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J Content

Publications and source records attributed to J Content.

At least 37 records · Page 2Linked to original sources
PubMed1998

DNA vaccines against tuberculosis.

DNA plasmids encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in animal models. Ag85 DNA induced relevant immune responses (i.e. T helper (Th) cells, Th1 cytokines and cytotoxic T lymphocytes) and was protective in mouse and guinea pig models of mycobacterial disease. Therefore, DNA vaccination holds promise as an effective means of preventing tuberculosis in humans. Furthermore, this technique is amenable to identifying the protective antigens of M. tuberculosis.

Acyltransferases↗
PubMed1998

The proximal interferon-stimulated response elements are essential for interferon responsiveness: a promoter analysis of the antiviral MxA gene.

Interferon (IFN)-inducible human MxA protein mediates resistance against influenza and several other RNA viruses. The MxA gene is under the control of type I IFN and, in certain cell types, is also directly activated by viruses. Here we show that in human macrophages, MxA mRNA levels are upregulated by very low doses of IFN-alpha in a dose-dependent manner. A similar, albeit much weaker, dose-dependent induction was seen with IFN-gamma. The induction was rapid and independent of protein synthesis. Interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-alpha) did not influence MxA mRNA levels alone or in combination with IFNs, in spite of the presence of putative response elements of these cytokines in the MxA promoter. We show that the promoter of the MxA gene contains two functional IFN-stimulated response elements (ISRE) near the transcription start site and one homologous ISRE-like element, which is apparently nonfunctional, further upstream. The two proximal ISRE sites are essential for IFN-alpha-induced transcription and appear to be binding sites for IFN-stimulated gene factor 3 complex. In addition, EMSA and DNAse I footprinting analysis demonstrated that Spl binds with high affinity to a region encompassing nucleotides -25 and -50 and, thus, may provide means of interaction with the basal transcriptional machinery.

Antiviral Agents↗
PubMed1998

Interferon-gamma and interleukin-6 inhibit proliferation in human melanoma cells by different signalling pathways.

Interferon regulatory factor-1 (IRF-1) is a cell growth inhibitor, induced by cytokines, which transactivates downstream effector genes. The role of IRF-1 in the antiproliferative effect of interleukin-6 (IL-6) was investigated using the A375 human melanoma cell line. IL-6 is a stronger inhibitor of A375 proliferation compared with interferon-gamma (IFNgamma). However, in contrast to IFNgamma, IL-6 triggered lower IRF-1 DNA binding activity and induced barely detectable IRF-1-dependent transactivation activity. Furthermore, although IFNgamma induces only activation of signal transducer and activator of transcription (STAT) 1, IL-6 activates mainly STAT3. These data suggest that IRF-1 plays a minor role in the antiproliferative effect of IL-6, which uses alternative signalling events to induce growth inhibition in A375 melanoma cells.

Cell Division↗
PubMed1998

Vaccination with plasmid DNA encoding mycobacterial antigen 85A stimulates a CD4+ and CD8+ T-cell epitopic repertoire broader than that stimulated by Mycobacterium tuberculosis H37Rv infection.

Vaccination of mice with plasmid DNA carrying the gene for the major secreted mycobacterial antigen 85A (Ag85A) from Mycobacterium tuberculosis is a powerful technique for generating robust specific Thl helper T-cell responses, CD8+-mediated cytotoxicity, and protection against M. tuberculosis challenge (K. Huygen et al., Nat. Med. 2:893-898, 1996). We have now analyzed in more detail the antigen-specific immune CD4+- and CD8+-T-cell responses induced in BALB/c mice vaccinated with Ag85A DNA and have compared these responses to those generated by intravenous infection with M. tuberculosis. T-cell-epitope mapping, as measured by interleukin-2 and gamma interferon secretion from splenic T cells restimulated in vitro with synthetic 20-mer peptides spanning the complete mature sequence of Ag85A, demonstrated that DNA vaccination stimulated a stronger and broader T-cell response than did M. tuberculosis infection. Moreover, elevated cytotoxic T lymphocyte (CTL) activity against Ag85A-transfected and peptide-pulsed P815 target cells could be generated exclusively by vaccination with plasmid DNA, not following M. tuberculosis infection. By using DNA vaccination, three Ag85A CTL epitopes with predicted major histocompatibility complex class I binding motifs were defined. One of them was previously reported as a dominant, promiscuously recognized T-cell epitope in healthy humans with primary infections. These data strengthen the potential of DNA vaccination with respect to inducing antituberculous immunity in humans.

Amino Acid Sequence↗
PubMed1997

Analysis of the mechanism of action of anti-human interleukin-6 and anti-human interleukin-6 receptor-neutralising monoclonal antibodies.

Anti-human interleukin-6 (human IL-6) and anti-human IL-6 receptor (IL-6R)-neutralising monoclonal antibodies (mAbs) are among the most promising human IL-6-specific inhibitors and have been shown to exert short-term beneficial effects in clinical trials. Simultaneous treatment with different anti-human IL-6 or anti-human IL-6R mAbs was recently suggested to be a potent way to inhibit the action of the cytokine in vivo. Although some of these mAbs are already used, their mechanisms of action and the location of their epitopes on the surface of human IL-6 and human IL-6R are still unknown. Here, we analysed the capacity of several anti-human IL-6 and anti-human IL-6R mAbs to inhibit the interaction between human IL-6, human IL-6R, and human glycoprotein 130 (gp130). We mapped the epitopes of several of these mAbs by studying their binding to human IL-6 and human IL-6R mutant proteins. Our results show that several anti-human IL-6 and anti-human IL-6R-neutralising mAbs block the binding between human IL-6 and human IL-6R, whereas others block the binding to gp130. We provide evidence that some of the latter mAbs inhibit interaction with gp130beta1, whereas others interfere with the binding to gp130beta2. Our results suggest that residues included in the C'D' loop of human IL-6R interact with gp130beta2.

Animals↗
PubMed1997

A serine/threonine protein kinase from Mycobacterium tuberculosis.

Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein. The size of the protein was confirmed by in vitro coupled transcription/translation. Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring. The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase. Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis. Its expression was detected in cultures of M. bovis BCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.

Amino Acid Sequence↗
PubMed1997

Analysis of the human interleukin-6/human interleukin-6 receptor binding interface at the amino acid level: proposed mechanism of interaction.

The interaction between interleukin-6 (IL-6) and IL-6 receptor (IL-6R) is the initial and most specific step in the IL-6 signaling pathway. Understanding its mechanism at the amino acid level is the basis for developing small IL-6-inhibiting molecules. We studied the human IL-6 (hIL-6)/hIL-6R binding interface by a combination of molecular modelling and site-directed mutagenesis. Our model suggests that the center of the interface between the two molecules consists of hydrophobic contacts predicted to account for most of the binding-free energy. These contacts can be regarded as a hydrophobic core shielded by hydrophilic residues that are also needed for recognition. Following this hypothesis, we altered in hIL-6 and hIL-6R residues predicted to reside in the contact region and to interact with each other. We studied the capacity of these mutants to form an IL-6/IL-6R complex and their ability to transduce the signal. This combined approach has led to the identification of certain residue-clusters in the binding interface and to a rational explanation of their specific interactions, suggesting therein a likely mechanism of complex formation. The results confirm the predictive model and strongly support our hypothesis. Comparison with other cytokines and their alpha-subunit receptors suggests that the structural location of certain binding sites are conserved.

Amino Acid Sequence↗
PubMed1997

Cloning of the antigen 85A from Mycobacterium gordonae and its use for the specific PCR identification of these mycobacteria.

The complete nucleotide sequence of 85A antigen of Mycobacterium gordonae was determined. This gene encodes 339 amino acids, including 43 amino acids for the signal peptide, followed by a mature protein of 296 amino acids. A polymerase chain reaction (PCR) assay for the rapid detection of M. gordonae DNA using two pairs of oligonucleotide primers, derived from our sequence, is described. This one-step PCR has been used successfully to amplify 38 strains of M. gordonae. Conversely, the primers did not amplify DNA from any of the 25 mycobacterial species tested. The results suggest that this PCR assay could be a good alternative to existing commercial assays for the specific identification of M. gordonae on early culture on solid medium or on early BACTEC broth culture.

Amino Acid Sequence↗
PubMed1997

Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination.

Plasmid DNA expression vectors encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in preclinical animal models. Expression of secreted and nonsecreted forms of Ag85 was observed after transient transfection of cells in vitro. In mice, both types of Ag85 DNA constructs induced strong humoral and cell-mediated immune responses, as measured by ELISA of sera and recall responses of spleen cells restimulated in vitro, respectively, Therefore, DNA vaccination is an effective means of expressing mycobacterial proteins in eukaryotic cells leading to the induction of potent immune responses.

Acyltransferases↗
PubMed1997

Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex.

BALB/c and C57BL/6 mice were injected intramuscularly with plasmid DNA encoding the three components of the immunodominant 30-32 kDa antigen 85 complex (Ag85A, Ag85B, and Ag85C) from Mycobacterium tuberculosis culture filtrate, in order to investigate the utility of nucleic acid vaccination for induction of immune responses against mycobacterial antigens. Ag85A and Ag85B encoding plasmids induced a robust Th1-like response towards native Ag85, characterized by elevated levels of interleukin (IL)-2, interferon-gamma, and TNF-alpha. Levels of IL-4, IL-6, and IL-10 were low or undetectable. Plasmid encoding Ag85C was not effective. Cytotoxic T cell activity was also generated in in vitro restimulated splenocyte cultures from Ag85A and Ag85B DNA vaccinated mice. Finally, Ag85A and Ag85B DNA vaccination conferred significant protection against mycobacterial replication in lungs from B6 mice, subsequently challenged. Therefore, this technique may be useful for the definition of protective antigens of M. tuberculosis and the development of a more effective tuberculosis vaccine.

Animals↗
PubMed1997

Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Im [corrected] associated with a sulI-type integron.

The amikacin resistance gene aac(6')-Im [corrected] from Citrobacter freundii Cf155 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 521 bp located down-stream from the 5' conserved segment of an integron. The sequence of this aac(6')-Im [corrected] gene corresponded to a protein of 173 amino acids which possessed 64.2% identity in a 165-amino-acid overlap with the aac(6')-Ia gene product (F.C. Tenover, D. Filpula, K.L. Phillips, and J. J. Plorde, J. Bacteriol. 170:471-473, 1988). By using PCR, the aac(6')-Im [corrected] gene could be detected in 8 of 86 gram-negative clinical isolates from two Belgian hospitals, including isolates of Citrobacter, Klebsiella spp., and Escherichia coli. PCR mapping of the aac(6')-Im [corrected] gene environment in these isolates indicated that the gene was located within a sulI-type integron; the insert region is 1,700 bases long and includes two genes cassettes, the second being ant (3")-Ib.

Acetyltransferases↗
PubMed1997

Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum.

By using appropriate Corynebacterium glutamicum-Escherichia coli shuttle plasmids, the gene encoding the fibronectin-binding protein 85A (85A) from Mycobacterium tuberculosis was expressed in C. glutamicum, also an actinomycete and nonsporulating gram-positive rod bacterium, which is widely used in industrial amino acid production. The 85A gene was weakly expressed in C. glutamicum under the control of the ptac promoter from E. coli, but it was produced efficiently under the control of the promoter of the cspB gene encoding PS2, one of the two major secreted proteins from C. glutamicum. The 85A protein was produced in various forms, with or without its own signal sequence and with or without the signal sequence and the NH2-terminal (18-amino-acid) mature sequence of PS2. Western blot analysis with monoclonal antibodies raised against the M. tuberculosis antigen 85 complex showed that recombinant 85A protein was present in the corynebacterial cell wall extract and also released in extracellular culture medium. NH2-terminal microsequencing of recombinant 85A secreted by C. glutamicum showed that signal peptide was effectively cleaved off at the predicted site. The recombinant 85A protein was biologically active in vitro, inducing significant secretion of Th1 T-cell cytokines, particularly interleukin-2 and gamma interferon, in spleen cell cultures from mice vaccinated with live Mycobacterium bovis BCG. Heterologous expression of mycobacterial antigens in C. glutamicum now offers a potent tool for further immunological characterization and large scale preparation of these recombinant proteins.

Animals↗
PubMed1997

Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG.

A gene encoding a protein homologous to the periplasmic ABC phosphate binding receptor PstS from Escherichia coli was cloned and sequenced from a lambda gt11 library of Mycobacterium tuberculosis by screening with monoclonal antibody 2A1-2. Its degree of similarity to the E. coli PstS is comparable to those of the previously described M. tuberculosis phosphate binding protein pab (Ag78, Ag5, or 38-kDa protein) and another M. tuberculosis protein which we identified recently. We suggest that the three M. tuberculosis proteins share a similar function and could be named PstS-1, PstS-2, and PstS-3, respectively. Molecular modeling of their three-dimensional structures using the structure of the E. coli PstS as a template and their inducibility by phosphate starvation support this view. Recombinant PstS-2 and PstS-3 were produced and purified by affinity chromatography. With PstS-1, these proteins were used to demonstrate the specificity of three groups of monoclonal antibodies. Using these antibodies in flow cytometry and immunoblotting analyses, we demonstrate that the three genes are expressed and their protein products are present and accessible at the mycobacterial surface as well as in its culture filtrate. Together with the M. tuberculosis genes encoding homologs of the PstA, PstB, and PstC components we cloned before, the present data suggest that at least one, and possibly several, related and functional ABC phosphate transporters exist in mycobacteria. It is hypothesized that the mycobacterial gene duplications presented here may be a subtle adaptation of intracellular pathogens to phosphate starvation in their alternating growth environments.

Amino Acid Sequence↗
PubMed1997

Induction of humoral and cellular immune responses by vaccination with M. tuberculosis antigen 85 DNA.

DNA vaccines have been demonstrated to be effective in inducing protective cell-mediated immune responses in animal models of infectious disease. In order to investigate this approach for potential use as a vaccine for tuberculosis, DNA constructs encoding Mycobacterium tuberculosis antigen 85A (Ag85A) were prepared. Expression of Ag85A in mammalian cells was demonstrated by transient transfection of cells in vitro. Intramuscular injection of Ag85A DNA vaccines resulted in the generation of anti-Ag85A antibodies and robust cell-mediated immune responses, as measured by lymphoproliferation of spleen cells in vitro upon specific antigen restimulation, leading to protection in animal challenge models. Therefore, the technique of DNA vaccination is effective in inducing relevant immune responses for protection against tuberculosis and may be used to identify the protective antigens of M. tuberculosis.

Animals↗
PubMed1997

Induction of immunity by DNA vaccination: application to influenza and tuberculosis.

DNA vaccination is an effective means of inducing both humoral and cell-mediated immunity in animal models of infectious disease. Presented here are data generated in two distinct disease models; one viral (influenza) and one bacterial (tuberculosis). Specifically, plasmid DNA encoding an influenza virus antigen (nucleoprotein; NP) and a Mycobacterium tuberculosis antigen (antigen 85; Ag85) were prepared and tested as DNA vaccines in mice. In both cases, high titer antibody responses and robust cell-mediated immune responses were induced against the respective antigens. With respect to the latter, lymphocyte proliferation, Th1-type cytokine secretion, and cytotoxic T lymphocyte responses were observed upon restimulation with antigen in vitro. Furthermore, protective efficacy in animal challenge models was demonstrated in both systems. The data support the hypothesis that DNA vaccination will prove to be a broadly applicable technique for inducing immunity against various infectious diseases.

Animals↗
PubMed1996

A Mycobacterium tuberculosis gene cluster encoding proteins of a phosphate transporter homologous to the Escherichia coli Pst system.

We report the cloning and sequencing of three M. tuberculosis genes encoding proteins homologous to E. coli PstA, PstC and PstB. They are tentatively called pstA-2, pstC-1 and pstB. They encode proteins of 302, 336 and 275 amino acids, respectively. In E. coli, PstB is the ATP binding component and PstA/PstC are the two hydrophobic subunits of a phosphate permease belonging to the family of ABC (ATP-binding cassette) transporters. In mycobacteria, PstS-1, the phosphate binding subunit (Andersen and Hansen, 1989), is encoded by a gene directly surrounded by pstB, pstC-1 and pstA-2 within a potential operon (pstB, pstS-1, pstC-1, pstA-2). Phosphate uptake by whole, suspension grown, M. bovis BCG cells was measured and could be inhibited by a monoclonal antibody directed against the PstS-1 subunit, suggesting that these genes encode subunits of a functional mycobacterial phosphate permease.

ATP-Binding Cassette Transporters↗
PubMed1996

Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter.

Following the identification of a M. tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS-3 and pstC-2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively. Together with the previously isolated M. tuberculosis gene similar to the E. coli pstA, these are included in a cluster encoding a second putative phosphate transport system. We demonstrate that pstS-3 encodes the previously described Ag 88, a 40 kDa M. bovis BCG culture filtrate antigen (immunodominant in H-2b haplotype type mice). Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding-dependent permeases is proposed.

ATP-Binding Cassette Transporters↗