The clinical assessment of changes in bone density in rheumatoid arthritis patients': Role of DEXA scan and bone turnover biomarkers.

In addition to generalised of bone loss and a higher fracture risk, rheumatoid arthritis (RA) causes periarticular bone erosions. Improvements in bone density/erosion and turnover may not go hand in hand with a positive clinical response to biological anti-inflammatory drugs assesed by disease activity score 28 (DAS28) in RA patients. This study aimed to understand how biologic anti-inflammatory drugs affect bone density, erosion, and turnover in RA patients. We examined bone mineral density (BMD) and bone turnover biomarkers. The study population consisted of 62 RA patients, 49 (79%) of whom were female and 13 (21%) of whom were male. The patients ranged in age from 40 to 79 years old. The patients' BMD was measured using a DEXA scan, and their plasma levels of bone turnover biomarkers CTX and osteocalcin were quantified utilizing an ELISA. BMD of the hip and lumbar spine in responder patients rose after therapy by 0.001g/cm2 (0.11 percent, p0.001 vs. before treatment) and 0.0396g/cm2 (3.96 percent, p0.001 vs. before treatment), respectively. Clinically non-responder patients' DAS28 revealed minor reductions in hip BMD values of -0.008g/cm2 (-0.78 percent, p0.001 vs. before therapy), as well as an improvement in lumbar spine BMD of 0.03g/cm2 (3.03 percent, p0.001 vs. before treatment). After 12 weeks of therapy, the CTX levels in responder patients dropped from 164 125 pg/ml to 131 129 pg/ml. Osteocalcin levels in non-responder patients increased substantially from 11.6 ng/ml to 14.9 ng/ml after 12 weeks of therapy compared to baseline (p = 0.01). Treatment with biologic anti-inflammatory medicines decreases widespread bone loss in RA patients' hip and lumbar spine. The beneficial effects of therapy on BMD were not associated with changes in disease activity of RA patients. Changes in plasma levels of bone turnover biomarkers such as sCTX and osteocalcin confirmed the treatment's beneficial effects.

Comparison of bone mineral density changes between male and female osteoporosis patients using dual energy X-ray absorptiometry scan.

The goal of the current research was to define the impact of individual characteristics on the response of osteoporosis patients to bisphosphonate medication, as well as the influence of gender on changes in the bone mineral density (BMD).The DXA scan was used to assess a group of 647 osteoporosis patients (533 females and 114 males) who visited the St Bartholomew's Hospitals and Royal London osteoporosis clinics. All male subjects received statistically substantial increases in BMD relative to baseline values after two years of therapy. When compared to prior therapy, men's BMD of the lumbar spine (LS) and hip joint (HJ) rose by 0.057 g/cm2 (6.9%, p0.001) and 0.021 g/cm2 (2.48 percent, p0.001), respectively.. Female patients had BMD changes of 0.028 g/cm2 (3.58 percent, p0.001 vs. prior therapy) and -0.006 g/cm2 (-0.78 percent, p0.001 vs. before treatment) in the lumbar spine and hip, respectively. Male patients exhibited a greater increase in BMD than female patients due to ovarian failure and significant oestrogen loss, which speeds up bone resorption by 90% following menopause, according the research findings.

Complete amino acid sequences of variable regions of two human IgM rheumatoid factors, BOR and KAS of the Wa idiotypic family, reveal restricted use of heavy and light chain variable and joining region gene segments.

Evidence derived from the complete amino acid sequences of the variable regions of both the heavy and light chains of two members (BOR and KAS) of the Wa idiotypic family of human rheumatoid factors suggests that not only are the light chains of these molecules derived from possibly one variable region gene segment, but the heavy chain variable regions are all derived from the VHI subgroup of human V region genes. These molecules exhibit a surprising conservation in the size of D region, and all use the JH4 gene element. This restriction in use of VL, VH, D, and JH suggests all of these elements may play a crucial role in either antigen binding and/or expression of the crossreactive idiotype.

Selective expression of a VHIV subfamily of immunoglobulin genes in human CD5+ B lymphocytes from cord blood.

Human B lymphocytes expressing the CD5 surface antigen (CD5+ B cells) constitute a subset capable of producing polyspecific antibodies recognizing a variety of self antigens. The repertoire of antibodies produced by CD5+ and CD5- B cells is different. However, it is not yet established whether this distribution is reflected in different immunoglobulin variable region gene (IgV) use. Rearrangement of heavy chain IgV (IgVH) genes represents one of the first identifiable stages in the maturation of B cells, and occurs in a developmentally ordered fashion. The repertoire of IgVH gene expression is highly restricted during fetal life but diversifies progressively after birth. A high frequency of VH gene use from the relatively small VHIV gene family has previously been demonstrated in human fetal liver B cells. In the present study, 102 B cell lines established by Epstein-Barr Virus-transformation of separated CD5+ and CD5- cord blood B cells, were examined for the frequency of IgV expression using monoclonal antibodies to cross-reactive idiotypes (CRI). The results demonstrate a relatively high frequency of VHIV gene use (30%) in B cells from cord blood. Furthermore, two mutually exclusive CRI associated with distinct subgroups of the VHIV family are segregated in their association with either subset of B cells. One CRI is exclusively expressed in lines established from CD5+ B cells while the other is associated with lines established from CD5- B cells.

Enhanced propensity of T lymphocytes in patients with systemic lupus erythematosus to apoptosis in the presence of tumour necrosis factor alpha.

OBJECTIVE: To determine the effect of inflammation through exposure to tumour necrosis factor (TNF)alpha on T lymphocytes in patients with systemic lupus erythematosus (SLE). METHODS: We studied the effect of TNFalpha on T-lymphocyte apoptosis in patients with SLE, rheumatoid arthritis (RA), and in healthy controls. Apoptosis of CD4 and CD8 T lymphocytes and naive and memory subpopulations was determined by flow cytometry using 7-amino-actinomycin D (7AAD) and propidium iodide (PI). In SLE, apoptosis was studied in patients with active and inactive disease and in patients on different medications. RESULTS: TNFalpha enhanced apoptosis of anti-CD3-activated T lymphocytes. The percentage of apoptotic cells was significantly higher in T lymphocytes from patients with SLE than RA patients and healthy controls. After 3 days of culture, 38% of CD4+ and 37% of CD8+ cells from SLE patients underwent apoptosis in the presence of TNFalpha compared with 25% CD4+ and 26% CD8+ T cells from the controls (p<0.001). In healthy controls, more memory than naive T lymphocytes underwent apoptosis. By contrast, in patients with SLE, more naive T cells underwent apoptosis with TNFalpha (p<0.01). Enhanced apoptosis of T cells in SLE was independent of disease activity or medication. Finally, inhibition experiments showed that apoptosis in the presence of TNFalpha was only partly blocked with anti-Fas ligand (FasL) antibody. CONCLUSIONS: This study demonstrates that T lymphocytes in patients with SLE are more prone to apoptosis in the presence of TNFalpha than T lymphocytes from healthy controls. Defects in TNFalpha signalling pathways rather than distribution of TNF receptors (TNFRs) probably explain the enhanced apoptosis in SLE.

Repopulation of B-lymphocytes with restricted gene expression using haematopoietic stem cells engineered with lentiviral vectors.

B-lymphocytes play a key role in the pathogenesis of many immune-mediated diseases, such as autoimmune and atopic diseases. Therefore, targeting B-lymphocytes provides a rationale for refining strategies to treat such diseases for long-term clinical benefits and minimal side effects. In this study we describe a protocol for repopulating irradiated mice with B-lymphocytes engineered for restricted expression of transgenes using haematopoietic stem cells. A self-inactivating lentiviral vector, which encodes enhanced green fluorescence protein (EGFP) from the spleen focus-forming virus (SFFV) promoter, was used to generate new vectors that permit restricted EGFP expression in B-lymphocytes. To achieve this, the SFFV promoter was replaced with the B-lymphocyte-restricted CD19 promoter. Further, an immunoglobulin heavy chain enhancer (Emu) flanked by the associated matrix attachment regions (MARs) was inserted upstream of the CD19 promoter. Incorporation of the Emu-MAR elements upstream of the CD19 promoter resulted in enhanced, stable and selective transgene expression in human and murine B-cell lines. In addition, this modification permitted enhanced selective EGFP expression in B-lymphocytes in vivo in irradiated mice repopulated with transduced bone marrow haematopoietic stem cells (BMHSCs). The study provides evidence for the feasibility of targeting B-lymphocytes for therapeutic restoration of normal B-lymphocyte functions in patients with B-cell-related diseases.

Idiotypic and subgroup analysis of human monoclonal rheumatoid factors. Implications for structural and genetic basis of autoantibodies in humans.

Rheumatoid factors (RFs) in humans have been studied intensively because of their association with autoimmune and lymphoproliferative diseases. Many human IgM-RFs express cross-reactive idiotypes (CRIs) and have homologous light chains, some of which are encoded by a single V kappa gene, termed V kappa 325. However, although antibody activity generally requires the interaction between heavy and light chain variable regions, much less is known about structural relationships among RF heavy chains. To delineate further the structural and genetic basis of RF autoantibody synthesis, we generated "sequence-dependent" reagents specific for the human heavy and kappa light chain subgroups, and used them to analyze a panel of 27 monoclonal RFs. In addition, these proteins were tested for the expression of a heavy chain-associated CRI (G6), and a light chain-associated CRI (17.109). The results showed that most 17.109-reactive RFs contain heavy chains of the VHI subgroup, which bear the G6 idiotypic marker. However, among the 14 17.109-reactive RFs, two have heavy chains of the VHII subgroup, and another two contain heavy chains of the VHIII subgroup. Previously, we have shown that 17.109 is a phenotypic marker of the human V kappa 325 gene. Accordingly, these results demonstrate that the same human V kappa gene can combine with several VH genes from different VH gene subgroups to generate RF activity.

Restricted immunoglobulin variable region gene usage by hybridoma rheumatoid factors from patients with systemic lupus erythematosus and rheumatoid arthritis.

Rheumatoid factors (RFs) are autoantibodies that are produced by approximately 75% of patients with rheumatoid arthritis (RA). Their role in pathogenesis is not well understood. In this study of 81 human hybridoma IgM antibodies derived from unstimulated peripheral blood B-cells of patients with RA and systemic lupus erythematosus (SLE), we have demonstrated that idiotypes associated with RFs derived from patients with mixed cryoglobulinemia were expressed by approximately 60% of RFs and 6% of IgM antibodies lacking RF activity. The specificity of the RFs for the Fc portion of IgG only (monospecificity) or for Fc and additional self antigens (polyreactivity) was found to correlate with the expression of specific heavy chain associated idiotypes. The VH3 associated RF idiotypes, D12 and B6, were expressed by 0/16 (0%) of monospecific RFs compared with 6/22 (27%) of polyreactive RFs. The predominant use of VH3 was verified by analysis of the expressed Ig with VH family specific anti-peptide antibodies. The light chains expressed by both populations of IgM RFs were found to be predominantly VKIII, both by detection of specific epitopes/idiotypes and V family analysis. This non-random gene usage of both the heavy and light chains suggests that there is a selective expression of V regions in the RF producing B-cells in patients with RA and SLE. We suggest that different antigen-driven, clonal selection events may occur which result in either monospecific RFs or polyreactive RFs.

Immunogenic and antigenic epitopes of immunoglobulin--XX. Denaturation of human IgG3 by free radicals.

It has previously been demonstrated that exposure of polyclonal IgG to free radicals results in denaturation evidenced by aggregation, auto-fluorescence and destruction of cysteine, proline and aromatic amino acids. In the present study we have used a panel of monoclonal antibodies (McAb) to epitopes expressed on the IgG3 heavy chain to detect changes in antigenicity. When IgG3 was exposed to u.v. irradiation, as a source of free radicals, subclass specific epitopes were rapidly lost whilst other epitopes were unaffected. Prolonged exposure resulted in further denaturation and a progressive loss of expression of further epitopes. The IgG3 subclass specific McAb are specific to epitopes localized to the hinge region of IgG3. Thus, this exposed cysteine and proline rich region is shown to be particularly vulnerable to free radical attack; however, prolonged exposure results in structural alterations throughout the heavy chain.

Anti-idiotypic antibody D12 and superantigen SPA both interact with human VH3-encoded antibodies on the external face of the heavy chain involving FR1, CDR2 and FR3.

The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V(H)3 encoded antibodies. We used mutational analysis to determine the subregions of a V(H)3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FRI, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V(H)3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.

The incidence of a new human cross-reactive idiotype linked to subgroup VHIII heavy chains.

Cross-reactive idiotypes (CRI) on human rheumatoid factors (RF), which are identified by murine monoclonal antibodies (mAb), have proved useful in defining both the incidence and the structural characteristics of these autoantibodies. In this study, a new murine anti-idiotypic reagent, mAb B6, has been used to identify and define the expression of a distinct heavy chain CRI. The B6 CRI was found on 20% of monoclonal IgM (16 of 81), but on only 5% of monoclonal IgA (1 of 20) and on no monoclonal IgG. In addition, this CRI was expressed exclusively on a subset of Ig derived from the VHIII protein variable region subgroup. In immunoblotting experiments, the mAb B6 bound directly to the heavy (H) chains of CRI positive proteins. The B6 CRI was found frequently on monoclonal IgM-RF molecules, and the mAb B6 could inhibit the binding of the RF to its IgG antigen. It was also demonstrated that Staphylococcus aureus protein A (SpA), which has recently been shown to bind to the F(ab) region of VHIII molecules, could block the interaction of some B6 CRI positive IgM to the anti-CRI. These experiments suggest that the B6 CRI is a marker for one or a few VHIII genes and that it is expressed commonly on IgM paraproteins, many of which have RF activity.

CD5+ B cells and the immune system.

The CD5+ B-cell population is prominent in early life and may play a key role in the ontogeny of the immune system. Transplantation studies in mice are in support of CD5+ B cells as a separate lineage from CD5- B cells. In both mice and men there is evidence in favour of CD5 being an activation antigen rather than a lineage marker, but the jury is still out! The frequency of CD5+ B cells appears to be under genetic influence. CD5+ B cells are receptive to many cytokines including IL-2 and IL-5 and themselves produce a number of cytokines especially IL-10. The function of the CD5 molecule on B cells is presently unknown but it might be involved in interaction with CD72 on other B cells. CD5+ B cells generally utilise minimally mutated germ-line genes and produce low avidity auto- and polyreactive antibodies (natural antibodies) generally of the IgM class.

Immunophenotypic and idiotypic characterisation of the leukaemic B-cells from patients with prolymphocytic leukaemia: evidence for a selective expression of immunoglobulin variable region (IGV) gene products.

B-cell prolymphocytic leukaemia (B-PLL) is a rare chronic lymphoproliferative disease characterised by a massive splenomegaly associated with a mild or no lymphadenopathy and a high leukocyte count, mostly representing prolymphocytic features. We have studied membrane expression of certain Ig VK and VH gene products in five patients with B-PLL using a panel of monoclonal anti-subgroup and anti-cross-reactive idiotype (CRI) antibodies. Membrane expression of leukocyte-associated markers has also been investigated. The leukaemic cells from four patients expressed VKIII and VKIIIb subgroup and sub-subgroup kappa light chains. The VKIIIb and VHI-associated CRI identified by the monoclonal antibodies (MoAb) 17-109 and G8 were co-expressed in one patient. No B-cells from the patients expressed the VHIII-associated CRI. The same pattern of CRI expression was observed in a serum paraprotein collected from one of the patients. These results suggest a biased selection for the IG VKIII and VKIIIb light chains in B-PLL.

Rheumatoid lymphadenopathy: a morphological and immunohistochemical study.

Sixteen lymph nodes from 14 patients with rheumatoid arthritis were examined immunohistochemically and morphometrically and compared with 10 control nodes showing follicular hyperplasia from patients without rheumatoid disease. Frozen material was available from nine patients and all controls. The nodes from patients with rheumatoid arthritis seemed to share characteristic features. The most striking of these was follicular hyperplasia in which the germinal centres, in spite of being quite large, showed relatively sparse proliferative activity. The nodes often showed infiltration of germinal centres by CD8 positive T lymphocytes and contained fewer IL2R positive cells in the paracortex than controls. These and other features may have some correlation with disease activity. Lymphadenopathy in rheumatoid arthritis may not just be a manifestation of joint inflammation but an active component of this multisystem disease and may reflect a widespread immunological abnormality.

Specificity and idiotope expression of IgM produced by CD5+ and CD5- cord blood B-cell clones.

Epstein-Barr virus (EBV)-immortalized monoclonal B-cell lines were established from CD5+ and CD5- cord-blood B cells. IgM from many of both CD5+ and CD5- clones reacted with IgG-Fc, ssDNA, and a variety of other autoantigens. More CD5+ B cells that used light chains of the kappa isotype reacted with IgG-Fc and ssDNA than kappa-bearing CD5- B cells. Because many of the clones reacted with IgG-Fc, they were analyzed for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor and cold agglutinin paraproteins using murine antibodies (mAb) recognizing V kappa and VH subgroup-associated determinants. Expression of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17.109 mAb was expressed at significantly higher frequency (32%; p less than 0.05) and IgM antibodies derived from the CD5+ compared with the CD5- clones (5%). Both CD5+ and CD5- clones expressed the RF paraprotein-associated idiotope recognized by G8 mAb to the same extent. Similar results were obtained using binding to SpA as a marker of VH III family usage. Furthermore, no differences in frequency of expression of RF paraprotein-associated idiotopes recognized by B6 and/or D12, and characteristic of some antibodies using VH III family genes, were found between the CD5+ and CD5- populations. Although a higher than expected frequency of VH IV-gene expression was demonstrated (around 30%) in both CD5+ and CD5- cells, there were differences in expression of CRI recognized by mAb Lc1 and R2.1A2 with specificities for two VH IV subfamilies. While some CD5+ and CD5- clones were identified in which their IgM reacted with mAb Lc1, only CD5+ clones were recognized by another mAb R2.1A2. Analysis of the relationships between antigen specificities and V kappa- and VH-family gene usage indicated that auto- or polyreactivity was not associated with V kappa III nor any particular VH family. The higher frequency of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17-109 in the CD5+ clones and the association of CD5+ B cells with the VH IV subfamily recognized by mAb R2.1A2 and 9G4 may suggest that CD5+ B cells in cord blood are expanded as a result of recruitment within the fetal environment.

Rheumatoid factor autoantibodies in health and disease.

Recent advances in molecular biological and human cell hybridization technology have significantly advanced the knowledge of mechanisms that underlie human rheumatoid factor (RF) production. These advances have provided insight into the etiopathogenesis of synovial inflammation and lymphocyte recruitment in rheumatoid arthritis (RA) joints. We have examined the mechanisms that lead to RF production in RA patients and those that regulate RF production in normals. The studies revealed structural features that distinguish RF produced in normals from those produced in RA synovial tissue. There are significant differences in the use of VL and VH genes between the two RF populations. Furthermore, IgV genes encoding synovial RF in RA have extensive evidence for nucleotide changes, leading to amino acid replacement in the complementarity determining regions (CDRs). In addition, RF produced in RA synovia show evidence for affinity maturation, isotype switch to IgG RF, and repertoire shift indicative of a continued recruitment of B cells. Together with computer modeling and crystallographic studies, our data suggest that the mechanisms that operate on RF selection in RA synovia are similar to immune responses to exogenous antigens. In contrast, RF established from human immunized donors (HID) are characterized by a very low ratio of replacement to silent (R:S) nucleotide changes in the CDR1+2. In addition, there is little increase in affinity with increasing numbers of mutations. There is thus evidence for regulatory mechanisms that limit affinity maturation of RF in normals.

Probing of the rheumatoid factor (RF) V gene repertoire in rheumatoid arthritis (RA) by hybridoma clones.

Twenty human monoclonal antibodies with rheumatoid factor (RF) specificity were produced from fusions using B lymphocytes derived from the synovial tissue of two patients with rheumatoid arthritis (RA) and one with the polyarticular form of juvenile rheumatoid arthritis (JRA) (1). All the 20 monoclonal antibodies were IgM. Fourteen of these were classical RFs with specificity restricted to IgG, and included 12 kappa and 2 lambda proteins. When the fine specificity for IgG Fc determinants were investigated most of them showed the Ga specificity. In addition, 5 lambda and 1 kappa monoclonal RF antibodies showed polyreactivity and also reacted with various other antigens than IgG (1). The 14 monoreactive RFs were further studied for the expression of RF-related cross-reactive idiotypes (CRI) and variable heavy (VH) and light chain (VL) subgroups. Only four of the twelve kappa RFs expressed the V kappa III subgroup. Three of them belong to the V kappa IIIb sub-subgroup and expressed the CRI 17.109. One of these 3 clones in addition expressed the VH I associated CRI G6. Five other monoreactive RFs expressed either or both of the VH III associated CRI B6 and D12 (2). Using staphylococcal protein A (SPA) binding as well as Northern blotting techniques (2), studies indicated that 10 out of the 12 RFs studied expressed the VH III regions and 2 expressed the VH I region. These data, both for the heavy and light chains, indicated a different V gene usage by the RF derived from RA patients than by the RF M-components derived from patients with mixed cryoglobulinemia and Waldenström's macroglobulinemia but without RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene therapy for rheumatoid arthritis. Theoretical considerations.

Current understanding of the pathogenesis of rheumatoid arthritis has provided evidence that therapeutic benefit can be achieved by using antagonists targeted to the inflammatory cytokines involved, mainly tumour necrosis factor-alpha and interleukin-1. Gene delivery of antagonists, which can inhibit the production or action of these cytokines and other mediators, has been achieved in experimental animal models. This new method of delivery can produce therapeutic effects at lower concentrations and in a local environment, overcoming the adverse effects that often accompany protein therapy. However, several technological and biological restraints preclude the immediate adaptation of this method to human treatment. Based on the experimental evidence, possible target therapeutic genes, cell types and vector systems that could be used are discussed in this article.

Analysis of the expressed immunoglobulin variable region heavy chain gene products in paraproteins from Iranian patients with multiple myeloma.

The frequency of expression of immunoglobulin (Ig) variable region heavy (VH ) chain gene products was studied in 43 Iranian patients with mutiple myeloma (MM). The expressed VH gene families and associated cross-reactive idiotypes (CRI) were analysed by immunoblotting and ELISA, using peptide-induced polyclonal antibodies specific for VH 1-VH 6 gene families and monoclonal antibodies (MAb) recognising CRI linked to theVH 1, VH 3, VH 4 and VH 6 gene families. The results revealed that the VH 3 family (60. 5%) was the most predominant gene family. In contrast, no paraproteins were encoded by genes from the VH 2 gene family and only 2.3% were encoded by the VH 5 family. The panel of paraproteins tested rarely expressed the probed VH -associated CRI. Our results suggest that: 1-The Ig VH genes, may not be randomly expressed in the malignant plasma cells from Iranian patients with MM. 2- Some of the genes seem to be negatively selected or highly mutated, as evidenced by the lack of expression of the probed CRI.