Gabriella Lakos

Transglutaminase 2-/- mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells

Tissue transglutaminase (TGase2) is a protein-crosslinking enzyme known to be associated with the in vivo apoptosis program. Here we report that apoptosis could be induced in TGase2-/- mice; however, the clearance of apoptotic cells was defective during the involution of thymus elicited by dexamethasone, anti-CD3 antibody, or γ-irradiation, and in the liver after induced hyperplasia. The lack of TGase2 prevented the production of active transforming growth factor-β1 in macrophages exposed to apoptotic cells, which is required for the up-regulation of TGase2 in the thymus in vivo, for accelerating deletion of CD4+CD8+ cells and for efficient phagocytosis of apoptotic bodies. The deficiency is associated with the development of splenomegaly, autoantibodies, and immune complex glomerulonephritis in TGase2-/- mice. These findings have broad implications not only for diseases linked to inflammation and autoimmunity but also for understanding the interrelationship between the apoptosis and phagocytosis process.

Anti-citrullinated protein/peptide autoantibodies in association with genetic and environmental factors as indicators of disease outcome in rheumatoid arthritis.

Anti-citrullinated protein/peptide antibodies (ACPA) have recently emerged as sensitive and specific serological markers of rheumatoid arthritis (RA), providing superior alternative of the rheumatoid factor (RF) test in the laboratory diagnostics of RA. Citrullination is a post-translational modification of arginine by deimination, physiologically occurring during apoptosis, inflammation or keratinization. The presence of several citrullinated proteins has been demonstrated in the RA synovium. The identification of citrullinated epitopes as targets led to the development of the first and later second-generation anti-cyclic citrullinated peptide (anti-CCP) antibody assays. The anti-Sa antibody has been identified a decade ago; however, recent studies confirmed that anti-Sa is directed against citrullinated vimentin. The determination of ACPA may have important prognostic significance, since ACPA production can precede the onset of clinical RA symptoms by years. ACPA(+) individuals with early, undifferentiated arthritis may have higher risk to develop RA. ACPA has important prognostic role during the progression of RA and it has also been associated with pronounced radiographic progression. ACPA production has been associated with several genetic predisposing factors, including HLA-DRB1 and PTPN22 1858T alleles, as well as with environmental and lifestyle-related factors, primarily smoking and possibly, the use of oral contraceptives and excessive caffeine intake. Thus, the assessment of ACPA, in addition to clinical, radiographic and genetic outcome measures may be important to assess disease prognosis and aids to design effective, early therapeutic strategies.

‘Criteria’ aPL tests: Report of a Task Force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, Texas, April 2010:

Current classification criteria for definite antiphospholipid syndrome (APS) mandate the use of one or more of three positive ‘standardized’ laboratory assays to detect antiphospholipid antibodies (aPL) (viz: anticardiolipin [aCL] IgG and IgM; anti-β2glycoprotein I [anti-β2GPI] antibodies IgG and IgM; and/or a lupus anticoagulant [LAC]), when at least one of the two major clinical manifestations (thrombosis or pregnancy losses) are present. Although, efforts of standardization for these ‘criteria’ aPL tests have been conducted over the last 27 years, reports of inconsistencies, inter-assay and inter-laboratory variation in the results of aCL, LAC, and anti-β2GPI, and problems with the interpretation and the clinical value of the tests still exist, which affect the consistency of the diagnosis of APS. A Task Force of scientists and pioneers in the field from different countries, subdivided in three working groups, discussed and analyzed critical questions related to ‘criteria’ aPL tests in an evidence-based manner, during the 13th International Congress on Antiphospholipid Antibodies (APLA 2010, April 13–16, 2010, Galveston, TX). These included: review of the standardization and the need for international consensus protocol for aCL and anti-β2GPI tests; the use of monoclonal and/or polyclonal standards in the calibration curve of those tests; and the need for establishment of international units of measurement for anti-β2GPI tests. The group also reviewed the recently updated guidelines for LAC testing, and analyzed and discussed the possibility of stratification of ‘criteria’ aPL tests as risk factors for APS, as well as the clinical value of single positive vs. multiple aPL positivity. The group members presented, discussed, analyzed data, updated and re-defined those critical questions at a preconference workshop that was open to congress attendees. This report summarizes the findings, conclusions, and recommendations of this Task Force.

Heat shock protein 70 is a potent activator of the human complement system

Abstract According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.

Seroreactivity to microbial components in Crohn's disease is associated with ileal involvement, noninflammatory disease behavior and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patients

Background: Antibodies directed against Saccharomyces cerevisiae (ASCA), perinuclear components of neutrophils (pANCA), and porin protein C of Escherichia coli (anti‐OmpC) are reported to be associated with disease phenotype and may be of diagnostic importance in inflammatory bowel disease (IBD). Since limited data are available from Eastern Europe, we assessed the above antibodies in Hungarian IBD patients. Methods: In all, 653 well‐characterized, unrelated consecutive IBD patients (Crohns disease [CD]: 558, m/f: 263/295, duration: 8.1 ± 10.7 years; ulcerative colitis [UC]: 95, m/f: 44/51, duration: 8.9 ± 9.8 years) and 100 healthy subjects were investigated. Sera were assayed for anti‐Omp and ASCA by enzyme‐linked immunosorbent assay (ELISA) and ANCA by indirect immunofluorescence assay (IIF). TLR4 and NOD2/CARD15 variants were tested by polymerase chain reaction/restriction fragment length polymorphism (PCR‐RFLP). Detailed clinical phenotypes were determined by reviewing the medical charts. Results: Anti‐Omp, ASCA, and atypical pANCA antibodies were present in 31.2%, 59.3%, and 13.8% of CD, 24.2%, 13.7%, and 48.5% of UC patients, and in 20%, 16%, and 5.6% of controls, respectively. ASCA and anti‐Omp positivity were associated with increased risk for CD (odds ratio [OR]ASCA = 7.65, 95% confidence interval [CI]: 4.37–13.4; OROmp = 1.81, 95% CI: 1.08–3.05). In a logistic regression analysis, anti‐Omp and ASCA were independently associated with ileal and noninflammatory disease, but not with a risk for surgery or response to steroids or infliximab. A serology dosage effect was also observed. ASCA and anti‐Omp antibodies were associated with NOD2/CARD15, in addition to a gene dosage effect. No associations were found in UC. Conclusions: Serological markers were useful in the differentiation between CD and UC in an Eastern European IBD cohort. Reactivity to microbial components was associated with disease phenotype and NOD2/CARD15 genotype, further supporting the role of altered microbial sensing in the pathogenesis of CD.

Isotype distribution and clinical relevance of anti-β2-glycoprotein I (β2-GPI) antibodies: importance of IgA isotype

The aim of this study was to evaluate the prevalence of IgG, IgA and IgM anti‐β2‐GPI antibodies in anti‐phospholipid syndrome (APS), and to establish the clinical significance of IgA type antibodies compared with the other isotypes. Anti‐β2‐GPI antibodies were measured in the sera of 70 patients by solid‐phase enzyme immunoassay in γ‐irradiated polystyrene plates coated with human purified β2‐GPI. Thirty‐three out of the 70 patients were classified as having APS: three of them had primary, and 30 had secondary APS related to systemic lupus erythematosus (SLE). The remaining 37 patients had SLE without APS. Anti‐β2‐GPI antibodies of IgG, IgA and IgM isotypes were present in 84.8%, 59.3% and 51.5% of patients with APS. Both the frequency and the level of each isotype were significantly higher in patients with APS. This association was very strong for IgA (P = 0.0004 for the antibody frequency and P < 0.0001 for the antibody level), as well as for IgG type antibodies (P < 0.0001 and P < 0.0001), whereas it was weaker for IgM (P = 0.01 and P = 0.04). A strong relationship was demonstrated between increased IgA anti‐β2‐GPI antibody levels and a history of venous thrombosis, thrombocytopenia, heart valve disease, livedo reticularis and epilepsy. IgG anti‐β2‐GPI antibodies were associated with the presence of lupus anticoagulant (LA) in addition to the main features of APS. However, antibodies of IgM isotype were related only to thrombocytopenia and heart valve disease. We recommend the evaluation of anti‐β2‐GPI antibodies of IgA isotype in addition to IgG in patients with clinical suspicion of APS.

Evaluation of clinical and laboratory features of antiphospholipid syndrome: a retrospective study of 637 patients

We retrospectively analysed the data of 1519 antiphospholipid antibody (APLA) positive patients between 1986 and 1999. Among them 637 were considered to have antiphospholipid syndrome (APS) based on the 1999 preliminary classification criteria, while 704 patients had no clinical signs of the syndrome. Our aim was to compare the autoantibody profile and clinical characteristics of primary and secondary APS, moreover to evaluate the associations between different APLA and specific symptoms attributable to APS. In our results, the APLA profiles for primary and SLE-associated secondary APS were similar. Among the evaluated clinical symptoms, cerebrovascular thrombosis was found to be more frequent in the SLE-associated, than in the primary APS group (P = 0.04). We identified important differences in the clinical profile of patient populations with various types of APLA. Venous thrombosis occurred more frequently in subjects with lupus anticoagulant (LA), than in those with IgG or IgM type ACLA (P < 0.0001), while coronary, carotid and peripheral artery thrombosis occurred more often in subjects with IgG or IgM ACLA (P < 0.0001). These findings may support the role of antibodies to cardiolipin or its cofactor, b2glycoprotein I (b2-GPI) in the initiation and progression of atherosclerosis. Cerebrovascular thrombosis was detected in larger proportion of LA or IgG ACLA-positive patients compared with to IgM ACLA-positive subjects, while the occurrence of foetal loss was similar in all three groups.

Smad-Independent Transforming Growth Factor-β Regulation of Early Growth Response-1 and Sustained Expression in Fibrosis: Implications for Scleroderma

Transforming growth factor-beta (TGF-beta) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-beta and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-beta causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-beta to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-beta, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-beta enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-beta signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.

Analysis of risk factors for the development of thrombotic complications in antiphospholipid antibody positive lupus patients

The objective of this study was to characterize risk factors for thrombotic events in lupus patients. A total of 272 lupus patients were followed up for five years during which the presence of aPL antibodies [anticardiolipin (aCL), anti-beta2-glycoprotein I (aβ2GPI) and lupus anticoagulant (LAC)] were determined, and all thrombotic incidents and antithrombotic therapy-related data were collected. At baseline, three groups were constituted, an aPL–group with 107 aPL negative patients, an aPL+ group with 81 aPL positive patients without clinical thrombosis and a secondary antiphospholipid syndrome (APS) group with 84 aPL+ patients who met the Sapporo criteria. LAC was more common in the APS than the aPL+ group (32.1% versus 9.9%, P < 0.001). The prevalence of clinical thrombotic events was significantly higher when all three types of aPL were present compared to only aCL positive cases. During follow up, aPL appeared in 7.5% of the aPL - group, and 2.8% of this group had thrombotic complications. In the aPL + group, thrombotic events reoccurred in 1.9% of those receiving antithrombotic prophylaxis and 6.9% of those without primary prophylaxis. Despite anticoagulant therapy, thrombotic events reoccurred in 8.3% of the APS group. These findings indicate that LAC, constant and cumulative presence of aPL and previous thrombosis are positive predictors for the development of thrombotic complication in lupus patients.

Th1/Th2 imbalance, measured by circulating and intracytoplasmic inflammatory cytokines : Immunological alterations in acute coronary syndrome and stable coronary artery disease

To describe how peripheral immune‐parameters reflect the inflammatory alterations of the atherosclerotic plaques in coronary atherosclerosis. We measured general inflammatory markers C‐reactive protein (CRP) and granulocyte activity, lymphocyte subpopulations and their state of activation, evaluated circulating Th1/Th2‐type cytokines, and specific intracytoplasmic cytokines. We investigated the association of immune‐parameters with disease outcome and mortality. Thirty‐three patients with acute coronary syndrome (ACS), 62 with stable coronary artery disease (CAD) and 58 healthy controls were studied. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines and autoantibodies were assessed using enzyme‐linked immunosorbent assay (ELISA), while intracellular cytokine levels were measured by flow cytometry after intracellular staining. We found elevated levels of CRP and granulocyte activity in ACS versus CAD (P < 0.001, P = 0.017, respectively). Natural killer (NK) cell percentages were elevated, while percentage of T cells to the total lymphocyte count was slightly decreased in ACS compared to controls (P < 0.0001, P = 0.012, respectively). Both forms of coronary atherosclerosis showed significantly higher percentages of activated T cells than controls when stained for the activation markers HLA‐DR3 and CD69+ (ACS: P < 0.0001, P = 0.002, CAD: P < 0.0001, P = 0.018, respectively). IL‐1, IL‐4 and IL‐10 proved significantly higher in ACS versus controls (P = 0.036, P = 0.01, P < 0.0001 respectively). Th1 to Th2 ratio shifted towards a Th1 dominance in both diseases. Both general proinflammatory markers and activated T cells signify CAD. The orchestrated proinflammatory cascade eventually leads to the development of the disease.