Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermal injection in cattle.In vitro, such antigens stimulate the production of gamma interferon (IFN-␥) by bovine T cells in whole-blood culture (IFN-␥ assay). We have analyzed various parameters of the in vitro IFN-␥ assay, ranging from blood sampling to execution of the IFN-␥ test, in view of potential simplifications of the assay. Here, we show that IFN-␥ responses may be reduced under certain animal handling/holding conditions and that a delayed time from blood collection to culture may lead to a reduced in vitro IFN-␥ response. Delayed initiation of culture in a purified-protein-derivative-based assay (24 h compared to 8 h after blood collection), however, resulted in a significant improvement of specificity (97% compared to 85%), whereas there was only a modest reduction of sensitivity (from 96% to 90%), which was statistically not significant. Furthermore, we show that the stimulation temperature needs to be 33°C or higher; that carbon dioxide is not required for stimulation; and that various plate formats, ranging from 24 to 96 wells per plate, can be utilized. The produced IFN-␥ is stable at 4°C for 28 days as well as after repeated freeze-thaw cycles. Thus, stimulation of samples may be initiated in the field without the need for a carbon dioxide source, and bovine IFN-␥ is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-␥ test platform and flexibilities in test application.Bovine tuberculosis (TB), caused by Mycobacterium bovis, has an important and adverse effect on socioeconomic conditions, public health, and trade of animals and animal products (2). Eradication of bovine TB in cattle is based on detection and slaughter of infected animals or whole herds. The standard antemortem screening test for detection of TB is the intradermal tuberculin skin test (i.e., intradermal injection of tuberculin eliciting a cell-mediated immune response [CMI] at the site, which in turn leads to skin thickening). As an alternative, the CMI can be measured in vitro by stimulating blood cells with tuberculin, which in turn leads to production of gamma interferon (IFN-␥), which can then be quantified by an enzyme-linked immunosorbent assay (ELISA; Bovigam IFN-␥ assay) (15).The Bovigam assay constitutes a laboratory-based TB test and is widely used complementarily to the tuberculin skin test (4, 11), as it offers national TB control programs and industry an additional tool for curtailing the spread of TB in cattle and other Bovidae. The assay critically depends on the sample quality, culture conditions, and quality control of stimulation reagents. The CMI, both in vivo and in vitro, may be negatively affected by stress or corticosteroid application (5). Thus, parallel stimulation of blood leukocytes with mitogen or superantigen in the IFN-␥ assay is commonly used as an indicator of sample quality and potential for underlying CMI suppression, thereby re...

Background and Purpose-Vascular effects of mental stress are only partially understood. Therefore, we studied effects of chronic stress and heart rate (HR) on endothelial function and cerebral ischemia. Methods-129S6/SvEv mice were randomized to the I(f)-channel inhibitor ivabradine (10 mg/kg per day) or vehicle and underwent a chronic stress protocol for 28 days. Results-Stress increased HR from 514Ϯ10 bpm to 570Ϯ14 bpm, this was prevented by ivabradine (485Ϯ7 bpm).Endothelium-dependent relaxation of aortic rings was impaired in mice exposed to stress. HR reduction restored endothelial function to the level of naive controls. Vascular lipid hydroperoxides were increased to 333%Ϯ24% and vascular NADPH oxidase activity was upregulated to 223Ϯ38% in stressed mice, which was prevented by ivabradine. Stress reduced aortic endothelial nitric oxide synthase mRNA expression to 84%Ϯ3% and increased AT1 receptor mRNA to 168%Ϯ18%. Both effects were attenuated by HR reduction. In brain tissue, stress resulted in an upregulation of lipid hydroperoxides to 140%Ϯ11%, which was attenuated by HR reduction. Ivabradine increased brain capillary density in naive and in stressed mice.

Background: Impaired vascular compliance is associated with cardiovascular mortality. The effects of heart rate on vascular compliance are unclear. Therefore, we characterized effects of heart rate reduction (HRR) by I(f) current inhibition on aortic compliance and underlying molecular mechanisms in apolipoprotein E-deficient (ApoE^–/^–) mice. Methods: ApoE^–/^– mice fed a high-cholesterol diet and wild-type (WT) mice were treated with ivabradine (20 mg/kg/d) or vehicle for 6 weeks. Compliance of the ascending aorta was evaluated by MRI. Results: Ivabradine reduced heart rate by 113 ± 31 bpm (∼19%) in WT mice and by 133 ± 6 bpm (∼23%) in ApoE^–/^– mice. Compared to WT controls, ApoE^–/^– mice exhibited reduced distensibility and circumferential strain. HRR by ivabradine increased distensibility and circumferential strain in ApoE^–/^– mice but did not affect both parameters in WT mice. Ivabradine reduced aortic protein and mRNA expression of the angiotensin II type 1 (AT1) receptor and reduced rac1-GTPase activity in ApoE^–/^– mice. Moreover, membrane translocation of p47^phox was inhibited. In ApoE^–/^– mice, HRR induced anti-inflammatory effects by reduction of aortic mRNA expression of IL-6, TNF-alpha and TGF-beta. Conclusion: HRR by ivabradine improves vascular compliance in ApoE^–/^– mice. Contributing mechanisms include downregulation of the AT1 receptor, attenuation of oxidative stress and modulation of inflammatory cytokine expression.