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Biomedical subjects

D A Isenberg

Publications and source records attributed to D A Isenberg.

At least 253 records · Page 14Linked to original sources
PubMed1994

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.

Amino Acid Sequence↗
PubMed1994

Polymerase chain reaction fails to incriminate exogenous retroviruses HTLV-I and HIV-1 in rheumatological diseases although a minority of sera cross react with retroviral antigens.

OBJECTIVES: To investigate the presence of antibodies to HTLV and HIV retroviral antigens in the rheumatological diseases rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM), primary Sjögren's syndrome (pSS), and systemic lupus erythematosus (SLE), and to use polymerase chain reaction (PCR) to seek these exogenous retroviruses in proviral form in cellular DNA from these patients. METHODS: Thirty patients with active RA, 13 with PM, 14 with pSS and five with SLE were recruited and their sera tested for antibodies to HTLV-I in enzyme linked immunosorbent assay (ELISA) and Western blot analysis. Seropositivity to HIV-1 was also sought. DNA was extracted from peripheral blood lymphocytes, synovial tissue and muscle biopsies and tested by polymerase chain reaction using consensus primers for HTLV-I and HIV-1. RESULTS: In HTLV-I ELISA, nine rheumatological sera (4/30 RA, 3/13 PM/DM and 2/5 SLE patients) were considered positive; 14 from pSS patients and 30 from normal subjects were negative. In a control group which included osteoarthritis, Crohn's disease and bacterial endocarditis patients, only two of 80 proved positive in this system. Validation of these sera by Western blotting generally revealed weak reactivity against a variety of HTLV-I antigens. PCR of genomic DNA derived from patients' peripheral blood mononuclear cells did not reveal the presence of HTLV-I and HIV-1 target sequences. CONCLUSIONS: This study shows that PCR precludes HTLV-I and HIV-1 infection as causative agents in these rheumatological diseases although a minority of patients possess antibodies that are weakly cross-reactive with retroviral antigens.

Adolescent↗
PubMed1994

Serological and genetic characterization of a human monoclonal immunoglobulin G anti-DNA idiotype.

This study analyzed the distribution of an idiotype, B3-Id, in patients with active SLE, classified according to organ involvement, normal controls, and other autoimmune rheumatic diseases. A polyclonal anti-idiotype was raised by immunizing a rabbit with a monoclonal IgG anti-double-stranded (ds) DNA antibody, B3, generated from a patient with SLE who had active arthritis. The idiotype is present on the lambda chain and is at or near the binding site for double-stranded DNA. The lambda chain, which was characterized by nucleotide sequencing, was 90% homologous to the V lambda 2.1 germline, which is known to be involved in coding for nephritogenic anti-DNA antibodies carrying the 8.12 idiotype. There were four changes to positively charged amino acids, known to be involved in DNA binding, in the complementarity determining regions of B3 lambda chain compared with a non-DNA binding, 8.12 positive antibody, PV11. Only one change to a positively charged amino acid occurs in the heavy chain of B3, which is 93.5% homologous to VH-26. The B3-Id was present on IgG antibodies in the serum of 20% of patients with SLE but was not found in the normal controls. Within the SLE group, there is a statistically significant association of B3-Id on IgG in the arthritis group (42%) compared to the other manifestations (9%) (P < 0.001). In four B3-Id-positive SLE patients tested serially, the level of B3-Id reflected the arthritis disease activity more closely than the overall disease activity (P < 0.05). The B3-Id was also present on IgM antibodies in one third of patients with rheumatoid arthritis. This idiotype is the first to be derived from a human monoclonal anti-DNA antibody of the IgG class, the isotype associated with active disease. Sequence analysis shows that positively charged amino acids on the lambda chain may contribute to DNA binding.

Adult↗
PubMed1994

Identification and characterization of a new human DNA reactive monoclonal antibody and a common idiotype, WRI 176 Id beta.

We describe a human monoclonal antibody designated WRI 176 beta and a common idiotype that it carries. This antibody was derived from the spleen of a patient with SLE. WRI 176 is an IgM kappa monoclonal reacting with ssDNA, dsDNA, poly(dT) and it is likely that mAb WRI 176 beta is a representative of the so-called natural autoantibodies. The common Id designated WRI 176 Id beta is located on the heavy chain of the mAb WRI 176 beta molecule and appears to be located outside the binding site. Sequence analysis of the WRI 176 beta heavy chain showed it to be highly homologous (97.3%) with a germline gene 56PI derived from a human fetus. In a retrospective analysis, although 44% of SLE patients had raised levels of the WRI 176 beta no correlation was found with the activity of the disease. The idiotype was also expressed frequently in a range of autoimmune rheumatic and infectious diseases and in some healthy first-degree relatives of SLE patients.

Amino Acid Sequence↗
PubMed1994

The role of hsp90 in SLE.

The heat shock proteins are a group of evolutionarily conserved proteins with important physiological functions, whose synthesis is enhanced by elevated temperature or other stresses. A role for one or more of these proteins in human autoimmune disease has been extensively discussed. This review considers the evidence of a role for hsp90 in systemic lupus erythematosus (SLE) where overexpression of this protein, its surface localization and auto-antibodies to it have been observed in both human patients and in the MRL/lpr mouse model of SLE.

Animals↗
PubMed1994

Enhanced transcription of the gene encoding the SmN autoantigen in patients with systemic lupus erythematosus does not result in enhanced levels of the SmN protein.

The SmN, protein is closely related to the constitutively expressed SmB and SmB' autoantigens and can also act as a target for human autoimmune sera. In contrast to the single gene encoding SmB and SmB' which is expressed in all tissues, the distinct gene encoding SmN is expressed at high levels only in brain and heart tissue. We show that the SmN gene is transcribed at significantly elevated levels in peripheral blood mononuclear cells (PBMCs) from SLE patients compared to normal controls. In contrast no significant elevation in transcription of the genes encoding SmB/B' or the U1-associated 70kD RNP autoantigen is observed in these patients. The elevation in SmN gene transcription in patient PBMCs does not result however, in enhanced levels of the SmN protein in the PBMCs of these patients. The significance of transcriptional and post-transcriptional processes in regulating the expression in SLE patients of SmN and other autoantigens is discussed.

Adult↗
PubMed1994

Bcl-2 and Fas, molecules which influence apoptosis. A possible role in systemic lupus erythematosus?

Polyclonal B cell activation and the production of antibodies against a variety of autoantigens are features of systemic lupus erythematosus (SLE). Autoreactive B cells are found in healthy individuals but their numbers are probably regulated by cell death, after a few days, in the absence of proliferative stimuli. The process which achieves this regulation is known as apoptosis or programmed cell death. It has been postulated that in SLE patients dysfunction of apoptosis could result in the inappropriate longevity of autoreactive B cells, allowing autoantibody levels to reach pathogenic thresholds. This hypothesis has arisen as a result of studies revealing links between autoimmunity and two molecules which influence apoptosis. These are bcl-2 which enhances cell survival by inhibiting or delaying apoptosis and Fas, a cell surface molecule involved in the induction of apoptosis. Transgenic mice over expressing bcl-2 in their B cells showed polyclonal B cell expansion and their B cells showed extended survival in vitro. After a few months these mice developed an autoimmune syndrome resembling SLE. Mice that carry the lpr disorder have defects in the Fas gene. These mice, which do not express functional Fas molecules, suffer from an SLE-like autoimmune syndrome. Thus inappropriate expression of both bcl-2 and Fas can result in SLE-like autoimmune disease in mice. Research is now in progress to ascertain whether quantitative or functional abnormalities in these molecules exist in human SLE patients and contribute to the pathogenesis of the disease in some or all cases.

Animals↗
PubMed1994

The interaction of beta 2 glycoprotein-I and heparin and its effect on beta 2 glycoprotein-I antiphospholipid antibody cofactor function in plasma.

beta 2 glycoprotein-I (beta 2GPI), a cofactor for antiphospholipid antibody (aPA) binding, binds to many anionic macromolecules including heparin. The nature of this interaction with heparin is not well understood and its effect on the purported biological functions of beta 2GPI is unknown. We have examined the interactions of dermatan sulphate (DS) and different pharmaceutical preparations of heparin with beta 2GPI by crossed immunoelectrophoresis (CIE) and investigated the effect of these agents on plasma levels of beta 2GPI antigen (beta 2GPI:Ag) by a standardised enzyme linked immunosorbent assay (ELISA). beta 2GPI aPA cofactor activity (beta 2GPI:Cof) was also measured using a modified solid phase anti-phosphatidylserine (aPS) ELISA. CIE results confirmed a heparin-beta 2GPI interaction with unfractionated (UF) heparin. beta 2GPI:Ag levels were unaffected by any of the preparations investigated. There were no significant differences in beta 2GPI:Cof activities of the samples containing LMW heparins or DS but levels of beta 2GPI:Cof were increased in samples containing UF sodium and calcium heparin preparations (0.5 IU/ml Monoparin, p < 0.05, and 10 IU/ml Liquemin and Calciparine, p < 0.05).

Antibodies, Antiphospholipid↗
PubMed1994

Clinical utility and serological connections of anti-RA33 antibodies in systemic lupus erythematosus.

OBJECTIVE: To determine whether the autoantibody anti-RA33, first identified in patients with rheumatoid arthritis, could define a subset of patients with lupus. METHODS: Sera from 30 patients with systemic lupus erythematosus (SLE), including serial bleeds from 8 patients were tested for anti-RA33 antibodies by immunoblotting. The results were compared to a wide range of SLE clinical features using the British Isles Lupus Assessment Group system and serological abnormalities. RESULTS: 12 of the 30 patients had RA33 antibodies. Their presence was associated with erosive arthropathy, and a lack of dermatological involvement in patients less likely to be Caucasian. CONCLUSION: The presence of anti-RA33 antibodies appears to identify a subset of patients with SLE.

Antigens, Nuclear↗
PubMed1994

C3 nephritic factor and SLE: report of four cases and review of the literature.

We present four patients with C3 nephritic factor associated with partial lipodystrophy and/or mesangiocapillary glomerulonephritis type II. Each of these patients subsequently developed features of SLE, with an onset between 2 and 24 years after the development of the lipodystrophy or glomerulonephritis. All four patients had antinuclear antibodies and three of the four had anti-Ro antibodies. These patients bring to six the number of reported cases of this association. Possible explanations for the link between these two conditions are discussed.

Adult↗
PubMed1994

The tissue specific elevation in synthesis of the 90 kDa heat shock protein precedes the onset of disease in lupus prone MRL/lpr mice.

OBJECTIVE: Expression of the 90 kDa heat shock protein (HSP 90) is elevated in the peripheral blood lymphocytes of a subset of patients with active systemic lupus erythematosus (SLE). We therefore wished to test whether this effect would also be observed in the lupus prone MRL/lpr mouse strain. METHODS: Levels of the individual HSP were measured by Western blotting in various tissues of MRL/lpr mice at different ages. RESULTS: Elevation of HSP 90 is observed in the spleen but not other tissues of MRL/lpr mice compared to Balb/c mice. The elevation in HSP 90 levels in the spleen is independent of the age of the MRL/lpr mice and precedes the development of autoimmune disease as measured by the appearance of high levels of antibodies to double and single stranded DNA. In contrast no alterations in the levels of HSP 73 and 65 are observed in MRL/lpr mice while elevated levels of HSP 72 are observed in several tissues of these mice and the level increases in an age dependent manner as the disease progresses. CONCLUSION: MRL/lpr mice show an age independent and tissue specific increase in HSP 90 levels which, as in human patients with SLE, is specific for this HSP.

Animals↗
PubMed1994

Elevation of the 90 kDa heat-shock protein in specific subsets of systemic lupus erythematosus.

We have previously shown that the 90 kDa heat-shock protein (hsp90) is notably elevated in lupus patients with active neuro-psychiatric (NP) and/or cardio-respiratory (CR) disease. This elevation is dependent upon enhanced transcription of the hsp90 beta gene. Serial studies have shown that changes in hsp90 levels have a high level of sensitivity for changes in activity of NP, CR, haematological and renal SLE. We now present evidence that overexpression of hsp90 in lupus patients is associated with the presence of the anti-phospholipid syndrome, and with the absence of the HLA allo-/haplotypes most commonly found in this particular cohort of patients. We conclude that the upregulation of hsp90 expression in SLE has a genetic basis, and that this may be directly involved in pathogenesis in subsets of patients with this disease.

Adolescent↗
PubMed1993

Immunoelectrophoresis and ELISA techniques for assay of plasma beta 2 glycoprotein-1 and the influence of plasma lipids.

Beta 2 glycoprotein-1 (beta 2GP1) has been identified as a cofactor for the binding of some antiphospholipid antibodies to anionic phospholipids and has been demonstrated to possess anticoagulant properties in vitro. We have investigated Laurell rocket immunoelectrophoresis (IEP) and ELISA techniques for measurement of beta 2GP1. Western blotting and crossed immunoelectrophoresis (CIE) of plasma demonstrated free beta 2GP1 and beta 2GP1 complexed with unidentified plasma constituents. The free and complexed forms were not distinguished in immunoelectrophoresis assays, allowing measurement of total beta 2GP1. Standard and detergent modified IEP and ELISA techniques were compared: significant correlation was demonstrated between unmodified and detergent modified IEP, detergent modified IEP and ELISA and unmodified IEP and ELISA. The intra-assay co-efficients of variation (CVs) of the unmodified and modified IEPs and ELISA were 7.5, 3.7 and 7.9%. Inter-assay CVs determined for the modified IEP and ELISA were 5.8 and 9.1% respectively. The purified beta 2GP1 used to standardise the assays was shown to have subfraction selectivity and different calibration values were obtained for pooled normal plasma by the unmodified IEP (242 mg/l) and modified IEP and ELISA (201 mg/l). We have also investigated the influence of some pre-test variables on beta 2GP1 levels and shown that heparin, citrate or EDTA plasma and serum samples are suitable for assay of this glycoprotein and that levels are unaffected by repeated freeze/thawing. The influence of plasma lipids on beta 2GP1 measurement was also examined and we demonstrated no significant differences between pre and postprandial samples, which suggests that fasting status is not an important consideration for assay of beta 2GP1 in healthy subjects.

Apolipoproteins↗