Allogeneic adipose tissue-derived mesenchymal stem cells in combination with platelet rich plasma are safe and effective in the therapy of superficial digital flexor tendonitis in the horse.

Overstrain tendonitis are common pathologies in the sport horses. Therapeutic approaches to tendon healing do not always result in a satisfactory anatomical and functional repair, and healed tendon is often characterized by functional impairment and high risk of reinjury. Recently, mesenchymal stem cells (MSCs) and platelet rich plasma (PRP) have been proposed as novel therapeutic treatments to improve the tendon repair process. MSCs are multipotent, easy to culture and being originated from adult donors do not pose ethical issues. To date, autologous MSCs have been investigated mainly in the treatment of large bone defects, cardiovascular diseases, osteogenesis imperfecta and orthopaedic injuries both in human and veterinary medicine. The clinical applications in which autologous MSCs can be used are limited because patient-specific tissue collection and cell expansion require time. For clinical applications in which MSCs should be used right away, it would be more practical to use cells collected from a donor, expanded in vitro and banked to be readily available when needed. However, there are concerns over the safety and the efficacy of allogeneic MSCs. The safety and efficacy of a therapy based on the use of allogeneic adipose tissue-derived mesenchymal stem cells (ASCs) associated to platelet rich plasma (PRP) were evaluated in 19 horses affected by acute or subacute overstrain superficial digital flexor tendonitis (SDFT). The application of allogeneic ASCs neither raised clinical sign of acute or chronic adverse tissue reactions, nor the formation of abnormal tissue in the long-term. After a follow-up of 24 months, 89.5% horses returned to their previous level of competition, while the reinjury rate was 10.5%, comparable to those recently reported for SDFT treated with autologous bone marrow derived MSCs. This study suggests that the association between allogeneic ASCs and PRP can be considered a safe and effective strategy for the treatment of SDF tendonitis in the horse.

Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo.

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.

Focal canine tetanus: diagnostic value of electromyography.

A four-year-old, male Italian hound was presented with severe spasticity of both thoracic limbs that worsened with external stimuli. The remainder of the neurological and general physical examination was normal. Complete blood cell count, chemistry profile, and serology for protozoal diseases were within normal limits. Survey radiography of the cervicothoracic spine and abdominal ultrasonography showed no abnormalities. Electromyography of the thoracic limbs demonstrated the presence of "doublets" and simultaneous activity in both agonist and antagonist muscles. These abnormalities may be explained by a defective glycinergic inhibition at the spinal cord level. Together with the history, progression of signs, and clinical findings, electromyography supported a presumptive diagnosis of focal tetanus. The dog received tetanus antitoxin and antibiotic treatment and gradually improved over four months.

Incomplete humeral condylar fracture in two English Pointer dogs.

Incomplete humeral condylar fracture was diagnosed by means of radiology, CT, scintigraphy, arthroscopy and bone biopsy in two English Pointer dogs. In both cases an acute thoracic limb lameness, unrelated to a known episode of major trauma, was observed. Incomplete humeral condylar fracture, mainly described in the Spaniel breeds, has been recently diagnosed in Labrador retrievers, Rottweiler, German Wachtel and other breeds. The pathogenesis of the condition is still unknown, but incomplete ossification of the humeral condyle and mechanical stress, alone or associated, have to be considered. However, our clinical and histopathological data lead us to believe that in Pointers, high performance dogs, the mechanical stress can assume a critical ethiopathogenetic role.

Cyclin D1 expression in pre-cancerous and cancerous lesions of the canine mammary gland.

Overexpression of cyclin D1, the regulatory subunit of cyclin-dependent kinases (cdk4 and cdk6) involved in cell cycle control, has often been found in breast cancer and other types of human cancer. Increased expression, or stability, of cyclin D1 molecules may cause sufficient cdk4 activation to produce retinoblastoma protein phosphorylation independently of mitogenic signals; this results in commitment of cells to the G1 phase at mitosis. In the present study, cyclin D1 expression was investigated in pre-cancerous and cancerous lesions of the canine mammary gland by a complex experimental approach, which included Western blot and immunohistochemical analysis of cyclin D1 and the related molecular system. Furthermore, to define relationships between cell growth and expression of cyclin D1, proliferative activity was studied by the AgNOR technique. The study provided the following information. Cyclin D1 overexpression was largely independent of the type of proliferative anomaly. Indeed, cyclin D1 was expressed in 60% of the pre-cancerous lesions and in 44% of cancerous lesions. Mitotic activity and cyclin D1 expression were related: mammary lesions that expressed cyclin D1 showed a high proliferative ratio, the opposite being true of cyclin D1-negative cell populations. This study may contribute to the establishment of an animal model for anti-cancer research based on cyclin D1 suppression or cdk inactivation, or both.

A simplified technique for diagnostic and surgical arthroscopy of the shoulder joint in the dog.

A modified technique is presented for surgical and diagnostic arthroscopy of the shoulder joint in the dog. The technique involves access to the joint through two points only; one was created in place of the drainage needle-cannula, which was replaced with a portal, while the second was located more caudally compared with previous techniques. Using a changing guide rod system the two portals are completely interchangeable in order to perform easier arthroscopic surgery either in the cranial or caudal aspect of the joint. The presence of only one portal caudal to the lateral collateral ligament allows more freedom of movement and avoids interference between the arthroscope and the instruments. The modified procedure was performed on 33 joints affected by osteochondritis dissecans or tenoligament diseases and facilitated straightforward diagnostic examinations, and simple and rapid surgical procedures.

Extra-articular absorbable suture stabilization of coxofemoral luxation in dogs.

OBJECTIVE: To evaluate the effectiveness of an extra-articular surgical technique using absorbable suture material for the stabilization of traumatic coxofemoral luxation in dogs. STUDY DESIGN: Prospective, clinical study. ANIMALS: Fourteen client-owned dogs with recent and long-standing traumatic coxofemoral luxation (13 craniodorsal and 1 ventral). METHODS: Coxofemoral luxations were surgically reduced and maintained in place with an extra-articular iliofemoral multifilamentous absorbable suture (3 to 6 strands of 2 USP Polyglactin 910). No external support was employed, and all the dogs were encouraged to use the affected limb postoperatively. The average time of clinical and radiographic follow-up was 11.6 +/- 6.3 months (from 2 to 22 months). RESULTS: During the follow-up period, no reluxations occurred and no complications associated with the surgical technique were identified. The dogs started bearing weight from 1 to 10 days after the surgery (mean, 4.3 +/- 2.9 days) and the period of lameness ranged from 7 to 30 days (20 +/- 8.6 days). At the final clinical examination, the dogs did not demonstrate any lameness or pain during passive flexo-extension movements, and there was no significant limitation of the range of motion. CONCLUSION: Extra-articular stabilization with multifilamentous absorbable sutures is a simple, effective method of treatment for acute and chronic coxofemoral luxation. The absorbable material used is strong enough to maintain articular stability during the period of scar tissue formation even in large-breed dogs. CLINICAL RELEVANCE: Absorbable sutures avoid the possible complications related to the use of nonabsorbable material and seem to be sufficient to maintain articular stability during the capsular healing process.

[Tension-free laparoscopic hernioplasty]. / Ernioplastica "tension free" per via laparoscopica.

Hernia recurrence after traditional "open" hernioplasty is an observed event. The tension undergone by anatomical structures of the area is believed to be responsible in large part for these failures. Thus, techniques involving "tension free" hernioplasty have been developed, some of which involve laparoscopic access. Here an experience is reported regarding laparoscopic hernioplasty carried out to repair groin hernias, first of all on an animal model (pigs: 7 cases) then in the clinical field. The technique chosen was the endo-peritoneal positioning of a PTFE prosthesis. The results revealed several advantages over the traditional methods, basically the possibility of reinforcing the entire inguinal floor at the same time as repairing the hernia, and the decrease in groin area discomfort and less time off from work for the patient.

[Experimental intestinal laparoscopic resection]. / Resezione intestinale laparoscopica sperimentale: note di tecnica.

At present, there is a debate in the literature regarding the possibility of extending the laparoscopic approach to intestinal resection. Some techniques have been developed, but certain imperfections and problems remain. This experimental contribution suggests answers to some technical problems arising in the course of a small-bowel laparoscopic resection on a pig.

Intraarticular sodium hyaluronate injections in the Pond-Nuki experimental model of osteoarthritis in dogs. I. Biochemical results.

An established anterior cruciate ligament deficiency-induced articular cartilage degeneration was used to evaluate the effects of intrasynovial injection of hyaluronic acid upon cartilage destruction. In this study, proteoglycan solubility under associative and dissociative conditions was compared in two treatment protocols at intervals of seven, 13, and 17 weeks after surgical breakage of the anterior cruciate ligament in 2.5-year-old Beagle dogs. Untreated joints showed a marked increase in both total soluble glycosaminoglycan (GAG measured as uronic acid) and in the associative fraction. In both treated groups, there was a reduced amount of soluble GAG. Cessation of treatment after seven weeks caused gradual regression, with an increasing amount of CaCl2-soluble material in the associative fraction, while inception at seven weeks gave biochemical evidence of reversal, with increasing GAG present in the guanidine-soluble (dissociative) fraction on the insoluble residue. The protective effects of hyaluronic acid suggest the potential clinical application of this therapy in retarding the advance of osteoarthritis.

nprR1 and nprR2 regulatory regions for neutral protease expression in Bacillus subtilis.

The gene coding for the Bacillus subtilis extracellular neutral protease was isolated from strain BGSC 1A341, an overproducer carrying the nprR2 region, and from strain 168, a normal producer with the nprR1 sequence. The sequence of about 600 nucleotides upstream from the start codon of the protease gene was determined for both strains. The two regions are highly homologous except for a stretch of 66 base pairs close to the promoter region, which is absent in the BGSC 1A341 gene. Northern blot analysis of the in vivo RNAs indicated that the different levels of enzyme secreted by the two strains were due to different amounts of transcripts that accumulated in the cells. Furthermore, at the end of exponential growth, the amount of transcript increased dramatically in the overproducer strain but remained approximately constant in the normal producer strain. The start point(s) for transcription, however, as determined by S1 nuclease mapping of the in vivo transcripts, appeared to be the same for both genes.

New plasmid expression vectors for Bacillus subtilis.

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.

DNA sequence and regulation of ermD, a macrolide-lincosamide-streptogramin B resistance element from Bacillus licheniformis.

The DNA sequence of ermD , a macrolide-lincosamide-streptogramin B (MLS) resistance determinant cloned from the chromosome of Bacillus licheniformis, has been determined. ermD encodes an erythromycin inducible protein of molecular weight 32,796. S1 nuclease mapping of the ermD promoter has revealed the presence of an approximately 354 base leader sequence on the ermD transcript. This leader contains a short open reading frame sufficient to encode a 14 amino acid peptide, which is preceded by a potential ribosomal binding site. The leader sequence has the potential to fold into several base paired structures, in some of which the ribosomal binding site for the ermD product would be sequestered. Deletion analysis demonstrated that the leader contains regulatory sequences. Removal of the ermD promoter and fusion to an upstream promoter did not interfere with induction, strongly suggestion that ermD regulation is posttranscriptional. Based on these features it appears likely that ermD is regulated by a translational attenuation mechanism, analogous to that suggested for ermC , a resistance element from Staphylococcus aureus ( Gryczan et al. 1980; Horinouchi and Weisblum 1980). Comparison of the ermD sequence and that of its product to two other sequenced MLS determinants reveals substantial phylogenetic relatedness, although the three genes are not homologous by the criterion of Southern blot hybridization.

The glycoproteins of the complex surface coat of Trypanosoma lewisi.

Blood stream forms of Trypanosoma lewisi from rats previously infected were labelled in vitro by galactose-oxidase oxidation followed by NaB3H4 reduction and subjected to 7.5% SDS-polyacrylamide gel analysis, by this procedure 8 bands were detected on SDS polyacrylamide gel electrophoresis. We conclude that surface composition of Trypanosoma lewisi resulted more complex than that of Trypanosoma so far studied, suggesting a possible relation with induced immunity and reduced ability of the parasite to survive in its host.

Molecular cloning of the "minor" component of the kinetoplast DNA of Trypanosoma lewisi.

Kinetoplast DNA was cloned in the pBR322 plasmid using the BamHI site. In situ hybridization of iodinated recombinant DNA showed hybridization grains only in the kinetoplast region. The recombinant DNA was analyzed with restriction enzymes and its base ratio was determined by analytical CsCl density gradient.