Biomedical subjects

D N Wedlock

Publications and source records attributed to D N Wedlock.

21 records · Page 2Linked to original sources

PubMed1997

Sequential activation of alveolar macrophages by IFN-gamma and LPS is required for enhanced growth inhibition of virulent Mycobacterium bovis but not M. bovis BCG.

Alveolar macrophages (AM) form the first line of defence against most respiratory pathogens and, unlike tissue macrophages, are constantly exposed to a wide variety of antigenic stimuli. In this study we investigated the in vitro effects of IFN-gamma and LPS on growth of virulent Mycobacterium bovis and M. bovis bacille Calmette-Guerin (BCG) in bovine AM. Bovine AM were purified from bronchial lavage fluid and cultured in serum-free medium. Pretreatment of bovine AM with IFN-gamma resulted in growth inhibition of M. bovis BCG but only partially inhibited growth of virulent M.bovis. Enhanced inhibition of virulent M.bovis by bovine AM required sequential stimulation with IFN-gamma and LPS and was associated with increased induction of nitric oxide (NO) and IL-12 mRNa. Growth inhibition of M. bovis was not affected by treatment of macrophages with the L-arginine analogue, NG-monomethyl-L-arginine although this treatment decreased NO production. These results suggest that a second activation signal in the form of TNF-alpha or LPS may be required to induce bacteriostasis of virulent M. bovis by bovine AM in vivo. The ability of bovine AM to respond to activation stimuli in vitro suggests that these cells may play an important role in preventing establishment of intracellular bacterial infections in the lung.

Animals↗

PubMed1996

Molecular cloning and characterization of tumor necrosis factor alpha (TNF-alpha) from the Australian common brushtail possum, Trichosurus vulpecula.

Immune responses in the Australian common brushtail possum (Trichosurus vulpecula) and in particular the role of cytokines are poorly understood. We have undertaken to isolate cytokine genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and in this study describe the molecular cloning of TNF-alpha. Primers were designed from consensus sequences at the N-terminus end of eutherian mammalian TNF-alpha and the possum cDNA, derived from spleen RNA, identified by RT-PCR. The complete cDNA encoding possum TNF-alpha was amplified from lymphocyte RNA by 5' and 3' rapid amplification of cDNA ends (RACE). The nucleotide sequence of the protein coding region of this cDNA shared 66-69% identity with other mammalian TNF-alpha genes. The predicted protein of 233 amino acids shared 56-58% identity with eutherian mammalian TNF-alpha was expressed in both Saccharomyces cerevisiae and Escherichia coli by constructing expression plasmid derivatives of the vectors pYES2 and pGEX-2T respectively. Cell extracts prepared from transformants and the purified GST/TNF-alpha fusion protein exhibited cytotoxic activity on the TNF-alpha-sensitive murine fibroblast L929 cells and stimulated proliferation of possum thymocyte cells. The induction of possum TNF-alpha mRNA in alveolar macrophages was analysed by RT-PCR using possum-specific TNF-alpha primers. Macrophages cultured in the presence of LPS showed enhanced transcription of TNF-alpha mRNA. This is the first report of the cloning and sequence analysis of the cDNA encoding a marsupial cytokine gene.

Amino Acid Sequence↗

PubMed1996

Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis.

Mycobacterium bovis causes tuberculosis in cattle and many other animals including humans while BCG, an attenuated form of M. bovis, has been used widely as a safe vaccine. Both strains infect host macrophages and their fate is determined by their ability to survive within these phagocytic cells. We compared interactions of these two strains with bovine alveolar macrophages in order to gain an understanding of virulence mechanisms involved in the early pathogenesis of M. bovis infection. Macrophages were infected with bacilli at varying multiplicities of infection and cultured for 1-4 days. Bacterial metabolism within macrophages was assessed by [3H]-uracil uptake and bacterial growth was assessed by culture and acid-fast staining. Induction of TNF-alpha, IL-1 beta and IL-6 cytokine mRNA transcription in macrophages was determined by reverse transcriptase-polymerase chain reaction. Infection of macrophages by virulent M. bovis resulted in enhanced bacterial metabolism, enhanced induction of macrophage cytokines and reduced viability of macrophages when compared to M. bovis BCG-infected macrophages. These differences may reflect virulence mechanisms contributing to the early pathogenesis of bovine tuberculosis.

Animals↗