Thomas M. Buchanan

T cell activation by mycobacterial antigens in inflammatory synovitis

To define which mycobacterial antigens were responsible for the activation of synovial fluid T lymphocytes, acetone-precipitated Mycobacterium tuberculosis (AP-MT) antigens were separated into five fractions following polyacrylamide gel electrophoresis and added to the mononuclear cell cultures of patients with inflammatory synovitis. Fractions 2 (50 to 70 kDa) and 5 (less than 28 kDa) resulted in significantly more proliferation than that of fractions 1, 3, and 4. The response to a purified mycobacterial 65-kDa heat shock protein (hsp), which migrated in fraction 2, was highly correlated (r = 0.89, P less than 0.001) with the response to the crude AP-MT. The proliferative response to a different hsp. the Escherichia coli DnaK, by synovial fluid lymphocytes was marginal. Analysis of the synovial fluid T cell response to mycobacterial culture filtrates by T cell Western blotting revealed dominant responses to antigen(s) in the range of 31 to 21 kDa in each responding patient, although no other consistent pattern of T cell activation was noted. Three lines of evidence suggested that the response to the low molecular weight fractions was directed against degradation fragments of the 65-kDa protein. These observations suggest that the activation of T lymphocytes obtained from inflammatory synovial fluids by crude mycobacterial antigens was due in large part to recognition of the 65-kDa mycobacterial hsp.

Monoclonal antibody activity against native and denatured forms of gonococcal outer membrane proteins as detected within ultrathin, longitudinal slices of polyacrylamide gels

Monoclonal antibodies were used to analyze the antigenic properties of denatured and native forms of gonococcal outer membrane proteins. The protein samples were only partially dissociated by treatment for 30 min at 40 degrees C with 0.1% (w/v) SDS, 0.5% (v/v) Triton X-100, and then processed by polyacrylamide gel electrophoresis without boiling. The resulting pattern included the native aggregated and trimeric forms of protein I and III as they exist in the gonococcal outer membrane, as well as the denatured monomeric forms. Two methods were compared to analyze these gels: gel immunoradioassay (GIRA), and Western blotting. With GIRA longitudinal 50 micron thin slices, up to 40 identical copies per gel, were produced with a microtome cryostat. These slices were exposed to the monoclonal antibody and antibody binding was detected by 125I-protein A and autoradiography. Serotype-specific, monoclonal antibodies reacted most commonly with the native polymeric form of gonococcal protein I and less frequently recognized the denatured, monomeric form. Monoclonal antibodies that recognized the polymeric form of protein I frequently produced antibody-mediated, complement-dependent, bactericidal activity for gonococci bearing the same protein I serotype. The antigen specificity of these functionally relevant antibodies could not be characterized by the Western blotting procedure, which produced incomplete transfer to nitrocellulose paper of the polymeric, high molecular weight protein aggregates. A third technique, radioimmunoprecipitation using partial dissociating conditions, did not permit differentiation between proteins I- and III-specific monoclonals after analysis of the precipitated material by denaturing SDS electrophoresis.

Immune-enhanced phagocytosis of Neisseria gonorrhoeae by macrophages: characterization of the major antigens to which opsonins are directed.

antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (fron gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili greater than lipopolysaccharide greater than principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab)2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.

Use of a polysulfone membrane support for immunochemical analysid of a glycilipid from Mycobacterium leprae

Polysulfone membranes have been used as a solid support for chromatography and immunoblotting of phenolic glycolipid I from Mycobacterium leprae. These membranes have an advantage over other supports such as nitrocellulose and silica gel in that very little non-specific background binding of antibodies occurs and assays can readily be carried out with IgM antibodies from human sera. An example of use of the polysulfone chromatography system for detection of phenolic glycolipid I in sera from leprosy patients is described.

Determination of biogenic amines using heterocyclic cation and aromatic anion elution

Abstract Heterocyclic cation and aromatic anion are used in the chromatographic buffers for the analysis of monoamines and diamines on a sulfonated cation-exchange column using an amino acid analyzer. All elution buffers employed for these analyses had a pH of 5.0 to maximize the ninhydrin color reaction. These methods have been successfully used for biological samples. Using a two-column (0.8 × 12 cm) system, with a buffer flow rate of 50 ml/h, analysis can be carried out in 330 min for monoamine and diamine mixtures on 0.5 to 10 nmol of each amine.

Monoclonal Antibodies to Protein I for Serotyping of Neisseria gonorrhoeae: Correlation of Serotype with Bactericidal Activity

Seven monoclonal antibodies have been used for the serotyping of one hundred Neisseria gonorrhoeae wild strains, randomly selected from nine U.S. cities, and seven serotype reference strains by the co-agglutination method. As determined by gel-immunoradioassay, the monoclonal antibodies recognized the protein I trimer of a single or a limited subset of serotype reference strains. All but three strains were typable by one or two of the antibodies. The most common serotypes were 1.3 (26%), 1 (20%), 5 (17%), 5.7 (11%) and 9 (10%). To correlate typing results with ability for killing of these antibodies, susceptibility of typed and non-typed strains to killing was studied. Susceptibility was significantly associated with typing by the serotype 7 (p = 0.011) and serotype 9 (p = 0.033) specific monoclonal antibodies. Reaction of antibodies recognizing epitopes on the protein IB molecule with a given strain predicted in an average of 43% of strains (49% of strains of serotype 5, 62% of serotype 7, 29% of serotype 8, and 33% of serotype 9) its susceptibility to killing by the typing antibodies. In contrast, only 15% of the strains (15% of strains of serotype 1 and 15% of serotype 3) were killed by their typing antibodies, recognizing epitopes on the protein IA molecule. These monoclonal antibodies might prove to be important for the isolation and structural characterization of epitopes responsible for susceptibility of the gonococcus to killing and thus for the development of a vaccine against invasive gonococcal disease.

Interaction of Non-piliated Neisseria gonorrhoeae Strain 7122 and Protein IA with an Epithelial Cell Monolayer

Studies with [14C] uracil-labelled bacteria revealed that the interaction of Neisseria gonorrhoeae with epithelial cells occurred in a time-dependent reaction which is slightly pH-dependent and optimal at pH 6.5. Immunofluorescence tests and immunoelectron microscopy of ultrathin sections confirmed the attachment of these bacteria to the epithelial cell membrane. The interaction of purified protein I with epithelial cells was time-dependent and reached equilibrium after four hours as shown by tracer experiments with 125I-labeled protein I. Cleavage experiments with trypsin followed by SDS-PAGE and autoradiography indicated that protein I (labeled with 125I) was associated with the membrane of the epithelial cells and only partly accessible by trypsin after its interaction with these mammalian cells. Immunofluorescence tests as well as immunoelectron microscopy with the monoclonal antibody G7A2C and gold-labeled protein A confirmed a dense association pattern of protein I with the cell monolayer.

Surface immunolabeling of Neisseria gonorrhoeae employing monoclonal antibodies to proteins IA and IB

The technique of immunomarking intact bacteria with monoclonal antibodies (MAb) and gold-labeled anti-IgG provides a rapid and reliable method for localization of surface-exposed antigens. Cell suspensions of Neisseria gonorrhoeae were incubated with monoclonal antibodies directed against different epitopes of protein I as shown by ELISA inhibition test (unpublished data). The bound IgG molecules were detected with gold labeled anti-mouse IgG or protein A. MAbs against various epitopes of protein I showed differences in their reaction pattern with intact bacteria. We compared the results achieved by the gold immunomarking technique with coagglutination (CoA) and immunofluorescence (IF) tests. The results of the three methods correlated for some antibodies and were ambiguous for other MAbs. The advantage of the immunogold method over immunofluorescence and coagglutination is that distribution and localization of antigen on the surface of gonococci can be exactly determined using electron microscopy.

Interaction of Neisseria gonorrhoeae and protein IA with HEp-2 cells

We investigated the interaction of Neisseria gonorrhoeae strain 7122 and isolated protein IA with an epithelial cell monolayer (HEp-2). With radiolabeled protein IA a time dependent interaction with tissue culture cells was observed. Immune electron microscopy revealed the association of gonococci with epithelial cells and the insertion of protein IA in the mammalian cell membrane. The findings of these investigations as well as enzyme cleavage experimetns with 1 2 5I-labeled protein IA associated with the epithelial membrane and subsequent SDS-PAGE followed by autoradiography indicated that protein IA is partially inserted in the mammalian cell identically to its orientation in the gonococcal membrane.

Examination of a surface exposed epitope on the protein IA molecule of Neisseria gonorrhoeae

Proteolytic cleavage of purified PIA of strain 7122 transparent phenotype (0-) (serotype 1, serogroup I) resulted in two, four, and five fragments using (TPCK)-trypsin, alpha-PI A was subsequently cleaved into smaller fragments. Immunoblot experiments using a monoclonal antibody (MoAb) specific for a surface exposed epitope of PI A of strain 7122, as assessed by coagglutination, Western blot, and immunofluorescence assay revealed that all cleavage fragments 2≥ 15K were reactive with the MoAb after incubation with peroxidase conjugated goat anti-mouse IgG, whereas controls using normal mouse serum showed no nonspecific binding. These data indicate that whereas purified protein I is susceptible to proteolytic cleavage, the epitope domain is relatively protease resistant.