Adenoviral delivery of soluble ovine OX40L or CD70 costimulatory molecules improves adaptive immune responses to a model antigen in sheep.

The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressing Ovis aries (Oa)OX40L or OaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. While OaOX40L administration mainly increased TNF-α and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-γ producing cells, we detected greater effects on adaptive immunity after OaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-γ production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+ T cells (CD8+ CD62L- CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+ CD62L- CD27+). Moreover, recall anti-OVA CD8+ T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+ T cell effector differentiation and long-term activity. In this work we characterized the in vivo adjuvant potential on the humoral and cellular immune response of OaOX40L and OaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant.

Evaluation of perinuclear antineutrophilic cytoplasmic antibodies in sera from dogs with inflammatory bowel disease or intestinal lymphoma.

OBJECTIVE: To assess and compare the expression of perinuclear antineutrophilic cytoplasmic antibodies (pANCA) in sera obtained from dogs with inflammatory bowel disease (IBD) and dogs with intestinal lymphoma. ANIMALS: 104 dogs with IBD and 23 dogs with intestinal lymphoma. PROCEDURES: Each ill dog had persistent gastrointestinal signs (> 3 weeks in duration) and absence of response to diet changes or antimicrobial treatments. Gastrointestinal endoscopy was performed in ill dogs to obtain intestinal biopsy specimens for histologic confirmation of IBD or lymphoma. A serum sample was obtained from each ill dog. Neutrophils were isolated from a blood sample from the healthy dog; neutrophil-bearing slides were incubated with serum from each ill dog and examined for expression of pANCA by use of an indirect immunofluorescence technique. Detection of cells that had a perinuclear fluorescence pattern was considered a positive result. RESULTS: The 2 groups of dogs did not differ with regard to breed and sex but did differ with regard to age. Expression of pANCA was detected in 38 of the 104 (36.5%) dogs with IBD and 4 of the 23 (17.4%) dogs with intestinal lymphoma. Although the frequency of pANCA expression was higher in dogs with IBD, compared with findings in dogs with intestinal lymphoma, the difference was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that circulating pANCA are present in some dogs with IBD or intestinal lymphoma. However, pANCA detection does not seem to be useful for distinguishing dogs with IBD from dogs with intestinal lymphoma.

Detection of perinuclear antineutrophil cytoplasmic antibodies and antinuclear antibodies in the diagnosis of canine inflammatory bowel disease.

In recent years, serologic markers for diagnosis and classification of inflammatory bowel disease (IBD) have been used in human medicine. Perinuclear, antineutrophil, cytoplasmic antibodies (p-ANCA) are the most important of these markers. Because of their similar pattern of fluorescence, antinuclear antibodies (ANA) could cause misleading interpretations. The aim of the present study was to evaluate the use of an indirect fluorescent antibody test to detect p-ANCA in dogs with IBD, to compare the presence of p-ANCA in dogs with IBD with the presence of the same antibodies in other dogs, and to analyze the presence of ANAs in the p-ANCA-positive samples. Using a 110 dilution as a cutoff point, a sensitivity of 0.34 and a specificity of 0.86 was obtained when dogs with IBD were compared with the other groups as a whole, and specificity increased to 0.94 when dogs with IBD were compared with animals with other chronic gastrointestinal disorders. The lowest specificity value, 0.76, was obtained when the group of dogs with IBD was compared with that of dogs with different inflammatory and infectious disorders. Globally, 78 dogs were positive for p-ANCA when the cutoff was 110. Only 1 dog from these 78 animals was also seropositive to ANA. The results suggest that 1) detection of p-ANCA might be included in the IBD diagnostic protocol as another test to differentiate between this disease and other digestive diseases with similar clinical signs, and 2) most p-ANCA-positive dogs are not ANA positive.

Evaluation of perinuclear anti-neutrophilic cytoplasmic autoantibodies as an early marker of protein-losing enteropathy and protein-losing nephropathy in Soft Coated Wheaten Terriers.

OBJECTIVE: To evaluate perinuclear anti-neutrophilic cytoplasmic autoantibody (pANCA) status in Soft Coated Wheaten Terriers (SCWTs) and SCWT-Beagle crossbred dogs and to correlate pANCA status of dogs with clinicopathologic variables of protein-losing enteropathy (PLE), protein-losing nephropathy (PLN), or both. ANIMALS: 13 SCWTs and 8 SCWT-Beagle crossbred dogs in a research colony and a control group comprising 7 dogs with X-linked hereditary nephropathy and 12 healthy SCWTs > 9 years old. PROCEDURES: Samples were obtained from dogs in the research colony every 6 months. At each sample-collection time point, serum concentrations of albumin, globulin, creatinine, and urea nitrogen; fecal concentration of alpha-proteinase inhibitor; and urinary protein-to-creatinine ratios were determined and correlated with pANCA status. RESULTS: 20 of 21 dogs in the research colony had positive results for pANCAs at a minimum of 2 time points, and 18 of 21 dogs had definitive evidence of disease. None of the control dogs had positive results for pANCAs. A positive result for pANCAs was significantly associated with hypoalbuminemia, and pANCAs preceded the onset of hypoalbuminemia on an average of 2.4 years. Sensitivity and specificity for use of pANCAs to predict development of PLE or PLN were 0.95 (95% confidence interval, 0.72 to 1.00) and 0.8 (95% confidence interval, 0.51 to 0.95), respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Most dogs in this study affected with PLE, PLN, or both had positive results for pANCAs before clinicopathologic evidence of disease was detected. Thus, pANCAs may be useful as an early noninvasive test of disease in SCWTs.