Low-dose xenogeneic mesenchymal stem cells target canine osteoarthritis through systemic immunomodulation and homing.

BACKGROUND: As current therapies for canine osteoarthritis (OA) provide mainly symptomatic improvement and fail to address the complex pathology of the disease, mesenchymal stem cells (MSCs) offer a promising biological approach to address both aspects of OA through their immunomodulatory properties. METHODS: This study aimed to investigate the safety and efficacy of xenogeneic MSCs in dogs with OA at different dose levels after intravenous injection. OA was surgically induced in the right stifle joint. Thirty-two male and female dogs were divided into three treatment groups and a control group. Regular general physical examinations; lameness, joint, radiographic, and animal caretaker assessments; pressure plate analyses; and blood analyses were performed over 42 days. At study end, joint tissues were evaluated regarding gross pathology, histopathology, and immunohistochemistry. In a follow-up study, the biodistribution of intravenously injected 99mTc-labeled equine peripheral blood-derived MSCs was evaluated over 24h in three dogs after the cruciate ligament section. RESULTS: The dose determination study showed the systemic administration of ePB-MSCs in a canine OA model resulted in an analgesic, anti-inflammatory, and joint tissue protective effect associated with improved clinical signs and improved cartilage structure, as well as a good safety profile. Furthermore, a clear dose effect was found with 0.3 × 106 ePB-MSCs as the most effective dose. In addition, this treatment was demonstrated to home specifically towards the injury zone in a biodistribution study. CONCLUSION: This model-based study is the first to confirm the efficacy and safety of systemically administered xenogeneic MSCs in dogs with OA. The systemic administration of a low dose of xenogeneic MSCs could offer a widely accessible, safe, and efficacious treatment to address the complex pathology of canine OA and potentially slow down the disease progression by its joint tissue protective effect.

An exploratory study on the prevalence of neoplasms in two strains of laying hens during an extended production cycle.

There is a trend towards extended periods of lay in the laying hen industry. Extended cycles without a moulting stage gives the opportunity to obtain more eggs from a single hen. However, appropriate management and care for older laying hens is needed. In this trial we assessed the prevalence of conditions in old laying hens with a focus on neoplastic diseases. In total 150 ISA Brown and 150 Dekalb white laying hens were selected at 86 weeks of age. Of each hen line, 75 hens were necropsied at 86 weeks of age; the other half were monitored for 44 weeks after which they were necropsied. At week 86, 15.3% of the hens suffered from a neoplasm, ISA Brown being the most affected. During the follow up period, 50 birds died because of a natural cause of which 20 hens showed signs of a neoplasms. At the end of the follow up period, 43% of the hens were affected by a neoplasm. Adenocarcinoma was the most prevalent neoplasm and equally distributed among both hen lines. Leiomyomas were most frequently observed in ISA brown hens. Among causes of death, 19.05% of ISA brown and 20.69% of Dekalb White was attributed to a neoplasm. Furthermore, link with ovarian activity and other pathologies were made with significant correlations between adenocarcinomas and inactive ovaries. In conclusion, this study shows that the prevalence of adenocarcinoma and leiomyoma is a factor to be considered in longer laying cycles with 1/5th of the mortality caused by these processes.RESEARCH HIGHLIGHTSAt 86 weeks of age, the prevalence of neoplasms was 15.3%, mainly in brown hens.At 130 weeks of age, 43% of the hens were affected by a neoplasm.Adenocarcinoma was the most prevalent neoplasm equally distributed among hen lines.Leiomyoma was the second most prevalent neoplasm, mainly found in brown hens.

Histological tissue healing following high-power laser treatment in a model of suspensory ligament branch injury.

BACKGROUND: High-power laser therapy gained popularity recently as a regenerative treatment for tendinitis and desmitis in the horse. However, studies evaluating the effects of laser therapy on tissue repair at the histological level in large mammals are lacking. OBJECTIVES: To evaluate the effects of high-power laser therapy on suspensory desmitis healing, using a model of suspensory ligament branch injury. STUDY DESIGN: In vivo experiments. METHODS: Standardised lesions were surgically induced in all four lateral suspensory branches of 12 healthy Warmblood horses. Laser therapy (class 4, 15W) was applied daily on two of four induced lesions for four consecutive weeks. Horses were randomly assigned to either short-term study (horses were sacrificed after 4 weeks) or long-term study (6 months). Suspensory ligament samples were scored after staining with haematoxylin-eosin and immunostaining for collagen 1- collagen 3- and factor VIII. RESULTS: In the short-term study, significantly better (lower) scores for variation in density (17% above cut-off score in treated lesions vs. 31% above cut-off score in controls, P = .03), shape of nuclei (54% vs 92%, P = .02), fibre alignment (32% vs 75%, P = .003) and fibre structure (38% vs 71%, P = .02) were found in laser-treated lesions when compared to controls. Collagen 3 expression was significantly higher (32% vs 19%, P = .006) in control lesions. In both short- and long-term studies combined, parameters lesion size (44% vs 56%, P = .02) and shape of nuclei (53% vs 84%, P = .05) scored significantly better in treated lesions. Long-term, significantly better (lower) scores were found in the laser-treated group for lesion size (15% vs 45%, P = .008) and a higher percentage above cut-off score for density of the nuclei (27% vs 9%, P = .02), compared to controls. MAIN LIMITATIONS: The model of suspensory branch injury is not an exact representation of clinical overstrain lesions. CONCLUSIONS: These results suggest that high-power laser therapy enables better lesion healing than conservative treatment.

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.

Power Doppler ultrasound and contrast-enhanced ultrasound demonstrate non-invasive tumour vascular response to anti-vascular therapy in canine cancer patients.

Combretastatin A4-phosphate (CA4P) is an anti-vascular agent which selectively shuts down blood supply in tumours, resulting in extensive tumour necrosis. The aim of this study was to assess in vivo, non-invasive ultrasound techniques for the early evaluation of tumour perfusion following CA4P treatment of spontaneous tumours. Eight dogs that bore spontaneous tumours were enrolled and were subsequently treated with a single dose of intravenous CA4P. Perfusion of tumours was evaluated by power Doppler ultrasound (PDUS) pre-treatment (0 h), during the injection (10 min, 20 min, 30 min) and after CA4P infusion (24 and 72 h). Vascularity index (VI) of the tumour tissue was quantitatively analysed and accuracy was verified by correlation analysis with the results of immunohistochemical evaluation of microvessel density (MVD). Central and peripheral perfusion was evaluated by contrast-enhanced ultrasound (CEUS) pre-treatment and at 72 h post-treatment. Post-treatment, PDUS demonstrated a significant decrease in VI within 10 min of CA4P infusion. CEUS parameters demonstrated a significant decrease in blood velocity and volume in the central aspect of the tumour. Histology revealed a 4.4-fold reduction (p < 0.001, 95% CI [2.2,9.4]) in MVD and a 4.1-fold increase (p = 0.003, 95% CI [1.4,11.8]) in necrotic tumour tissue. A strong correlation between PDUS results and immunohistochemical results was found (Pearson R2 = 0.957, p < 0.001). Furthermore, the findings of PDUS were supported by the objective results of the CEUS analyses. These data suggest a role for ultrasound in real-time, non-invasive monitoring of tumour vascular response as an early indicator of CA4P treatment efficacy.

Histopathological lesions in reproductive organs, distal spinal cord and peripheral nerves of horses naturally infected with Trypanosoma equiperdum.

BACKGROUND: Dourine, a venereal transmitted trypanosomosis caused by Trypanosoma equiperdum, has different clinical signs related to the reproductive and nervous system. Pathologic tissue changes associated with the disease are poorly described. The present study describes the histopathological lesions in naturally T. equiperdum-infected horses in the chronical stage of dourine. RESULTS: Four chronically dourine diseased horses underwent a post-mortem examination. They were Woo test negative, but CATT/T. evansi positive, had a low packed cell volume (PCV) and exhibited obvious clinical signs of dourine. Post-mortem examination did not reveal gross lesions in the organs assumed to be responsible for the symptomatology. On histopathology, genital organs were affected, with mononuclear cell infiltration and erosions and degeneration of seminiferous tubules and perivascular lymphoplasmacytic cuffing in the uterus. In the nervous system, mononuclear cell infiltration was located in peripheral nerves, ganglia and in the spinal cord, leading to axonal degeneration. Real-time PCR using ITS primer revealed the presence of trypanosomes in these organs and conventional PCRs using maxicircle and RoTat1.2 primers further confirmed the involvement of T. equiperdum since the DNAs from the vagina, testicle, distal spinal cord, sciatic and obturator nerves found to be positive for maxicircle and negative for RoTat 1.2. CONCLUSIONS: The histopathological lesions in the spinal cord and peripheral nerves explain the incoordination of the hind legs in T. equiperdum-infected horses, whilst its presence in the genital tract exemplifies the venereal transmission.

Reduction of Trypanosoma equiperdum from equine semen by single layer centrifugation.

Trypanosoma equiperdum (T. equiperdum) causes dourine, a venereally transmitted infection in horses. Purification of semen by single layer centrifugation (SLC) has been proven to be successful in reducing venereally transmitted diseases when dealing with other pathogens. The objective of this study was to evaluate the purification of T. equiperdum spiked semen by SLC. Semen was spiked using cryopreserved T. equiperdum stabilates (Dodola strain isolate 943). In total, 6 concentrations, varying from 102 to >5 × 106 trypanosomes, were added to semen samples. Subsequently, SLC was performed following standard procedures. The presence of the parasite in the purified semen was checked by wet smear examination, ITS1 PCR and in vivo inoculation in mice. Before SLC, all spiked semen samples, except the negative controls, were positive on PCR analysis. After SLC, all the pellets were found to be negative for T. equiperdum on microscopic examinations. Examination of the pellet by PCR could also not detect any parasite-DNA in the SLC-pellet of semen spiked with the lower number of parasites (102 to104 trypanosomes). However, in the SLC pellets spiked with 104 - 5 × 104 trypanosomes, only 1 out of the 4 replicates was negative for parasite DNA. All groups spiked with >5 × 104 trypanosomes were found to be positive on PCR. All mice in the positive controls exhibited parasitaemia (5/5). Mice inoculated with SLC-purified semen that was spiked with lower than 5 × 104 trypanosomes, remained free of parasitaemia, similar to the negative controls. However inoculation with SLC-pellets from samples with a higher number of trypanosomes (>5 × 104 - 5 × 106 and > 5 × 106), induced parasitaemia in 2 out of 5 and 3 out of 5 mice, respectively. This study indicates that single layer centrifugation can be used to clear T. equiperdum infected semen but that the success is dependent on the number of parasites.

Tissue (re)distribution of Trypanosoma equiperdum in venereal infected and blood transfused horses.

Dourine, caused by Trypanosoma equiperdum, is a life-threatening venereal disease in equidae. So far, there is no clear evidence on how and when stallions become infectious, nor which tissues are affected by the parasite in diseased animals. Post-infection, after a transient, temporary phase of parasitaemia, the parasite disperses to different tissues in an unknown distribution pattern. This study describes the distribution of the parasite after infection by artificial insemination (AI) or blood transfusion. Mares (N = 4) were artificially inseminated with T. equiperdum spiked semen whereas stallions (N = 4) were infected by blood transfusion. The course of the disease was monitored by parasitological (Woo) and molecular (PCR) tests and clinical signs and haematological parameters were recorded. At 120 days post infection, horses had a full necropsy, histopathology and PCR. A similar pattern of parasitaemia, disease progression and tissue distribution were seen in all horses. Ejaculated semen in the preclinical stage and epididymal semen in the chronic stage of the disease was positive on PCR and caused infection in mice. Cymelarsan® treatment in the chronic stage did not result in a clinico-haematological or histopathological improvement. At necropsy, lesions were observed in the nervous and reproductive system. Histopathological lesions were most severe in the peripheral nerves and associated ganglia, the testicles and genital mucosae with multifocal infiltration of lymphocytes, plasma cells and histocytes. The parasites disseminated to several tissues including the nervous system, testicles and semen. The results indicate that transmission of T. equiperdum is possible through semen even from symptomless stallions post-treatment.

Evaluation of an osteochondral fragment-groove procedure for induction of metacarpophalangeal joint osteoarthritis in horses.

OBJECTIVE To evaluate lameness and morphological changes associated with an osteochondral fragment-groove procedure as a means of experimental induction of metacarpophalangeal (MCP) joint osteoarthritis within an 11-week period in horses. ANIMALS 6 nonlame adult warmbloods. PROCEDURES The right MCP joint of each horse underwent an osteochondral fragment-groove procedure (day 0). After 1 week of stall rest (ie, starting day 7), each horse was trained daily on a treadmill. Weekly, horses underwent visual and inertial sensor-based assessments of lameness. Both MCP joints were assessed radiographically on days 0 (before surgery), 1, 35, and 77. A synovial fluid sample was collected from the right MCP joint on days 0 (before surgery), 35, 36, 49, 63, and 77 for cytologic and biomarker analyses. On day 77, each horse was euthanized; both MCP joints were evaluated macroscopically and histologically. RESULTS Right forelimb lameness was detected visually and by the inertial sensor system when horses were moving on a straight line after distal forelimb flexion or circling left on days 14 to 77. Compared with presurgical values, synovial fluid interleukin-6, prostaglandin E2, hyaluronic acid, and interleukin-1 receptor antagonist protein concentrations were increased at 2 or 3 time points, whereas tumor necrosis factor-α and interleukin-10 concentrations were decreased at 1 time point. Gross examination of all right MCP joints revealed synovitis and wear lines; synovitis was confirmed histologically. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that a combined osteochondral fragment-groove procedure can be used to induce clinically and grossly observable early MCP joint osteoarthritis during an 11-week period in horses.

Immunological, anti-angiogenic and clinical effects of intratumoral interleukin 12 electrogene therapy combined with metronomic cyclophosphamide in dogs with spontaneous cancer: A pilot study.

The immunological, anti-angiogenic and clinical effects of metronomic cyclophosphamide and 3 consecutive intratumoral interleukin (IL)-12 gene therapy (electrogene therapy (EGT)) treatments were evaluated in 6 dogs with spontaneous cancer. In all dogs, a decrease in peripheral leukocytes 2 days after IL-12 EGT coincided with erythema and swelling of the tumor. In the tumor, a transient increase in IL-12 levels was measured, whereas a continuous increase in interferon γ (IFNγ) and thrombospondin 1 (TSP-1) were determined in contrast to a continuous decrease in vascular endothelial growth factor (VEGF). In the serum, a transient increase in IL-12 and IL-10 levels were noted in contrast to a transient decrease in VEGF and TSP-1. The treatment resulted in a significant anti-angiogenic effect. Although all primary tumors continued to progress in time, this progression was slower than before treatment according to the contrast-enhanced ultrasound data. Besides the encouraging immunostimulatory and anti-angiogenic effects observed in all dogs we also noticed in 4 out of 6 dogs clinically relevant improvements in quality of life and weight. These results hold great promise for combinatorial strategies of IL-12 EGT and metronomic chemotherapy with conventional antitumor (immuno)therapies.

A canine orthologue of the human GFAP c.716G>A (p.Arg239His) variant causes Alexander disease in a Labrador retriever.

Alexander disease (AxD) is a fatal neurodegenerative disorder of astrocyte dysfunction in man, for which already a number of causal variants are described, mostly de novo dominant missense variants in the glial fibrillary acidic protein (GFAP). A similar disorder was already phenotypically described in animals but without the identification of causal variants. We diagnosed a Labrador retriever with a juvenile form of AxD based on clinical (tetraparesis with spastic front limbs mimicking 'swimming puppy syndrome') and pathological (the detection of GFAP containing Rosenthal fibers in astrocytes) features. In order to identify a causal variant, the coding sequences of the four detected GFAP transcript variants (orthologues from human transcript variants α, γ, δ/ɛ and κ) were sequenced. From the five detected variants, a heterozygous c.719G>A nucleotide substitution resulting in a p.Arg240His substitution was considered to be causal, because it is orthologous to the heterozygous de novo dominant c.716G>A (p.Arg239His) hotspot variant in man, proven to cause a severe phenotype. In addition, the variant was not found in 50 unrelated healthy Labrador retrievers. Because the condition in dogs is morphologically similar to man, it could be a promising animal model for further elucidating the genotype/phenotype correlation in order to treat or prevent this disease.

Parenteral and enteral feeding in preterm piglets differently affects extracellular matrix proteins, enterocyte proliferation and apoptosis in the small intestine.

The preterm intestine is immature and responds differently to total parenteral nutrition (TPN) and enteral nutrition, compared with the term intestine. We hypothesised that in preterms, diet composition and feeding route affect mucosal morphology, enterocyte mitosis and apoptosis, and the distribution of laminin-1, fibronectin and collagen IV (extracellular matrix proteins (ECMP)). Preterm piglets (93.5 % of gestation) were delivered via caesarean section and birth weight-matched allocated to one of the four experimental groups: the piglets were either euthanised immediately after delivery, after 3 d of TPN or after 2 d enteral feeding with colostrum or milk formula, following 3 d of TPN. We combined immunohistochemistry, image analysis and stereological measurements to describe the intestinal mucosal layer. No significant changes occurred after 3 d of TPN. Feeding colostrum or milk replacer for 2 d after TPN was associated with an increased crypt depth. Only enteral feeding with colostrum resulted in an increased villus height and mitotic index. Neither TPN nor enteral feeding changed the distribution pattern of ECMP or the occurrence of bifid crypts. The immature distribution pattern of ECMP in TPN-fed piglets, coupled with unchanged enterocyte mitosis and apoptosis indices, illustrates that feeding preterm pigs 3 d TPN does not lead to mucosal atrophy. Despite the invariable distribution of ECMP, colostrum was associated with crypt hyperplasia resulting in an increased villus height. These data illustrate that some mechanisms regulating cell turnover are immature in preterms and may in part explain the abnormal gut responses to TPN and enteral feeding in prematurely born pigs.

Measurement of skin desmosine as an indicator of altered cutaneous elastin in draft horses with chronic progressive lymphedema.

BACKGROUND: Chronic progressive lymphedema in Clydesdale and Shire draft horses causes severe disability of the limbs which leads to premature death of these horses. Since appropriate function of lymph vessels is dependent on the presence of viable elastin fibers, the goal of this study was to document differences in skin elastin fibers in affected horse breeds, compared to a nonaffected draft horse breed. METHODS AND RESULTS: Biochemical analysis of cutaneous desmosine, a cross-linking amino acid found only in elastin, was used to measure elastin in the skin from 110 draft horses. This included 7 normal, 38 mildly affected, 30 moderately, and 15 severely affected horses, and 20 horses of a nonaffected draft breed. Desmosine concentrations in neck, considered a nonaffected skin region, and left forelimb, an affected skin region, were compared between the groups. A significantly lower desmosine concentration was found in the skin of the neck and limb of clinically normal animals of affected draft breeds compared to a nonaffected draft horse breed. During the progression of the disease in the affected breeds, cutaneous desmosine concentrations most prominently increased in the skin of the distal limbs. CONCLUSIONS: Chronic progressive lymphedema in draft horses was associated with an initially systemic lower cutaneous elastin level and a deposition of elastin during the progression of the disease. A failure of elastic fibers to appropriately support the skin and its lymphatics is proposed as a possible contributing factor for chronic progressive lymphedema in Shires and Clydesdales.