PubMed Health⌕ Search

Biomedical subjects

R J Shoberg

Publications and source records attributed to R J Shoberg.

8 recordsLinked to original sources
PubMed1995

Borrelia burgdorferi vesicle production occurs via a mechanism independent of immunoglobulin M involvement.

Borrelia burgdorferi produces extracellular vesicles containing various borrelial protein antigens when propagated in vitro in culture media. Commonly observed components of borrelial vesicle preparations are borrelial surface antigens, bovine serum albumin, and the heavy chains of rabbit immunoglobulin G and immunoglobulin M. This study employed ultracentrifugation to harvest borrelial vesicles and analyzed these preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. We demonstrated that the rabbit mu heavy-chain band observed was devoid of OspA or at most levels below those detectable by immunoblot. We also demonstrated the recovery of borrelial vesicles at relative centrifugal forces as low as 25,000 x g, compared with the force of > 200,000 x g normally employed. Further, the mu heavy-chain band was recovered from uninoculated growth media processed at 25,000 x g, suggesting that it behaves as a particle rather than as a soluble molecule under these conditions. Lastly, vesicles were demonstrated to be present in preparations harvested from growth media supplemented with fetal calf serum, suggesting that vesicle production by B. burgdorferi can occur in the absence of immunoglobulins.

Animals↗
PubMed1994

Identification of a highly cross-reactive outer surface protein B epitope among diverse geographic isolates of Borrelia spp. causing Lyme disease.

The outer surface lipoprotein B (OspB) of Borrelia burgdorferi is a major component of the borrelial protein profile and has been shown to be highly immunogenic in experimentally immunized and infected mammals. However, the ospB loci of different strains show considerable heterology at the nucleic acid sequence level, and the progeny of a clonal strain of B. burgdorferi exhibited OspB polymorphisms with respect to apparent molecular weights and reactivities with monoclonal antibodies. These data suggest that OspB is not a good candidate for vaccination or diagnostic purposes. The present study describes a monoclonal antibody, designated 84C, directed against a very highly conserved domain of the OspB lipoprotein. Western immunoblot analysis with 84C demonstrated reactivity in 84.2% of human, tick, and other vertebrate isolate strains examined from widely diverse geographic regions, including strains of B. burgdorferi sensu stricto and two closely related species, B. garinii and B. afzelii. The 84C-binding region was delimited to a highly conserved 11-amino-acid region in the carboxyl terminus of OspB as demonstrated by (i) DNA sequence analysis of wild-type and 84C-resistant mutant ospB alleles and (ii) deletion mutagenesis of a recombinant ospB gene in Escherichia coli. Finally, the 84C epitope was demonstrated to be exposed on the borrelial surface in situ as (i) the monoclonal antibody 84C was able to agglutinate borrelias in culture and (ii) 84C-resistant escape variants were isolated. These data suggest that the potential value of OspB as a vaccine candidate or diagnostic tool be examined more closely, in the context of the 84C-reactive domain.

Amino Acid Sequence↗
PubMed1993

A monoclonal antibody to OspA inhibits association of Borrelia burgdorferi with human endothelial cells.

Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B. burgdorferi.

Animals↗
PubMed1993

Specific adherence of Borrelia burgdorferi extracellular vesicles to human endothelial cells in culture.

Borrelia burgdorferi produces extracellular vesicles which contain some of the outer surface proteins of the bacterium (e.g., OspA and OspB). Borrelial vesicles, isolated by differential centrifugation and filtration, were tested for the ability to bind to cultured human umbilical vein endothelial (HUVE) cells in culture. The recently described lipoprotein OspD was expressed on vesicles. Vesicles exhibited differential expression of OspB and OspD in a relationship with passage number and medium serum supplement type, respectively. Qualitative immunoblotting analyses demonstrated dose-dependent, passage number-dependent adsorption of vesicles by HUVE cells. This adsorption was demonstrated to be dependent upon a borrelial component of the vesicle and not due to the presence of minor contamination with intact spirochetes. Quantitative experiments examining inhibition of B. burgdorferi-HUVE association as a function of prior vesicle-HUVE association demonstrated dependence upon (i) a borrelial component(s) in the vesicle, (ii) low passage number, and (iii) vesicle protein concentration. However, vesicle pretreatment of the HUVE cell monolayer was not requisite for this inhibition. Vesicles from highly passaged borrelias were noninhibitory for B. burgdorferi-HUVE cell association, regardless of the serum used to supplement the medium. The use of vesicles as a tool for studying B. burgdorferi pathogenesis and/or physiology is proposed.

Adsorption↗
PubMed1991

Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease.

The immunoglobulin A1 (IgA1) proteases of Neisseria gonorrhoeae have been defined as having human IgA1 as their single permissive substrate. However, in recent years there have been reports of other proteins which are susceptible to the proteolytic activity of these enzymes. To examine the possibility that gonococcal membrane proteins are potential substrates for these enzymes, isolated outer and cytoplasmic membranes of N. gonorrhoeae were treated in vitro with exogenous pure IgA1 protease. Analysis of silver-stained sodium dodecyl sulfate-polyacrylamide gels of outer membranes indicated that there were two outer membrane proteins of 78 and 68 kDa which were cleaved by IgA1 protease in vitro in GCM 740 (a wild-type strain) and in two isogenic IgA1 protease-negative variants. Similar results were observed with a second gonococcal strain, F62, and its isogenic IgA1 protease-negative derivative. When GCM 740 cytoplasmic membranes were treated with protease, three minor proteins of 24.5, 23.5, and 21.5 kDa were cleaved. In addition, when outer membranes of Escherichia coli DH1 were treated with IgA1 protease, several proteins were hydrolyzed. While the identities of all of these proteolyzed proteins are unknown, the data presented indicate that there are several proteins found in the isolated membranes of gram-negative bacteria which are permissive in vitro substrates for gonococcal IgA1 protease.

Bacterial Outer Membrane Proteins↗
PubMed1987

Restriction site polymorphism in genes encoding type 2 but not type 1 gonococcal IgA1 proteases.

Neisseria gonorrhoeae produces two phenotypically distinct types of IgA1 proteases, each of which cleaves a specific peptide bond in the hinge region of the human IgA1 heavy chain. The genes encoding IgA1 protease from twenty-eight different strains of N. gonorrhoeae, including twelve which produce type 1 enzyme, thirteen which produce type 2 enzyme, and three which are protease negative, were analyzed. Nine restriction site patterns were found in the iga genes. All twelve type 1 strains showed identical restriction maps of the iga gene, which differed from all the iga-2 variants. The three protease negative strains each contained DNA homologous to the probe. While strain to strain variation in restriction maps of specific genes is not unique and has been reported in N. gonorrhoeae previously, the existence of such restriction site polymorphism among iga-2 genes contrasts strongly with the lack of such variation among iga-1 genes. The basis for this lack of diversity among the iga-1 genes is under further investigation.

DNA Restriction Enzymes↗