Characterisation of anti-neutrophil cytoplasmic antibody target antigens using electrophoresis and western blotting techniques.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies which react with components of phagocytic cells, and are associated with vasculitis and other idiopathic inflammatory disorders. However, the antigenic targets of many of these autoantibodies have not been defined yet. In this study, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were evaluated for characterising the antigenic specificity of unidentified ANCA. The uncharacterised sera included those from patients with ulcerative colitis (n = 21), Crohn's disease (n = 5), cystic fibrosis (n = 16) and sarcoidosis (n = 2). In addition, sera from patients with antibodies to the phagocytic enzymes proteinase 3 (PR3) (n = 11) and myeloperoxidase (MPO) (n = 5) were also included. The sub-cellular localisation of antigens was determined by testing sera against crude neutrophil extract and sub-cellular fractions consisting of azurophilic granules, specific granules and cytosolic, fractions using enzyme-linked immunosorbent assays (ELISAs). All sera reacted with the crude and azurophilic granule extracts. The native system of IEF followed by capillary immunoblotting successfully detected anti-PR3 and anti-MPO in azurophilic granule extracts. In contrast, SDS-PAGE Western blotting failed to detect any reactivity, either to PR3 or MPO, in the crude extract or azurophilic granule extract. However, the antibody specificity of patient sera with uncharacterised autoantibodies could not be detected by IEF/capillary immunoblotting or SDS-PAGE. This study showed that the sub-cellular azurophilic granules are the antigenic target of a variety of uncharacterised ANCA. It also showed that IEF characterised both anti-PR3 and anti-MPO but failed to detect other forms of ANCA. In contrast, the majority of common ANCA were not detected by SDS-PAGE.     

The immunological consequences of gold therapy: a prospective study in patients with rheumatoid arthritis.

Gold sodium thiomalate (GST) is known to modify the disease process in patients with active rheumatoid arthritis (RA). To help understand the mechanism of action of GST, several immunological parameters were prospectively evaluated in 10 patients with active RA following the introduction of gold therapy. Before therapy, absolute numbers of peripheral blood T suppressor/cytotoxic lymphocytes were significantly depressed (P less than 0.01) and a raised T helper/T suppressor cell ratio was found. After 1 g of GST, an absolute reduction in total lymphocyte numbers including HLA/DR positive mononuclear cells, was evident (P less than 0.01). This lymphopenic effect was not selective for a single population since the proportions of T cells, T cell subsets and B cells remained unchanged. Lymphocyte function was also examined. Raised in vitro production of IgG (P less than 0.01) and IgA (P less than 0.05) was found before therapy. After GST, in vitro immunoglobulin synthesis was reduced and this was significant with respect to the IgM (P less than 0.001) and IgA (P less than 0.01) isotypes. Similarly, a parallel reduction in serum immunoglobulin levels developed. GST therapy was also associated with a reduced proliferative response to phytohaemagglutinin, concanavalin A and pokeweed mitogen in the initial phase of gold administration. The significant finding in this study suggest that the in vivo immunosuppressive effect of GST is explained not only by impaired mononuclear cell function but also by a significant reduction in T and B lymphocyte numbers.     

Characterization and quantification of solubilised HLA-DR antigens from circulating human monocytes using an immunoblotting procedure

An immunoblotting technique was modified to detect and biochemically characterize HLA-DR antigens expressed on circulating human monocytes. Membrane proteins of peripheral blood monocytes were solubilised using the mildly anionic detergent, sodium deoxycholate. These solubilised proteins were resolved by SDS-PAGE and transferred electrophoretically to nitrocellulose. The HLA-DR antigen was detected using a polyclonal antiserum and two monoclonal anti-HLA-DR antibodies. Both immunoperoxidase and ¹²⁵I autoradiography techniques were used for visualisation of the antigen. The resolution of HLA-DR reactive material was increased when proteins were renatured with 4M urea after blotting. Immunoprobing of a sample of solubilized membrane proteins showed three bands of HLA-DR antigenic reactivity at molecular weights 65kDa, 55kDa and 46kDa. After storage at –70°C for 2 months, only the 46kDa HLA-DR antigen band was detectable. Nonetheless, the 2-chain HLA-DR molecule was found to be an extremely stable complex which could not be dissociated by boiling in sample buffer containing 5% 2-mercaptoethanol and 2% SDS. A stronger reducing agent, 25 mM dithiotreitol, was required to split the HLA-DR molecule into its alpha and beta subunit chains. Finally, in a study of circulating monocytes from normal subjects, the immunoblotting technique was shown to quantitate solubilised HLA-DR antigen in a reproducible manner.     

Antibodies to prothrombin in antiphospholipid syndrome and inflammatory disorders

Antiphospholipid antibodies associated with the antiphospholipid syndrome (APS) have been shown to bind plasma proteins, particularly beta 2-glycoprotein I (β2-GPI). In this study the incidence of antibodies to solid-phase prothrombin was examined in patients with antiphospholipid syndrome and a variety of other inflammatory disorders.
Significantly elevated levels of IgG anti-prothrombin (anti-PT) antibodies were detected in 63% of patients with APS ( n  = 27, median 22 arbitrary units: AU), 33% with SLE ( n  = 92, median 14 AU), 45% with rheumatoid factor ( n  = 22, median 16 AU), 21% with carotid artery stenosis ( n  = 21, median 15 AU), 32% with stroke ( n  = 38, median 13 AU), 67% of patients with a false positive serology for syphilis ( n  = 21, median 24 AU), 37% with HIV ( n  = 30, median 14 AU), 29% with syphilis ( n  = 14, median 19 AU) and 3% with infectious mononucleosis ( n  = 30, median 9 AU). In addition, a group of lupus anticoagulant (LA) positive patients ( n  = 48) was examined for antibodies to prothrombin, β2-GPI and cardiolipin. 10 (21%) patients had raised levels of IgG anti-PT antibodies, 30 (62%) had significantly elevated levels of anti-β2-GPI antibodies and 15 (31%) had elevated levels of anticardiolipin antibodies (ACA). Of the LA-positive patients, 15 (43%) were identified with definite APS, eight (23%) with probable APS, two (6%) with possible APS and 10 (28%) patients had no clinical evidence of APS.
In conclusion, antibodies to prothrombin were found in a variety of inflammatory disorders and were therefore not specific for the APS. However, identification of the plasma proteins recognized by antibodies from patients with APS may provide insight into the pathogenic mechanisms involved in the heterogenous clinical manifestations of the APS.     

Increased concanavalin A induced suppression in treated and untreated coeliac disease.

The generation of suppression by concanavalin A in peripheral blood mononuclear cells in treated and untreated coeliac subjects using an in vitro assay was found to be significantly increased when compared with controls. The response of peripheral blood mononuclear cells to the plant mitogen concanavalin A (con A) was also significantly depressed in both groups of coeliac patients. It is proposed that the depressed cell mediated immunity found in this and other studies in coeliac patients is because of increased suppression. The possible connection between these findings and the increased incidence of malignancy also found in coeliac disease is discussed.     

Humoral response to alpha gliadin as serological screening test for coeliac disease.

The diagnostic value of measuring alpha gliadin antibodies in children with suspected coeliac disease has been evaluated prospectively. Jejunal biopsy and alpha gliadin antibody measurements were performed in 77 consecutive children who were being investigated for a suspected malabsorption syndrome. The typical small intestinal histological lesion of coeliac disease was found, and this diagnosis was subsequently confirmed clinically in 20 children. Raised IgG alpha gliadin antibody concentrations were found in 19 (95%). Fifty of 57 patients (88%) with a normal jejunal mucosa had normal alpha gliadin antibody concentrations. These results are similar to those previously reported in a prospective study of adult patients with coeliac disease and indicate that measurement of alpha gliadin antibody is a highly sensitive and specific screening test for childhood coeliac disease.     

The prevalence of coeliac disease among female subjects having bone densitometry

Background  

Osteoporosis frequently complicates coeliac disease but most studies focus on symptomatic patients at the time of diagnosis. Screening tests have revealed that many individuals with coeliac disease have mild, atypical, or absent symptoms.     

Effects of an advocacy intervention to reduce smoking among teenagers

OBJECTIVES: To test whether high school students' participation in advocacy activities related to the advertising, availability, and use of tobacco in their communities would prevent or reduce their own tobacco use. DESIGN: Ten continuation high schools in northern California, randomly assigned to a semester-long program in which students either carried out advocacy activities to counter environmental-level smoking influences in their communities (treatment) or learned about drug and alcohol abuse prevention (control). PARTICIPANTS: Eleventh and 12th grade high school students; 5 (advocacy) treatment and 5 control schools over 4 semesters from 2000 through 2002. MAIN OUTCOME MEASURES: Self-reported smoking defined as nonsmokers (those who had never smoked tobacco or those who were former smokers), light smokers (those who smoked <1 pack per week), or regular smokers (those who smoked >or=1 pack per week), and confirmed by carbon monoxide level readings. The following 3 constructs related to social cognitive theory- perceived incentive value, perceived self-efficacy, and outcome expectancies-were assessed. RESULTS: There was a significant net change from baseline to the end of the semester (after the intervention) between treatment and control schools for students who were regular smokers, but not for students who were nonsmokers or light smokers. Regular smoking decreased 3.8% in treatment schools and increased 1.5% in control schools (P<.001). Regular smoking continued to decrease at 6 months after the intervention in treatment schools, with a total change in prevalence from 25.1% to 20.3%. Involvement in community-advocacy activities and the 3 social constructs-perceived incentive value, perceived self-efficacy, and outcome expectancies-also showed significant net changes between treatment and control schools (all P values <.01). CONCLUSION: Student engagement in community-advocacy activities that addressed environmental influences of cigarette smoking resulted in significant decreases in regular smoking.     

Association of retail tobacco marketing with adolescent smoking

A survey of 2125 middle-school students in central California examined adolescents' exposure to tobacco marketing in stores and its association with self-reported smoking. Two thirds of sixth-, seventh-, and eighth-grade students reported at least weekly visits to small grocery, convenience, or liquor stores. Such visits were associated with a 50% increase in the odds of ever smoking, even after control for social influences to smoke. Youth smoking rates may benefit from efforts to reduce adolescents' exposure to tobacco marketing in stores.