Doherty Institute - Research Publications
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ItemThe interaction of macrophage and non-macrophage tropic isolates of HIV-1 with thymic and tonsillar dendritic cells in vitro(ROCKEFELLER UNIV PRESS, 1996-04-01)Dendritic cells isolated from thymus and tonsil were tested for susceptibility to HIV-1 strains that are tropic for macrophages or for T cell lines. DCs were purified by cell sorting and before infection expressed high levels of CD4 and HLA-DR and lacked markers for T, B, NK cells, or macrophages. Viral entry and reverse transcription was found after pulsing with strains of HIV-1 that could infect macrophages. During the first 36 h the PCR signals for gag sequences increased in DCs and macrophages. In contrast little if any viral DNA was found after pulsing macrophages or DCs with HIV-1 that was able to infect T cell lines. DCs pulsed with HIV-1 were able to transmit infection to responding T cells during an allogeneic or superantigen response. Selection for virus able to infect lymphoid DCs and other DCs expressing CD4 and its transfer to T cells during subsequent immune responses may provide a mechanism for the observed predominance of macrophage-tropic HIV-1 after in vivo transmission.
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ItemDENDRITIC CELLS FRESHLY ISOLATED FROM HUMAN BLOOD EXPRESS CD4 AND MATURE INTO TYPICAL IMMUNOSTIMULATORY DENDRITIC CELLS AFTER CULTURE IN MONOCYTE-CONDITIONED MEDIUM(ROCKEFELLER UNIV PRESS, 1993-09-01)A procedure has been developed to isolate dendritic cells to a high degree of purity from fresh blood. Prior enrichment methods have relied upon an initial 1-2-d culture period. Purified fresh isolates lack the characteristic morphology, phenotype, and immunostimulatory function of dendritic cells. The purified cells have the appearance of medium sized lymphocytes and express substantial levels of CD4, but lack the T cell molecules CD3, CD8, and T cell receptor. When placed in culture, the cells mature in a manner resembling the previously described, cytokine-dependent maturation of epidermal dendritic cells (Langerhans cells). The cells enlarge and exhibit many cell processes, express much higher levels of major histocompatibility complex class II and a panel of accessory molecules for T cell activation, and become potent stimulators of the mixed leukocyte reaction. Among the many changes during this maturation process are a fall in CD4 and the appearance of high levels of B7/BB1, the costimulator for enhanced interleukin 2 production in T cells. These changes are not associated with cell proliferation, but are dependent upon the addition of monocyte-conditioned medium. We suggest that the freshly isolated CD4-positive blood dendritic cells are recent migrants from the bone marrow, and that subsequent maturation of the lineage occurs in tissues in situ upon appropriate exposure to cytokines.
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ItemDIFFERENCES IN GENE COPY NUMBER CARRIED BY DIFFERENT MHC ANCESTRAL HAPLOTYPES - QUANTITATION AFTER PHYSICAL SEPARATION OF HAPLOTYPES BY PULSED FIELD GEL-ELECTROPHORESIS(ROCKEFELLER UNIV PRESS, 1990-06-01)We have examined the hypothesis that MHC ancestral haplotypes have a specific content of genes regulating the extent of autoimmune reactions. Gene copy number was quantitated by objective densitometry after PFGE was used to separate heterozygous AHs of different lengths. Initially we analyzed examples of known gene copy number at the C4 and 21 hydroxylase loci and showed that the approach provides predictable results. We then studied heterozygotes containing one characterized and one uncharacterized AH with particular attention to the gene copy number at the C4, Cyp21, and DRB loci. Each AH studied has a characteristic gene copy number at each locus studied. The same may be true of TNF, but other possibilities must be considered. AHs are markers for extensive chromosomal segments including particular numbers of several functional genes. Since AHs mark susceptibility to autoimmune disease, differences in gene copy number may be implicated.
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ItemDevelopment of recombinant S. Typhimurium, as a model for S. Typhi-based vaccine vectors(Faculty of Medicine, Universitas Indonesia, 1998-01-01)
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ItemStudies of the pathogenesis and immunology of attenuated mutants of Salmonella enterica var. Typhimurium: Lessons for human typhoid fever?(Faculty of Medicine, Universitas Indonesia, 1998-01-01)
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ItemVaccine potential of attenuated mutants of Corynebacterium pseudotuberculosis in sheep(AMER SOC MICROBIOLOGY, 1998-02)Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C. pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable specific gamma interferon (IFN-gamma)-secreting lymphocytes and induced low levels of antibodies to C. pseudotuberculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-gamma-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines or as vaccine vectors.
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ItemDNA vaccines for bacterial infections(BLACKWELL SCIENCE, 1997-08)DNA vaccines are an exciting development in vaccine technology which may have a special role in preventing viral infections and as 'theracines' for cancer. Their use in preventing bacterial infections has, by comparison, been less well documented. While it is unlikely that traditional, highly successful and cheap vaccines for diseases such as diphtheria will be replaced by DNA vaccines, naked DNA may be particularly appropriate for preventing bacterial infections where cytotoxic T cells confer protection, or where a Th1 type T cell response mediates resistance. For example, DNA vaccines containing different mycobacterial antigens have been shown to inhibit overt infections by Mycobacterium tuberculosis in rodent models. The use of DNA vaccines in bacterial infections may be complicated by fundamental differences between prokaryotic and eukaryotic genes and gene products, including mRNA stability, codon bias, secondary structures surrounding native start sequences and glycosylation. These problems can be solved by re-synthesis of bacterial genes to produce 'new' sequences which are more highly expressed by eukaryotic cells.
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ItemUse of in vivo-regulated promoters to deliver antigens from attenuated Salmonella enterica var. typhimurium(AMER SOC MICROBIOLOGY, 1999-10)This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the DeltaaroAD mutant of Salmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding beta-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimurium in vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more beta-galactosidase and luciferase in S. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in the aroAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from the pagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutive trc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimurium as a vaccine vector.