The sequence of addition of IL-3 and IgE-dependent/IgE-independent stimuli regulates RANKL expression in human basophils

Background: Receptor Activator of Nuclear Factor kappaB Ligand (RANKL), a member of the TNF superfamily, contributes to the imbalance of bone resorption and immunoregulation in rheumatoid arthritis. In mice, collagen induced arthritis was exacerbated by IL-3 and anti-IgER antibodies, two mediators activating basophils that are known as effector cells of allergy. Interestingly, our unpublished microarray data revealed that IL-3 induces RANKL mRNA in human basophils. Here we further investigate under which conditions human basophils express surface and/or soluble RANKL. Methods: One part of purified human basophils was co-stimulated with IL-3 and either IgE-dependent or IgE-independent stimuli. The other part of purified basophils was first primed with IL-3 and subsequently triggered with IgE-dependent or IgE-independent stimuli. Expression of surface and soluble RANKL were detected by flow cytometry, ELISA and real-time PCR. Results: By flow cytometry we show that IL-3 induces de novo expression of surface RANKL on human basophils in a time and dose dependent manner. Co-stimulation of basophils with IL-3 and an IgE-dependent stimulus reduces IL-3-induced expression of surface RANKL in a dose dependent manner while IgE-independent stimuli have no effect. In contrast, both IgE-dependent and IgE-independent stimuli enhance expression of surface and soluble RANKL in basophils that were first primed with IL-3 and then triggered. Real-time PCR analysis shows that surface hRANKL1 and soluble hRANKL3 are induced by IL-3 and reduced by co-stimulation with IL-3 and an IgE-dependent stimulus and thus confirms our flow cytometry data. Conclusion: RANKL expression in human basophils is not only dependent on IL-3 and IgE-dependent/IgE-independent stimuli but also on the sequence of their addition to cell culture. Based on our data, we suggest that basophils might have previously unidentified functions in bone resorption or immunoregulation via RANKL.

Human IgA Fc receptor FcαRI (CD89) triggers different forms of neutrophil death depending on the inflammatory microenvironment

FcαRI (CD89), the human Fc receptor for IgA, is highly expressed on neutrophil granulocytes. In this study, we show that FcαRI induces different forms of neutrophil death, depending on the inflammatory microenvironment. The susceptibility of inflammatory neutrophils from sepsis or rheumatoid arthritis toward death induced by specific mAb, or soluble IgA at high concentrations, was enhanced. Although unstimulated cells experienced apoptosis following anti-FcαRI mAb stimulation, preactivation with cytokines or TLR agonists in vitro enhanced FcαRI-mediated death by additional recruitment of caspase-independent pathways, but this required PI3K class IA and MAPK signaling. Transmission electron microscopy of FcαRI-stimulated cells revealed cytoplasmic changes with vacuolization and mitochondrial swelling, nuclear condensation, and sustained plasma membrane. Coculture experiments with macrophages revealed anti-inflammatory effects of the partially caspase-independent death of primed cells following FcαRI engagement. Our data suggest that FcαRI has the ability to regulate neutrophil viability and to induce different forms of neutrophils depending on the inflammatory microenvironment and specific characteristics of the ligand-receptor interactions. Furthermore, these findings have potential implications for FcαRI-targeted strategies to treat neutrophil-associated inflammatory diseases.

State of the art: Reproduction and pregnancy in rheumatic diseases

Throughout the last decade, increasing awareness has been raised on issues related to reproduction in rheumatic diseases including basic research to clarify the important role of estrogens in the etiology and pathophysiology of immune/inflammatory diseases. Sub- or infertility is a heterogeneous condition that can be related to immunological mechanisms, to pregnancy loss, to disease burden, to therapy, and to choices in regard to family size. Progress in reproductive medicine has made it possible for more patients with rheumatic disease to have children. Active disease in women with rheumatoid arthritis (RA) affects their children's birth weight and may have long-term effects on their future health status. Pregnancy complications as preeclampsia and intrauterine growth restriction are still increased in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS), however, biomarkers can monitor adverse events, and several new therapies may improve outcomes. Pregnancies in women with APS remain a challenge, and better therapies for the obstetric APS are needed. New prospective studies indicate improved outcomes for pregnancies in women with rare diseases like systemic sclerosis and vasculitis. TNF inhibitors hold promise for maintaining remission in rheumatological patients and may be continued at least in the first half of pregnancy. Pre-conceptional counseling and interdisciplinary management of pregnancies are essential for ensuring optimal pregnancy outcomes.

Double seronegative myasthenia gravis with antiphospholipid syndrome: a case report

INTRODUCTION Myasthenia gravis is an autoimmune disease characterized by fluctuating muscle weakness. It is often associated with other autoimmune disorders, such as thyroid disease, rheumatoid arthritis, systemic lupus erythematosus, and antiphospholipid syndrome. Many aspects of autoimmune diseases are not completely understood, particularly when they occur in association, which suggests a common pathogenetic mechanism. CASE PRESENTATION We report a case of a 42-year-old Caucasian woman with antiphospholipid syndrome, in whom myasthenia gravis developed years later. She tested negative for both antibodies against the acetylcholine receptor and against muscle-specific receptor tyrosine-kinase, but had typical decremental responses at the repetitive nerve stimulation testing, so that a generalized myasthenia gravis was diagnosed. Her thromboplastin time and activated partial thromboplastin time were high, anticardiolipin and anti-β2 glycoprotein-I antibodies were slightly elevated, as a manifestation of the antiphospholipid syndrome. She had a good clinical response when treated with a combination of pyridostigmine, prednisone and azathioprine. CONCLUSIONS Many patients with myasthenia gravis test positive for a large variety of auto-antibodies, testifying of an immune dysregulation, and some display mild T-cell lymphopenia associated with hypergammaglobulinemia and B-cell hyper-reactivity. Both of these mechanisms could explain the occurrence of another autoimmune condition, such as antiphospholipid syndrome, but further studies are necessary to shed light on this matter.Clinicians should be aware that patients with an autoimmune diagnosis such as antiphospholipid syndrome who develop signs and neurological symptoms suggestive of myasthenia gravis are at risk and should prompt an emergent evaluation by a specialist.

Incidence and risk factors for early adjacent vertebral fractures after balloon kyphoplasty for osteoporotic fractures: analysis of the SWISSspine registry

PURPOSE The SWISSspine registry (SSR) was launched in 2005 to assess the safety and effectiveness of balloon kyphoplasty (BKP). In the meantime, repeated reports on high rates of adjacent vertebral fractures (ASF) after BKP of vertebral insufficiency fractures were published. The causes for ASF and their risk factors are still under debate. The purpose of this study was to report the incidence and potential risk factors of ASF within the SSR dataset. METHODS The SSR data points are collected perioperatively and during follow-ups, with surgeon- and patient-based information. All patients documented with a monosegmental osteoporotic vertebral insufficiency fracture between March 2005 and May 2012 were included in the study. The incidence of ASF, significant associations with co-variates (patient age, gender, fracture location, cement volume, preoperative segmental kyphosis, extent of kyphosis correction, and individual co-morbidities) and influence on quality of life (EQ-5D) and back pain (VAS) were analyzed. RESULTS A total of 375 patients with a mean follow-up of 3.6 months was included. ASF were found in 9.9 % (n = 37) and occurred on average 2.8 months postoperatively. Preoperative segmental kyphosis >30° (p = 0.026), and rheumatoid arthritis (p = 0.038) and cardiovascular disease (p = 0.047) were significantly associated with ASF. Furthermore, patients with ASF had significantly higher back pain at the final follow-up (p = 0.001). No further significant associations between the studied co-variates and ASF were seen in the adjusted analysis. CONCLUSIONS The findings suggest that patients with a preoperative segmental kyphosis >30° or patients with co-morbidities like rheumatoid arthritis and a cardiovascular disease are at high risk of ASF within 6 months after the index surgery. In case of an ASF event, back pain levels are significantly increased. LEVEL OF EVIDENCE IV.

In human basophils, IL-3 selectively induces RANKL expression that is modulated by IgER-dependent and IgER-independent stimuli

BACKGROUND Receptor activator of NF-κB ligand (RANKL) is expressed as either surface (hRANKL1, hRANKL2) or soluble (hRANKL3) form. RANKL is involved in multifaceted processes of immunoregulation and bone resorption such as they occur in rheumatoid arthritis (RA). Interestingly, activated basophils, which are effector cells in allergic inflammation, contribute to the progress of collagen-induced arthritis (CIA), a mouse model for RA. Here, we investigate under which conditions human basophils express RANKL. METHODS Among other stimuli, basophils were cultured with IL-3 alone. Alternatively, as a secondary stimulus, IgER-dependent or IgER-independent agents were added simultaneously either with IL-3 or after prolonged IL-3 culturing. Expression of RANKL protein and mRNA was analyzed by flow cytometry, ELISA, and real-time PCR. A coculture system was applied to investigate biological activity of basophil-derived RANKL. RESULTS We show that in human basophils, IL-3 but no other stimulus induces de novo expression of soluble and surface RANKL, of which the latter enhances survival of MoDC. Upon simultaneous stimulation, IgER cross-linking reduces surface RANKL expression, while IgER-independent stimuli have no effect. This is in contrast to consecutive stimulation, as triggering with both IgER-dependent and IgER-independent stimuli enhances RANKL expression, particularly in its soluble form. Real-time PCR analysis shows that RANKL expression is mainly regulated at the mRNA level. CONCLUSION This study identifies IL-3 as a potent inducer of RANKL expression in human basophils, suggesting them to interact with bone physiology and activation of immune cells. IgER-dependent and IgER-independent stimuli modulate the IL-3-mediated RANKL expression in a time- and stimulus-dependent fashion.

A novel organ-slice culturing system to simulate meniscal repair: Proof of concept using a synovium-based pool of meniscoprogenitor cells.

Meniscal injuries can occur secondary to trauma or be instigated by the changes in knee-joint function that are associated with aging, osteo- and rheumatoid arthritis, disturbances in gait and obesity. Sixty per cent of persons over 50 years of age manifest signs of meniscal pathology. The surgical and arthroscopic measures that are currently implemented to treat meniscal deficiencies bring only transient relief from pain and effect but a temporary improvement in joint function. Although tissue-engineering-based approaches to meniscal repair are now being pursued, an appropriate in-vitro model has not been conceived. The aim of this study was to develop an organ-slice culturing system to simulate the repair of human meniscal lesions in vitro. The model consists of a ring of bovine meniscus enclosing a chamber that represents the defect and reproduces its sequestered physiological microenvironment. The defect, which is closed with a porous membrane, is filled with fragments of synovial tissue, as a source of meniscoprogenitor cells, and a fibrin-embedded, calcium-phosphate-entrapped depot of the meniscogenic agents BMP-2 and TGF-ß1. After culturing for 2 to 6 weeks, the constructs were evaluated histochemically and histomorphometrically, as well as immunohistochemically for the apoptotic marker caspase 3 and collagen types I and II. Under the defined conditions, the fragments of synovium underwent differentiation into meniscal tissue, which bonded with the parent meniscal wall. Both the parent and the neoformed meniscal tissue survived the duration of the culturing period without significant cell losses. The concept on which the in-vitro system is based was thus validated. This article is protected by copyright. All rights reserved.

Alcoholic Cirrhosis Increases Risk for Autoimmune Diseases: A Nationwide Registry-Based Cohort Study

BACKGROUND & AIMS Alcoholic cirrhosis is associated with hyperactivation and dysregulation of the immune system. In addition to its ability to increase risk for infections, it also may increase the risk for autoimmune diseases. We studied the incidence of autoimmune diseases among patients with alcoholic cirrhosis vs controls in Denmark. METHODS We collected data from nationwide health care registries to identify and follow up all citizens of Denmark diagnosed with alcoholic cirrhosis from 1977 through 2010. Each patient was matched with 5 random individuals from the population (controls) of the same sex and age. The incidence rates of various autoimmune diseases were compared between patients with cirrhosis and controls and adjusted for the number of hospitalizations in the previous year (a marker for the frequency of clinical examination). RESULTS Of the 24,679 patients diagnosed with alcoholic cirrhosis, 532 developed an autoimmune disease, yielding an overall increased adjusted incidence rate ratio (aIRR) of 1.36 (95% confidence interval [CI], 1.24-1.50). The strongest associations were with Addison's disease (aIRR, 2.47; 95% CI, 1.04-5.85), inflammatory bowel disease (aIRR, 1.56; 95% CI, 1.26-1.92), celiac disease (aIRR, 5.12; 95% CI, 2.58-10.16), pernicious anemia (aIRR, 2.35; 95% CI, 1.50-3.68), and psoriasis (aIRR, 4.06; 95% CI, 3.32-4.97). There was no increase in the incidence rate for rheumatoid arthritis (aIRR, 0.89; 95% CI, 0.69-1.15); the incidence rate for polymyalgia rheumatica decreased in patients with alcoholic cirrhosis compared with controls (aIRR, 0.47; 95% CI, 0.33-0.67). CONCLUSIONS Based on a nationwide cohort study of patients in Denmark, alcoholic cirrhosis is a risk factor for several autoimmune diseases.

Blocking of LFA-1 enhances expansion of Th17 cells induced by human CD14(+) CD16(++) nonclassical monocytes.

Among human peripheral blood (PB) monocyte (Mo) subsets, the classical CD14(++) CD16(-) (cMo) and intermediate CD14(++) CD16(+) (iMo) Mos are known to activate pathogenic Th17 responses, whereas the impact of nonclassical CD14(+) CD16(++) Mo (nMo) on T-cell activation has been largely neglected. The aim of this study was to obtain new mechanistic insights on the capacity of Mo subsets from healthy donors (HDs) to activate IL-17(+) T-cell responses in vitro, and assess whether this function was maintained or lost in states of chronic inflammation. When cocultured with autologous CD4(+) T cells in the absence of TLR-2/NOD2 agonists, PB nMos from HDs were more efficient stimulators of IL-17-producing T cells, as compared to cMo. These results could not be explained by differences in Mo lifespan and cytokine profiles. Notably, however, the blocking of LFA-1/ICAM-1 interaction resulted in a significant increase in the percentage of IL-17(+) T cells expanded in nMo/T-cell cocultures. As compared to HD, PB Mo subsets of patients with rheumatoid arthritis were hampered in their T-cell stimulatory capacity. Our new insights highlight the role of Mo subsets in modulating inflammatory T-cell responses and suggest that nMo could become a critical therapeutic target against IL-17-mediated inflammatory diseases.

Slow release antibiotics for treatment of septic arthritis in large animals

The search for an effective treatment for septic arthritis is ongoing. Current therapies are expensive since they require repeated joint lavage and long term antibiotic treatment. Local application of antimicrobial drugs is advantageous because high concentrations can be attained at the infection site, although repeated injections increase the risk of superinfection of the joint. Thus, slow release formulations, which have the advantage of local treatment yet single application of the drug, are appealing. Antibiotics used in slow release formulations are selected for tissue compatibility, an appropriate antibacterial spectrum, and stability both during the mixing procedure and within the carrier during the release period. Ideally the carriers should be bioresorbable. Promising reports on the clinical use of poly(methyl methacrylate) (PMMA) mixed with several different antibiotics, and of collagen sponges impregnated with gentamicin, should encourage the search for formulations optimally adapted to veterinary medical requirements.

Limited value of cyclosporine A for the treatment of patients with uveitis associated with juvenile idiopathic arthritis

AIMS: Juvenile idiopathic arthritis (JIA) is often associated with severe chronic anterior uveitis (CAU), and immunosuppressive therapy may be required. In this study, the value of cyclosporine A (CsA) as monotherapy or as combination therapy for treating uveitis was studied in a large cohort of JIA children. METHODS: Multicentre retrospective study including 82 JIA children (girls n=60) suffering from unilateral or bilateral (n=55) CAU. The indication for CsA was active uveitis, although patients were on topical or systemic corticosteroids, MTX, or other immunosuppressive drugs. RESULTS: Inactivity of uveitis during the entire treatment period (mean 3.9 years) was obtained with CsA monotherapy in 6 of 25 (24%) patients, but more often when CsA was combined with the immunosuppressives (35/72 patients; 48.6%, P=0.037), or MTX (18/37 patients, 48.6%, P=0.065), which had already been given. With CsA (mean dosage 2.9 mg/kg), systemic immunosuppressive drugs and steroids could be reduced by >or=50% (n=19) or topical steroids reduced to treatment in any of the patients. In nine patients (11%), CsA was discontinued because of systemic hypertension (n=1), elevated creatinine levels (n=3), or other adverse effects (n=5). CONCLUSIONS: These observations suggest that CsA has limited value as a second-line immunosuppressive drug for the treatment of JIA-associated CAU. The efficacy was better as the combination therapy in patients not responding to other immunosuppressives (eg, MTX) than the systemic monotherapy.

Abatacept in the Treatment of Severe, Longstanding, and Refractory Uveitis Associated with Juvenile Idiopathic Arthritis.

OBJECTIVE Abatacept (ABA), a selective T cell costimulation modulator that binds to CD80 and CD86 on antigen-presenting cells, was investigated for its antiinflammatory effect in treating severe chronic uveitis associated with juvenile idiopathic arthritis (JIA). METHODS Our retrospective study was conducted by members of the Multinational Interdisciplinary Working Group for Uveitis in Childhood (MIWGUC). Patients with JIA who are receiving ABA treatment for active uveitis were included. In all patients, uveitis had been refractory to previous topical and systemic corticosteroids, immunosuppressives, and at least 1 tumor necrosis factor-α inhibitor. A standardized protocol was used to document uveitis (MIWGUC) and arthritis. Baseline visit and visits at 3, 6, 9, and 12 months before and after ABA start were evaluated. Primary outcome measure was defined as achievement of uveitis inactivity; secondary outcome measures were tapering of corticosteroid and/or immunosuppressive treatment, and occurrence of complications. RESULTS In all, 21 patients (16 female) with active uveitis (n = 21) and arthritis (n = 18) were included (mean age 11.8 ± 3.6 yrs). In 7 of 18 patients with active arthritis at baseline, inactivity was achieved following ABA treatment. Uveitis inactivity was achieved in 11 patients, but recurred later in 8 of them, and remained active in another 10 cases. Systemic corticosteroids or immunosuppression were tapered in 3 patients, but uveitis recurred in all of them during further followup. Ocular complications secondary to uveitis were present in 17 patients at baseline, while 3 patients developed new ocular complications during followup. CONCLUSION A sustained response to ABA was uncommon in patients with severe and refractory uveitis.

Impact of anti-inflammatory treatment on the onset of uveitis in juvenile idiopathic arthritis: Longitudinal analysis from a nation-wide paediatric rheumatological database

PURPOSE Based on a nation-wide database, this study analysed the influence of methotrexate (MTX), TNF inhibitors and a combination of the two on uveitis occurrence in JIA patients. METHODS Data from the National Paediatric Rheumatological Database in Germany were used in this study. Between 2002 and 2013, data from JIA patients were annually documented at the participating paediatric rheumatological sites. Patients with JIA disease duration of less than 12 months at initial documentation and ≥2 years of follow-up were included in this study. The impact of anti-inflammatory treatment on the occurrence of uveitis was evaluated by discrete-time survival analysis. RESULTS A total of 3,512 JIA patients (mean age 8.3±4.8 years, female 65.7%, ANA-positive 53.2%, mean age at arthritis onset 7.8±4.8 years) fulfilled the inclusion criteria. Mean total follow-up time was 3.6±2.4 years. Uveitis developed in a total of 180 patients (5.1%) within one year after arthritis onset. Uveitis onset after the first year was observed in another 251 patients (7.1%). DMARD treatment in the year before uveitis onset significantly reduced the risk for uveitis: MTX (HR 0.63, p=0.022), TNF inhibitors (HR 0.56, p<0.001) and a combination of the two (HR 0.10, p<0.001). Patients treated with MTX within the first year of JIA had an even a lower uveitis risk (HR 0.29, p<0.001). CONCLUSION The use of DMARDs in JIA patients significantly reduced the risk for uveitis onset. Early MTX use within the first year of disease and the combination of MTX with a TNF inhibitor had the highest protective effect. This article is protected by copyright. All rights reserved.