Arne Hansen

EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI): development of a consensus patient index for primary Sjögren's syndrome

Objectives To develop a score for assessment of patients symptoms in primary Sjögrens syndrome (SS): the EULAR SS Patient Reported Index (ESSPRI). Methods Dryness, pain, somatic and mental fatigue were identified as the main symptoms of patients with primary SS, in studies developing the Profile of Fatigue and Discomfort (PROFAD) and Sicca Symptoms Inventory (SSI). It was suspected that a single 0–10 numerical scale for each domain was sufficient to assess these symptoms. These four scales were gathered to form the ESSPRI. 230 patients, from 12 countries completed the ESSPRI, SSI and PROFAD questionnaires and a 0–10 patient global assessment (PGA). Correlations between each symptom and PGA were obtained. Multiple regression modelling, using PGA as ‘gold standard’ was used to select domains and estimate their weights. Results PGA had good correlation with dryness, limb pain, fatigue and mental fatigue (r=0.49–0.59, all p<0.0001), but correlated less well with individual dryness features. In multivariate analysis, dryness, limb pain and fatigue, but not mental fatigue, were significantly associated with PGA; weights derived from the regression were identical for these three domains. Thus, ESSPRI was redefined as the mean of the three scales: dryness, limb pain and fatigue. Lastly, ESSPRI significantly correlated with PGA (r=0.70), PROFAD (r=0.73) and SSI (r=0.66). Conclusion ESSPRI is a very simple index designed to measure patients symptoms in primary SS. It has good construct validity and is well correlated with SSI and PROFAD. ESSPRI should now be validated for use as an outcome measure in clinical trials.

Immunopathogenesis of primary Sjögren's syndrome: implications for disease management and therapy.

Purpose of reviewRecent studies have broadened our understanding of the etiopathogenesis and immunopathology of primary Sjögrens syndrome. This review highlights recent advances in understanding the underlying mechanisms of the disease as well as their implications for clinical handling and therapeutic options. Recent findingsIt becomes increasingly apparent that certain disturbances of the immune system (i.e. B-cell hyperreactivity and enhanced levels of B-cell-activating factor/B-lymphocyte stimulator) play a central role in this entity. Whether this is a primary abnormality or the result of predisposing factors or infectious, e.g. viral, agents remains uncertain. New insights into the pathogenesis also provide candidates for better diagnosis and classification of disease severity, such as flow cytometric analysis, measurement of soluble cell surface molecules, autoantibodies, cytokines, and ligands (B-cell-activating factor/B-lymphocyte stimulator). Whether B-cell-directed therapies (i.e. blocking B-cell-activating factor/B-lymphocyte stimulator, anti-CD20 therapy) will have an impact on primary Sjögrens syndrome needs to be shown in clinical trials. Alternative therapeutic approaches such as organ-targeted gene transfer are in development but must be carefully evaluated for safety and efficacy in preclinical models that resemble human primary Sjögrens syndrome. SummaryThe pathogenesis of primary Sjögrens syndrome is complex and the factors initiating and driving autoimmunity in this entity are largely unknown. Recent studies provide new insights into potential pathogenetic mechanisms of the disease and, thereby, the chance for improved strategies in disease management and therapy.

New concepts in the pathogenesis of Sjögren syndrome: many questions, fewer answers.

Although a modified European–American consensus classification of Sjögren syndrome has been introduced during the last year, the etiopathogenesis of this disease characterized by chronic lymphocytic inflammation, impaired function, and, finally, destruction of the salivary and lacrimal glands as well as systemic manifestations remains to be elucidated. Recent insights into the pathogenesis of Sjögren syndrome resulting from immunogenetic, hormonal, and epidemiologic evaluations as well as animal and in vitro studies are highlighted by this review. Evidence confirms that lymphocytic disturbances, including ectopic germinal center formation and aberrations of cellular signaling play a significant role in Sjögren syndrome. Although some of these features are unique to Sjögren syndrome, others are also found in a number of systemic autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis. The underlying cause of Sjögren syndrome remains largely enigmatic. However, distinct characteristics may provide the basis for the classification of the disease entities. Finally, an enhanced risk of lymphomagenesis is a well-known hallmark of primary Sjögren syndrome, indicating the central role of derangement of lymphocyte regulation. As demonstrated by the introduction of the new targeted therapeutic approaches in rheumatoid arthritis, solid insights into the pathogenesis of Sjögren syndrome may pave the way toward new therapeutic approaches.

Human hybridomas derived from CDS+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies

Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B‐CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT‐sensitive heteromye‐loma line (CB‐F7). A fusion frequency of up to 10‐5 allowed us to analyse hundreds of initial hybridoma lines per fusion. In all culture supernatants in three out of five fusions IgM lambda antibodies were detected, in two experiments only IgM kappa was measured, suggesting monoclonality of the primary hybridoma cell lines. The later fusions resulted in hybridomas producing multi‐specific antibodies against both an autoantigen and an infectious agent: (i) dsDNA/influenza virus haemagglutinin; (ii) dsDNA/class V outer membrane protein type C from Neisseria meningitidis. However, no antibodies of the described specificity were detected in blood sera of patients, indicating a ‘switch‐on’ of the immunoglobulin secretion capacity of malignant B cells during fusion to a myeloma partner. We discuss the results as further evidence for the natural multi‐reactive antibody repertoire of CD5+ B cells.

Autoantibodies in normals – the value of predicting rheumatoid arthritis

The cause of rheumatoid arthritis (RA) remains elusive, however, a study demonstrating synovitis in clinically uninflamed joints [1] and several studies reporting the presence of characteristic autoantibodies (IgM rheumatoid factor [RF] and anti-cyclic citrullinated peptide [CCP] antibodies) prior to the appearance of disease manifestations [2-7] have provided evidence of a preclinical, asymptomatic, phase of the disease. Detection of autoimmune T cells has not yet reached routine diagnostics, but with the development of tetramer and ELISPOT technologies it seems likely that autoantibody detection will serve as the method of choice for the identification of autoimmunity and breaches of tolerance (Fig. u200b(Fig.1).1). In patients with early RA, the frequency of RF is 50–66% and the prevalence of anti-CCP 41–48%, compared to 7–13% and 3–9% respectively in normals [8-10]. Several recent studies have regenerated interest in the value of positive titres of autoantibodies as markers of rheumatic diseases [11-15]. Autoantibody positivity prior to symptom development/disease manifestation has also been identified in other autoimmune diseases, such as systemic lupus erythematosus, insulin-dependent diabetes mellitus (IDDM), autoimmune polyendocrine syndromes and celiac disease [11-20]. n n n nFigure 1 n nProposed model of the development of autoimmunity influenced by genetic and environmental factors at certain stages. n n n n nPutative role of protein citrullination in rheumatoid arthritis nCeliac disease is a chronic intestinal disease with an immune response to antigens in wheat gluten. Disease occurs after the target antigen gliadin has been modified by the enzyme tissue transglutaminase, which allows subsequent presentation in the context of specific HLA molecules [19]. It is likely that RA is also the result of post-translational modifications of antigens by enzymatic activity (deiminases), and subsequent immune-mediated destruction of the synovium [9]. Regulation of peptidyl arginine deiminase (PADI) activity appears also to be involved in the maintenance of immunological tolerance to antigens. Thus, citrullination of proteins in RA patients may unmask essential epitopes that bind strongly to HLA class II molecules and result in initiation of an autoimmune response.

A human monoclonal antibody with the capacity to neutralize Staphylococcus aureus alpha-toxin

A human monoclonal antibody, CB STL-1, against staphylococcal alpha-toxin has been established by hybridoma technology. It is of IgG1 subclass with lambda light chain and possesses a dissociation constant of 8 x 10(-10) mol/l. 1 mg of purified antibody neutralizes the hemolytic activity of 800 micrograms/ml alpha-toxin in an in vitro hemolysis assay using rabbit erythrocytes. The antibody does not bind to overlapping (7 residues) decapeptides spanning the sequence of alpha toxin, thus it might bind to a conformational epitope. The epitope recognized by the antibody is not accessible in oligomeric toxin. The antibody binds both to the hydrophilic and amphipathic forms of the monomeric toxin Fab fragments of the antibody are stable and show no significant loss of activity. CB STL-1 was able to protect mice in vivo from i.p. challenge with alpha toxin. Thus, the antibody is a candidate for passive immunotherapy. The variable regions of the antibody secreted by CB STL-1 were sequenced and found to be encoded by a VH gene segment belonging to the VH1 family, and a Vlambda segment most likely belonging to the VlambdaIII subgroup. Further analysis concerning the third complementarity determining region (CDR3) of the heavy chain is presented.

Immunoglobulin V? light chain gene usage in patients with Sjgren's syndrome

Objective n nTo determine whether patients with Sjogrens syndrome (SS) have abnormalities in Ig Vλ and Jλ gene usage, differences in somatic hypermutation, defects in selection, or indications for perturbations of B cell maturation. n n n nMethods n nIndividual peripheral B cells from SS patients were analyzed for their Vλ gene usage by single-cell polymerase chain reaction amplification of genomic DNA and compared with those from normal controls. n n n nResults n nMolecular differences from controls in Vλ–Jλ recombination were identified that were reflected by findings in the nonproductive Vλ repertoire of the patients, including enhanced rearrangement of Vλ10A and Jλ2/3 gene segments. In addition, a number of abnormalities in the productive repertoire were identified, indicating disordered selection. A greater usage of 4 Vλ genes (2A2, 2B2, 2C, and 7A), representing 56% of all productive Vλ rearrangements, was observed, suggesting positive selection of these genes. Overutilization of Jλ2/3 and underutilization of Jλ7 in both nonproductive and productive Vλ rearrangements of SS patients compared with controls suggested decreased receptor editing in SS. The mutational frequency did not differ from that in controls, and positive selection of mutations into the productive V gene repertoire was found, similar to that in controls, although mutational targeting toward RGYW/WRCY motifs, typically found in controls, was not found in SS patients. n n n nConclusion n nDisturbed regulation of B cell maturation with abnormal selection, defects in editing Ig receptors, and abnormal mutational targeting may contribute to the emergence of autoimmunity in SS.

An anti-lipid a antibody obtained from the human fetal repertoire is encoded by VH6-Vλ1 genes

Abstract The human hybridoma cell line CB-201 has been obtained from a fusion of fetal spleen lymphocytes (31st gestational week) with heteromyeloma cells. The IgM(λ) secreted was found to bind to lipid A, whereas other endogenous and exogenous antigens were not recognized. The CB-201 antibody is encoded by the unmutated V H 6 gene recombined with DN4 and J H 3 elements and a V λ subgroup 1 gene. Therefore, a V H gene, which was previously described to be over-represented in the fetal repertoire and to be expressed in autoantibody-producing B cells, may encode an anti-bacterial specificity.

Cryopreserved human B cells as an alternative source for single cell mRNA analysis.

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO] – stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the ‘house-keeping gene’ glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B␣cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.