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J B Natvig

Publications and source records attributed to J B Natvig.

At least 37 records · Page 2Linked to original sources
PubMed1999

Change in the Th1/Th2 phenotype of memory T-cell clones from rheumatoid arthritis synovium.

The stability of established memory T helper (Th)1/Th2 cells in chronic inflammatory diseases is not clear, and a shift of the cytokine balance could control chronic inflammation. In order to study the regulation of the Th phenotype of memory T cells, polyclonal T-cell lines and clones with a Th1, Th0 or Th2 phenotype were developed from rheumatoid synovial tissue. Th1 [interleukin (IL)-12 + anti-IL-4] and Th2 (IL-4 + anti-IL-12) promoting environments and IL-2 were used to manipulate the cytokine profile. Polyclonal T-cell lines of predominantly Th1 type could be shifted to produce Th2 cytokines, and polyclonal Th2/Th0 lines could be shifted to produce Th1 cytokines. However, this shift was due to an amplification of CD8+ T cells with a memory phenotype and a loss of the CD4+ T cells, giving Tc2 or Tc1 profiles, respectively. Th2 clones cultured repeatedly with IL-2 switched to either a Th0 or a Th1 phenotype, while both Th1 and Th0 memory clones kept a stable phenotype. Addition of Th2-promoting conditions strongly reduced the production of both interferon-gamma and IL-17, while Th1-promoting conditions increased the production of these cytokines. These results demonstrate that RA Th2 clones readily switch, while Th1 and Th0 clones are stable. However, induction of Th2 cytokines can be obtained in polyclonal polarized memory T cells due to amplification of Tc2 cells.

Adult↗
PubMed1999

Light chain variable (VL) sequences of rheumatoid factors (RF) in patients with primary Sjögren's syndrome (pSS): moderate contribution of somatic hypermutation.

We have characterized and sequenced the variable (V) region genes of the light (L) chains of 10 immunoglobulin (IgM) rheumatoid factor (RF) monoclonal antibodies (MoAb) derived by the hybridoma/Epstein-Barr virus (EBV) technique from the peripheral blood of patients with primary Sjögren's syndrome (pSS). Six out of 10 RFs used lambda (lambda) L chains, while four RFs used kappa (kappa) L chains. Five out of the six lambda RFs were encoded by Vlambda3 gene segments, the sixth one was encoded by a Vlambda1 gene segment. This preferential utilization of the Vlambda3 family genes suggests selective expansion of the B cell in pSS. Three of the kappa RFs used Vkappa3 gene segments, while the fourth used a Vkappa2 gene segment. Half of the RFs were found as unmutated copies of their closest germline (GL) gene. Interestingly these RFs were previously shown to use heavy (H) chains in GL gene configuration. Three RFs have very few mutations (2-3) and only two RFs have substantial numbers of mutations (6 and 11). This also correlated with the number of mutations in the respective H chains. In contrast to RFs in normal and RA these results further suggest the somatic mutation to be of moderate importance in the generation of RF from the peripheral blood of pSS patients.

Amino Acid Sequence↗
PubMed1998

Mapping IgG epitopes bound by rheumatoid factors from immunized controls identifies disease-specific rheumatoid factors produced by patients with rheumatoid arthritis.

We have mapped the specificity of 28 monoclonal IgM rheumatoid factors (RFs) produced by heterohybridomas derived from five healthy blood donors immunized with mismatched human red blood cells (HID). The HID-RFs did not differ in their binding specificity for IgG epitopes from RFs that we previously analyzed from patients with Waldenström's macroglobulinemia. However, IgM RFs produced by HID differed in their specificity for IgG compared with RFs expressed by patients with rheumatoid arthritis (RA-RFs). Only 1 of 28 HID-RFs bound all IgG subclasses (pan binding pattern) compared with 7 of 19 RA-RFs (p = 0.006). Three HID-RFs bound IgG3 compared with 9 RA-RFs (p = 0.007). Fine specificity differences were also identified between HID- and RA-RFs. Therefore, some RA-RFs show novel specificities for IgG not found among RFs from HID or individuals with Waldenström's macroglobulinemia who do not have joint disease. These Abs with unique specificities may represent disease-specific autoantibodies in patients with RA. Nine of the HID-RFs from the same individual were clonally related, and several contained somatic mutations. Even when the clonally related HID-RFs were considered as one RF for comparison, the reactivity of the HID-RFs differed significantly from RA-RFs in their inability to recognize all IgG subclasses (p = 0.044) and recognize IgG3 (p = 0.041). Interestingly, among the clonally related RFs, considerable differences in the specificity for IgG were also observed, with the RF containing the most somatic mutations in VH and VL showing the most distinctive specificity changes. Therefore, these studies also demonstrate a correlation between somatic mutation and binding specificity.

Amino Acids↗
PubMed1998

Human monoclonal antibodies encoded by the V4-34 gene segment show cold agglutinin activity and variable multireactivity which correlates with the predicted charge of the heavy-chain variable region.

We have characterized the reactivities of a panel of V4-34-encoded human IgM monoclonal antibodies (mAb) which bind the erythrocyte Rh D antigen, derived from an immunized individual. These were compared with the specificities of V4-34-encoded autoantibodies with I/i reactivity produced from patients with cold agglutinin disease (CAD), and other V4-34-encoded autoantibodies. The antibodies were evaluated for cold agglutinin activity using haemagglutination tests, immunofluorescence microscopy for reactivity with tissue components, and in solid phase radiobinding assays with purified antigens. We found that (i) cold agglutinin activity was a property of all the V4-34-encoded mAb (ii) the cold agglutinins from CAD patients were generally monospecific for I/i whereas most of the anti-D and the other V4-34-encoded mAb displayed multireactive properties, frequently binding to strongly acidic antigens (iii) computation of the net charge of the heavy-chain V regions showed that the multireactive mAb were generally more positively charged than the monospecific cold agglutinins, which could contribute to their multireactive phenotype. The involvement of charge interactions was further indicated by the effects of pH and ionic strength on the immunofluorescence staining patterns.

Adult↗
PubMed1997

Rheumatoid factor autoantibodies in health and disease.

Recent advances in molecular biological and human cell hybridization technology have significantly advanced the knowledge of mechanisms that underlie human rheumatoid factor (RF) production. These advances have provided insight into the etiopathogenesis of synovial inflammation and lymphocyte recruitment in rheumatoid arthritis (RA) joints. We have examined the mechanisms that lead to RF production in RA patients and those that regulate RF production in normals. The studies revealed structural features that distinguish RF produced in normals from those produced in RA synovial tissue. There are significant differences in the use of VL and VH genes between the two RF populations. Furthermore, IgV genes encoding synovial RF in RA have extensive evidence for nucleotide changes, leading to amino acid replacement in the complementarity determining regions (CDRs). In addition, RF produced in RA synovia show evidence for affinity maturation, isotype switch to IgG RF, and repertoire shift indicative of a continued recruitment of B cells. Together with computer modeling and crystallographic studies, our data suggest that the mechanisms that operate on RF selection in RA synovia are similar to immune responses to exogenous antigens. In contrast, RF established from human immunized donors (HID) are characterized by a very low ratio of replacement to silent (R:S) nucleotide changes in the CDR1+2. In addition, there is little increase in affinity with increasing numbers of mutations. There is thus evidence for regulatory mechanisms that limit affinity maturation of RF in normals.

Arthritis, Rheumatoid↗
PubMed1997

Differences in mutational patterns between rheumatoid factors in health and disease are related to variable heavy chain family and germ-line gene usage.

The sequences of the heavy chain variable (V(H)) segment and dissociation constants (Kd) of 14 IgM rheumatoid factors (RF) derived from 11 different germline gene segments from five healthy immunized donors (HID) are described. We extend a previous analysis of two clones from one donor using only the germline segment DP-10. In the present study, the mutation patterns of these new RF and the two earlier reported HID RF clones are analyzed in relation to V(H) family, germ-line origin, and Kd. The panel of HID RF is further compared with 33 previously described IgM RF from patients with rheumatoid arthritis (RA). There is a high rate of mutation in the panel of HID RF (mean of ten mutations/V(H)). RF originating in RA patients have a comparable mutation rate (mean of 11 mutations/V(H)), suggesting that hypermutation of IgM RF is not disease related. The HID RF have, however, a significantly lower affinity for IgG than the RA RF. We found that the structural basis of the differences between HID and RA RF is related to V(H) family usage. RF of the V(H)1 family use very similar germ-line genes in HID and RA patients. HID RF of the V(H)1 family have, however, a low ratio of replacement-to-silent (R:S) mutations of only 0.41 in the heavy chain complementarity region (CDR(H))1 and 2. This is statistically significantly lower than the corresponding ratio of 3.14 in the V(H)1 RA RF. In contrast, RF of the V(H)3 family from HID and RA patients have very similar R:S ratios of 1.75 and 1.71 in CDR(H)1 and 2, respectively. The V(H)3 RA RF are, however, predominantly encoded by genes not encoding any HID RF. Thus, both repertoire differences and hypermutation resulting in significantly lower R:S ratios can be observed in RF from HID compared with RA RF.

Amino Acid Sequence↗
PubMed1995

Structural restriction in the heavy chain CDR3 of human rheumatoid factors.

We have compared the variable regions of 14 new IgM rheumatoid factors (RFs), produced in healthy human immunized donors (HIDs) with RFs originating from patients with rheumatoid arthritis (RA) and monoclonal Ig RFs (paraproteins or M-components, MC). Two groups with very restricted variable region structures were found. Twelve RFs (3 HID, 3 MC, and 6 RA) encoded by variable heavy (VH) chain germ-line genes with closest homology to DP-10 co-express the Kv325 variable light (VL) chain germ-line gene. These RFs have a remarkable restriction in the length (12-14 amino acids) and structure of the CDRH3. One HID RF has a CDRH3 only two amino acids different from the CDRH3 of a MC RF. Two sets of clonally related RFs, one from an RA patient and one from an HID, have CDRH3s that differ by only three amino acids. Five RFs (3 HID, 1 MC, and 1 RA) encoded by VH germ-line gene segments with closest homology to DP-54 all use the Kv328 VL germ-line gene combined to J kappa 1. Four are rearranged to the D21/9 D segment in the same reading frame, with CDRH3s of 16 to 17 amino acids. Three RFs (1 HID, 1 RA, and 1 MC) have CDRH3s differing by only three amino acids. The highly homologous V-regions in RFs from these two groups imply an initial selection to very similar, if not identical, epitopes. However, it remains to be seen whether somatic hypermutation alters the fine specificity of these autoantibodies.

Amino Acid Sequence↗
PubMed1995

V-gene repertoire and hypermutation of rheumatoid factors produced in rheumatoid synovial inflammation and immunized healthy donors.

We have compared RF from normal, immunized donors and RF derived from the synovial tissues of RA patients. We have found a difference in the preferential use of VL and VH gene families. In both conditions, RFs were found to have accumulated somatic mutations. However, there was a striking difference in the patterns of mutation. RFs from normals were characterized by a very low R:S ratio in the CDR1+2, considerably lower than seen among the RARFs. In addition, there was little increase in affinity with increasing numbers of mutations in a group of clonally related RFs from an immunized normal. This contrasts with RF from RA, where there is evidence of both affinity maturation and class switching. Together these data suggest that in healthy persons there is a controlling mechanism to limit the affinity of RF autoantibodies, and that this is lost in RA. The higher affinity of the RA-derived RF may be of significance in the pathology of the disease.

Antibody Affinity↗
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

The identification of germinal centres and follicular dendritic cell networks in rheumatoid synovial tissue.

We document here the identification of germinal centres with dark and light zones, a follicular dendritic cell network and clonal expansion in the synovium of rheumatoid arthritis patients. Synovial tissue from 24 patients suffering from rheumatoid arthritis or the polyarticular form of juvenile rheumatoid arthritis were screened for the presence of lymphoid follicles. The synovial tissues of 14 patients contained follicles and four of these had germinal centres and a follicular dendritic cell network. There was a statistically significant association between follicles in the synovium and the presence of rheumatoid factor autoantibodies in the patients' serum indicating a link between local germinal centre formation and the presence of pathological rheumatoid factors. Nucleotide sequencing of monoclonal rheumatoid factors from one of the patients' synovial tissue which contained germinal centres clearly supports the possibility that these rheumatoid factors have gone through a germinal centre reaction. While rheumatoid factors from healthy immunized donors are regulated through a tolerization mechanism which selects against replacement mutations and does not allow affinity maturation, synovial rheumatoid factors seem to lack this tolerization mechanism. The formation of germinal centres where B cells affinity mature and expand at the central site of disease in rheumatoid arthritis may explain why rheumatoid factors in rheumatoid arthritis develop into auto-aggressive antibodies.

Adolescent↗
PubMed1995

Structural analysis of VH4-21 encoded human IgM allo- and autoantibodies against red blood cells.

We have sequenced the variable heavy chain regions of a number of VH4-21 encoded monoclonal IgM anti-Rh(D) antibodies produced in response to deliberate immunization. These were compared with the sequences of similarly encoded IgM anti-I cold agglutinins (CA) derived from patients with lympho-proliferative diseases. The anti-Rh(D) antibodies show evidence of clonal expansion and somatic diversification. Even though they are produced in response to an antigenic stimulus, they demonstrate limited hypermutation in the variable heavy chain (VH) segments and there is no evidence of selective pressure acting on the complementarity determining regions (CDRs). The CA demonstrate a higher rate of mutation and yet this results in a lower ratio of replacement to silent mutations (R:S) in the CDRs than seen in the anti-Rh(D) antibodies. It is not clear whether the different pattern of mutations seen in the CA is related to their auto-reactivity or their tumour origin. In both groups of antibodies the region encoded by the VH4-21 segment can be found in germline configuration at the amino-acid level indicating that other V-gene structures, i.e. light chains or CDRH3s, are crucial to the generation of either specificity. A role of the CDRH3 is indicated by the identification of a motif shared by four CAs and one Rh(D) antibody which also demonstrates CA activity independent of its anti-Rh(D) specificity. Amongst the anti-Rh(D) antibodies there seems to be an obligatory combination with VL having closest homology to the DPL16 germline segment indicating this as particularly important in generation anti-Rh(D) specificity.

Agglutinins↗
PubMed1994

Control of autoantibody affinity by selection against amino acid replacements in the complementarity-determining regions.

Rheumatoid factor (RF) autoantibodies can be produced in healthy individuals after infections or immunizations and thus escape normal tolerization mechanisms. It has not been clear whether such autoantibodies can undergo somatic hypermutation and affinity maturation similar to antibodies to exogenous antigens. We have investigated how these autoantibodies are regulated in normal individuals by analyzing the sequences of monoclonal IgM RFs obtained as hybridomas from donors after immunization. The variable regions undergo extensive hypermutation, but in contrast to antibodies against exogenous antigens, there is a strong selection against mutations that result in replacement of amino acids in the hypervariable, or complementarity-determining, regions. Furthermore, we found no increase in affinity of these RFs with the accumulation of mutations. This suggests that high-affinity variants are tolerized during the hypermutation process and there is a peripheral mechanism operating on certain autoreactive B cells that, while not deleting or anergizing all autoreactive cells, prevents the generation of high-affinity autoantibodies. Comparison of RFs by using the VH1 DP-10 heavy chain variable region segment from both normal individuals and rheumatoid arthritis (RA) patients suggests that RF from RA patients may not be subject to such a controlling mechanism.

Antibody Affinity↗
PubMed1994

Variable region gene usage of human monoclonal rheumatoid factors derived from healthy donors following immunization.

A study was performed to compare the use of immunoglobulin V gene segments by rheumatoid factors (RF) produced in physiological responses following a defined antigenic stimulus, with RF produced in rheumatoid arthritis (RA) and RF produced as monoclonal (M)-components in certain lympho-proliferative diseases. A panel of 46 monoclonal RF was produced, using hybridoma techniques, from healthy individuals following immunization with foreign antigens (mis-matched red blood cells). A panel of previously characterized monoclonal RF from RA synovial tissues was extended to a total of 24 and included in the study. The variable heavy (VH) and variable light (VL) chain gene families used by these RF were determined using idiotypic markers and polymerase chain reaction amplification with VH-specific primers. The frequencies of expression of the various gene families was compared between the two groups, and compared with the published expression frequencies seen amongst M-component RF. The majority (87%) of RF from healthy donors were found with light chains using V gene segments of the V chi 3 family, in conjunction with VH gene segments belonging to the VH1, VH3 and VH4 families. The over-expression of V chi 3, together with the distribution of VH families, demonstrates close similarities with RF found as M-components in lympho-proliferative diseases. In contrast, RF from RA patients showed a predominant use of VH3 gene segments (82%) and an unbiased expression of V chi 3 segments (29% of the chi light chains). These data suggest that RF found as M-components are representative of RF used in normal physiological responses, but have undergone neoplastic or other transformation. RF found in the synovial tissue of RA patients appear to be driven by different mechanisms than RF seen in physiological responses in healthy individuals.

Arthritis, Rheumatoid↗
PubMed1994

Different cytokine production profiles of gamma delta T cell clones: relation to inflammatory arthritis.

This study was performed to investigate whether gamma delta T cells could also be divided into subsets, identified by a cytokine profile, as described for alpha beta T helper (Th) cell subsets. Cytokine production was studied in 22 gamma delta T cell clones obtained from the synovial fluid and peripheral blood of one patient with inflammatory arthritis and compared to that of 26 alpha beta T cell clones of the same and different patients. Interferon-gamma (IFN-gamma) was produced by 18 (82%) and interleukin-4 (IL-4) by 17 (77%) out of 22 gamma delta T cell clones, respectively. In contrast, IL-10 was not produced, except at very low level in one case. The mean levels of IL-4 were lower for clones derived from synovial fluid. When considering the production of IFN-gamma as an indicator of Th1 and that of IL-4 as an indicator of Th2, respectively, the most common pattern was a gamma delta Th1-like pattern, with the combination of high levels of IFN-gamma and low levels of IL-4. This pattern was found in V delta 1+ clones, all from synovial fluid. Additional patterns were also observed: a mixed, probably gamma delta Th0-like pattern with a more balanced production of both IFN-gamma and IL-4; a gamma delta Th1 pattern with the production of IFN-gamma alone; a gamma delta Th2 pattern with the production of IL-4 alone. These three patterns were also seen in blood gamma delta T cells which were all V delta 2, indicating that these patterns were independent of the V delta phenotype. gamma delta T cell clones produced lower levels of IFN-gamma (p = 0.001) and higher levels of IL-4 than alpha beta clones (p < 0.02). These differences in cytokine production between alpha beta and gamma delta subsets and within these subsets may contribute to their respective role in chronic inflammation.

Adolescent↗
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↗