Non-opioid analgesics and post-operative pain following transoral robotic surgery for oropharyngeal cancer.

OBJECTIVE: To investigate associations between multimodal analgesia and post-operative pain among patients undergoing transoral robotic surgery for oropharyngeal squamous cell carcinoma. METHODS: Records of patients who underwent surgery from 5 September 2012 to 30 November 2016 were abstracted. Associations were assessed using multivariable analysis. RESULTS: A total of 216 patients (mean age of 59.1 years, 89.4 per cent male) underwent transoral robotic surgery (92.6 per cent were human papilloma virus positive, 87.5 per cent had stage T1-T2 tumours, and 82.9 per cent had stage N0-N1 nodes). Gabapentin (n = 86) was not associated with a reduction in severe pain. Ibuprofen (n = 72) was administered less often in patients with severe pain. Gabapentin was not associated with increased post-operative sedation (p = 0.624) and ibuprofen was not associated with increased bleeding (p = 0.221). Post-operative opioid usage was not associated with surgical duration, pharyngotomy, bilateral neck dissections, tumour stage, tumour size, subsite or gabapentin. CONCLUSION: Scheduled low-dose gabapentin was not associated with improved pain control or increased respiratory depression. Ibuprofen was not associated with an increased risk of bleeding and may be under-utilised.

Differential Effects of Polyphenols on Insulin Proteolysis by the Insulin-Degrading Enzyme.

The insulin-degrading enzyme (IDE) possesses a strong ability to degrade insulin and Aß42 that has been linked to the neurodegeneration in Alzheimer's disease (AD). Given this, an attractive IDE-centric strategy for the development of therapeutics for AD is to boost IDE's activity for the clearance of Aß42 without offsetting insulin proteostasis. Recently, we showed that resveratrol enhances IDE's activity toward Aß42. In this work, we used a combination of chromatographic and spectroscopic techniques to investigate the effects of resveratrol on IDE's activity toward insulin. For comparison, we also studied epigallocatechin-3-gallate (EGCG). Our results show that the two polyphenols affect the IDE-dependent degradation of insulin in different ways: EGCG inhibits IDE while resveratrol has no effect. These findings suggest that polyphenols provide a path for developing therapeutic strategies that can selectively target IDE substrate specificity.

Intrinsic properties of primary hippocampal neurons contribute to PIP2 depletion during nsEP-induced physiological response.

High-energy, short-duration electric pulses (EPs) are known to be effective in neuromodulation, but the biological mechanisms underlying this effect remain unclear. Recently, we discovered that nanosecond electric pulses (nsEPs) could initiate the phosphatidylinositol4,5-bisphosphate (PIP2) depletion in non-excitable cells identical to agonist-induced activation of the Gq11 coupled receptors. PIP2 is the precursor for multiple intracellular second messengers critically involved in the regulation of intracellular Ca2+ homeostasis and plasma membrane (PM) ion channels responsible for the control of neuronal excitability. In this paper we demonstrate a novel finding that five day in vitro (DIV5) primary hippocampal neurons (PHNs) undergo significantly higher PIP2 depletion after 7.5 kV/cm 600 ns EP exposure than DIV1 PHNs and day 1-5 (D1-D5) non-excitable Chinese hamster ovarian cells with muscarinic receptor 1 (CHO-hM1). Despite the age of development, the stronger 15 kV/cm 600 ns or longer 7.5 kV/cm 12 µs EP initiated profound PIP2 depletion in all cells studied, outlining damage of the cellular PM and electroporation. Therefore, the intrinsic properties of PHNs in concert with nanoporation explain the stronger neuronal response to nsEP at lower intensity exposures. PIP2 reduction in neurons could be a primary biological mechanism responsible for the stimulation or inhibition of neuronal tissues.

Validation of a new fluidic device for mechanical stimulation and characterization of microspheres: A first step towards cartilage characterization.

Articular cartilage is made of chondrocytes surrounded by their extracellular matrix that can both sense and respond to various mechanical stimuli. One of the most widely used in vitro model to study cartilage growth is the model of mesenchymal stromal cells-derived cartilage micropellet. However, mechanical stimulation of micropellets has never been reported probably because of their small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of cartilage micropellets. The fluidic-based device was designed for the concomitant stimulation or characterization of six microspheres placed into the conical wells of a tank. In the present study, the device was validated using alginate-, collagen- and crosslinked collagen-based microspheres. Different types and ranges of pressure signals (square, sinusoidal and constant) were applied. The mechanical properties of microspheres were equivalent to those determined by a conventional compression test. Accuracy, repeatability and reproducibility of all types of pressure signals were demonstrated even though square signals were less accurate and sinusoidal signals were less reproducible than the others. The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with diameters in the range of 900 to 1500 µm. Mechanical stimulation can be performed on six microspheres in parallel allowing the mechanical and molecular characterization of the same group of cartilage micropellets. The device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli.

Helix Dipole and Membrane Electrostatics Delineate Conformational Transitions in the Self-Assembly of Amyloidogenic Peptides.

The self-assembly of amyloidogenic peptides on membrane surfaces is associated with the death of neurons and ß-cells in Alzheimer's disease and type 2 diabetes, respectively. The early events of self-assembly in vivo are not known, but there is increasing evidence for the importance of the α-helix. To test the hypothesis that electrostatic interactions involving the helix dipole play a key role in membrane-mediated peptide self-assembly, we studied IAPP[11-25(S20G)-NH2] (R11LANFLVHSGNNFGA25-NH2), which under certain conditions self-assembles in hydro to form ß-sheet assemblies through an α-helix-containing intermediate. In the presence of small unilamellar vesicles composed solely of zwitterionic lipids, the peptide does not self-assemble presumably because of the absence of stabilizing electrostatic interactions between the membrane surface and the helix dipole. In the presence of vesicles composed solely of anionic lipids, the peptide forms a long-lived α-helix presumably stabilized by dipole-dipole interactions between adjacent helix dipoles. This helix represents a kinetic trap that inhibits ß-sheet formation. Intriguingly, when the amount of anionic lipids was decreased to mimic the ratio of zwitterionic and anionic lipids in cells, the α-helix was short-lived and underwent an α-helix to ß-sheet conformational transition. Our work suggests that the helix dipole and membrane electrostatics delineate the conformational transitions occurring along the self-assembly pathway to the amyloid.

Mesenchymal stem cells-derived cartilage micropellets: A relevant in vitro model for biomechanical and mechanobiological studies of cartilage growth.

The prevalence of diseases that affect the articular cartilage is increasing due to population ageing, but the current treatments are only palliative. One innovative approach to repair cartilage defects is tissue engineering and the use of mesenchymal stem/stromal cells (MSCs). Although the combination of MSCs with biocompatible scaffolds has been extensively investigated, no product is commercially available yet. This could be explained by the lack of mechanical stimulation during in vitro culture and the absence of proper and stable cartilage matrix formation, leading to poor integration after implantation. The objective of the present study was to investigate the biomechanical behaviour of MSC differentiation in micropellets, a well-defined 3D in vitro model of cartilage differentiation and growth, in view of tissue engineering applications. MSC micropellet chondrogenic differentiation was induced by exposure to TGFß3. At different time points during differentiation (35 days of culture), their global mechanical properties were assessed using a very sensitive compression device coupled to an identification procedure based on a finite element parametric model. Micropellets displayed both a non-linear strain-induced stiffening behaviour and a dissipative behaviour that increased from day 14 to day 29, with a maximum instantaneous Young's modulus of 179.9 ± 18.8 kPa. Moreover, chondrocyte gene expression levels were strongly correlated with the observed mechanical properties. This study indicates that cartilage micropellets display the biochemical and biomechanical characteristics required for investigating and recapitulating the different stages of cartilage development.

Inhibition of the Self-Assembly of Aß and of Tau by Polyphenols: Mechanistic Studies.

The amyloid-ß (Aß) peptide and tau protein are thought to play key neuropathogenic roles in Alzheimer's disease (AD). Both Aß and tau self-assemble to form the two major pathological hallmarks of AD: amyloid plaques and neurofibrillary tangles, respectively. In this review, we show that naturally occurring polyphenols abundant in fruits, vegetables, red wine, and tea possess the ability to target pathways associated with the formation of assemblies of Aß and tau. Polyphenols modulate the enzymatic processing of the amyloid-ß precursor protein and inhibit toxic Aß oligomerization by enhancing the clearance of Aß42 monomer, modulating monomer-monomer interactions and remodeling oligomers to non-toxic forms. Additionally, polyphenols modulate tau hyperphosphorylation and inhibit tau ß-sheet formation. The anti-Aß-self-assembly and anti-tau-self-assembly effects of polyphenols increase their potential as preventive or therapeutic agents against AD, a complex disease that involves many pathological mechanisms.

TGFßi is involved in the chondrogenic differentiation of mesenchymal stem cells and is dysregulated in osteoarthritis.

OBJECTIVE: Transforming growth factor-ß (TGFß) is a major regulator of cartilage homeostasis and its deregulation has been associated with osteoarthritis (OA). Deregulation of the TGFß pathway in mesenchymal stem cells (MSCs) has been proposed to be at the onset of OA. Using a secretome analysis, we identified a member of the TGFß family, TGFß-induced protein (TGFßi or ßIGH3), expressed in MSCs and we investigated its function and regulation during OA. DESIGN: Cartilage, bone, synovium, infrapatellar fat pad and bone marrow-MSCs were isolated from patients with OA or healthy subjects. Chondrogenesis of BM-MSCs was induced by TGFß3 in micropellet culture. Expression of TGFßi was quantified by RT-qPCR, ELISA or immunohistochemistry. Role of TGFßi was investigated in gain and loss of function experiments in BM-MSCs and chondrocytes. RESULTS: TGFßi was up-regulated in early stages of chondrogenesis and its knock-down in BM-MSCs resulted in the down-regulation of mature and hypertrophic chondrocyte markers. It likely occurred through the modulation of adhesion molecules including integrin (ITG)ß1, ITGß5 and N-cadherin. We also showed that TGFßi was upregulated in vitro in a model of OA chondrocytes, and its silencing enhanced the hypertrophic marker type X collagen. In addition, TGFßi was up-regulated in bone and cartilage from OA patients while its expression was reduced in BM-MSCs. Similar findings were observed in a murine model of OA. CONCLUSIONS: Our results revealed a dual role of TGFßi during chondrogenesis and pointed its deregulation in OA joint tissues. Modulating TGFßi in BM-MSCs might be of interest in cartilage regenerative medicine.

Resveratrol Sustains Insulin-Degrading Enzyme Activity toward Aß42.

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is the sixth leading cause of death in the United States. We hypothesize that the impaired clearance of Aß42 from the brain is partly responsible for the onset of sporadic AD. In this work, we evaluated the activity of insulin-degrading enzyme (IDE) toward Aß42 in the presence of resveratrol, a polyphenol found in red wine and grape juice. By liquid chromatography/mass spectrometry, we identified initial cleavage sites in the absence and presence of resveratrol that carry biological relevance connected to the amyloidogenic properties of Aß42. Incubation with resveratrol results in a substantial increase in Aß42 fragmentation compared to the control, signifying that the polyphenol sustains IDE-dependent degradation of Aß42 and its fragments. Our findings suggest that therapeutic and/or preventative approaches combining resveratrol and IDE may hold promise for sporadic AD.

Enzyme kinetics from circular dichroism of insulin reveals mechanistic insights into the regulation of insulin-degrading enzyme.

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that selectively degrades biologically important substrates associated with type 2 diabetes and Alzheimer's disease (AD). As such, IDE is an attractive target for therapeutic innovations. A major requirement is an understanding of how other molecules present in cells regulate the activity of the enzyme toward insulin, IDE's most important physiologically relevant substrate. Previous kinetic studies of the IDE-dependent degradation of insulin in the presence of potential regulators have used iodinated insulin, a chemical modification that has been shown to alter the biological and biochemical properties of insulin. Here, we present a novel kinetic assay that takes advantage of the loss of helical circular dichroic signals of insulin with IDE-dependent degradation. As proof of concept, the resulting Michaelis-Menten kinetic constants accurately predict the known regulation of IDE by adenosine triphosphate (ATP). Intriguingly, we found that when Mg2+ is present with ATP, the regulation is abolished. The implication of this result for the development of preventative and therapeutic strategies for AD is discussed. We anticipate that the new assay presented here will lead to the identification of other small molecules that regulate the activity of IDE toward insulin.

The Longest Amyloid-ß Precursor Protein Intracellular Domain Produced with Aß42 Forms ß-Sheet-Containing Monomers That Self-Assemble and Are Proteolyzed by Insulin-Degrading Enzyme.

Alzheimer's disease (AD) is the most common neurodegenerative disease resulting in dementia. It is characterized pathologically by extracellular amyloid plaques composed mainly of deposited Aß42 and intracellular neurofibrillary tangles formed by hyperphosphorylated tau protein. Recent clinical trials targeting Aß have failed, suggesting that other polypeptides produced from the amyloid-ß precursor protein (APP) may be involved in AD. An attractive polypeptide is AICD57, the longest APP intracellular domain (AICD) coproduced with Aß42. Here, we show that AICD57 forms micelle-like assemblies that are proteolyzed by insulin-degrading enzyme (IDE), indicating that AICD57 monomers are in dynamic equilibrium with AICD57 assemblies. The N-terminal part of AICD57 monomer is not degraded, but its C-terminal part is hydrolyzed, particularly in the YENPTY motif that has been associated with the hyperphosphorylation of tau. Therefore, sustaining IDE activity well into old age holds promise for regulating levels of not only Aß but also AICD in the aging brain.

Mechanistic Studies of the Inhibition of Insulin Fibril Formation by Rosmarinic Acid.

The self-assembly of insulin to form amyloid fibrils has been widely studied because it is a significant problem in the medical management of diabetes and is an important model system for the investigation of amyloid formation and its inhibition. A few inhibitors of insulin fibrillation have been identified with potencies that could be higher. Knowledge of how these work at the molecular level is not known but important for the development of more potent inhibitors. Here we show that rosmarinic acid completely inhibits amyloid formation by dimeric insulin at pH 2 and 60 °C. In contrast to other polyphenols, rosmarinic acid is soluble in water and does not degrade at elevated temperatures, and thus we were able to decipher the mechanism of inhibition by a combination of solution-state 1H NMR spectroscopy and molecular docking. On the basis of 1H chemical shift perturbations, intermolecular nuclear Overhauser effect enhancements between rosmarinic acid and specific residues of insulin, and slowed dynamics of rosmarinic acid in the presence of insulin, we show that rosmarinic acid binds to a pocket found on the surface of each insulin monomer. This results in the formation of a mixed tetramolecular aromatic network on the surface of insulin dimer, resulting in increased resistance of the amyloidogenic protein to thermal unfolding. This finding opens new avenues for the design of potent inhibitors of amyloid formation and provides strong experimental evidence for the role of surface aromatic clusters in increasing the thermal stability of proteins.

Long-term mortality in outpatients with type 2 diabetes in a reference hospital in Cameroon: a retrospective cohort study.

OBJECTIVES: There are limited data on mortality in patients with type 2 diabetes mellitus (T2DM) in Sub-Saharan Africa. We aimed at determining the mortality rate, and the causes and the predictors of death in patients with T2DM followed as outpatients in a reference hospital in Cameroon. DESIGN: Retrospective cohort study. SETTING: A reference hospital in Cameroon. PARTICIPANTS: From December 2015 to March 2016, patients with T2DM aged 18 years and older and who consulted between January 2009 and December 2014, were contacted directly or through their next of kin, and included in this study. All participants with less than 75% of desired data in files, those who could not be reached on the phone and those who refused to provide consent were excluded from the study. Of the 940 eligible patients, 628 (352 men and 276 women) were included and completed the study, giving a response rate of 66.8%. OUTCOME MEASURES: Death rate, causes of death and predictors of death. RESULTS: Of the 628 patients (mean age: 56.5 years; median diabetes duration: 3.5 years) followed up for a total of 2161 person-years, 54 died, giving a mortality rate of 2.5 per 100 person-years and a cumulative mortality rate of 8.6%. Acute metabolic complications (22.2%), cardiovascular diseases (16.7%), cancers (14.8%), nephropathy (14.8%) and diabetic foot syndrome (13.0%) were the most common causes of death. Advanced age (adjusted HR (aHR) 1.06, 95% CI 1.02 to 1.10; P=0.002), raised glycated haemoglobin (HbA1c) (aHR 1.16, 95% CI 1.00 to 1.35; P=0.051), low blood haemoglobin (aHR 1.06, 95% CI 1.02 to 1.10; P=0.002) and proteinuria (aHR 2.97, 95% CI 1.40 to 6.28; P=0.004) were identified as independent predictors of death. CONCLUSIONS: The mortality rate in patients with T2DM is high in our population, with acute metabolic complications as the leading cause. Patients with advanced age, raised HbA1c, anaemia or proteinuria are at higher risk of death and therefore represent the target of interest to prevent mortality in T2DM.

Cutaneous nerve biomarkers in transthyretin familial amyloid polyneuropathy.

OBJECTIVE: To determine the utility of skin biopsies as a biomarker of disease severity in subjects with amyloid neuropathy. METHODS: Five groups of patients were studied: (1) transthyretin (TTR) familial amyloidotic polyneuropathy (FAP; n = 20), (2) TTR mutation carriers without peripheral neuropathy (TTR-noPN; n = 10), (3) healthy controls (n = 20), (4) diabetic neuropathy disease controls (n = 20), and (5) patients with light-chain (AL) amyloid (n = 2). All subjects underwent neurological examination and 3mm skin biopsies. Sections were stained with anti-PGP9.5, anti-TTR, and Congo red. Intraepidermal (IENFD), sweat gland (SGNFD), and pilomotor nerve fiber densities (PMNFD) were measured. Correlations between the amount of amyloid present (amyloid burden), fiber subtype, and Neuropathy Impairment Score in the Lower Limbs (NIS-LL) were evaluated. RESULTS: IENFD, SGNFD, and PMNFD were all significantly reduced in TTR-FAP patients versus healthy controls, whereas TTR-noPN subjects had intermediate reductions. Lower nerve fiber densities were associated with NIS-LL (p < 0.001). Congo red staining revealed brilliant red amyloid deposits confirmed by apple-green birefringence within dermal collagen, sweat glands, and arrector pili that engulfed axons. The diagnostic sensitivity and specificity to detect amyloid in skin were 70% and 100%. Both AL amyloidosis and 2 of 10 TTR-noPN subjects were Congo red-positive. Amyloid burden correlated with IENFD (r = -0.63), SGNFD (r = -0.67), PMNFD (r = -0.50), and NIS-LL (r = -0.57). Wild-type TTR staining was less prominent in TTR-FAP patients. INTERPRETATION: Cutaneous amyloid was detected in 70% of TTR-FAP and 20% of TTR-noPN subjects. Amyloid burden correlated strongly with reductions in IENFD, SGNFD, PMNFD, and NIS-LL. Skin is an attractive tissue to establish an amyloid diagnosis, and amyloid burden has potential as a biomarker to detect treatment effect in TTR-FAP drug trials. Ann Neurol 2017;82:44-56.

Systemic sclerosis and exposure to heavy metals: A case control study of 100 patients and 300 controls.

OBJECTIVE: This case control study assessed: 1) the relationship of systemic sclerosis (SSc) related to exposure to heavy metals; and 2) the risk of SSc related to occupational exposure in male and female patients. METHODS: From 2005 to 2008, 100 patients with a definite diagnosis of SSc were included in the study; 3 age, gender, and smoking habit matched controls were selected for each patient. All SSc patients and controls underwent detection and quantification of heavy metal traces in hair samples, using multi-element inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: SSc patients exhibited higher median levels of the following metals: antimony (p=0.001), cadmium (p=0.0003), lead (p=0.02), mercury (p=0.02), molybdenum (p=0.04), palladium (p<0.0001) and zinc (p=0.0003). A marked association between SSc and occupational exposure was further found for: 1) antimony (p=0.008) and platinum (p=0.04) in male patients; and 2) antimony (p=0.02), cadmium (p=0.001), lead (p=0.03), mercury (p=0.03), palladium (p=0.0003) and zinc (p=0.0001) in female patients CONCLUSION: The results show the impact of occupational risk factors in the development of SSc for: antimony, cadmium, lead, mercury, molybdenum, palladium and zinc. Thus, occupational exposure should be systematically checked in all SSc patients at diagnosis. Finally, the association between SSc and occupational exposure may be variable according to patients' gender.

PPARß/δ directs the therapeutic potential of mesenchymal stem cells in arthritis.

OBJECTIVES: To define how peroxisome proliferator-activated receptor (PPAR) ß/δ expression level in mesenchymal stem cells (MSCs) could predict and direct both their immunosuppressive and therapeutic properties. PPARß/δ interacts with factors such as nuclear factor-kappa B (NF-κB) and regulates the expression of molecules including vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1. Since these molecules are critical for MSC function, we investigated the role of PPARß/δ on MSC immunosuppressive properties. METHODS: We either treated human MSCs (hMSCs) with the irreversible PPARß/δ antagonist (GSK3787) or derived MSCs from mice deficient for PPARß/δ (PPARß/δ-/- MSCs). We used the collagen-induced arthritis (CIA) as model of immune-mediated disorder and the MSC-immune cell coculture assays. RESULTS: Modulation of PPARß/δ expression in hMSCs either using GSK3787 or hMSCs from different origin reveals that MSC immunosuppressive potential is inversely correlated with Ppard expression. This was consistent with the higher capacity of PPARß/δ-/- MSCs to inhibit both the proliferation of T lymphocytes, in vitro, and arthritic development and progression in CIA compared with PPARß/δ+/+ MSCs. When primed with proinflammatory cytokines to exhibit an immunoregulatory phenotype, PPARß/δ-/- MSCs expressed a higher level of mediators of MSC immunosuppression including VCAM-1, ICAM-1 and nitric oxide (NO) than PPARß/δ+/+ MSCs. The enhanced NO2 production by PPARß/δ-/- MSCs was due to the increased retention of NF-κB p65 subunit on the κB elements of the inducible nitric oxide synthase promoter resulting from PPARß/δ silencing. CONCLUSIONS: Our study is the first to show that the inhibition or knockdown of PPARß/δ in MSCs primes their immunoregulatory functions. Thus, the regulation of PPARß/δ expression provides a new strategy to generate therapeutic MSCs with a stable regulatory phenotype.

Mesenchymal stem cells for the management of inflammation in osteoarthritis: state of the art and perspectives.

Osteoarthritis (OA) is the most common form of degenerative arthritis, mainly characterized by the degradation of articular cartilage and associated with subchondral bone lesions. Novel therapeutic approaches for OA include cell-based therapies that have become thriving areas of research and development. In this context, mesenchymal stem or stromal cells (MSCs) have gained much interest based on their trophic and immunomodulatory properties that can help tissue repair/regeneration. The present review article discusses the interest of using MSCs in cell-therapy approaches with a focus on the mechanisms by which MSCs might exhibit a therapeutic potential in OA. Special attention is given to the anti-inflammatory function of MSCs and on miRNA modulation in OA for possible future innovative strategies. The paper also presents the current data on the undergoing MSCs-based clinical trials in OA.

Lack of anti-inflammatory and anti-catabolic effects on basal inflamed osteoarthritic chondrocytes or synoviocytes by adipose stem cell-conditioned medium.

OBJECTIVE: To define whether good manufacturing practice (GMP)-clinical grade adipose stem cell (ASC)-derived conditioned medium (CM) is as effective as GMP-ASC in modulating inflammatory and catabolic factors released by both osteoarthritis (OA) chondrocytes or synoviocytes. METHODS: OA chondrocytes and synoviocytes were treated with ASC-CM or co-cultured with ASC. Inflammatory factors (IL6, CXCL1/GROα,CXCL8/IL8, CCL2/MCP-1, CCL3/MIP-1α and CCL5/RANTES) and proteinases, such as metalloproteinase (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS4, ADAMTS5) and their tissue metalloproteinase inhibitors (TIMP1, TIMP3) were evaluated by qRT-PCR or immunoassays. The involvement of prostaglandin E2 (PGE2) was also analyzed. RESULTS: Most ASC-CM ratios tested did not decrease IL6, CCL2/MCP-1, CCL3/MIP1-α, CCL5/RANTES on basal inflamed chondrocytes or synoviocytes in contrast to what we found using ASC in co-culture. CXCL8/IL8 and CXCL1/GROα were not decreased by ASC-CM on synoviocytes but were only partially reduced on chondrocytes. Moreover, ASC-CM was less efficient both on basal inflamed OA chondrocytes and synoviocytes in reducing proteinases, such as MMP13, ADAMTS4, ADAMTS5 and increasing TIMP1 and TIMP3 compared to ASC in co-culture. The different ratios of ASC-CM contain lower amounts of PGE2 which were not sufficient to reduce inflammatory factors. CONCLUSIONS: These data show that ASC-CM has a limited ability to decrease inflammatory and proteinases factors produced by OA chondrocytes or synoviocytes. ASC-CM is not sufficient to recapitulate the beneficial effect demonstrated using ASC in co-culture with inflamed OA chondrocytes and synoviocytes and shows that their use in clinical trials is fundamental to counteract OA progression.

Glucocorticoid-induced leucine zipper governs the therapeutic potential of mesenchymal stem cells by inducing a switch from pathogenic to regulatory Th17 cells in a mouse model of collagen-induced arthritis.

OBJECTIVE: Mesenchymal stem cells (MSCs) are potent immunosuppressive cells that have shown promise in the treatment of rheumatoid arthritis (RA). Deciphering the intrinsic characteristics of MSCs that correlate with their biologic activity will facilitate their clinical use. Recently, the role of glucocorticoid-induced leucine zipper (GILZ) in the development of RA has been documented. The aim of this study was to evaluate whether GILZ expression by MSCs may contribute to their therapeutic effect. METHODS: MSCs were isolated from GILZ-deficient (GILZ(-/-) ) mice and wild-type mice. MSCs (1 × 10(6) cells) were injected twice via the tail vein into mice with collagen-induced arthritis (CIA). RESULTS: In vitro, we showed that GILZ is a key factor involved in the immunosuppressive potential of MSCs. MSCs derived from GILZ(-/-) mice did not suppress the proliferation of CD4+ T cells and were less efficient than MSCs derived from WT mice in altering Th17 cell polarization. Thus, we investigated the role of GILZ in an experimental model of arthritis and demonstrated that although WT MSCs significantly reduced paw swelling in arthritic mice, GILZ(-/-) MSCs did not. Moreover, the magnitude of the effects of GILZ(-/-) MSCs on Th17 cell frequency was significantly lower than that of WT MSCs. The therapeutic effect of MSCs correlated with the generation of Treg cells bearing the CD4 + RORγt+IL-17(low) IL-10+ signature, and Th17 cell polarization was GILZ dependent. CONCLUSION: This study demonstrates that GILZ has an essential role in the therapeutic effectiveness of MSCs in arthritis by favoring Th17 cell polarization toward a regulatory phenotype. Therefore, potentiation of GILZ expression in MSCs could represent a means to enhance their therapeutic effect in autoimmune diseases.