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.

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.

The role of antigen in the selection of the human V3-23 immunoglobulin heavy chain variable region gene.

The immune system mounts antibody responses using few of the available immunoglobulin variable region (IgV) genes with some, such as the V3-23 heavy chain gene, regularly over-represented in responses to many antigens. The reasons for the over-representation of some V genes have not been established; the process could be either stochastic or selective. We demonstrated previously that the V3-23 gene, which is over-represented in the primary B lymphocyte repertoire in humans, encodes antibodies with differing antigen-binding reactivities in transgenic mice that express the human V3-23 gene. The aim of the current study was to assess if V3-23 gene over-representation is stochastic or could be influenced by antigen exposure. Transgenic mice were immunized with human IgG-Fc (hIgG-Fc), bovine collagen type II (bCII) or tetanus toxoid (TT), and hybridomas secreting human mu chain-containing antibodies generated. These were tested for binding to the immunogens and a panel of self- and exogenous antigens. In hybridomas derived from hIgG-Fc-immunized mice, 53% secreted antibodies specific for hIgG-Fc. A similar proportion (54%) of hybridomas from bCII-immunized mice secreted antibody that bound to collagen. By contrast, only 21% of hybridomas from mice immunized with TT bound to tetanus toxoid. Intriguingly, chimaeric antibodies generated from mice immunized with bCII or TT were mainly polyreactive, similar to antibodies generated from naive transgenic mice. However, hybridomas generated from mice immunized with hIgG-Fc were mainly specific, reacting exclusively with hIgG-Fc. These results suggest that selection and eventual expansion of B lymphocytes expressing the V3-23 gene are likely to be determined by exposure to self- and/or environmental antigens.

Immunopathology and the gene therapy of lupus.

Lupus is a chronic autoimmune inflammatory disease with complex clinical manifestations. In humans, lupus, also known as systemic lupus erythematosus (SLE), affects between 40 and 250 individuals, mostly females, in each 100 000 of the population. There are also a number of murine models of lupus widely used in studies of the genetics, immunopathology, and treatment of lupus. Human patients and murine models of lupus manifest a wide range of immunological abnormalities. The most pervasive of these are: (1) the ability to produce pathogenic autoantibodies; (2) lack of T- and B-lymphocyte regulation; and (3) defective clearance of autoantigens and immune complexes. This article briefly reviews immunological abnormalities and disease mechanisms characteristic of lupus autoimmunity and highlight recent studies on the use of gene therapy to target these abnormalities.

Polymorphism in the immunoglobulin VH gene V1-69 affects susceptibility to rheumatoid arthritis in subjects lacking the HLA-DRB1 shared epitope.

OBJECTIVE: To investigate the contribution of polymorphism in the immunoglobulin heavy chain variable region V1-69 gene set to genetic susceptibility to rheumatoid arthritis (RA) in Czech and British patients. METHODS: We used V1-69 gene sequence-specific polymerase chain reaction (PCR) and restriction enzyme digestion to study polymorphism in the V1-69 gene set in germline DNA of 109 Czech and 159 British RA patients and 164 ethnically matched controls. Polymorphism was further studied by nucleotide sequencing of the V1-69 gene locus in germline DNA. RESULTS: We found that all patients and controls had at least one V1-69 gene copy. In the Czech RA cohort, the dimorphic nucleotide in codon 73 of V1-69 (GAA or AAA) was present in the homozygous form 73(A/A) in 31 of 109 (28.4%) RA patients vs 12 of 79 (15.2%) controls [odds ratio (OR)=2.22, P<0.001]. When the RA patients and controls were classified according to HLA shared epitope (SE) status, 73(A/A) was found in 18 of 76 (23.7%) SE(+) patients compared with 13 of 38 (34.2%) SE(-) patients, four of 12 (18.2) SE(+) controls and eight of 57 (14%) SE(-) controls. This suggests that homozygosity for the dimorphic sequence 73(A) contributed to susceptibility to RA in SE(-) Czech individuals (OR=3.2, P<0.001). The most striking observation was that none of the 38 SE(-) Czech patients, compared with 11 of 76 (14.5%) SE(+) RA patients, three of 22 (13.6%) SE(+) and 11 of 57 (19.3%) SE(-) ethnically matched controls, were homozygous for the alternative dimorphic sequence 73(G/G) (OR=9.1, P<0.05). These data, however, were not replicated in a Caucasoid British RA population. CONCLUSION: The dimorphic sequence at codon 73 (73(A/A)) of the V1-69 gene contributes to genetic susceptibility in SE(-) Czech RA patients.

Cross-reactivity and pathogenicity of anti-DNA autoantibodies in systemic lupus erythematosus.

Autoantibodies to DNA were discovered over 40 years ago following the discovery a few years earlier of the 'LE' cell phenomenon by Hargraves and colleagues in 1948. These investigators noted that, when leucocytes were incubated with serum from lupus patients, changes in the nucleus could be seen together with phagocytosis of nuclear remnants by polymorphonuclear leucocytes. Since that time numerous studies in many laboratories have investigated almost every aspect of anti-DNA antibodies, partly to identify what determines their pathology. Whilst a subset of anti-DNA antibodies, especially anti-native, or double-stranded DNA (dsDNA) antibodies constitutes a hallmark of lupus disease and a diagnostic criterion, it is now clear that not all anti-DNA autoantibodies are of pathogenic relevance. Moreover, anti-DNA autoantibodies may also be found in other connective tissue disorders. Here we briefly review studies presented at the fifth international workshop on anti-DNA autoantibodies held in London to highlight relevant properties of pathogenic anti-DNA antibodies.

Tumour necrosis factor alpha in systemic lupus erythematosus and anti-DNA autoantibody production.

Tumour necrosis factor (TNFalpha) is a cytokine with a wide range of diverse and at times paradoxical effects. These include immunoregulatory, lymphoid organogenesis and pro-inflammatory effects. In recent years, TNFalpha has become a focus of interest more for its inflammatory effects in a number of chronic autoimmune diseases. This interest culminated in the successful treatment of patients with rheumatoid arthritis, Crohn's diseases and ankylosing spondylitis with blocking antibodies or soluble TNFalpha receptors. Paradoxically, however, TNFalpha also has immunomodulatory effects in some autoimmune conditions such as lupus in some mouse models of the disease and in diabetes in the none-obese diabetic mouse. The role TNFalpha plays in human systemic lupus erythematosus is, however, controversial. In this article we review some of the studies carried out to elucidate the effects of TNFalpha in lupus disease and likely mechanisms of action. Further, we discuss recent data on the likely effects of blocking TNFalpha on anti-DNA autoantibody production.

Higher frequency of allele 2 of the interleukin-1 receptor antagonist gene in patients with juvenile idiopathic arthritis.

OBJECTIVE: An increased incidence of allele 2 of the interleukin-1 receptor antagonist gene (IL1RN*2) in several inflammatory diseases has recently been reported. The aim of this study was to examine a variable number tandem repeat (VNTR) polymorphism of the IL1RN gene in patients with juvenile idiopathic arthritis (JIA). METHODS: Findings in 185 Czech patients with JIA were compared with those in 168 Czech controls, 50 JIA patients and 52 controls of Turkish origin, and 79 controls from central England. VNTR polymorphism analysis of IL1RN was performed by polymerase chain reaction using 2 flanking primers to amplify an 86-bp tandem repeat region in intron 2. RESULTS: The frequency and carriage rate of IL1RN*2 were significantly increased in Czech JIA patients compared with the Czech controls (frequency 27.6% versus 15.8%; carriage rate 44.3% versus 26.2%). Increased frequency and carriage rate of IL1RN*2 were found in 23.3% and 40.0% of Turkish JIA patients and in 17.3% and 34.6% of ethnically matched controls. The high representation of IL1RN*2 in 52.3% of the 22 patients with extended oligoarthritis, 31.3% of the 56 patients with enthesitis-related arthritis, and 42.9% of the 14 patients with other arthritis was particularly responsible for the increased frequency of IL1RN*2 in the Czech JIA patients. We found no association of IL1RN*2 with disease activity or severity parameters. CONCLUSION: Inheritance of IL1RN*2 may contribute to genetic susceptibility in several forms of autoimmune diseases, including JIA. The IL1RN*2 allele may be useful as a prognostic indicator of the evolution of an extended oligoarticular course of JIA.

The effects of a human IgM monoclonal anticardiolipin antibody on pregnancy in a transgenic mouse model.

The clinical features of antiphospholipid (or Hughes') syndrome (APS) are most commonly seen in individuals who have raised levels of IgG anticardiolipin antibodies. Most murine models of the syndrome have involved the administration of such antibodies to normal mice. However, APS can occur in the presence of raised levels of serum IgM anticardiolipin antibodies alone. The present study was designed to see if an IgM monoclonal antibody can induce changes in mice similar to those seen in human APS. This antibody, BH1, has previously been derived from a patient with primary APS. In its ligand-binding and idiotypic characteristics it is representative of antiphospholipid antibodies (aPL) found in the serum of patients with APS. In order to minimise the immune response to human IgM, we used transgenic mice (F15) which express, and are predicted to be tolerant of, human immunoglobulin mu chains. The features of APS may develop more readily in individuals who have an existing autoimmune disorder, such as systemic lupus erythematosus (SLE). We therefore crossed these transgenic mice with New Zealand Black (NZB, SLE-prone) mice, and used the progeny (F15 x NZB/F1) in our experiments. Twenty-four F15 x NZB/F1 mice were given BH1, or a control IgM antibody, (A5566) immediately preceding and then three times during pregnancy. There was a reduction in the mean number of foetuses in animals given BH1 compared with those given A5566 (8.6 vs 11.0; P < 0.05), and a similar reduction in mean total foetal weight per pregnancy (9.05 vs 12.73 g; P < 0.05). Two mice showed a marked reduction in platelet count. No evidence of thrombosis was detected macroscopically or histologically. Our results show a lower incidence of APS-type features compared to previous studies in which mice have been administered aPL. This may be because BH1 is an IgM antibody. Nevertheless, the data support the concept that IgM aPL of particular ligand-binding specificities may have a direct pathogenetic role in certain cases of human APS.

Rearrangement of the human heavy chain variable region gene V3-23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region.

To formulate a 'logic' for how a single immunoglobulin variable region gene generates antibodies with different antigen specificity and polyreactivity, we analysed chimeric antibodies produced in transgenic mice carrying the germ-line human V3-23 gene, multiple diversity (D) and joining (J) gene segments. Hybridomas producing antibodies encoded by the V3-23 gene in combination with different mouse Vkappa genes were obtained by fusion of splenocytes from transgenic mice. All antibodies had human mu-chains and mouse light chains, were multimeric in structure and expressed the human V3-23 gene. Nucleotide sequence analyses of genes encoding the heavy and light chains of 12 antibodies in relation to antigen specificity highlighted the importance of heavy chain variable region CDR3 in determining reactivity with different antigens. However, the results also suggest that non-CDR3 sequences intrinsic to the V3-23 gene itself may be involved in, or determine, the binding of the chimeric antibodies to some of the antigens tested in the current study.

CD4+ T lymphocytes with constitutive CD40 ligand in preautoimmune (NZB x NZW)F1 lupus-prone mice: phenotype and possible role in autoreactivity.

Lupus disease is marked by B lymphocyte hyperactivity and the production of Abs to dsDNA. The production of these anti-dsDNA Abs is T lymphocyte dependent. However, it is not clear how CD4+ T lymphocytes provide help for B lymphocytes to produce IgG anti-dsDNA Abs. One possible mechanism is suggested by studies showing that human patients with systemic lupus erythematosus and lupus mice have increased numbers of CD40 ligand (CD40L)+ T and B lymphocytes. The results described in this study reveal that young, clinically healthy lupus-prone New Zealand Black x New Zealand White F1 (BWF1) mice have naive CD4+ T cells with preformed CD40L. These cells contribute to a brisk response to immunization and to the production of anti-dsDNA Abs. In vitro experiments revealed that CD4+ T cells with preformed CD40L could, upon stimulation, provide antiapoptotic signals for B cells but could not induce proliferation or reduce activation threshold. These results suggest that the direct target cells for the effect of T cells with preformed CD40L in lupus may not be B lymphocytes.

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.

Molecular analysis of human immunoglobulin heavy chain variable region associated determinants recognized by anti-VH3 antibodies 7B4, B6 and D12.

7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin heavy chain products of the closely related V3-30, V3-30.3 and V3-33 genes from the VH3 family. B6 and D12 have additional reactivities with some immunoglobulins (Ig) encoded by the V3-11 and V3-7 genes; D12 also reacts with some V3-43 gene Ig. We show here, by site-directed mutagensis, that the lysine at position 57 in the complementarity-determining region 2 (CDR-2) of the V3-30 gene product is crucial for epitope recognition by all three anti-VH3 MoAbs. Further analysis of the amino-acid sequences of a large panel of Ig reactive, or nonreactive, with MoAb 7B4 indicates that the determinant recognized by 7B4 is dependent on the presence of the tetrapeptide sequence NKYY between positions 56 and 59 in the CDR-2. Comparing the efficiency of 7B4 reactivity with VH3 gene-encoded human Ig indicates that amino-acid position 4 in the frame region 1 (FR-1) may also influence the binding of 7B4 to Ig encoded by three very closely related germline genes, V3-30, V3-30.3 and V3-33. NKYY is also found on the gp120 V3 region of human immunodeficiency virus (HIV)-2, SIV and HTLV-4. We also report that other tetrapeptide sequences found on the 56-59 motif of heavy chain variable regions encoded by germline genes are expressed on the solvent exposed V2 region of gp120 of HIV-1 isolates. The possible significance of these observations is discussed.

Immuno- and genetic therapy in autoimmune diseases.

Animal models of autoimmune disease have been developed that mimic some aspects of the pathophysiology of human disease. These models have increased our understanding of possible mechanisms of pathogenesis at the molecular and cellular level and have been important in the testing, development and validation of new immunotherapies. The susceptibility to develop disease in the majority of these models is polygenic as is the case in humans. The exceptions to this rule are gene knock outs and transgenic models of particular genes which, in particular genetic backgrounds, have also contributed to the understanding of single gene function and their possible contribution to pathogenesis. Gene therapy approaches that target immune functions are being developed with encouraging results, despite the polygenic nature of these diseases. Basically this novel immuno-genetic therapy harnesses the knowledge of immunology with the myriad of biotechnological breakthroughs in vector design and delivery. Autoimmune disease is the result of genetic dysregulation which could be controlled by gene therapy. Here we summarize the genetic basis of these human diseases as well as some of the best characterized murine models. We discuss the strategies for their treatment using immuno- and gene therapy.

Increased IgA rheumatoid factor and V(H)1 associated cross reactive idiotype expression in patients with Lyme arthritis and neuroborreliosis.

OBJECTIVE: To investigate whether autoreactive mechanisms occur in Lyme disease (LD) by determining IgA, IgG and IgM rheumatoid factor (RF) concentrations and RF associated cross reactive idiotype (CRI) expression in the serum of LD patients, with comparison to patients with rheumatoid arthritis (RA). METHODS: The RF isotype profiles were determined in 59 patients with LD; erythema migrans (EM) (n=19), neuroborreliosis (NB) (n=20) and Lyme arthritis (LA) (n=20). Mouse monoclonal antibodies (mAbs) G6 and G8 (V(H)1 gene associated), D12 (V(H)3 gene associated) and C7 (V(kappa)III gene associated) were then used to determine the RF associated CRI expression on IgM antibodies in 16 of these LD patients (eight seropositive for RF); (EM (n=3), NB (n=6), LA (n=7)). RESULTS: Seven (18%) patients with either NB or LA had increased concentrations of IgA RF compared with none with EM. Significant differences in the number of patients with raised concentrations of IgG RF or IgM RF were not found between the LD patient groups. Five (3NB, 1LA and 1 EM) (31%) and three (2NB and 1LA) (19%) of LD patients had raised concentrations of the CRIs recognised by mAbs G6 and G8, respectively. These CRIs were detected in LD sera both with and without raised concentrations of RF and were not demonstrated on anti-Borrelia burgdorferi antibodies using ELISA. No LD sera tested had raised concentrations of the determinants recognised by mAbs C7 or D12. CONCLUSION: Significantly raised concentrations of IgA RF and increased use of V(H)1 germline gene associated CRIs are found on IgM antibodies in the serum of LD patients. These data indicate the recruitment of autoreactive B lymphocytes in some patients with the later stages of LD.

Rheumatoid factor isotype switch and somatic mutation variants within rheumatoid arthritis synovium.

The presence of clonally-related B-lymphocyte aggregates within synovial lining tisue of rheumatoid arthritis (RA) patients suggests a germinal centre-like reaction, which may hold implications for disease pathogenesis and the causes of chronic inflammation. We studied 250 rheumatoid factor (RF) heavy-chain sequences cloned from the synovium of three patients with RA, to determine whether they undergo both somatic mutation and isotype switching consistent with this hypothesis. Size analysis of immunoglobulin heavy-chain cDNAs from synovial RF+ B cells revealed oligoclonal RF+ populations and identically-sized VH-D-JH transcripts of different immunoglobulin isotypes. Sequencing of individual inserts selected from cloned immunoglobulin heavy-chain cDNAs demonstrated a clonal relationship between immunoglobulin M (IgM) RF and IgA RF, suggesting that this isotype switch occurred in synovium. Furthermore, most somatic mutations were found to have occurred after this isotype switch. This finding suggests that the RA synovial microenvironment sustains somatic mutation and isotype switching in RF-specific B lymphocytes akin to secondary lymphoid organs.