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 investigate the relationship between runt-related transcription factor 2 (RUNX2) expression in canine nucleus pulposus (NP) cells and intervertebral disk aging in chondrodystrophoid dogs. Animals: 7 healthy Beagles (mean age, 35.6 months) and 11 Dachshunds with herniated disks (mean age, 61 months). Procedures: All dogs underwent MRI examination of the thoracic and lumbar vertebral column immediately before sample collection under general anesthesia. The disk center–to–CSF T2-weighted signal intensity ratio was determined for healthy Beagles. Samples of NP were obtained from nonherniated disks in healthy Beagles and from herniated disks during surgical treatment of hospitalized Dachshunds. Samples were evaluated for RUNX2 and matrix metalloproteinase 13 transcript expression via reverse transcriptase PCR assay; RUNX2 protein expression was evaluated via immunohistochemical analysis, and correlation between these variables and age of dogs was evaluated. A 3′ and 5′ rapid amplification of cDNA ends method was used to identify the RUNX2 coding region. Results: RUNX2 cDNA had > 97% conservation with the human cDNA sequence and approximately 95% conservation with the mouse cDNA sequence; RUNX2 and matrix metalloproteinase 13 mRNA expression and RUNX2 protein expression in NP cells were positively correlated with age. The disk center–to–CSF T2-weighted signal intensity ratio was negatively correlated with RUNX2 protein expression in the NP of healthy dogs. Conclusions and Clinical Relevance: Results indicated that RUNX2 mRNA and protein expression in the NP are enhanced in aging intervertebral disks in dogs.

OBJECTIVE To compare gene expression patterns of T cells in porcine colostrum and peripheral blood. ANIMALS 10 multiparous sows. PROCEDURES Cytotoxic and CD4-CD8 double-positive T cells were separated from porcine colostrum and peripheral blood. Total RNA was extracted. The cDNA prepared from RNA was amplified, labeled, fragmented, and competitively hybridized to DNA microarray slides. The DNA microarray data were validated by use of a real-time reverse-transcription PCR assay, and expression of the genes FOS, NFKBI, IFNG, CXCR6, CCR5, ITGB2, CCR7, and SELL was assessed. Finally, DNA microarray data were validated at the protein level by use of flow cytometry via expression of c-Fos and integrin β-2. RESULTS Evaluation of gene expression profiles indicated that in contrast to results for peripheral blood, numerous cell-signaling pathways might be activated in colostrum. Profile analysis also revealed that FOS and NFKBI (genes of transcription factors) were involved in most cell-signaling pathways and that expression of these genes was significantly higher in colostral T cells than in peripheral blood T cells. Furthermore, CCR7 and SELL (genes of T-cell differentiation markers) in colostral T cells had expression patterns extremely similar to those found in effector or effector memory T cells. CONCLUSIONS AND CLINICAL RELEVANCE All or most of the T cells in colostrum had an effector-like phenotype and thus were more activated than those in peripheral blood. This gene expression profile would enable T cells to migrate to mammary glands, be secreted in colostrum, and likely contribute to passive immunity provided by sows to newborn pigs.

Mannheimia haemolytica is an important cause of pneumonia in feedlot cattle. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor responsible for the induction of antioxidant enzymes, such as heme oxygenase 1 (HO-1), within the lung. The expression of Nrf2 and HO-1 was immunohistochemically evaluated in 4 calves 24 h after experimental infection with M. haemolytica. Calves receiving normal saline served as controls. In the infected lungs, cytoplasmic Nrf2 expression was high in macrophages and bronchioles and low in alveolar epithelium, whereas nuclear expression was high in endothelial cells, macrophages, and bronchioles and lowest in alveolar epithelium. Normal lung samples displayed only faint Nrf2 cytoplasmic staining within bronchiolar epithelium. Expression of HO-1 was detected within the cytoplasm of macrophages and bronchiolar epithelial cells in all infected lung samples, whereas normal lungs displayed only weak cytoplasmic staining in bronchiolar epithelial cells. These findings suggest that bronchiolar epithelial cells and macrophages up-regulate Nrf2 expression early in the course of infection, which results in increased expression of HO-1 within these cells.

Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm2) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

Immunohistochemistry has been used extensively to evaluate protein expression in clinical and research settings. However, immunohistochemistry is not always successful in veterinary medicine due to the lack of reliable antibody options, poor tissue preservation, labor-intensive staining, and antigen-retrieval optimization processes. RNAscope in-situ hybridization (ISH) is a powerful technology that uses a specific sequence probe to identify targeted mRNA. In this study, we demonstrate RNAscope ISH in 4 common canine malignancies, which are traditionally diagnosed by histopathology and immunohistochemistry. Probes were designed for commonly targeted mRNA markers of neoplastic tumors; these included c-kit in mast cell tumor, microphthalmia-associated transcription factor in malignant melanoma, ionized calcium-binding adapter molecule(-1) in histiocytic sarcoma, and alkaline phosphatase in osteosarcoma. A strong staining signal was obtained by these 4 targets in each canine malignancy. These results support the use of RNAscope ISH for definitive diagnosis in canine malignancies.

Objective: To isolate and characterize mesenchymal stem cells (MSCs) from canine muscle and periosteum and compare proliferative capacities of bone marrow-, adipose tissue-, muscle-, and periosteum-derived MSCs (BMSCs, AMSCs, MMSCs, and PMSCs, respectively). Sample: 7 canine cadavers. Procedures: MSCs were characterized on the basis of morphology, immunofluorescence of MSC-associated cell surface markers, and expression of pluripotency-associated transcription factors. Morphological and histochemical methods were used to evaluate differentiation of MSCs cultured in adipogenic, osteogenic, and chondrogenic media. Messenger ribonucleic acid expression of alkaline phosphatase, RUNX2, OSTERIX, and OSTEOPONTIN were evaluated as markers for osteogenic differentiation. Passage-1 MSCs were counted at 24, 48, 72, and 96 hours to determine tissue-specific MSC proliferative capacity. Mesenchymal stem cell yield per gram of tissue was calculated for confluent passage-1 MSCs. Results: Successful isolation of BMSCs, AMSCs, MMSCs, and PMSCs was determined on the basis of morphology; expression of CD44 and CD90; no expression of CD34 and CD45; mRNA expression of SOX2, OCT4, and NANOG; and adipogenic and osteogenic differentiation. Proliferative capacity was not significantly different among BMSCs, AMSCs, MMSCs, and PMSCs over a 4-day culture period. Periosteum provided a significantly higher MSC yield per gram of tissue once confluent in passage 1 (mean ± SD of 19,400,000 ± 12,800,000 of PMSCs/g of periosteum obtained in a mean ± SD of 13 ± 1.64 days). Conclusions and Clinical Relevance: Results indicated that canine muscle and periosteum may be sources of MSCs. Periosteum was a superior tissue source for MSC yield and may be useful in allogenic applications.