Wayel H. Abdulahad

Effectiveness of Rituximab Treatment in Primary Sjogren's Syndrome A Randomized, Double-Blind, Placebo-Controlled Trial

OBJECTIVE To study the efficacy and safety of B cell depletion with rituximab, a chimeric murine/human anti-CD20 monoclonal antibody, in patients with primary Sjögrens syndrome (SS) in a double-blind, randomized, placebo-controlled trial. METHODS Patients with active primary SS, as determined by the revised American-European Consensus Group criteria, and a rate of stimulated whole saliva secretion of > or =0.15 ml/minute were treated with either rituximab (1,000 mg) or placebo infusions on days 1 and 15. Patients were assigned randomly to a treatment group in a ratio of 2:1 (rituximab:placebo). Followup was conducted at 5, 12, 24, 36, and 48 weeks. The primary end point was the stimulated whole saliva flow rate, while secondary end points included functional, laboratory, and subjective variables. RESULTS Thirty patients with primary SS (29 female) were randomly allocated to a treatment group. The mean +/- SD age of the patients receiving rituximab was 43 +/- 11 years and the disease duration was 63 +/- 50 months, while patients in the placebo group were age 43 +/- 17 years and had a disease duration of 67 +/- 63 months. In the rituximab group, significant improvements, in terms of the mean change from baseline compared with that in the placebo group, were found for the primary end point of the stimulated whole saliva flow rate (P = 0.038 versus placebo) and also for various laboratory parameters (B cell and rheumatoid factor [RF] levels), subjective parameters (Multidimensional Fatigue Inventory [MFI] scores and visual analog scale [VAS] scores for sicca symptoms), and extraglandular manifestations. Moreover, in comparison with baseline values, rituximab treatment significantly improved the stimulated whole saliva flow rate (P = 0.004) and several other variables (e.g., B cell and RF levels, unstimulated whole saliva flow rate, lacrimal gland function on the lissamine green test, MFI scores, Short Form 36 health survey scores, and VAS scores for sicca symptoms). One patient in the rituximab group developed mild serum sickness-like disease. CONCLUSION These results indicate that rituximab is an effective and safe treatment strategy for patients with primary SS.

Skewed distribution of Th17 lymphocytes in patients with Wegener's granulomatosis in remission.

OBJECTIVE The recently characterized interleukin-17 (IL-17)-producing T helper cell lineage (Th17), rather than the Th1 lineage, is involved in several autoimmune diseases. The possible role of Th17 cells in Wegeners granulomatosis (WG) has not yet been elucidated. We undertook this study to assess the distribution of Th1/Th2/Th17 cells and to investigate the presence of Th17 cells specific for the WG autoantigen proteinase 3 (PR3) in WG. METHODS Peripheral blood from patients with WG in remission (n = 26) and healthy controls (n = 10) was stimulated in vitro with PR3 or with the control stimuli staphylococcal enterotoxin B (SEB), tetanus toxoid (TT), or phorbol myristate acetate/calcium ionophore, together with anti-CD28 and anti-CD49d. The frequencies of the various CD4+ T cell phenotypes responsive to stimuli were determined by 7-color flow cytometric detection of CD3, CD8, an early activation marker (CD69), and intracellular cytokines (IL-2, interferon-gamma [IFNgamma], IL-17, and IL-4). RESULTS The percentage of CD69+,CD4+ T cells in patients with WG in remission was significantly decreased in response to PR3 and tended to be lower in response to other stimuli compared with the percentage in healthy controls. The percentages of Th17 cells (IL-4-,IL-17+,IFNgamma-) and Th2 cells (IL-4+,IL-17-,IFNgamma-) within the activated CD69+,CD4+ T cell population were significantly increased in patients with WG in remission, while no difference was found in Th1 cells (IL-4-,IL-17-,IFNgamma+) compared with the percentage in healthy controls. Increased percentages of Th17 cells in response to TT and SEB were found both in antineutrophil cytoplasmic antibody (ANCA)-positive and in ANCA-negative patients, while an increased frequency of PR3-specific Th17 cells was restricted to ANCA-positive patients. CONCLUSION A skewed Th17 response found in ANCA-positive WG patients following stimulation with the autoantigen PR3 suggests that IL-17 is involved in disease pathogenesis and could constitute a new therapeutic target.

Abatacept treatment reduces disease activity in early primary Sjögren's syndrome (open-label proof of concept ASAP study)

Objective To assess the efficacy and safety of abatacept in patients with early and active primary Sjögrens syndrome (pSS). Methods All 15 patients (12 women, three men) included in the open-label Active Sjögren Abatacept Pilot study met the revised American-European Consensus Group criteria for pSS and were biological disease-modifying antirheumatic drug-naive. Patients were treated with eight intravenous abatacept infusions on days 1, 15 and 29 and every 4 weeks thereafter. Follow-up was conducted at 4, 12, 24 (on treatment), 36 and 48 weeks (off treatment). Disease activity was assessed with European League Against Rheumatism (EULAR) Sjögrens Syndrome Disease Activity Index (ESSDAI) and EULAR Sjögrens Syndrome Patient Reported Index (ESSPRI). Several other functional, laboratory and subjective variables were analysed. Generalised estimating equations were used to analyse parameters over time. Results ESSDAI, ESSPRI, rheumatoid factor and IgG levels decreased significantly during abatacept treatment and increased post-treatment. Salivary and lacrimal gland function did not change during treatment. Fatigue and health-related quality of life (HR-QoL) improved significantly during treatment. No serious side effects or infections were seen. Conclusions In this open-label study, abatacept treatment is effective, safe and well tolerated, and results in improved disease activity, laboratory parameters, fatigue and HR-QoL in patients with early and active pSS. Trial registration number 2009-015558-40.

Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is an autoimmune disease accompanied by a disturbed T-cell balance skewed towards effector T-cells, in particular Th17-cells. The novel cytokine interleukin-21 (IL-21) is suggested to be crucial for triggering T-cell responses towards IL-17 producing cells. Thus, we aimed to investigate the ability of T-cells to produce IL-21 and IL-17 in SLE patients.MethodsPeripheral blood of 34 SLE patients and 18 healthy controls (HC) was stimulated with phorbol myristate acetate (PMA) and calcium ionophore (Ca-Io). Percentages of IL-21- and IL-17A expressing T-cells were analysed by flow cytometry. The expression levels of the transcription factors B-cell lymphoma-6 (BCL-6) and factors retinoid-related orphan receptor (ROR-γt) were assessed in T-cells by real-time RT-PCR and flow cytometry. Additionally, IL-21 receptor (IL-21R) expression on B- and T-cells of patients and HC was analyzed.ResultsSignificantly increased percentages of IL-21 expressing CD4+ T-cells and CD8+ T-cells were found in SLE patients as compared to HC. The percentages of IL-21+ CD4+ T-cells and CD8+ T-cells correlated significantly with the percentages of IL-17A+ CD4+ T-cells and CD8+ T-cells, respectively. The relative expression of BCL-6 and ROR-γt did not differ between SLE patients and HC. IL-21R expression occurred mainly on B-cells and was not different comparing SLE patients and HC.ConclusionsThis study demonstrates an increased proportion of IL-21+ T-cells in SLE patients correlating with the proportion of IL-17+ T-cells. This suggests a pivotal role of IL-21 in the pathogenesis of SLE.

Disturbed Th1, Th2, Th17 and T-reg balance in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease accompanied by disturbed T-cell homeostasis. Dysbalance of T-helper-cell (Th) subsets (Th1/Th2/Th17) and regulatory T-cells (T(regs)) is suggested to contribute to the pathogenesis of SLE. Recent reports suggest functional deviation of T(regs) in terms of producing IL-17A, a process that may be aberrant in SLE. Therefore, we analyzed these T-cell subsets in SLE to test the hypothesis that aberrant T-cell subset skewing is present in SLE-patients. We investigated simultaneously the intracellular cytokines IFN-γ, IL-4 and IL-17A in CD4(+)T-cells as well as in T(regs). Skewing of T-cell subsets towards Th17 cells was observed in SLE-patients. Although the proportion of T(regs) was similar between SLE-patients and healthy controls, the ability of T(regs) to express IFN-γ and IL17A was impaired in SLE-patients. Even in quiescent SLE-patients T-cell homeostasis is aberrant in terms of skewing towards IL-17 producing T-cells.

Frequencies and role of regulatory T cells in patients with (pre)malignant cervical neoplasia

Oncogenic human papillomavirus (HPV)‐infection is crucial for developing cervical cancer and its precursor lesions [cervical intraepithelial neoplasia (CIN)]. Regulatory T cells (Tregs) might be involved in the failure of the immune system to control the development of HPV‐induced cancer. We investigated frequencies, phenotype and activity of Tregs in patients with cervical neoplasia. CIN and cervical cancer patients showed increased CD4+/CD25high T cell frequencies in peripheral blood and CD4+ T cell fraction. These CD4+/CD25high T cells represent Tregs as demonstrated by their low proliferation rate, low interferon (IFN)‐γ/interleukin (IL)‐10 ratio, high expression of CD45RO, GITR, CTLA‐4, forkhead box P3 (FoxP3) and low CD45RA expression. Moreover, in HPV16+ cervical cancer patients, in‐vitro depletion of CD25+ T cells resulted in increased IFN‐γ T cell responses against HPV16 E6‐ and E7 peptides. Thus, increased frequencies of Tregs in cervical cancer patients may indeed suppress HPV‐specific immunity. Longitudinal analysis of CD4+/CD25high T cell frequencies in patients showed a modest decline 1 year after curative surgery or chemoradiation. This study demonstrates increased frequencies and suppressive activity of Tregs in cervical cancer. These results imply that Tregs may suppress the immune control of cervical neoplasia and furthermore that suppression of immunity by Tregs will be another hurdle to overcome in therapeutic immunization strategies against cervical neoplasia.

Urinary CD4+ effector memory T cells reflect renal disease activity in antineutrophil cytoplasmic antibody–associated vasculitis

OBJECTIVE Numbers of circulating CD4+ effector memory T cells are proportionally increased in patients with proteinase 3 antineutrophil cytoplasmic antibody-associated vasculitis (AAV) whose disease is in remission and are decreased during active disease, which presumably reflects their migration toward sites of inflammation. Since renal infiltrating cells may appear in urine, we investigated the presence of CD4+ effector memory T cells in urinary sediment as a reflection of renal disease activity in AAV. METHODS CD4+ effector memory (CD45RO+CCR7-CD3+CD4+) T cells were quantitated in the urine and peripheral blood of patients with AAV with renal involvement (n = 33), patients with AAV without renal involvement (n = 18), patients with AAV whose disease was in remission (n = 29), and patients with active disease (n = 22), using 4-color flow cytometric analysis. Numbers and percentages of urine CD4+ effector memory T cells in 12 patients with AAV with active renal disease were obtained over several weeks of followup during remission induction. RESULTS A notable increase in urine CD4+ effector memory T cell numbers was observed in patients with active renal AAV compared with patients whose disease was in remission and patients with active disease without renal involvement. The increase in these cells in the urine of patients with active renal AAV was accompanied by a reciprocal decrease in these cells in peripheral blood. Results from followup analysis showed a clear reduction in urine CD4+ effector memory T cells following treatment. Moreover, a negative correlation was observed between percentages of circulating and urine CD4+ effector memory T cells, consistent with their migration toward sites of inflammation. CONCLUSION Our findings indicate that the presence of CD4+ effector memory T cells in urine reflects renal involvement in AAV. Flow cytometric analysis of these cells in urine may contribute to assessing renal disease activity in patients with AAV.

Bacterial DNA motifs trigger ANCA production in ANCA-associated vasculitis in remission

OBJECTIVES CpG motifs, which are highly prevalent in bacterial DNA, have been shown to trigger the production of ANCA in vitro by B lymphocytes from patients with active ANCA-associated vasculitis (AAV). Staphylococcus aureus is associated with relapses in AAV, and CpG motifs from staphylococcal DNA may trigger ANCA production in AAV patients in remission. We investigated the presence of ANCA-producing B lymphocytes during quiescent disease and tested the capacity of these cells to produce ANCA in response to CpG. METHODS Expression of Toll-like receptor 9 (TLR9) by B lymphocytes from AAV patients and controls was assessed. Peripheral blood mononuclear cells were isolated from 23 PR3-ANCA and 15 MPO-ANCA patients (33 quiescent, 5 active disease) and 14 healthy controls, and cultured for 12 days in the presence of cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) and IL-2. B-lymphocyte activation, differentiation, immunoglobulin production and in vitro ANCA production were studied. RESULTS TLR9 expression by B lymphocytes was comparable in AAV patients and controls. B lymphocytes were activated and differentiated towards a plasma cell phenotype in response to CpG-ODN and IL-2. ANCA were produced in vitro by 13 out of 23 PR3-ANCA patients and 3 out of 15 MPO-ANCA patients. CONCLUSIONS We conclude that ANCA-producing B lymphocytes can be present in the peripheral blood of AAV patients during remission. These autoreactive B lymphocytes are triggered by CpG-ODN and IL-2 to produce ANCA in vitro. CpG motifs may trigger the production of ANCA in vivo, contributing to the development of relapses in AAV.

Expression profiles of long non-coding RNAs located in autoimmune disease-associated regions reveal immune cell-type specificity

BackgroundAlthough genome-wide association studies (GWAS) have identified hundreds of variants associated with a risk for autoimmune and immune-related disorders (AID), our understanding of the disease mechanisms is still limited. In particular, more than 90% of the risk variants lie in non-coding regions, and almost 10% of these map to long non-coding RNA transcripts (lncRNAs). lncRNAs are known to show more cell-type specificity than protein-coding genes.MethodsWe aimed to characterize lncRNAs and protein-coding genes located in loci associated with nine AIDs which have been well-defined by Immunochip analysis and by transcriptome analysis across seven populations of peripheral blood leukocytes (granulocytes, monocytes, natural killer (NK) cells, B cells, memory T cells, naive CD4+ and naive CD8+ T cells) and four populations of cord blood-derived T-helper cells (precursor, primary, and polarized (Th1, Th2) T-helper cells).ResultsWe show that lncRNAs mapping to loci shared between AID are significantly enriched in immune cell types compared to lncRNAs from the whole genome (α <0.005). We were not able to prioritize single cell types relevant for specific diseases, but we observed five different cell types enriched (α <0.005) in five AID (NK cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, and psoriasis; memory T and CD8+ T cells in juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis; Th0 and Th2 cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis). Furthermore, we show that co-expression analyses of lncRNAs and protein-coding genes can predict the signaling pathways in which these AID-associated lncRNAs are involved.ConclusionsThe observed enrichment of lncRNA transcripts in AID loci implies lncRNAs play an important role in AID etiology and suggests that lncRNA genes should be studied in more detail to interpret GWAS findings correctly. The co-expression results strongly support a model in which the lncRNA and protein-coding genes function together in the same pathways.

Increased Expression of Toll-Like Receptors by Monocytes and Natural Killer Cells in ANCA-Associated Vasculitis

Introduction Toll-like receptors (TLRs) are a family of receptors that sense pathogen associated patterns such as bacterial cell wall proteins. Bacterial infections are associated with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Here, we assessed the expression of TLRs 2, 4, and 9 by peripheral blood leukocytes from patients with AAV, and investigated TLR mediated responses ex vivo. Methods Expression of TLRs was determined in 38 AAV patients (32 remission, 6 active disease), and 20 healthy controls (HC). Membrane expression of TLRs 2, 4, and 9, and intracellular expression of TLR9 by B lymphocytes, T lymphocytes, NK cells, monocytes and granulocytes was assessed using 9-color flowcytometry. Whole blood from 13 patients and 7 HC was stimulated ex vivo with TLR 2, 4 and 9 ligands and production of cytokines was analyzed. Results In patients, we observed increased proportions of TLR expressing NK cells. Furthermore, patient monocytes expressed higher levels of TLR2 compared to HC, and in a subset of patients an increased proportion of TLR4+ monocytes was observed. Monocytes from nasal carriers of Staphylococcus aureus expressed increased levels of intracellular TLR9. Membrane expression of TLRs by B lymphocytes, T lymphocytes, and granulocytes was comparable between AAV patients and HC. Patients with active disease did not show differential TLR expression compared to patients in remission. Ex vivo responses to TLR ligands did not differ significantly between patients and HC. Conclusions In AAV, monocytes and NK cells display increased TLR expression. Increased TLR expression by these leukocytes, probably resulting from increased activation, could play a role in disease (re)activation.