Tenogenically induced allogeneic mesenchymal stem cells for the treatment of proximal suspensory ligament desmitis in a horse

Suspensory ligament injuries are a common injury in sport horses, especially in competing dressage horses. Because of the poor healing of chronic recalcitrant tendon injuries, this represents a major problem in the rehabilitation of sport horses and often compromises the return to the initial performance level. Stem cells are considered as a novel treatment for different pathologies in horses and humans. Autologous mesenchymal stem cells (MSCs) are well known for their use in the treatment of tendinopathies; however, recent studies report a safe use of allogeneic MSCs for different orthopedic applications in horses. Moreover, it has been reported that pre-differentiation of MSCs prior to injection might result in improved clinical outcomes. For all these reasons, the present case report describes the use of allogeneic tenogenically induced peripheral blood-derived MSCs for the treatment of a proximal suspensory ligament injury. During conservative management for 4 months, the horse demonstrated no improvement of a right front lameness with a Grade 2/5 on the American Association of Equine Practitioners (AAEP) scale and a clear hypo-echoic area detectable in 30% of the cross sectional area. From 4 weeks after treatment, the lameness reduced to an AAEP Grade 1/5 and a clear filling of the lesion could be noticed on ultrasound. At 12 weeks (T-4) after the first injection, a second intra-lesional injection with allogeneic tenogenically induced MSCs and platelet rich plasma was given and at 4 weeks after the second injection (T-5), the horse trotted sound under all circumstances with a close to total fiber alignment. The horse went back to previous performance level at 32 weeks after the first regenerative therapy and is currently still doing so (i.e., 20 weeks later or 1 year after the first stem cell treatment). In conclusion, the present case report demonstrated a positive evolution of proximal suspensory ligament desmitis after treatment with allogeneic tenogenically induced MSCs

A feasibility study on the use of equine chondrogenic induced mesenchymal stem cells as a treatment for natural occurring osteoarthritis in dogs

Conventional treatments of osteoarthritis (OA) reduce pain and the inflammatory response but do not repair the damaged cartilage. Xenogeneic peripheral blood-derived equine chondrogenically induced mesenchymal stem cells (ciMSC) could thus provide an interesting alternative. Six client-owned dogs with confirmed elbow OA were subjected to a baseline orthopedic examination, pressure plate analysis, general clinical examination, hematological analysis, synovial fluid sampling, and radiographic examination, and their owners completed two surveys. After all examinations, a 0.9% saline solution (placebo control product=CP) was administered intra-articularly. After 6 weeks, all examinations were repeated, owners again completed two surveys, and equine ciMSCs were administered in the same joint. After another 6 weeks, dogs were returned for a final follow-up. No serious adverse events or suspected adverse drug reactions were present during this study. No significant differences in blood analysis were noted between the CP and ciMSC treatment. Two adverse events were observed, both in the same dog, one after CP treatment and one after ciMSC treatment. The owner surveys revealed significantly less pain and lameness after ciMSC treatment compared to after CP treatment. There was no significant difference in the orthopedic examination parameters, the radiographic examination, synovial fluid sampling, and pressure plate analysis between CP treatment and ciMSC treatment. A single intra-articular administration of equine ciMSCs proved to be a well-tolerated treatment, which reduced lameness and pain according to the owner's evaluations compared to a placebo treatment

Equine allogeneic chondrogenic induced mesenchymal stem cells are an effective treatment for degenerative joint disease in horses

Degenerative joint disease is one of the main causes of equine early retirement from pleasure riding or a performance career. The disease is initially triggered by an abnormal loading of normal cartilage or a normal loading of abnormal cartilage. This primary insult is accompanied with joint inflammation, which leads to further progressive degeneration of the articular cartilage and changes in the surrounding tissues. Therefore, in search for an effective treatment, 75 adult horses with early signs of degenerative fetlock joint disease were enrolled in a randomized, multicenter, double-blinded, and placebo-controlled study. Fifty animals were injected intra-articularly with the investigational veterinary product (IVP) consisting of allogeneic chondrogenic induced mesenchymal stem cells (ciMSCs) with equine allogeneic plasma, and 25 horses were injected with 0.9% NaCl (saline) control product. From week 3 to 18 after treatment, lameness scores (P<0.001), flexion test responses (P<0.034), and joint effusion scores (P<0.001) were remarkably superior in IVP-treated horses. Besides nasal discharge in both treatment groups, no adverse events were observed during the entire study period. On long-term follow-up (1 year), significantly more investigational product-treated horses were working at training level or were returned to their previous level of work (P<0.001)

Chondrogenic priming at reduced cell density enhances cartilage adhesion of equine allogeneic MSCs : a loading sensitive phenomenon in an organ culture study with 180 explants

Background: Clinical results of regenerative treatments for osteoarthritis are becoming increasingly significant. However, several questions remain unanswered concerning mesenchymal stem cell (MSC) adhesion and incorporation into cartilage. Methods: To this end, peripheral blood (PB) MSCs were chondrogenically induced and/or stimulated with pulsed electromagnetic fields (PEMFs) for a brief period of time just sufficient to prime differentiation. In an organ culture study, PKH26 labelled MSCs were added at two different cell densities (0.5 x10(6) vs 1.0 x10(6)). In total, 180 explants of six horses (30 per horse) were divided into five groups: no lesion (i), lesion alone (ii), lesion with naive MSCs (iii), lesion with chondrogenically-induced MSCs (iv) and lesion with chondrogenically-induced and PEMF-stimulated MSCs (v). Half of the explants were mechanically loaded and compared with the unloaded equivalents. Within each circumstance, six explants were histologically evaluated at different time points (day 1, 5 and 14). Results: COMP expression was selectively increased by chondrogenic induction (p = 0.0488). PEMF stimulation (1mT for 10 minutes) further augmented COL II expression over induced values (p = 0.0405). On the other hand, MSC markers remained constant over time after induction, indicating a largely predifferentiated state. In the unloaded group, MSCs adhered to the surface in 92.6% of the explants and penetrated into 40.7% of the lesions. On the other hand, physiological loading significantly reduced surface adherence (1.9%) and lesion filling (3.7%) in all the different conditions (p < 0.0001). Remarkably, homogenous cell distribution was characteristic for chondrogenic induced MSCs (+/- PEMFs), whereas clump formation occurred in 39% of uninduced MSC treated cartilage explants. Finally, unloaded explants seeded with a moderately low density of MSCs exhibited greater lesion filling (p = 0.0022) and surface adherence (p = 0.0161) than explants seeded with higher densities of MSCs. In all cases, the overall amount of lesion filling decreased from day 5 to 14 (p = 0.0156). Conclusion: The present study demonstrates that primed chondrogenic induction of MSCs at a lower cell density without loading results in significantly enhanced and homogenous MSC adhesion and incorporation into equine cartilage. Copyright (C) 2015 S. Karger AG, Base

The evaluation of equine allogeneic tenogenic primed mesenchymal stem cells in a surgically induced superficial digital flexor tendon lesion model

Background: Tendon injuries are very common in horses and jeopardize the athletic performance, and due to the high risk of reinjury may lead to early retirement. The use of mesenchymal stem cells for the treatment of equine tendon disease is widely investigated because of their regenerative potential. The objective of this study is to investigate the safety and efficacy of equine allogeneic tenogenic primed mesenchymal stem cells (tpMSCs) for the management of tendinitis in horses. Methods: A core lesion was surgically induced in the superficial digital flexor tendon of both forelimbs of eight horses. After 7 days, one forelimb was treated with tpMSCs, while the contralateral forelimb served as an intra-individual control and was treated with saline. A prescribed exercise program was started. All horses underwent a daily clinical evaluation throughout the entire study period of 112 days. Blood samples were taken at different time points for hematological and biochemical analysis. Tendon assessment, lameness examination, ultrasound assessment and ultrasound tissue characterization (UTC) were performed at regular time intervals. At the end of the study period, the superficial digital flexor tendons were evaluated macroscopically and histologically. Results: No suspected or serious adverse events occurred during the entire study period. There was no difference in local effects including heat and pain to pressure between a single intralesional injection of allogeneic tpMSCs and a single intralesional injection with saline. A transient moderate local swelling was noted in the tpMSC treated limbs, which dissipated by day 11. Starting at a different time point depending on the parameter, a significant improvement was observed in the tpMSC treated limbs compared to the placebo for echogenicity score, fiber alignment score, anterior-posterior thickness of the tendon and echo type by UTC assessment. Immunohistochemistry 112 days post-injection revealed that the amount of collagen type I and Von Willebrand factor were significantly higher in the tendon tissue of the tpMSC group, while the amount of collagen type III and smooth muscle actin was significantly lower. Conclusion: Equine allogeneic tenogenic primed mesenchymal stem cells were shown to be well-tolerated and may be effective for the management of tendon injuries

Pitfalls in machine learning‐based assessment of tumor‐infiltrating lymphocytes in breast cancer: a report of the international immuno‐oncology biomarker working group

The clinical significance of the tumor-immune interaction in breast cancer (BC) has been well established, and tumor-infiltrating lymphocytes (TILs) have emerged as a predictive and prognostic biomarker for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2 negative) breast cancer (TNBC) and HER2-positive breast cancer. How computational assessment of TILs can complement manual TIL-assessment in trial- and daily practices is currently debated and still unclear. Recent efforts to use machine learning (ML) for the automated evaluation of TILs show promising results. We review state-of-the-art approaches and identify pitfalls and challenges by studying the root cause of ML discordances in comparison to manual TILs quantification. We categorize our findings into four main topics; (i) technical slide issues, (ii) ML and image analysis aspects, (iii) data challenges, and (iv) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns, or design choices in the computational implementation. To aid the adoption of ML in TILs assessment, we provide an in-depth discussion of ML and image analysis including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial- and routine clinical management of patients with TNBC

Image-based multiplex immune profiling of cancer tissues: translational implications. A report of the International Immuno-oncology Biomarker Working Group on Breast Cancer

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer

Spatial analyses of immune cell infiltration in cancer : current methods and future directions. A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer

Modern histologic imaging platforms coupled with machine learning methods have provided new opportunities to map the spatial distribution of immune cells in the tumor microenvironment. However, there exists no standardized method for describing or analyzing spatial immune cell data, and most reported spatial analyses are rudimentary. In this review, we provide an overview of two approaches for reporting and analyzing spatial data (raster versus vector-based). We then provide a compendium of spatial immune cell metrics that have been reported in the literature, summarizing prognostic associations in the context of a variety of cancers. We conclude by discussing two well-described clinical biomarkers, the breast cancer stromal tumor infiltrating lymphocytes score and the colon cancer Immunoscore, and describe investigative opportunities to improve clinical utility of these spatial biomarkers. © 2023 The Pathological Society of Great Britain and Ireland.http://www.thejournalofpathology.com/hj2024ImmunologySDG-03:Good heatlh and well-bein

Pitfalls in machine learning-based assessment of tumor-infiltrating lymphocytes in breast cancer: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer

Cellular and Humoral Immunogenicity Investigation of Single and Repeated Allogeneic Tenogenic Primed Mesenchymal Stem Cell Treatments in Horses Suffering From Tendon Injuries

The use of mesenchymal stem cells (MSCs) for the treatment of equine tendon disease is widely investigated because of their regenerative and immunomodulatory potential. However, questions have been raised concerning the immunogenic properties of allogeneic MSCs. Therefore, two studies were conducted to assess the safety of equine allogeneic peripheral blood-derived tenogenic primed MSCs (tpMSCs). The objective was to evaluate if a single and repeated tpMSC administration induced a cellular and humoral immune response in horses suffering from tendon injuries. Horses enrolled in the first study (n = 8) had a surgically induced superficial digital flexor tendon core lesion and were treated intralesionally with tpMSCs. Before and after treatment the cellular immunogenicity was assessed by modified mixed lymphocyte reactions. The humoral immune response was investigated using a crossmatch assay. Presence of anti-bovine serum albumin (BSA) antibodies was detected via ELISA. Horses enrolled in the second study (n = 6) suffered from a naturally occurring tendon injury and were treated twice with tpMSCs. Blood was collected after the second treatment for the same immunological assays. No cellular immune response was found in any of the horses. One out of eight horses in the first study and none of the horses in the second study had anti-tpMSC antibodies. This particular horse had an equine sarcoid and further investigation revealed presence of antibodies against sarcoid cells and epithelial-like stem cells before treatment, which increased after treatment. Additionally, formation of antibodies against BSA was observed. These findings might indicate a non-specific immune response generated after treatment. Serum from the other horses revealed no such antibody formation. These two studies showed that the administration of tpMSCs did not induce a cellular or humoral immune response following an intralesional single or repeated (two consecutive) allogeneic tpMSC treatment in horses with tendon injury, except for one horse. Therefore, a larger field study should confirm these findings and support the safe use of tpMSCs as a therapeutic for horses suffering from tendon injuries