Enhanced expression of natural cytotoxicity receptors on cytokine-induced memory-like natural killer cells correlates with effector function.

Introduction: Natural killer (NK) cells are a key component of the innate immune system, involved in defending the host against virus-infected cells and tumor immunosurveillance. Under in vitro culture conditions, IL-12/15/18 can induce a memory-like phenotype in NK cells. These cytokine-induced memory-like (CIML) NK cells possess desirable characteristics for immunotherapies, including a longer lifespan and increased cytotoxicity. Methods: In this study, NK cells were isolated from peripheral blood of healthy donors and stimulated with IL-12/15/18 to induce a memory-like phenotype or with IL-15 alone as a control. After seven days of culture, multiparametric flow cytometry analysis was performed to evaluate the phenotypic and functional profiles of CIML and control NK cells. Results: Our results showed a significantly higher expression of CD25, CD69, NKG2D, NKp30, NKp44, NKp46, TACTILE, and Granzyme B in CIML NK cells compared to control NK cells. In contrast, KIR2D expression was significantly lower in CIML NK cells than in control NK cells. Moreover, functional experiments demonstrated that CIML NK cells displayed enhanced degranulation capacity and increased intracellular IFN-γ production against the target cell line K562. Interestingly, the degranulation capacity of CIML NK cells was positively correlated with the expression of the activating receptors NKp46 and NKp30, as well as with the inhibitory receptor TACTILE. Discussion: In conclusion, this study provides a deep phenotypic characterization of in vitro-expanded CIML NK cells. Moreover, the correlations found between NK cell receptors and degranulation capacity of CIML NK cells allowed the identification of several biomarkers that could be useful in clinical settings.

Menstrual blood-derived mesenchymal stromal cells: impact of preconditioning on the cargo of extracellular vesicles as potential therapeutics.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia). METHODS: MenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O2), proinflammatory stimuli (IFNγ and TNFα, 21% O2), physioxia (1-2% O2), and acute hypoxia (< 1% O2) for 48-72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties. RESULTS: No differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response. CONCLUSIONS: Priming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.

A Fibrin Coating Method of Polypropylene Meshes Enables the Adhesion of Menstrual Blood-Derived Mesenchymal Stromal Cells: A New Delivery Strategy for Stem Cell-Based Therapies.

Polypropylene (PP) mesh is well-known as a gold standard of all prosthetic materials of choice for the reinforcement of soft tissues in case of hernia, organ prolapse, and urinary incontinence. The adverse effects that follow surgical mesh implantation remain an unmet medical challenge. Herein, it is outlined a new approach to allow viability and adhesion of human menstrual blood-derived mesenchymal stromal cells (MenSCs) on PP surgical meshes. A multilayered fibrin coating, based on fibrinogen and thrombin from a commercial fibrin sealant, was optimized to guarantee a homogeneous and stratified film on PP mesh. MenSCs were seeded on the optimized fibrin-coated meshes and their adhesion, viability, phenotype, gene expression, and immunomodulatory capacity were fully evaluated. This coating guaranteed MenSC viability, adhesion and did not trigger any change in their stemness and inflammatory profile. Additionally, MenSCs seeded on fibrin-coated meshes significantly decreased CD4+ and CD8+ T cell proliferation, compared to in vitro stimulated lymphocytes (p < 0.0001). Hence, the proposed fibrin coating for PP surgical meshes may allow the local administration of stromal cells and the reduction of the exacerbated inflammatory response following mesh implantation surgery. Reproducible and easy to adapt to other cell types, this method undoubtedly requires a multidisciplinary and translational approach to be improved for future clinical uses.

IFN-Gamma and TNF-Alpha as a Priming Strategy to Enhance the Immunomodulatory Capacity of Secretomes from Menstrual Blood-Derived Stromal Cells.

Mesenchymal stromal cells isolated from menstrual blood (MenSCs) exhibit a potent pro-angiogenic and immunomodulatory capacity. Their therapeutic effect is mediated by paracrine mediators released by their secretomes. In this work, we aimed to evaluate the effect of a specific priming condition on the phenotype and secretome content of MenSCs. Our results revealed that the optimal condition for priming MenSCs was the combination of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) that produced a synergistic and additive effect on IDO1 release and immune-related molecule expression. The analyses of MenSC-derived secretomes after IFNγ and TNFα priming also revealed an increase in EV release and in the differentially expressed miRNAs involved in the immune response and inflammation. Proliferation assays on lymphocyte subsets demonstrated a decrease in CD4+ T cells and CD8+ T cells co-cultured with secretomes, especially in the lymphocytes co-cultured with secretomes from primed cells. Additionally, the expression of immune checkpoints (PD-1 and CTLA-4) was increased in the CD4+ T cells co-cultured with MenSC-derived secretomes. These findings demonstrate that the combination of IFNγ and TNFα represents an excellent priming strategy to enhance the immunomodulatory capacity of MenSCs. Moreover, the secretome derived from primed MenSCs may be postulated as a therapeutic option for the regulation of adverse inflammatory reactions.

The epicardial delivery of cardiosphere derived cells or their extracellular vesicles is safe but of limited value in experimental infarction.

The epicardial administration of therapeutics via the pericardial sac offers an attractive route, since it is minimally invasive and carries no risks of coronary embolization. The aim of this study was to assess viability, safety and effectiveness of cardiosphere-derived cells (CDCs), their extracellular vesicles (EVs) or placebo administered via a mini-thoracotomy 72 h after experimental infarction in swine. The epicardial administration was completed successfully in all cases in a surgery time (knife-to-skin) below 30 min. No significant differences between groups were found in cardiac function parameters evaluated using magnetic resonance imaging before therapy and at the end of the study, despite a trend towards improved function in CDC-treated animals. Moreover, infarct size at 10 weeks was smaller in treated animals, albeit not significantly. Arrhythmia inducibility did not differ between groups. Pathological examination showed no differences, nor were there any pericardial adhesions evidenced in any case 10 weeks after surgery. These results show that the epicardial delivery of CDCs or their EVs is safe and technically easy 3 days after experimental myocardial infarction in swine, but it does not appear to have any beneficial effect on cardiac function. Our results do not support clinical translation of these therapies as implemented in this work.

Characterization of encapsulated porcine cardiosphere-derived cells embedded in 3D alginate matrices.

Myocardial infarction is caused by an interruption of coronary blood flow, leading to one of the main death causes worldwide. Current therapeutic approaches are palliative and not able to solve the loss of cardiac tissue. Cardiosphere derived cells (CDCs) reduce scarring, and increase viable myocardium, with safety and adequate biodistribution, but show a low rate engraftment and survival after implantation. In order to solve the low retention, we propose the encapsulation of CDCs within three-dimensional alginate-poly-L-lysine-alginate matrix as therapy for cardiac regeneration. In this work, we demonstrate the encapsulation of CDCs in alginate matrix, with no decrease in viability over a month, and showing the preservation of CDCs phenotype, differentiation potential, gene expression profile and growth factor release after encapsulation, moving a step forward to clinical translation of CDCs therapy in regeneration in heart failure.

Laparoscopy for the Treatment of Congenital Hernia: Use of Surgical Meshes and Mesenchymal Stem Cells in a Clinically Relevant Animal Model.

More than a century has passed since the first surgical mesh for hernia repair was developed, and, to date, this is still the most widely used method despite the great number of complications it poses. The purpose of this study was to combine stem cell therapy and laparoscopy for the treatment of congenital hernia in a swine animal model. Porcine bone marrow-derived mesenchymal stem cells (MSCs) were seeded on polypropylene surgical meshes using a fibrin sealant solution as a vehicle. Meshes with (cell group) or without (control group) MSCs were implanted through laparoscopy in Large White pigs with congenital abdominal hernia after the approximation of hernia borders (implantation day). A successive laparoscopic biopsy of the mesh and its surrounding tissues was performed a week after implantation, and surgical meshes were excised a month after implantation. Ultrasonography was used to measure hernia sizes. Flow cytometry, histological, and gene expression analyses of the biopsy and necropsy samples were performed. The fibrin sealant solution was easy to prepare and preserved the viability of MSCs in the surgical meshes. Ultrasonography demonstrated a significant reduction in hernia size 1 week after implantation in the cell group relative to that on the day of implantation (p < 0.05). Flow cytometry of the mesh-infiltrated cells showed a non-significant increase of M2 macrophages when the cell group was compared with the control group 1 week after implantation. A significant decrease in the gene expression of VEGF and a significant increase in TNF expression were determined in the cell group 1 month after implantation compared with gene expressions in the control group (p < 0.05). Here, we propose an easy and feasible method to combine stem cell therapy and minimally invasive surgical techniques for hernia repair. In this study, stem cell therapy did not show a great immunomodulatory or regenerative effect in overcoming hernia-related complications. However, our clinically relevant animal model with congenital hernia closely resembles the clinical human condition. Further studies should be focused on this valuable animal model to evaluate stem cell therapies in hernia surgery.

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Dose-dependent improvement of cardiac function in a swine model of acute myocardial infarction after intracoronary administration of allogeneic heart-derived cells.

BACKGROUND: Allogeneic cardiac-derived progenitor cells (CPC) without immunosuppression could provide an effective ancillary therapy to improve cardiac function in reperfused myocardial infarction. We set out to perform a comprehensive preclinical feasibility and safety evaluation of porcine CPC (pCPC) in the infarcted porcine model, analyzing biodistribution and mid-term efficacy, as well as safety in healthy non-infarcted swine. METHODS: The expression profile of several pCPC isolates was compared with humans using both FACS and RT-qPCR. ELISA was used to compare the functional secretome. One week after infarction, female swine received an intracoronary (IC) infusion of vehicle (CON), 25 × 106 pCPC (25 M), or 50 × 106 pCPC (50 M). Animals were followed up for 10 weeks using serial cardiac magnetic resonance imaging to assess functional and structural remodeling (left ventricular ejection fraction (LVEF), systolic and diastolic volumes, and myocardial salvage index). Statistical comparisons were performed using Kruskal-Wallis and Mann-Whitney U tests. Biodistribution analysis of 18F-FDG-labeled pCPC was also performed 4 h after infarction in a different subset of animals. RESULTS: Phenotypic and functional characterization of pCPC revealed a gene expression profile comparable to their human counterparts as well as preliminary functional equivalence. Left ventricular functional and structural remodeling showed significantly increased LVEF 10 weeks after IC administration of 50 M pCPC, associated to the recovery of left ventricular volumes that returned to pre-infarction values (LVEF at 10 weeks was 42.1 ± 10.0% in CON, 46.5 ± 7.4% in 25 M, and 50.2 ± 4.9% in 50 M, p < 0.05). Infarct remodeling was also improved following pCPC infusion with a significantly higher myocardial salvage index in both treated groups (0.35 ± 0.20 in CON; 0.61 ± 0.20, p = 0.04, in 25 M; and 0.63 ± 0.17, p = 0.01, in 50 M). Biodistribution studies demonstrated cardiac tropism 4 h after IC administration, with substantial myocardial retention of pCPC-associated tracer activity (18% of labeled cells in the heart), and no obstruction of coronary flow, indicating their suitability as a cell therapy product. CONCLUSIONS: IC administration of allogeneic pCPC at 1 week after acute myocardial infarction is feasible, safe, and associated with marked structural and functional benefit. The robust cardiac tropism of pCPC and the paracrine effects on left ventricle post-infarction remodeling established the preclinical bases for the CAREMI clinical trial (NCT02439398).

Extracellular vesicles derived from endometrial human mesenchymal stem cells enhance embryo yield and quality in an aged murine model†.

Advanced age is a risk factor undermining women's fertility. Hence, the optimization of assisted reproduction techniques is an interdisciplinary challenge that requires the improvement of in vitro culture systems. Here, we hypothesize that supplementation of embryo culture medium with extracellular vesicles from endometrial-derived mesenchymal stem cells (EV-endMSCs) may have a positive impact on the embryo competence of aged oocytes. In this work, 24 weeks old B6D2 female mice were used as egg donors and in vitro fertilization assays were performed using males from the same strain (8-12 weeks); the presumptive zygotes were incubated in the presence of 0, 10, 20, 40, or 80 µg/ml of EV-endMSCs. The results from the proteomic analysis of EV-endMSCs and the classification by Reactome pathways allowed us to identify proteins closely related with the fertilization process. Moreover, in our aged murine model, the supplementation of the embryo culture medium with EV-endMSCs improved the developmental competence of the embryos as well as the total blastomere count. Finally, gene expression analysis of murine blastocysts showed significant changes on core genes related to cellular response to oxidative stress, metabolism, placentation, and trophectoderm/inner cell mass formation. In summary, we demonstrate that EV-endMSCs increase the quality of the embryos, and according to proteomic and genomic analysis, presumably by modulating the expression of antioxidant enzymes and promoting pluripotent activity. Therefore, EV-endMSCs could be a valuable tool in human assisted reproduction improving the developmental competence of aged oocytes and increasing the odds of implantation and subsequent delivery.

Ischemia-reperfusion injury in a rat microvascular skin free flap model: A histological, genetic, and blood flow study.

Ischemia reperfusion injury is associated with tissue damage and inflammation, and is one of the main factors causing flap failure in reconstructive microsurgery. Although ischemia-reperfusion (I/R) injury is a well-studied aspect of flap survival, its biological mechanisms remain to be elucidated. To better understand the biological processes of ischemia reperfusion injury, and to develop further therapeutic strategies, the main objective of this study was to identify the gene expression pattern and histological changes in an I/R injury animal model. Fourteen rats (n = 7/group) were randomly divided into control or ischemia-reperfusion group (8 hours of ischemia). Microsurgical anastomoses were objectively assessed using transit-time-ultrasound technology. Seven days after surgery, flap survival was evaluated and tissue samples were harvested for anatomopathological and gene-expression analyses.The I/R injury reduced the survival of free flaps and histological analyses revealed a subcutaneous edema together with an inflammatory infiltrate. Interestingly, the Arginase 1 expression level as well as the ratio of Arginase 1/Nitric oxide synthase 2 showed a significant increase in the I/R group. In summary, here we describe a well-characterized I/R animal model that may serve to evaluate therapeutic agents under reproducible and controlled conditions. Moreover, this model could be especially useful for the evaluation of arginase inhibitors and different compounds of potential interest in reconstructive microsurgery.

Extracellular vesicles derived from endometrial human mesenchymal stem cells improve IVF outcome in an aged murine model.

Advanced age reduces the success of in vitro fertilization (IVF) being this effect partly mediated by an overproduction of reactive oxygen species (ROS) that trigger apoptosis. It has been demonstrated that extracellular vesicles derived from endometrial mesenchymal stem cells (EV-endMSCs) exert an antioxidant effect and can be used as IVF coadjutants. In this work, endMSCs were isolated from human menstrual blood (n = 4) and characterized according to multipotentiality and surface marker expression prior EV-endMSCs isolation. Oocytes were obtained from 21 B6D2 mice (24 weeks) and coincubated with sperm from young males (8-12 weeks). Presumptive zygotes were incubated in the presence of 0, 10, 20, 40 or 80 µg/ml of EV-endMSCs in KSOM medium. Blastocyst yield was evaluated, and 25 blastocysts per group were used for qPCR. Blastocyst rate was 29.4% in control; 45.2% for 10 µg/ml, 62.9% for 20 µg/ml, 55.5% for 40 µg/ml and 53.8% in the 80 µg/ml (n = 124-130 oocytes) being all the increases significantly different when compared against control (p < 0.05). The 20-80 µg/ml treatments decreased the expression of glutathione peroxidase (Gpx1), and the 10-40 µg/ml treatments reduced the expression of superoxide dismutase (Sod1; p < 0.05) compared to control; Bax mRNA expression did not vary. Our results suggest that the increased developmental competence of the embryos could be partly mediated by the EV-endMSCs' ROS scavenger activity.

The immunomodulatory activity of extracellular vesicles derived from endometrial mesenchymal stem cells on CD4+ T cells is partially mediated by TGFbeta.

Endometrial mesenchymal stem cells (endMSCs) reside in the basal and functional layer of human endometrium and participate in tissue remodelling, which is required for maintaining the regenerative capacity of the endometrium. The endMSCs are multipotent stem cells and exhibit immunomodulatory effects. This paper aimed to evaluate the regulatory effects of extracellular vesicles derived from endMSCs (EV-endMSCs) in the setting of T cell activation. In vitro stimulations of lymphocytes were performed in the presence of EV-endMSCs. These in vitro-stimulated lymphocytes were functionally and phenotypically characterized to distinguish CD4+ and CD8+ T cell differentiation subsets. Moreover, the inhibition of TGFß was performed with neutralizing antibodies. The phenotype and nanoparticle tracking analysis of the EV-endMSCs demonstrated that they are similar in terms of size distribution to other mesenchymal stem cells-derived exosomes. The in vitro assays showed an immunomodulatory potential of these vesicles to counteract the differentiation of CD4+ T cells. The quantification of active TGFß in EV-endMSCs was found to be very high when compared with extracellular vesicles-free concentrated supernatants. Finally, the neutralization of TGFß significantly attenuated the immunomodulatory activity of EV-endMSCs. In summary, this is the first report demonstrating that EV-endMSCs exhibit a potent inhibitory effect against CD4+ T cell activation, which is partially mediated by TGFß signalling.

Adipose-Derived Stem Cells Ameliorate Ischemia-Reperfusion Injury in a Rat Skin Free Flap Model.

BACKGROUND: Ischemia-reperfusion (I/R) injury is inevitable during free tissue transfers. When the period of ischemia exceeds the tissue tolerance, it causes necrosis and flap failure. The aim of this study was to investigate the effects of adipose-derived stem cells (ASCs) embedded in a collagen type I scaffold on the survival of free skin flaps to counteract I/R injury. METHODS: Left superficial caudal epigastric skin flaps (3 × 6 cm) were performed in 28 Wistar rats that were divided into four groups. The flaps elevated in the animals of the control group did not suffer any ischemic insult, and the vascular pedicle was not cut. All other flaps were subjected to 8 hours of ischemia prior to revascularization: I/R control group (8 hours of ischemia), I/R scaffold group (8 hours of ischemia + collagen type I scaffold), and I/R scaffold-ASCs group (8 hours of ischemia + collagen type I scaffold with rat ASCs embedded). Transit-time ultrasound blood flow measurements were performed. After 7 days, the areas of flap survival were measured and tissues were stained with hematoxylin/eosin and Masson's trichrome stain for histological analysis. RESULTS: The mean percentage flap survival area was significantly higher in the ASCs-treated flaps (I/R scaffold-ASCs group) compared with the ischemic controls (I/R control group and I/R scaffold group). Higher vascular proliferation and lower severity of necrosis and inflammatory changes were seen histologically in the samples of the ASCs-treated group. No significant difference in blood flow was detected between groups. CONCLUSION: Subcutaneous administration of ASCs embedded on a collagen type I scaffold reduces tissue damage after I/R injury in microvascular free flaps.

Murine embryos exposed to human endometrial MSCs-derived extracellular vesicles exhibit higher VEGF/PDGF AA release, increased blastomere count and hatching rates.

Endometrial Mesenchymal Stromal Cells (endMSCs) are multipotent cells with immunomodulatory and pro-regenerative activity which is mainly mediated by a paracrine effect. The exosomes released by MSCs have become a promising therapeutic tool for the treatment of immune-mediated diseases. More specifically, extracellular vesicles derived from endMSCs (EV-endMSCs) have demonstrated a cardioprotective effect through the release of anti-apoptotic and pro-angiogenic factors. Here we hypothesize that EV-endMSCs may be used as a co-adjuvant to improve in vitro fertilization outcomes and embryo quality. Firstly, endMSCs and EV-endMSCs were isolated and phenotypically characterized for in vitro assays. Then, in vitro studies were performed on murine embryos co-cultured with EV-endMSCs at different concentrations. Our results firstly demonstrated a significant increase on the total blastomere count of expanded murine blastocysts. Moreover, EV-endMSCs triggered the release of pro-angiogenic molecules from embryos demonstrating an EV-endMSCs concentration-dependent increase of VEGF and PDGF-AA. The release of VEGF and PDGF-AA by the embryos may indicate that the beneficial effect of EV-endMSCs could be mediating not only an increase in the blastocyst's total cell number, but also may promote endometrial angiogenesis, vascularization, differentiation and tissue remodeling. In summary, these results could be relevant for assisted reproduction being the first report describing the beneficial effect of human EV-endMSCs on embryo development.

Fibrin glue mesh fixation combined with mesenchymal stem cells or exosomes modulates the inflammatory reaction in a murine model of incisional hernia.

Surgical meshes are effective and frequently used to reinforce soft tissues. Fibrin glue (FG) has been widely used for mesh fixation and is also considered an optimal vehicle for stem cell delivery. The aim of this preclinical study was to evaluate the therapeutic effect of MSCs and their exosomes combined with FG for the treatment of incisional hernia. A murine incisional hernia model was used to implant surgical meshes and different treatments with FG, MSCs and exo-MSCs were applied. The implanted meshes were evaluated at day 7 by anatomopathology, cellular analysis of infiltrating leukocytes and gene expression analysis of TH1/TH2 cytokines, MMPs, TIMPs and collagens. Our results demonstrated a significant increase of anti-inflammatory M2 macrophages and TH2 cytokines when MSCs or exo-MSCs were used. Moreover, the analysis of MMPs, TIMPs and collagen exerted significant differences in the extracellular matrix and in the remodeling process. Our in vivo study suggests that the fixation of surgical meshes with FG and MSCs or exo-MSCs will have a beneficial effect for the treatment of incisional hernia in terms of improved outcomes of damaged tissue, and especially, in the modulation of inflammatory responses towards a less aggressive and pro-regenerative profile. STATEMENT OF SIGNIFICANCE: The implantation of surgical meshes is the standard procedure to reinforce tissue defects such as hernias. However, an exacerbated and persistent inflammatory response secondary to this implantation is frequently observed, leading to a strong discomfort and chronic pain in the patients. In many cases, an additional surgical intervention is needed to remove the mesh. This study shows that mesenchymal stem cells and their exosomes, combined with a fibrin sealant, can be used for the successful fixation of these meshes. This new therapeutic approach, assayed in a murine model of incisional hernia, favors the modulation of the inflammatory response towards a less aggressive and pro-regenerative profile.

Evaluation of antigen-induced synovitis in a porcine model: Immunological, arthroscopic and kinetic studies.

BACKGROUND: Synovitis is an inflammation-related disease linked to rheumatoid arthritis, osteoarthritis, infections and trauma. This inflammation is accompanied by immune cells infiltration which initiates an inflammatory response causing pain, discomfort and affecting the normal joint function. The treatment of synovitis is based on the administration of anti-inflammatory drugs or biological agents such as platelet rich plasma and mesenchymal stem cells. However, the evaluation and validation of more effective therapies of synovitis requires the establishment of clinically relevant animal models. RESULTS: In this study, Large White pigs were pre-immunized to evaluate an antigen-induced synovitis. The immune monitoring of synovial fluids in this model allowed us the identification of IL-12p40 and T cell subsets as immune biomarkers. Moreover, the evolution of synovitis was performed by arthroscopic procedures and kinetic analysis. In summary, this paper describes an animal model of antigen-induced synovitis to be used in the evaluation of anti-inflammatory therapies. CONCLUSIONS: The novelty of this paper lies in the development of a clinically relevant model of synovitis which permits the simultaneous evaluation of synovitis from an immunological, surgical and kinetic point of view.

Immunosenescence: limitations of natural killer cell-based cancer immunotherapy.

Cancer is primarily considered a disease of old age. Immunosenescence refers to the age-associated changes in the immune system, and its contribution to the increased risk of cancer in old individuals has been discussed for many years. Natural killer (NK) cells are cytotoxic innate immune cells specialized in defence against tumour and virus-infected cells. NK cell cytotoxicity is the result of a fine balance between activating and inhibitory receptors. Several activating receptors have been identified that recognize different ligands frequently found over-expressed on tumour cells or virus-infected cells. The most important NK cell inhibitory receptors interact with major histocompatibility complex class I molecules expressed on almost all nucleated cells preventing NK cell-mediated lysis of healthy cells. NK cell immunosenescence is characterized by a redistribution of NK cell subsets, a diminished expression of several activating receptors and lower per-cell cytotoxicity. Altered expression of activating receptors has also been described in young and elderly cancer patients probably due to chronic exposure to ligands on tumour cells. Thus, the effect of both age and cancer may act synergistically to diminish NK cell-mediated tumour immunosurveillance. Different strategies harnessing the power of NK cells to target tumour cells have been designed including adoptive therapy with autologous or allogeneic expanded NK cells. In addition, checkpoint blockade of inhibitory receptors and the use of agonist antibodies to stimulate activating receptors are emerging areas of research. In this context, the effect of immunosenescence should be considered to improve the efficiency of cancer immunotherapy.

Comparison of mesenchymal stem cells and leukocytes from Large White and Göttingen Minipigs: Clues for stem cell-based immunomodulatory therapies.

The mesenchymal stem cells (MSCs) are one of the most promising cell types for human and veterinary use and their therapeutic effect is associated with their immunomodulatory properties. Farm animal models, such as pigs, have become a valuable tool to evaluate the safety and efficacy of adoptively transferred MSCs in the setting of veterinary medicine. In order to evaluate the immunomodulatory effect of stem cell-based therapies in porcine breeds, a deep analysis and comparison of MSCs and leukocyte subsets are absolutely necessary. Here we provide a detailed analysis of bone-marrow derived MSCs and leukocyte subsets from Large White pigs and Göttingen Minipigs. Significant differences were observed between the two pig breeds in terms of T cell subsets that need to be considered for immune monitoring of stem cell-based therapies.

Changes on Adipose Tissue Distribution After Laparoscopic Roux-en-Y Gastric Bypass in Obese Göttingen Minipig. Effects on Glucose Metabolism.

BACKGROUND: The objective of the study was to assess Roux-en-Y independent-to-weight metabolic effects on an obese Gottingen minipig. METHODS: Eleven castrated Göttingen minipigs were enrolled. Minipigs became obese after a 9-month period of experimental diet. The animals were then subjected to a laparoscopic Roux-en-Y gastric bypass (period P1) followed by 4 months of follow-up (period P2). During this last period, they were fed to maintain similar body weight to that observed at the day of surgery. Registered parameters during each period included biometric variables; abdominal adipose tissue (AT) distribution; lipid profile; HOMA-IR; and fasting plasma leptin, adiponectin, glucose, insulin and fructosamine. RESULTS: Two pigs were unable to complete the study due to postoperative complications. Four months after surgery, the body weight (kg) was maintained (P1 51.90 ± 5.78 vs P2 51.47 ± 5.68). On the contrary, intraperitoneal AT (p < 0.0001), glucose (mmol/L) (P1: 5.53 ± 0.37 vs 4.66 ± 0.72, p = 0.019), fructosamine (µmol/L) (p = 0.0001) and insulin (pmol/L) (P1: 36.66 ± 13.94 vs P2: 21.42 ± 12.62, p = 0.001) significantly decreased after surgery. Lipid profile showed a significant increase after high-fat feeding period and a slight reduction at P2, which continued increasing. At the end of the study, leptin plasma levels (ng/mL) showed a significant reduction compared to basal value (p = 0.001). CONCLUSIONS: The improvement in glucose metabolism seems to occur by means of the redistribution of body fat. In order to fully confirm this hypothesis, more studies including the evaluation of incretins and inflammatory molecules, as well as a glycaemia control study against glucose load, are needed.