Biomedical subjects

A Dybwad

Publications and source records attributed to A Dybwad.

12 recordsLinked to original sources

PubMed2003

Probing the specificity of human myeloma proteins with a random peptide phage library.

Human myeloma proteins (HMPs) from 10 patients with multiple myeloma (MM) were used to affinity-select peptides from a random phage-display peptide library. Binding peptides were identified for the 10 analysed antibodies (eight, immunoglobulin G (IgG), and two, immunoglobulin A (IgA)). The specificity of the binding was confirmed by competitive experiments using phages and chemically synthesized peptides. Interestingly, some phage-displayed peptides were immuno-selected with HMPs isolated from different patients. Sequence alignments and homology searches revealed a significant homology with human proteins (e.g. neural cell adhesion proteins) and pathogen-derived proteins (e.g. herpes simplex virus capsid proteins). The selected peptides could be useful as targeting agents for myeloma cells expressing surface immunoglobulins.

Amino Acid Sequence↗

PubMed2000

Profiling the immune responses in patient sera with peptide and cDNA display libraries.

Peptide and cDNA phage display libraries can be used to determine the specificities of antibodies present in the whole sera of patients where information about the parental antigens is unknown. In this respect, patient serum antibody binding ligands have been identified. Such ligands would facilitate the design of diagnostic assays and therapeutic vaccines. In the case of cancer, this novel technology is expected to improve our understanding of the immune responses against tumour cells and to discriminate between autoantigen and true tumour specific antigens. Here, we review work on how peptide and cDNA phage display libraries can be used to address the specificity of the immune responses in patients with autoimmune diseases and cancer.

Animals↗

PubMed1997

Probing for cerebrospinal fluid antibody specificities by a panel of random peptide libraries.

In a state of health the central nervous system is nearly devoid of macrophages, T and B cells. However, such cells are required to initiate the inflammation associated with multiple sclerosis (MS). An important issue in understanding the pathogenesis of such conditions is to fully define the T and B cell specificities. Using random peptide libraries, we have analysed the cerebrospinal fluid antibody specificities within one oligoclonal "band" from a patient with MS. The selected peptides from a 6-mer library revealed two peptide motifs S (S/T/Q) (R/S) (N/G) FP and PRn (G/P) FF. Interestingly, the first motif was also selected from a 15-mer library, while the second motif was selected from the 9-mer library. In addition, distinct but structurally related peptides to the mentioned motifs were also selected. Surprisingly, a SwissProt search with these motifs revealed a significant linear homology with collagen proteins, the 68 kDa neurofilament protein, versican and other proteins from viruses such as herpes simplex virus, human cytomegalovirus and human papillomavirus. Thus, the observation that antibodies present within one oligoclonal immunoglobulin band would recognize peptides, some that might be related by only the structure, raises the question of the involvement of multiple pathogens in MS.

Adolescent↗

PubMed1996

Selection of ligands for polyclonal antibodies from random peptide libraries: potential identification of (auto)antigens that may trigger B and T cell responses in autoimmune diseases.

The development of random peptide libraries has increased our possibility for analyzing the structural features involved in binding events. Recently, reports have appeared in which these libraries have been successfully used to investigate binding properties of homogeneous proteins such as monoclonal antibodies. However, a more general application of peptide libraries would be the use of polyclonal sera or fluids from patients with autoimmune diseases in biopanning experiments. This would subsequently allow the identification of (auto)antigen leads responsible for the initiation and/or perpetuation of the immune response in these patients. Moreover, the strategy allows the structural characterization of autoantibody specificities in body fluids that have been produced in vivo without the introduction of bias due to preferential B cell growth under in vitro conditions. The application of this novel strategy for selection of antibody ligands for polyclonal sera as well as to study the nature of immune responses to defined proteins will be discussed with emphasis on the development of peptide reagents for diagnostic and vaccine use.

Amino Acid Sequence↗

PubMed1996

Increased serum and synovial fluid antibodies to immunoselected peptides in patients with rheumatoid arthritis.

OBJECTIVES: To investigate the role of potential immunoselected phages displaying random peptides in addition to possible antigen leads in rheumatoid arthritis (RA) by assaying the levels of synovial fluid (SF) and serum antibodies to synthetic peptides. METHODS: Serum and SF antibodies from patients and controls were measured using an enzyme linked immunosorbent assay (ELISA). RESULTS: Sera and SF from RA patients reacted significantly more strongly to a 12 amino acid peptide, EFHELGDIAIAA, that shares a significant homology with collagen type IX, than did SF and sera from control groups (p < 0.0209 and p < 0.0115, respectively). In addition, the humoral responses to a 15 amino acid peptide, GGYGDGGAHGGGYGG, derived from the glycine-rich cell wall protein (GRP) 1.8, and to a 16 amino acid synthetic peptide, LGSISESRRALQDSQR, derived from the Proteus haemolysin protein were significantly stronger in RA patients compared with healthy individuals (p < 0.0001 and p < 0.0011, respectively). CONCLUSION: Our data indicate that peptide phage libraries can be used as tools for the identification of the (auto)antigen leads that may be responsible for the initiation, perpetuation, or both, of the immune response in patients with RA.

Amino Acid Sequence↗

PubMed1995

Structural characterization of peptides that bind synovial fluid antibodies from RA patients: a novel strategy for identification of disease-related epitopes using a random peptide library.

Phage epitope library technology has become a powerful tool for identifying determinants recognized by homogenous antibodies. Screening of human sera or fluids with random peptide phage libraries is a novel approach that can dissect common features at the amino acid level in individuals infected by the same etiological agent(s). In this study we have analyzed the specificity of antibodies in synovial fluids (SF) obtained from patients with rheumatoid arthritis (RA). We found that a high proportion of the peptides binding SF antibodies differ from those binding serum antibodies, suggesting that synovial B cells are expanded as a result of local antigen stimulation. When all the selected phages (enriched library) that bind the SF antibodies from a seropositive RA patient were further screened by the use of different SF antibodies with or without rheumatoid factor activity, we identified common SF antibody specificities (IRRSETPRA, RRRVRNSDT, PVSSTRGNM). Antibodies against these common specificities are also found in SF from other RA patients. Taken together, the present results open the possibility of defining the entire set of synovium antibody specificities and also common SF specificities between RA patients.

Amino Acid Sequence↗

PubMed1995

Peptide phage libraries can be an efficient tool for identifying antibody ligands for polyclonal antisera.

We have examined the potential of isolating ligands for polyclonal antibodies from a nanopeptide phage library. The library was screened with a rabbit polyclonal antiserum raised against a synthetic peptide (ALWFRNHFVFGGGTKVT). Following screening, the positive phages were tested in an ELISA for their reactivity with the antiserum. Phages that showed positive reactivity with the antiserum compared with a normal rabbit serum were selected and their displayed peptides were determined. Among the 36 random positive clones, 31 clones carried the sequence AVFGGGTKL, PFFGGGSRA or APTGGSKRT that have a significant homology to the immunizing peptide. Five positive phages displayed the ATNIFIEGT sequence, which has no obvious linear homology with either the other selected peptides or with the peptide used for immunization. In contrast to the control peptide, the immunizing peptide inhibited binding of the antiserum to the peptide-displaying phages in a dose-dependent manner, thus demonstrating the specificity of the interaction. Furthermore, the rabbit B cell response to the peptide was found to be limited and focused on its C-terminal. Taken together, our data demonstrate the potential of random peptide phage libraries for defining epitopes for polyclonal antisera as well as for investigation of the nature of B cell responses to any given antigen.

Amino Acid Sequence↗

PubMed1995

Decrease in anti-Proteus mirabilis but not anti-Escherichia coli antibody levels in rheumatoid arthritis patients treated with fasting and a one year vegetarian diet.

OBJECTIVE: To measure Proteus mirabilis and Escherichia coli antibody levels in patients with rheumatoid arthritis (RA) during treatment by vegetarian diet. METHODS: Sera were collected from 53 RA patients who took part in a controlled clinical trial of fasting and a one year vegetarian diet. P mirabilis and E coli antibody levels were measured by an indirect immunofluorescence technique and an enzyme immunoassay, respectively. RESULTS: The patients on the vegetarian diet had a significant reduction in the mean anti-proteus titres at all time points during the study, compared with baseline values (all p < 0.05). No significant change in titre was observed in patients who followed an omnivorous diet. The decrease in anti-proteus titre was greater in the patients who responded well to the vegetarian diet compared with diet non-responders and omnivores. The total IgG concentration and levels of antibody against E coli, however, were almost unchanged in all patient groups during the trial. The decrease from baseline in proteus antibody levels correlated significantly (p < 0.001) with the decrease in a modified Stoke disease activity index. CONCLUSION: The decrease in P mirabilis antibody levels in the diet responders and the correlation between the decrease in proteus antibody level and decrease in disease activity supports the suggestion of an aetiopathogenetic role for P mirabilis in RA.

Antibodies, Bacterial↗

PubMed1995

Evidence for monoclonal expansion of synovial T cells bearing V alpha 2.1/V beta 5.5 gene segments and recognizing a synthetic peptide that shares homology with a number of putative autoantigens.

A peptide of 15 amino acids derived from the cereal glycine-rich cell wall protein (GRP), sharing a significant homology with Epstein-Barr virus nuclear antigen-1 (EBNA-1), fibrillar and procollagen, stimulated synovial fluid (SF) T cells from juvenile (JRA) and adult (RA) rheumatoid arthritis patients. An overexpression of the V alpha 2 gene family was found in the SF from patients who responded significantly to the peptide. To investigate in more detail the SF T-cell responses to the GRP peptide, we established peptide-specific T-cell lines and clones from a DR8+ positive JRA patient with pauciarticular form. The T-cell clones were phenotyped as T-cell receptor (TCR)alpha beta+/CD4+ and their clonality was investigated by polymerase chain reaction (PCR) and flow cytometric analysis. TCR sequences from different clones demonstrated that the clones were identical and used the V alpha 2.1/J alpha 6 combined with V beta 5.5/J beta 2.7 gene segments. Interestingly, direct sequencing of the V alpha 2 family PCR product obtained from cDNA prepared from freshly isolated SF mononuclear cells identified the same TCR sequence as that used by the clones, suggesting the monoclonality of SF CD4+ T cells bearing V alpha 2.1/J alpha 6 gene products. The present data suggest a recruitment and expansion of a SF T-cell subpopulation, and also support the hypothesis that autoimmune diseases can be triggered by protein epitopes with crucial amino acids homologous to self-proteins.

Amino Acid Sequence↗

PubMed1994

Characterization of naturally occurring autoantibodies against tumour necrosis factor-alpha (TNF-alpha): in vitro function and precise epitope mapping by phage epitope library.

Naturally occurring autoantibodies against cytokines exist in the sera of patients with autoimmune diseases as well as in the sera of normal individuals. We report here that affinity-purified autoantibodies against human TNF-alpha from one rheumatoid arthritis (RA) patient inhibited the cytotoxic effect of TNF-alpha on the mouse fibrosarcoma cell line WEHI 164, by 50%. In an attempt to predict the autoantibodies' recognition site on TNF-alpha protein we screened a random nanopeptide phage library with the affinity-purified TNF-alpha autoantibodies. Among 63 random selected clones, 46 clones carried the sequence ASSLLASSP, NSSPYLNTK or PQSPGSSFP. Frequency analysis of the relative occurrence of the 20 amino acids in the nanopeptides displayed by 50 random bacteriophages picked before selection and 63 after selection to bind to TNF-alpha autoantibodies indicated that proline (P < 0.0003) and serine (P < 0.04) are involved in the binding of the autoantibodies to the phages. Furthermore, we demonstrated that three synthetic peptides (ASSLLASSP, NSSPYLNTK and PPLKPVIDE) displayed by the selected phages reduced the binding of the autoantibodies to TNF-alpha protein by 50%. Interestingly, the sera of mice (BALB/c) immunized with phages displaying ASSLLASSP and NSSPYLNTK peptide showed an anti-TNF-alpha response as detected by ELISA. This response was not found in mice immunized with the wild type phage. Thus, the recombinant phages selected from the phage libraries could be used as carrier for immunization, and therefore as a tool for vaccine development. This work sets the stage for experiments designed to isolate ligands for protective antibodies.

Antibody Specificity↗

PubMed1993

Identification of new B cell epitopes in the sera of rheumatoid arthritis patients using a random nanopeptide phage library.

A random nanopeptide phage library was used to screen a pool of immunoglobulin fractions obtained from rheumatoid arthritis (RA) patients. After three rounds of panning, random individual phages were selected by their capacity to react with individual sera from RA patients. By sequencing the inserts corresponding to the peptides displayed on the surface of the phages, we found that phages displaying particular peptides were overrepresented in the selected libraries. The peptides displayed by these phages were: pep1 = Ala-Asp-Gly-Gly-Ala-Gln-Gly-Thr-Ala; pep2 = Pro-Gly-Pro-Ser-Arg-Ala-His-Phe-Leu; pep3 = Leu-Ser-Ser-Arg-Glu-Pro-Gln-Ala-Arg; pep4 = Arg-Leu-Thr-Arg-Glu-Leu-Tyr-Ala-Gln and pep5 = Tyr-Thr-Gln-Lys-His-Gln-Ala. The percentage of sera positive for pep1 was higher in RA patients as compared to the normal adults (p < 0.0004) and the reacting antibody was mainly of IgG isotype. The specificity of binding to the phage displaying pep1 was confirmed by competition experiments using both isolated phages and a synthetic peptide. Interestingly, a mutated phage displaying only Ala-Asp-Gln-Gly-Thr-Ala had no significant reactivity with the sera, indicating that the amino acids (Gly-Gly-Ala) of pep1 are the vital for the binding. Taken together this study demonstrates that it is possible to select specific ligands from a random phage library using sera from RA patients. In addition, this approach could be useful for identifying peptide antigens that might be part of causitive agents in autoimmune diseases.

Amino Acid Sequence↗

PubMedDate not supplied

Probing antinuclear antibody specificities by peptide phage display libraries.

OBJECTIVE: To uncover the specificities of autoantibodies to nuclear proteins (ANA) in patients with juvenile rheumatoid arthritis (JRA). METHODS: Peptide ligands for ANA were selected by panning random peptide phage display libraries on antibodies binding to HEp-2 cells. Positive phage clones were identified by the immunoscreening technique. RESULTS: Groups of peptides were identified, some of which share the core motifs of KTTTnPY, RVADnL/I or RnNSPL. Perinuclear and nuclear staining of HEp-2 cells were obtained with patient serum antibodies binding to the phage displaying the core peptide motifs. In contrast, no significant reactivity was seen with the antibodies binding to the wild type phage. Antibodies to the phage displaying peptides containing some of the core motifs were detected more frequently in ANA-positive as compared to ANA-negative JRA patients. Homology search with the selected core motifs revealed a significant homology with a number of human nuclear proteins and proteins from potential infectious agents that could serve as trigger in the breakdown of tolerance. CONCLUSION: Panning of phage display libraries on antibodies reacting with cellular structures can lead to the identification of their specificities. Thus, the peptide epitopes reported here constitute additional information that may lead to the development of diagnostic tests and the identification of the parental antigens that initiated the B cell responses in patients with JRA.

Amino Acid Sequence↗