The systemic cellular immune response against allogeneic mesenchymal stem cells is influenced by inflammation, differentiation and MHC compatibility: in vivo study in the horse.

The effectiveness and safety of allogeneic mesenchymal stem/stromal cells (MSCs) can be affected by patient's immune recognition. Thus, MSC immunogenicity and their immunomodulatory properties are crucial aspects for therapy. Immune responses after allogeneic MSC administration have been reported in different species, including equine. Interactions of allogenic MSCs with the recipient's immune system can be influenced by factors like matching or mismatching for the major histocompatibility complex (MHC) between donor-recipient, and by the levels of MHC expression in MSCs. The latter can vary upon MSC inflammatory exposure or differentiation, such as chondrogenic induction, making both priming and differentiation interesting therapeutic strategies. This study investigated the systemic in vivo immune cellular response against allogeneic equine MSCs in these situations. Either MSCs in basal conditions (MSC-naïve), pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were repeatedly administered subcutaneously into autologous, MHC-matched or MHC-mismatched allogeneic equine recipients. At different time-points after each administration, lymphocytes were obtained from recipient horses and exposed in vitro to the same type of MSCs to assess the proliferative response of different T cell subsets (cytotoxic, helper, regulatory), B cells, and interferon gamma (IFNγ) secretion. Higher proliferative response of helper and cytotoxic T lymphocytes and IFNγ secretion was observed in response to all types of MHC-mismatched MSCs over MHC-matched ones. MSC-primed produced the highest immune response, followed by MSC-naïve, and MSC-chondro. However, MSC-primed activated Treg and had a mild effect on B cells, and the response after their second administration was similar to the first one. On the other hand, both MSC-chondro and MSC-naïve barely induced Treg response but promoted B lymphocyte activation, and proportionally induced a higher cell response after the second administration. In conclusion, both the type of MSC conditioning and the MHC compatibility influenced systemic immune recognition of equine MSCs after single and repeated administrations, but the response was different. Selecting MHC-matched donors would be particularly recommended for MSC-primed and repeated MSC-naïve administrations. While MHC-mismatching in MSC-chondro would be less critical, B cell response should not be ignored. Comprehensively investigating the in vivo immune response against equine allogeneic MSCs is crucial for advancing veterinary cell therapies.

Use of Knotless Barbed Sutures in Laparoscopic Inguinal Hernioplasty in Horses: 40 Cases.

Inguinal hernias (IHs) and ruptures are a relatively common condition in horses, occurring in foals (congenital) and adult (acquired) animals. A retrospective observational analysis was conducted on 40 cases that underwent laparoscopic surgery to close the VRs using barbed sutures alone or combined with other techniques. Signalment, clinical presentation, surgery, and follow-up data were obtained. In total, fifty-nine VRs were closed using barbed sutures (alone or in combination with other methods), with six cases performed prophylactically and forty-four due to acquired IH. Of the forty-four cases with IH, four were non-strangulated hernias, while thirty presented with strangulated small intestines (twenty-eight acquired and two congenital). The results obtained in this study suggest that laparoscopic hernioplasty with barbed sutures is an effective and safe surgical procedure that could be recommended as a standard practice for managing inguinal hernias in horses, particularly when sparing testicles or preserving reproductive capabilities is a priority.

Laparoscopically Assisted Percutaneous Inguinal Ring Closure for Resolution of Inguinal/Scrotal Hernias in Rams: Cadaveric Study and Three Cases Report.

The aim of this study is to evaluate a laparoscopically assisted percutaneous suture (LAPS) procedure to treat inguinal hernia (IH) while preserving testicles in rams. An ex vivo experiment with six ram cadavers and a report of three clinical cases are discussed. In cadavers, both internal inguinal rings (IIR) were partially closed by LAPS. Two LAPS methods were tested: (1) using a laparoscopic portal closure device and (2) using a suture loop inserted through needles in each IIR. After each procedure, the closure was laparoscopically evaluated and the number of U- sutures was recorded. The procedure was also performed on three client-owned rams with unilateral non-strangulated IH and the occurrence of re-herniation was followed up. In cadavers, LAPS of the IIRs could be easily and satisfactorily performed with either of the two systems, requiring one to three U-sutures per IIR. No differences were observed between the two surgical procedures. In two clinical cases, the procedure was successfully performed without reoccurrence of herniation or alterations in reproductive behavior in the following 3 and 6 months. In the third case, the hernia was reduced but a retroperitoneal emphysema during laparoscopy prevented hernioplasty and the animal herniated again. In conclusion, LAPS of IIR can be used as a simple and feasible treatment to preserve testicles in rams with IH.

Development of a laparoscopic technique for inguinal hernioplasty in standing horses.

BACKGROUND: Most previously described techniques for laparoscopic inguinal hernioplasty (IH) in horses require advanced laparoscopic skills. Our objective was to describe a new laparoscopic IH technique using a surgical anchoring system. METHODS: Standing laparoscopic IH was performed unilaterally in eight experimental stallions, using the contralateral inguinal canal (IC) as a control. A polyether ether ketone harpoon was anchored in the craniolateral aspect of the vaginal ring, and an extracorporeal knot was used to fix the device. Clinical evaluation, including testicular palpation and lameness examination, was conducted before and for 4 weeks after surgery. Repeat laparoscopy was performed 28 days later. RESULTS: Standing laparoscopic IH was performed in all horses with a surgical time of 38 ± 12.85 minutes. In two animals, a small peritoneal tear occurred that did not require repair. No other complications were recorded. On repeat laparoscopy, all devices were in place, and the IC remained partially closed in all horses. LIMITATIONS: The procedure was performed on normal experimental horses and has not been employed on horses that have had an inguinal hernia. CONCLUSIONS: This new standing laparoscopic hernioplasty technique provides another potential method for simple partial closure of the IC in stallions at risk of or with history of inguinal herniation.

Complications in Laparoscopic Access in Standing Horses Using Cannula and Trocar Units Developed for Human Medicine.

First cannulation is a critical manoeuvre in equine laparoscopy. This retrospective study aimed at the comparison of the frequency and type of complications detected when using different human laparoscopy devices for laparoscopic access in standing horses, and the influence of body condition in such complications. Forty-four procedures were included, and retrieved data comprised cannula insertion technique, body condition, and type and frequency of complications. Laparoscopic access techniques were classified into five groups: P: pneumoperitoneum created using Veress needle prior to cannulation; T: sharp trocar; D: direct access via surgical incision; V: Visiport optical trocar and H: optical helical cannula (OHC). In groups T, D, V and H, access was achieved without prior induction of pneumoperitoneum. Complications were registered in 13/44 procedures, of which retroperitoneal insufflation was the most common (6/13). Statistically significant association was found between the complication incidence and the type of access, with group D showing the highest complication frequency (80%) and group H the lowest frequency (0%). The majority of complications (9/13) were observed in overweight horses. We conclude that devices designed for human patients can be used for laparoscopic access in standing horses, with the use of OHC minimizing the appearance of complications, especially in overweight horses with OW.

The immunomodulation-immunogenicity balance of equine Mesenchymal Stem Cells (MSCs) is differentially affected by the immune cell response depending on inflammatory licensing and major histocompatibility complex (MHC) compatibility.

The immunomodulatory properties of equine mesenchymal stem cells (MSCs) are important for their therapeutic potential and for their facilitating role in their escape from immune recognition, which may also be influenced by donor-recipient major histocompatibility complex (MHC) matching/mismatching and MHC expression level. Factors such as inflammation can modify the balance between regulatory and immunogenic profiles of equine MSCs, but little is known about how the exposure to the immune system can affect these properties in equine MSCs. In this study, we analyzed the gene expression and secretion of molecules related to the immunomodulation and immunogenicity of equine MSCs, either non-manipulated (MSC-naive) or stimulated by pro-inflammatory cytokines (MSC-primed), before and after their exposure to autologous or allogeneic MHC-matched/-mismatched lymphocytes, either activated or resting. Cytokine priming induced the immunomodulatory profile of MSCs at the baseline (MSCs cultured alone), and the exposure to activated lymphocytes further increased the expression of interleukin 6 (IL6), cyclooxygenase 2, and inducible nitric oxide synthase, and IL6 secretion. Activated lymphocytes were also able to upregulate the regulatory profile of MSC-naive to levels comparable to cytokine priming. On the contrary, resting lymphocytes did not upregulate the immunomodulatory profile of equine MSCs, but interestingly, MSC-primed exposed to MHC-mismatched lymphocytes showed the highest expression and secretion of these mediators, which may be potentially linked to the activation of lymphocytes upon recognition of foreign MHC molecules. Cytokine priming alone did not upregulate the immunogenic genes, but MSC-primed exposed to activated or resting lymphocytes increased their MHC-I and MHC-II expression, regardless of the MHC-compatibility. The upregulation of immunogenic markers including CD40 in the MHC-mismatched co-culture might have activated lymphocytes, which, at the same time, could have promoted the immune regulatory profile aforementioned. In conclusion, activated lymphocytes are able to induce the equine MSC regulatory profile, and their effects seem to be additive to the priming action. Importantly, our results suggest that the lymphocyte response against MHC-mismatched MSC-primed would promote further activation of their immunomodulatory ability, which eventually might help them evade this reaction. Further studies are needed to clarify how these findings might have clinical implications in vivo, which will help developing safer and more effective therapies.

Percutaneous Ultrasound-Guided Carotid Access and Puncture Closure with Angio-Seal in Horses.

Background: There are different indications for endovascular surgery in horses, mainly the treatment of guttural pouch mycosis. Traditionally, these procedures are carried out by open arteriotomy of the common carotid artery (CCA), although less invasive percutaneous ultrasound-guided carotid access (PUGCA) has been described in experimental horses. In human medicine, commercial closure systems are used to seal these arterial puncture sites and reduce complications. The aims of this study are to retrospectively describe our experience with PUGCA in clinical cases and to report, for the first time, the use of the commercial vascular closure device Angio-Seal after PUGCA in horses. Methods: Retrospective study of clinical case records. Collected parameters, including the feasibility of the PUGCA and variables related to the safety and efficacy of the use of the Angio-Seal. Results: Twelve PUGCA procedures in 11 horses were included. In all cases, the artery was effectively accessed, and the planned procedure could be performed. In two cases, haematoma/bleeding due to incorrect use of the Angio-Seal was recorded. This complication rate (16.66%) was lower than that obtained in other studies using PUGCA in horses, but where the puncture was sealed by manual compression only. Main limitations: A control group of clinical cases with PUGCA but without using Angio-Seal is not available. Conclusions: Clinical data confirm previous experimental results, which showed that PUGCA is safe and effective in horses. The Angio-Seal system, regardless of possible complications due to incorrect use, can be used safely and effectively in horses. Further studies comparing arterial access site management using manual compression or Angio-Seal would be necessary to state if its routine use in horses is advisable.

Equine Mesenchymal Stem Cells Influence the Proliferative Response of Lymphocytes: Effect of Inflammation, Differentiation and MHC-Compatibility.

Immunomodulation and immunogenicity are pivotal aspects for the therapeutic use of mesenchymal stem cells (MSCs). Since the horse is highly valuable as both a patient and translational model, further knowledge on equine MSC immune properties is required. This study analysed how inflammation, chondrogenic differentiation and compatibility for the major histocompatibility complex (MHC) influence the MSC immunomodulatory-immunogenicity balance. Equine MSCs in basal conditions, pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were co-cultured with either autologous or allogeneic MHC-matched/mismatched lymphocytes in immune-suppressive assays (immunomodulation) and in modified one-way mixed leukocyte reactions (immunogenicity). After co-culture, frequency and proliferation of T cell subsets and B cells were assessed by flow cytometry and interferon-É£ (IFNÉ£) secretion by ELISA. MSC-primed showed higher regulatory potential by decreasing proliferation of cytotoxic and helper T cells and B cells. However, MHC-mismatched MSC-primed can also activate lymphocytes (proliferative response and IFNÉ£ secretion), likely due to increased MHC-expression. MSC-chondro maintained their regulatory ability and did not increase their immunogenicity, but showed less capacity than MSC-primed to induce regulatory T cells and further stimulated B cells. Subsequent in vivo studies are needed to elucidate the complex interactions between MSCs and the recipient immune system, which is critical to develop safe and effective therapies.

Allo-antibody production after intraarticular administration of mesenchymal stem cells (MSCs) in an equine osteoarthritis model: effect of repeated administration, MSC inflammatory stimulation, and equine leukocyte antigen (ELA) compatibility.

BACKGROUND: Antibody production after allogeneic administration of mesenchymal stem cells (MSCs) could impact their clinical application. Proinflammatory priming of MSCs can potentiate their regulatory ability in vivo but increased expression of major histocompatibility complex (MHC) might augment their immunogenicity, potentially leading to immune memory thus limiting repeated allogeneic administration. This study aimed at evaluating the production of cytotoxic allo-antibodies directed against donor's ELA (equine leukocyte antigen) in mismatched and halfmatched horses receiving repeated intraarticular administration of stimulated MSCs (MSC-primed) and unstimulated MSCs (MSC-naïve) in pathologic joints. METHODS: From available stored samples from a previous in vivo study, cells from one donor and serially collected sera (five time-points) from three groups of recipients were used based on their ELA haplotypes to perform microcytotoxicity assays: Group 1 recipients mismatched with the donor that received MSC-naïve (naïve-mismatched recipients); Group 2 recipients mismatched with the donor that received MSC-primed (primed-mismatched recipients); Group 3 recipients halfmatched with the donor (sharing 1/2 haplotypes) that received MSC-primed (primed-halfmatched recipients). Sera from recipients (neat, 1:2 and 1:16 dilution) were tested against target cells from the donor (cryopreserved and expanded MSC-naïve and MSC-primed) or from one animal presenting the same ELA haplotypes than the donor (fresh peripheral blood lymphocytes as control). RESULTS: One to three weeks after first MSC administration, all recipient groups produced allo-antibodies regardless of MSC received (naïve or primed) and matching degree with donor. However, secondary response after MSC re-exposure was less evident in halfmatched recipients (MSC-primed) than in mismatched ones (both MSC-naïve and MSC-primed). Recipients of MSC-primed (both mismatched and halfmatched) tended towards developing lower antibody response than MSC-naïve recipients in vivo, but MSC-primed were targeted to death in higher percentage in vitro in the microcytoxicity assay. CONCLUSIONS: After first intraarticular allogeneic administration, the immunomodulatory profile of MSC-primed would have led to lower antibody production, but these antibodies would target more easily MSC-primed after second injection (re-exposure), likely because of their higher MHC expression.

Effect of allogeneic platelet lysate on equine bone marrow derived mesenchymal stem cell characteristics, including immunogenic and immunomodulatory gene expression profile.

Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MSCs) as well as on their proliferation ability, phenotype markers, and viability post-cryopreservation. The eBM-MSCs (n = 3) cultures were supplemented with 20% of allogeneic pooled concentrated PL (CPL; 591 × 103 platelets/µL) or basal PL (BPL; 177 × 103 platelets/µL) from three donors, using 10% FBS supplementation as control. The proliferative ability of eBM-MSCs under the three conditions was evaluated by calculating the cell doubling times (DT) up to passage 3 (P3) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P3. Viability of eBM-MSCs post-cryopreserved with CPL or FBS was assessed at 15, 30 and 60 days. The gene expression profile of eBM-MSCs was evaluated in P3 by RT-qPCR for characterization, immunogenic and immunomodulatory markers. The cells cultured in CPL had significantly higher ability to proliferate than with FBS or BPL (P < 0.001) in the MTT assay. Post-cryopreserved viability was similar between cells cultured and preserved in FBS and CPL at all time-points. Gene expression of MSC characterization markers was similar among the three conditions. The gene expression of the immunogenic markers MHC-I, MHC-II and CD40 was slightly (non-significant) increased in CPL condition compared to FBS and BPL. The CPL condition showed higher expression of the genes coding for the immunomodulatory molecules VCAM-1 (non-significant) and IL-6 (P < 0.05), and similar for COX-2; whereas iNOS and IDO were not expressed under any condition. In conclusion, the replacement of FBS by allogeneic CPL as a supplement for ex vivo propagation of eBM-MSCs provides appropriate proliferation and cryopreservation, and mildly upregulates the gene expression of immunomodulatory markers, thus constituting a potentially suitable alternative to the use of FBS. Further studies are needed to clarify the composition and effects of CPL supplementation on equine MSCs immunological profile.

Application of a laparoscopic technique for vasectomy in standing horses.

This report describes a technique for standing laparoscopic vasectomy in stallions through a prospective descriptive study. A preliminary study was carried out with two experimental intact male horses and subsequently the procedure was performed in two clinical cases. These horse owners want to keep their animals in the most possible natural way, preserving its stallion behaviour in a herd without generating offspring. The horses were sedated and restrained in stocks and laparoscopic vasectomy was performed using three portal sites in both paralumbar fossae recording surgical times. A 4-cm segment of each ductus deferens (DD) was occluded with laparoscopic vessel sealing devices and subsequently excised. Semen collection was performed using an artificial vagina before the laparoscopic procedure and at 15 and 60 days postoperatively. Sexual behaviour and spermiogram were analysed. Two months after vasectomy, control laparoscopy was performed in experimental horses to assess the surgical site. Bilateral vasectomy could be performed without intraoperative complications in a mean surgical time of 20 min per DD. Success of the procedure was confirmed in all cases by azoospermic ejaculates 60 days after vasectomy. This is the first time that the technique for laparoscopic vasectomy is described in horses.

Assessment of effectiveness and safety of repeat administration of proinflammatory primed allogeneic mesenchymal stem cells in an equine model of chemically induced osteoarthritis.

BACKGROUND: This study aimed at assessing the effectiveness and safety of repeated administrations of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) primed with tumor necrosis factor (TNF)-α and interferon-γ in an equine model of chemically-induced osteoarthritis. Arthritis was induced in both radio-carpal (RC)-joints by amphotericin-B in 18 ponies, divided into three groups depending on the treatment injected: MSC-naïve (n = 7), MSC-primed (n = 7) and control (n = 4). The study consisted of two phases and used one RC-joint of each animal in each phase, with four months time-lapse, in order to assess two end-points. Clinical, synovial, radiological and ultrasonographic follow-up was performed. At six months, animals were euthanized and both carpi were assessed by magnetic resonance imaging (MRI), gross anatomy, histopathology, histochemistry and gene expression. RESULTS: Clinical and synovial inflammatory signs were quicker reduced in MSC-treated groups and repeated allogeneic administration did not produce adverse reactions, but MSC-primed group showed slight and transient local inflammation after second injection. Radiology and MRI did not show significant differences between treated and control groups, whereas ultrasonography suggested reduced synovial effusion in MSC-treated groups. Both MSC-treated groups showed enhanced cartilage gross appearance at two compared to six months (MSC-naïve, p < 0.05). Cartilage histopathology did not reveal differences but histochemistry suggested delayed progression of proteoglycan loss in MSC-treated groups. Synovium histopathology indicated decreased inflammation (p < 0.01) in MSC-primed and MSC-naïve at two and six months, respectively. At two months, cartilage from MSC-primed group significantly (p < 0.05) upregulated collagen type II (COL2A1) and transforming growth factor (TGF)-ß1 and downregulated cyclooxygenase-2 and interleukin (IL)-1ß. At six months, MSC-treatments significantly downregulated TNFα (p < 0.05), plus MSC-primed upregulated (p < 0.05) COL2A1, aggrecan, cartilage oligomeric protein, tissue inhibitor of metalloproteinases-2 and TGF-ß1. In synovium, both MSC-treatments decreased (p < 0.01) matrix metalloproteinase-13 expression at two months and MSC-primed also downregulated TNFα (p < 0.05) and IL-1ß (p < 0.01). CONCLUSIONS: Both MSC-treatments provided beneficial effects, mostly observed at short-term. Despite no huge differences between MSC-treatments, the findings suggested enhanced anti-inflammatory and regulatory potential of MSC-primed. While further research is needed to better understand these effects and clarify immunogenicity implications, these findings contribute to enlarge the knowledge about MSC therapeutics and how they could be influenced.

Acute phase protein haptoglobin as inflammatory marker in serum and synovial fluid in an equine model of arthritis.

Acute phase proteins are useful inflammatory markers in horses. Haptoglobin (Hp) serum level is increased in horses undergoing different inflammatory processes, including arthritis. However, Hp concentration has not been assessed in inflammatory synovial fluid (SF). The aim of the present study was to investigate the Hp response in serum and SF in horses undergoing experimentally induced arthritis. For this purpose, serum and SF samples were collected from 12 animals before amphotericin B-induced arthritis was created (T0, healthy) and 15days after the lesion induction (T1, joint inflammation) and Hp was determined by single radial immunodiffusion. The Hp increase between T0 and T1 was significant in both serum and SF, and serum Hp concentration at T0 was significantly higher than in SF, but significant differences were not found at T1, indicating a higher Hp increase in SF. A significant positive correlation for Hp concentration between serum and SF samples was found. These results highlight the potential usefulness of Hp as inflammatory marker in horses, showing for the first time the increase of Hp in SF from joint inflammation in the horse.