Ole Petter Rekvig

European League Against Rheumatism recommendations for monitoring patients with systemic lupus erythematosus in clinical practice and in observational studies

Objectives To develop recommendations for monitoring patients with systemic lupus erythematosus (SLE) in clinical practice and observational studies and to develop a standardised core set of variables to monitor SLE. Methods We followed the European League Against Rheumatism (EULAR) standardised procedures for guideline development. The following techniques were applied: nominal groups, Delphi surveys for prioritisation, small group discussion, systematic literature review and two Delphi rounds to obtain agreement. The panel included rheumatologists, internists, dermatologists, a nephrologist and an expert related to national research agencies. The level of evidence and grading of recommendations were determined according to the Levels of Evidence and Grades of Recommendations of the Oxford Centre for Evidence-Based Medicine. Results A total of 10 recommendations have been developed, covering the following aspects: patient assessment, cardiovascular risk factors, other risk factors (osteoporosis, cancer), infection risk (screening, vaccination, monitoring), frequency of assessments, laboratory tests, mucocutaneous involvement, kidney monitoring, neuropsychological manifestations and ophthalmology assessment. A ‘core set’ of minimal variables for the assessment and monitoring of patients with SLE in clinical practice was developed that included some of the recommendations. In addition to the recommendations, indications for specific organ assessments that were viewed as part of good clinical practice were discussed and included in the flow chart. Conclusions A set of recommendations for monitoring patients with SLE in routine clinical practice has been developed. The use of a standardised core set to monitor patients with SLE should facilitate clinical practice, as well as the quality control of care for patients with SLE, and the collection and comparison of data in observational studies.

BK and JC Viruses in Patients with Systemic Lupus Erythematosus: Prevalent and Persistent BK Viruria, Sequence Stability of the Viral Regulatory Regions, and Nondetectable Viremia

A role for polyomaviruses in the pathogenesis of systemic lupus erythematosus (SLE) has been suggested. BK virus (BKV) and JC virus (JCV) were demonstrated in single urine specimens from 7 (16%) of 44 and 5 (11%) of 44 patients with SLE and 0/88 and 18 (21%) of 88 matched healthy controls, respectively. During a 1-year follow-up study, episodes of polyomaviruria were detected in 16 (80%) of 20 patients, BKV in 13, and JCV in 3 patients. A group of 12 (60%) of 20 patients demonstrated persistent or recurrent polyomaviruria, BKV viruria (n=9), or JCV viruria (n=3) in 180 (70%) of 256 specimens. Polyomaviruria was not significantly associated with immunosuppressive therapy. The BKV and JCV isolates revealed predominantly stable archetypal regulatory regions over 3 years, indicating viral persistence rather than reinfection as a cause for urinary shedding. The demonstration of nondetectable viremia and stable archetypal BKV and JCV noncoding control regions during persistent viruria argue against the urinary tract as a focus for the creation of rearranged regulatory region variants.

Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity

Background: The presence of “anti-DNA antibodies in abnormal titres” is a well established criterion for SLE classification, but there is no agreement on the performance of this test. Objective: To study the correlation between clinical findings and five different solid and solution phase anti-DNA antibody assays. Methods: 158 consecutively collected ANA positive sera were studied in a double blind fashion. Anti-DNA antibodies were determined by different solid phase assays (ssDNA-, dsDNA- specific ELISA, EliA anti-dsDNA assay, Crithidia luciliae assay), and by an experimental solution phase anti-DNA assay using biotinylated pUC18 plasmid, human, calf thymus, and E coli DNA. Antibody affinity was determined by surface plasmon resonance. Clinical data were obtained independently of the laboratory analyses and later related to the anti-dsDNA findings. Results: Anti-dsDNA antibodies were most frequently detected by ELISA, but were not specific for SLE as they were present in up to 30% of other disease groups. Those detected by the Crithidia luciliae assay were predictive for SLE, while antibodies binding in solution phase ELISA using the pUC18 correlated strongly with the Crithidia luciliae assay. Surface plasmon resonance analysis showed that antibody binding to pUC18 was not due to higher relative affinity for dsDNA in general, but apparently to specificity for that plasmid DNA. Serum samples from three patients with lupus nephritis were positive in both pUC18 solution phase and Crithidia luciliae assays. Conclusions: Assay principle selection is decisive for the detection of clinically significant anti-DNA antibodies. Revision of the anti-DNA antibody criterion in the SLE classification may be needed.

Nephritogenic Potential of Anti-DNA Antibodies against Necrotic Nucleosomes

Systemic lupus erythematosus is an inflammatory autoimmune syndrome of unknown cause. Kidney disease is a central and serious complication in this syndrome. Deposition of chromatin-containing immune complexes within glomerular membranes is considered a key event in the pathogenesis of lupus nephritis. One set of autoantibodies that participate in these complexes is directed against components of chromatin, particularly against double-stranded DNA (dsDNA). Matzingers danger model implicates chromatin fragments as both inducers and glomerular targets for nephritogenic anti-dsDNA and anti-nucleosome antibodies. In context of this model, apoptosis, secondary necrosis, and exposure of chromatin fragments may causally trigger autoimmunity and subsequent lupus nephritis. The exposure of glomerular basement membrane-associated extracellular chromatin depends on an observed acquired downregulation of renal DNase1 transcription and loss of nuclease activity preceding development of severe nephritis; this downregulation would result in reduced fragmentation and clearance of chromatin fragments. These fragments bind glomerular basement membrane structures with high affinity. In addition, exposed chromatin fragments contain structures that stimulate the innate immune system through Toll-like receptors and the adaptive immune system to produce affinity-maturated pathogenic anti-chromatin and anti-dsDNA antibodies that are central to the development of lupus nephritis.

Lupus Nephritis : The Central Role of Nucleosomes Revealed

Systemic lupus erythematosus (SLE) is an autoimmune syndrome characterized by autoantibodies to nuclear constituents. Some of these antibodies are diagnostically important, whereas others act as disease-modifying factors. One clinically important factor is autoantibodies against dsDNA and nucleosomes, which have overlapping diagnostic and nephritogenic impact in SLE. Although a scientific focus for 5 decades, the molecular and cellular origin of these antibodies, and why they are associated with lupus nephritis, is still not fully understood. A consensus has, however, evolved that antibodies to dsDNA and nucleosomes are central pathogenic factors in the development of lupus nephritis. In contrast, no agreement has been reached as to which glomerular structures are bound by nephritogenic anti-nucleosome antibodies in vivo. Mutually contradictory paradigms and models have evolved simply because we still lack precise and conclusive data to provide definitive insight into how autoantibodies induce lupus nephritis and which specificity is critical in the nephritic process(es). In this review, data demonstrating the central role of nucleosomes in inducing and binding potentially nephritogenic antibodies to DNA and nucleosomes are presented and discussed. These autoimmune-inducing processes are discussed in the context of Matzingers danger model (Matzinger P: Friendly and dangerous signals: is the tissue in control? Nat Immunol 2007, 8:11-13; Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305; Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991-1045) and Medzhitovs and Janeways (Medzhitov R, Janeway CA Jr: Decoding the patterns of self and nonself by the innate immune system. Science 2002, 296:298-300; Medzhitov R, Janeway CA Jr: How does the immune system distinguish self from nonself? Semin Immunol 2000, 12:185-188; Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 2002, 20:197-216) distinction of noninfectious self (NIS) and infectious nonself (INS). The mechanisms leading to production of potentially nephritogenic anti-nucleosome antibodies and to overt lupus nephritis are interpreted in the context of these paradigms.

Autoantibodies in lupus: culprits or passive bystanders?

Several autoantibodies are culprits in the pathogenesis of organ damage in systemic lupus erythematosus, by means of established or postulated mechanisms, whereby inducing a perturbation of cell structure and function, with consequent tissue-organ impairment. Common autoantibody-mediated mechanisms of damage include cell surface binding with or without cytolysis, immune complex-mediated damage, penetration into living cells, binding to cross-reactive extracellular molecules. Experimental data from both murine models and humans have recently clarified the key role of autoantibodies in severe organ involvements, including nephritis, neuropsychiatric (NP) dysfunction, and cerebrovascular disease (CVD). In lupus nephritis early and late phases are distinguishable and mediated by different processes in which anti-chromatin antibodies are both inducing and perpetuating agents, by immune-complex formation and massive deposition in mesangial matrix at first, and in glomerular basement membrane at end-stage. Also NP abnormalities occur very early, much earlier than other systemic manifestations, and exacerbate with the increase in autoantibody titers. Among the autoantibodies mainly implicated in neurolupus, anti-β2 glycoprotein I (β2GPI) antibodies are preferentially involved in focal NP events which are a consequence of non-inflammatory microangiopathy; otherwise, anti-ribosomal P protein antibodies and N-methyl-d-aspartate receptor (NMDAR) antibodies cause diffuse NP events through a direct cytotoxic effect on neuronal cells at specific brain zones.

Glomerular Targets of Nephritogenic Autoantibodies in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibodies directed against various nuclear autoantigens, particularly against components of chromatin, such as doublestranded DNA (dsDNA), histones, nucleosomes, and ribonucleoproteins. Lupus nephritis, a severe clinical manifestation of SLE, is generally associated with high titers of antinuclear antibodies, especially antibodies against chromatin, and with immunoglobulin and complement deposits in the glomerulus. Historically, the formation of antibodies against dsDNA has been considered the serologic hallmark of SLE (1), and anti-dsDNA antibodies are found in the majority of SLE patients (2). In recent years, it has become clear that the nucleosome is the driving autoantigen in SLE (3), and some investigators claim that the presence of antinucleosome antibodies could serve as a better marker for SLE than anti-dsDNA antibodies, since the former are present in up to 90% of lupus patients (4,5). In SLE patients and in lupus-prone mice, nucleosomes can be found in the circulation; they are released from apoptotic cells and are present as a result of either disturbed apoptosis or a defective mechanism for clearing of apoptotic cells (3). Chromatin is basically defined as the entire complex of compacted DNA and associated proteins. The nucleosome is the fundamental unit of chromatin. The core nucleosome particle consists of 146 bp of dsDNA, wrapped twice around a histone octamer (2 copies of each of the core histones H2A, H2B, H3, and H4) (6). Antibodies against epitopes on the nucleosome, called nucleosome-specific antibodies, have no or very low reactivity against individual histones or naked dsDNA. The formation of nucleosome-specific antibodies precedes the appearance of anti-dsDNA and antihistone antibodies as a result of epitope spreading. Anti-dsDNA and antihistone antibodies bind to dsDNA and histones, respectively, but also to nucleosomes (7). A serious topic of debate for several decades involves the pathogenicity, and especially the nephritogenicity, of anti-dsDNA antibodies. DNA itself is not immunogenic, and immunization of mice with naked dsDNA leads to rapid removal, mainly through the liver. Nevertheless, anti-dsDNA antibody levels often show a high correlation with disease activity, especially in lupus nephritis, and elucidating their pathogenic properties is thus of great interest. Therefore, the targets of antidsDNA antibodies have been intensively studied to gain clues to mechanisms that could explain the pathogenicity of anti-dsDNA antibodies in SLE. With regard to their role in the development of lupus nephritis, many investigators postulate that antidsDNA antibodies localize in the glomerular basement membrane (GBM) due to direct cross-reactive binding to intrinsic glomerular antigens (mechanism 1), whereas, in our opinion, glomerular localization of anti-dsDNA antibodies is mediated via nucleosomes, which bind to heparan sulfate (HS) in the GBM (mechanism 2). These two hypotheses may not be mutually exclusive, and until recently, there was no unequivocal proof for either of the two proposed mechanisms. In this review, we will discuss the pros and cons of each of these mechanisms, taking into account recent pertinent observations by Supported by the Dutch Kidney Foundation (grant C05.2119), the PhD student program of the Radboud University Nijmegen Medical Centre, the Health and Rehabilitation Organization, Norway (grant 2001/2/0235), and Oslo Sanitetsforening and Skibsreder Tom Wilhelmsens Stiftelse. Casandra C. van Bavel, MSc, Johan van der Vlag, PhD, Jo H. Berden, MD, PhD: Nijmegen Centre for Molecular Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Kristin A. Fenton, PhD: University of Tromso, Tromso, Norway; Ole P. Rekvig, MD, PhD: University of Tromso, and University Hospital of North Norway, Tromso, Norway. Address correspondence and reprint requests to Jo H. Berden, MD, PhD, Division of Nephrology (464), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: J.Berden@nier.umcn.nl. Submitted for publication November 8, 2007; accepted in revised form April 14, 2008.

Anti-dsDNA antibodies promote initiation, and acquired loss of renal Dnase1 promotes progression of lupus nephritis in autoimmune (NZBxNZW)F1 mice.

Background Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease. Methodology/Principals In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis. Findings Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs. Significance These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure.

Mechanisms of Transcriptional Regulation of Cellular Genes by SV40 Large T- and Small T-Antigens

During the past decade a number of virus-encoded transcriptional trans-activators that regulate the expression of viral genes have been reported. These trans-activators may also affect the expression or activity of several cellular genes or gene products to create an optimal cellular environment that favors viral replication. Among the better-studied viral trans-activating proteins are the Simian virus 40 large T- and small t-antigens. During the last few years, mechanisms by which these two viral proteins influence cellular gene expression start to emerge. They are grouped provisionally and reflect the methods used to determine the effects of large T-antigen. Large T-antigen may influence cellular gene expression by: i. altering mRNA levels of cellular transcription factors; ii. interacting with and regulating the DNA-binding or transcriptional activity of specific transcription factors; iii. functionally substitution of eukaryotic transcription factors; iv. direct binding to DNA; or v. regulating components of signaling transduction pathways. Small t-ag seems to exert its effect mainly through inhibiting a cellular phosphatase, protein phosphatase 2A, thereby modulating components of signal transduction pathways and preventing dephosphorylation of several transcription factors. However, small t-ag may also control cellular gene expression by regulating mRNA levels of transcription factors or by interacting with other transcription factors.

Acid Elution of Blood Group Antibodies from Intact Erythrocytes

Abstract. Human IgM and IgG antibodies against blood group antigens (A, B, D, C, c, E, e, Fya, K), autoantibodies and mouse IgM and IgG antibodies against sheep erythrocytes have been eluted from intact human and sheep red cells by glycine‐HC1 buffer, pH 3.0. The yield of human antibodies was higher with acid than with heat and ether elution, and the contamination of hemoglobin in the eluate was negligible. The acid elution method is very simple and rapid and, therefore, highly suitable for experimental as well as routine immuno‐hematological work.