Ingrid Randen

Nucleotide Sequence Analysis of Rheumatoid Factors and Polyreactive Antibodies derived from Patients with Rheumatoid Arthritis Reveals Diverse Use of VH and VL Gene Segments and Extensive Variability in CDR‐3

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogrens syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of VH and VL gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain ‘restrictions’ do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of VH3 genes beyond that one would expect based on random utilization.

Rheumatoid factor V genes from patients with rheumatoid arthritis are diverse and show evidence of an antigen-driven response

Since the discovery of autoantibodies and autoimmune disease, one of the most important challenges in the field has been to understand the nature of the genes which encode autoantibodies. Between 1930-1950 a number of antibodies to autoantigens such as thyroglobuHn, immunoglobulin and DNA-nucleoprotein were described (Mackay & Bumet 1963). Through the 1960 70s. many studies were primarily concerned with the IgG class and subclass of such autoantibodies as this was particularly relevant for understanding the effector mechanisms involved in the autoimmune process. During the same period Kunkel et al. (1963) and Oudin & Michel (1963) independently defined idiotypes. and anti-idiotypic antisera were the first and most widely used immunological tools to study the structure ofthe antigen combining site and its variable region genes. Such idiotypic studies in humans were, however, primarily restricted to rare M-components with antibody activity, most of which were not associated with autoimmune disease. Early studies with V-region family-specific antisera (Eorre et al. 1976,

Prenatal genotyping of RHD and SRY using maternal blood

Background and Objectives  The aim of the study was to perform fetal RHD genotyping in maternal plasma using a fluorescent polymerase chain reaction (PCR) technique. Duplex PCR, amplifying RHD and SRY in the same tube, was undertaken. The effect of varying storage temperatures on the concentration of fetal DNA was investigated in a separate study involving 10 RhD‐negative pregnant women.

Rapid and reliable genotyping of human platelet antigen (HPA)‐1, ‐2, ‐3, ‐4, and ‐5 a/b and Gov a/b by melting curve analysis

BACKGROUND: The probability for occurrence of neonatal alloimmune thrombocytopenic purpura (NAITP) depends largely on the frequency of each individual phenotype in various populations. In caucasians, antibodies to human platelet antigen (HPA)‐1a are the major cause of neonatal alloimmune thrombocytopenic purpura, whereas in the Japanese population, antibodies to HPA‐4b is most frequently involved in NAITP. Conventional PCR techniques for platelet antigen genotyping rely on sequence‐specific primers (SSPs) and detection by gel electrophoresis, a method which is laborious and time consuming. New PCR technology, measuring the match of a hybridization probe with its target and thereby allowing simultaneous detection of both alleles, provides an efficient tool for genotyping of the HPA systems.

Glycogen synthase kinase-3 (GSK-3) inhibition induces apoptosis in leukemic cells through mitochondria-dependent pathway

The roles of glycogen synthase kinase-3 (GSK-3) in cell survival and apoptosis are controversial. We examined the effect of a specific GSK-3 inhibitor (SB-415286) on the regulation of leukemic cells proliferation and apoptosis. SB-415286 (40 μM) induced cell growth inhibition, β-catenin stabilization, cell cycle arrest in G(2)/M phase, cyclin B1 downregulation, and apoptosis in leukemic cell lines KG1a, K562, and CMK. Blocking the death receptor pathway by using a specific inhibitor of caspase-8, did not inhibit SB-415286-induced apoptosis. This indicates that activation of caspase-8 is part of the intrinsic apoptotic pathway and occurs downstream of mitochondria membrane potential depolarization mediated by other caspases. Furthermore, we found that depolarization of mitochondria membrane caused by GSK-3 inhibition is regulated by dephosphorylation of anti-apoptotic protein Bcl-2 and downregulation of Bcl-xL. Thus, inhibition of GSK-3-induced apoptosis of leukemic cells could be an attractive target for treatment of leukemia.

Human monoclonal IgG rheumatoid factors from the synovial tissue of patients with rheumatoid arthritis

We have established IgG rheumatoid factor (RF)‐secreting hybridoma cell lines from the synovial tissues of two patients (TS and SJ) with rheumatoid arthritis (RA) und one (KL) with the polyarlicular form of juvenile rheumatoid arthritis (JRA). The IgG RF bind human and rubbit IgG and all except one form intracellular complement‐fixing complexes indicative of a self‐association process. The possibility of IgG RF for self association and immune complex formation is a feature thought to be important for the inflammatory processes in RA. Of the IgG RF‐secretingcell lines established, three clones from patient SJ and one from patient KL are of the IgG Ik isotype while five clones from patient TS are of the IgG2λ isotype.

V‐GENE Repertoire and Hypermutation of Rheumatoid Factors Produced in Rheumatoid Synovial Inflammation and Immunized Healthy Donorsa

We have compared RF from normal, immunized donors and RF derived from the synovial tissues of RA patients. We have found a difference in the preferential use of VL and VH gene families. In both conditions, RFs were found to have accumulated somatic mutations. However, there was a striking difference in the patterns of mutation. RFs from normals were characterized by a very low R:S ratio in the CDR1+2, considerably lower than seen among the RARFs. In addition, there was little increase in affinity with increasing numbers of mutations in a group of clonally related RFs from an immunized normal. This contrasts with RF from RA, where there is evidence of both affinity maturation and class switching. Together these data suggest that in healthy persons there is a controlling mechanism to limit the affinity of RF autoantibodies, and that this is lost in RA. The higher affinity of the RA-derived RF may be of significance in the pathology of the disease.

Three New Cross-Reacting Idiotopes as Markers for the Products of Two Distinct Human VH3 Genes Expressed in the Early Repertoire

This article describes characterization of three new cross‐reacting idiotopes, as recognized by mouse MoAbs, on human antibodies utilizing VH3 genes that are expressed in the early repertoire. Two of the mouse MoAbs (3H7 and 3H1) were raised against a human MoAb utilizing the DP47 (VH26) VH3 gene, whilst the third (7B4) was raised against a DP46 (GLSJ2) gene product. Evidence for the anti‐idiotypic specificity of the mouse MoAbs was provided by their reactivity with the immunizing IgM, but not with Fcα, and by their specific inhibition of the binding between each immunizing antibody and its antigen. The three anti‐idiotypic MoAbs were shown to be VH‐specific reagents by the independence of their reactivity upon the L‐chain type, or the untigenic specificity of the human MoAbs tested. Specificity of each mouse MoAb for VH3 gene‐products was demonstrated by its sole cross‐reactivity with VH3 proteins. Each anti‐Id had a different reactivity pattern with a panel of MoAbs utilizing different VH3 genes. By relating the VH sequences of the tested VH3 proteins to their germline counterparts, 3H7 and 3H1 appeared to be specific for DP47‐encoded proteins, although 3H1 had weak cross‐reactivities with a few other VH3 gene‐products. 7B4 appeared to be specific for antibodies utilizing DP46‐related genes. Both 3H7 and 3H1 were also completely different to B6 and D12, two previously described MoAbs that also recognize VH3 proteins. Although 7B4 was similar to B6 and D12 in its binding to DP46‐related gene products, B6 and D12 additionally recognized non DP46‐related proteins and were thus different to 7B4.

Accurate Rh phenotype determination by reticulocyte mRNA typing shortly after multiple transfusions

This study describes a method for correctly typing patients’ own Rh-antigens after multiple transfusions. Alloimmunization is a common phenomenon after transfusion, with an estimated incidence of 0.5% increasing to 20–60% in chronically transfused patients. In recently transfused patients, serological typing can be hampered by mixed field agglutination. We established RT-PCR methods for RHD, RHC/c and RHE/e typing using mRNA from reticulocytes. Molecular typing was performed soon after 51 separate mismatched transfusion events involving 30 patients. Accurate identification of the transfused patients’ phenotype was confirmed in all cases. Reticulocyte maturation studies revealed that temperature is a crucial parameter for transition into mature red blood cells.

The B cell system in the rheumatoid inflammation

ConclusionRA is an immunological disorder characterized by a genetic predisposition related to HLA DR4 (Dw4, Dw14 and Dw15) and DR1 antigen-presenting molecules. The disease processes result from an immune response due to interaction between these genetically determined antigen-presenting HLA class II molecules on accessory cells and T helper cells. This gives rise to a strong delayed-type hypersensitivity reaction and a T helper cell-induced B cell proliferation with RF production, which in turn may initiate the classical immune complex disease. To perform a detailed study on the structure of RF and other antibodies in RA we have focussed on making hybridomas between autoantibody-producing cells derived from synovial tissues from patients with RA and mouse myeloma cells. This also provides an opportunity to determine the genes that govern the synthesis of these autoantibodies in RA. Similar studies in patients with M-components with RF activity have led to the characterization of two VkIII subgroup germ-line genes, HumKv325 and HumKv328 [7]. These genes are frequently used in patients with RF M-components, but are used only in a small percentage of RF antibodies in patients with RF. There are, therefore, several indications that RA patients also use a wider reportoir of genes for RF autoantibody production than those used in the production of RF M-components.