatments, as determined by flow cytometry. For each treatment, the design was: treated allografts, nontreated allografts, treated isografts, and nontreated isografts. After 21 d, morphometric and immunohistochemical analyses were performed. We found that the three treatments significantly reduced intimal proliferation, compared with nontreated allografts. This was associated with intimal re-endothelialization of the grafts. Further, in chimeric mice that had previously received GFP-transgenic bone marrow transplantation, GFP-positive cells were found in the vascular allograft intima, indicating that re-endothelialization was, at least partly, due to the recruitment of bone marrow-derived, presumably endothelial progenitor circulating cells.
In this aortic allograft model, three different mobilizing treatments were found to partially prevent vascular transplant rejection. Bone marrow-derived progenitor cells mobilized by the three treatments may play a direct role in the endothelial repair process and in the suppression of intimal proliferation.
Journal of Surgical Research 12/2011; 176(2):657-65. · 2.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Olfactory ensheathing cells (OECs) play a crucial role during neurogenesis of primary olfactory neurons. Transplantation of OECs is considered as a promising new therapy for central nervous system repair. Nevertheless, OECs are constituted of distinct subpopulations and their role during neurogenesis is not clearly understood. In particular, OECs from the olfactory bulb (OB) constitute a heterogeneous, but not yet isolated and characterized, population of cells. In our study, flow cytometry analyses of primary OB cultures, based on cell surface expression of low-affinity nerve growth factor receptor (p75), reveal the presence of two distinct populations of OECs. Indeed, some of them express a high level of p75 (P75High) and the other a low level of p75 (P75Low). Effects of OB microenvironment were assessed, and we were able to show that fibroblasts mediate the induction of these two populations through the secretion of soluble factors. To characterize P75High and P75Low OECs, cells were sorted based on their differential expression of p75. Microarray analyses revealed that P75High OECs overexpress genes implicated in modulation of extracellular matrix and cell sorting, whereas P75Low OECs overexpress genes involved in regulation of the inflammatory response and axonal guidance. These results permit, for the first time, to isolate the two distinct subpopulations of OECs from OB, and suggest their specific role during neurogenesis.
[show abstract][hide abstract] ABSTRACT: Previous studies have shown that the glucose supply reduces postoperative insulin resistance and improves patient outcomes. However, the effects of luminal glucose on intestinal mucosal proteins remain unknown.
We aimed to assess the effects of an enteral glucose supply on protein synthesis, proteolytic pathways, and proteome in human duodenal mucosa.
Twenty healthy volunteers received a 5-h enteral infusion of either saline or glucose (0.12 g · kg(-1) · h(-1)). Simultaneously, a continuous intravenous infusion of l-[1-(13)C]leucine (12 μmol · kg(-1) · h(-1)) was maintained until endoscopy. The duodenal mucosal protein fractional synthesis rate (FSR) was calculated from leucine enrichments assessed in protein and free amino acid pools by gas chromatography-mass spectrometry. Cathepsin D, calpains, and chymotrypsin-like proteasome mucosal activities were evaluated by using specific fluorogenic substrates. A 2-dimensional PAGE-based comparative proteomics analysis was also performed on additional duodenal mucosal biopsy samples to identify differentially expressed proteins.
Duodenal mucosal protein FSR and protease activities were not affected by glucose infusion relative to saline. Nevertheless, the comparative proteomics analysis indicated that 10 protein spots were significantly differentially expressed (ie, at least ±1.5-fold modulated; Student's t test, P < 0.05) in response to the glucose infusion relative to saline. Of the 8 proteins identified by mass spectrometry, α-enolase, cytoplasmic aconitate hydratase, and glutathione S-transferase ω-1 were upregulated, whereas epoxide hydrolase 2 was downregulated.
Enteral glucose supply affected neither duodenal mucosal protein FSR nor activities of mucosal proteases but altered the duodenal mucosal proteome by modulating the expression of several enzymes involved mainly in carbohydrate and xenobiotic metabolism. This trial is registered at clinicaltrials.gov as NCT00213551.
American Journal of Clinical Nutrition 09/2011; 94(3):784-94. · 6.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: To analyse the therapeutic effects of etanercept (ETA) or adalimumab (ADA) on the numbers and phenotypes of CD4+CD25hi Tregs in RA patients.
RA patients received ADA (n = 28) or ETA (n = 20) and stable-dose MTX or LEF. Therapeutic responses were assessed with the 28-joint DAS (DAS-28) criteria after 12 weeks of treatment. Treg numbers and phenotypes, determined by flow cytometry using different gating strategies, were compared between responders and non-responders before and after 6 and 12 weeks of treatment.
The percentages of good, moderate and non-responders among patients given ADA or ETA, respectively, were 46.5, 35.7 and 17.8% or 30, 20 and 50%, with respective mean (s.d.) pre-treatment CD4+CD25hi Treg percentages of 5.5 (0.04)% or 4.95 (0.02)%. Overall, for patients with active RA given ADA or ETA, neither TNF-α-blocking agent had an effect on Tregs percentage and absolute number. Moreover, CD4+CD25hi Treg counts remained unaffected in RA responders to ADA or ETA, compared with RA non-responders. Furthermore, the CD4+CD25hiCD45RA+, CD4+CD25hiCD45RO+ and CD4+CD25hiCD62L+ cell populations were unchanged by TNF-α-blocking agents.
Neither ADA nor ETA modified the percentages or absolute numbers of circulating CD4+CD25hi Tregs and their phenotypes after being administered for 6 and 12 weeks to RA patients. Trial Registration: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00234234.