Biomedical subjects

J B Natvig

Publications and source records attributed to J B Natvig.

At least 55 records · Page 3Linked to original sources

PubMed1994

The binding of synovial tissue-derived human monoclonal immunoglobulin M rheumatoid factor to immunoglobulin G preparations of differing galactose content.

There are several sites on IgG Fc that have been reported to be the epitopes for binding rheumatoid factors (RF). It is now established that there are alterations in the oligosaccharides on IgG from patients with rheumatoid arthritis and it has been suggested that these changes may enhance immune complex and cryoglobulin formation. We have used a series of IgG preparations differing in their content of oligosaccharide chains lacking galactose from 18 to 86% to determine whether changes in sugar content affect the binding of rheumatoid factor. Five of 16 monoclonal rheumatoid factors prepared from synovial tissue, from patients with juvenile or adult rheumatoid arthritis, bound better to IgG which was deficient in galactose. Six of the 16 rheumatoid factors from the same patients bound independently of the galactose content. Four of the 16 rheumatoid factors could not be absolutely grouped in this manner but seemed to demonstrate a preference for agalactosyl IgG. One rheumatoid factor bound better to fully galactosylated IgG. There was an association between enhanced binding to galactose-deficient IgG and monoreactivity and a very strong association between the functional affinity of the rheumatoid factors and the dependent binding.

Antibodies, Anti-Idiotypic↗

PubMed1994

Three new cross-reacting idiotopes as markers for the products of two distinct human VH3 genes expressed in the early repertoire.

This article describes characterization of three new cross-reacting idiotopes, as recognized by mouse MoAbs, on human antibodies utilizing VH3 genes that are expressed in the early repertoire. Two of the mouse MoAbs (3H7 and 3H1) were raised against a human MoAb utilizing the DP47 (VH26) VH3 gene, whilst the third (7B4) was raised against a DP46 (GLSJ2) gene product. Evidence for the anti-idiotypic specificity of the mouse MoAbs was provided by their reactivity with the immunizing IgM, but not with Fc mu, and by their specific inhibition of the binding between each immunizing antibody and its antigen. The three anti-idiotypic MoAbs were shown to be VH-specific reagents by the independence of their reactivity upon the L-chain type, or the antigenic specificity of the human MoAbs tested. Specificity of each mouse MoAb for VH3 gene-products was demonstrated by its sole cross-reactivity with VH3 proteins. Each anti-Id had a different reactivity pattern with a panel of MoAbs utilizing different VH3 genes. By relating the VH sequences of the tested VH3 proteins to their germline counterparts, 3H7 and 3H1 appeared to be specific for DP47-encoded proteins, although 3H1 had weak cross-reactivities with a few other VH3 gene-products. 7B4 appeared to be specific for antibodies utilizing DP46-related genes. Both 3H7 and 3H1 were also completely different to B6 and D12, two previously described MoAbs that also recognize VH3 proteins. Although 7B4 was similar to B6 and D12 in its binding to DP46-related gene products, B6 and D12 additionally recognized non DP46-related proteins and were thus different to 7B4.

Animals↗

PubMed1994

Human monoclonal rheumatoid factors: incidence of cross-reactions with tissue components and correlation with VH gene usage.

Human monoclonal antibodies with rheumatoid factor (RF) activity, derived from lymphocytes from the synovial tissue of rheumatoid arthritis (RA) patients and the peripheral blood of healthy individuals were examined for cross-reactivity with tissue and cellular antigens. The majority of IgM RF from RA patients (68%) showed reactivity with at least one component, and were frequently multispecific. A very significantly smaller proportion (28%) of the RF derived from healthy individuals demonstrated reactivities against tissue/cellular antigens (P = 0.004). RF from RA patients most commonly reacted with gastric glands (61%), nuclei (50%) and smooth muscle (50%), whereas RF from healthy donors most commonly reacted with gastric glands (20%), smooth muscle (16%), endothelium (16%) and glomeruli (16%). The most striking difference between the two groups was the reactivity with nuclear components, demonstrated by 50% of the RA RF, but by none of the healthy donor RF. As the two groups of antibodies share the same specificity for IgG Fc, but show differences in variable region segment usage, we investigated the relationship between VH gene usage and tissue/cell cross-reactivity using these antibodies and anti-blood group antibodies. Antibodies using VH3 or VH4 gene segments showed a very significantly greater frequency of tissue/cell reactions than those using VH1 (P = 0.0095 and 0.0004 respectively).

Antibodies, Monoclonal↗

PubMed1993

Mapping studies reveal unique epitopes on IgG recognized by rheumatoid arthritis-derived monoclonal rheumatoid factors.

We have used chimeric IgG antibodies and their genetically engineered variants prepared by a combination of site-directed mutagenesis and exon exchange to define the structure(s) on IgG recognized by monoclonal rheumatoid factor (RF) autoantibodies from rheumatoid arthritis (RA) patients. Nineteen RF produced by EBV-transformed cell lines from the synovium or blood of RA patients were analyzed. Their binding patterns differ significantly from those seen with RF obtained from patients with Waldenstrom's macroglobulinemia (WMac). Half of the RA-derived RF bound IgG1, 2, and 4, but not 3 (Ga specificity), the common pattern in WMac. However, heterogeneity in fine specificity within the Ga reactivity pattern was observed. Moreover, seven others bound all four IgG subclasses, a pattern observed for only one WMac-derived RF from a patient who also had RA. Three RF had subclass specificities unlike any observed with WMac-derived RF. Most RA-derived RF bound IgG at a discontinuous epitope comprised of residues from both the CH2 and CH3 H chain constant regions. However, unlike any WMac-derived RF, one RA-derived RF bound IgG in CH2, another in CH3, and a third at an undetermined site outside of the CH2-CH3 interface. Some RA-derived RF bound aglycosylated IgG4 less well than glycosylated IgG4, suggesting that the carbohydrate moiety was important in establishing their binding epitope in CH2. These studies demonstrate that the repertoire of RF expressed by RA patients contains some unique binding specificities for IgG epitopes not found among our panel of WMac-derived RF. Our results therefore call into question whether WMac-derived RF with their limited diversity are appropriate models for disease-related RF. In addition, RF with their multiple specificities can serve as probes of antibody structure.

Animals↗

PubMed1993

Differentiation of human lambda I variable regions with a monoclonal antibody to a defined, germ-line-encoded idiotope correlates with V lambda Ia chemical structure.

This article describes the characterization of a mouse monoclonal anti-idiotypic antibody (1H7B) prepared against a human monoclonal rheumatoid factor (RFSJ2) whose L chain utilized a V lambda I subgroup gene. The mAb 1H7B reacted with 6 of the tested 12 human V lambda I proteins, as well as with a newly produced lambda mAb whose V lambda gene usage has not as yet been determined. Because six of the 1H7B-positive mAb were heterogeneous with respect to both their VH gene utilization and antigenic specificity, and because mAb 1H7B did not react with any of the tested 43 kappa or 12 lambda proteins belonging to various subgroups other than V lambda I, mAb 1H7B appeared to be a V lambda I subgroup-specific reagent. The L chain specificity of mAb 1H7B was confirmed by Western blotting, and the inhibition of RFSJ2 binding to human Fc gamma by 1H7B provided additional evidence for the V region specificity of mAb 1H7B. The 11 sequenced V lambda I proteins used in this study were assigned to sub-subgroups by comparison with the previously published germ-line V lambda Ia, V lambda Ib, and V lambda Ic sequences. The mAb 1H7B only appeared to recognize V lambda Ia proteins as it reacted with five of the seven V lambda Ia, but not with three V lambda Ib or with one V lambda Ic protein. Because mAb 1H7B reacted with at least one V lambda Ia sequence in germ-line configuration, it appeared to be a marker for V lambda Ia sub-subgroup germ-line gene(s). The idiotope recognized by 1H7B was localized to the first framework region by inhibiting its binding to RFSJ2 with a synthetic peptide to the 11-24 amino acid region of V lambda Ia L chains. Comparison of the V lambda 1-24 region sequence of RFSJ2 with those of the two 1H7B-negative V lambda Ia mAb revealed a single amino acid difference at position 17, suggesting that the idiotope recognized by 1H7B encompassed this position.

Amino Acid Sequence↗

PubMed1993

Synovial IgG rheumatoid factors show evidence of an antigen-driven immune response and a shift in the V gene repertoire compared to IgM rheumatoid factors.

We have established IgG rheumatoid factor (RF)-secreting hybridoma cell lines from the synovial tissues of three patients from whom we have previously characterized several IgM RF. The IgG RF bind human and rabbit IgG and form intracellular complement-fixing complexes indicative of a self association process in vivo. Nucleotide sequence analysis revealed that two IgG RF used VHIII gene segments, while one used a VHI gene segment. The VL gene usage consisted of a V kappa 1, a V lambda 2 and a V kappa 4/V kappa 6 hybrid, confirming our previous findings that many different VL genes can contribute to RF specificity. Although the IgG RF used VH genes from the same families that dominated the IgM RF response, two of the actual gene segments employed were not found among the IgM RF. In contrast to IgM RF, which in general were not very mutated, the IgG RF showed somatic mutations characteristic of an antigen-driven immune response.

Amino Acid Sequence↗

PubMed1993

Complementarity-determining region 2 is implicated in the binding of staphylococcal protein A to human immunoglobulin VHIII variable regions.

Staphylococcal protein A (SPA) has two distinct binding sites on human immunoglobulins. In addition to binding to the Fc region of most IgG molecules, an "alternative" binding site has been localized to the Fab region of human immunoglobulins encoded by heavy chain variable gene segments belonging to the VHIII family. Comparison of amino acid sequences of closely related SPA-binding and -non-binding proteins suggested that VHIII-specific residues in the second complementarity-determining region (CDR2) were likely responsible for SPA binding activity. Site-directed mutagenesis of a single amino acid residue in CDR2 converted an IgM rheumatoid factor which did not bind SPA to an SPA binder. These findings, therefore, locate a critical site involved in SPA binding to the CDR2 of human immunoglobulins encoded by VHIII family gene segments.

Amino Acid Sequence↗

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↗

PubMed1993

A high proportion of the V delta 1+ synovial fluid gamma delta T cells in juvenile rheumatoid arthritis patients express the very early activation marker CD69, but carry the high molecular weight isoform of the leucocyte common antigen (CD45RA).

We have previously shown that gamma delta T cells in the synovial compartment of patients with juvenile rheumatoid arthritis (JRA) express activation antigens (CD69 and HLA-DR) and that they are predominantly of the V delta 1 subset. In this study we have analysed the expression of activation antigens (CD69 and HLA-DR) and different isoforms of the leucocyte common antigen (CD45RO and CD45RA) on the V delta 1 and the V delta 2 subsets of gamma delta T cells in paired samples of synovial fluid and peripheral blood of nine patients with JRA, and in the peripheral blood of five children with idiopathic scoliosis. In the synovial fluid of children with JRA, there were significantly more V delta 1+CD69+ and V delta 2+CD69+ cells compared with the peripheral blood of the same patients. In contrast, however, in the synovial fluid the V delta 1 and the V delta 2 subsets differed with respect to the expression of the two isoforms of the leucocyte common antigen. The majority of the V delta 1+ cells expressed the high molecular weight isoform (CD45RA+) while most of the V delta 2+ cells carried the low molecular weight variant (CD45RO+) of this molecule.

Adolescent↗

PubMed1993

Human monoclonal IgG rheumatoid factors from the synovial tissue of patients with rheumatoid arthritis.

We have established IgG rheumatoid factor (RF)-secreting hybridoma cell lines from the synovial tissues of two patients (TS and SJ) with rheumatoid arthritis (RA) and one (KL) with the polyarticular form of juvenile rheumatoid arthritis (JRA). The IgG RF bind human and rabbit IgG and all except one form intracellular complement-fixing complexes indicative of a self-association process. The possibility of IgG RF for self association and immune complex formation is a feature thought to be important for the inflammatory processes in RA. Of the IgG RF-secreting cell lines established, three clones from patient SJ and one from patient KL are of the IgG1 kappa isotype while five clones from patient TS are of the IgG2 lambda isotype.

Antibody Specificity↗

PubMed1993

Rheumatoid inflammatory T-cell clones express mostly Th1 but also Th2 and mixed (Th0-like) cytokine patterns.

This study was performed in order to characterize whether T cells from rheumatoid synovial inflammation belong to the Th1- or Th2-like functional subsets. Cytokine production was studied in 26 CD4+ alpha beta+ and 2 CD8+ alpha beta T-cell clones from the synovial fluid, the synovial membrane and peripheral blood of 5 patients. Fifteen of the CD4+ clones were raised against various mycobacterial antigens and 11 CD4+ clones and 2 CD8+ clones were raised unspecifically using PHA and/or IL-2. The specificities of these clones are not known. In the mycobacterial antigen-specific group, all CD4+ alpha beta T-cell clones produced IFN-gamma at high levels, while the production of IL-4 was generally absent or low (< 1 ng/ml), consistent with a Th1-like profile. Some of these clones, however, also produced various amounts of IL-10 which has been regarded as a Th2 product but can be produced also in lower amounts by Th1 cells. One HSP-65-specific clone produced levels of IL-4 and IL-10 in the same order as that of IFN-gamma, thus appearing to be Th0-like. Among the 11 unspecific CD4+ clones, 7 showed a Th1-like pattern but with lower levels of IFN-gamma than the antigen-specific clones. However, three clones did not produce any IFN-gamma activity but produced IL-4 and one of them also produced distinct amounts of IL-10, compatible with a Th2-like pattern. In addition, one of the clones also showed an almost equally strong IFN-gamma and IL-4 production, thus most likely representing a Th0-like clone.

Adolescent↗

PubMed1993

Heterogeneity of the TCR repertoire in synovial fluid T lymphocytes responding to BCG in a patient with early rheumatoid arthritis.

T lymphocytes have been implicated in the pathogenesis of rheumatoid arthritis. Interestingly, many of the activated T cells isolated from the synovial fluid of individuals with rheumatoid arthritis react with antigens from Mycobacterium tuberculosis or BCG. This response is seen to a much lesser extent in the peripheral blood of these patients. To investigate the nature of the T-cell response to BCG in RA, we isolated T cells from the synovial fluid of a patient with early-stage rheumatoid arthritis, stimulated them with BCG and cloned by limiting dilution. Staining with monoclonal antibodies specific for different V beta gene families revealed a statistically significant greater proportion of synovial-derived T-cell clones expressing the V beta 8 gene family product compared with peripheral blood clones. While the antigen specificity of some of the clones could not be determined, several of the clones displayed distinct antigen reactivities. Sequencing the TCR beta chain genes of these T cells suggested that although the V beta 8 gene products appeared to be over-represented in these BCG-specific clones, each clone utilized distinct J beta gene segments and used N segment addition to different extents. In addition, no common motifs were identified in the beta chain CDR3s of the clones sequenced. Analysis of bulk cultured BCG-specific SF T cells and unstimulated peripheral blood T cells for V beta 8 gene expression also revealed a large amount of diversity within the CDR3 region. Thus, the T-lymphocyte response to BCG in this patient with early rheumatoid arthritis appears to be quite heterogeneous.

Adult↗

PubMed1992

Clonally related IgM rheumatoid factors undergo affinity maturation in the rheumatoid synovial tissue.

Using hybridoma technology we established a panel of human monoclonal rheumatoid factors (RF) from the synovial tissues of two patients with rheumatoid arthritis (RA), and one patient with polyarticular juvenile RA. Nucleotide sequence analysis of the V regions of these RF indicates that two independently derived antibodies from one of the RA patients are clonally related. One of these antibodies appears to be close to germ-line configuration, whereas the other has accumulated a total of 36 substitutions in both H and L chains. Measurements of the affinity for human IgG of the two RF show that the extensively mutated RF has 100-fold higher affinity for IgG than the RF close to germline. These findings indicate that IgM RF in RA can undergo affinity maturation and suggest that certain RF may be the product of an Ag-driven immune response.

Amino Acid Sequence↗

PubMed1992

Preformed ribozyme destroys tumour necrosis factor mRNA in human cells.

Maintaining RNA stability is a major problem in the delivery of preformed inhibitory RNA to target cells. In this study, we delivered a hammerhead ribozyme directed against tumour necrosis factor alpha into human promyelocytic leukaemia cells by cationic liposome-mediated transfection. Delivering a ribozyme in this manner reduced by 90% and 85% tumour necrosis factor alpha mRNA and protein, respectively. A modified ribozyme with a bacteriophage T7 transcription terminator at its 3' end was more stable than one lacking this sequence. This indicates that ribozyme stability can be improved by the addition of terminal sequences expected to protect against cellular nucleases.

Base Sequence↗

PubMed1992

Mycobacteria and human heat shock protein-specific cytotoxic T lymphocytes in rheumatoid synovial inflammation.

OBJECTIVE: To study the cytotoxic capacity of mycobacteria-specific T lymphocyte lines and clones from sites of inflammation in patients with rheumatoid arthritis (RA). We also studied antigen specificity, surface phenotype, expression of T cell receptors (TCR), and HLA restriction. METHODS: Autologous macrophages (M phi) from the synovial membrane (SM), synovial fluid (SF), or peripheral blood (PB) were used as target cells in cytotoxicity assays. RESULTS: All SM and SF cell lines tested thus far have shown specific lysis of the autologous M phi from SM or PB that had been pulsed with BCG (bacillus Calmette-Guerin), but no cytotoxicity when the targets were pulsed with irrelevant antigens such as tetanus toxoid and Chlamydia. Both CD4+ and CD8+ cells were shown to be involved in the specific cytolysis. The majority of the cytotoxic T lymphocyte (CTL) lines were TCR alpha/beta + cells. However, both TCR alpha/beta + and TCR gamma/delta + clones (TCR delta 1+) from one RA patient showed antigen-specific lysis. Antigen-specific recognition by a number of CTL lines and clones generated from SF and SM was restricted by HLA-DR molecules. Two Mycobacterium bovis 65-kd heat shock protein (HSP)-specific TCR alpha/beta + SF T cell clones also lysed M phi that had been pulsed with a recombinant human 65-kd HSP. CONCLUSION: Joint inflammation and destruction might be partly attributable to a cross-reaction of mycobacteria-induced cytotoxic T cells with self HSP.

Antibodies, Monoclonal↗

PubMed1992

Peptide recognition, T cell receptor usage and HLA restriction elements of human heat-shock protein (hsp) 60 and mycobacterial 65-kDa hsp-reactive T cell clones from rheumatoid synovial fluid.

A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65-kDa heat-shock protein (hsp) as a putative causal agent. The 65-kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65-kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65-kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino-acid sequence 241-255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA-DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251-265) which is also highly homologous with the human sequence, but this clone was restricted by HLA-DR. The clones utilized different V beta gene segments but the same D beta and J beta gene elements, and both exhibited specific cytotoxicity against autologous antigen-pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.

Adult↗

PubMed1992

Limited heterogeneity of T cell receptor variable region gene usage in juvenile rheumatoid arthritis synovial T cells.

The aim of this study was to determine whether synovial fluid (SF) T cells in patients with juvenile rheumatoid arthritis (JRA) are restricted in their T cell receptor (TcR) gene repertoire. The quantitative polymerase chain reaction (QPCR) was used to compare the transcription of V beta and V alpha gene families in freshly isolated SF T cells, in interleukin-2 receptor-positive (IL-2R+) T cells and in peripheral blood (PB) T cells from 18 patients. Significantly less V beta families are detected in SF when compared with PB (p greater than 0.0003). The TcR V beta gene usage by IL-2R+ T cells was even less heterogeneous when compared with freshly isolated SF T cells (p greater than 0.0002). Freshly isolated SF T cells from the left and the right knees of four patients transcribed the same V beta families. Furthermore, we demonstrate that in SF the distribution of certain TcR V beta gene segments in CD4+ and CD8+ T cells differed from that in PB of the same patient. The TcR V alpha usage was studied in IL-2R+ T cells from six patients who had shown restriction in their SF TcR V beta gene usage. Only two to five TcR alpha transcripts were detected in three of these patients while a broad TcR V alpha usage was seen in the other three patients. Sequence analysis of the SF V beta 20 cDNA clones generated from the IL-2R+ T cells of two patients demonstrated an oligoclonal expansion. Taken together, our data could indicate an antigen- and/or superantigen-driven expansion of selected T cells in the synovial compartment.

Adolescent↗

PubMed1992

Nucleotide sequence analysis of rheumatoid factors and polyreactive antibodies derived from patients with rheumatoid arthritis reveals diverse use of VH and VL gene segments and extensive variability in CDR-3.

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of VH and VL gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain 'restrictions' do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of VH3 genes beyond that one would expect based on random utilization.

Amino Acid Sequence↗