Conserved nucleotide sequences at the 5' end of T cell receptor variable genes facilitate polymerase chain reaction amplification.

Alignment of all available nucleotide sequences of mouse and rat alpha/beta T cell receptor (TcR) variable (V) regions revealed the presence of relatively conserved sequences at the 5' end of the V gene segments. Based on these conserved sequences, degenerate primers were developed for use in the polymerase chain reaction (PCR). The degenerate primers developed on the basis of the conserved sequences at the 5' end of rat and mouse V gene segments are expected to enable the amplification of all mouse and rat TcR alpha/beta chain V regions.

Efficient recognition by rat T cell clones of an epitope of mycobacterial hsp 65 inserted in Escherichia coli outer membrane protein PhoE.

PhoE is a pore-forming protein, abundantly expressed in the Escherichia coli outer membrane. Previous investigations have shown the possibility of inserting antigenic determinants in cell surface-exposed regions of PhoE by recombinant DNA techniques without disturbing the biogenesis and the functioning of the protein. This method proved to be successful for foot-and-mouth disease virus B cell determinants.

Antibodies to the mycobacterial 65-kd heat-shock protein are reactive with synovial tissue of adjuvant arthritic rats and patients with rheumatoid arthritis and osteoarthritis.

The expression of 65-kd mycobacterial heat-shock protein (HSP)-related antigens in synovial membrane from rats and humans with arthritis was studied using three monoclonal antibodies and one polyclonal antiserum directed to antigens of mycobacteria. The antibodies labeled synovial tissue sections from both adjuvant arthritis (AA) rats and from patients with either rheumatoid arthritis (RA) or osteoarthritis (OA); especially the synovial lining cells appeared to be positive. The cytoplasmic staining patterns in rats and humans were essentially the same and were not related to the extent of inflammation ie, the size of lymphoid infiltration.

Heat shock proteins: immunity and immunopathology.

Much interest has been generated in heat shock proteins (hsps) since they were implicated as dominant antigens of infectious microorganisms and suggested to have a role in the development of adjuvant arthritis (AA) in rats. Advances in the understanding of the role of hsps in immunity and immunopathology were recently discussed at a meeting held in Utrecht, as part of the European Community concerted action on immunopathogenesis and immunotherapy of chronic arthritis.

Heat-shock proteins in autoimmune arthritis: a critical contribution based on the adjuvant arthritis model.

Recognition of self protein epitopes, apart from those engaged in idiotypic network interactions and MHC restriction, is probably a physiological event in the normal functioning immune system. Furthermore T and B cells recognizing self antigens can be easily cloned from healthy individuals and sometimes be shown to confer autoimmune disease by passive transfer in the experimental situation. The issue is how potentially autoaggressive cells can become activated and how such activity can be contained safely.

Mycobacterial antigens stimulate rheumatoid mononuclear cells to cartilage proteoglycan depletion.

In a coculture with porcine articular cartilage explants unstimulated blood mononuclear cells (BMC) from patients with rheumatoid arthritis (RA), but not from healthy controls, induced proteoglycan depletion of dead cartilage. Specific stimulation of the RA BMC with Mycobacterium tuberculosis (MT), in comparison with concanavalin A (Con-A), strongly enhanced the proteoglycan depletion of living cartilage; this was not found with the BMC of healthy controls. However, the MT induced proliferative responses of the same BMC were similar in healthy controls and patients with RA.

Protection against streptococcal cell wall-induced arthritis by pretreatment with the 65-kD mycobacterial heat shock protein.

We report that streptococcal cell wall (SCW)-induced arthritis in rats, a T cell-dependent chronic, erosive polyarthritis, can be prevented by pretreatment of the rats with the mycobacterial 65-kD heat shock protein. This 65-kD protein shows extensive amino acid homology with prokaryotic and eukaryotic 65-kD heat shock proteins and is a ubiquitous bacterial common antigen. Both the clinical and histopathologic manifestations of the arthritis were prevented completely when rats were pretreated with 50 micrograms of 65-kD protein intraperitoneally at 35, 25, 15, or 5 d before administration of SCW.

Properties of monoclonal antibodies against Berne virus (Toroviridae).

Seven hybridomas that secreted monoclonal antibodies (MAB) against the peplomer protein and one that secreted MAB against the nucleocapsid protein of Berne virus (proposed family Toroviridae) were isolated. All MAB directed against the peplomer protein neutralized virus infectivity and, with the exception of MAB 6A7, inhibited each other's binding in competition assays. Neutralization of Berne virus infectivity was potentiated when some MAB were used in pairs.

The mycobacterial 65 kD heat-shock protein and autoimmune arthritis.

Arthritis - induced experimentally in rats by immunization with mycobacteria has been shown to depend on specific T cell recognition of an epitope present on the mycobacterial 65-kD heat-shock protein. This particular epitope has been observed to have a structural mimicry with a cartilage-associated molecule present in the joints. Since the bacterial heat-shock proteins and the cartilage-associated molecules are of a conserved nature, one might infer from the experimental model that in humans similar mimicry could play a role in the initiation of autoimmune arthritis.

Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides.

Prediction, identification and analysis of T cell epitopes in protein antigens has become a central theme in fundamental and applied immunology. However, while for the characterization of linear B cell epitopes the so-called Pepscan procedure was found to be extremely effective, no such technique has so far been available for T cell studies. Recently, we described the identification and localization of a T cell epitope in a mycobacterial 65-kDa shock protein in the model of adjuvant arthritis.

Synovial fluid T cell reactivity against 65 kD heat shock protein of mycobacteria in early chronic arthritis.

The in vitro proliferative response against a recombinant 65 kD Mycobacterium bovis protein that has 100% homology with the 65 kD protein of M tuberculosis was tested in synovial fluid and peripheral blood mononuclear cells from patients with rheumatoid arthritis (RA) and other types of chronic arthritis. An acetone precipitate (AP) of M tuberculosis, and a purified protein derivative (PPD) of M tuberculosis were also tested. Responsiveness of synovial fluid lymphocytes to the mycobacterial antigens was found both in patients with RA and in patients with other forms of chronic inflammatory arthritis, but not among controls.

Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis.

Adjuvant arthritis (AA) is a chronic disease inducible in rats by immunization with an antigen of Mycobacterium tuberculosis. After the isolation of arthritogenic T-cell lines and clones, it became possible to demonstrate that the critical M. tuberculosis antigen contained an epitope cross-reactive with a self-antigen in joint cartilage.

Antigenic relatedness of a strongly immunogenic 65 kDA mycobacterial protein antigen with a similarly sized ubiquitous bacterial common antigen.

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae recombinants were frequently found expressing an immunodominant 65 kDa protein antigen. In this study polyclonal and monoclonal antibodies against the 65 kDa antigen were found to react with a variety of different bacteria. Furthermore it is shown that the 65 kDa mycobacterial protein belongs to the family of antigens previously designated 'common antigen' due to their presence in a large variety of bacterial species.

An enzyme-linked immunosorbent assay for the determination of chloramphenicol using a monoclonal antibody. Application to residues in swine muscle tissue.

A competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody is described for the determination of chloramphenicol (CAP) residues in swine muscle tissue. The limit of detection of standard solutions is established to be 25 ng ml-1 = 2.0 ng CAP per well).

Histopathology of arthritis induced in rats by active immunization to mycobacterial antigens or by systemic transfer of T lymphocyte lines. A light and electron microscopic study of the articular surface using cationized ferritin.

We analyzed the histopathologic findings of arthritis in 3 rat models: adjuvant arthritis induced by active immunization to Mycobacterium tuberculosis (MT) antigens, arthritis produced by passive transfer of an intrinsically arthritogenic line of anti-MT T lymphocytes, and bystander arthritis produced by intraarticular injection of a foreign antigen, ovalbumin, into rats with T lymphocyte line cells specific for the ovalbumin antigen. The histopathology of the tibiotarsal and knee joints was studied by light microscopy and the articular surface of the cartilage by electron microscopy after labeling with cationized ferritin. The lesions in the 3 models of arthritis were compared.

T lymphocytes of rheumatoid arthritis patients show augmented reactivity to a fraction of mycobacteria cross-reactive with cartilage.

An acetone-precipitable fraction of Mycobacterium tuberculosis cross-reacts with human cartilage. Immune responses to this antigen were assessed in 34 patients with rheumatoid arthritis, 16 patients with degenerative joint disease, and 15 healthy controls. The RA patients differed from the other two groups in having more pronounced T lymphocyte responses to the antigen; their serum antibody levels were not higher.

Evidence for an HLA-DR4-associated immune-response gene for Mycobacterium tuberculosis. A clue to the pathogenesis of rheumatoid arthritis?

Antigens of Mycobacterium tuberculosis, M leprae, M scrofulaceum, and M vaccae were injected intradermally in 86 caucasoid leprosy patients, and skin responses (measured in mm of induration at 72 h) were analysed in relation to HLA class II phenotypes. HLA-DR4 was associated with high responsiveness to antigens specific to M tuberculosis but not to antigens shared with other mycobacteria (p = 0.0005).