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.

Introduction: Due to their immunostimulatory properties TLR ligands are used prophylactically to protect against a variety of viral and bacterial pathogens in mammals. Knowledge of the molecular and functional aspects of TLRs is essential for a better understanding of the immune system and resistance to diseases in birds. For that reason, this study attempted to determine the impact of TLR21 stimulation by its synthetic ligand (CpG ODN, class B) on the chicken immune system. Material and Methods: Sixty embryonated chicken eggs were randomly allocated into three groups (control and two experimental groups). On day 18 of embryonic development, chickens in one experimental group were administered in ovo a low dose of CpG ODN and the birds of the second experimental group were given a high dose of the ligand. Spleens were collected at 1, 2, 5, and 10 days post-hatching (dph) for analysis of IFN-α, IFN-β, IFN-γ, IL-6, and IL-10 expression using qRT-PCR. Results: Significant differences were observed in mRNA expression levels of all the measured cytokines associated with the modulation and regulation of the immune response at different time points. Conclusion: The obtained data clearly demonstrate that immune response induction takes place after in ovo administration of class B CpG ODN, and that the ligand has the ability to induce cytokine responses in neonatal chicken spleen.

OBJECTIVE: To investigate the distribution of T-cell markers (CD4 and CD8α) in lymphoid organs of newborn, juvenile, and adult yaks. ANIMALS: 15 healthy male yaks of various ages from highland plateaus. PROCEDURES: Yaks were allocated to groups on the basis of age (newborn [1 to 7 days old; n = 5], juvenile [5 to 7 months old; 5], and adult [3 to 4 years old; 5]). The thymus, spleen, 5 mesenteric lymph nodes, and 5 hemal nodes were harvested from each yak within 10 minutes after euthanasia. Morphological characteristics of those lymphoid organs were assessed by histologic examination; expression of CD4 and CD8α mRNAs and proteins were measured by quantitative real-time PCR assay and immunohistochemical staining. RESULTS: Among the lymphoid organs evaluated, expressions of CD4 and CD8α mRNAs were highest in the thymus in all age groups. In newborn lymphoid organs, CD4 mRNA expression and CD4+ cell distribution were more predominant, whereas in juvenile and adult lymphoid organs, CD8α mRNA expression and CD8α+ cell distribution were more predominant. The CD4+ and CD8α+ cells were mainly located in the cortex and medulla of the thymus, the medulla of the hemal nodes and mesenteric lymph nodes, the periarteriolar lymphoid sheaths, and the red pulp of the spleen. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the CD4 mRNA expression and CD4+ T-cell distribution in yak lymphoid organs decreased and CD8α mRNA expression and CD8α+ T-cell distribution increased with age. Moreover, CD8α+ cells were present in the follicles of yaks’ secondary lymphoid organs, which differs from findings for other mammals.

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 quantify plasma fentanyl concentrations (PFCs) and evaluate antinociceptive and respiratory effects following application of transdermal fentanyl patches (TFPs) and assess cerebrospinal μ-opioid receptor mRNA expression in ball pythons (compared with findings in turtles). ANIMALS 44 ball pythons (Python regius) and 10 turtles (Trachemys scripta elegans). PROCEDURES To administer 3 or 12 μg of fentanyl/h, a quarter or whole TFP (TFP-3 and TFP-12, respectively) was used. At intervals after TFP-12 application in snakes, PFCs were measured by reverse-phase high-pressure liquid chromatography. Infrared heat stimuli were applied to the rostroventral surface of snakes to determine thermal withdrawal latencies after treatments with no TFP (control [n = 16]) and TFP-3 (8) or TFP-12 (9). Breathing frequency was measured in unrestrained controls and TFP-12–treated snakes. μ-Opioid receptor mRNA expression in brain and spinal cord tissue samples from snakes and turtles (which are responsive to μ-opioid receptor agonist drugs) were quantified with a reverse transcription PCR assay. RESULTS Mean PFCs were 79, 238, and 111 ng/mL at 6, 24, and 48 hours after TFP-12 application, respectively. At 3 to 48 hours after TFP-3 or TFP-12 application, thermal withdrawal latencies did not differ from pretreatment values or control treatment findings. For TFP-12–treated snakes, mean breathing frequency significantly decreased from the pretreatment value by 23% and 41% at the 24- and 48-hour time points, respectively. Brain and spinal cord tissue μ-opioid receptor mRNA expressions in snakes and turtles did not differ. CONCLUSIONS AND CLINICAL RELEVANCE In ball pythons, TFP-12 application resulted in high PFCs, but there was no change in thermal antinociception, indicating resistance to μ-opioid-dependent antinociception in this species.

OBJCTIVE To investigate the effects of dietary lipid overload on bile acid metabolism and gallbladder motility in healthy dogs. ANIMALS 7 healthy Beagles. PROCEDURES In a crossover study, dogs were fed a high-fat–high-cholesterol diet (HFCD) or a low-fat diet (LFD) for a period of 2 weeks. After a 4-month washout period, dogs were fed the other diet for 2 weeks. Before and at the end of each feeding period, the concentrations of each of the gallbladder bile acids, cholecystokinin (CCK)-induced gallbladder motility, and bile acid metabolism–related hepatic gene expression were examined in all dogs. RESULTS The HFCD significantly increased plasma total cholesterol concentrations. The HFCD also increased the concentration of taurochenodeoxycholic acid and decreased the concentration of taurocholic acid in bile and reduced gallbladder contractility, whereas the LFD significantly decreased the concentration of taurodeoxycholic acid in bile. Gene expression analysis revealed significant elevation of cholesterol 7α-hydroxylase mRNA expression after feeding the HFCD for 2 weeks, but the expression of other genes was unchanged. CONCLUSIONS AND CLINICAL RELEVANCE Feeding the HFCD and LFD for 2 weeks induced changes in gallbladder bile acid composition and gallbladder motility in dogs. In particular, feeding the HFCD caused an increase in plasma total cholesterol concentration, an increase of hydrophobic bile acid concentration in bile, and a decrease in gallbladder sensitivity to CCK. These results suggested that similar bile acid compositional changes and gallbladder hypomotility might be evident in dogs with hyperlipidemia.

OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis. SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram. PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti–human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed. RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C. CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.

OBJECTIVE To determine effects of transforming growth factor (TGF)-β and interleukin (IL)-1β on inflammatory markers in cultured canine chondrocytes to clarify the role of these cytokines in osteoarthritis of dogs. SAMPLE Pooled chondrocytes isolated from the stifle joints of 4 adult dogs. PROCEDURES Chondrocytes were isolated, cultured, and frozen at −80°C. Pooled cells were incubated in medium with or without TGF-β (1 or 10 ng/mL) and subsequently stimulated with IL-1β (10 ng/mL). Concentrations of nitric oxide (NO) and prostaglandin (PG) E were measured in culture supernatants. Gene expression of matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-2, inducible NO synthase (iNOS), and cyclooxygenase (COX)-2 was quantified by use of real-time quantitative PCR assays. RESULTS Stimulation with IL-1β increased gene expression of all inflammatory markers, except for TIMP-2. Incubation with TGF-β resulted in a significant decrease in MMP-3 and TIMP-2 mRNA concentrations but had no effect on PGE and NO concentrations. For cells treated with TGF-β followed by IL-1β, concentrations of PGE and NO were lower, compared with concentrations for IL-1β control cells. Furthermore, IL-1β–induced gene expression of iNOS, MMP-3, and COX-2 was downregulated. However, the IL-1β–induced downregulation of TIMP-2 gene expression was partially restored by pretreatment with TGF-β. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that IL-1β increased the expression of inflammatory genes and mediators, and TGF-β largely attenuated the IL-1β–mediated inflammatory response. Therefore, TGF-β might be a novel target for use in the prevention and treatment of cartilage breakdown in dogs with osteoarthritis.

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII–deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in factor VII–deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.