Duration of immunity against Mycobacterium bovis following neonatal vaccination with bacillus Calmette-Guérin Danish: significant protection against infection at 12, but not 24, months.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against bovine tuberculosis, caused by infection with virulent M. bovis. In two independent experiments, we assessed the duration of the protective immunity induced in calves by neonatal vaccination with BCG Danish. Protection from disease was assessed at 12 and 24 months postvaccination in cattle challenged via the endotracheal route with M. bovis. We also assessed antigen-specific immune responses to assess their utility as correlates of protection. At 12 months postvaccination, significant reductions in lung and lymph node pathologies were observed compared to nonvaccinated M. bovis-challenged control cattle. At 24 months post-BCG vaccination, there was a reduction in lung and lymph node pathology scores and in bacterial burden. However, when comparing vaccinated and control groups, this did not reach statistical significance. Vaccination induced long-lived antigen (purified protein derivative [PPD])-specific gamma interferon (IFN-γ) release in whole-blood cultures, which remained above baseline levels for more than 20 months (approximately 90 weeks). The number of antigen-specific IFN-γ-secreting central memory T cells present at the time of M. bovis challenge was significantly higher in vaccinated than in control animals at 12 months postvaccination, but not at 24 months. Vaccination of neonatal calves with BCG Danish induced protective immune responses against bovine TB which were maintained for at least 12 months postvaccination. These studies provide data on the immunity induced by BCG vaccination in calves; the results could inform vaccination strategies for the control of bovine TB in United Kingdom cattle herds.

Identification of surrogates and correlates of protection in protective immunity against Mycobacterium bovis infection induced in neonatal calves by vaccination with M. bovis BCG Pasteur and M. bovis BCG Danish.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against infection with virulent M. bovis, the causative agent of bovine tuberculosis (bTB). We compared two strains of BCG, Pasteur and Danish, in order to confirm that the current European human vaccine strain (BCG Danish) induced protective immunity in calves, and we assessed immune responses to determine correlates of protection that could assist future vaccine evaluation in cattle. Both vaccine strains induced antigen (purified protein derivate [PPD])-specific gamma interferon (IFN-γ) in whole-blood cultures. These responses were not significantly different for BCG Pasteur and BCG Danish and peaked at week 2 to 4 postvaccination. Vaccination with either BCG Danish or BCG Pasteur induced significant protection against bTB, with reductions in both lesion score and bacteriological burden evident in both groups of vaccinated calves compared with nonvaccinated control calves. Measurement of IFN-γ-expressing T lymphocytes postvaccination and postchallenge revealed both correlates and surrogates of protective efficacy. The frequency of central memory T lymphocytes present at 12 weeks postvaccination (at the time of M. bovis challenge) correlated significantly with protection. Conversely, the number of IFN-γ-expressing effector T cells present after M. bovis challenge was correlated with disease. These results demonstrate that vaccination of neonatal calves with either BCG Pasteur or BCG Danish induces protective immune responses against TB. In addition, we show that measurement of antigen-specific T lymphocyte populations may provide a reliable means for identifying protective vaccine candidates.

Influence of the nature of the antigen on the boosting of responses to mycobacteria in M. bovis-BCG vaccinated cattle.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB) and of a proportion of human TB. Protection against TB requires Th1 responses and worsening of disease is associated with Th2 responses. To help clarify the nature of the response to mycobacteria, the responses from M. bovis-BCG vaccinated cattle boosted with live mycobacteria (BCG), bacterial soluble antigens (PPD) or PBS were evaluated. The results indicated that macrophages may be the major cell population ingesting and presenting mycobacteria in BCG boosted animals, while B-cells seem able to ingest and present PPD to T-cells in PPD boosted animals.

The effect of tuberculin testing on the development of cell-mediated immune responses during Mycobacterium bovis infection.

Protection against tuberculosis (TB) is associated with Th1-type cell-mediated immunity (CMI). Whilst the intradermal injection of partially purified derivatives of tuberculin (PPD) represents the classic test assessing the delayed type hypersensitivity (DTH) response used in both humans and cattle for diagnosing TB, it has been suggested that the test may modulate host CMI responses. To investigate the kinetics of the development of the DTH response and its subsequent effect on CMI responses, groups of 6-month old calves were inoculated intranasally with 8 x 10(4) cfu of Mycobacterium bovis, subjected to the comparative intradermal tuberculin test (TT) using bovine and avian PPD (PPD-B, PPD-A) at various time intervals post-infection, and immune responses compared. These included DTH, lymphocyte proliferation, IgG production, and synthesis of the cytokines: IFNgamma, IL-10, IL-4, IL-6, and IL-13. All animals were subjected to post-mortem examination. The kinetics of the development of the DTH response assessed in the TT was such that infected cattle could be identified as early as 3 weeks post-infection, which correlated with the detection of an antigen-specific IFNgamma response. Transient increases in plasma-derived IFNgamma as a result of TT during an established TB infection were more pronounced when blood was stimulated with PPD-A compared with PPD-B stimulation. This has the potential to mask diagnosis of infection as a result of the stronger avian-bias if the IFNgamma test is used the week following TT. Disease pathology was not affected by TT. A transient failure to a second TT was observed in 1 of 30 animals and the time (post-infection) at which the TT is administered may be of significance. In serum, IgG responses to PPD-B, which were undetectable prior to TT, were elevated after TT and were most pronounced in cattle that were TT at 6 weeks post-infection. Other cytokines were also affected by the TT; IL-4 mRNA levels increased and IL-6 mRNA levels decreased, whilst PPD-B specific IL-10 protein synthesis was enhanced. These observations may offer the potential for further diagnostic assays that could complement the TT and IFNgamma test.

Investigation of the role of CD8+ T cells in bovine tuberculosis in vivo.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), and it has the potential to induce disease in humans. CD8(+) T cells (CD8 cells) have been shown to respond to mycobacterial antigens in humans, cattle, and mice. In mice, CD8 cells have been shown to play a role in protection against mycobacterial infection. To determine the role of CD8 cells in bovine TB in vivo, two groups of calves were infected with the virulent M. bovis strain AF2122/97. After infection, one group was injected with a CD8 cell-depleting monoclonal antibody (MAb), and the other group was injected with an isotype control MAb. Immune responses to mycobacterial antigens were measured weekly in vitro. After 8 weeks, the animals were killed, and postmortem examinations were carried out. In vitro proliferation responses were similar in both calf groups, but in vitro gamma interferon (IFN-gamma) production in 24-h whole-blood cultures was significantly higher in control cattle than in CD8 cell-depleted calves. Postmortem examination showed that calves in both groups had developed comparable TB lesions in the lower respiratory tract and associated lymph nodes. Head lymph node lesion scores, on the other hand, were higher in control calves than in CD8 cell-depleted calves. Furthermore, there was significant correlation between the level of IFN-gamma and the head lymph node lesion score. These experiments indicate that CD8 cells play a role in the immune response to M. bovis in cattle by contributing to the IFN-gamma response. However, CD8 cells may also play a deleterious role by contributing to the immunopathology of bovine TB.

Upstream tissue inhibitor of metalloproteinases-1 (TIMP-1) element-1, a novel and essential regulatory DNA motif in the human TIMP-1 gene promoter, directly interacts with a 30-kDa nuclear protein.

Elevated expression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein and mRNA has been reported in human diseases including cancers and tissue fibrosis. Regulation of TIMP-1 gene expression is mainly mediated at the level of gene transcription and involves the activation of several well known transcription factors including those belonging to the AP-1, STAT, and Pea3/Ets families. In the current study, we have used DNase-1 footprinting to identify a new regulatory element (5'-TGTGGTTTCCG-3') present in the human TIMP-1 gene promoter. Mutagenesis and transfection studies in culture-activated rat hepatic stellate cells and the human Jurkat T cell line demonstrated that the new element named upstream TIMP-1 element-1 (UTE-1) is essential for transcriptional activity of the human TIMP-1 promoter. Electrophoretic mobility shift assay studies revealed that UTE-1 can form protein-DNA complexes of distinct mobilities with nuclear extracts from a variety of mammalian cell types and showed that induction of a high mobility UTE-1 complex is associated with culture activation of freshly isolated rat hepatic stellate cells. A combination of UV-cross-linking and Southwestern blotting techniques demonstrated that UTE-1 directly interacts with a 30-kDa nuclear protein that appears to be present in all cell types tested. We conclude that UTE-1 is a novel regulatory element that in combination with its cellular binding proteins may be an important component of the mechanisms controlling TIMP-1 expression in normal and pathological states.

Interleukin-10 expression and function in experimental murine liver inflammation and fibrosis.

Kupffer cells (KC) play a central role in the initiation and perpetuation of hepatic inflammation, which, if uncontrolled, can result in tissue damage, fibrosis, and cirrhosis. Interleukin-10 (IL-10) can inhibit a range of macrophage functions. We hypothesized that the transcription, synthesis, and release of IL-10 may influence the development of liver injury. Rat KC were activated in vitro with lipopolysaccharide (LPS), and expression of IL-10 mRNA compared with IL-13 and IL-1beta by reverse-transcription polymerase chain reaction (RT-PCR). The effects of pretreatment with recombinant IL-10 (rIL-10) on KC phagocytosis, production of superoxide (SO), and tumor necrosis factor (TNF-) were examined by fluorescent activated cell sorter (FACS), reduction of ferricytochrome C, and bioassay, respectively. Rats were administered intraperitoneal carbon tetrachloride (CCl4), and expression of IL-10 mRNA and protein in vivo compared with IL-13 and IL-1beta by RT-PCR and immunoblotting. Results were correlated with histological inflammatory changes. Finally, IL-10 gene-deleted (IL-10-/-) mice and wild-type (WT) controls were administered intraperitoneal CCl4 biweekly for up to 70 days, and the development of inflammation and fibrosis compared by scoring histological changes. IL-10 mRNA was up-regulated early, both in KC in vitro and in whole liver in vivo, concurrent with that of IL-1beta. IL-10 was able to inhibit KC production of both SO and TNF- in vitro, and this was achieved more effectively than IL-4 or IL-13; no such effects were seen on KC phagocytosis. After 70 days of treatment with CCl4, IL-10-/- mice showed significantly more severe fibrosis and exhibited higher hepatic TNF- levels than WT controls. These results suggest that IL-10 synthesized during the course of liver inflammation and fibrosis may modulate KC actions, and influence subsequent progression of fibrosis.