Borut Bozic

The basis of the synovial fluid analysis.

Abstract Synovial fluid fills the spaces in the joint cavities. Many diseases can affect the joints and frequently only a direct examination of the tissue or synovial fluid will yield the correct diagnosis. The article presents the basis of the synovial fluid analysis and a relevant decision- making scheme. The reliability and applicability of synovial fluid tests are commented upon. The synovial fluid analysis undoubtedly plays an important role in the management of patients with joint diseases. Due to a lack of interlaboratory comparisons, and therefore expert opinions on the interpretation of results, this knowledge and the acquisition of relevant experience should be encouraged to enable evaluation of the clinical applicability of recent discoveries in synovial fluid pathology.

Anti-β2-Glycoprotein I ELISA : Methodology, determination of cut-off values in 434 healthy caucasians and evaluation of monoclonal antibodies as possible international standards

Abstract Antibodies against β2-glycoprotein I are among the most commonly detected subset of antiphospholipid antibodies. The inter-laboratory comparability of results is hardly possible due to methodological differences, lack of international standards and different cut-off values. We evaluated an ELISA for the detection of anti-β2-glycoprotein I using the analytical goals based on biological variations for similar analytes (immunoglobulins). By our ELISA we fulfilled the optimal (IgA, IgM) or minimal (IgG) analytical goals in longterm imprecision. The determination of cut-off values was based on the frequency distribution of results obtained on 434 healthy Caucasians. To aim at a better inter-laboratory comparability we tested two monoclonal antibodies as possible calibrators: HCAL (IgG) and EY2C9 (IgM). Binding properties determined by dilutional curves showed great similarities with polyclonal sera, used as in-house standards. Cut-off values were expressed by concentrations of IgG and IgM monoclonal antibodies (4.5 and 25.3 μg/l). Our study shows the possibility for a successful application of analytical goals based on biological variation even when data for a particular analyte are not available. The expression of cut-off values, obtained on a large scale Caucasian population, by the concentration of IgG and IgM monoclonal antibodies could make possible a more reliable inter-laboratory comparison of data.

The Dissociation of Arterial Hypertension and Lupus Glomerulonephritis in Systemic Lupus Erythematosus

In spite of several articles questioning the general opinion that arterial hypertension in patients with systemic lupus erythematosus (SLE) is only the consequence of lupus glomerulonephritis (LGN), this still remains the usual pathophysiologic explanation. The purpose of this study was to explore the correlations between hypertension and LGN and to assess the importance of hypertension control for the prognosis of patients. A retrospective analysis of 173 patients with SLE over a period of 14 years was performed. For most of the patients, data were available from regular follow-up visits over an average of 6 years. Our results show a dissociation of hypertension and LGN and an association of hypertension and renal dysfunction. Severe hypertensive renal vascular lesions correlated well with a decrease of renal function. Successful treatment of hypertension is therefore essential in order to prevent deterioration of renal function in patients with LGN.

Antibodies against annexin A5: Detection pitfalls and clinical associations

Antiphospholipid syndrome (APS) has been defined as a clinical and laboratory entity. Laboratory criteria include the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA), collectively termed as antiphospholipid antibodies (aPL). However, there has been a rising interest in antibodies against so-called protein cofactors, particularly in β2-glycoprotein I. In the early 90s, annexins were considered as target antigens for aPL, but at present the exact role of antibodies against annexins (aANX) remains puzzling. This review is concerned with annexin V or annexin A5 (ANXA5), a widespread member of the annexin family, and antibodies directed towards it. We have endeavoured to summarise essential information about the detection of anti-annexin V antibodies (aANXA5) and their clinical relevance. This review has also brought together some relevant published data concerning the structure, physiological role and therapeutic potential of ANXA5.

Concomitant isolation of protein C inhibitor and unnicked β2-glycoprotein I

Abstract β2-Glycoprotein I (β2GPI) is the major target molecule for so-called anticardiolipin antibodies. We evaluated the isolation procedure of β2GPI from human plasma with special emphasis on the time of precipitation, composition of different isolated fractions and their antigenic properties. The isolation was initiated by perchloric acid precipitation for either 3, 18 or 50 min, followed by heparin affinity and cationic exchange chromatography. The properties of isolated proteins were tested by rocket electrophoresis, enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, immunoblotting and N-terminal sequencing. Each isolation procedure, regardless of the perchloric acid precipitation duration, resulted in three distinct protein peaks, differing in protein composition qualitatively. Comparing sequential peaks between the isolations of different precipitation times, we found that all the three first peaks (set of peaks No. 1), all the three second peaks (set No. 2) as well as the three third peaks (set No. 3) consisted of identical proteins but in different quantities. Set No. 1 was composed of immunoglobulins and a lesser amount of β2GPI. In set No. 2 only unnicked β2GPI was detected. Protein C inhibitor was found in addition to smaller amounts of unnicked β2GPI in set No. 3. Oxidation or degradation of β2GPI during the isolation procedure did not result in a mixture of different forms of β2GPI but rather in a lower yield of wild-type β2GPI. The co-existence of β2GPI and protein C inhibitor in the isolated fractions may suggest their protein-protein interactions in vivo.

Serum amyloid A activation of human coronary artery endothelial cells exhibits a neutrophil promoting molecular profile.

BACKGROUND Serum amyloid A (SAA) has been shown to be an active participant in atherosclerosis and cardiovascular diseases. SAA-stimulated human coronary artery endothelial cells (HCAEC) were reported to release pro-inflammatory cytokines, chemokines and adhesion molecules; however it remains unclear which putative SAA receptors are present in these cells and how they act. We investigated the effects of inflammatory stimuli on the expression of SAA receptors, signaling pathways and molecular profiles in HCAEC. METHODOLOGY/PRINCIPLE FINDINGS HCAEC were cultured in vitro and stimulated with SAA (1000nM) or IL-1β (1000pg/ml). Expression of mRNA was determined by qPCR, and expression and quantification of proteins were assessed by dot array blots and ELISA, respectively. Protein phosphorylation was determined by dot blot arrays and Western blots. We report that all potential SAA receptors tested (FPR2/ALX, RAGE, TANIS, TLR2, TLR4 and CLA-1/hSR-B1) are expressed in HCAEC. Importantly, IL-1β or SAA significantly increased solely the expression of the innate immune receptor TLR2. SAA upregulated the phosphorylation of ERK1/2, NF-κB (p65, p105) and JNK, as well as expression/release of IL-6, IL-8, G-CSF, GM-CSF, ICAM-1 and VCAM-1, all potent molecules involved in neutrophil-related activities. A TLR2-dependent positive feedback mechanism of SAA expression was found. CONCLUSION/SIGNIFICANCE SAA stimulated responses in HCAEC target neutrophil rather than monocyte/macrophage activation.

Autoimmune reactions after electro-oxidation of IgG from healthy persons: relevance of electric current and antioxidants.

Abstract:  Proteins, including immunoglobulins, can be modified by oxidation. Extensive oxidation of immunoglobulins leads to denaturation and loss of biological activity, while initial steps of oxidation may change their specificity due to chemical alteration of the paratope. Electro‐oxidation of the IgG fraction from healthy persons progress to auto‐immunoreactivity, as shown for several autoantibodies including anti‐β2‐glycoprotein I. Changes in immunoreactivity of IgG due to oxidative reactions highly depend on electric current and levels of serum antioxidants. Autoimmune reactions, leading to certain autoimmune diseases, may be partially a consequence of unbalanced antioxidative status of an individual.

Atherogenesis, Inflammation and Autoimmunity - An Overview

In the 16th century, Leonardo Da Vinci had described “the narrowing of the passage of blood vessels, thickening of the coats of these vessels and hardening of arteries” in his work (Boon, 2009). This is the first known documentation of atherosclerosis (AS). Today, our understanding of atherogenesis as a process of a chronic inflammatory disease has been greatly promoted by many theories, such as, the cellular cycle of cholesterol and hypercholesterolemia, dysfunction of endothelial cells, oxidized lipoproteins, discovery of scavenger receptors and response to injury theory, among others. The factors leading to the elucidation of atherogenesis are still not all known, since AS can persist silently (subclinically) without showing any serious symptoms for a longer period. That, together with the fact that AS is not limited to warm-blooded vertebrates and the occurrence of intimal thickening of coronary arteries in rabbitfish (Duran et al., 2010), as well as evidence of coronary AS in salmonids (Farrell, 2002), brings proof that AS is still a largely elusive, complicated and multi-component disease. AS is not a modern world disease as determined by recent images obtained from coronary arteries and the aorta of ancient mummies. Atheromatous lesions, as well as aortic AS were identified in an Aleutian mummy from Alaska originating from 400 AD (Zimmerman, 1998). In 2009, images generated by computer tomography of vascular calcifications in examined Egyptian mummies found that 16 out of the 22 examined mummies had identifiable cardiovascular tissue, with definite AS present in 5 and AS found in additional 4 mummies (together 56% of total) (Allam et al., 2009). This study was recently expanded to 52 ancient Egyptian mummies from the Middle Kingdom to the Greco-Roman period and identified cardiovascular structures in 44 mummies, with 20 of these showing either definite AS (n=12, as defined by calcification within the wall of an identifiable artery) or probable AS (n=8, as defined by calcifications along the expected course of an artery) (Allam et al., 2011).

Counterimmunoelectrophoresis: fast, easy and cost-effective method for the detection of autoantibodies to intracellular antigens.

We read with interest the short communication of Bossuyt et al. about the evaluation of automated enzyme immunoassays for the detection of antibodies to extractable nuclear antigens, published in Clinical Chemistry and Laboratory Medicine 2001; 39(7): 658–659. It was one of many attempts to find an easy and as-fast-as-possible way to detect autoantibodies against intracellular antigens (1). Unfortunately, nobody has yet found the best solution to meet all the needs of rheumatologists and demands of insurance companies. We all seek the best screen at the shortest time possible in the most cost-effective way. Therefore, we believe it is impossible to overlook the ongoing effort of a group of experts constituing the European Consensus Study Group for Autoantibodies who meet annually at the European Workshop for Rheumatology Research (EWRR). The group was formed in 1988 to examine the sensitivity and reproducibility of different methodologies for the detection of autoantibodies to intracellular antigens such as immunofluorescence, gel techniques (radial immunodiffusion (RID) and counterimmunoelectrophoresis (CIE)), ELISA, Western blotting, and then to improve the performance of these tests. Therefore, every year 10 different sera samples are sent to 35–40 leading laboratories from 21 different European countries to test for as many autoantibodies against intracellular antigens as possible, and the results of these exercises show a tremendous improvement in detection rates (2, 3). A significant step in the life of the EWRR has been reached when laboratory experts in the consensus group agreed to submit their protocols, of which the best were chosen and published in the Manual of Biological Markers of Disease (4). Also, participating laboratories are obliged to function as national expert laboratories to disseminate the knowledge from the consensus meetings to other laboratories and clinicians. One of the main conclusions of the consensus group was that different detection techniques could not be directly compared. Immunofluorescence as a good screening method should preceed all the more specific ones. Results obtained by ELISA or immunoblotting tests should be further confirmed by one of the gel techniques (RID or CIE). The Immunology Laboratory at the Department of Rheumatology, University Medical Centre, Ljubljana, Slovenia has been a part of the consensus group since the very beginning, contributing to the CIE protocol for the detection of autoantibodies against different intracellular antigens (5). In the last 10 years, sera from over 30,000 patients have been tested with this version of CIE with most satisfactory results at the lowest possible cost. Because immune complexes formed in CIE gel precipitate as lines, all the reactions between autoantibodies and relevant antigens are actually seen. This includes also those with unknown autoantibodies. Therefore, many rare, but sometimes diagnostically important autoantibodies (PCNA, SL, Ku, PM/Scl, etc.) were discovered by simple routine testing. Considering the results, CIE is neither expensive nor complicated: it can be performed at very low cost in a very reasonable time, 24 hours for the overall positive/negative result and additional 24 hours to test for specific autoantibodies. This is, for this type of analytes, more than acceptable. A trained technician can test over 100 sera in just 2 hours needed for screening for positivity with two antigen substrates and for further characterisation of all positives using relevant standard antisera. The negative CIE test means that the tested serum is negative for all autoantibodies against most common antigens (Ro, La, Sm, U1RNP, Jo-1, Scl-70) and also against some rare, but very important ones (PCNA, PM/Scl, PL-7, PL-4, Ku, SL), as well as against many unknown antigens. Therefore, there is no need to run separate tests for each autoantibody separately as is the case with ELISA technique. Based on over 20 years of experience, we have summarised major practical advantages and disadvantages of the two-step CIE procedure.