Reinhard Bos

LRegulation of cytokine-induced HW-1 alpha expression in rheumatoid synovial fibroblasts

Abstract:  The transcription factor hypoxia‐inducible factor (HIF)‐1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilization of the subunit HIF‐1α. Activation can also occur by proinflammatory cytokines during inflammation. Hypoxia, as well as proinflammatory cytokines, plays an important role in the synovia in rheumatoid arthritis (RA) patients. Expression of HIF‐1α has been demonstrated in RA synovial lining layer. The aim of the study was to investigate the regulation of the intracellular signal transduction pathways, involved in the expression of HIF‐1α, and in the expression of genes regulated by HIF‐1α in rheumatoid synovial fibroblasts (RSF). RSF were cultured under proinflammatory conditions (IL‐1β and TNF‐α stimulation) and under chemical hypoxia (CoCl2 treatment). Expression of HIF‐1α was analyzed in nuclear extracts by Western blotting. The effect of inhibitors of the PI3K and the ERK pathway on HIF‐1α protein expression was measured. mRNA expression of HIF‐1α, COX‐2, vascular endothelial growth factor (VEGF), and stromal cell‐derived factor (SDF)‐1 was determined by real‐time RT‐PCR, and protein production of VEGF and SDF‐1 by ELISA. Treatment of the synovial fibroblasts with 150 mM CoCl2 as well as stimulation with 10 ng/mL IL‐1β or TNF‐α resulted in strong protein expression of HIF‐1α, measured with Western blotting. Pretreatment with the MEK1/2 inhibitor PD98059 as well as the PI3K inhibitor LY294002 resulted in inhibition of the cytokine‐induced HIF‐1α expression. Furthermore, it was shown that cytokine‐induced mRNA expression of HIF‐1α was inhibited by the PI3K inhibitor. We found that cytokine stimulation induced VEGF mRNA and protein production, but no significant effect of kinase inhibition was found on VEGF production in cytokine‐stimulated RSF. Both the ERK pathway and the PI3K pathway are involved in the cytokine‐induced HIF‐1α expression in RSF and in the expression of proangiogenic factors.

Stopping Tumor Necrosis Factor Inhibitor Treatment in Patients With Established Rheumatoid Arthritis in Remission or With Stable Low Disease Activity: A Pragmatic Multicenter, Open‐Label Randomized Controlled Trial

Tumor necrosis factor inhibitor (TNFi) biologic agents are an effective treatment for rheumatoid arthritis (RA). It is unclear whether patients whose disease is in remission or who have stable low disease activity need to continue use of TNFi or can stop this treatment. This study was undertaken to assess whether patients with established RA who are in remission or have stable low disease activity can effectively and safely stop their TNFi therapy.

The effect of three years of TNF alpha blocking therapy on markers of bone turnover and their predictive value for treatment discontinuation in patients with ankylosing spondylitis: a prospective longitudinal observational cohort study

IntroductionThe aim of this study was to investigate the effect of three years of tumor necrosis factor-alpha (TNF-α) blocking therapy on bone turnover as well as to analyze the predictive value of early changes in bone turnover markers (BTM) for treatment discontinuation in patients with ankylosing spondylitis (AS).MethodsThis is a prospective cohort study of 111 consecutive AS outpatients who started TNF-α blocking therapy. Clinical assessments and BTM were assessed at baseline, three and six months, as well as at one, two, and three years. Z-scores of BTM were calculated to correct for age and gender. Bone mineral density (BMD) was assessed yearly.ResultsAfter three years, 72 patients (65%) were still using their first TNF-α blocking agent. In these patients, TNF-α blocking therapy resulted in significantly increased bone-specific alkaline phosphatase, a marker of bone formation; decreased serum collagen-telopeptide (sCTX), a marker of bone resorption; and increased lumbar spine and hip BMD compared to baseline. Baseline to three months decrease in sCTX Z-score (HR: 0.394, 95% CI: 0.263 to 0.591), AS disease activity score (ASDAS; HR: 0.488, 95% CI: 0.317 to 0.752), and physicians global disease activity (HR: 0.739, 95% CI: 0.600 to 0.909) were independent inversely related predictors of time to treatment discontinuation because of inefficacy or intolerance. Early decrease in sCTX Z-score correlated significantly with good long-term response regarding disease activity, physical function and quality of life.ConclusionsThree years of TNF-α blocking therapy results in a bone turnover balance that favors bone formation, especially mineralization, in combination with continuous improvement of lumbar spine BMD. Early change in sCTX can serve as an objective measure in the evaluation of TNF-α blocking therapy in AS, in addition to the currently used more subjective measures.

Spinal Radiographic Progression in Patients with Ankylosing Spondylitis Treated with TNF-α Blocking Therapy: A Prospective Longitudinal Observational Cohort Study

Objectives To evaluate spinal radiographic damage over time and to explore the associations of radiographic progression with patient characteristics and clinical assessments including disease activity in ankylosing spondylitis (AS) patients treated with tumor necrosis factor-alpha (TNF-α) blocking therapy in daily clinical practice. Methods Consecutive outpatients from the Groningen Leeuwarden AS (GLAS) cohort were included based on the availability of cervical and lumbar radiographs before start of TNF-α blocking therapy and after 2, 4, and/or 6 years of follow-up. Clinical data were assessed at the same time points. Radiographs were scored by two independent readers using the modified Stoke AS Spine Score (mSASSS). Spinal radiographic progression in relation to clinical assessments was analyzed using generalized estimating equations. Results 176 AS patients were included, 58% had syndesmophytes at baseline. Median mSASSS increased significantly from 10.7 (IQR: 4.6–24.0) at baseline to 14.8 (IQR: 7.9–32.8) at 6 years. At the group level, spinal radiographic progression was linear with a mean progression rate of 1.3 mSASSS units per 2 years. Both spinal radiographic damage at baseline and radiographic progression were highly variable between AS patients. Male gender, older age, longer disease duration, higher BMI, longer smoking duration, high CRP, and high ASDAS were significantly associated with syndesmophytes at baseline. Significantly more radiographic progression was seen in patients with versus without syndesmophytes (2.0 vs. 0.5 mSASSS units per 2 years) and in patients >40 versus ≤40 years of age (1.8 vs. 0.7 mSASSS units per 2 years). No longitudinal associations between radiographic progression and clinical assessments were found. Conclusions This prospective longitudinal observational cohort study in daily clinical practice shows overall slow and linear spinal radiographic progression in AS patients treated with TNF-α blocking therapy. At the individual level, progression was highly variable. Patients with syndesmophytes at baseline showed a 4-fold higher radiographic progression rate than patients without syndesmophytes.

Higher Bone Turnover Is Related to Spinal Radiographic Damage and Low Bone Mineral Density in Ankylosing Spondylitis Patients with Active Disease: A Cross- Sectional Analysis

Introduction Ankylosing spondylitis (AS) is characterized by excessive bone formation and bone loss. Our aim was to investigate the association of bone turnover markers (BTM) with spinal radiographic damage and bone mineral density (BMD) in AS patients with active disease. Methods 201 consecutive AS outpatients of the Groningen Leeuwarden AS (GLAS) cohort were included. Serum markers of bone resorption (C-telopeptides of type-I collagen, sCTX) and bone formation (procollagen type-I N-terminal peptide, PINP; bone-specific alkaline phosphatase, BALP) were measured. Z-scores were used to correct for the normal influence that age and gender have on bone turnover. Radiographs were scored by two independent readers according to modified Stoke AS Spinal Score (mSASSS). The presence of complete bridging (ankylosis of at least two vertebrae) was considered as measure of more advanced radiographic damage. Low BMD was defined as lumbar spine and/or hip BMD Z-score ≤ −1. Results Of the 151 patients with complete data, 52 (34%) had ≥1 complete bridge, 49 (33%) had ≥1 syndesmophyte (non-bridging), and 50 (33%) had no syndesmophytes. 66 (44%) had low BMD. Patients with bridging had significantly higher sCTX and PINP Z-scores than patients without bridging (0.43 vs. −0.55 and 0.55 vs. 0.04, respectively). Patients with low BMD had significantly higher sCTX Z-score than patients with normal BMD (−0.08 vs. −0.61). After correcting for gender, symptom duration, and CRP, sCTX Z-score remained significantly related to the presence of low BMD alone (OR: 1.60), bridging alone (OR: 1.82), and bridging in combination with low BMD (OR: 2.26). Conclusions This cross-sectional study in AS patients with active and relatively long-standing disease demonstrated that higher serum levels of sCTX, and to a lesser extent PINP, are associated with the presence of complete bridging. sCTX was also associated with low BMD. Longitudinal studies are needed to confirm that serum levels of sCTX can serve as objective marker for bone-related outcome in AS.

Thalidomide and dexamethasone followed by autologous stem cell transplantation for scleromyxoedema

Thalidomide and dexamethasone followed by autologous stem cell transplantation for scleromyxoedema SIR, Scleromyxoedema is a rare disease, which mimics SSc. Subtle differences are a waxy non-pruritic eruption over the thickened skin, and the more proximal onset of skin thickening. Systemic manifestations include gastrointestinal, lung or heart involvement. In scleromyxoedema, the fibroblast is affected by an unrecognized mechanism, resulting in excessive mucin deposition [1]. Most intriguing is the finding of a paraprotein type immunoglobulin G lambda (IgG) in 80% of cases, which is the basis on which patients are frequently treated with regimens according to myeloma patients. A 45-year-old woman was referred to our hospital for progressive thickening and tightening of the skin. Her medical history revealed a transient ischaemic attack and hypertension 3 years before presentation. During the past 2 years she had noted a progressive skin thickening. Due to the thickening of the skin, she could not make a tight fist. She had recently noticed RP without other complaints. A skin biopsy revealed a diagnosis of scleromyxoedema. In the same period, a ductal carcinoma of the right breast was diagnosed (T1N0M0). Treatment consisted of lumpectomy and radiotherapy, but skin abnormalities progressed in the following 6 months. At presentation, her general physical examination was unremarkable. Her musculoskeletal and skin examination revealed a thickened skin of the fingers, wrists, upper arms, shoulders, around the mouth and forehead, and the upper and lower legs, which resulted in a modified Rodnan total skin score (mRTSS) of 34 out of 51 [2]. A skin-coloured papular eruption was present at the wrists. She was not able to make a tight fist, and had a reduced oral aperture of 4 cm. Laboratory investigations showed no abnormalities apart from an IgGof 5.7 g/l. Free light-chain lambda and kappa concentrations were within the reference ranges. ANA and ENA were negative, complement C3 1.1 g/l, complement C4 0.2 g/l, RF 10 kIU/l. Further investigations of internal organs showed reduced digital perfusion after cooling, a normal pattern at nailfold capillary microscopy, a disturbed oesophagus scintigraphy indicating dysmotility and a slightly reduced lung diffusion capacity of 78% predicted. Bone marrow biopsy and cytogenetic analysis, s.c. fat biopsy for amyloidosis, radiology of the skeleton and cardiac US did not show any abnormalities. In view of the clinical findings and the relation between scleromyxoedema and IgGparaprotein, we decided to treat the patient according a multiple myeloma protocol. This consisted for 3 months of the combination of thalidomide (100 mg daily) and dexamethasone (40 mg Days 1 4 and Days 16 20 each month) [3]. There was a significant improvement of clinical symptoms. The mRTSS decreased from 34 to 2, and the paraprotein disappeared. Peripheral blood stem cell mobilization and collection was performed with CYC, adriamycine and dexamethasone (CAD) [3], followed by G-CSF 10 mg/kg s.c. until stem cell harvest. Subsequently, the patient was treated with high-dose melphalan (200 mg/m) and autologous stem cell reinfusion without severe complications. Current follow-up until 31 months showed an mRTSS of 0 without paraprotein. Her RP disappeared and the ranges of motion of her oral aperture and the wrists returned to normal. ANA screening remains negative. For scleomyxoedema different treatment regimens have been applied, including CSs, HCQ, AZA, IFN, CYC, oral melfalan and IVIG, with disappointing results. However, treatment with high-dose chemotherapy directed against the aberrant monoclonal plasma cell population showed especially positive results in line with results obtained in light chain (AL) amyloidosis and polyneuropathy, organomegaly, endocrinopathy, monoclonal gammapathy and skin changes (POEMS) disease [4]. In 2001, Donato et al were the first to describe the positive results of high-dose melphalan in the setting of autologous stem cell transplantation (ASCT) in scleromyxoedema. In 2006, the same group published a cohort of seven patients treated with ASCT [5]. In this study, no induction regimen was applied before ASCT. Also, most of the included patients had extensive disease, some requiring tube feeding due to gastrointestinal dysfunction. From 2001 until now, ASCT has been described in 17 scleromyxoedema patients including our patient as depicted in Table 1. Complete remission (CR) defined as disappearance of all theclinical symptoms,skinabnormality andserumparaprotein was obtained in 58% and partial remission (PR) defined as significant improvement but without normalization was obtained in 29% of the patients. At a median follow-up of >40 months (range 14 to >60 months) persistent CR was only demonstrated in 12% of patients. These data demonstrate the positive effects of intensive chemotherapy directed against the aberrant plasma cell population in scleromyxoedema. However, a high number of patients relapsed, which might be due to the fact that the transplantation was performed at a late stage of the disease. Further improvement might be obtained by applying a pre-induction regimen with thalidomide and dexamethasone prior to ASCT, thereby reducing the total tumour mass before transplantation, which might further improve the long-term outcome.

Clinical risk factors for the presence and development of vertebral fractures in patients with ankylosing spondylitis

To investigate the prevalence and incidence of radiographic vertebral fractures and the association with patient characteristics, clinical assessments, and medication use in a large prospective cohort of patients with ankylosing spondylitis (AS) in daily clinical practice.

Stopping Tumor Necrosis Factor-inhibitors in Patients with Established Rheumatoid Arthritis in Remission or Stable Low Disease Activity: A Pragmatic Randomized Multicenter Open-Label Controlled Trial

Tumor necrosis factor inhibitor (TNFi) biologic agents are an effective treatment for rheumatoid arthritis (RA). It is unclear whether patients whose disease is in remission or who have stable low disease activity need to continue use of TNFi or can stop this treatment. This study was undertaken to assess whether patients with established RA who are in remission or have stable low disease activity can effectively and safely stop their TNFi therapy.

Tapering and discontinuation of methotrexate in patients with RA treated with TNF inhibitors: data from the DREAM registry.

Objectives To study the number of patients that taper or discontinue concomitant methotrexate (MTX) in daily practice in patients with rheumatoid arthritis (RA) treated with tumour necrosis factor inhibitor (TNFi) and to analyse the effects of that adaption on disease activity and drug survival. Methods Data were collected from the Dutch Rheumatoid Arthritis Monitoring (DREAM) registry. Patients who started their first TNFi were included in the study. Treatment effectiveness after MTX tapering or discontinuation was analysed using Disease Activity Score of 28 joints (DAS28). Drug survival of the TNFi was analysed using the Cox proportional hazard model with a time-dependent covariate. Results In 458 patients (34%), MTX was tapered, 126 patients (10%) discontinued MTX and 747 patients (56%) continued MTX at the same dose. On average, DAS28 improved after tapering MTX (−0.40, −0.45) and after stopping MTX (−0.28, −0.12) at 6 and 12 months. In the taper group, 21% of the patients relapsed (DAS28 increase >0.6), and in the discontinuation group this was 21% and 24% at 6 and 12 months, respectively. Patients who taper and discontinue MTX have a similar DAS28 score over time as patients who continue MTX. Moreover, there was no influence of tapering or discontinuation of MTX on long-term drug survival of TNFi. Conclusions In daily practice, tapering or discontinuation of concomitant MTX in patients with RA treated with TNFi frequently occurs and it does not seem to influence the average DAS28 over time or the long-term TNFi drug survival. It appears that in daily clinical practice the correct patients are selected to taper or discontinue MTX.

Ankylosing spondylitis patients at risk of poor radiographic outcome show diminishing spinal radiographic progression during long-term treatment with TNF-a inhibitors

Objective To investigate the influence of patient characteristics on the course of spinal radiographic progression in a large prospective longitudinal cohort study of ankylosing spondylitis (AS) patients treated long-term with TNF-α inhibitors. Methods Consecutive patients from the Groningen Leeuwarden AS (GLAS) cohort starting TNF-α inhibitors with spinal radiographs at least available at baseline and 6 years of follow-up were included. Radiographs were scored using mSASSS by two independent readers. Generalized estimating equations (GEE) were used to explore the associations between baseline characteristics and spinal radiographic progression. The course of radiographic progression in patients with and without risk factors for poor radiographic outcome was investigated using different time models (linear and non-linear). Single linear imputation was used in case of missing radiographic data at the intermediate (2 or 4 years) follow-up visits. Results 80 AS patients were included with mean baseline mSASSS 8.7±13.3. Baseline syndesmophytes, male gender, older age, longer symptom duration, smoking, and higher BMI were significantly associated with more radiographic damage over time. GEE analysis in patients with these risk factors revealed that radiographic progression followed a non-linear course with mean mSASSS progression rates reducing from max. 2.8 units over 0–2 years to min. 0.9 units over 4–6 years. The GEE model revealed a linear course with overall very low progression (≤1 mSASSS units/2yrs) in patients without risk factors. Complete case analysis in 53 patients showed similar results. Conclusion AS patients at risk of poor radiographic outcome showed the highest but diminishing spinal radiographic progression during long-term treatment with TNF-α inhibitors.