Publications by authors named "Olivier Boyer"

110 Publications

RITUXIMAB AND CORTICOSTEROID EFFECT ON DESMOGLEIN-SPECIFIC B-CELLS AND DESMOGLEIN-SPECIFIC T-FOLLICULAR-HELPER-CELLS IN PEMPHIGUS.

J Invest Dermatol 2021 Mar 22. Epub 2021 Mar 22.

Department of Dermatology, Rouen University Hospital, Centre de référence des maladies bulleuses auto-immunes, Normandie University, Rouen, France; Normandie University, UNIROUEN, Inserm U1234, Rouen, France.

Pemphigus is an autoimmune blistering disease mediated by autoantibodies directed against desmogleins. We recently showed that first-line treatment with rituximab enables more patients to achieve long-lasting remission off therapy than corticosteroids alone. To understand the immunological mechanisms that mediate the long-lasting clinical remission after rituximab treatment, we analyzed the phenotype of desmoglein-specific memory B cells and desmoglein-specific T-follicular-helper cells by flow cytometry, and measured antibody-secreting-cells by ELISPOT in patients treated with corticosteroids alone or rituximab. This post hoc analysis of the RITUX3 trial showed that rituximab induced a significant decrease of IgG-switched desmoglein-specific memory B cells. Accordingly, anti-desmoglein antibody-secreting-cells were no longer detected in patients in complete remission after rituximab. In contrast, corticosteroids did not modify the frequency or the phenotype of desmoglein-specific memory B cells, and anti-desmoglein antibody-secreting-cells were still detected after treatment, even in patients in remission. Using peptide-HLADRB1*0402 tetramer staining, we identified desmoglein-3-specific T-follicular-helper cells, which dramatically decreased after rituximab, while remaining stable after corticosteroid treatment. Our findings suggest that the long-lasting response to rituximab in pemphigus relies on the decrease of desmoglein-specific circulating T-follicular-helper cells, which correlates with a sustained depletion of IgG-switched memory autoreactive B cells, leading to the disappearance of anti-desmoglein antibody-secreting-cells.
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http://dx.doi.org/10.1016/j.jid.2021.01.031DOI Listing
March 2021

TRIM33 gene somatic mutations identified by next generation sequencing in neoplasms of patients with anti-TIF1γ positive cancer-associated dermatomyositis.

Rheumatology (Oxford) 2021 Mar 25. Epub 2021 Mar 25.

Normandie University, UNIROUEN, IRIB, Inserm, U1234, Rouen, France.

Objective: To deep sequence the TRIM33 gene in tumours from patients with cancer-associated anti-TIF1γ autoantibody-positive dermatomyositis (DM) since TRIM33 somatic mutations in tumours may trigger this auto-immune disease.

Methods: Next generation sequencing of tumour DNA samples from patients with cancer-associated anti-TIF1γ autoantibody-positive DM. Fourteen tumours from 13 anti-TIF1γ autoantibody-positive DM individuals were sequenced along with 2 control tumours from non-DM individuals.

Results: Fourteen probable somatic variants from 4 tumours were identified in the TRIM33 gene.

Conclusion: These results are in accordance with the previous report of Pinal-Fernandez et al. and support the hypothesis of a role of TRIM33 gene mutations in the pathophysiology of anti-TIF1γ autoantibody-positive DM.
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http://dx.doi.org/10.1093/rheumatology/keab260DOI Listing
March 2021

Evaluation of Humoral Immunity to SARS-CoV-2: Diagnostic Value of a New Multiplex Addressable Laser Bead Immunoassay.

Front Microbiol 2020 26;11:603931. Epub 2020 Nov 26.

Normandie University, UNIROUEN, INSERM, U1234, Rouen, France.

Despite efforts to develop anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody (Ab) immunoassays, reliable serological methods are still needed. We developed a multiplex addressable laser bead immunoassay (ALBIA) to detect and quantify anti-Spike S1 and nucleocapsid N Abs. Recombinant S1 and N proteins were bound to fluorescent beads (ALBIA-IgG-S1/N). Abs were revealed using class-specific anti-human Ig Abs. The performances of the test were analyzed on 575 serum samples including 192 from SARS-CoV-2 polymerase chain reaction-confirmed patients, 13 from seasonal coronaviruses, 70 from different inflammatory/autoimmune diseases, and 300 from healthy donors. Anti-S1 IgM were detected by monoplex ALBIA-IgM-S1. Comparison with chemiluminescent assays or enzyme-linked immunosorbent assays was performed using commercial tests. Multiplex ALBIA-IgG-S1/N was effective in detecting and quantifying anti-SARS-CoV-2 IgG Abs. Two weeks after first symptoms, sensitivity and specificity were 97.7 and 98.0% (anti-S1), and 100 and 98.7% (anti-N), respectively. Agreement with commercial tests was good to excellent, with a higher sensitivity of ALBIA. ALBIA-IgG-S1/N was positive in 53% of patients up to day 7, and in 75% between days 7 and 13. For ALBIA-IgM-S1, sensitivity and specificity were 74.4 and 98.7%, respectively. Patients in intensive care units had higher IgG Ab levels (Mann-Whitney test, < 0.05). ALBIA provides a robust method for exploring humoral immunity to SARS-CoV-2. Serology should be performed after 2 weeks following first symptoms, when all COVID-19 (coronavirus disease 2019) patients had at least one anti-S1 or anti-N IgG Ab, illustrating the interest of a multiplex test.
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http://dx.doi.org/10.3389/fmicb.2020.603931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726470PMC
November 2020

Syngeneic Transplantation of Rat Olfactory Stem Cells in a Vein Conduit Improves Facial Movements and Reduces Synkinesis after Facial Nerve Injury.

Plast Reconstr Surg 2020 12;146(6):1295-1305

From the Institute for Research and Innovation in Biomedicine and the Department of Immunology and Biotherapy, Normandie University; the Department of Otorhinolaryngology and Head Neck Surgery, Rouen University Hospital; the Department Otorhinolaryngology and Head Neck Surgery, Conception University Hospital; Aix Marseille University, INP, CNRS UMR 7051; and APHM, Culture and Cell Therapy Laboratory.

Background: Posttraumatic facial paralysis is a disabling condition. Current surgical management by faciofacial nerve suture provides limited recovery. To improve the outcome, the authors evaluated an add-on strategy based on a syngeneic transplantation of nasal olfactory stem cells in a rat model of facial nerve injury. The main readouts of the study were the recording of whisking function and buccal synkinesis.

Methods: Sixty rats were allocated to three groups. Animals with a 2-mm facial nerve loss were repaired with a femoral vein, filled or not with olfactory stem cells. These two groups were compared to similarly injured rats but with a faciofacial nerve suture. Olfactory stem cells were purified from rat olfactory mucosa. Three months after surgery, facial motor performance was evaluated using video-based motion analysis and electromyography. Synkinesis was assessed by electromyography, using measure of buccal involuntary movements during blink reflex, and double retrograde labeling of regenerating motoneurons.

Results: The authors' study reveals that olfactory stem cell transplantation induces functional recovery in comparison to nontransplanted and faciofacial nerve suture groups. They significantly increase (1) maximal amplitude of vibrissae protraction and retraction cycles and (2) angular velocity during protraction of vibrissae. They also reduce buccal synkinesis, according to the two techniques used. However, olfactory stem cell transplantation did not improve axonal regrowth of the facial nerve, 3 months after surgery.

Conclusions: The authors show here that the adjuvant strategy of syngeneic transplantation of olfactory stem cells improves functional recovery. These promising results open the way for a phase I clinical trial based on the autologous engraftment of olfactory stem cells in patients with a facial nerve paralysis.
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http://dx.doi.org/10.1097/PRS.0000000000007367DOI Listing
December 2020

Immune-mediated necrotizing myopathy: clinical features and pathogenesis.

Nat Rev Rheumatol 2020 12 22;16(12):689-701. Epub 2020 Oct 22.

Normandie University, UNIROUEN, Inserm U1234, Department of Immunology and Biotherapy, Rouen University Hospital, Rouen, France.

Immune-mediated necrotizing myopathy (IMNM) is a group of inflammatory myopathies that was distinguished from polymyositis in 2004. Most IMNMs are associated with anti-signal recognition particle (anti-SRP) or anti-3-hydroxy-3-methylglutaryl-coA reductase (anti-HMGCR) myositis-specific autoantibodies, although ~20% of patients with IMNM remain seronegative. These associations have led to three subclasses of IMNM: anti-SRP-positive IMNM, anti-HMGCR-positive IMNM and seronegative IMNM. IMNMs are frequently rapidly progressive and severe, displaying high serum creatine kinase levels, and failure to treat IMNMs effectively may lead to severe muscle impairment. In patients with seronegative IMNM, disease can be concomitant with cancer. Research into IMNM pathogenesis has shown that anti-SRP and anti-HMGCR autoantibodies cause weakness and myofibre necrosis in mice, suggesting that, as well as being diagnostic biomarkers of IMNM, they may play a key role in disease pathogenesis. Therapeutically, treatments such as rituximab or intravenous immunoglobulins can now be discussed for IMNM, and targeted therapies, such as anticomplement therapeutics, may be a future option for patients with refractory disease.
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http://dx.doi.org/10.1038/s41584-020-00515-9DOI Listing
December 2020

T cell and antibody responses to SARS-CoV-2: Experience from a French transplantation and hemodialysis center during the COVID-19 pandemic.

Am J Transplant 2021 02 6;21(2):854-863. Epub 2020 Nov 6.

Department of Nephrology, Transplantation and hemodialysis, Rouen University Hospital, Rouen, France.

Immunosuppressed organ-transplanted patients are considered at risk for severe forms of COVID-19. Moreover, exaggerated innate and adaptive immune responses might be involved in severe progression of the disease. However, no data on the immune response to SARS-CoV-2 in transplanted patients are currently available. Here, we report the first assessment of antibody and T cell responses to SARS-CoV-2 in 11 kidney-transplanted patients recovered from RT-PCR-confirmed (n = 5) or initially suspected (n = 6) COVID-19. After reduction of immunosuppressive therapy, RT-PCR-confirmed COVID-19 transplant patients were able to mount vigorous antiviral T cell and antibody responses, as efficiently as two nontherapeutically immunosuppressed COVID-19 patients on hemodialysis. By contrast, six RT-PCR-negative patients displayed no antibody response. Among them, three showed very low numbers of SARS-CoV-2-reactive T cells, whereas no T cell response was detected in the other three, potentially ruling out COVID-19 diagnosis. Low levels of T cell reactivity to SARS-CoV-2 were also detected in seronegative healthy controls without known exposure to the virus. These results suggest that during COVID-19, monitoring both T cell and serological immunity might be helpful for the differential diagnosis of COVID-19 but are also needed to evaluate a potential role of antiviral T cells in the development of severe forms of the disease.
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http://dx.doi.org/10.1111/ajt.16348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675512PMC
February 2021

Lack of association between chilblains outbreak and severe acute respiratory syndrome coronavirus 2: Histologic and serologic findings from a new immunoassay.

J Am Acad Dermatol 2020 11 16;83(5):1434-1436. Epub 2020 Jul 16.

Normandie University, UNIROUEN, Inserm, U1234, FOCIS Center of Excellence PAn'THER, Department of Immunology and Biotherapy, Rouen University Hospital, Rouen, France.

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http://dx.doi.org/10.1016/j.jaad.2020.07.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365061PMC
November 2020

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.

Mol Ther 2020 09 30;28(9):2056-2072. Epub 2020 May 30.

INTEGRARE, Genethon, INSERM, Université d'Evry, Université Paris-Saclay, 91002 Evry, France; Sorbonne Université, Paris, France; Spark Therapeutics, Philadelphia, PA 19103, USA. Electronic address:

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa mouse model via liver gene transfer of secGAA.
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http://dx.doi.org/10.1016/j.ymthe.2020.05.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474269PMC
September 2020

Mapping of proteomic profile and effect of the spongy layer in the human amniotic membrane.

Cell Tissue Bank 2020 Jun 12;21(2):329-338. Epub 2020 Mar 12.

Faculty of Medicine and Pharmacy, University of Rouen Normandy, Inserm U1234, Rouen, France.

The graft of human amniotic membrane (HAM) contributes to the healing of corneal perforating ulcers and so to save a large number of eyes suffering of severe chemical burns. This biological material is used for the treatment of ocular surface diseases because of its capacity to reduce inflammation and promote a quicker wound healing. For clinical use, the HAM is denuded from its spongy layer, but this layer can be an important source of growth factors which promote re-epithelialization. The aim of our study is to provide a general view of protein expression of the HAM and the spongy layer and therefore to determine if the spongy layer and/or a specific part of HAM have a beneficial role in the process of wound healing in patients with corneal ulcers. For this study, human placentas were obtained from healthy women after vaginal delivery or caesarean section after signing the consent form. Mapping of protein expression is done by dividing the placenta in 2 equal parts, one with spongy layer and another without (conventional HAM). Each part is also divided in 3 zones depending on the distance from the umbilical cord. The proteomic analysis was done by ELISA, targeting growth factors (EGF, HGF, KGF, NGF and TGF-beta1) and pro inflammatory cytokine TNF-α in the HAM without spongy layer and in the spongy layer. In this study we observed significant difference in the total amount of protein extract between the different donors. We do not observe a significant difference in the growth factor level between the conventional HAM and the spongy layer. No variation was observed in the expression of HGF, KGF and NGF in different zone of HAM and neither between conventional HAM and spongy layer in each zone. (*p value < 0.05, **p value<0.01,***p value < 0.001). We do detect very low dose of TNF-α and no correlation with the amount of growth factors. In our study we demonstrated that keeping the spongy layer in conventional method of handling HAM can add more GF, and so probably have a positive affect the wound healing process. Variation in some growth factors expression has been observed between the placentas and therefore this may explain the variation in clinical results. No indicator for the selection of placentas with a higher rate of growth factor was found.
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http://dx.doi.org/10.1007/s10561-020-09821-8DOI Listing
June 2020

CD11c B Cells Are Mainly Memory Cells, Precursors of Antibody Secreting Cells in Healthy Donors.

Front Immunol 2020 25;11:32. Epub 2020 Feb 25.

INSERM U1234, Normandy University, Rouen, France.

CD11c B cells have been reported to be increased in autoimmune diseases, but they are detected in the blood of healthy individuals as well. We aimed to characterize CD11c B cells from healthy donors by flow cytometry, microarray analysis, and functional assays. Here, we report that CD11c B cells are a distinct subpopulation of B cells, enriched in the memory subpopulation even if their phenotype is heterogeneous, with overexpression of genes involved in B-cell activation and differentiation as well as in antigen presentation. Upon activation, CD11c B cells can differentiate into antibody-secreting cells, and CD11c could be upregulated in CD11c B cells by B-cell receptor activation. Finally, we show that patients with pemphigus, an autoimmune disease mediated by B cells, have a decreased frequency of CD11c B cell after treatment, relative to baseline. Our findings show that CD11c B cells are mainly memory B cells prone to differentiate into antibody secreting cells that accumulate with age, independently of gender.
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http://dx.doi.org/10.3389/fimmu.2020.00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051942PMC
February 2021

Hyaluronan-based hydrogels as versatile tumor-like models: Tunable ECM and stiffness with genipin-crosslinking.

J Biomed Mater Res A 2020 05 17;108(5):1256-1268. Epub 2020 Feb 17.

Normandie Université, PBS UMR 6270, UFR de Sciences et Techniques, FR3038, UNIROUEN, INSA Rouen, CNRS, Evreux Cedex, France.

Three-dimensional (3D) biomimetic cell culture platforms offer more realistic microenvironments that cells naturally experience in vivo. We developed a tunable hyaluronan-based hydrogels that could easily be modified to mimic healthy or malignant extracellular matrices (ECMs). For that, we pre-functionalized our hydrogels with an adhesive polypeptide (poly-l-lysine, PLL) or ECM proteins (type III and type IV collagens), naturally present in tumorous tissues, and next, we tuned their stiffness by crosslinking with gradual concentrations of genipin (GnP). Then, we thoroughly characterized our substrates before testing them with glioblastoma and breast cancer cells, and thereafter with endothelial cells. Overall, our hydrogels exhibited (a) increasing stiffness with GnP concentration for every pre-functionalization and (b) efficient enzyme resistance with PLL treatment, and also with type IV collagen but to a lesser extent. While PLL-treated hydrogels were not favorable to the culture of any glioblastoma cell lines, they enhanced the proliferation of breast cancer cells in a stiffness-dependent manner. Contrary to type III collagen, type IV collagen pre-treated hydrogels supported the proliferation of glioblastoma cells. The as-desired HA-based 3D tumor-like models we developed may provide a useful platform for the study of various cancer cells by simply tuning their biochemical composition and their mechanical properties.
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http://dx.doi.org/10.1002/jbm.a.36899DOI Listing
May 2020

Modifications of the Transcriptomic Profile of Autoreactive B Cells From Pemphigus Patients After Treatment With Rituximab or a Standard Corticosteroid Regimen.

Front Immunol 2019 7;10:1794. Epub 2019 Aug 7.

INSERM U1234, Normandie University, Rouen, France.

Pemphigus Vulgaris is an autoimmune disease of the skin and mucous membranes, which is due to the production of pathogenic autoantibodies targeting desmoglein (DSG) 1 and 3, which are adhesion proteins of the keratinocytes. Rituximab is an anti-CD20 mAb which induces a prolonged depletion of blood B cells. We recently showed that rituximab was more effective than a standard oral corticosteroid (CS) treatment, allowing 90% of patients to achieve complete remission (CR). Additionally, we showed that DSG-specific-B (DSG positive) cells were still detectable during the B cell recovery which follows the initial rituximab-induced B cell depletion, even in patients in CR. In order to characterize DSG positive B cells in patients in CR after rituximab or CS treatment relative to those detectable at baseline in patients with an active pemphigus, we studied the expression profile of 31 genes of interest related to inflammatory cytokines, TNF receptors and activation markers. Using quantitative Polymerase Chain Reaction performed on one cell with a microfluidic technique, we found that patients' autoreactive B cells collected at baseline had a significantly higher expression of genes encoding for IL-1β, IL-23p19, and IL-12p35 pro-inflammatory cytokines and the IRF5 transcription factor, than non-autoreactive B cells. Surprisingly, the gene expression profile of DSG positive B cells collected after rituximab treatment in patients in CR was close to that of DSG positive B cells at baseline in patients with active pemphigus, except for the IL-1β and the CD27 memory marker genes, which were under-expressed after rituximab compared to baseline. Conversely, we observed a decreased expression of genes encoding for IL-1β and IL-23p19 in patients treated with CS relative to baseline. This study showed that: (i) DSG positive autoreactive B cells have a different gene expression profile than non-autoreactive B cells; (ii) rituximab and CS have different effects on the genes' expression in autoreactive DSG positive B cells from pemphigus patients.
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http://dx.doi.org/10.3389/fimmu.2019.01794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693356PMC
October 2020

HLA-Class II Artificial Antigen Presenting Cells in CD4 T Cell-Based Immunotherapy.

Front Immunol 2019 17;10:1081. Epub 2019 May 17.

Inserm U1237, Physiopathology and Imaging of Neurological Disorders, Caen University Hospital, Caen, France.

CD4 T cells differentiate into various T helper subsets characterized by distinct cytokine secreting profiles that confer them effector functions adapted to a variety of infectious or endogenous threats. Regulatory CD4 T cells are another specialized subset that plays a fundamental role in the maintenance of immune tolerance to self-antigens. Manipulating effector or regulatory CD4 T cells responses is a promising immunotherapy strategy for, respectively, chronical viral infections and cancer, or severe autoimmune diseases and transplantation. Adoptive cell therapy (ACT) is an emerging approach that necessitates defining robust and efficient methods for the expansion of antigen-specific T cells then infused into patients. To address this challenge, artificial antigen presenting cells (AAPCs) have been developed. They constitute a reliable and easily usable platform to stimulate and amplify antigen-specific CD4 T cells. Here, we review the recent advances in understanding the functions of CD4 T cells in immunity and in immune tolerance, and their use for ACT. We also describe the characteristics of different AAPC models and the way to improve their stimulating functions. Finally, we discuss the potential interest of these AAPCs, both as fundamental tools to decipher CD4 T cell responses and as reagents to generate clinical grade antigen-specific CD4 T cells for immunotherapy.
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http://dx.doi.org/10.3389/fimmu.2019.01081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533590PMC
September 2020

The IgG2 Isotype of Anti-Transcription Intermediary Factor 1γ Autoantibodies Is a Biomarker of Cancer and Mortality in Adult Dermatomyositis.

Arthritis Rheumatol 2019 08 8;71(8):1360-1370. Epub 2019 Jul 8.

Normandy University, University of Rouen, INSERM U1234, Rouen University Hospital, Rouen, France.

Objective: Anti-transcription intermediary factor 1γ (anti-TIF1γ) antibodies are the main predictors of cancer in dermatomyositis (DM). Yet, a substantial proportion of anti-TIF1γ-positive DM patients do not develop cancer. This study was undertaken to identify biomarkers to better evaluate the risk of cancer and mortality in DM.

Methods: This multicenter study was conducted in adult anti-TIF1γ-positive DM patients from August 2013 to August 2017. Anti-TIF1γ autoantibody levels and IgG subclasses were identified using a newly developed quantitative immunoassay. Age, sex, DM signs and activity, malignancy, and creatine kinase (CK) level were recorded. Risk factors were determined by univariate and multivariate analysis according to a Cox proportional hazards regression model.

Results: Among the 51 adult patients enrolled (mean ± SD age 61 ± 17 years; ratio of men to women 0.65), 40 (78%) had cancer and 21 (41%) died, with a mean ± SD survival time of 10 ± 6 months. Detection of anti-TIF1γ IgG2 was significantly associated with mortality (P = 0.0011) and occurrence of cancer during follow-up (P < 0.0001), with a 100% positive predictive value for cancer when the mean fluorescence intensity of anti-TIF1γ IgG2 was >385. None of the patients developed cancer after 24 months of follow-up. Univariate survival analyses showed that mortality was also associated with age >60 years (P = 0.0003), active DM (P = 0.0042), cancer (P = 0.0031), male sex (P = 0.011), and CK level >1,084 units/liter (P = 0.005). Multivariate analysis revealed that age >60 years (P = 0.015) and the presence of anti-TIF1γ IgG2 (P = 0.048) were independently associated with mortality.

Conclusion: Our findings indicate that anti-TIF1γ IgG2 is a potential new biomarker of cancer that should be helpful in identifying the risk of mortality in anti-TIF1γ-positive DM patients.
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http://dx.doi.org/10.1002/art.40895DOI Listing
August 2019

Systemic administration of orexin A ameliorates established experimental autoimmune encephalomyelitis by diminishing neuroinflammation.

J Neuroinflammation 2019 Mar 20;16(1):64. Epub 2019 Mar 20.

University of Rouen Normandy, INSERM U1234 PANTHER, Institute for Research and Innovation in Biomedicine (IRIB), Faculté de Médecine et Pharmacie, 22 Boulevard Gambetta, 76183, Rouen, France.

Background: Orexins (hypocretins, Hcrt) A and B are GPCR-binding hypothalamic neuropeptides known to regulate sleep/wake states and feeding behavior. A few studies have shown that orexin A exhibits anti-inflammatory and neuroprotective properties, suggesting that it might provide therapeutic effects in inflammatory and neurodegenerative diseases like multiple sclerosis (MS). In MS, encephalitogenic Th1 and Th17 cells trigger an inflammatory response in the CNS destroying the myelin sheath. Here, we investigated the effects of peripheral orexin A administration to mice undergoing experimental autoimmune encephalomyelitis (EAE), a widely used model of MS.

Methods: Mice were subcutaneously immunized with myelin oligodendrocyte glycoprotein peptide (MOG) in CFA. Mice were treated intraperitoneally for five consecutive days with either PBS or 300 μg of orexin A starting at a moderate EAE score. Molecular, cellular, and histological analysis were performed by real-time PCR, ELISA, flow cytometry, and immunofluorescence.

Results: Orexin A strongly ameliorated ongoing EAE, limiting the infiltration of pathogenic CD4 T lymphocytes, and diminishing chemokine (MCP-1/CCL2 and IP-10/CXCL10) and cytokine (IFN-γ (Th1), IL-17 (Th17), TNF-α, IL-10, and TGF-β) expressions in the CNS. Moreover, orexin A treatment was neuroprotective, decreasing demyelination, astrogliosis, and microglial activation. Despite its strong local therapeutic effects, orexin A did not impair peripheral draining lymph node cell proliferation and Th1/Th17 cytokine production in response to MOG in vitro.

Conclusions: Peripherally-administered orexin A ameliorated EAE by reducing CNS neuroinflammation. These results suggest that orexins may represent new therapeutic candidates that should be further investigated for MS treatment.
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http://dx.doi.org/10.1186/s12974-019-1447-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425555PMC
March 2019

Myositis-specific autoantibodies, a cornerstone in immune-mediated necrotizing myopathy.

Autoimmun Rev 2019 Mar 11;18(3):223-230. Epub 2019 Jan 11.

Department of Internal Medicine and Clinical Immunology, Sorbonne Université, Pitié-Salpêtrière University Hospital, Paris, France; Institut National de la Santé et de la Recherche Médicale, Association Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France. Electronic address:

Over the past few years, myositis-specific autoantibodies played an increasing role in the inflammatory idiopathic myositis definition. They became the critical immunological marker for immune-mediated necrotizing myopathy diagnosis (IMNM) since the paradigm switch from histological to serological criteria. This review is focused on the key role of the anti-signal recognition particle (anti-SRP) and the anti-3-Hydroxy-3-MethylGlutaryl-Coenzyme A Reductase (anti-HMGCR) antibodies in immune-mediated necrotizing myopathy. Anti-SRP and anti-HMGCR antibodies are robust diagnostic tools in case of both the classical subacute form and the slowly progressive form of IMNM that may mimic muscular dystrophy. Anti-SRP and anti-HMGCR patients share clinical, biological and histological features with some antibody-associated specificity. Anti-SRP patients harbour more severe muscle weakness and atrophy with severe muscle damage on magnetic resonance imaging study. Approximately 10-20% of anti-SRP patients develop extramuscular symptoms, especially lung interstitial disease. Conversely, anti-HMGCR patients are often associated with statin exposure. In both cases, patients have a poor outcome with frequent relapse and the use of combined immunotherapy. Of note, various data suggest a direct pathogenic role of these antibodies reinforcing the interest in targeted therapeutic strategy.
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http://dx.doi.org/10.1016/j.autrev.2018.09.008DOI Listing
March 2019

pathogenicity of IgG from patients with anti-SRP or anti-HMGCR autoantibodies in immune-mediated necrotising myopathy.

Ann Rheum Dis 2019 01 11;78(1):131-139. Epub 2018 Oct 11.

Normandie Univ, UNIROUEN, IRIB, Inserm, U1234, Departement of Immunology, Rouen University Hospital, Rouen, France

Objectives: In autoimmunity, autoantibodies (aAb) may be simple biomarkers of disease or true pathogenic effectors. A form of idiopathic inflammatory myopathy associated with anti-signal recognition particle (SRP) or anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) aAb has been individualised and is referred to as immune-mediated necrotising myopathy (IMNM). The level of aAb correlates with IMNM activity and disease may respond to immunosuppression, suggesting that they are pathogenic. We aimed to evaluate the pathogenicity of IgG from patients with anti-SRP or anti-HMGCR aAb by developing the first mouse model of IMNM.

Methods: IgG from patients suffering from anti-SRP or anti-HMGCR associated IMNM were passively transferred to wild-type, Rag2 or complement C3 mice. Muscle deficiency was evaluated by muscle strength on electrostimulation and grip test. Histological analyses were performed after haematoxylin/eosin staining or by immunofluorescence or immunohistochemistry analysis. Antibody levels were quantified by addressable laser bead assay (ALBIA).

Results: Passive transfer of IgG from patients suffering from IMNM to C57BL/6 or Rag2 mice provoked muscle deficiency. Pathogenicity of aAb was reduced in C3 mice while increased by supplementation with human complement. Breakage of tolerance by active immunisation with SRP or HMGCR provoked disease.

Conclusion: This study demonstrates that patient-derived anti-SRP and anti-HMGCR IgG are pathogenic towards muscle through a complement-mediated mechanism, definitively establishing the autoimmune character of IMNM. These data support the use of plasma exchanges and argue for evaluating complement-targeting therapies in IMNM.
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http://dx.doi.org/10.1136/annrheumdis-2018-213518DOI Listing
January 2019

AMPK Activation of PGC-1α/NRF-1-Dependent SELENOT Gene Transcription Promotes PACAP-Induced Neuroendocrine Cell Differentiation Through Tolerance to Oxidative Stress.

Mol Neurobiol 2019 Jun 28;56(6):4086-4101. Epub 2018 Sep 28.

UNIROUEN, Inserm U1239, Neuronal and Neuroendocrine Differentiation and Communication Laboratory, Rouen-Normandie University, 76821, Mont-Saint-Aignan, France.

Several cues including pituitary adenylate cyclase-activating polypeptide (PACAP), which acts through cAMP stimulation, specify the conversion of sympathoadrenal (SA) precursors toward different cell phenotypes by promoting their survival and differentiation. Selenoprotein T (SELENOT) is a PACAP-stimulated ER oxidoreductase that exerts an essential antioxidant activity and whose up-regulation is associated with SA cell differentiation. In the present study, we investigated the transcriptional cascade elicited by PACAP/cAMP to trigger SELENOT gene transcription during the conversion of PC12 cells from SA progenitor-like cells toward a neuroendocrine phenotype. Unexpectedly, we found that PACAP/cAMP recruits the canonical pathway that regulates mitochondrial function in order to elicit SELENOT gene transcription and the consequent antioxidant response during PC12 cell differentiation. This cascade involves LKB1-mediated AMPK activation in order to stimulate SELENOT gene transcription through the PGC1-α/NRF-1 complex, thus allowing SELENOT to promote PACAP-stimulated neuroendocrine cell survival and differentiation. Our data reveal that a PACAP and cAMP-activated AMPK-PGC-1α/NRF-1 cascade is critical for the coupling of oxidative stress tolerance, via SELENOT gene expression, and mitochondrial biogenesis in order to achieve PC12 cell differentiation. The data further highlight the essential role of SELENOT in cell metabolism during differentiation.
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http://dx.doi.org/10.1007/s12035-018-1352-xDOI Listing
June 2019

Efficacy of Rituximab in Refractory Generalized anti-AChR Myasthenia Gravis.

J Neuromuscul Dis 2018 ;5(2):241-249

AP-HP, Hôpital Pitié-Salpêtrière, Department of Internal Medicine and ClinicalImmunology, Inflammation-Immunopathology-Biotherapy Department (I2B), East Paris Neuromuscular Diseases Reference Center, Inserm U974, Sorbonne Université, Paris 6, Paris, France.

Background: Several retrospective case series have suggested rituximab (RTX) might improve patients with refractory Myasthenia Gravis (MG).

Objective: In this study, we aimed to evaluate prospectively the efficacy of RTX on muscle function in refractory generalized anti-acetylcholine receptor (AChR) MG patients.

Methods: Enrolled patients received 1 g of RTX at day 0, day 14, and 6-month follow-up (M6). The primary endpoint was improvement of muscle function at 12-month (M12) based on myasthenic muscle score (MMS). Secondary endpoints were an improvement of the MG Foundation of America Postintervention Status (MGFA-PIS), respiratory forced vital capacity, occurrences of acute MG exacerbation and requirement of associated immunosuppressants and immunomodulatory agents.

Results: Twelve patients were enrolled, and 11 completed the study. Only a single patient presented an improvement of at least 20 points on MMS at M12, although 2 patients displayed an increase of at least 18 points at M12. MGFA-PIS had improved in 55% of patients by M12. The clinical improvement was not associated with a reduction of immunosuppressant burden.

Conclusions: These results provide data on the effect of RTX in patients with severe, refractory anti-AChR Abs generalized MG. Even though primary outcome was only reached in a single patient at M12, a beneficial effect of RTX on muscle function was seen in half of the patients at M12 and persisted in a third of patients at M18.
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http://dx.doi.org/10.3233/JND-180300DOI Listing
November 2018

Influence of Pre-existing Anti-capsid Neutralizing and Binding Antibodies on AAV Vector Transduction.

Mol Ther Methods Clin Dev 2018 Jun 13;9:119-129. Epub 2018 Feb 13.

University Pierre and Marie Curie - Paris 6 and INSERM U974, 75005 Paris, France.

Pre-existing immunity to adeno-associated virus (AAV) is highly prevalent in humans and can profoundly impact transduction efficiency. Despite the relevance to AAV-mediated gene transfer, relatively little is known about the fate of AAV vectors in the presence of neutralizing antibodies (NAbs). Similarly, the effect of binding antibodies (BAbs), with no detectable neutralizing activity, on AAV transduction is ill defined. Here, we delivered AAV8 vectors to mice carrying NAbs and demonstrated that AAV particles are taken up by both liver parenchymal and non-parenchymal cells; viral particles are then rapidly cleared, without resulting in transgene expression. , imaging of hepatocytes exposed to AAV vectors pre-incubated with either NAbs or BAbs revealed that virus is taken up by cells in both cases. Whereas no successful transduction was observed when AAV was pre-incubated with NAbs, an increased capsid internalization and transgene expression was observed in the presence of BAbs. Accordingly, AAV8 vectors administered to mice passively immunized with anti-AAV8 BAbs showed a more efficient liver transduction and a unique vector biodistribution profile compared to mice immunized with NAbs. These results highlight a virtually opposite effect of neutralizing and binding antibodies on AAV vectors transduction.
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http://dx.doi.org/10.1016/j.omtm.2018.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948224PMC
June 2018

Prevalence and long-term monitoring of humoral immunity against adeno-associated virus in Duchenne Muscular Dystrophy patients.

Cell Immunol 2019 08 16;342:103780. Epub 2018 Mar 16.

Genethon and INSERM U951, Evry 91000 France. Electronic address:

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans.
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http://dx.doi.org/10.1016/j.cellimm.2018.03.004DOI Listing
August 2019

Synergistic promoting effects of pentoxifylline and simvastatin on the apoptosis of triple-negative MDA-MB-231 breast cancer cells.

Int J Oncol 2018 Apr 9;52(4):1246-1254. Epub 2018 Feb 9.

National Institute of Health and Medical Research, Medical Research Unit S-1165/Paris Diderot University, University Institute of Hematology, Saint-Louis Hospital, 75010 Paris, France.

Pentoxifylline (PTX), a xanthine family molecule and simvastatin (SIM), an anti-hypercholesterolemic agent, have recently been considered as sensitizers to chemotherapy and radiotherapy. The present in vitro study evaluated their antitumor synergistic effects on MDA‑MB‑231 breast cancer cells characterized by the triple‑negative phenotype (TNP). The anti-proliferative effects of these two agents were evaluated by MTT and clonogenic assays. Cell cycle progression was examined using propidium iodide staining. Apoptosis was investigated by Annexin V labeling, and by examining caspase 3 activity and DNA fragmentation. Autophagic vesicles and reactive oxygen species (ROS) levels were monitored by flow cytometry. Western blot analysis was performed to evaluate molecular targets. Our results revealed that when used alone, PTX and SIM exerted antitumor effects. Nevertheless, used in combination, the inhibition of cell proliferation was synergistically superior (80% vs 42%) than that observed following treatment with each agent alone after 48 h. PTX alone (0.5 mM) induced both apoptosis (25%) and autophagy (25%); however, when used in combination with SIM (0.5 µM), the balance between these processes was disrupted and the cells underwent apoptosis (>65%) as opposed to autophagy (<13%). This imbalance was associated with an increase in ERK1/2 and AKT activation, but not with an increase in mTOR phosphorylation, and with the suppression of the NF-κB pathway. In addition, in the cells treated with both agents, almost 78% of the cells were arrested at the G0/G1 phase and lost their colony-forming ability (38±5%) compared to the cells treated with PTX alone (115±5%). On the whole, these results suggest that the induction of autophagy may be a protective mechanism preventing MDA‑MB‑231 cancer cell death. The combined use of PTX and SIM may drive dormant autophagic cancer cells to undergo apoptosis and thus this may be a novel treatment strategy for breast cancer characterized by the TNP.
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http://dx.doi.org/10.3892/ijo.2018.4272DOI Listing
April 2018

Necrosis in anti-SRP and anti-HMGCRmyopathies: Role of autoantibodies and complement.

Neurology 2018 02 12;90(6):e507-e517. Epub 2018 Jan 12.

From the Departments of Neuropathology (Y.A., C.P., H.R., H.-H.G., W.S.) and Pathology (P.H., N.Z.), Charité-Universitätsmedizin, Berlin, Germany; Internal Medicine Department (Y.A., N.C., K.M., A.R., O. Benveniste), Reference for Neuro-muscular Diseases, Paris Est, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris; Myology Research Center (Y.A., L.A.-D., G.B.-B., D.A., O. Benveniste), Sorbonne Universités UPMC Univ Paris 06, INSERM UMRS974, Pitié-Salpêtrière University Hospital; Department of Neuropathology (T.M., S.L.-L., C.D.) and Institut de Myologie (B.E.), Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University Hospital, Paris; and Department of Immunology (C.B., L.D., O. Boyer), UNIROUEN, INSERM, U1234, Normandie University, Rouen University Hospital, France.

Objective: To characterize muscle fiber necrosis in immune-mediated necrotizing myopathies (IMNM) with anti-signal recognition particle (SRP) or anti-3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) antibodies and to explore its underlying molecular immune mechanisms.

Methods: Muscle biopsies from patients with IMNM were analyzed and compared to biopsies from control patients with myositis. In addition to immunostaining and reverse transcription PCR on muscle samples, in vitro immunostaining on primary muscle cells was performed.

Results: Creatine kinase levels and muscle regeneration correlated with the proportion of necrotic fibers ( = 0.6, < 0.001). CD68iNOS macrophages and a Th-1 immune environment were chiefly involved in ongoing myophagocytosis of necrotic fibers. T-cell densities correlated with necrosis but no signs of cytotoxicity were detected. Activation of the classical pathway of the complement cascade, accompanied by deposition of sarcolemmal immunoglobulins, featured involvement of humoral immunity. Presence of SRP and HMGCR proteins on altered myofibers was reproduced on myotubes exposed to purified patient-derived autoantibodies. Finally, a correlation between sarcolemmal complement deposits and fiber necrosis was observed ( = 0.4 and = 0.004). Based on these observations, we propose to update the pathologic criteria of IMNM.

Conclusion: These data further corroborate the pathogenic role of anti-SRP and anti-HMGCR autoantibodies in IMNM, highlighting humoral mechanisms as key players in immunity and myofiber necrosis.
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http://dx.doi.org/10.1212/WNL.0000000000004923DOI Listing
February 2018

Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors.

Blood Adv 2017 Oct 16;1(23):2019-2031. Epub 2017 Oct 16.

University Pierre and Marie Curie-Paris 6 and INSERM U974, Paris, France.

Results from clinical trials of liver gene transfer for hemophilia demonstrate the potential of the adeno-associated virus (AAV) vector platform. However, to achieve therapeutic transgene expression, in some cases high vector doses are required, which are associated with a higher risk of triggering anti-capsid cytotoxic T-cell responses. Additionally, anti-AAV preexisting immunity can prevent liver transduction even at low neutralizing antibody (NAb) titers. Here, we describe the use of exosome-associated AAV (exo-AAV) vectors as a robust liver gene delivery system that allows the therapeutic vector dose to be decreased while protecting from preexisting humoral immunity to the capsid. The in vivo efficiency of liver targeting of standard AAV8 or AAV5 and exo-AAV8 or exo-AAV5 vectors expressing human coagulation factor IX (hF.IX) was evaluated. A significant enhancement of transduction efficiency was observed, and in hemophilia B mice treated with 4 × 10 vector genomes per kilogram of exo-AAV8 vectors, a staggering ∼1 log increase in hF.IX transgene expression was observed, leading to superior correction of clotting time. Enhanced liver expression was also associated with an increase in the frequency of regulatory T cells in lymph nodes. The efficiency of exo- and standard AAV8 vectors in evading preexisting NAbs to the capsid was then evaluated in a passive immunization mouse model and in human sera. Exo-AAV8 gene delivery allowed for efficient transduction even in the presence of moderate NAb titers, thus potentially extending the proportion of subjects eligible for liver gene transfer. Exo-AAV vectors therefore represent a platform to improve the safety and efficacy of liver-directed gene transfer.
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http://dx.doi.org/10.1182/bloodadvances.2017010181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728288PMC
October 2017

Dermatomyositis and Immune-Mediated Necrotizing Myopathies: A Window on Autoimmunity and Cancer.

Front Immunol 2017 21;8:992. Epub 2017 Aug 21.

Normandie University, UNIROUEN, INSERM, U1234, Rouen University Hospital, Department of Immunology, Rouen, France.

Autoimmune myopathies (myositides) are strongly associated with malignancy. The link between myositis and cancer, originally noticed by Bohan and Peter in their classification in 1975 (1), has been evidenced by large population-based cohort studies and a recent meta-analysis. The numerous reports of cases in which the clinical course of myositis reflects that of cancer and the short delay between myositis and cancer onset support the notion that myositis may be an authentic paraneoplastic disorder. Thus, cancer-associated myositis raises the question of cancer as a cause rather than a consequence of autoimmunity. Among myositides, dermatomyositis and more recently, although to a lesser extent, immune-mediated necrotizing myopathies are the most documented forms associated with cancer. Interestingly, the current diagnostic approach for myositis is based on the identification of specific antibodies where each antibody determines specific clinical features and outcomes. Recent findings have shown that the autoantibodies anti-TIF1γ, anti-NXP2 and anti-HMGCR are associated with cancers in the course of myositis. Herein, we highlight the fact that the targets of these three autoantibodies involve cellular pathways that intervene in tumor promotion and we discuss the role of cancer mutations as autoimmunity triggers in adult myositis.
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http://dx.doi.org/10.3389/fimmu.2017.00992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566616PMC
August 2017

IFN-β-induced reactive oxygen species and mitochondrial damage contribute to muscle impairment and inflammation maintenance in dermatomyositis.

Acta Neuropathol 2017 Oct 16;134(4):655-666. Epub 2017 Jun 16.

Institut de Physiologie EA 3072, Service de Physiologie et d'Explorations Fonctionnelles, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.

Dermatomyositis (DM) is an autoimmune disease associated with enhanced type I interferon (IFN) signalling in skeletal muscle, but the mechanisms underlying muscle dysfunction and inflammation perpetuation remain unknown. Transcriptomic analysis of early untreated DM muscles revealed that the main cluster of down-regulated genes was mitochondria-related. Histochemical, electron microscopy, and in situ oxygraphy analysis showed mitochondrial abnormalities, including increased reactive oxygen species (ROS) production and decreased respiration, which was correlated with low exercise capacities and a type I IFN signature. Moreover, IFN-β induced ROS production in human myotubes was found to contribute to mitochondrial malfunctions. Importantly, the ROS scavenger N-acetyl cysteine (NAC) prevented mitochondrial dysfunctions, type I IFN-stimulated transcript levels, inflammatory cell infiltrate, and muscle weakness in an experimental autoimmune myositis mouse model. Thus, these data highlight a central role of mitochondria and ROS in DM. Mitochondrial dysfunctions, mediated by IFN-β induced-ROS, contribute to poor exercise capacity. In addition, mitochondrial dysfunctions increase ROS production that drive type I IFN-inducible gene expression and muscle inflammation, and may thus self-sustain the disease. Given that current DM treatments only induce partial recovery and expose to serious adverse events (including muscular toxicity), protecting mitochondria from dysfunctions may open new therapeutic avenues for DM.
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http://dx.doi.org/10.1007/s00401-017-1731-9DOI Listing
October 2017

Neuron-to-Neuron Transfer of FUS in Drosophila Primary Neuronal Culture Is Enhanced by ALS-Associated Mutations.

J Mol Neurosci 2017 May 20;62(1):114-122. Epub 2017 Apr 20.

Inserm, U1245, IRIB, Rouen, France.

The DNA- and RNA-binding protein fused in sarcoma (FUS) has been pathologically and genetically linked to amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Cytoplasmic FUS-positive inclusions were identified in the brain and spinal cord of a subset of patients suffering with ALS/FTLD. An increasing number of reports suggest that FUS protein can behave in a prion-like manner. However, no neuropathological studies or experimental data were available regarding cell-to-cell spread of these pathological protein assemblies. In the present report, we investigated the ability of wild-type and mutant forms of FUS to transfer between neuronal cells. We combined the use of Drosophila models for FUS proteinopathies with that of the primary neuronal cultures to address neuron-to-neuron transfer of FUS proteins. Using conditional co-culture models and an optimized flow cytometry-based methodology, we demonstrated that ALS-mutant forms of FUS proteins can transfer between well-differentiated mature Drosophila neurons. These new observations support that a propagating mechanism could be applicable to FUS, leading to the sequential dissemination of pathological proteins over years.
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http://dx.doi.org/10.1007/s12031-017-0908-yDOI Listing
May 2017

Autologous Myoblasts for the Treatment of Fecal Incontinence: Results of a Phase 2 Randomized Placebo-controlled Study (MIAS).

Ann Surg 2018 03;267(3):443-450

Rouen University Hospital, Department of Digestive Surgery, Rouen, France.

Objective: The aim of this study was to evaluate the efficacy of intrasphincteric injections of autologous myoblasts (AMs) in fecal incontinence (FI) in a controlled study.

Summary Of Background Data: Adult stem cell therapy is expected to definitively cure FI by regenerating damaged sphincter. Preclinical data and results of open-label trials suggest that myoblast therapy may represent a noninvasive treatment option.

Methods: We conducted a phase 2 randomized, double-blind, placebo-controlled study of intrasphincteric injections of AM in 24 patients. The study compared outcome after AM (n = 12) or placebo (n = 12) injection using Cleveland Clinic Incontinence (CCI), score at 6 and 12 months. Patients in the placebo group were eligible to receive frozen AM after 1 year.

Results: At 6 months, the median CCI score significantly decreased from baseline in both the AM (9 vs 15, P = 0.02) and placebo (10 vs 15, P = 0.01) groups. Hence, no significant difference was found between the 2 groups (primary endpoint) at 6 months. At 12 months, the median CCI score continued to ameliorate in the AM group (6.5 vs 15, P = 0.006), while effect was lost in the placebo group (14 vs 15, P = 0.35). Consequently, there was a higher response rate at 12 months in the treated than the placebo arm (58% vs 8%, P = 0.03). After delayed frozen AM injection in the placebo group, the response rate was 60% (6/10) at 12 months.

Conclusions: Intrasphincteric AM injections in FI patients have shown tolerance, safety, and clinical benefit at 12 months despite a transient placebo effect at 6 months.
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http://dx.doi.org/10.1097/SLA.0000000000002268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805121PMC
March 2018

The Spontaneous Autoimmune Neuromyopathy in ICOSL NOD Mice Is CD4 T-Cell and Interferon-γ Dependent.

Front Immunol 2017 31;8:287. Epub 2017 Mar 31.

INSERM U1016, Cochin Institute, Paris Descartes University, Sorbonne Paris Cité, Paris, France.

Abrogation of ICOS/ICOS ligand (ICOSL) costimulation prevents the onset of diabetes in the non-obese diabetic (NOD) mouse but, remarkably, yields to the development of a spontaneous autoimmune neuromyopathy. At the pathological level, ICOSL NOD mice show stronger protection from insulitis than their ICOS counterparts. Also, the ICOSL NOD model carries a limited C57BL/6 region containing the nul mutation, but, in contrast to ICOS NOD mice, no gene variant previously reported as associated to NOD diabetes. Therefore, we aimed at providing a detailed characterization of the ICOSL NOD model. The phenotype observed in ICOSL NOD mice is globally similar to that observed in ICOS and ICOSICOSL double-knockout NOD mice, manifested by a progressive locomotor disability first affecting the front paws as observed by catwalk analysis and a decrease in grip test performance. The pathology remains limited to peripheral nerve and striated muscle. The muscle disease is characterized by myofiber necrosis/regeneration and an inflammatory infiltrate composed of CD4 T-cells, CD8 T-cells, and myeloid cells, resembling human myositis. Autoimmune neuromyopathy can be transferred to NOD. recipients by CD4 but not by CD8 T-cells isolated from 40-week-old female ICOSL NOD mice. The predominant role of CD4 T-cells is further demonstrated by the observation that neuromyopathy does not develop in CIITAICOSL NOD in contrast to β2microglobulinICOSL NOD mice. Also, the cytokine profile of CD4 T-cells infiltrating muscle and nerve of ICOSL NOD mice is biased toward a Th1 pattern. Finally, adoptive transfer experiments show that diabetes development requires expression of ICOSL, in contrast to neuromyopathy. Altogether, the deviation of autoimmunity from the pancreas to skeletal muscles in the absence of ICOS/ICOSL signaling in NOD mice is strictly dependent on CD4 T-cells, leads to myofiber necrosis and regeneration. It provides the first mouse model of spontaneous autoimmune myopathy akin to human myositis.
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http://dx.doi.org/10.3389/fimmu.2017.00287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371727PMC
March 2017

Pathogenic role of anti-signal recognition protein and anti-3-Hydroxy-3-methylglutaryl-CoA reductase antibodies in necrotizing myopathies: Myofiber atrophy and impairment of muscle regeneration in necrotizing autoimmune myopathies.

Ann Neurol 2017 Apr;81(4):538-548

Pierre and Marie Curie University, Sorbonne Universities, National Institute of Health and Medical Research, National Center for Scientific Research, Myology Research Center, Pitié-Salpêtrière University Hospital, Paris, France.

Objective: Immune-mediated necrotizing myopathies (IMNM) may be associated with either anti-signal recognition protein (SRP) or anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibodies (Abs), and the titer of these Abs is correlated with disease activity. We investigated whether anti-SRP and anti-HMGCR Abs could be involved in muscle damage.

Methods: Muscle biopsies of patients were analyzed for atrophy and regeneration by measuring fiber size and by performing immunostaining of neonatal myosin heavy chain. To further understand the role of the Abs in the pathology, we performed muscle cell coculture with the Abs. Atrophy and regeneration were evaluated based on the myotube surface area as well as gene and cytokine profiles.

Results: In muscle biopsies of patients with anti-SRP and anti-HMGCR Abs, a large number of small fibers corresponding to both atrophic and regenerating fibers were observed. In vitro, anti-SRP and anti-HMGCR Abs induced muscle fiber atrophy and increased the transcription of MAFbx and TRIM63. In addition, the muscle fiber atrophy was associated with high levels of inflammatory cytokines: tumor necrosis factor, interleukin (IL)-6, and reactive oxygen species. In the presence of anti-SRP or anti-HMGCR Abs, mechanisms involved in muscle regeneration were also impaired due to a defect of myoblast fusion. This defect was associated with a decreased production of IL-4 and IL-13. The addition of IL-4 and/or IL-13 totally rescued fusion capacity.

Interpretation: These data show that molecular mechanisms of atrophy and regeneration are affected and contribute to loss of muscle function occurring in IMNM. This emphasizes the potential interest of targeted therapies addressing these mechanisms. Ann Neurol 2017;81:538-548.
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http://dx.doi.org/10.1002/ana.24902DOI Listing
April 2017