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J Ivanyi

Publications and source records attributed to J Ivanyi.

At least 73 records · Page 4Linked to original sources
PubMed1994

A versatile system for the production of recombinant chimeric peptides.

Production of chimeric and multimeric peptides is of interest for the analysis of topographic relationships between T and B cell stimulatory epitopes. Recombinant DNA technology has certain advantages over conventional chemical peptide synthesis for the production of peptide constructs of large size (more than 40 amino acid residues). We describe a methodology which is versatile and independent of the expression vector used because it only relies on the incorporation of appropriate restriction enzyme sites in oligonucleotides. The method was verified using two 20mer sequences from the 38 kDa antigen of M. tuberculosis. Peptide 201-220, containing an antibody binding linear epitope, has been made immunogenic in vivo when combined with T cell stimulatory peptide 350-369 in a chimeric peptide. The results demonstrate that a distinct orientation of the constituent peptides was essential for achieving optimal immunogenicity.

Amino Acid Sequence↗
PubMed1994

Promiscuous T cell recognition of an H-2 IA-presented mycobacterial epitope.

Genetically permissive T cell epitopes are an important prerequisite for the development of peptide-based vaccines or immunodiagnostic reagents. We have investigated the structural requirements of permissive T cell recognition of peptide p350-369 from the 38-kDa antigen of Mycobacterium tuberculosis. This peptide was found to be immunogenic in mice of the H-2b, bm12, d, s and k, but not of the H-2f genotype. T cell responses were restricted by I-A class II molecules. The same epitope core was recognized in the H-2b, d and k genotypes. T cell hybrids from BALB/c and C57BL/10 mice were used to determine: (i) the critical residues using substituted peptide derivatives and (ii) the degree of T cell promiscuity. Two out of five BALB/c (H-2d)-derived hybridomas tested displayed promiscuous peptide recognition in the context of H-2b and H-2bm12 antigen-presenting cells. The recognition of critical residues was found to be uniform for all five hybridomas when tested with syngeneic antigen-presenting cells; additional critical residues were identified when the peptide was recognized in the context of allogeneic antigen-presenting cells. Only one of the four tested C57BL/10 (H-2b) hybridomas showed promiscuity in the context of H-2bm12. Each of these C57BL/10-derived clones had a distinct response profile toward the critical residues. We propose that the demonstrated T cell promiscuity involves peptide interaction with polymorphic H-2 I-A residues.

Amino Acid Sequence↗
PubMed1994

The modulation of tumour necrosis factor-alpha, interleukin-1 alpha and glucose levels with GMDP and other analogues of muramyl dipeptide.

Immunomodulatory agent muramyl dipeptide (MDP) and seven of its analogues were tested for ability to counteract the toxic actions of lipopolysaccharide (LPS) in experimental mouse models. Female BALB/c mice were presensitized with Corynebacterium parvum (P. acnes) and given MDP or equimolar doses of one of its analogues after 2 weeks, followed by intravenous challenge with LPS 18 h later. This treatment produced a sharp increase in blood cytokine (TNF-alpha, IL-1 alpha) levels 4 h after LPS administration followed by a decline to control values after 6 h. Four analogues, GMDP, threonylMDP, GMDPBenz and GMDPOBut, were able to reduce the level of cytokines induced with LPS. For most of the analogues, the higher doses reduced the levels of TNF-alpha but slightly increased the concomitant IL-1 alpha levels. GMDP was the most effective compound tested in terms of reduction of TNF-alpha and IL-1 alpha levels, as well as for reduction of the hypoglycaemia caused by the administration of LPS.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1994

Epitope specificity and isoforms of the mycobacterial 19-kilodalton antigen.

The topography and specificity of B- and T-cell stimulatory epitopes from the 19-kDa protein of Mycobacterium tuberculosis were investigated by using overlapping synthetic peptides. Murine antisera identified two cryptic epitopes (residues 11 to 30 and 61 to 80) and one species-specific immunodominant epitope (residues 140 to 159). Immunoglobulins G1 and G2a antibody isotypes varied for the respective peptide immunogens but without relationship to the T-cell cytokine profiles which were characterized by high gamma interferon and low interleukin 5 levels. Antisera to recombinant M. tuberculosis 19-kDa protein (rGST-19) cross-reacted with homologous proteins of similar size from organisms of the Mycobacterium avium-intracellulare complex. Two-dimensional gel electrophoresis revealed differences in the number, relative mobility, and charge of isoforms of the 19-kDa protein, possibly reflecting posttranslational modifications. The immunodominant T-cell epitope from the M. tuberculosis 19-kDa protein (residues 61 to 80) and the corresponding peptide sequence from Mycobacterium avium subsp. intracellulare (residues 64 to 83), differing at five residues, were both recognized in a genetically permissive manner. Peptides 61-80 and 64-83 stimulated cross-reactive responses in BALB/c (H-2d) mice, while in the C57BL/10 (H-2b) strain, responses to peptide 61-80 were species specific. In purified protein derivative-positive healthy individuals, the M. avium subsp. intracellulare peptide stimulated stronger responses than did the M. tuberculosis peptide, whereas patients with active tuberculosis had enhanced in vitro T-cell responses to both peptides.

Amino Acid Sequence↗
PubMed1994

V delta 5+ T cells of BALB/c mice recognize the murine heat shock protein 60 target cell specificity.

The purpose of this paper was to study the gamma delta T-cell receptor repertoire and target specificity following stimulation of peripheral T cells of BALB/c mice with autologous or bacterial ligands. The expression of V gamma and V delta chain families in T cells that had been expanded by stimulation in syngeneic mixed lymphocyte culture or with purified protein derivative (PPD) was determined by the semiquantitative DNA polymerase chain reaction (PCR) method. Responder T cells to either of these stimuli strongly expressed both V delta 5 and V delta 6 genes. However, addition of the ML 30 anti-murine heat shock protein (hsp) 60 monoclonal antibody (mAb) to the cell culture selectively inhibited only the expansion of V delta 5 T cells. A V delta 5 T-cell hybridoma (KMT-5), which recognized syngeneic splenic and fibrosarcoma Meth A cells but not allogeneic cells, was produced by cell fusion from autoreactive blast cells. Incubation of the KMT-5 hybridoma in the presence of ML 30 antibody blocked the stimulation of interleukin-2 (IL-2) secretion by syngeneic target cells. It was also found that the DNA of KMT-5 hybridoma and of the autoreactive gamma delta T cells contained the BALB/c invariant delta (BID) chain sequence. It is concluded from these results that BALB/c peripheral V delta 5 T cells recognize an autologous hsp 60 target specificity in a V delta-gene restricted manner. We also propose that T cells of this V gene family may be involved in the immune surveillance of certain tumours and intracellular infections.

Animals↗
PubMed1994

Th1/Th2 profiles in tuberculosis, based on the proliferation and cytokine response of blood lymphocytes to mycobacterial antigens.

Proliferation and cytokine production profiles by blood mononuclear cells in response to in vitro stimulation with mycobacterial antigens were compared in patients with active tuberculosis and in sensitized healthy controls. Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were detected at single-cell level using the ELISPOT assay. Patients showed significantly (P < 0.01) increased numbers of IL-4-secreting cells and decreased thymidine incorporation, but no significant difference in IFN-gamma-producing cells in response to the 38,000 MW or 19,000 MW antigens and their immunodominant peptide epitopes. Pronounced individual variations were found in both patient and control groups, when comparing the responsiveness to the mycobacterial extract, two protein antigens and five synthetic peptides. None of the antigens or peptides tested showed preferential stimulation of either IL-4- or IFN-gamma-secreting T cells, and proliferation was not correlated with either IL-4 or IFN-gamma production. In particular, cytokine responsiveness was of similar frequency in subjects who did or did not show positive proliferation, indicating that the latter test was not fully representative of the active T-cell repertoire. It is concluded that the demonstrated Th2 type of profile in response to two prominent mycobacterial antigens may play a role in the mechanisms of defective host resistance in tuberculosis.

Adolescent↗
PubMed1993

Genetically permissive recognition of adjacent epitopes from the 19-kDa antigen of Mycobacterium tuberculosis by human and murine T cells.

The specificity of the T cell-immune repertoire at the level of individual antigenic determinants could play a fundamental role in the immunopathogenesis of tuberculous infections. Therefore, we analyzed the immunogenicity, genetic restriction, and epitope core structure of two adjacent, yet distinct, immunodominant T cell determinants from the 19-kDa Ag of Mycobacterium tuberculosis. After immunization with two peptides comprising residues 45 to 64 and 61 to 80, vigorous in vitro proliferative responses to the homologous peptide were elicited in five different strains of C57BL/10 mice (H-2b,k,d,s,f), indicating that both epitopes were recognized in a genetically permissive manner. When immunized with intact 19-kDa protein, lymph node cells from the same mouse strains responded to both peptides, with the exception of H-2b mice, which did not respond to p45-64. Delayed-type hypersensitivity responses in C57BL/10 (H-2b) mice were elicited by p61-80 only, whereas in H-2d mice both peptides were delayed-type hypersensitivity negative, despite eliciting pronounced proliferative responses. Analysis of the proliferative responses of human PBMC in purified protein derivative-positive healthy subjects and tuberculosis patients revealed significant differences in the antigenicity to the two peptides. All purified protein derivative-positive healthy and diseased individuals manifested strong responses to p45-64, indicating HLA permissive recognition. In contrast, pronounced responses to p61-80 were detected only in patients with lymphatic tuberculosis. Epitope core structures, composed of 6 or 7 residues within each peptide, have been mapped with peptides of overlapping sequence. Significantly, for both epitopes, the core sequences recognized by both human and murine T cells were almost identical. We conclude that despite many similarities between murine and human T cell epitope recognition, distinct differences in the responsiveness of the infected host could play a role in pathogenesis. Furthermore, the genetically permissive nature of the identified epitopes is a potentially important attribute for the development of peptide-based diagnostic reagents and vaccines.

Adult↗
PubMed1993

Prenatal expression of the 65-kDa heat-shock protein homologue in pig tissues.

A monoclonal antibody (ML30) recognizing the 65-kDa heat-shock protein of mycobacteria and reacting with homologous human protein was found to stain various porcine tissues. Development of this reactivity was studied. The first ML30-positive cells were embryonic hepatocytes. The protein reacting with the ML30 antibody was localized predominantly in the Golgi area and mitochondria of hepatocytes. Cell membranes of some peripheral blood lymphocytes were also found to bind ML30.

Animals↗
PubMed1993

Antigen-dependent in vitro culture of protective T cells from BCG-primed mice.

Induction of protective immunity against pathogenic mycobacteria depends on vaccination with live organisms such as Bacille Calmette-Guérin (BCG). However, it is not known how many and which antigens are involved in the protective host response. In this study, we developed a system of antigen-dependent in vitro culture which is suitable for the analysis of protective subunits, presented in a soluble form. Spleen cells from Mycobacterium bovis BCG-immune mice, enriched for T cells and depleted of adherent cells on a column of G-10 Sephadex, were cultured for periods varying between 3 and 14 days before transfer and challenge with M. tuberculosis in irradiated hosts. Following 10 days in culture, immune T cells sustained their capacity to transfer protection to tuberculous infection when incubated in the presence of either live BCG or a soluble extract from M. tuberculosis, but lost this ability when cultured in the absence of antigen, or in the presence of the polyclonal mitogen concanavalin A. One immunodominant antigen, represented by the recombinant 38-kD antigen, failed to sustain the adoptive protection, despite pronounced stimulation of lymphoproliferation in culture. Antigenic in vitro stimulation of protective T cells was accompanied by enhanced responsiveness to exogenous IL-2. The experimental system described may be generally suitable to test in vitro the protective potentials of soluble molecular subunits of mycobacteria.

Animals↗
PubMed1993

Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation.

The gene encoding a 19-kDa antigen from Mycobacterium tuberculosis was expressed as a recombinant protein in the rapid-growing species Mycobacterium smegmatis. The recombinant antigen was expressed at a level approximately ninefold higher than in M. tuberculosis and, like the native antigen, was found in the pellet fraction after high-speed centrifugation of bacterial extracts. The 19-kDa antigen in crude bacterial extracts, and the purified recombinant antigen, bound strongly to concanavalin A, indicating the possibility of posttranslational glycosylation. The recombinant antigen stimulated T-cell proliferation in vitro when added to assays either in the form of whole recombinant bacteria or as a purified protein. Homologous expression of mycobacterial antigens in a rapid-growing mycobacterial host may be particularly useful for the immunological characterization of proteins which are subject to posttranslational modification.

Amino Acid Sequence↗
PubMed1993

Recognition of peptide epitopes of the 16,000 MW antigen of Mycobacterium tuberculosis by murine T cells.

The T-cell repertoire to a prominent immunogen of Mycobacterium tuberculosis has been investigated on the assumption that differences in epitope specificity could influence the protective and pathogenic host reactions. Proliferative responses of lymph node and spleen cells to overlapping peptides, spanning the entire sequence of the 16,000 MW protein antigen were analysed in C57BL/10 and B10.BR mice. Following footpad priming and in vitro challenge with homologous peptide, 12 out of the 14 peptides tested were found to be immunogenic. However, only two peptides of residues 31-40 and 71-91 stimulated strong proliferative responses of T cells from mice which had been presensitized with either killed or live M. tuberculosis organisms; another three peptides were only weakly stimulatory. These epitopes have been immunodominant in both H-2b and H-2k mouse strains, indicating the genetically permissive nature of their recognition. Furthermore, both major immunodominant epitopes were found to be species specific for the M. tuberculosis complex and therefore potentially suitable for the early diagnosis of tuberculous infection.

Amino Acid Sequence↗
PubMed1993

The effect of lipoylation on CD4 T-cell recognition of the 19,000 MW Mycobacterium tuberculosis antigen.

The mechanisms contributing to the dominant and degenerate recognition of the N-terminal region of the Mycobacterium tuberculosis 19,000 MW protein have been investigated. Using polyclonal and cloned T cells it was found that the apparently promiscuous response to the N-terminal peptide was due to the presence of multiple epitopes recognized in the context of different HLA determinants. This finding did not, however, explain the concentration of T-cell recognition on this part of the molecule. The 19,000 MW antigen is a lipoprotein, which raised the possibility that presence of a lipidation motif preceded by a signal peptide influenced the processing and presentation of the protein. Modified peptides covalently attached to lipid moieties were, therefore, tested on both polyclonal and cloned T cells. It was found that whilst lipoylation enhanced polyclonal T-cell recognition, the effect on cloned T cells was variable and depended on their epitope specificity and restricting HLA determinants. This suggested that whilst lipoylation may enhance some aspect of signalling for polyclonal T cells it does not affect the presentation of peptide to T-cell clones. The explanation for the immunodominance of the N-terminal region may, therefore, lie in some aspect of its processing.

Amino Acid Sequence↗
PubMed1992

T cell repertoire in tuberculosis: selective anergy to an immunodominant epitope of the 38-kDa antigen in patients with active disease.

It is generally accepted that both host protection and pathogenic reactions in tuberculosis are mediated by T lymphocytes. However, little is known about the structures and discreet functions of epitopes stimulating the immune response. In this study, proliferative responses of blood T lymphocytes to synthetic peptides derived from the sequence of the 38-kDa antigen from Mycobacterium tuberculosis have been investigated in 41 healthy individuals and in 36 patients with active tuberculosis. Of the healthy purified protein derivative (PPD)-positive donors, 90% responded to a permissively recognized peptide, 38.G (residues 350-359), located at the carboxy terminus of the molecule. Four other permissively recognized epitopes of this molecule (38.A, 38.I, 38.E, 38.K) were stimulatory for more than 50% of healthy PPD-positive individuals. Patients with lymphatic tuberculosis responded to these peptides in a similar manner. In contrast, we observed a selective anergy to stimulation with peptide 38.G in the majority of patients with pulmonary (11% responders) and nonlymphatic extrapulmonary tuberculosis (25% responders). The lack of responsiveness to 38.G was epitope specific since the degree of responsiveness to the other four permissively recognized peptide epitopes was similar for patients and PPD-positive controls. Using the PEPSCAN technology and truncated peptides, the core epitope of 38.G was localized to a peptide 10 amino acids long (HFQPLPPAVV). This minimal structure was capable of inducing a proliferative response in all healthy 38.G responders tested. The mechanisms influencing this epitope-specific anergy in patients could give new insights into the immunopathogenesis of tuberculosis.

Amino Acid Sequence↗
PubMed1992

Morphometric analysis of Mycobacterium tuberculosis infection in mice suggests a genetic influence on the generation of the granulomatous inflammatory response.

There is evidence in natural human disease and experimental infection in mice that host genetic factors influence susceptibility to infection with Mycobacterium tuberculosis and the progress of the disease. In mouse models, both H-2 and non-H-2 genes have been implicated. In this study, four inbred strains of mice (Balb/b, Balb/k, B10, B10.BR), selected for combinations of two different H-2 haplotypes on two different non-H-2 backgrounds, were inoculated with M. tuberculosis, strain H37Rv, by intraperitoneal injection. The histological features of the granulomatous inflammatory response in the liver and lungs were investigated during the first 18 weeks of the infection. Granuloma fraction, mean granuloma area, bacillary load, and the density of acid-fast bacilli within granulomata were measured. Animals of all four strains showed the same general pattern of infection with an early, and later self-limiting, infection of the liver and delayed onset, but progressive, infection of the lung. The non-H-2 related genetic background appears to influence the morphology of the granulomatous inflammatory response. In comparison, H-2 differences appeared to be small and inconsistent.

Animals↗
PubMed1992

Serodiagnostic value of the 19 kilodalton antigen of Mycobacterium tuberculosis in Indian patients.

Previous studies in UK subjects suggested that the 19 kDa protein antigen of Mycobacterium tuberculosis might be valuable in the serodiagnosis of paucibacillary tuberculosis. In this study, antibody titres for the 19 kDa antigen were higher in healthy controls in India than in the UK. Consequently, the diagnostic sensitivity of this antigen and its TB23 epitope was negligible in Indian patients with tuberculosis. However, a diagnostic sensitivity of 50% was achieved in patients with skin tuberculosis on the basis of a high ratio between antibody titres for the whole antigen and its TB23 epitope.

Antibodies, Bacterial↗
PubMed1992

Localisation of linear epitopes at the carboxy-terminal end of the mycobacterial 71 kDa heat shock protein.

Four distinct linear epitopes localized within species-specific sequences at the carboxy-terminal end of the 71 kDa heat shock protein of M. tuberculosis have been identified by scanning 94 overlapping peptides with 13 human sera. One epitope ("C") of entirely M. tuberculosis-specific core sequence (GEAGPG) has been found immunogenic in smear-negative tuberculosis, but not in non-tuberculous mycobacterial diseases. This peptide appears to be a valuable candidate for further serodiagnostic evaluation.

Amino Acid Sequence↗