Introduction: Antimicrobial peptides (AMP) are a large group of innate immune effectors, which apart from antimicrobial activity show immunomodulative properties. Platelet-rich plasma (PRP) is a source of autologous growth factors and is used for stimulation of bone and soft tissue healing. The purpose of this study was to assess the influence of PRP and AMP extract on ovine monocyte-derived macrophage cultures. Material and Methods: The study was conducted on ovine macrophages (Mfs) previously stimulated with LPS or dexamethasone and then with preparations of PRP or AMP. Following activation of the Mfs their morphological and functional features were assessed. Results: The study revealed pro-inflammatory influence of both examined preparations on Mfs cultures on the basis of morphology, ROS generation and arginase activity. Both preparations enhanced the pro-inflammatory response of cultured Mfs. Conclusion: This activity may intensify the antimicrobial action of Mfs, however, in cases of excessive and prolonged inflammation the use of these preparations should be limited.

This study was conducted with the purpose of investigating the distribution of the Activating Transcription Factor 6 (ATF6) and the Nerve Growth Factor (NGF) in the duodenum tissue of diabetic and non-diabetic rats. Eighteen female Sprague dawley rats were randomly divided into three groups as thecontrol, sham and diabetes groups. Routine histological and immunohistochemical methods were appliedon the duodenum tissues collected at the end of the study.Results: It was determined that the villus length measurements showed a statistically significant differencebetween the control and diabetes groups. There was NGF immunoreactivity which was moderate anddiffuse cytoplasmic in the villus intestinalis and muscularis layer in all groups, weak in the crypts andglands in the control and sham groups and moderate and diffuse cytoplasmic in the diabetes group. ATF6immunoreactivity was determined moderate in the villus intestinalis, crypts, glands and muscularis layerin the control and sham groups and strong diffuse cytoplasmic in the diabetes group. It wasdetermined that both NGF and ATF6 immunoreactivity increased in the duodenum tissue of the rats onwhich diabetes was induced experimentally.

Objective-To evaluate intra-articular autologous protein solution (APS) for the treatment of osteoarthritis in horses. Animals-40 client-owned horses with naturally occuring osteoarthritis. Procedures-APS was generated from a dual-device system that concentrated plasma and WBC proteins and enriched platelet growth factors. Horses were randomly assigned to receive an intra-articular injection of 5 mL of saline (0.9% NaCl) solution (n = 20) or APS (20), exercised on a treadmill, and evaluated on the basis of lameness grades, kinetic gait analysis, joint circumference, and range of motion for 14 days. Horses that received saline solution were administered APS at termination of the study, and clients scored horses for lameness and discomfort before, 12 weeks after, and 52 weeks after the APS injection. Results-The APS group had significant improvements in lameness grade, asymmetry indices of vertical peak force, and range of joint motion by 14 days, compared with baseline or control group values. No adverse effects associated with APS treatment were evident. Clients assessed lameness and comfort as improved at 12 and 52 weeks. The APS had greater likelihood (OR, 4.3 to 30.0) of a therapeutic response in horses with a lameness score < 4, < 10% vertical force asymmetry, or absence of marked osteophyte formation, subchondral sclerosis, or joint space narrowing. Concentration of interleukin-1 receptor antagonist in APS was 5.8 times that in blood. Conclusions and Clinical Relevance-Intra-articular administration of APS can be considered an effective treatment option for equine osteoarthritis, with the potential for disease-modifying effects.

Objective—To determine the differentiation of canine adipose tissue–derived mesenchymal stem cells (ASCs) into endothelial progenitor cells (EPCs). Animals—3 healthy adult Beagles. Procedures—Canine ASCs were isolated and cultured from adipose tissue, and endothelial differentiation was induced by culturing ASCs in differentiation medium. Morphological and immunophenotypic changes were monitored. Expression of endothelial-specific markers was analyzed by conventional and real-time RT-PCR assay. The in vitro and in vivo functional characteristics of the endothelial-like cells induced from canine ASCs were evaluated by use of an in vitro solubilized basement membrane tube assay, low-density lipoprotein uptake assay, and in vivo solubilized basement membrane plug assay. Results—After differentiation culture, the cells developed morphological changes, expressed EPC markers such as CD34 and vascular endothelial growth factor receptor 2, and revealed functional characteristics in vitro. Furthermore, the induced cells allowed vessel formation in solubilized basement membrane plugs in vivo. Conclusion and Clinical Relevance—Results indicated that canine ASCs developed EPC characteristics when stimulated by differentiation medium with growth factors. Thus, differentiated canine ASC-EPCs may have the potential to provide vascularization for constructs used in regenerative medicine strategies.

Objective-To determine the efficiency of a novel point-of-care gravitational marrow separator and bone marrow aspiration needle for concentrated bone marrow production and bone marrow-derived mesenchymal stem cell (MSC) separation and assess the effect of repeated bone marrow collections in horses. Animals-8 healthy adult horses. Procedures-Bone marrow aspiration was performed twice (1 month apart) from sternebral bodies with a standard or prototype multidirectional needle. Concentrated bone marrow was obtained by gravitational marrow separation and evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, MSCs, and cell viability. Results-Concentrated bone marrow samples obtained with the marrow separator had 5- to 19-fold bone marrow-derived MSC, WBC, and platelet counts, compared with original bone marrow samples. Use of a multidirectional needle increased the frequency of obtaining MSC-richer concentrated bone marrow. Repeating bone marrow aspiration at 1 month yielded greater MSC numbers but slightly lower cell viability after processing. Conclusions and Clinical Relevance-The gravitational bone marrow separator and multidirectional needle were used to effectively harvest bone marrow and improve the quality of concentrated bone marrow. Comparable, or even greater, numbers of bone marrow-derived MSCs were collected by repeated bone marrow aspiration after a 1-month interval from the same aspiration sites. Use of the marrow separator and multidirectional bone marrow aspiration needle can facilitate a 1-step, point-of-care, nonlaboratory method to obtain concentrated bone marrow as a mixture of bone marrow-derived MSCs and growth factors from platelets and plasma.

Objective: To isolate and characterize neural stem and progenitor cell populations in the brain of adult dogs. Animals: 7 healthy adult dogs. Procedures: Dogs (age, 10 to 60 months) were euthanized for reasons unrelated to the study. The subventricular zone (SVZ) adjacent to the lateral ventricles and subgranular zone (SGZ) of the hippocampus were isolated and used to generate single cell suspensions for nonadherent culture. The resulting primary neurospheres were serially passaged to assess self-renewal capacity. Neurospheres were differentiated by the withdrawal of growth factors and the addition of serum. Differentiated and undifferentiated neurospheres were analyzed via reverse transcriptase PCR assay or immunocytochemical staining for markers of pluripotency and neural lineage. Results: Neurospheres were generated from the SVZ and SGZ in all dogs. The SVZ generated more primary neurospheres than did the SGZ. Serial passage was successful, although few neurospheres could be generated after the fifth passage. Undifferentiated neurospheres were positive for SOX2, nestin, and glial fibrillary acidic protein (GFAP) and negative for OCT4 and NANOG. After differentiation, GFAP, neuronal class III β-tubulin, and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase–positive progeny were noted migrating out of the neurospheres. Conclusions and Clinical Relevance: Results suggested the persistence of SOX2-positive, nestin-positive, GFAP-positive, OCT4-negative, and NANOG-negative neural progenitor cells in the SVZ and SGZ regions of mature canine brains, which are capable of producing multiple cell lineages. This study may serve as a basis for future studies investigating the role of these cells in various disease processes, such as neoplasia, or for regenerative purposes.

Objective-To evaluate canine histiocytic sarcoma cell lines and tumor samples for dysregulation of the Kit/stem-cell factor (SCF), Flt3/Flt3 ligand (Flt3L), and Met/hepatocyte growth factor (HGF) receptor tyrosine kinase signaling pathways, as these are known to contribute to the differentiation and survival of normal dendritic cells as well as malignant transformation of dendritic cells in mouse models. Sample Population-4 histiocytic sarcoma tumor cell lines and 35 formalin-fixed histiocytic sarcoma specimens obtained from dogs. Procedure-Histiocytic sarcoma cell lines were evaluated for expression of Kit/SCF, Flt3/Flt3L, and Met/HGF by use of reverse transcriptase-PCR procedures. Histiocytic sarcoma cell lines and tumor samples were evaluated for mutations in Kit, Flt3, and Met by use of PCR analysis of genomic DNA, followed by both sequencing and fluorescent PAGE for deletions or internal tandem duplications. The ability of the multitargeted split-kinase inhibitor SU11654 to block proliferation and induce apoptosis of histiocytic sarcoma cell lines was also evaluated. Results-No mutations in Kit, Flt3, and Met were identified in any of the cell lines or tumor samples evaluated. Furthermore, SU11654 did not induce cellcycle arrest or apoptosis of histiocytic sarcoma lines, even at supratherapeutic doses. Conclusions and Clinical Relevance-These data suggest that dysregulation of Kit/SCF, Flt3/Flt3L, and Met/HGF signaling pathways is unlikely to occur in histiocytic sarcomas of dogs and that inhibitors of the Kit, Flt3, and Met pathways are unlikely to provide clinical benefit to dogs with histiocytic sarcomas.

The effect of ingested epidermal growth factor (EGF) on the small intestinal mucosa of conventionally weaned pigs was determined. At 21 days of age, 39 pigs were randomly distributed into suckling and weaned treatment groups that were administered 124 micrograms of EGF, 372 micrograms of EGF, or the dosing compound daily. Fecal water content was determined daily. On postweaning days 0, 3, 6, and 9, representative pigs from each group were euthanatized, and jejunal mucosa samples were collected for determination of villus-to-crypt ratio, total protein content, disaccharidase activities, and microbiological populations. At postweaning day 3, the 372-micrograms dose of EGF significantly (P less than or equal to 0.05) increased jejunal lactase and sucrase activities in the weaned pigs. Increased lactase activity was not greater than that of the suckling pig controls, whereas sucrase activity was significantly (P less than or equal to 0.05) higher than that of the suckling pig controls. Significant changes were not observed in villus-to-crypt ratio, mucosal protein content, or disaccharidase activities on other collection days.

Canine circulating neutrophils, isolated by a blood lysing technique, were incubated with 7-S nerve growth factor (NGF), at final concentrations between 12.5 and 800 ng/ml, for 30 minutes at 37 C. Neutrophil cytosolic H2O2 production, measured by flow cytometry, after 7-S NGF incubation was not significantly different from that produced at 37 C (baseline temperature controls) alone. Phorbol myristate acetate (PMA; 100 ng/ml) stimulation of neutrophils produced cytosolic H2O2 concentrations almost 13 times that of baseline temperature control neutrophils. Preincubation of neutrophils with 7-S NGF (100 to 800 ng/ml, 30 minutes, 37 C) and subsequent stimulation by PMA resulted in augmented H2O2 production in excess of twice that of neutrophils treated with PMA alone, and almost 30 times that of baseline temperature controls.

Epidermal growth factor was injected intracamerally into the anterior chamber of the right eye of 9 cats. The central portion of the cornea in 8 of the 9 cats that had been cryoinjured. Effect of epidermal growth factor on the repair of endothelial cells in cats was evaluated by endothelial specular microscopy. Endothelial cell density and corneal thickness were studied quantitatively, as a measure of endothelial cell function. The repair process also was evaluated qualitatively by studying morphologic changes, developing as a result of reendothelialization and return to normal function. Seemingly, differences between rate of healing of cryoinjured eyes injected with epidermal growth factor and that in nontreated eyes were not significant (P = 0.86). The endothelial repair process was characterized by enlargement and migration of adjacent noninjured cells.