Introduction: Vascular cell adhesion molecule 1 (VCAM-1) is a member of Ig superfamily. The aim of this study was to prepare highly specific polyclonal antibodies against bovine VCAM-1 and to evaluate the expression of VCAM-1 in the mammary lymph nodes of cows with subclinical mastitis.Material and Methods: The VCAM-1 gene was cloned from bovine Peyer’s patches and inserted into the pGEX-4T-1 and pET-28a vectors. The recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1 were transferred into Escherichia coli BL21 and the recombinant strains were induced by isopropyl-D-thiogalactoside to produce fusion proteins tagged with polyhistidine (His) and glutathione S-transferase (GST), respectively. The expressed fusion proteins His-VCAM-1 and GST-VCAM-1 were identified by SDS-PAGE and Western blot. His-VCAM-1 protein was used as an antigen to immunise Wistar rats and polyclonal antibody serum against VCAM-1 was obtained.Results: The serum titre tested by indirect ELISA was 128,000 using GST-VCAM-1 as the well coating antigen. Western blots indicated that the antibody recognised recombinant VCAM-1 protein as well as endogenous VCAM-1. In addition, using qPCR and Western blot, VCAM-1 mRNA and protein expression levels were measured in dairy cows with subclinical mastitis. It was demonstrated that VCAM-1 levels in the mammary lymph nodes of the cows were significantly higher than those from healthy controls (P < 0.05).Conclusion: These results are to our knowledge the first report that VCAM-1 expression in the mammary lymph nodes is elevated in dairy cows with subclinical mastitis.

OBJECTIVE To examine whether expression of extracellular matrix metalloproteinase inducer (EMMPRIN) can be detected in equine lungs and whether it correlates with matrix metalloproteinase (MMP)-2 and -9 expression in bronchoalveolar lavage fluid (BALF) of horses with chronic inflammation of the lungs (ie, lower airway inflammation [LAI]). ANIMALS 29 horses with signs of chronic respiratory tract disease, which were classified as the LAI (n = 17) and LAI with respiratory distress (RDLAI [12]) groups, and 15 control horses. PROCEDURES BALF, tracheal aspirate, and blood samples were obtained, and EMMPRIN expression was determined from BALF cells and RBCs by use of western blotting. Activities of MMP-2 and -9 were determined with zymography. RESULTS Expression of EMMPRIN protein was identified in BALF cells of all horses. Expression of EMMPRIN protein was highest for the RDLAI group and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that EMMPRIN was involved in the pathophysiologic processes of asthma in horses. However, additional studies of horses and other species are warranted to elucidate the regulation of EMMPRIN expression in asthmatic lungs.

In order to study the ability of Lactobacillus casei to ameliorate murine enteritis, 18 mice were randomly divided into 3 groups: the enteritis group, intervention group, and control group. The interleukin (IL)-6 and transforming growth factor-β (TGF)-β content in mouse peripheral blood and duodenum was detected using an enzyme-linked immunosorbent assay (ELISA). The number of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) and CD4+IL-17A+ Th17 cells in the mesenteric lymph nodes (MLN) and spleen were detected using flow cytometry, and quantitative reverse transcription polymerase chain reaction (PCR) and western blot analysis were used to measure Foxp3 and retinoid-related orphan receptor-γ (RORγt) mRNA and protein expression in the MLN. Histological changes in the duodenum were observed. Results indicate that in the intervention group, IL-6 content in mouse peripheral blood and duodenum was significantly lower than in the enteritis group (P < 0.05), while TGF-β content was significantly increased compared to the enteritis group (P < 0.05). For the intervention group, the percentages of CD4+CD25+Foxp3+ Tregs in spleen and MLN were increased (P < 0.05), while the percentages of CD4+IL-17A+ Th17 cells were decreased compared to the enteritis group (P < 0.05). The expression of Foxp3 mRNA and protein in the intervention group was higher than in the enteritis group, while RORγt mRNA and protein were significantly lower (P < 0.05). After mice in the enteritis group were treated with L. casei, duodenal inflammation was relieved. This study demonstrated that L. casei could have possible implications for the enterotoxigenic Escherichia coli (ETEC) induced intestinal inflammation by regulating the ratio imbalance of Th17/Treg cells.

OBJECTIVE To investigate protein kinase CK2 (CK2) expression in squamous cell carcinoma (SCC) of cats and to examine effects of CK2 downregulation on in vitro apoptosis and viability in SCC. SAMPLE Biopsy specimens of oral mucosa and testis and blood samples from clinically normal cats, biopsy specimens of oral SCC from cats, and feline SCC (SCCF1) and mammary gland carcinoma (K12) cell lines. PROCEDURES Immunohistochemically labeling for CK2α was performed on biopsy specimens. Sequences of the CK2α subunit gene and CK2α’ subunit gene in feline blood and feline cancer cell lines were determined by use of PCR and reverse-transcription PCR assays followed by direct Sanger sequencing. Specific small interfering RNAs (siRNAs) were developed for feline CK2α and CK2α'. The SCCF1 cells were treated with siRNA and assessed 72 hours later for CK2α and CK2α’ expression and markers of apoptosis (via western blot analysis) and for viability (via 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium assays). RESULTS CK2α was expressed in all feline oral mucosa samples and 7 of 8 oral SCC samples. Expression of CK2α and CK2α’ was successfully downregulated in SCCF1 cells by use of siRNAs, which resulted in decreased viability and induction of apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE In this study, CK2 appeared to be a promising therapeutic target for SCCs of cats. A possible treatment strategy for SCCs of cats would be RNA interference that targets CK2.