Age-dependent prevalence of antibodies cross-reactive to the influenza A(H3N2) variant virus in sera collected in Norway in 2011.

Antibody cross-reactivity to the influenza A(H3N2) variant virus recently reported in the United States, was investigated in Norwegian sera. Seroprevalence was 40% overall, and 71% in people born between 1977 and 1993. The most susceptible age groups were children and people aged around 50 years. The high immunity in young adults is likely to be due to strong priming infection with similar viruses in the 1990s. More research is needed to explain the poor immunity in 45­54 year-olds.

High prevalence of antibodies to the 2009 pandemic influenza A(H1N1) virus in the Norwegian population following a major epidemic and a large vaccination campaign in autumn 2009.

The prevalence of antibodies reactive to the 2009 pandemic influenza A(H1N1) was determined in sera collected before the start of the pandemic, during the early phase, and after the main epidemic wave and nationwide vaccination campaign in Norway. A substantial rise in prevalence of antibodies at protective titres, from 3.2% to 44.9%, was observed between August 2009 and January 2010. The highest prevalence, 65.3%, was seen in the age group of 10-19 year-olds.

Neutralizing human antibodies to varicella-zoster virus (VZV) derived from a VZV patient recombinant antibody library.

Varicella-zoster virus (VZV), the causative agent of chickenpox and herpes zoster, can be life-threatening in prematurely born children and in children with immune defects or who are under immunosuppressive treatment. Therefore agents for passive immunization, such as VZV-specific immunoglobulin preparations (VZIG) derived from convalescent plasma, are crucial in the prophylaxis of VZV infection. This study describes the isolation of human VZV-neutralizing recombinant antibodies. A human single-chain variable fragment (scFv) phage display library was generated from RNA extracted from peripheral blood lymphocytes of a convalescent varicella patient. Specific phage antibodies were selected against VZV-infected human fibroblasts, and eight unique clones were further expressed as soluble scFv in Escherichia coli. They all showed binding characteristics to varicella antigens with affinities in the K(D) range 0.1-0.2 muM. Two of the scFv antibodies, VZV4 and VZV5, showed dose-dependent in vitro neutralization of VZV. VZV39 also showed a neutralizing effect as scFv, an effect that was increased 4000-fold by conversion into IgG and was further increased by the addition of complement. This is possibly the first time that monovalent scFv antibodies have been shown to neutralize VZV in vitro. This finding will have an impact on the production of new prophylactic antibodies, as such antibody fragments can be cost-effectively produced in E. coli. The antibodies isolated bind both complement-dependent and -independent epitopes for neutralization, thus they may prove useful tools for the study of VZV virulence mechanisms.

Spontaneous in vivo gene transcription of interleukin-2, interleukin-3, interleukin-4, interleukin-6, interferon-gamma, interleukin-2 receptor (CD25) and proto-oncogene c-myc by rheumatoid synovial T lymphocytes.

Rheumatoid synovial T lymphocytes were investigated for the presence of mRNA for the cytokines interleukin-2, -3, -4, -6, interferon-gamma, the interleukin-2 receptor (CD25) and the proto-oncogene c-myc. The isolated RNAs were analysed by dot blot and Northern blot hybridization. Our results show that synovial T lymphocytes from patients with rheumatoid arthritis (n = 12) had spontaneous in vivo gene transcription of interleukin-2 (93%), interleukin-4 (67%), interleukin-6 (92%), interleukin-2 receptor (92%) and the proto-oncogene c-myc (67%). Only a few of the RA patients had synovial T cells with increased expression of mRNA for interleukin-3 (25%) and interferon-gamma (25%). The amounts of mRNA for the various cytokines and activation molecules produced by the rheumatoid synovial T lymphocytes were in most instances comparable to those of normal peripheral blood T lymphocytes activated in vitro by the mitogen phytohaemagglutinin. The data thus indicate that the synovial T lymphocytes are activated in vivo in the majority of rheumatoid arthritis patients.

The V delta gene usage by freshly isolated T lymphocytes from synovial fluids in rheumatoid synovitis: a preliminary report.

Taking advantage of the polymerase chain reaction we have studied the usage of variable delta-(V delta) region genes in freshly isolated synovial fluid T cells from patients with rheumatoid synovitis. Amplified mRNA from one patient with rheumatoid arthritis (RA) was cloned into an SmaI-cleaved pUC19 vector and colonies were screened with probes for three of the known human variable delta-gene families (V delta 1, V delta 2, V delta 3). Of 10 clones, seven used V delta 1, two V delta 2 and one V delta 3. This pattern of distribution is different from that of normal peripheral blood, where approximately 60% of T gamma delta cells are reported to use the V delta 2 gene. Furthermore, Northern blot hybridization analyses of mononuclear cells from two additional synovial fluids derived from another patient with RA and one with juvenile rheumatoid arthritis (JRA) also showed significant hybridization only with V delta 1. In summary, these preliminary results suggest a usage of V delta gene families in T gamma delta lymphocytes in synovial fluid of rheumatoid patients different to that found in normal peripheral blood.

Human monoclonal rheumatoid factors derived from the polyclonal repertoire of rheumatoid synovial tissue: production and characterization.

A panel of 20 human monoclonal antibodies was produced from cells derived from the synovial tissue of two rheumatoid arthritis patients and one polyarticular juvenile rheumatoid arthritis patient. Fourteen IgM monoclonal antibodies were classical rheumatoid factors (RFs) with specificities restricted to IgG and included twelve kappa and two lambda proteins. Two of them were pan-specific for IgG and reacted with all four human subclasses. Nine showed the Ga specificity, i.e. they reacted with IgG1, IgG2 and IgG4 subclass proteins. Two showed the 'new Ga-related' specificity, i.e. they reacted with IgG1, IgG2 and IgG4 subclass proteins, and also with the Ge3m(st) allotype proteins. One RF demonstrated an anti-G3m(u) anti-allotypic specificity. Five IgM(lambda) monoclonal antibodies and one IgM(kappa) monoclonal antibody demonstrated polyreactivity against various antigens including DNA, human thyroglobulin, human serum albumin and tetanus toxoid.

Inflammatory synovial T cells in different activity subgroups of patients with rheumatoid arthritis and juvenile rheumatoid arthritis.

Mononuclear cells were eluted from synovial membranes of 39 patients with rheumatoid arthritis and 12 patients with juvenile rheumatoid arthritis. A considerable cell loss, about 50% or more, was seen during the various isolation steps. The CD4/CD8 ratio just after enzyme treatment (stage I) was significantly higher than at later stages, i.e. after removal of adherent cells (stage II, p less than 0.05) and after Isopaque Ficoll gradient centrifugation (stage III, p less than 0.01). This indicates a selective loss of CD4+ cells during isolation. In addition, stages I and II had higher CD4/CD8 ratios than peripheral blood of normal controls (p less than 0.01 and p less than 0.03), but not significantly higher than in peripheral blood of patients (p greater than 0.05). The CD4/CD8 ratio in eluted synovial membrane cells did not differ between patients with high and patients with low disease activity (p greater than 0.05). No correlation was found between any of the CD4/CD8 ratios and individual disease activity variables. Furthermore, a laboratory activity index and a disease outcome index were determined for each patient and no correlation was found between these indices and the CD4/CD8 ratios.

Phenotypes and spontaneous cell cytotoxicity of mononuclear cells from patients with seronegative spondyloarthropathies: ankylosing spondylitis, psoriatic arthropathy and pauciarticular juvenile chronic arthritis--analysis of mononuclear cells from peripheral blood, synovial fluid and synovial membranes.

T-cell subpopulations and natural killer (NK) cells from peripheral blood, synovial fluid and synovial membranes from patients with seronegative spondyloarthropathies were investigated. Thirty-four patients with ankylosing spondylitis, sixteen patients with psoriatic arthropathy and six patients with pauciarticular juvenile chronic arthritis were studied. All the patient groups had normal proportions of T4+ and T8+ cells as well as normal T4/T8 ratios in peripheral blood. In the synovial fluids the T4/T8 ratios were reduced in ankylosing spondylitis and psoriatic arthropathy (p less than 0.05). Although both the T4 and T8 subpopulations were reduced, the T4/T8 ratios in the synovial membranes of patients with these two disorders tended to be within the normal range of that of peripheral blood. Increased numbers of T-cells in the synovial fluid from patients with ankylosing spondylitis expressed class II MHC antigens. The natural killer cell activity was normal in peripheral blood and synovial fluids of patients with ankylosing spondylitis and psoriatic arthropathy while it tended to be reduced, although not significantly, in pauciarticular juvenile chronic arthritis. Synovial membranes were almost devoid of NK cell activity. The number of Leu 7+ cells were reduced in synovial fluid of patients with psoriatic arthropathy (p less than 0.04), but not as significantly as in the two other patient groups.

Evidence for activation of rheumatoid synovial T lymphocytes--development of rheumatoid T cell clones.

Rheumatoid arthritis joints are infiltrated by numerous T cells. These T cells coexist in close proximity to HLA class I and class II bearing accessory cells. A great proportion of the rheumatoid T cells are activated in vivo as shown by their microscopic appearance, expression of surface activation antigens, spontaneous proliferation and their spontaneous production and consumption of interleukin-2 (IL-2). T cells from the rheumatoid lesions also express high levels of mRNA for IL-2, for IL-2-receptor and for other mediators. The expression of IL-2-receptors by activated synovial T cells make it easy to propagate them in IL-2 containing media which is the basis for the subsequent development of T cell clones. The rheumatoid T cells are hypo-responsive after stimulation with antigens and anti-CD3 antibodies which indicate that the CD3-TCR complex has been modulated in vivo.

B lymphocytes, B cell clones and rheumatoid factor antibodies in rheumatoid inflammation.

This report focuses on the B lymphocytes and plasma cells in rheumatoid inflammation, and discusses the major autoantibodies in the pathogenetic mechanisms, i.e. the rheumatoid factor (RF) antibodies. We describe ways of raising human hybridomas that produce RF antibodies in rheumatoid arthritis (RA) in order to elucidate how these antibodies differ from the RF antibodies that are part of the normal immune response in man and animals, and from those in diseases other than RA, e.g. in M-components seen in mixed cryoglobulinaemia and in Waldenström's macroglobulinaemia. The preliminary results of these studies are presented and discussed.

Rheumatoid lymphoid dendritic cells--characteristics and functions.

Dendritic cells have been isolated from peripheral blood and inflamed synovial tissue and synovial fluid of patients with rheumatoid arthritis and from normal peripheral blood. Synovial and blood dendritic cells are strongly positive for CD45 and MHC class II antigens, and lack almost all other mononuclear cell markers. Thus, in most respects they have the same characteristics as lymphoid dendritic cells in mice. Synovial and blood dendritic cells are very potent accessory cells for T lymphocyte responses, and much more effective than monocytes. Synovial dendritic cells also spontaneously produce interleukin 1. The accessory function is inhibited by an antibody to interleukin 1. Synovial dendritic cells may thus be critical for starting and perpetuating the chronic inflammation seen in rheumatoid arthritis.

Partial amino acid sequence analysis and variable subgroup determination (VH and VL) of a monoclonal rheumatoid factor derived from a rheumatoid arthritis patient.

Continuous cell lines secreting monoclonal rheumatoid factors (RF) were derived from rheumatoid arthritis (RA) patients by cloning Epstein-Barr virus (EBV) transformed B cells and by hybridoma techniques. We studied five different clones with stable RF secretion. All were IgM, 4 kappa and 1 lambda. One of these clones, RFAN was extensively studied, and the partial amino acid sequences of the variable regions of both heavy and light chains were determined. After affinity purification, the IgM lambda RF antibody derived from the EBV clone was run under reducing conditions on SDS-polyacrylamide gel electrophoresis. The separated heavy and light chains were blotted and then sequenced by a gas-phase sequenator. The N-terminal sequence of the lambda light chain corresponded to that of the V lambda III subgroup. The heavy chain of the same IgM RF clone had a blocked N-terminus, but a cyanogen bromide peptide starting after methionine at position 82 showed a sequence typical of the VHIII subgroup. Heavy and light chains were also prepared by gel filtration after reduction and carboxymethylation from the same EBV clone made into a hybridoma. After this preparation, the heavy chain was not blocked and the N-terminal sequence confirmed that the heavy chain variable region belonged to the VHIII subgroup. We believe this to be the first amino acid sequence study of a monoclonal RF derived from the repertoire of an RA patient.

Characteristics of human rheumatoid synovial and normal blood dendritic cells. Retention of class II major histocompatibility complex antigens and accessory function after short-term culture.

Dendritic cells (DC) were isolated from synovial tissue and synovial fluids of patients with rheumatoid arthritis and from peripheral blood of healthy donors. The cells were analysed for various surface antigens in indirect immunofluorescence by means of monoclonal antibodies. Surface antigen expression and accessory activity of the DC during short-term cultures were also investigated. Both the rheumatoid synovial and the normal blood DC were strongly positive for panleucocyte antigen and class II major histocompatibility complex (MHC) antigens (HLA-DP, HLA-DQ, and HLA-DR). The DC suspensions (purity approximately 80-85%) showed very low percentage of cells staining for various other cell membrane markers, including B cell, T cell, natural killer (NK) cell, and various monocyte/macrophage markers as well as markers specific for dendritic reticulum cells and Reed Sternberg cells. Moreover, neither rheumatoid nor normal DC reacted with the RFD1 monoclonal antibody, which is specific for interdigitating cells of human thymus. In contrast to Langerhans' cells, the DC lacked the thymocyte (T6) marker. The various DC expressed neither complement receptors (CR1, CR3), transferrin receptors, nor Fc receptors. They also lacked enzyme markers like peroxidase and nonspecific esterase. The DC formed clusters with autologous T cells. Cluster formation was readily inhibited by anti-HLA-DR and anti-CD2 (T11) monoclonal antibodies. After 3 to 5 days in culture the DC still expressed class II MHC antigens and were potent stimulators in allogeneic mixed lymphocyte reactions (MLR). Only a small number of cells in the DC suspensions from synovial tissue expressed fibroblast antigens before and after culture.

Human rheumatoid synovial and normal blood dendritic cells as antigen presenting cell--comparison with autologous monocytes.

Rheumatoid dendritic cells (DC) from synovial membrane and synovial fluid and normal dendritic cells from peripheral blood were compared with autologous monocytes concerning accessory activities for T cell antigen responses. Purified protein derivative of tuberculin (PPD), herpes simplex virus type 1 antigen (HSV) and Chlamydia trachomatis antigen were used as antigens. The dendritic cells were shown to be efficient antigen presenting cells for the various antigens and were much more potent than monocytes. The DC were strongly positive for major histocompatibility complex (MHC) class II antigens. The T cell responses both to bacterial and viral antigens induced by DC could all be inhibited by monoclonal antibodies against HLA-DQ and HLA-DR antigens. Dendritic cells may thus play an important role as accessory cells in presenting antigens for T cells in the context fo class II MHC molecules in the rheumatoid inflammation.

Natural killer (NK) cells at inflammatory sites of patients with rheumatoid arthritis and IgM rheumatoid factor positive polyarticular juvenile rheumatoid arthritis.

NK cell activity and Leu 7+ cells were determined in mononuclear cells (MNC) from patients with rheumatoid arthritis (RA) and IgM rheumatoid factor positive polyarticular juvenile rheumatoid arthritis (JRA). NK cell activity was measured in a 51Cr release assay and the Leu 7 positive cells were enumerated in indirect immunofluorescence. The mean NK cell activity +/- SEM was reduced in MNC from peripheral blood (PB), synovial fluid (SF) and synovial membranes (SM's) of patients with RA, with the values of 19.5 +/- 1.4 (p less than 0.00003), 18.3 +/- 3.1 (p less than 0.009) and 2.9 +/- 0.5 (p less than 0.0003) respectively, compared with 26.1 +/- 1.4 in MNC from the PB of healthy controls. The mean percentages of Leu 7 positive cells in MNC from PB and SF on patients with RA were normal while the mean percentage of Leu 7+ cells in MNC eluted from SM's was significantly reduced as compared to that of MNC from PB of healthy controls (p less than 0.0006). In JRA similar results concerning NK activity and Leu 7 positive cells were found but the number of experiments was too low for statistical analysis. MNC from the SF, in contrast to that of BP and SM, had a significant cytotoxicity against the Raji cell line which is a non-NK cell target.