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.

OBJECTIVE To evaluate a feline-specific multiplex, bead-based assay system for detection of recombinant and native proteins in serum samples and in EDTA-treated and heparinized plasma samples. SAMPLE Serum samples and EDTA-treated and heparinized plasma samples from 30 sick cats and 9 healthy client-owned cats and heparinized whole blood samples from 5 healthy purpose-bred cats. PROCEDURES Ability of the assay system to detect 19 recombinant and native immunologically active proteins in plasma and serum samples from healthy and purpose-bred cats was evaluated via spike-and-recovery tests, assessments of inter- and intra-assay variation, linearity results, and leukocyte stimulation. Effects of various concentrations of heparin and serum matrix solution on percentages of analytes recovered were also evaluated. Analyte concentrations in samples from healthy and sick cats were measured and compared between groups. RESULTS Percentages of analytes recovered were unsatisfactory for most assays. Serum and heparinized plasma samples yielded better recovery results than did EDTA-treated plasma samples. Use of serum matrix solution did not improve results. Use of heparin concentrations greater than the recommended range affected the results. Linearity of results was difficult to assess because of the poor recovery. For the analytes that were recovered sufficiently for assessment, linearity appeared to be reasonable despite the limited detection. CONCLUSIONS AND CLINICAL RELEVANCE Poor percentages of analytes recovered and adverse effects of sample protein matrix limited the usefulness of the multiplex, bead-based assay system for measurement of immunologically active proteins in solutions with high protein content; however, recovery results were fairly linear, potentially allowing evaluation of feline plasma or serum samples with high analyte concentrations.

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.

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.

The proliferation of mesangial cells has been associated with the development of diabetic nephropathy. The cell proliferation has been regulated by diverse growth factors. Among them, insulin like growth factors(IGFs) are also involved in the pathogenesis of diabetic nephropathy. However, it is not yet known about the effect of high glucose on IGF-I and IGF-Ⅱ secretion and the relationship between high glucose-induced secretion of IGFs and PKC or oxidative stress in the mesangial cells.

OBJECTIVE To evaluate 2 processing methods (commercial kit vs conical tube centrifugation) for preparing platelet rich plasma (PRP) for use in llamas and alpacas. SAMPLES Blood samples (30 mL each) aseptically collected from 6 healthy llamas and 6 healthy alpacas. PROCEDURES PRP was prepared from blood samples by use of a commercial kit and by double-step conical tube centrifugation. A CBC was performed for blood and PRP samples. Platelets in PRP samples were activated by means of a freeze-thaw method with or without 23mM CaCl2, and concentrations of platelet-derived growth factor-BB and transforming growth factor-β1 were measured. Values were compared between processing methods and camelid species. RESULTS Blood CBC values for llamas and alpacas were similar. The commercial kit yielded a significantly greater degree of platelet enrichment (mean increase, 8.5 fold vs 2.8 fold) and WBC enrichment (mean increase, 3.7 fold vs 1.9 fold) than did conical tube centrifugation. Llamas had a significantly greater degree of platelet enrichment than alpacas by either processing method. No difference in WBC enrichment was identified between species. Concentrations of both growth factors were significantly greater in PRP samples obtained by use of the commercial kit versus those obtained by conical tube centrifugation. CONCLUSIONS AND CLINICAL RELEVANCE For blood samples from camelids, the commercial kit yielded a PRP product with a higher platelet and WBC concentration than achieved by conical tube centrifugation. Optimal PRP platelet and WBC concentrations for various applications need to be determined for llamas and alpacas.

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 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.

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.