Debamestrocel multimodal effects on biomarker pathways in amyotrophic lateral sclerosis are linked to clinical outcomes.

INTRODUCTION/AIMS: Biomarkers have shown promise in amyotrophic lateral sclerosis (ALS) research, but the quest for reliable biomarkers remains active. This study evaluates the effect of debamestrocel on cerebrospinal fluid (CSF) biomarkers, an exploratory endpoint. METHODS: A total of 196 participants randomly received debamestrocel or placebo. Seven CSF samples were to be collected from all participants. Forty-five biomarkers were analyzed in the overall study and by two subgroups characterized by the ALS Functional Rating Scale-Revised (ALSFRS-R). A prespecified model was employed to predict clinical outcomes leveraging biomarkers and disease characteristics. Causal inference was used to analyze relationships between neurofilament light chain (NfL) and ALSFRS-R. RESULTS: We observed significant changes with debamestrocel in 64% of the biomarkers studied, spanning pathways implicated in ALS pathology (63% neuroinflammation, 50% neurodegeneration, and 89% neuroprotection). Biomarker changes with debamestrocel show biological activity in trial participants, including those with advanced ALS. CSF biomarkers were predictive of clinical outcomes in debamestrocel-treated participants (baseline NfL, baseline latency-associated peptide/transforming growth factor beta1 [LAP/TGFß1], change galectin-1, all p < .01), with baseline NfL and LAP/TGFß1 remaining (p < .05) when disease characteristics (p < .005) were incorporated. Change from baseline to the last measurement showed debamestrocel-driven reductions in NfL were associated with less decline in ALSFRS-R. Debamestrocel significantly reduced NfL from baseline compared with placebo (11% vs. 1.6%, p = .037). DISCUSSION: Following debamestrocel treatment, many biomarkers showed increases (anti-inflammatory/neuroprotective) or decreases (inflammatory/neurodegenerative) suggesting a possible treatment effect. Neuroinflammatory and neuroprotective biomarkers were predictive of clinical response, suggesting a potential multimodal mechanism of action. These results offer preliminary insights that need to be confirmed.

Machine Learning Identifies Key Proteins in Primary Sclerosing Cholangitis Progression and Links High CCL24 to Cirrhosis.

Primary sclerosing cholangitis (PSC) is a rare, progressive disease, characterized by inflammation and fibrosis of the bile ducts, lacking reliable prognostic biomarkers for disease activity. Machine learning applied to broad proteomic profiling of sera allowed for the discovery of markers of disease presence, severity, and cirrhosis and the exploration of the involvement of CCL24, a chemokine with fibro-inflammatory activity. Sera from 30 healthy controls and 45 PSC patients were profiled with proximity extension assay, quantifying the expression of 2870 proteins, and used to train an elastic net model. Proteins that contributed most to the model were tested for correlation to enhanced liver fibrosis (ELF) score and used to perform pathway analysis. Statistical modeling for the presence of cirrhosis was performed with principal component analysis (PCA), and receiver operating characteristics (ROC) curves were used to assess the useability of potential biomarkers. The model successfully predicted the presence of PSC, where the top-ranked proteins were associated with cell adhesion, immune response, and inflammation, and each had an area under receiver operator characteristic (AUROC) curve greater than 0.9 for disease presence and greater than 0.8 for ELF score. Pathway analysis showed enrichment for functions associated with PSC, overlapping with pathways enriched in patients with high levels of CCL24. Patients with cirrhosis showed higher levels of CCL24. This data-driven approach to characterize PSC and its severity highlights potential serum protein biomarkers and the importance of CCL24 in the disease, implying its therapeutic potential in PSC.

Evaluation of neurotrophic factor secreting mesenchymal stem cells in progressive multiple sclerosis.

BACKGROUND: Autologous mesenchymal stem cell neurotrophic factor-secreting cells (NurOwn®) have the potential to modify underlying disease mechanisms in progressive multiple sclerosis (PMS). OBJECTIVE: This open-label phase II study was conducted to evaluate safety/efficacy of three intrathecal cell treatments. METHODS: Eighteen participants with non-relapsing PMS were treated. The primary endpoint was safety. Secondary endpoints included: cerebrospinal fluid (CSF) biomarkers; timed 25-foot walk speed, nine-hole peg test (9-HPT), low-contrast letter acuity, symbol digit modalities test, and 12-item multiple sclerosis (MS) walking scale. Seventeen participants received all treatments. RESULTS: No deaths/adverse events related to worsening of MS, clinical/magnetic resonance imaging (MRI) evidence of disease activation, and clinically significant changes in safety lab results were reported. Two participants developed symptoms of low back and leg pain, consistent with a diagnosis of arachnoiditis, occurring in one of three intrathecal treatments in both participants. Nineteen percent of treated participants achieved pre-specified ⩾ 25% improvements in timed 25-foot walk speed/nine-HPT at 28 weeks compared to baseline, along with consistent efficacy signals for pre-specified response criteria across other secondary efficacy outcomes. CSF neuroprotective factors increased, and inflammatory biomarkers decreased after treatment, consistent with the proposed mechanism of action. CONCLUSION: Based on these encouraging preliminary findings, further confirmation in a randomized study is warranted.

A randomized placebo-controlled phase 3 study of mesenchymal stem cells induced to secrete high levels of neurotrophic factors in amyotrophic lateral sclerosis.

INTRODUCTION/AIMS: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative illness with great unmet patient need. We aimed to evaluate whether mesenchymal stem cells induced to secrete high levels of neurotrophic factors (MSC-NTF), a novel autologous cell-therapy capable of targeting multiple pathways, could safely slow ALS disease progression. METHODS: This randomized, double-blind, placebo-controlled study enrolled ALS participants meeting revised El Escorial criteria, revised ALS Functional Rating Scale (ALSFRS-R) ≥25 (screening) and ≥3 ALSFRS-R points decline prior to randomization. Participants received three treatments of MSC-NTF or placebo intrathecally. The primary endpoint evaluated efficacy of MSC-NTF through a responder analysis and safety. A change in disease progression post-treatment of ≥1.25 points/mo defines a clinical response. A pre-specified analysis leveraged baseline ALSFRS-R of 35 as a subgroup threshold. RESULTS: Overall, MSC-NTF treatment was well tolerated; there were no safety concerns. Thirty-three percent of MSC-NTF and 28% of placebo participants met clinical response criteria at 28 wk (odds ratio [OR] = 1.33, P = .45); thus, the primary endpoint was not met. A pre-specified analysis of participants with baseline ALSFRS-R ≥ 35 (n = 58) showed a clinical response rate at 28 wk of 35% MSC-NTF and 16% placebo (OR = 2.6, P = .29). Significant improvements in cerebrospinal biomarkers of neuroinflammation, neurodegeneration, and neurotrophic factor support were observed with MSC-NTF, with placebo unchanged. DISCUSSION: The study did not reach statistical significance on the primary endpoint. However, a pre-specified subgroup suggests that MSC-NTF participants with less severe disease may have retained more function compared to placebo. Given the unmet patient need, the results of this trial warrant further investigation.

Suppression of experimental autoimmune myasthenia gravis by autologous T regulatory cells.

Adoptive transfer of regulatory T (Treg) cells have been employed effectively for suppression of several animal models for autoimmune diseases. In order to employ Treg cell therapy in patients, it is necessary to generate Treg cells from the patient's own cells (autologous) that would be able to suppress effectively the disease in vivo, upon their reintroduction to the patient. Towards this objective, we report in the present study on a protocol for a successful immune-regulation of experimental autoimmune myasthenia gravis (EAMG) by ex vivo--generated autologous Treg cells. For this protocol bone marrow (BM) cells, are first cultured in the presence of GM-CSF, giving rise to a population of CD11c(+)MHCII(+)CD45RA(+)CD8(-) DCs (BMDCs). Splenic CD4(+) T cells are then co-cultured with the differentiated BM cells and expand to 90% of Foxp3(+) Treg cells. In vitro assay exhibits a similar dose dependent manner in the suppression of T effector cells proliferation between Treg cells obtained from either healthy or sick donors. In addition, both Treg cells inhibit similarly the secretion of IFN-γ from activated splenocytes. Administration of 1 × 10(6) ex-vivo generated Treg cells, I.V, to EAMG rats, modulates the disease following a single treatment, given 3 days or 3 weeks after disease induction. Similar disease inhibition was achieved when CD4 cells were taken from either healthy or sick donors. The disease suppression was accompanied by reduced levels of total AChR specific antibodies in the serum. Moreover, due to the polyclonality of the described Treg cell, we have examined whether this treatment approach could be also employed for the treatment of other autoimmune diseases involving Treg cells. Indeed, we demonstrated that the ex-vivo generated autologous Treg cells suppress Adjuvant Arthritis (AA) in rats. This study opens the way for the application of induced autologous Treg cell therapy for myasthenia gravis, as well as for other human autoimmune diseases involving Treg cells.

Experimental Autoimmune Myasthenia Gravis (EAMG): from immunochemical characterization to therapeutic approaches.

Myasthenia Gravis (MG) is an organ-specific autoimmune disease. In high percentage of patients there are autoantibodies to the nicotinic acetylcholine receptor (AChR) that attack AChR on muscle cells at the neuromuscular junction, resulting in muscle weakness. Experimental Autoimmune Myasthenia Gravis (EAMG) is an experimental model disease for MG. EAMG is induced in several animal species by immunization with acetylcholine receptor (AChR), usually isolated from the electric organ of electric fish, which is a rich source for this antigen. Our lab has been involved for several decades in research of AChR and of EAMG. The availability of an experimental autoimmune disease that mimics in many aspects the human disease, provides an excellent model system for elucidating the immunological nature and origin of MG, for studying various existing treatment modalities and for attempting the development of novel treatment approaches. In this review in honor of Michael Sela and Ruth Arnon, we report first on our early pioneering contributions to research on EAMG. These include the induction of EAMG in several animal species, early attempts for antigen-specific treatment for EAMG, elicitation and characterization of monoclonal antibodies and anti-idiotypic antibodies, measuring humoral and cellular AChR-specific immune responses in MG patient and more. In the second part of the review we discuss more recent studies from our lab towards developing and testing novel treatment approaches for myasthenia. These include antigen-dependent treatments aimed at specifically abrogating the humoral and cellular anti-AChR responses, as well as immunomodulatory approaches that could be used either alone, or in conjunction with antigen-specific treatments, or alternatively, serve as steroid-sparing agents.

Serum CCL24 as a Biomarker of Fibrotic and Vascular Disease Severity in Systemic Sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a heterogeneous disease, characterized by variable tissue and vascular fibrosis in the context of autoimmune activation. CCL24 (or Eotaxin2) has been shown to promote microangiopathic, proinflammatory, and profibrotic processes in preclinical models of SSc. Here, we study serum CCL24 levels in a real-life cohort of patients with SSc, to determine its distribution across disease features and its value in predicting disease progression and related mortality. METHODS: Serum CCL24 was assessed in an observational cohort of consecutively enrolled patients with SSc. A high CCL24 cutoff was defined based on its distribution in a matched cohort of healthy controls. Disease progression and mortality were analyzed from the date of serum assessment. RESULTS: Two-hundred thirteen consecutively enrolled patients with SSc were included in this analysis. Median disease duration was six years (interquartile range 3-14), 28.6% of patients presented with interstitial lung disease (ILD), 46.9% had digital ulcers, and 25.3% showed high CCL24 serum concentration. High-CCL24 patients were more frequently male and positive for anti-scl-70, with a diagnosis of ILD and synovitis (P < 0.05 for all). Notably, high-CCL24 patients had lower diffusion of carbon monoxide and higher prevalence of digital ulcers, telangiectasias, and calcinosis (P < 0.05 for all). In a longitudinal setting, high CCL24 was associated with greater lung function decline and with higher disease-related mortality. CONCLUSION: Serum CCL24 is a biomarker of disease severity across fibrotic and vascular disease manifestations. These data support the development of therapies targeting CCL24 as a novel comprehensive therapeutic target in SSc.

The Role of CCL24 in Primary Sclerosing Cholangitis: Bridging Patient Serum Proteomics to Preclinical Data.

Primary sclerosing cholangitis (PSC) is an inflammatory and fibrotic biliary disease lacking approved treatment. We studied CCL24, a chemokine shown to be overexpressed in damaged bile ducts, and its involvement in key disease-related mechanisms. Serum proteomics of PSC patients and healthy controls (HC) were analyzed using the Olink® proximity extension assay and compared based on disease presence, fibrosis severity, and CCL24 levels. Disease-related canonical pathways, upstream regulators, and toxicity functions were elevated in PSC patients compared to HC and further elevated in patients with high CCL24 levels. In vitro, a protein signature in CCL24-treated hepatic stellate cells (HSCs) differentiated patients by disease severity. In mice, CCL24 intraperitoneal injection selectively recruited neutrophils and monocytes. Treatment with CM-101, a CCL24-neutralizing antibody, in an α-naphthylisothiocyanate (ANIT)-induced cholestasis mouse model effectively inhibited accumulation of peribiliary neutrophils and macrophages while reducing biliary hyperplasia and fibrosis. Furthermore, in PSC patients, CCL24 levels were correlated with upregulation of monocyte and neutrophil chemotaxis pathways. Collectively, these findings highlight the distinct role of CCL24 in PSC, influencing disease-related mechanisms, affecting immune cells trafficking and HSC activation. Its blockade with CM-101 reduces inflammation and fibrosis and positions CCL24 as a promising therapeutic target in PSC.

CCL24 regulates biliary inflammation and fibrosis in primary sclerosing cholangitis.

ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2-knockout (Mdr2-/-) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis.

Blocking of IL-6 suppresses experimental autoimmune myasthenia gravis.

Suppressive regulatory T cells (Treg) and pathogenic T helper 17 (Th17) cells are two lymphocyte subsets with opposing activities in autoimmune diseases. The proinflammatory cytokine IL-6 is a potent factor in switching immune responses in vivo from the induction of Treg to pathogenic Th17 cells. We studied the Treg and Th17 cell compartments in experimental autoimmune myasthenia gravis (EAMG) and healthy control rats in order to assess whether the equilibrium between Treg and Th17 cells is perturbed in the disease. We found that Th17 cell-related genes are upregulated and Treg-related genes are downregulated in EAMG. The shift in favor of Th17 cells in EAMG could be reversed by antibodies to IL-6. Administration of anti-IL-6 antibodies to myasthenic rats suppressed EAMG when treatment started at the acute or at the chronic phase of disease. Suppression of EAMG by anti-IL-6 antibodies was accompanied by a decrease in the overall rat anti-AChR antibody titer and by a reduced number of B cells as compared with control treatment. Administration of anti-IL-6 antibodies led to down-regulation of several Th17 related genes including IL-17, IL-17R, IL-23R and IL-21 but did not affect the number of Treg cells in the lymph nodes. These data identify IL-6 as an important target for modulation of autoimmune responses.

The susceptibility of Aire(-/-) mice to experimental myasthenia gravis involves alterations in regulatory T cells.

The autoimmune regulator (Aire) is involved in the prevention of autoimmunity by promoting thymic expression of tissue restricted antigens which leads to elimination of self-reactive T cells. We found that Aire knockout (KO) mice as well as mouse strains that are susceptible to experimental autoimmune myasthenia gravis (EAMG) have lower thymic expression of acetylcholine receptor (AChR- the main autoantigen in MG), compared to wild type (WT) mice and EAMG-resistant mouse strains, respectively. We demonstrated that Aire KO mice have a significant and reproducible lower frequency of CD4+Foxp3+ cells and a higher expression of Th17 markers in their thymus, compared to wild type (WT) mice. These findings led us to expect that Aire KO mice would display increased susceptibility to EAMG. Surprisingly, when EAMG was induced in young (2 month-old) mice, EAMG was milder in Aire KO than in WT mice for several weeks until the age of about 5 months. However, when EAMG was induced in relatively aged (6 month-old) mice, Aire KO mice presented higher disease severity than WT controls. This age-related change in susceptibility to EAMG correlated with an elevated proportion of Treg cells in the spleens of young but not old KO, compared to WT mice, suggesting a role for peripheral Treg cells in the course of disease. Our observations point to a possible link between Aire and Treg cells and suggest an involvement for both in the pathogenesis of myasthenia.

MSC-NTF (NurOwn®) exosomes: a novel therapeutic modality in the mouse LPS-induced ARDS model.

BACKGROUND: One of the most severe complications of the current COVID-19 pandemic is acute respiratory distress syndrome (ARDS). ARDS is caused by increased amounts of pro-inflammatory cytokines, leading to lung damage and loss of lung function. There are currently no effective therapies for combatting ARDS. Mesenchymal stem cells (MSCs) have been suggested as a potential treatment for ARDS due to their significant immunomodulatory properties. MSC small extracellular vesicles (sEVs), including exosomes, modulate the immune response as effectively as MSCs themselves, with the added advantages of increased safety and tissue penetration. METHODS: We isolated sEVs from MSCs induced to secrete increased levels of neurotrophic and immunomodulatory factors, termed Exo MSC-NTF, and compared their ability to treat ARDS, in a lung injury LPS mouse model, to sEVs isolated from naïve MSCs (Exo MSC). Measurments of lung histopathological changes and neutrophil infiltration, blood oxygen saturation, and bronchoalveolar lavge fluid (BALF) proinflammatory cytokines and coagulation related factors were performed. RESULTS: We found that Exo MSC-NTF was superior to Exo MSC in reducing LPS-induced ARDS markers, including physiological lung damage such as alveolar wall thickness, fibrin presence, and neutrophil accumulation, as well as increasing oxygenation levels. Furthermore, Exo MSC-NTF reversed the imbalance in the host immune response, seen as decreased IFN-γ, IL-6, TNF-α, and RANTES levels in the bronchoalveolar lavage fluid. CONCLUSIONS: These positive preclinical results suggest that Exo MSC-NTF may be suitable as a therapy for COVID-19-induced ARDS and are more effective at combatting ARDS physiological, pathological, and biochemical symptoms than sEVs isolated from non-induced MSCs.

MuSK EAMG: Immunological Characterization and Suppression by Induction of Oral Tolerance.

Myasthenia gravis (MG) with antibodies to the muscle-specific receptor tyrosine kinase (MuSK) is a distinct sub-group of MG, affecting 5-8% of all MG patients. MuSK, a receptor tyrosine kinase, is expressed at the neuromuscular junctions (NMJs) from the earliest stages of synaptogenesis and plays a crucial role in the development and maintenance of the NMJ. MuSK-MG patients are more severely affected and more refractory to treatments currently used for MG. Most patients require long-term immunosuppression, stressing the need for improved treatments. Ideally, preferred treatments should specifically delete the antigen-specific autoimmune response, without affecting the entire immune system. Mucosal tolerance, induced by oral or nasal administration of an auto-antigen through the mucosal system, resulting in an antigen-specific immunological systemic hyporesponsiveness, might be considered as a treatment of choice for MuSK-MG. In the present study we have characterized several immunological parameters of murine MuSK-EAMG and have employed induction of oral tolerance in mouse MuSK-EAMG, by feeding with a recombinant MuSK protein one week before disease induction. Such a treatment has been shown to attenuate MuSK-EAMG. Both induction and progression of disease were ameliorated following oral treatment with the recombinant MuSK fragment, as indicated by lower clinical scores and lower anti-MuSK antibody titers.

NurOwn, phase 2, randomized, clinical trial in patients with ALS: Safety, clinical, and biomarker results.

OBJECTIVE: To determine the safety and efficacy of mesenchymal stem cell (MSC)-neurotrophic factor (NTF) cells (NurOwn®, autologous bone marrow-derived MSCs, induced to secrete NTFs) delivered by combined intrathecal and intramuscular administration to participants with amyotrophic lateral sclerosis (ALS) in a phase 2 randomized controlled trial. METHODS: The study enrolled 48 participants randomized 3:1 (treatment: placebo). After a 3-month pretransplant period, participants received 1 dose of MSC-NTF cells (n = 36) or placebo (n = 12) and were followed for 6 months. CSF was collected before and 2 weeks after transplantation. RESULTS: The study met its primary safety endpoint. The rate of disease progression (Revised ALS Functional Rating Scale [ALSFRS-R] slope change) in the overall study population was similar in treated and placebo participants. In a prespecified rapid progressor subgroup (n = 21), rate of disease progression was improved at early time points (p < 0.05). To address heterogeneity, a responder analysis showed that a higher proportion of treated participants experienced ≥1.5 points/month ALSFRS-R slope improvement compared to placebo at all time points, and was significant in rapid progressors at 4 and 12 weeks (p = 0.004 and 0.046, respectively). CSF neurotrophic factors increased and CSF inflammatory biomarkers decreased in treated participants (p < 0.05) post-transplantation. CSF monocyte chemoattractant protein-1 levels correlated with ALSFRS-R slope improvement up to 24 weeks (p < 0.05). CONCLUSION: A single-dose transplantation of MSC-NTF cells is safe and demonstrated early promising signs of efficacy. This establishes a clear path forward for a multidose randomized clinical trial of intrathecal autologous MSC-NTF cell transplantation in ALS. CLASSIFICATION OF EVIDENCE: This phase II study provides Class I evidence.

Suppression of experimental autoimmune myasthenia gravis by combination therapy: pentoxifylline as a steroid-sparing agent.

Myasthenia gravis (MG) is frequently treated by corticosteroids such as methylprednisolone. However, continuous treatment with steroids often results in adverse effects. In the present study we evaluated the therapeutic potential of a combination of suboptimal doses of methylprednisolone (Solumedrol) and Pentoxifylline (PTX), a general phosphodiesterase (PDE) inhibitor, in rat experimental autoimmune MG (EAMG). This combined treatment resulted in a pronounced suppressive effect on EAMG and was by far more effective than each of the drugs administered separately at these low doses. The suppressive effect on EAMG was accompanied by decreased humoral and cellular responses to AChR as well as down-regulated mRNA expression levels of Th1 cytokines and IL-10 in lymph node cells and of PDE-4 and cathepsin-l in the muscle. This study demonstrates the potential of PTX as a steroid-sparing agent in the management of myasthenia gravis.

Multifunctional tellurium molecule protects and restores dopaminergic neurons in Parkinson's disease models.

In Parkinson's disease (PD) dopaminergic neurons in the substantia nigra (SN) become dysfunctional and many ultimately die. We report that the tellurium immunomodulating compound ammonium trichloro(dioxoethylene-O,O'-)tellurate (AS101) protects dopaminergic neurons and improves motor function in animal models of PD. It is effective when administered systemically or by direct infusion into the brain. Multifunctional activities of AS101 were identified in this study. These were mainly due to the peculiar Tellur(IV)-thiol chemistry of the compound, which enabled the compound to interact with cysteine residues on both inflammatory and apoptotic caspases, resulting in their inactivation. Conversely, its interaction with a key cysteine residue on p21(ras), led to its activation, an obligatory activity for AS101-induced neuronal differentiation. Furthermore, AS101 inhibited IL-10, resulting in up-regulation of GDNF in the SN. This was associated with activation of the neuroprotective kinases Akt and mitogen-activated protein kinases, and up-regulation of the antiapoptotic protein Bcl-2. Inhibition of caspase-1 and caspase-3 activities were associated with decreased neuronal death and inhibition of IL-1beta. We suggest that, because multiple mechanisms are involved in the dysfunction and death of neurons in PD, use of a multifunctional compound, exerting antiapoptotic, anti-inflammatory, and neurotrophic-inducing capabilities may be potentially efficacious for the treatment of PD.

Overexpression of phosphodiesterases in experimental autoimmune myasthenia gravis: suppression of disease by a phosphodiesterase inhibitor.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent antibody-mediated autoimmune disorders, in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. DNA microarray analysis revealed increased levels of several phosphodiesterase (PDE) subtypes in lymph node cells (LNC) and muscles of EAMG rats compared with healthy controls. Quantitative real-time PCR analysis indicated that EAMG is characterized by an increase of PDE subtypes 1, 3, 4, and 7 in LNC and of PDE subtypes 2, 3, 4, and 7 in muscles. Pentoxifylline (PTX), a general PDE inhibitor, inhibited the progression of EAMG when treatment started at either the acute or chronic stages of disease. This suppression was associated with down-regulation of humoral and cellular AChR-specific responses, as well as down-regulation of PDE4, TNF-alpha, IL-18, IL-12, and IL-10 in LNC and of PDEs 1, 4, 7, and TNF-alpha in muscles. The expression of Foxp3, a transcription factor essential for CD4+CD25+ regulatory T cell function, was increased in splenocytes although the number of these cells remained unchanged. PTX also reduced the expression of the endopeptidase cathepsin-l, a marker of muscle damage, in EAMG muscles. This study demonstrates the involvement of PDE regulation in EAMG pathogenesis and suggests that PDE inhibitors may be considered for immunotherapy of MG.

DNA microarray in search of new drug targets for myasthenia gravis.

DNA microarray technology was used to identify new potential drug targets for myasthenia gravis (MG), to delineate genes involved in the pathogenesis of the disease and to possibly target their protein products for immunotherapy. In this study we compared the gene expression in lymph node cells (LNC) and muscles of rats with experimental autoimmune MG (EAMG) to those of control, healthy rats. Of the genes that were found to be deregulated in EAMG, we chose to elaborate on two gene systems: (a) The chemokine IFN-gamma-inducible protein 10 (IP-10, CXCL10), and its receptor (CXCR3) and (b) phosphodiesterases.

miRNA profiling of NurOwn®: mesenchymal stem cells secreting neurotrophic factors.

BACKGROUND: MSC-NTF cells are Mesenchymal Stromal Cells (MSC) induced to express high levels of neurotrophic factors (NTFs) using a culture-medium based approach. MSC-NTF cells have been successfully studied in clinical trials for Amyotrophic Lateral Sclerosis (ALS) patients. MicroRNAs (miRNA) are short non-coding RNA molecules that coordinate post-transcriptional regulation of multiple gene targets. The purpose of this study was to determine whether the miRNA profile could provide a tool for MSC-NTF cell characterization and to distinguish them from the matched MSC from which they are derived. METHODS: NTF secretion in the culture supernatant of MSC-NTF cells was evaluated by ELISA assays. The Agilent microarray miRNA platform was used for pairwise comparisons of MSC-NTF cells to MSC. The differentially expressed miRNAs and putative mRNA targets were validated using qPCR analyses. RESULTS: Principal component analysis revealed two distinct clusters based on cell type (MSC and MSC-NTFs). Nineteen miRNAs were found to be upregulated and 22 miRNAs were downregulated in MSC-NTF cells relative to the MSC cells of origin. Further validation of differentially expressed miRNAs confirmed that miR-3663 and miR-132 were increased 18.5- and 4.06-fold, respectively while hsa-miR-503 was reduced more than 15-fold, suggesting that miRNAs could form the basis of an MSC-NTF cell characterization assay. In an analysis of the miRNA mRNA targets, three mRNA targets of hsa-miR-132-3p (HN-1, RASA1 and KLH-L11) were found to be significantly downregulated. CONCLUSIONS: We have demonstrated that MSC-NTF cells can be distinguished from their MSCs of origin by a unique miRNA expression profile. TRIAL REGISTRATION: Clinicaltrial.gov identifier NCT01777646 . Registered 12 December 2012.

Novel CXCL13 transgenic mouse: inflammation drives pathogenic effect of CXCL13 in experimental myasthenia gravis.

Abnormal overexpression of CXCL13 is observed in many inflamed tissues and in particular in autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disease mainly mediated by anti-acetylcholine receptor autoantibodies. Thymic hyperplasia characterized by ectopic germinal centers (GCs) is a common feature in MG and is correlated with high levels of anti-AChR antibodies. We previously showed that the B-cell chemoattractant, CXCL13 is overexpressed by thymic epithelial cells in MG patients. We hypothesized that abnormal CXCL13 expression by the thymic epithelium triggered B-cell recruitment in MG. We therefore created a novel transgenic (Tg) mouse with a keratin 5 driven CXCL13 expression. The thymus of Tg mice overexpressed CXCL13 but did not trigger B-cell recruitment. However, in inflammatory conditions, induced by Poly(I:C), B cells strongly migrated to the thymus. Tg mice were also more susceptible to experimental autoimmune MG (EAMG) with stronger clinical signs, higher titers of anti-AChR antibodies, increased thymic B cells, and the development of germinal center-like structures. Consequently, this mouse model finally mimics the thymic pathology observed in human MG. Our data also demonstrated that inflammation is mandatory to reveal CXCL13 ability to recruit B cells and to induce tertiary lymphoid organ development.