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Abstract: Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3−CD2+ and NKp46+ largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3−CD2+NKp46+. The remainder of the NK-like cells comprised two minor populations, CD3−CD2+NKp46− and CD3−CD2−NKp46+; the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3−CD2+ and NKp46+ NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8αα and CD8αβ and did not express CD21, WC1, CD14 or γδ TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3−CD2−NKp46+ population expressed CD8α compared to CD3−CD2+NKp46+ cells. Furthermore, a significantly greater proportion of the CD3−CD2+NKp46− population expressed CD8 compared to total CD3−CD2+ cells. Adult cattle had a significantly higher proportion of perforin+ cells compared to calves aged ≤6 weeks. In this age group, the majority of perforin+ cells expressed NKp46, while in adults the majority of perforin+ cells were NKp46−. However, the proportion of NKp46+ and CD3−CD2+ cells that expressed perforin was not significantly different in any age group tested. [Copyright &y& Elsevier]

Abstract: Inducible nitric oxide (iNOS) is an enzyme that catalyzes the production of the reactive nitrogen intermediate nitric oxide (NO). NO is an important signalling molecule, released by numerous cells, that acts in many tissues to regulate a diverse range of physiological and biological processes, including neurotransmission, immune defence and the regulation of cell death (apoptosis). NO plays a major role in the killing of intracellular pathogens as part of the innate immune response. iNOS is known to be induced by a number of stimuli including cytokines as well as pathogens and their components. As yet, a full-length bovine iNOS sequence has only been predicted from the genome, although partial sequences from cDNA are available. Here, we have identified a 3471bp transcript for bovine iNOS, isolated from RNA from bovine alveolar macrophages stimulated with the intracellular pathogen Mycobacterium bovis. When translated this gives a protein of 1156 amino acids. Bovine iNOS shows a high degree of similarity to iNOS from other species, and also shares a common protein domain structure. [Copyright &y& Elsevier]

Abstract: Tuberculosis (TB) remains a major threat to public health. The identification of safe TB vaccine candidates beyond Mycobacterium bovis BCG, is an exciting prospect for control of human TB and necessary in the context of the human immunodeficiency virus (HIV) pandemic. Selection of vaccine candidates for human trials which are ultimately targeted for use in children less than 5 years of age or in newborns will require an animal model that closely approximates immune function and disease. We propose that the bovine neonate and adolescent is a robust animal model for preclinical safety and efficacy evaluation of TB candidate vaccines targeting this special human population. Parallel studies conducted in bovine neonates and non-human primates with a leading auxotrophic mutant with demonstrated efficacy/safety in a rodent TB model of TB demonstrated similar findings with respect to gross pathology scoring relative to BCG. The findings indicated more numerous and severe lesions in the lung in addition to higher levels of IFN-gamma producing cells. BCG vaccinates demonstrated higher levels of FoxP3 transcripts and lower levels of IL-4 mRNA. [Copyright &y& Elsevier]

The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge. The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells. We report here that bovine DC are also induced to mature following lipopeptide stimulation. Exposure of DC to the model lipopeptide Pam3CSK4 was associated with increased expression of MHC, costimulatory molecules, and enhanced secretion of IL-12 and TNFα. Lipopeptide-matured DC were superior in their ability to induce T cell activation and IFNγ secretion. In contrast, exposure of MΦ to lipopeptides induced down-regulation of MHC expression and much lower increases in IL-12 secretion. A lipopeptide derived from the sequence of a relevant mycobacterial lipoprotein, MPB83, also influenced bovine DC by stimulating increases in IL-12 and TNFα secretion. These different changes in bovine DC and MΦ may have important implications for immune responses induced following bacterial infection with uptake of microbes by DC resulting in potentiation of their immunostimulatory capacity and uptake by MΦ having a much less marked effect on immune responses. [Copyright &y& Elsevier]