Decrease in 14-3-3η protein levels is correlated with improvement in disease activity in patients with rheumatoid arthritis treated with Tofacitinib.

14-3-3η protein is a proinflammatory mediator that may represent a novel diagnostic and prognostic biomarker for rheumatoid arthritis (RA). We assessed the correlation between changes in serum 14-3-3η levels and changes in clinical disease activity measures in RA patients treated with Tofacitinib (TOF). Paired serum samples from 35 patients with RA were obtained at baseline and 5 months after the initiation of treatment with TOF. The levels of 14-3-3η were measured by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). The cut-off was defined as 0.19 ng/ml. 14-3-3η positivity was found in 57% of the patients at baseline and in 37% of the patients after 5 months of treatment. Mean ± SD baseline 14-3-3η levels [4.92 ± 8.86 ng/ml] were significantly higher (p < 0.005) than 14-3-3η levels following treatment [1.97 ± 4.59 ng/ml]. A statistically significant improvement (p < 0.001) of CDAI, SDAI, DAS4ESR and DAS4CRP was achieved after 5 month of treatment. Decrease in 14-3-3η protein levels was highly correlated with improvement in DAS4ESR (r = 0.50, p < 0.01), DAS4CRP (r = 0.46, p < 0.01) and ESR (r = 0.36, p = 0.03) and moderately correlated with improvement in CDAI (r = 0.32, p = 0.065) and SDAI (r = 0.33, p = 0.051). The correlation between decrease in 14-3-3η levels and improvement in DAS4ESR remained significant in a partial correlation analysis controlling for ESR (r = 0.39, p = 0.02). This study demonstrates that in RA patients who were treated with TOF, decrease in 14-3-3η levels is correlated with improvement in clinical disease activity parameters. The 14-3-3η protein may serve as an objective biomarker for monitoring of TOF therapy response.

Coincidence of granulomatosis and polyangiitis with atypical clinical manifestation and antiphospholipid syndrome.

Granulomatosis and angiitis (GPA) is a multisystemic disease characterized by a granulomatous inflammation, tissue necrosis, and vasculitis of small and medium-sized blood vessels. Although the disease has a predilection for the upper respiratory tract, lungs, and kidneys, any organ system may be affected. Here, we present a case of generalized GPA manifested initially by necrotizing isolated parotitis and later by pulmonary-renal syndrome. Simultaneously with pulmonary hemorrhage, our patient developed an antiphospholipid syndrome (APS) presenting with deep vein thrombosis and strongly positive lupus anticoagulant. To the best of our knowledge the coincidence of parotitis and pulmonary-renal syndrome due to GPA and APS has never been reported previously. Concomitant venous thromboembolism may be life-threatening in a patient with GPA. Early diagnosis and institution of the proper therapy are critical in order to prevent organ damage.

Improved accuracy in DFS pattern interpretation using a novel HEp-2 ELITE system.

INTRODUCTION/OBJECTIVES: Accurate interpretation of DFS70 (dense fine speckled 70) and mixed antinuclear antibodies (ANAs) patterns can be challenging using conventional HEp-2 immunofluorescence (IIF) method. We evaluated a novel HEp-2 IIF substrate (HEp-2 ELITE/DFS70-KO) composed of a mixture of engineered HEp-2 devoid of the DFS70 autoantigen and conventional HEp-2 cells. The study assessed the utility of the new substrate in ANA screening and its advantages. METHOD: One thousand and five consecutive routine samples sent for ANA screening were tested on both standard HEp-2 and the HEp-2 ELITE DFS70 KO substrates (ImmuGlo ANA HEp-2 and HEp-2 ELITE/DFS70-KO, Trinity Biotech, Buffalo, NY). Anti-DFS70 antibody specificity was additionally determined by immunoblot (IB). Clinical and serological data were included in the analysis of the overall impact of the novel HEp-2 substrate on DFS pattern interpretation. RESULTS: Of the 22 cases suspected as positive for DFS pattern alone or in combination with homogeneous or speckled patterns on conventional HEp-2 cells, 17 were interpreted with a higher accuracy using the new HEp-2 ELITE method as positive for DFS70 (monospecific DFS70 (10), mixed DFS70 (7)), speckled (3), and DFS (2) patterns. CONCLUSIONS: The new substrate was not only useful in deciphering unclear mixed ANA patterns but also highly sensitive in detecting DFS70 pattern in comparison to the DFS70 positivity obtained using IB.

A better definition of the anti-DFS70 antibody screening by IIF methods.

BACKGROUND: Anti-DFS70 antibodies have been recently included in a new testing algorithm for patients with suspicion of connective tissue diseases (CTDs). This algorithm enables to assess the probability of having a CTD in patients with a positive antinuclear antibodies (ANA) result. The aim of the study was to analyze the the inter-method agreement between three different HEp-2 cell substrates for anti-DFS70 detection, focusing on two novel IIF methods that assess the presence of monospecific anti-DFS70 antibodies. METHODS: Immunological and clinical records of 29 patients who were double positive for anti-DFS70 autoantibodies using chemiluminescence assay (CIA) and Immunoblot (IB) were studied. The IIF on HEp-2 cells were determined using slides from Inova Diagnostics, Euroimmun and Immco. The capability to detect isolated anti-DFS70 antibodies was compared using immunoadsorption on NOVA Lite HEp-2 Select (Inova Diagnostics) and the HEp-2 ELITE/DFS70 knockout test (Immco). RESULTS: The three substrates had very good sensitivity for detecting patients with anti-DFS staining pattern (93.1%, 79.3% and 72.4% for Euroimmun, Immco and Inova respectively). Most of the patients had full inhibition of DFS pattern (65.5%) by immunoabsorption test. Also, the 55.2% of the subjects were positive for monospecific DFS pattern using HEp-2 ELITE/DFS70 knockout test. However, the correlation between the full inhibition by immunoadsorption and the monospecific DFS pattern in knockout cells was very low (kappa: 0.22). CONCLUSION: The evaluation of monospecific anti-DFS70 antibodies is clinically fundamental and challenging using traditional HEp-2 IIF. Results obtained in this study support the hypothesis that the lack of standardization across IIF kits along with the subjectivity of user interpretation among other factors contribute to the overall reduction in the agreement.

The diagnostic value of 14-3-3η protein levels in patients with rheumatoid arthritis.

14-3-3η may represent a useful diagnostic biomarker for rheumatoid arthritis (RA). We assessed the prevalence and serum levels of 14-3-3η in patients with RA and in patients with other rheumatic diseases. Serum levels of 14-3-3η were measured in 96 patients with RA, in 101 patients with other rheumatic diseases, and in 66 healthy subjects. All of the sera samples were evaluated by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). Median (IQR) 14-3-3η levels were significantly higher in the early RA group [0.25 ng/ml (0.075-3.11)] and in patients with established RA [0.15 ng/ml (0.08-1.26)] than in healthy subjects [0 ng/ml (0-0)] and disease controls: SLE [0.01 ng/ml (0-0.055)], AS [0.05 ng/ml (0-0.255)], and PsA [0.01 ng/ml (0-0.065)]. The prevalence of 14-3-3η positivity in patients with early RA was 58%, significantly higher than that in disease controls and healthy subjects (p < 0.001). In patients with established RA, this prevalence was 43%, and it was significantly higher than that in patients with other rheumatic diseases and healthy subjects (p < 0.05), excluding the AS group (p = 0.054). In the early RA cohort, the positivity for 14-3-3η, RF, and anti-CCP was 58%, 67%, and 71%, respectively. Eighty-two percent of the patients in this cohort were positive for at least one of these biomarkers. The concentration of 14-3-3η protein may be used to distinguish between patients with early RA and patients with other rheumatic diseases.

Probiotic supplementation with Lactobacillus casei (Actimel) induces a Th1 response in an animal model of antiphospholipid syndrome.

Probiotic fermented milk products have the capacity to modulate many immunological mechanisms. Several attempts have been made to alter the progression of various atopic and inflammatory disorders in which the immune system plays a major role. We studied this issue in an animal model of the antiphospholipid syndrome (APS) by supplementing the animals' daily intake with a probiotic mixture. We studied the effects of nutritional supplementation of a commercial product that consists of 10(8)/ml Lactobacillus casei (Actimel) on Balb/c mice that were immunized with beta-2- glycoprotein (beta2GPI) in order to induce a familiar murine model of APS. As controls, we used similar animals that were fed with either yogurt or sham solution as a supplement. We analyzed the effect of Actimel on the concentrations of interleukin (IL)-10 interferon gamma (IFNgamma) as well as the extent of the primary T cell response to beta2GPI, and the levels of autoantibodies to beta2GPI determined by ELISA. Two weeks after priming (in the hind footpad) of Balb/c mice with beta2GPI, we analyzed the cytokine profile of the animals by measuring the concentration of IL-10 and IFNgamma in the supernatants of lymphocytes that were extracted from the popliteal lymph nodes. Following stimulation with 10 microg/mL of beta2GPI, we noticed significant (P < 0.05) suppression of IL-10 production by the stimulated lymphocytes in the animals fed with Actimel and yogurt in comparison to sham solution (73.42 +/- 29.4, 84.7 +/- 8, 196 +/- 41.62 pg/mL, respectively). Both dairy products enhanced the secretion of IFNgamma from 657 +/- 47.09 pg/mL to 896 +/- 78.1, and 933 +/- 76.7 (P < 0.01), respectively; similarly they also accelerated by a mild degree the level of the T cell primary response to beta2GPI measured by [3H]thymidine incorporation. The level of autoantibodies to beta2GPI was suppressed in mice fed with actimel and yogurt in a significant manner (P < 0.05). Actimel as well as yogurt confer an immunological impact on Balb/c mice immunized with beta2GPI. Actimel was able not only to enhance IFNgamma secretion but also to inhibit IL-10 production.

Novel insights into associations of antibodies against cardiolipin and beta2-glycoprotein I with clinical features of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a clinical autoimmune disorder characterized by arterial and/or venous thrombosis and/or pregnancy morbidity, associated with the persistence of lupus anticoagulant or anticardiolipin (aCL) antibodies. Accumulating evidence indicates that phospholipid binding protein, beta2-glycoprotein I (beta2GPI) represents the major target antigen for antiphospholipid (aPL) antibodies and plays a role in the pathogenesis of APS. It is widely accepted that aPL antibodies detected by conventional solid phase assays in patients with APS are mainly directed against a complex of aCL and anti-beta2GPI, although antibodies against beta2GPI protein can now also be detected by specific ELISA using purified proteins in solid phase. Despite the fact that these antibodies are not listed in the new diagnostic criteria, a high specificity of anti-beta2GPI assay for the clinical features of APS was established. During the last decade, numerous studies have investigated the clinical link between aCL and/or anti-beta2GPI antibodies and diverse features of APS. This manuscript reviews the current studies published recently in this field and discusses the relationship between the existence of aCL and anti-beta2GPI antibodies and the main and unusual manifestations of APS.

Increased prevalence of anti-third generation cyclic citrullinated peptide antibodies in patients with rheumatoid arthritis and CREST syndrome.

To investigate the prevalence of anti-third generation cyclic citrullinated peptide antibodies (anti-CCP3) in patients with systemic connective tissue diseases, we assembled a training set consisting of 115 patients with rheumatoid arthritis (RA), 52 with Calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly, telangiectasia (CREST) syndrome, 21 with scleroderma, 20 with ankylosing spondylitis, 18 with reactive arthritis, 25 with juvenile rheumatoid arthritis (RA), 51 with osteoarthritis, 26 with mixed connective tissue disease, 23 with primary Sjogren's syndrome, 74 with systemic lupus erythematosus, 49 with Polymyalgia rheumatica, and 39 with polymyositis/dermatomyositis. The commercial enzyme-linked immunosorbent assay (ELISA) was used to detect anti-CCP antibodies, including anti-CCP2 (regular, second generation of CCP antigen) and anti-CCP3 (third generation of CCP antigen) in disease-related specimens and normal controls. These serum samples were also evaluated for anti-centomere antibodies by anti-centromere ELISA kit. The higher frequencies of anti-CCP3 and anti-CCP2 were detected in 75.6 and 70.4% patients with RA, respectively. At the same time, anti-CCP3 (not anti-CCP2) was significantly increased in samples isolated from patients with CREST syndrome. The clinical sensitivity of IgG anti-CCP3 for the patients with CREST syndrome was 29% (15 of 52) and the specificity was 96% (384 of 397), with the exception of the RA group. The anti-centromere antibodies were significantly higher in patients with CREST only. The results of our study suggest that compared to anti-CCP2 assay, the new anti-CCP3 assay can enhance the clinical sensitivity for diagnosis of RA and, as an associate marker combined with anticentromere, can distinguish CREST syndrome from other systemic connective tissue diseases, especially RA. The clinical specificity of anti-CCP3 was lower than anti-CCP2 assay in diagnosis of RA because of the crossreaction to the patients with CREST syndrome.

Anti-HMGCR antibodies demonstrate high diagnostic value in the diagnosis of immune-mediated necrotizing myopathy following statin exposure.

Anti-HMGCR antibodies represent a characteristic serological feature of statin-exposed and statin-unexposed patients with immune-mediated necrotizing myopathy (IMNM). We assessed anti-HMGCR antibodies in patients with suspected IMNM following statin exposure and patients with other autoimmune rheumatic diseases. We evaluated the presence of anti-HMGCR autoantibodies in sera samples from 13 statin-exposed patients who were suspected of having IMNM, 38 patients with different inflammatory and autoimmune rheumatic diseases and 29 healthy subjects. The autoantibodies were evaluated by two assays: a new chemiluminescence QUANTA Flash HMGCR kit utilizing BIO-FLASH system and QUANTA Lite® HMGCR ELISA kit. Twelve samples from patients with suspicion for IMNM were found positive for anti-HMGCR antibodies by both assays. Only one of the 13 samples that were found positive by ELISA was negative by CIA. A very good qualitative correlation (κ = 0.95; 95 % CI 0.85-1.0) and quantitative agreement (Spearman's rho 0.87; P value < 0.0001; 95 % CI 0.62-0.96) were found between these two assays. All samples from healthy subjects and from the disease-controlled patient cohort were negative for anti-HMGCR antibodies. In comparison with ELISA results, the CIA exhibited high sensitivity and specificity values of 92.3 and 100 %, respectively. Receiver operating characteristic analysis for CIA and ELISA yielded area under the curve values of 0.99. The presence of anti-HMGCR antibodies may be a useful biomarker of IMNM in statin-exposed patients. There is a good correlation between the two anti-HMGCR antibody assays evaluated in the present study.

Requisite role for interleukin-4 in the acceleration of fatty streaks induced by heat shock protein 65 or Mycobacterium tuberculosis.

Atherosclerotic lesions can be induced in rabbits and mice immunized with heat shock protein 65 (HSP65). In the current study, we investigated the role of interleukin (IL)-4 in the HSP65- and Mycobacterium tuberculosis (MT)-induced models that exhibit an inflammatory phenotype. Fatty streak formation in IL-4-knockout (IL-4 KO) mice immunized with HSP65 or MT was significantly reduced when compared with lesions in wild-type C57BL/6 mice. However, when injected with control (HSP-free) adjuvant, no differences were evident in the lesion size between wild-type and the IL-4 KO mice. Next, we studied comparatively the extent of humoral and cellular immune responses to HSP65 in the IL-4 KO and wild-type mice, as those are thought to be influential in murine atherosclerosis. Anti-HSP65 antibody levels were reduced in the HSP65-immunized IL-4 KO mice as compared with their wild-type littermates, whereas no differences were evident between the groups with respect to the primary cellular immune response to HSP65. Other than the absence of IL-4 in the knockout mice, the pattern of secreting cytokines interferon-gamma and IL-10 in concanavalin A-primed splenocytes was similar between the groups. HSP65-primed inguinal lymphocytes from IL-4 KO mice immunized with HSP65 secreted higher levels of interferon-gamma (previously shown to be proatherogenic in vivo) as compared with their wild-type controls. 12-/15-Lipoxygenase expression, known to be regulated by IL-4 and to contribute to murine atherosclerosis, in the lesions was not influenced by the immunization protocol used or by IL-4 disruption. Thus, IL-4 may prove a principal cytokine in the progression of early "inflammatory" atherosclerotic lesions and may serve as a target for immunomodulation.

Restricted specificity of anti-ribosomal P antibodies to SLE patients in Israel.

OBJECTIVE: Anti-ribosomal P antibodies (aRib-P Ab) are highly specific for systemic lupus erythematosus (SLE), but their correlatation with disease activity and manifestations including renal, hepatic and central nervous system (CNS) involvement is still controversial. The aim of our study was to evaluate the prevalence of aRib-P Ab and their correlation with clinical manifestations and anti-dsDNA antibodies in SLE patients from Israel. METHODS: Elevated titers of aRib-P Ab utilizing the ELISA method were analyzed in 141 sera samples from 44 SLE patients, 20 Familial Mediterranean Fever (FMF) patients, 22 primary antiphospholipid syndrome (PAPS) patients, 12 patients with infections, and 43 healthy individuals. The SLEDAI score was utilized for assessing SLE disease activity. RESULTS: Elevated titers of aRib-P Ab were present in 11% of SLE patients (n = 6). The mean SLEDAI was 7 (range: 3-10). No statistically significant association was observed between the presence of aRib-P Ab and disease manifestations present in the SLEDAI. The 6 SLE patients had renal disease (n = 1), leucopenia (n = 1), rash (n = 3), and CNS involvement manifested as psychosis (n = 1) or depression (n = 1). Elevated titers of anti-dsDNA antibodies were found in 50% of patients with elevated titers of aRib- P Ab. Patients with PAPS, FMF, infections or healthy controls did not harbor elevated titers of aRib-P Ab. CONCLUSION: Elevated titers of aRib-P Ab were restricted to SLE patients. We confirm previously reported associations of aRib-P Ab reactivity with disease activity and elevated anti-dsDNA Ab titers. No significant correlation with a specific manifestation described on the SLEDAI score was established in this small cohort of patients.

Adoptive transfer of beta(2)-glycoprotein I-reactive lymphocytes enhances early atherosclerosis in LDL receptor-deficient mice.

BACKGROUND: It has been proposed that autoimmune factors can influence the progression of atherosclerosis. We have previously shown that immunization of LDL receptor-deficient (LDL-RD mice) with beta(2)-glycoprotein I (beta2GPI; a principal target of "autoimmune" antiphospholipid antibodies) enhances early atherosclerosis. In the present study, we tested the hypothesis that adoptive transfer of beta2GPI-reactive T cells can accelerate fatty streak formation in LDL-RD mice. METHODS AND RESULTS: LDL-RD mice were immunized with human beta2GPI. An additional group of mice were immunized with beta2GPI and boosted with the same antigen 3 weeks later. Control mice with immunized with human serum albumin. Lymphocytes obtained from the draining lymph node cells or from splenocytes of beta2GPI- or human serum albumin-immunized mice were stimulated in vitro with beta2GPI or with the mitogen concavalin A, respectively. The cultured lymphocytes were transferred intraperitoneally to syngenic LDL-RD mice, and the mice were fed a high-fat "Western" diet for 5 weeks until death. Mice injected with lymphocytes from draining lymph nodes or spleens of beta2GPI-immunized animals displayed larger fatty streaks than those induced by control treated animals. T-cell-depleted splenocytes from beta2GPI were unable to promote lesion formation in the mice. CONCLUSIONS: The present study provides the first direct evidence for a role of antigen (beta2GPI)-reactive T cells in the promotion of fatty streaks in mice.

Immunolocalization of beta2-glycoprotein I (apolipoprotein H) to human atherosclerotic plaques: potential implications for lesion progression.

BACKGROUND: beta2-Glycoprotein I (beta2GPI) is a major antigenic target of antiphospholipid antibodies, which possesses natural anticoagulant properties. The aim of the present study was to determine its presence and localization within human atherosclerotic plaques and to study its association with endothelial cells and monocyte macrophages in vitro. METHODS AND RESULTS: Human atherosclerotic lesions were obtained after carotid endarterectomies and studied immunohistochemically with anti-beta2GPI as well as antibodies to CD4/CD8, macrophages, and adhesion molecules. In vitro, human umbilical vein endothelial cells (HUVECs) and U937 (myelomonocytic cell line) cells were investigated for their ability to associate with radiolabeled beta2GPI. We found beta2GPI to be abundantly expressed within the subendothelial regions and intimal-medial borders of human atherosclerotic plaques and to colocalize with CD4-positive lymphocytes. This observation was confirmed by Western blot applied on homogenates of atherosclerotic lesions with anti-beta2GPI antibodies. Both HUVECs and U937 cells bound labeled beta2GPI, and the process was inhibited by oxidized LDL and not by native LDL. CONCLUSIONS: The abundant presence of human beta2GPI within the lesions, its association with endothelial cells and macrophages, and its colocalization with CD4-positive lymphocytes suggests that it may serve as a target for an immune-mediated reaction that can influence lesion progression.

Cellular and humoral immune responses to heat shock protein 65 are both involved in promoting fatty-streak formation in LDL-receptor deficient mice.

OBJECTIVES: This study was designed to determine the role of cellular and humoral immune responses to heat shock protein 65 (HSP65) in murine atherosclerosis. BACKGROUND: Inflammatory processes appear to influence the progression of atherosclerosis. Immunization with HSP65 was previously shown to induce arteriosclerosis in rabbits and to enhance fatty-streak formation in mice. However, it has not been demonstrated directly whether HSP65-reactive antibodies and lymphocytes are separately capable of influencing lesion formation. METHODS: Low density lipoprotein-receptor deficient (LDL-RD) mice were immunized with HSP65 or control bovine serum albumin (BSA). Lymph-node cells, splenocytes and immunoglobulin G (IgG) were obtained from the immunized mice and transferred separately to six groups of syngenic LDL-RD mice. RESULTS: Adoptive transfer of HSP65-reactive lymph node cells increased fatty-streak formation in comparison with mice treated with BSA-primed cells. Similarly, transfer of splenocytes reactive with HSP65 led to enhanced fatty-streak generation compared with mice injected with BSA-sensitized splenocytes. Repeated intraperitoneal administration of IgG from serum of HSP65-immunized mice (every 10 days) enhanced fatty-streak formation in mice in comparison with their anti-BSA-IgG injected littermates. CONCLUSIONS: Antibodies and lymphocytes reactive to HSP65 promote fatty-streak formation in mice, providing direct evidence for the proatherogenic properties of cellular and humoral immunity to HSP65.

Multiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseases.

BACKGROUND: Multiplexed assays using fluorescence microspheres is an exciting technology with multiple applications including the detection of antinuclear autoantibodies (ANA) and autoantibody profiles. It is a rapid, sensitive and automatic method for simultaneous quantitative detection of several autoantibodies. The aim of our study was to determinate ANA and other autoantibodies to the nine extractable nuclear antigens by the AtheNA Multi-Lyte ANA system and compare the results achieved by this method to the routinely used enzyme immunoassay. METHODS: Four hundred eighteen serum samples were tested utililizing the multiplexed method: 96 healthy donors, 86 requested ANA specimens obtained from routine lab, and 236 samples from patients with known autoimmune diseases (43-scleroderma, 113-systemic lupus erythematosus, 38-Sjogren's syndrome, and 42 rheumatoid arthritis). The ANA and antibodies to nine different analytes (SS/A, SS/B, Sm, RNP, Jo-1, Scl-70, dsDNA, Centromere B and Histone) were tested. RESULTS: ANA screening by AtheNA system revealed high concordance of 99 and 97.7% with the enzyme immunoassay test in samples obtained from healthy donors and ANA requested samples, respectively. Evaluation of autoimmune disease-related samples for ANA by AtheNA technology also confirmed a high rate of concordance of 92-97.7% and correlated with the enzyme immunoassay. Positive discrepant results were found for Scl-70 specificity in 12.7% of SLE specimens by AtheNA technology, while all tested sera were negative for this antibody by enzyme immunoassay. Negative discrepant results were observed by the AtheNA system for anti-dsDNA. The sera (15 randomly obtained samples from SLE patients) were positive for anti-dsDNA in 50% of samples in Farr assay and 55% in enzyme immunoassay, respectively. CONCLUSION: We suggest that the AtheNA technology may be a useful diagnostic tool for ANA screening. Additional investigations are required to compare an analytic performance between AtheNA and routine methods in determination of the individual autoantibody profile.

Evaluation of the BioPlex 2200 ANA screen: analysis of 510 healthy subjects: incidence of natural/predictive autoantibodies.

The BioPlex 2200 ANA Screen is a fully automated system that determines levels for 13 different autoimmune antibodies of established clinical significance. The objective of this study was to determine the specificity of the BioPlex 2200 ANA Screen assay and to analyze the antibody profile samples collected from healthy subjects against comparative ELISA and IIF screening methods. A total of 510 specimens were randomly selected from a cohort of apparently healthy blood bank donors. Samples were distributed to five age brackets. All samples were tested using Bio-Rad's ANA Screen kit. Specificity was compared to IIF and ELISA results. Most of the samples were found negative in all ANA screening systems (84.5% by IIF, 92.5% by BioPlex 2200 ANA Screen kit, and 94.5% by ELISA). The frequency of positive results was highest (15.5%) using IIF, in comparison to almost similar results (5.5% vs. 7.5%) achieved by ANA ELISA and BioPlex 2200 ANA Screen kits. The positive rate of autoantibodies was significantly reduced when analyzed by different combinations of ANA screen assays (from 2.35% using IIF + BioPlex ANA Screen tests to 0.98% by using all three tests). Using the BioPlex 2200 ANA Screen system, we were able to identify samples with high levels of individual antibodies: anti-dsDNA at 20-63 IU/mL, antichromatin at 4-8 AI, anti-SmRNP at 2-6 AI, and anti-RNPA at 2-4.5 AI. Importantly, from 7 IIF and ELISA positive sera, 5 of these were also BioPlex 2200 positive, suggesting that the BioPlex is seeing the samples that are of the greatest interest, using the established techniques. The specificity of the BioPlex 2200 ANA Screen analysis of 13 different analytes (dsDNA, centromere B, chromatin, Jo1, ribosomal P, RNP 68, RNP A, Scl-70, Sm, SmPNP, SS-A52, SS-A60, SS-B) is comparable (P < 0.252) to the ELISA ANA screening test. Like the ELISA, the BioPlex 2200 has a lower (P < 0.001) positive rate than IIF for the autoantibody screening.

A fully automated IIF system for the detection of antinuclear antibodies and antineutrophil cytoplasmic antibodies.

Indirect immunofluorescence (IIF) is the main technique for the detection of antinuclear antibodies (ANA) and antineutrophil cytoplasmic antibodies (ANCA). The fully automated IIF processor HELIOS(®) is the first IIF processor that is able to automatically prepare slides and perform automatic reading. The objective of the present study was to determine the diagnostic performance of this system for ANA and ANCA IIF interpretation, in comparison with visual IIF. ANA detection by visual IIF or HELIOS(®) was performed on 425 sera samples including: 218 consecutive samples submitted to a reference laboratory for routine ANA testing, 137 samples from healthy subjects and 70 ANA/ENA positive samples. For ANCA determination, 170 sera samples were collected: 40 samples for routine testing, 90 samples from healthy blood donors and 40 anti-PR3/anti-MPO positive subjects. Good correlation was found for the visual and automated ANA IIF approach regarding positive/negative discrimination of these samples (kappa = 0.633 for ANA positive samples and kappa = 0.657 for ANA negative samples, respectively). Positive/negative IIF ANCA discrimination by HELIOS(®) and visual IIF revealed a complete agreement of 100% in sera from healthy patients and PR3/MPO positive samples (kappa = 1.00). There was 95% agreement between the ANCA IIF performed by automated and visual IIF on the investigation of routine samples. Based on these results, HELIOS(®) demonstrated a high diagnostic performance for the automated ANA and ANCA IIF interpretation that was similar to a visual reading in all groups of samples.

Emerging cross-regulatory roles of immunity and autoimmunity in atherosclerosis.

Atherosclerosis is a histopathological process of a multifactorial origin. Whereas the genetic and biochemical causes received considerable attention, the involvement of the immune system has generally been considered negligible. In recent years evidence has been presented to support the dominant role played by the immune system in atherosclerosis. Two major antigenic determinants against which the immune response may be triggered have been suggested, namely the heat shock protein 60/65 and oxidized low density lipoprotein. The current paper reviews the data regarding the involvement of the immune system in atherogenesis with respect to the antigenic candidates mentioned above.

Hyperimmunization of apo-E-deficient mice with homologous malondialdehyde low-density lipoprotein suppresses early atherogenesis.

The role of the immune system in modulating atherosclerosis has recently been the subject of intensive research. Several previous authors have put forward a paradigm of the autoimmune process occurring in the vicinity of the plaque. Two recent studies have shown that immunization of rabbits with homologous modified low-density lipoprotein (LDL) led to suppression of atherosclerosis. In the current study we evaluated the effects of homologous malondialdehyde (MDA)-LDL immunizations on atherogenesis in apo-E-deficient mice. Two groups of female chow-diet-fed, apo-E-deficient mice (n = 10) were either immunized with homologous MDA-LDL or with phosphate buffer saline (PBS) at 2-week intervals. The mice were sacrificed 12 weeks following the primary immunization. The MDA-LDL-immunized mice were shown to develop high titers of anti-MDA-LDL antibodies. Atherosclerosis, determined by the lesion size at the aortic sinus, was significantly suppressed in the MDA-LDL-immunized mice as compared with their littermates immunized with PBS (mean area +/- S.D.; 74000 +/- 17300 microm2 versus 158000 +/- 12800 microm2; P < 0.01). No differences were found between the groups with respect to the cellular composition of the atherosclerotic plaques. The results of this study show that immunization with MDA-LDL has a protective effect in apo-E-deficient mice, and further suggests that this mouse model is suitable for studies of immunomodulation.

10th International Congress on Antiphospholipid Antibodies--summary.

The 10th International Congress on Antiphospholipid Antibodies (Sicily, Italy, September 29-October 3, 2002) (Fig. 1) provided enlightening aspects on the recent developments in antiphospholipid syndrome (APS) and antiphospholipid antibodies in more than 150 lectures and posters. Researchers from all aspects of medicine attended the meeting, implicating the systemic characteristics of APS. The important breakthroughs are summarized.