PLOD2, a key factor for MRL MSC metabolism and chondroprotective properties.

BACKGROUND: Initially discovered for its ability to regenerate ear holes, the Murphy Roth Large (MRL) mouse has been the subject of multiple research studies aimed at evaluating its ability to regenerate other body tissues and at deciphering the mechanisms underlying it. These enhanced abilities to regenerate, retained during adulthood, protect the MRL mouse from degenerative diseases such as osteoarthritis (OA). Here, we hypothesized that mesenchymal stromal/stem cells (MSC) derived from the regenerative MRL mouse could be involved in their regenerative potential through the release of pro-regenerative mediators. METHOD: To address this hypothesis, we compared the secretome of MRL and BL6 MSC and identified several candidate molecules expressed at significantly higher levels by MRL MSC than by BL6 MSC. We selected one candidate, Plod2, and performed functional in vitro assays to evaluate its role on MRL MSC properties including metabolic profile, migration, and chondroprotective effects. To assess its contribution to MRL protection against OA, we used an experimental model for osteoarthritis induced by collagenase (CiOA). RESULTS: Among the candidate molecules highly expressed by MRL MSC, we focused our attention on procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2). Plod2 silencing induced a decrease in the glycolytic function of MRL MSC, resulting in the alteration of their migratory and chondroprotective abilities in vitro. In vivo, we showed that Plod2 silencing in MRL MSC significantly impaired their capacity to protect mouse from developing OA. CONCLUSION: Our results demonstrate that the chondroprotective and therapeutic properties of MRL MSC in the CiOA experimental model are in part mediated by PLOD2.

Large mercury release from the Greenland Ice Sheet invalidated.

The major input of mercury (Hg) to the Arctic is normally ascribed to long-range transport of anthropogenic Hg emissions. Recently, alarming concentrations of Hg in meltwater from the Greenland Ice Sheet (GrIS) were reported with bedrock as the proposed source. Reported Hg concentrations were 100 to 1000 times higher than in known freshwater systems of Greenland, calling for independent validation of the extraordinary concentrations and conclusions. Here, we present measurements of Hg at 21 glacial outlets in West Greenland showing that extreme Hg concentrations cannot be reproduced. In contrast, we find that meltwater from below the GrIS is very low in Hg, has minor implications for the global Hg budget, and pose only a very limited risk for local communities and the natural environment of Greenland.

Mesenchymal stromal cells for improvement of cardiac function following acute myocardial infarction: a matter of timing.

Acute myocardial infarction (AMI) is the leading cause of cardiovascular death and remains the most common cause of heart failure. Reopening of the occluded artery, i.e., reperfusion, is the only way to save the myocardium. However, the expected benefits of reducing infarct size are disappointing due to the reperfusion paradox, which also induces specific cell death. These ischemia-reperfusion (I/R) lesions can account for up to 50% of final infarct size, a major determinant for both mortality and the risk of heart failure (morbidity). In this review, we provide a detailed description of the cell death and inflammation mechanisms as features of I/R injury and cardioprotective strategies such as ischemic postconditioning as well as their underlying mechanisms. Due to their biological properties, the use of mesenchymal stromal/stem cells (MSCs) has been considered a potential therapeutic approach in AMI. Despite promising results and evidence of safety in preclinical studies using MSCs, the effects reported in clinical trials are not conclusive and even inconsistent. These discrepancies were attributed to many parameters such as donor age, in vitro culture, and storage time as well as injection time window after AMI, which alter MSC therapeutic properties. In the context of AMI, future directions will be to generate MSCs with enhanced properties to limit cell death in myocardial tissue and thereby reduce infarct size and improve the healing phase to increase postinfarct myocardial performance.

PAHs in high Arctic copepods Calanus hyperboreus following exposure of residues from in situ burning of oil spill.

In situ burning of marine oil spills reduces the total amount of oil in the environment, but a negative side effect may be the generation of environmentally hazardous polycyclic aromatic hydrocarbons (PAHs) that may pose a risk for bioaccumulation, particularly in organisms having a high lipid content. In this study uptake of PAHs from oil and burn residue were examined in the high arctic copepod Calanus hyperboreus. A major part of the low ring number petrogenic PAHs in the oil was removed during burning and relative higher concentrations of pyrogenic high ring number PAHs was found in the burn residue. This suggests that burning markedly reduces the general PAH exposure load. Furthermore, the pyrogenic PAHs generated during the burn were not bioconcentrated to quantifiable levels in the copepods. We conclude that in situ burning can mitigate the potential risk of PAH uptake for copepods and other pelagic organisms in the marine environment as the pyrogenic PAHs only pose low risk for uptake from the water by the copepods and other pelagic organisms.

Evidence of 11-Hydroxy-hexahydrocannabinol and 11-Nor-9-carboxy-hexahydrocannabinol as Novel Human Metabolites of Δ9-Tetrahydrocannabinol.

(-)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) is the primary psychoactive compound in the Cannabis sativa plant. Δ9-THC undergoes extensive metabolism, with the main human phase I metabolites being 11-hydroxy-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH). Early animal studies have indicated that the 9-10 double bond may be reduced in vivo to yield 11-hydroxy-hexahydrocannabinol (11-OH-HHC) and 11-nor-9-carboxy-hexahydrocannabinol (HHC-COOH). These metabolites have not been confirmed in humans. In this study, we aimed to investigate whether this metabolic transformation occurs in humans. A range of cannabinoids and metabolites, including 11-OH-HHC and HHC-COOH, were measured in whole blood from 308 authentic forensic traffic cases, of which 222 were positive for Δ9-THC. HHC-COOH and 11-OH-HHC were detected in 84% and 15% of the Δ9-THC positive cases, respectively, and the estimated median concentration of HHC-COOH was 7%, relative to that of THC-COOH. To corroborate the in vivo findings, Δ9-THC and its metabolites 11-OH-THC and THC-COOH were incubated with pooled human liver microsomes. HHC-COOH was detected in both the Δ9-THC and 11-OH-THC incubations, while 11-OH-HHC was only detectable in the 11-OH-THC incubation. Hexahydrocannabinol was not detected in any of the incubations, indicating that it is 11-OH-THC or the corresponding aldehyde that undergoes double bond reduction with subsequent oxidation of the aliphatic alcohol to HHC-COOH. In summary, the presented data provide the first evidence of HHC-COOH and 11-OH-HHC being human phase I metabolites of Δ9-THC. These findings have implications for interpretation of analytical results from subjects exposed to Δ9-THC or HHC.

Synthetic Nucleic Acid Antigens in Localized Scleroderma.

We investigated the impact of synthetic nucleic acid antigens on the autoantibody profiles in patients with localized scleroderma, an autoimmune skin disease. Anti-DNA antibodies, including double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), are common among autoimmune diseases, such as systemic lupus erythematosus and localized scleroderma. Based on recent studies, we hypothesized that the sequence of nucleic acid antigens has an impact on the autoimmune reactions in localized scleroderma. To test our hypothesis, we synthesized a panel of DNA and RNA antigens and used them for autoantibody profiling of 70 children with localized scleroderma compared with the healthy controls and patients with pediatric systemic lupus erythematosus (as a disease control). Among the tested antigens, dsD4, which contains the sequence of the human oncogene BRAF, showed a particularly strong presence in localized scleroderma but not systemic lupus erythematosus. Disease activity in patients was significantly associated with dsD4 autoantibody levels. We confirmed this result in vivo by using a bleomycin-induced mouse model of localized scleroderma. When administered intraperitoneally, dsD4 promoted an active polyclonal response in the mouse model. Our study highlights sequence specificity for nucleic acid antigens in localized scleroderma that could potentially lead to developing novel early-stage diagnostic tools.

Modeling Substrate Entry into the P-Glycoprotein Efflux Pump at the Blood-Brain Barrier.

We report molecular dynamics simulations of rhodamine entry into the central binding cavity of P-gp in the inward open conformation. Rhodamine can enter the inner volume via passive transport across the luminal membrane or lateral diffusion in the lipid bilayer. Entry into the inner volume is determined by the aperture angle at the apex of the protein, with a critical angle of 27° for rhodamine. The central binding cavity has an aqueous phase with a few lipids, which significantly reduces substrate diffusion. Within the central binding cavity, we identified regions with relatively weak binding, suggesting that the combination of reduced mobility and weak substrate binding confines rhodamine to enable the completion of the efflux cycle. Tariquidar, a P-gp inhibitor, aggregates at the lower arms of the P-gp, suggesting that inhibition involves steric hindrance of entry into the inner volume and/or steric hindrance of access of ATP to the nucleotide-binding domains.

What is the rational for mesenchymal stromal cells based therapies in the management of hemophilic arthropathies?

Hemophilia A and B are rare X-linked genetic bleeding disorders due to a complete or partial deficiency in the coagulation factors VIII or IX, respectively. The main treatment for hemophilia is prophylactic and based on coagulation factor replacement therapies. These treatments have significantly reduced bleeding and improved the patients' quality of life. Nevertheless, repeated joint bleedings (hemarthroses), even subclinical hemarthroses, can lead to hemophilic arthropathy (HA). This disabling condition is characterized by chronic pain due to synovial inflammation, cartilage and bone destruction requiring ultimately joint replacement. HA resembles to rheumatoid arthritis because of synovitis but HA is considered as having similarities with osteoarthritis as illustrated by the migration of immune cells, production of inflammatory cytokines, synovial hypertrophy and cartilage damage. Various drugs have been evaluated for the management of HA with limited success. The objective of the review is to discuss new therapeutic approaches with a special focus on the studies that have investigated the potential of using mesenchymal stromal cells (MSCs) in the management of HA. A systematic review of the literature has been made. Most of the studies have focused on the interest of MSCs for the delivery of missing factors VIII or IX but in some studies, more insight on the effect of MSC injection on synovial inflammation or cartilage structure were provided and put in perspective for possible clinical applications.

Chronological interplay, clinical features, and treatments among patients with cancer and primary Sjögren's syndrome.

OBJECTIVE: While the incidence and type of blood malignancies are well documented amid primary Sjögren's syndrome patients (pSS), data focusing on solid neoplasms are more conflicting. We aimed to describe clinical, pathological, and immunological characteristics of pSS patients with cancers, along with the chronological interplay between the two conditions. METHODS: Outcomes concerning both pSS and cancer were retrospectively collected from Montpellier University Hospital (tertiary center) between 2019 and 2020. pSS characteristics were compared to a control group of pSS patients without cancer. RESULTS: A total of 165 patients with pSS were included: 55 patients with cancer (52 female, mean age 58.4 ± 10.4 years at pSS diagnosis; mean follow-up 10.5 ± 10.1 years, 12 patients had multiple cancers) and 110 controls without cancer. Characteristics of pSS patients with cancers were different from controls mostly for lymphoma prognosis factors. Among the 70 cancers, we recorded 55 solid neoplasms (whom 27 breast cancers and 8 lung cancers, and 82% of adenocarcinomas), with no evidence of disease at the end of follow-up in 85% of them. Among the 15 recorded blood malignancies, ten were lymphomas with an excellent prognosis. Regarding chronological interplay between cancer and pSS, most cancers (43%) were diagnosed close (± 5 years) to pSS diagnosis. Breast cancers were diagnosed before or close to pSS diagnosis (mean delay - 1.8 ± 13.0 years), at an early stage, with only two relapses (no cancer-related death), while lung cancers were diagnosed late after. CONCLUSIONS: The tight chronological interplay between breast cancer and pSS and the intriguing pathological and immunological pattern of pSS in these patients suggest a hypothesis of immune control of cancer.

Methylotrophic Communities Associated with a Greenland Ice Sheet Methane Release Hotspot.

Subglacial environments provide conditions suitable for the microbial production of methane, an important greenhouse gas, which can be released from beneath the ice as a result of glacial melting. High gaseous methane emissions have recently been discovered at Russell Glacier, an outlet of the southwestern margin of the Greenland Ice Sheet, acting not only as a potential climate amplifier but also as a substrate for methane consuming microorganisms. Here, we describe the composition of the microbial assemblage exported in meltwater from the methane release hotspot at Russell Glacier and its changes over the melt season and as it travels downstream. We found that a substantial part (relative abundance 27.2% across the whole dataset) of the exported assemblage was made up of methylotrophs and that the relative abundance of methylotrophs increased as the melt season progressed, likely due to the seasonal development of the glacial drainage system. The methylotrophs were dominated by representatives of type I methanotrophs from the Gammaproteobacteria; however, their relative abundance decreased with increasing distance from the ice margin at the expense of type II methanotrophs and/or methylotrophs from the Alphaproteobacteria and Betaproteobacteria. Our results show that subglacial methane release hotspot sites can be colonized by microorganisms that can potentially reduce methane emissions.

Prevalence of anti-neutrophil cytoplasmic antibody-associated vasculitis in the south of France, using the capture-recapture method.

OBJECTIVES: This study aimed to estimate the prevalence of ANCA-associated vasculitis (AAV), ie granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), in Southern France in 2018, and evaluate differences among Europeans and non-Europeans. METHODS: This population-based, cross-sectional study used four sources (hospitals, community-based physicians, laboratories, National Health Insurance) to identify adults ≥ 15 years diagnosed with GPA, MPA or EGPA, living in Hérault and Gard in 2018. Cases were defined using the ACR/EULAR classification criteria, and if necessary, the European Medicines Agency algorithm. Prevalence estimates were standardised to the world population and capture-recapture analysis was used to assess the comprehensiveness of the estimation. The influence of geographical origin was evaluated. RESULTS: 202 patients were selected, with 86 cases of GPA (42.6%), 85 cases of MPA (42.1%), and 31 cases of EGPA (15.3%). The standardised prevalence estimates per million inhabitants for 2018 were: 103 (95%CI 84 - 125) for AAV, 48 (95%CI 35 - 64) for GPA, 39 (95%CI 28 - 53) for MPA and 16 (95%CI 9 - 26) for EGPA, 36 (95%CI 25 - 50) for anti-PR3 positive AAV, 46 (95%CI 34 - 61) for anti-MPO positive AAV, and 16 (95%CI 9 - 26) for ANCA-negative AAV. The global estimation of comprehensiveness by capture-recapture analysis was 80.5%. The number of AAV cases was higher for non-European residents (P=0.001), particularly for MPA (P<0.0001). CONCLUSION: We provide a new estimate of AAV prevalence in France and show a higher prevalence of MPA in non-European patients.

Distinct laccase expression and activity profiles of Trametes versicolor facilitate degradation of benzo[a]pyrene.

A Trametes versicolor isolate from the Changbai Mountain showed promising activity in degrading benzo[a]pyrene (BaP), which is a high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH) compound. It was hypothesized that the T. versicolor isolate encode BaP-degrading enzymes, among which laccase is mostly sought after due to significant commercial potential. Genome of the T. versicolor isolate was sequenced and assembled, and seven laccase homologues were identified (TvLac1-7) as candidate genes potentially contributing to BaP degradation. In order to further identify the BaP responsive laccases, time-course transcriptomic and proteomic analyses were conducted in parallel on the T. versicolor isolate upon BaP treatment. Homologous laccases showed distinct expression patterns. Most strikingly, TvLac5 was rapidly induced in the secreted proteomes (secretomes), while TvLac2 was repressed. Recombinant laccase expression and biochemical characterization further showed corresponding enzymatic activity profiles, where TvLac5 was 21-fold more effective in BaP degradation compared to TvLac2. Moreover, TvLac5 also showed 3.6-fold higher BaP degrading activity compared to a commercial laccase product of T. versicolor origin. Therefore, TvLac5 was concluded to be a BaP-responsive enzyme from T. versicolor showing effective BaP degradation activity.

Therapeutic potential in rheumatic diseases of extracellular vesicles derived from mesenchymal stromal cells.

The incidence of rheumatic diseases such as rheumatoid arthritis and osteoarthritis and injuries to articular cartilage that lead to osteochondral defects is predicted to rise as a result of population ageing and the increase in high-intensity physical activities among young and middle-aged people. Current treatments focus on the management of pain and joint functionality to improve the patient's quality of life, but curative strategies are greatly desired. In the past two decades, the therapeutic value of mesenchymal stromal cells (MSCs) has been evaluated because of their regenerative potential, which is mainly attributed to the secretion of paracrine factors. Many of these factors are enclosed in extracellular vesicles (EVs) that reproduce the main functions of parental cells. MSC-derived EVs have anti-inflammatory, anti-apoptotic as well as pro-regenerative activities. Research on EVs has gained considerable attention as they are a potential cell-free therapy with lower immunogenicity and easier management than whole cells. MSC-derived EVs can rescue the pathogenetic phenotypes of chondrocytes and exert a protective effect in animal models of rheumatic disease. To facilitate the therapeutic use of EVs, appropriate cell sources for the production of EVs with the desired biological effects in each disease should be identified. Production and isolation of EVs should be optimized, and pre-isolation and post-isolation modifications should be considered to maximize the disease-modifying potential of the EVs.

Chapitre 12. Impact sociétal et éthique de la thérapie cellulaire et des biotechnologies.

Cell therapy is becoming established in many fields, including oncology with CAR-Ts or in regenerative medicine for cardiovascular diseases, diabetes or musculoskeletal disorders with mesenchymal stromal cells. These therapeutic cells are called advanced therapy medicinal products (ATMPs) and include all processes including cells manipulated to obtain reprogramming (iPS), to induce gene expression or by genome editing to modify the expression of a gene. The development of new biomaterial supports that can be 3D printed and take the desired shape of the target tissue before being colonised by the cellular elements necessary for their biological functions and replace the failing organ. All of these new technologies are driving innovation and the development of tomorrow’s bio-medicines. These new biotherapies will profoundly modify patient care in all areas, changing medical practices but with a considerable societal impact. Thus, the development and clinical research on cellular biotherapies are essential health issues but with a major ethical, societal and economic impact.

A least-squares-fitting procedure for an efficient preclinical ranking of passive transport across the blood-brain barrier endothelium.

The treatment of various disorders of the central nervous system (CNS) is often impeded by the limited brain exposure of drugs, which is regulated by the human blood-brain barrier (BBB). The screening of lead compounds for CNS penetration is challenging due to the biochemical complexity of the BBB, while experimental determination of permeability is not feasible for all types of compounds. Here we present a novel method for rapid preclinical screening of libraries of compounds by utilizing advancements in computing hardware, with its foundation in transition-based counting of the flux. This method has been experimentally validated for in vitro permeabilities and provides atomic-level insights into transport mechanisms. Our approach only requires a single high-temperature simulation to rank a compound relative to a library, with a typical simulation time converging within 24 to 72 h. The method offers unbiased thermodynamic and kinetic information to interpret the passive transport of small-molecule drugs across the BBB.

The two enantiomers of 2-hydroxyglutarate differentially regulate cytotoxic T cell function.

2-Hydroxyglutarate (2HG) is a byproduct of the tricarboxylic acid (TCA) cycle and is readily detected in the tissues of healthy individuals. 2HG is found in two enantiomeric forms: S-2HG and R-2HG. Here, we investigate the differential roles of these two enantiomers in cluster of differentiation (CD)8+ T cell biology, where we find they have highly divergent effects on proliferation, differentiation, and T cell function. We show here an analysis of structural determinants that likely underlie these differential effects on specific α-ketoglutarate (αKG)-dependent enzymes. Treatment of CD8+ T cells with exogenous S-2HG, but not R-2HG, increased CD8+ T cell fitness in vivo and enhanced anti-tumor activity. These data show that S-2HG and R-2HG should be considered as two distinct and important actors in the regulation of T cell function.

Complex deformation of cartilage micropellets following mechanical stimulation promotes chondrocyte gene expression.

BACKGROUND: Articular cartilage (AC)'s main function is to resist to a stressful mechanical environment, and chondrocytes are responding to mechanical stress for the development and homeostasis of this tissue. However, current knowledge on processes involved in response to mechanical stimulation is still limited. These mechanisms are commonly investigated in engineered cartilage models where the chondrocytes are included in an exogeneous biomaterial different from their natural extracellular matrix. The aim of the present study is to better understand the impact of mechanical stimulation on mesenchymal stromal cells (MSCs)-derived chondrocytes generated in their own extracellular matrix. METHODS: A fluidic custom-made device was used for the mechanical stimulation of cartilage micropellets obtained from human MSCs by culture in a chondrogenic medium for 21 days. Six micropellets were positioned into the conical wells of the device chamber and stimulated with different signals of positive pressure (amplitude, frequency and duration). A camera was used to record the sinking of each micropellet into their cone, and micropellet deformation was analyzed using a finite element model. Micropellets were harvested at different time points after stimulation for RT-qPCR and histology analysis. RESULTS: Moderate micropellet deformation was observed during stimulation with square pressure signals as mean von Mises strains between 6.39 and 14.35% were estimated for amplitudes of 1.75-14 kPa superimposed on a base pressure of 50% of the amplitude. The compression, tension and shear observed during deformation did not alter micropellet microstructure as shown by histological staining. A rapid and transient increase in the expression of chondrocyte markers (SOX9, AGG and COL2B) was measured after a single 30-min stimulation with a square pressure signal of 3.5 kPa amplitude superimposed on a minimum pressure of 1.75 kPa, at 1 Hz. A small change of 1% of cyclical deformations when using a square pressure signal instead of a constant pressure signal induced a fold change of 2 to 3 of chondrogenic gene expression. Moreover, the expression of fibrocartilage (COL I) or hypertrophic cartilage (COL X, MMP13 and ADAMTS5) was not significantly regulated, except for COL X. CONCLUSIONS: Our data demonstrate that the dynamic deformation of cartilage micropellets by fluidic-based compression modulates the expression of chondrocyte genes responsible for the production of a cartilage-like extracellular matrix. This lays the foundations for further investigating the chondrocyte mechanobiology and the cartilage growth under mechanical stimulation.

Adaptive host responses to infection can resemble parasitic manipulation.

Using a dynamic optimisation model for juvenile fish in stochastic food environments, we investigate optimal hormonal regulation, energy allocation and foraging behaviour of a growing host infected by a parasite that only incurs an energetic cost. We find it optimal for the infected host to have higher levels of orexin, growth and thyroid hormones, resulting in higher activity levels, increased foraging and faster growth. This growth strategy thus displays several of the fingerprints often associated with parasite manipulation: higher levels of metabolic hormones, faster growth, higher allocation to reserves (i.e. parasite-induced gigantism), higher risk-taking and eventually higher predation rate. However, there is no route for manipulation in our model, so these changes reflect adaptive host compensatory responses. Interestingly, several of these changes also increase the fitness of the parasite. Our results call for caution when interpreting observations of gigantism or risky host behaviours as parasite manipulation without further testing.

Senescence-Driven Inflammatory and Trophic Microenvironment Imprints Mesenchymal Stromal/Stem Cells in Osteoarthritic Patients.

Senescent cells promote progressive tissue degeneration through the establishment of a combined inflammatory and trophic microenvironment. The cellular senescence state has therefore emerged as a central driving mechanism of numerous age-related diseases, including osteoarthritis (OA), the most common rheumatic disease. Senescence hallmarks are detectable in chondrocytes, synoviocytes and sub-chondral bone cells. This study investigates how the senescence-driven microenvironment could impact the cell fate of resident osteoarticular mesenchymal stromal/stem cells (MSCs) that are hence contributing to OA disease progression. For that purpose, we performed a comparative gene expression analysis of MSCs isolated from healthy donors that were in vitro chronically exposed either to interferon-gamma (IFN-γ) or Transforming Growth Factor beta 1 (TGFß1), two archetypical factors produced by senescent cells. Both treatments reduced MSC self-renewal capacities by upregulating different senescence-driven cycle-dependent kinase inhibitors. Furthermore, a common set of differentially expressed genes was identified in both treated MSCs that was also found enriched in MSCs isolated from OA patients. These findings highlight an imprinting of OA MSCs by the senescent joint microenvironment that changes their matrisome gene expression. Altogether, this research gives new insights into OA etiology and points to new innovative therapeutic opportunities to treat OA patients.