Biomedical subjects
Publications and source records attributed to A R Coates.
Molecular chaperones are intracellular protein-folding proteins which form part of an ancient cellular response to stress called the heat shock response. They have been the focus for attention during the last decade because of the discovery of their vital role in cell functioning. In very recent years additional roles for these 'topologically-active' proteins in the process of tissue pathology and its treatment have been indicated and are reviewed.
The chaperonin activity of sequence-related chaperonin 10 proteins requires their aggregation into heptameric structures. We describe size-exclusion chromatography and ultracentrifugation studies that reveal that while Escherichia coli chaperonin 10 exists as a heptamer, the Mycobacterium tuberculosis chaperonin 10 is tetrameric in dilute solutions and in whole M. tuberculosis lysate. At high protein concentration and in the presence of saturating amounts of divalent ions, the protein is heptameric. Human chaperonin 10 is predominantly heptameric, although smaller oligomers were detected. These differences in structural assembly between species may explain differences in biological activity such as antigenicity. Using C-terminal and N-terminal fragments, sequence 1-25 was identified as indispensable for aggregation. CD spectroscopy studies revealed that (i) a minimum at 202-204 nm correlates with aggregation and characterizes not only the spectrum of the mycobacterial protein, but also those of E. coli and human chaperonin 10 proteins; (ii) the interactions between subunits are of the hydrophobic type; and (iii) the anti-parallel beta-pleated sheet is the main secondary structure element of subunits in both tetrameric and heptameric proteins.
The peptide corresponding to the C-terminal half of M.tuberculosis hsp10 was synthesised based on the prediction that it might represent an independent structural region of the protein. This hypothesis was confirmed by aggregation and CD studies using this peptide and longer sequences of the protein. The peptide shares about 40-50% sequence homology with alpha 2 and beta 1 chains of MHC class I and II antigens. This and the CD results which indicated that the peptide at acidic pHs folds into an anti-parallel beta-sheet were used to generate a 3D model which has the same "W" fold contained in the MHC peptide binding groove. These data suggest that the hypothesis of molecular mimicry proposed to be one of the mechanisms which triggers autoimmune diseases may be extended to hsp10 proteins. Furthermore the suggested evolutionary relationship between hsp's and MHC antigens may find support from these data.
Six hundred and ninety-two specimens, each consisting of a suspension of the residual free cells in samples of clotted blood (but not the clots themselves) from 680 patients at high risk for exposure to human immunodeficiency virus (HIV), were tested by a nested polymerase chain reaction (PCR) procedure which had been optimized to give a sensitivity of detection of one copy of HIV-1 proviral plasmid DNA, and the results were compared to those of testing for antibody to HIV on the same specimens. Fifty-one of the specimens were positive for antibody to HIV and 49 of these were also positive by the PCR; the two samples which gave discordant results were found to be PCR-positive when the test was repeated on DNA extracted from the clots themselves. Two specimens were found to be negative for antibody to HIV but were positive by the PCR (53 positive specimens in all). Direct sequencing of the PCR DNA products confirmed their specificity in all cases and demonstrated that no two patients gave the same predicted amino acid sequence for the V3 loop region. The sequences revealed both European/North American and African motifs at the crown of the V3 loop thus indicating a diversity of HIV strains in the SW Thames Region of South London. The results show that the confirmatory PCR for HIV-1 can be carried out efficiently on the same clotted blood specimens as used for routine HIV serology on patients undergoing diagnostic evaluation.
The overexpression of the Mycobacterium tuberculosis chaperonin 10 (Cpn10)-encoding gene was accomplished using baculovirus expression vectors. The product was immunoreactive with a Cpn10 monoclonal antibody (mAb) and had an electrophoretic mobility identical to authentic Cpn10. The baculovirus system was most successful in terms of reaching nearly the full expression potential of the system. Recombinant Cpn10 was purified from recombinant baculovirus-infected Spodoptera frugiperda cells by isoelectrofocussing and size-exclusion chromatography. The baculovirus vector and purification methodology described represent a very powerful system for the large-scale production of the M. tuberculosis Cpn10 which may allow us to undertake structure-function analysis.
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OBJECTIVES: To assess the effect of an intra-articular presentation of stress (heat shock) proteins (hsp) on joint inflammation. METHODS: Wistar rats were sensitised with a suspension of heat killed Mycobacterium tuberculosis in oil in the scruff of the neck and challenged intra-articularly with stress protein or M tuberculosis preparations. Inflammation was assessed by joint swelling and, using immunohistology, cellular infiltration of the synovium and antibody induction by an enzyme-linked immunosorbent method. RESULTS: It was shown, for the first time, that the intra-articular administration of a recombinant myobacterial 65 kDa hsp can induce joint inflammation in M tuberculosis sensitised recipients; both powdered M tuberculosis and the purified protein derivative of tuberculin (PPD) produced a similar response, with T cell infiltration of the synovium and a time course typical of delayed type hypersensitivity. This response was specific to the 65 kDa protein as another immunodominant mycobacterial stress protein of 10 kDa was ineffective. Furthermore, intra-articular injection of the 65 kDa hsp induced an antibody response against both the 65 kDa and 10 kDa proteins and the antibody titres continued to rise when knee swelling had subsided. CONCLUSIONS: These results support the hypothesis that 60 kDa proteins are a relevant arthritogenic stimulus in an M tuberculosis background. Moreover, when antigen presentation occurs in the synovium of previously sensitised individuals, circulating antibodies are generated which persist and recognise cross-reactive epitopes on several stress proteins.
A 65-kDa protein and a 10-kDa protein are two of the more strongly immunoreactive components of Mycobacterium tuberculosis, the causative agent of tuberculosis. The 65-kDa antigen has homology with members of the GroEL or chaperonin-60 (Cpn60) family of heat shock proteins. The 10-kDa antigen has homology with the GroES or chaperonin-10 family of heat shock proteins. These two proteins are encoded by separate genes in M. tuberculosis. The studies reported here reveal that M. tuberculosis contains a second Cpn60 homolog located 98 bp downstream of the 10-kDa antigen gene. The second Cpn60 homolog (Cpn60-1) displays 61% amino acid sequence identity with the 65-kDa antigen (Cpn60-2) and 53% and 41% identity with the Escherichia coli GroEL protein and the human P60 protein, respectively. Primer-extension analysis revealed that transcription starts 29 bp upstream of the translation start of the Cpn60-1 homolog and protein purification studies indicate that the cpn60-1 gene is expressed as an approximately 60-kDa polypeptide.
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Previously we have shown the ability to induce experimental systemic lupus erythematosus (SLE) in naive mice with pathogenic antibodies carrying the 16/6 idiotype (Id) and with the T-cell line specific for the 16/6 Id. In the present study we established and characterized a series of T-cell clones that react against diverse autoantibodies carrying the 16/6 Id and show that they are capable of inducing a SLE-like disease in mice. The T-cell clones were generated from BALB/c mice immunized with the human mAb anti-DNA antibody (SA-1) and the mouse monoclonal anti-tuberculous Ab (TB/68), both carrying the 16/6 Id. The T-cell clones proliferated only in the presence of either human or mouse mAb carrying the 16/6 Id. All the T-cell clones were found to be of the helper type (L3T4) and were H-2 restricted in their function. The injection of the clones to BALB/c mice resulted in serological findings (e.g., anti-DNA, anti-Sm), clinical manifestations (e.g., proteinuria, low white blood cell counts, increased erythrocyte sedimentation rate), and renal insult typical of SLE disease. Our data support the role attributed to pathogenic idiotypes in SLE on the one hand and that played by cellular immunity on the other. The mechanism by which Id-specific T-helper cells may induce SLE is currently not clear. The immunogenicity of the T-cell receptor (anti-16/6) and the cells themselves acting as effector/helper cells, thus leading to damage, may play a role in initiating a chain of events that ends in the production of a panoply of autoantibodies, some of which may also have a regulatory function.
A set of seven murine monoclonal antibodies were generated against a chemically synthesized 11-kDa 104-mer peptide covering the C-terminal residues 270-373 of the p24 gag protein (HIV-1BRU strain). All monoclonal antibodies recognized HIV-1IIIB infected MOLT3 cells by fluorescence and gave positive Western blot signals with viral gag peptides (p55 and/or p24). Oligopeptide binding regions were located with competitive enzyme-linked immunosorbent assays. Detailed epitope scanning analyses (the Geysen technique) were performed by serological testing of the monoclonal antibodies against 99 overlapping hexapeptides which corresponded to the entire 104-mer region. The antibodies bound to p24 peptide sequences located within the 275-293 and 351-368 regions. One antibody (LH104-B) which reacted with residues 357-362 bound to p55 alone. In contrast, another antibody (LH104-I), which recognized the residues 358-363, i.e. with five out of six residues in common with antibody LH104-B for its epitope region, reacted exclusively with p24. At least two of the antibodies (LH104-C and -A) which bound to p24 alone, apparently recognized conformational epitopes. They gave positive reactions with the regions 288-293/351-356 and 284-289/351-356, respectively. This work shows that chemical synthesis of large peptides is a viable alternative approach to immunochemical studies of viral proteins.
Only "fair" agreement has been shown between the Abbott and DuPont enzyme linked immunosorbent assays when used for the detection of human immunodeficiency virus (HIV) antigen in serum samples from asymptomatic HIV antibody positive homosexual men. To investigate the discrepancies between the two ELISA results, further experiments were performed. The rabbit detector antibody solutions of both tests were western blotted and showed that the DuPont test was specific for p24; the Abbott detector antibody had bands for p18, p41-43, gp120 as well as p24. By using dilutions of a known amount of HIV antigen, the Abbott test could detect 20 pg/ml p24; the DuPont test could detect 30 pg/ml p24. The DuPont test was also more sensitive than the Abbott test at detecting a synthetic 104mer peptide of p24. Within the 104mer sequence two regions (294-318, 334-348 amino acids) inhibited the binding of the DuPont detector antibody, but no blocking was observed with the Abbott antibody. Although the Abbott test was slightly more sensitive at detecting HIV protein than the DuPont test, the major difference between the tests was in the molecular specificity, in that the Abbott test detected proteins other than p24. This may not be important for detecting antigen in cell culture, but it may affect the detection of antigenaemia in patients' sera.
We have previously demonstrated the pathogenicity of the common anti-DNA idiotype designated 16/6 Id. Immunization of naive mice with the 16/6 Id induced SLE-like disease characterized by serological (e.g. anti-dsDNA and anti-Sm auto-antibodies), clinical (increased ESR, leucopenia and proteinuria), and pathological (16/6 Id deposition in kidneys) parameters. To elucidate further the role of the 16/6 Id in SLE induction the following studies were carried out: BALB/c mice were immunized with SA-1, a human anti-DNA monoclonal antibody carrying the 16/6 Id; TB-68, a mouse monoclonal anti-tuberculosis (TB) glycolipid, which binds dsDNA and carries the 16/6 Id; TB-72, a mouse monoclonal anti-TB glycolipid that binds DNA and does not harbour the 16/6 Id; and 4B4, a human anti-Sm antibody that carries the 16/6 Id. SLE was induced in BALB/c mice only when immunized with SA-1, TB-68, and 4B4, namely antibodies with diverse binding capacities albeit having the 16/6 Id. Our studies further support previous evidence on the pathogenic role attributed to the 16/6 Id in SLE, and suggest that SLE is most probably an idiotype-induced disease.
Three hundred and ninety eight serum samples from 270 human immunodeficiency virus (HIV) antibody positive asymptomatic homosexual men were tested in the Abbott and Dupont HIV antigen ELISA tests. In the Abbott test 62 (16%) of the sera were positive, according to the manufacturer's instructions, compared with 55 (14%) in the Dupont test. Twenty six sera were positive with the Abbott test but negative with the Dupont test and 19 sera were positive only by the Dupont test. Only 36 (9%) of the sera were positive in both tests. The Abbott confirmatory neutralisation test gave excellent agreement with the initial Abbott HIV antigen ELISA test; the Dupont confirmatory test was only in agreement with the initial positive Dupont antigen ELISA test in one third of the sera tested. Although the overall sensitivity of each of the two commercial assays tested was similar, the Abbott method may be preferable for clinical purposes if confirmation of an initial ELISA positive test result by neutralisation assay is required.
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The rapid identification of anti-HIV compounds in the laboratory following the isolation of the causative virus in 1983 and their subsequent use in the clinic was not unexpected. Three decades of previous work had established a scientific basis for the evaluation of antiviral compounds. However, no antiviral yet discovered can cause total blockade of a virus replicating in a cell. The combination of properties of HIV including latency, antigenic and biochemical variation is unusual and the virus represents a daunting challenge for chemotherapy. But at least 90 antiviral compounds have been discovered, many inhibiting the virus reverse transcriptase. Other targets for inhibition are possible including viral regulatory gene products, viral protease and endonuclease enzymes but compounds for initial study will have to be found by random searching. X-ray crystallography of HIV proteins will shortly be possible, enabling the commencement of a more molecular specific search for inhibitors. Meanwhile, advantage can be taken of comparative nucleotide sequences of the HIV-1 and -2 genomes to test short oligonucleotides as potential inhibitors of mRNA transcription. The pol gene also has a zinc finger amino acid sequence suggesting that chelation chemotherapy may have a potential role. In the absence of HIV vaccines, and associated theoretical problems in their development, antiviral chemotherapy is expected to occupy a central role in combating the AIDS epidemic.
The gene encoding a major protein antigen of Mycobacterium tuberculosis has been cloned and sequenced using oligonucleotide probes derived from the N-terminal sequence of the analogous protein from Mycobacterium bovis BCG. The gene analysis revealed a sequence encoding a protein of 99 amino acid residues, with a molecular mass of 10.7 kDa. Computer prediction suggests that the protein contains three T-cell-determined epitopes (of which one has been demonstrated experimentally) and three B-cell-determined epitopes. The protein sequence was homologous to two prokaryote heat-shock proteins and the gene possessed heat-shock-like promoter sequences upstream of the initiation codon. A hairpin loop identified in the upstream sequence may also be important in regulation of the gene.
The binding of human lupus anti-DNA antibodies and murine anti-mycobacterial antibodies to human cortical brain tissue sections was assessed by the indirect immunofluorescent technique. Prior adsorption of the reactive monoclonal antibodies on nuclear extracts, ss-DNA, synthetic polynucleotide polymers, histones, mycobacterial glycolipids, and bovine brain extracts abrogated the monoclonal antibodies' binding to the brain. Intermediate blocking activity was conferred also by ribonucleic components as RNP, Ro(SSA), and La(SSB). Specificity to neuronal tissue was demonstrated by failure of non-neuronal tissue extracts to abolish antibodies' reaction to the cortical tissue sections in competition assays. The anti-TB and anti-DNA antibodies seemed to compete on their binding to a common neuronal membranal epitope. These results indicate that mycobacteria share antigens with DNA and human brain tissue. Furthermore, these data support the concept that anti-DNA antibodies may play a pathogenetic role in SLE patients with neuropsychiatric involvement via crossing of a "leaky" blood brain barrier and attachment to brain tissue components.