Association between antibodies to heat shock protein 65 and coronary atherosclerosis. Possible mechanism of action of Helicobacter pylori and other bacterial infections in increasing cardiovascular risk.

INTRODUCTION: There is growing evidence that the immune response is involved in atherosclerosis. Antibodies to heat shock protein 60/65 have been shown to be a risk factor for carotid atherosclerosis and been proposed as a diagnostic marker of atherosclerosis. In addition, it has been suggested that the immune response to heat shock protein 60/65 may be a link between exposure to microorganisms and increased cardiovascular risk. AIMS: (1) To investigate the association between anti-shock protein 65 titre and coronary atherosclerosis. (2) To assess whether anti-mhsp65 titre is a useful diagnostic marker of atherosclerosis; (3) To examine the influence of Helicobacter pylori infection on anti-heat shock protein 65 titre. METHODS AND RESULTS: In the first study we measured anti-heat shock protein 65 titres in 136 consecutive male subjects admitted for routine coronary angiography. Anti-heat shock protein 65 titres correlated with both the severity and extent of coronary atherosclerosis and the relationship remains statistically significant for the presence of atherosclerosis (P = 0.012) after adjustment for possible confounding influences. However the association had insufficient sensitivity to be a useful clinical test. In the second study we recruited 100 patients with confirmed active H. pylori infection and double blindly randomized them to eradication therapy or placebo. Successful eradication of H. pylori led to a significant fall in anti-heat shock protein 65 titres (from a mean of 256.4 AU.ml-1 to 137.5 AU. ml-1. P = 0.033). CONCLUSION: These results raise the possibility that exposure to H. pylori and other micro-organisms lead to an increased risk of clinically manifest coronary artery disease by an autoimmune process.

Systemic complement activation in psoriasis vulgaris.

Nineteen patients with psoriasis vulgaris and no other cause for systemic complement activation were studied for evidence of such activation. There was a marked elevation in serum C5b-9 complexes with no other significant complement abnormalities, and no correlation between C5b-9 levels and disease activity. This is the most detailed study of complement in psoriasis yet attempted and confirms that complement activation is a feature of psoriasis vulgaris.

Antimycobacterial hsp65 and rheumatoid factor titres in a population of normal twins: evidence of genetic control of rheumatoid factor.

Rheumatoid arthritis (RA) is an autoimmune disease and rheumatoid factor (RF), anti-IgG, has been implicated in the pathogenesis, but the exact etiology remains unclear. There are data to suggest and infectious trigger to the autoimmune process, and mycobacteria are considered a candidate. Immunization of various animals with mycobacterial heat shock protein 65 (mhsp65) protects against subsequent autoimmune arthritis in a number of experimental models. Elevated anti-mhsp65 titres have been demonstrated in RA patients, together with specific T cells isolated from inflamed synovium. Mycobacterial hsp65 has also been implicated in other autoimmune disease and in atherosclerosis. The anti-mhsp65 and RF (IgG, IgM and IgA isotypes) titres were assayed by ELISA in 123 pairs of normal twins (61 monozygotic and 62 dizygotic, age 14-79 years), to examine the population distribution and inter-relationship of these antibodies. In addition, we studied the effects of age, sex, genetics and environment on antibody titres. IgG-RF and IgM-RF were detectable in all subjects and IgA-RF in 41 subjects. None of the RF isotypes showed any significant dependence on age or sex. There was a statistically significant correlation between twins for the IgG-RF and IgM-RF, and a positive but not significant correlation for the IgA-RF. All three correlations were stronger for monozygotic than dizygotic twins, reaching statistical significance for IgM-RF (P < 0.001), and this indicates that there is a genetic influence on RF titres. Anti-mhsp65 titres were detectable in 90.5% of the study group with a range of 0.15-19.7 AU/ml. There were weak correlations between twins, stronger for dizygotic than monozygotic twins. This suggests that familial influences on anti-mhsp65 titres are very small, with no evidence of any genetic influence at all. There was no significant relationship of anti-mhsp65 titre with age, sex or RF titres.

Purification and characterization of RHP (factor H) and study of its interactions with the first component of complement.

RHP has been purified from the plasma of both normal individuals and patients with rheumatoid arthritis (RA). RHP from both these sources was shown to be identical with Factor H by reaction with antisera and N-terminal amino acid sequence analysis. Factor H, from both normal and RA sera, inhibited the solubilization of immune precipitates but did not affect prevention of immune precipitation. Factor H was shown to inhibit the haemolytic activity of fluid-phase C1, but unlike C1-inhibitor, it had little effect on C1 bound to EA (EAC1). Factor H was shown to complex with intact C1, to isolated C1q and to the C1r:C1s tetramer. However, binding of factor H to C1 did not dissociate the C1 macromolecule. A C1-Factor H complex was detected in the serum and plasma from normal individuals and patients with systemic lupus erythematosus and RA. Serum levels of this complex were reduced, by EDTA-treatment of serum and by activation of complement by the classical pathway.

The relative roles of C4A and C4B in prevention of immune precipitation, solubilisation and immune adherence.

C4A and C4B levels were measured in serum from 246 normal individuals. Complement-mediated solubilisation, assayed using alkaline phosphatase anti-alkaline phosphatase immune complexes (IC), correlated with both C4A and C4B levels. However, C4A and C4B levels showed no correlation with solubilisation of bovine serum albumin (BSA) ICs, or with the prevention of immune precipitation of BSA or alkaline phosphatase ICs, nor with immune adherence assayed using thyroglobulin and BSA ICs.

Immunoalkaline phosphatase technique applied to paraffin wax embedded tissues in diagnostic renal pathology.

An indirect immunoalkaline phosphatase (IAP) technique was used to evaluate the glomerular deposition of immunoglobulins, C3, C1q and fibrinogen. In 80 renal biopsy specimens the results obtained using this technique were compared with those obtained by direct immunofluorescence to see if it could be used as a viable alternative. The IAP technique was straightforward to perform, it yielded quick results, and was highly reproducible, provided that a standardised short fixation period of two and a half hours was used. For the detection of immunoglobulin deposits, the IAP results correlated well with those of immunofluorescence. Despite poorer performance in identifying complement components and fibrinogen it could, within certain limits, provide an adequate diagnostic alternative to immunofluorescence. Each technique gave false negative results, those of immunofluorescence being related to its failure to identify mesangial deposition of IgA in two cases where its distribution seemed to be focal, and those of IAP to a failure to detect linear deposition of IgG in all three cases of anti-glomerular basement membrane disease.

Measurement of complement activation products in patients with chronic rheumatic diseases.

Measurement of the complement activation products C1s:C1-inh, C3bP and C5b-9 by ELISA in plasma samples from normals, rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE) patients showed significantly elevated levels in the two patient groups (P less than 0.0001 for C1s:C1-inh, C3bP and C5b-9) compared to normals. In seropositive RA patients there were significant correlations between the levels of the three complement activation complexes and IgM-RF, IgG-RF and IgA-RF. However, IgM-RF did not interfere with any of the ELISA systems. Mean levels of C1s:C1-inh, C3bP and C5b-9 were the same in paired plasma and synovial fluids; however, C3bP levels in the paired samples did not correlate with one another by rank. Our conclusions are that: (a) elevated plasma levels of these complement activation products are detectable in rheumatic diseases; (b) plasma levels of these complement activation products are related to Rheumatoid factor (RF) levels in seropositive RA patients; and (c) IgM-RF does not influence these solid-phase ELISA procedures.

Complement and related clinical disorders.

The complement system, composed of 20 plasma proteins and several membrane receptors, plays an essential role in humoral immune responses. The activation of the classical and/or alternative pathways by specific and non-specific stimuli leads to the generation of chemotactic and anaphylatoxic inflammatory mediators, the formation of the cytolytic membrane attack complex, and the formation of C3 breakdown fragments which are opsonic. There is now evidence that the membrane receptors play important immunoregulatory functions. The link between various disease states and deficiencies of complement components and membrane receptors further supports the important immunological role this system plays. This review briefly introduces the components of the system, their biological roles and diseases associated with deficiency states.

Familial properdin deficiency associated with chronic discoid lupus erythematosus.

A large family comprised of 18 members is described. Four male members are properdin-deficient, all are healthy bar the index patient who presented with chronic discoid lupus erythematosus. Serum from properdin-deficient males had a reduced ability to lyse rabbit erythrocytes via the alternative pathway or solubilize pre-formed immune complexes. Addition of purified properdin restored these activities. Classical pathway activity was normal. Definite, probable and possible female carriers had normal classical and alternative pathway activities.

The relative roles of genetic and environmental factors in the regulation of erythrocyte C3b receptor (ECR1) numbers in normal individuals.

Erythrocyte C3b receptors (ECR1) have been measured in 122 pairs of twins (60 monozygotic, 62 dizygotic) using an [125I]-labelled monoclonal antibody (E11). The range of ECR1 numbers was wide, 99-4179 sites/cell, with a log-normal distribution around a geometric mean of 837 sites/cell. The intra-pair variance in dizygotic twins was no greater than that in monozygotic twins. These data indicate that ECR1 numerical expression is governed by environmental rather than genetic factors.

C3b receptor (CR1) expression on the polymorphonuclear leukocytes from patients with systemic lupus erythematosus.

Polymorphonuclear leukocytes (PMN) C3b receptor (CR1) numbers have been measured in 14 normal individuals and 15 patients with SLE. The results in the normals showed that PMN possess three distinct pools of CR1. CR1 expression was lowest at 0 degrees C (mean 86,000 +/- s.e.m. 7,000), but increased when the cells were incubated at 37 degrees C (125,000 +/- 16,000) or when the cells were exposed to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 10(-5) mol 1) at 37 degrees C (207,000 +/- 21,000). The increased expression at 37 degrees C was not dependent upon protein synthesis, an intact cytoskeleton or energy. Although the response to FMLP did not require de novo protein synthesis, increased CR1 expression was dependent upon an intact cytoskeleton and energy. All three PMN CR1 pools were reduced in patients with active SLE, but were normal in those in whom the disease was inactive. Serial studies performed on three SLE patients showed that PMN CR1 numbers were low during periods of disease activity and increased during remission. These data suggest that low PMN CR1 numbers in SLE are a consequence of the disease.