A. Sturgess

Novel Assays of Thrombogenic Pathogenicity in the Antiphospholipid Syndrome Based on the Detection of Molecular Oxidative Modification of the Major Autoantigen β2-Glycoprotein I

Objective Beta-2-glycoprotein I (β2GPI) constitutes the major autoantigen in the antiphospholipid syndrome (APS), a common acquired cause of arterial and venous thrombosis. We recently described the novel observation that β2GPI may exist in healthy individuals in a free thiol (biochemically reduced) form. The present study was undertaken to quantify the levels of total, reduced, and posttranslationally modified oxidized β2GPI in APS patients compared to various control groups. Methods In a retrospective multicenter analysis, the proportion of β2GPI with free thiols in serum from healthy volunteers was quantified. Assays for measurement of reduced as well as total circulating β2GPI were developed and tested in the following groups: APS (with thrombosis) (n = 139), autoimmune disease with or without persistent antiphospholipid antibodies (aPL) but without APS (n = 188), vascular thrombosis without APS or aPL (n = 38), and healthy volunteers (n = 91). Results Total β2GPI was significantly elevated in patients with APS (median 216.2 μg/ml [interquartile range 173.3–263.8]) as compared to healthy subjects (median 178.4 μg/ml [interquartile range 149.4–227.5] [P < 0.0002]) or control patients with autoimmune disease or vascular thrombosis (both P < 0.0001). The proportion of total β2GPI in an oxidized form (i.e., lacking free thiols) was significantly greater in the APS group than in each of the 3 control groups (all P < 0.0001). Conclusion This large retrospective multicenter study shows that posttranslational modification of β2GPI via thiol-exchange reactions is a highly specific phenomenon in the setting of APS thrombosis. Quantification of posttranslational modifications of β2GPI in conjunction with standard laboratory tests for APS may offer the potential to more accurately predict the risk of occurrence of a thrombotic event in the setting of APS.

Consensus guidelines on anti‐cardiolipin antibody testing and reporting

UNLABELLED Consensus guidelines on anti-cardiolipin antibody (aCL) testing have been developed to help minimise laboratory variation in the performance and reporting of aCL assays. These guidelines include minimum, optimum and optional recommendations for the following aspects of aCL testing and reporting: (1) isotype of aCL tested; (2) specimen type; (3) controls and assay precision; (4) calibrators; (5) patient samples; (6) rheumatoid factors and IgM aCL testing; (7) reporting of results; (8) cut-off values; and (9) interpretative comments. ABBREVIATIONS aCL, anti-cardiolipin antibodies; APS, anti-phospholipid antibody syndrome; ASCIA, Australasian Society of Clinical Immunology and Allergy; ASTH, Australasian Society of Thrombosis and Haemostasis; beta2-GPI=beta2-glycoprotein I; ELISA, enzyme-linked immunosorbent assay; NCCLS, National Committee for Clinical Laboratory Standards; HSANZ, Haematology Society of Australia and New Zealand; QAP, Quality Assurance Program; RCPA, Royal College of Pathologists of Australasia; %CV, inter-assay inter-run coefficient of variation.

Antigen-induced IL-17 response in the peripheral blood mononuclear cells (PBMC) of healthy controls

IL‐17 is a T cell cytokine with a complex and important role in the immune system. It has been detected in rheumatoid arthritis (RA) synovial membrane and found to stimulate the production of the proinflammatory cytokines IL‐6, IL‐8, tumour necrosis factor‐alpha (TNF‐α) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in vitro. To date, there are few data available on the agents that stimulate IL‐17 production. We therefore investigated the in vitro IL‐17 response to a variety of mitogens and antigens, and compared the IL‐17 response to interferon‐gamma (IFN‐γ), IL‐4, IL‐10 and TNF‐α. In this study we used a type‐0 antigen, tetanus toxoid (TT), a type‐1 antigen, PPD from Mycobacterium tuberculosis, a potential type‐2 rye grass (RG) antigen (Lol I) and an autoantigen SS.B (La), to stimulate PBMC from healthy controls. Cytokine mRNA was measured using semiquantitative reverse transcriptase‐polymerase chain reaction and cytokine protein measured using specific ELISA techniques, while the frequency of IL‐17‐producing T cells was determined by flow cytometry. The mitogens concanavalin A, phytohaemagglutinin and phorbol myristate acetate/ionomycin induced a significant increase in IL‐17, with the highest levels being produced by anti‐CD3/anti‐CD28 stimulation. The antigens TT and PPD significantly increased IL‐17 mRNA expression over time, but failed to have such an effect at the protein level. IL‐17 protein was also detectable in both antigen‐specific (TT, SS.B) and non‐specific T cell clones, but at levels lower than IFN‐γ. IL‐17 production did not correlate with either the type‐1 cytokine IFN‐γ or TNF‐α or the type‐2 cytokine IL‐4 or IL‐10 at either the mRNA or protein level.

A double blind randomised trial of low power laser treatment in rheumatoid arthritis.

OBJECTIVES--To define the value of low power laser treatment in small joint rheumatoid arthritis. METHODS--Twenty five women with active disease were recruited. The metacarpophalangeal and proximal interphalangeal joints of one hand were treated with 12 J/cm2 for 30 s with a gallium-aluminium-arsenate laser. The other hand received a sham laser treatment designed so that neither therapist nor patient could distinguish the active laser from the sham laser. Each patient received 12 treatments over four weeks. The following parameters were measured: pain as assessed by visual analogue scale; range of joint movements; grip strength; duration of early morning stiffness, joint circumference, Jebsens hand assessment; drug usage; total swollen joint counts; Arthritis Impact Measurement Scales; three phase bone scans; haematological and serological tests. RESULTS--A total of 72% of patients reported pain relief but this reduction was reported equally in both hands. No significant changes were seen in other clinical, functional, scintigraphic, or laboratory features. Neither patients nor staff were able to detect which hand was treated with the active laser. CONCLUSION--When this specific laser and dose regimen was used, low power laser treatment had no objective effect on patients with rheumatoid arthritis. It did appear to produce analgesia through a powerful placebo effect.

Evidence for chronically elevated serum protein oxidation in systemic lupus erythematosus patients.

Serum protein oxidation levels in people with the autoimmune disease systemic lupus erythematosus (SLE) have previously been shown to (a) be elevated at a single time point and (b) correlate with disease activity. This study investigates whether this elevation is a chronic phenomenon, by analysis of multiple serum samples collected from 21 SLE patients and nine controls over a period of up to 38 months. Protein thiols were chronically decreased in SLE patients with stable or variable disease activity compared to controls, whilst protein-bound carbonyls and glycine were chronically increased. 2D-gel analysis of carbonyl distribution showed albumin and immunoglobulins to be particularly affected. In SLE patients with stable disease activity, higher long-term protein oxidation correlated with higher long-term disease activity. SLE patients with variable disease activity exhibited varying correlations between protein oxidation and disease activity markers. These results further support a role for oxidative stress in the pathogenesis of SLE.

Detection of ‘antiphospholipid’ antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding β2-glycoprotein I and prothrombin

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against β2‐glycoprotein I (β2GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and β2GPI‐dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with β2GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0·05) and at lower antibody concentrations (12·5 µg/ml versus 100 µg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to β2GPI (r = 0·90; P < 0·001) but not to CL (r = 0·06; P = 0·76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti‐β2GPI or antiprothrombin antibodies. All MoAbs binding β2GPI showed inhibition of thrombin generation, while MoAbs binding domain I of β2GPI had more LA effect.

A multi-centre evaluation of the intra-assay and inter-assay variation of commercial and in-house anti-cardiolipin antibody assays

Aims: To assess the intra‐assay (intra‐run) and inter‐assay (inter‐run) variation of commercial and in‐house IgG and IgM anti‐cardiolipin antibody (aCL) assays/kits, and to determine an appropriate maximum value for inclusion in consensus guidelines. Methods: Frozen aliquots of two patient specimens and one commercial control were sent to nine laboratories for the evaluation of eight commercial kits and one in‐house assay. Intra‐assay and inter‐assay evaluations were performed with all three samples for IgG aCL, and one patient specimen for IgM aCL. Results: The IgG and IgM aCL values varied considerably between the nine assays/kits. The majority of assays/kits demonstrated less than 20% intra‐assay and inter‐assay variation, with lower intra‐assay and inter‐assay variation observed with the commercial control. Single calibrator assays were not consistently associated with higher inter‐assay variation than multi‐point calibrator assays. Conclusions: An inter‐assay coefficient of variation of 20% was determined to be an appropriate maximum value for inclusion in the Australasian aCL Working Party consensus guidelines. Improved standardisation between different assay/kits is still required.

Serum protein oxidation and apolipoprotein CIII levels in people with systemic lupus erythematosus with and without nephritis.

Increased oxidative stress is a hallmark of the autoimmune disease systemic lupus erythematosus (SLE). This study compares serum protein oxidation levels in SLE patients without and with renal involvement (lupus nephritis); the latter have a significantly poorer prognosis. Similar increases in protein carbonyls and decreases in protein thiols were observed in both SLE groups compared to controls. Protein carbonyl distribution, determined by Western blotting of 2D gels, was similar in both SLE groups, suggesting factors other than oxidation also play a role in SLE complications. 2D electrophoresis examined the serum proteome further. Six proteins were significantly decreased in non-renal SLE patients compared to controls; five were identified by mass spectrometry, including one isoform of pro-atherogenic apoCIII. Total apoCIII levels (assessed by ELISA) in lupus nephritis patients were significantly elevated compared to controls or non-renal SLE patients. Thus, levels of oxidized proteins and apoCIII may be useful biomarkers in SLE studies.

Fabry disease in a heterozygote presenting as hand ischaemia and painful acroparaesthesia.

A 48‐year‐old woman presented with acute unilateral ischaemia of the left hand. She had a background of chronic peripheral neuropathic pain, palpitations, anaemia and an episode of superficial thrombophlebitis. Physical examination revealed non‐blanching purple discoloration of her left fingers and her left thumb, index finger and thenar eminance appeared ischaemic. Digital subtraction angiography of the left hand demonstrated reduced flow. Skin punch biopsy histology was unremarkable. The diagnosis of Fabry disease was made on urine lipid profile analysis and confirmed by reduced peripheral blood leukocyte α‐galactosidase A activity.

Salmonella typhimurium mediastinal abscess in a patient with systemic lupus erythematosus.

We describe a case of a Salmonella typhimurium mediastinal abscess in a patient with systemic lupus erythematosus (SLE). Patients with SLE are predisposed to nontyphoidal salmonella infection with a high incidenceof bacteraemiaand abscessformation.Our case is the first report of a mediastinalabscess from Salmonella typhimurium in an SLE patient and highlights the need for thorough assessment and treatment of SLE patients who have this organism identified.