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R B Raybourne

Publications and source records attributed to R B Raybourne.

At least 37 records · Page 2Linked to original sources
PubMed1994

Testing the importance of each residue in a HLA-B27-binding peptide using monoclonal antibodies.

When a peptide derived from histone 3.3 was incubated with mouse L cells transfected with HLA-B27, the cells became highly reactive with Ye-2, an anti-HLA-B27 mAb. The critical residues were analyzed by testing analogues in which each of the nine residues in the peptide was consecutively substituted by 19 other amino acids. The conclusions were separately verified using a different HLA-B27-positive cell line. The ability of some of these peptides to bind to HLA-B27 was also assayed by their ability to stabilize HLA-B27 in a mutant cell line which required HLA-B27-binding peptides to express HLA-B27 at 37 degrees C. These experiments showed that in P4, P5, P6, P7, P8, and P9, all 20 different amino acids could be substituted without eliminating the ability of the analogues to bind to HLA-B27. The residues which were responsible for the HLA-B27-peptide complex reacting with the Ye-2 antibody were P8 and P9. The latter might mediate its effect by altering either the surface conformation of the closely associated HLA-B27 heavy chain or the conformation of the peptide itself.

Amino Acid Sequence↗
PubMed1994

A monoclonal antibody that recognizes HLA-B27 in the context of peptides.

The T2 mutant cell line is unable to load peptides into the MHC class I Ags inside the cells. These "empty" MHC class I Ags are not expressed on the cell surface unless the cells are cultured at low temperatures. Expression will occur at 37 degrees C only in the presence of peptides that bind to and stabilize the class I Ags. T2 cells transfected with the B*2705 gene were tested with a panel of anti-HLA-B27 mAb. Two of the antibodies, ME1 and KS3, reacted with the "empty" HLA-B27 expressed at low culture temperatures. Three antibodies, B27.M1, B27.M2, and Ye-2, were unreactive with these "empty" HLA-B27. The cells were then incubated with a panel of HLA-B27-binding peptides. One of the antibodies, Ye-2, became reactive when the cells were incubated with a peptide derived from HIV gp120 and to a less degree with a peptide derived from histone H3.3. Mouse L cells transfected with the B*2705 and the human beta 2m genes also reacted very poorly with B27.M1, B27.M2, and Ye-2. Those two peptides were also able to induce high increase in Ye-2 reactivity. Alternately, increase in Ye-2 reactivity was also observed when the L cells were incubated with IFN-gamma or TNF-alpha. These experiments indicate that the Ye-2 anti-HLA-B27 mAb recognizes HLA-B27 in the context of certain residing peptides either added exogenously or expressed endogenously. The B27.M1 and B27.M2 antibodies might share similar characteristics.

Amino Acid Sequence↗
PubMed1994

Imipramine reduces the local inflammatory response to carrageenin.

Imipramine was administered chronically to LEW/N, outbred and F344/N rats which were then exposed to the aseptic irritant carrageenin in order to determine whether the decreased hypothalamic expression of CRH m-RNA previously shown to be associated with imipramine affects peripheral immune processes. Both LEW/N and outbred but not F344/N rats had vigorous inflammatory responses to carrageenin, and imipramine was associated with significant decreases in the local cellular inflammatory response to carrageenin. Imipramine was also associated with changes in the expression of peripheral blood cell MHC class II expression in LEW/N and outbred rats. These results suggest that at doses comparable to those used clinically imipramine has significant effects on response to an inflammatory stimulus.

Animals↗
PubMed1994

Serum antibodies from patients with ankylosing spondylitis and Reiter's syndrome are reactive with HLA-B27 cells transfected with the Mycobacterium tuberculosis hsp60 gene.

HLA-B27-related arthritis is probably mediated by an immune response against HLA-B27 complexed with peptides derived from proteins of arthritis-causing bacteria. Immunogenic proteins with a high degree of homology among bacteria, such as in the hsp60 family, are likely candidates. To create such complexes experimentally, we transfected an HLA-B27 cell line with the Mycobacterium tuberculosis hsp60 gene. Because of previous observations that HLA-B27-peptide complexes can be distinguished by antibodies, we tested the transfected cell line with a panel of sera from 24 HLA-B27+ arthritis patients. Significant antibodies were detected in at least eight of the sera. Several cell lines and peptides were used as negative controls to ensure that the antibody reactivity was specific to HLA-B27-peptide complexes. A panel of nine peptides derived from the sequence of the Mycobacterium hsp60 were synthesized and tested. At least three were identified as being responsible for the serological activities.

Amino Acid Sequence↗
PubMed1994

Bacterium-host cell interactions at the cellular level: fluorescent labeling of bacteria and analysis of short-term bacterium-phagocyte interaction by flow cytometry.

Flow cytometry is a potentially powerful tool for analyzing the interactions of facultative intracellular bacteria and macrophages on a cellular level, particularly when fluorochromes are used to label the bacteria. We labeled Listeria monocytogenes and Salmonella typhimurium with a lipophilic dye, PKH-2, and used flow cytometry to investigate phagocytosis by J774A.1 cells and short-term bacterial survival. Labeled and unlabeled bacteria were identical in terms of viability, growth kinetics, and survival within macrophages, although recovery per macrophage was much greater for L. monocytogenes than for S. typhimurium. Using L. monocytogenes as a prototypical facultative intracellular bacterium, we estimated bacterial survival during phagocytosis on the basis of linear fluorescence measurements of infected J774A.1 cells and recovery of L. monocytogenes from sorted cells. The lower percentage of surviving L. monocytogenes in macrophages containing higher bacterial loads indicated the accumulation of nonviable bacteria within phagocytes. Removal of the external source of viable bacteria by washes and gentamicin treatment reduced the percentage of surviving intracellular L. monocytogenes to a baseline level, and all baseline levels were similar, regardless of bacterial load. Listeria enrichment recoveries, derived from individually sorted J774A.1 cells, demonstrated the heterogeneity of macrophages in intracellular bacterial survival, especially within heavily infected cells. These results indicated that survival of L. monocytogenes was dependent on the adaptations of a small fraction of bacteria within a population of macrophages which permit intracellular growth.

Adaptation, Physiological↗
PubMed1993

Pathological and immunological effects of ingesting L-tryptophan and 1,1'-ethylidenebis (L-tryptophan) in Lewis rats.

The eosinophilia-myalgia syndrome (EMS) has been associated with ingestion of L-tryptophan (L-TRP) produced by a single manufacturer. Epidemiological data implicated 1,1'-ethylidenebis (L-tryptophan) (EBT) (peak 97 or peak E) as a possible etiologic agent. We showed previously that Lewis rats treated with the L-TRP implicated in EMS develop fasciitis and perimyositis similar to those seen in human EMS. We now report the pathology associated with the treatment of Lewis rats with synthetic EBT and/or L-TRP. All animals treated for 6 wk with case-associated L-TRP or EBT developed significant myofascial thickening, compared with animals in the vehicle control and control L-TRP groups. However, even those animals receiving the control L-TRP showed a mild but significant increase in the thickness of the myofascia, compared with vehicle-treated control animals. All animals except vehicle controls also exhibited significant pancreatic pathology, including fibrosis and acinar changes. Only animals treated with case-associated L-TRP for 6 wk showed evidence of immune activation with increased frequency of CD8, Ia, and IL-2 receptor-positive cells in the peripheral blood. Animals receiving L-TRP or EBT for < 6 wk did not show significant differences in myofascial thickness, although these animals did show pancreatic acinar changes. Although these results demonstrate for the first time the pathological effects of EBT, they do not rule out the possibility that other impurities in the EMS-case-associated L-TRP may also contribute to some of the features of EMS.

Animals↗
PubMed1991

The nucleotide and deduced amino acid sequence of the cationic 19 kDa outer membrane protein OmpH of Yersinia pseudotuberculosis.

The OmpH proteins of enteric bacteria are recently described, small (16 kDa), cationic outer membrane proteins. Because a Yersinia pseudotuberculosis cell envelope protein of this size has been found to cross-react serologically with the human histocompatibility antigen HLA-B27 (B*2701), the sequence of Y. pseudotuberculosis OmpH was determined by sequencing the gene region which encodes mature OmpH. A protein consisting of 143 amino acid residues was found. It was 96% homologous with the OmpH of Y. enterocolitica and 62% homologous with that of Escherichia coli. Two separate OmpH regions had sequence similarity with B*2701; they were identical in both Yersinia species.

Amino Acid Sequence↗
PubMed1990

Monoclonal antibodies against an HLA-B27-derived peptide react with an epitope present on bacterial proteins.

The role of molecular mimicry in the spondyloarthropathies was investigated with respect to the epitopes involved. mAb were produced against a synthetic peptide whose sequence was derived from a polymorphic region of the HLA-B27 molecule (amino acids 63-83). Two antibodies (J7F2 and H2B6) were selected for study on the basis of their ability to react with bacterial envelope proteins (ELISA) and B27-positive cells (immunofluorescence). J7F2 reacted preferentially with B27-positive cells and neither antibody reacted with MHC class I negative cells. Based on SDS-PAGE blot analysis of bacterial envelope proteins, the pattern of reactivity for both antibodies (against 36- and 19-kDa proteins) was the same as that for a third monoclonal produced against bacterial envelope and reactive with B27-positive cells. This apparent epitope similarity was investigated by using synthetic peptides to inhibit binding of the monoclonals. The B27 synthetic peptide and a smaller peptide derived from it were efficient inhibitors of antipeptide and antibacterial antibody binding to bacterial Ag and B27-positive cells. These studies provide insight into the molecular basis of cross-reactivity between bacterial proteins and MHC class I molecules.

Amino Acid Sequence↗
PubMed1990

Demonstration of cross-reactivity between bacterial antigens and class I human leukocyte antigens by using monoclonal antibodies to Shigella flexneri.

Bacterial envelope proteins which share immunodeterminants with the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 may invoke spondyloarthritic disease through the process of molecular mimicry in patients expressing this phenotype. Monoclonal antibodies generated by the immunization of BALB/c mice with envelope proteins of Shigella flexneri type 2a were tested for reactivity against cultured lymphoblastoid cell lines of defined HLA phenotype. As measured by flow microfluorometry, four immunoglobulin M monoclonal antibodies reacted preferentially with HLA-B27-positive lymphocytes (HOM-2, MM) as compared with a B27-loss mutant line (1065) or cells lacking major histocompatibility complex class I antigen (Daudi, K562). Monoclonal antibodies also reacted with mouse EL-4 cells transfected with and expressing the HLA-B7 gene. Western immunoblot analysis of isolated enterobacterial envelopes demonstrated that the reactive epitope was present on bacterial proteins with an apparent relative molecular mass of 36 and 19 kilodaltons. The structural basis for the cross-reactivity of bacterial antigen and HLA-B27 appeared to reside in the portion of the HLA molecule that is responsible for allotypic specificity (amino acids 63 through 83), since monoclonal antibodies were positive by enzyme-linked immunosorbent assay with synthetic polypeptides corresponding to this segment.

Amino Acid Sequence↗
PubMed1990

Demonstration of shared epitopes between bacterial proteins and HLA class-I proteins using monoclonal antibodies.

Monoclonal antibodies were used to identify the presence of epitopes on bacterial proteins which are cross-reactive with HLA-B27. Anti-HLA-B27 monoclonals, B27M2 and YE-2 reacted with a major protein of approximately 35 kDa, which was found to be OMPA. On some strains a 19 KDa protein was also seen. Anti-Shigella flexneri monoclonals were also developed which reacted with a 36 kDa protein and a 19 kDa protein. The 36 kDa protein was OMPF. These antibodies also reacted with a synthetic peptide representing amino acids 63-83 of the B*2705 sequence. These results support a potential role for molecular mimicry in the spondyloarthropathies.

Antibodies, Monoclonal↗
PubMed1989

Induction of ornithine decarboxylase in the stomach mucosa of swine with NaCl or 12-O-tetradecanoylphorbol-13-acetate.

The effect of sodium chloride and 12-O-tetradecanoylphorbol-13-acetate on ornithine decarboxylase (ODC) activity in gastric mucosa of miniature swine was investigated as a model for gastric inflammation. The level of the enzyme was lower in the pylorus than in the fundic or cardiac regions of the stomach in untreated animals. Treatment with sodium chloride at 1 g/kg produced large increases in all three regions, with the greatest relative increase in the pylorus. Treatment with sodium chloride at 0.25 g/kg or the phorbol ester at 2.0 mg/pig produced significant but less dramatic increases. ODC activity in control and treated mucosal extracts was inhibited by the specific ODC inhibitor difluoromethylornithine. Most of the enzyme activity was associated with superficial and exfoliated cells that could be scraped from the mucosal surface. No increase in the inflammatory mediator leukotriene B4 was observed in the mucosal extracts. Ornithine decarboxylase appears to be a useful enzymatic marker for the regenerative events that occur after tissue damage and may correlate with the putative tumor-promoting function of sodium chloride in gastric tissues.

Animals↗
PubMed1989

Synthetic oligonucleotide probes complementary to the HLA-B variable region hybridize with Klebsiella pneumoniae.

Three oligonucleotide probes complementary to base sequences of the HLA-B variable region were used to probe clinical and foodborne isolates of Klebsiella spp. by DNA colony hybridization. One oligonucleotide (RR-3) corresponding to amino acid residues 66-74 of the HLA-B27.1 sequence and one corresponding to residues 66-74 of the HLA-B7 sequence (RR-5) hybridized with K. pneumoniae under conditions of high stringency. A genomic library was constructed using K. pneumoniae K43 chromosomal DNA, and a 1 kb PstI restriction fragment was found to contain the sequence associated with specific binding of the oligonucleotide probes. After purification and radiolabeling, the cloned fragment hybridized with 96.2% of the K. pneumoniae isolates by DNA colony hybridization. These results confirm the presence of sequence similarities between bacterial DNA and the MHC Class I variable region.

Antigens, Bacterial↗
PubMed1988

Reaction of anti-HLA-B monoclonal antibodies with envelope proteins of Shigella species. Evidence for molecular mimicry in the spondyloarthropathies.

Immunologic cross-reactivity between enteric bacteria and the HLA-B27 protein may play a role in the etiology of Reiter's syndrome and reactive arthritis. The reactivity of two anti-B27 mAb (B27M1 and B27M2) with envelope proteins of Shigella flexneri isolated from Reiter's syndrome patients was studied by Western blot analysis. Proteins with an apparent Mr of approximately 36 and 23 kDa reacted with both mAb in ascites. mAb against related HLA class I Ag B7 and B40 did not react with the 23 kDa protein. Relatively high concentrations of antibody were required for reactivity, suggesting a low affinity interaction. Additional evidence for cross-reactive epitopes was obtained by ELISA against whole envelope and by using unsolubilized envelope to inhibit binding of M1 and M2 to B27-positive cell lines, as measured by quantitative flow microfluorimetry. The presence of cross-reactive proteins was not related to the presence of the intact virulence-associated plasmid or the invasive phenotype. Two Shigella sonnei isolates not implicated as causative agents of Reiter's syndrome or reactive arthritis showed a similar pattern of cross-reactivity. These results indicate that cross-reactive epitopes may be present on "arthritogenic" bacteria, but their presence is not a unique feature of such strains and is not the sole factor in induction of arthritis in B27-positive individuals.

Animals↗
PubMed1988

Intestinal anisakiasis. A case diagnosed by morphologic and immunologic methods.

The diagnosis of the fourth reported case of intestinal anisakiasis in the United States was based on the morphologic characteristics of the worm in histologic sections of resected ileum. Detection of antibodies to the worm by a radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA), and an immunofluorescent antibody assay (IFA) support the diagnosis. IgE antibodies in patient serum reacted specifically to larval Anisakis antigens but not to larval Ascaris antigens in the RAST. IgG and IgM antibodies to larval Anisakis antigen could be detected by ELISA up to six months after infection. IFA findings demonstrated that patient serum recognized the excretory-secretory products produced by the worm and showed their presence in vivo. These products appear to originate from the excretory pore and dorsal esophageal gland of the larval parasite.

Adult↗
PubMed1986

Invasive anisakiasis. A case report from Hawaii.

A confirmed case of penetration into the stomach wall by an anisakine nematode, to our knowledge the first in the United States, is reported. A viable third-stage larva of Anisakis simplex was removed from a woman in Hawaii who had eaten fresh, raw salmon. A serodiagnostic test showed the serum of the patient to be positive for A. simplex-specific IgE at days 11 and 60 postinfection. Our results indicate that eating fresh, raw salmon exposes consumers to a significant health risk.

Adult↗