Interval prolongation of etanercept in rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis: a randomized controlled trial.

OBJECTIVE: The majority of patients with a rheumatic disease treated with etanercept may be overexposed. Data regarding etanercept tapering are scarce, particularly in psoriatic arthritis (PsA) and ankylosing spondylitis (AS). We compared extending the dose interval to continuation of the standard dose and studied the success rate of etanercept discontinuation. Etanercept concentrations were measured throughout the study. METHOD: 160 patients with rheumatoid arthritis (RA), PsA, or AS with sustained minimal disease activity (MDA) were enrolled in this 18-month, open-label, randomized controlled trial. The intervention group doubled the dosing interval at baseline and discontinued etanercept 6 months later. The control group continued the standard dose for 6 months and doubled the dosing-interval thereafter. The primary outcome was the proportion of patients maintaining MDA at 6 month follow-up. RESULTS: At 6 months, MDA status was maintained in 47 patients (63%) in the intervention group and 56 (74%) in the control group (p = 0.15), with comparable results in all rheumatic diseases. And median etanercept concentrations decreased from 1.50 µg/mL (interquartile range 1.06- 2.65) to 0.46 µg/mL (0.28-0.92). In total, 40% discontinued etanercept successfully with maintained MDA for at least 6 months. CONCLUSION: Etanercept tapering can be done without losing efficacy in RA, PsA, and AS patients in sustained MDA. A substantial proportion of patients could stop etanercept for at least 6 months. In many patients, low drug concentrations proved sufficient to control disease activity. However, the risk of minor and major flares is substantial, even in patients continuing standard dosing.

The effect of anti-TNF treatment on body composition and insulin resistance in patients with rheumatoid arthritis.

Given the link between systemic inflammation, body composition and insulin resistance (IR), anti-inflammatory therapy may improve IR and body composition in inflammatory joint diseases. This study assesses the IR and beta cell function in rheumatoid arthritis (RA) patients with active disease compared to osteoarthritis (OA) patients and investigates the effect of anti-TNF treatment on IR, beta cell function and body composition in RA. 28 Consecutive RA patients starting anti-TNF treatment (adalimumab), and 28 age, and sex-matched patients with OA were followed for 6 months. Exclusion criteria were use of statins, corticosteroids, and cardiovascular or endocrine co-morbidity. Pancreatic beta cell function and IR, using the homeostasis model assessment (HOMA2), and body composition, using dual-energy X-ray absorptiometry (DXA) were measured at baseline and 6 months. At baseline, IR [1.5 (1.1-1.8) vs. 0.7 (0.6-0.9), 100/%S] and beta cell function (133% vs. 102%) were significantly (p < 0.05) higher in RA patients with active disease as compared to OA patients. After 6 months of anti-TNF treatment, IR [1.5 (1.1-1.8) to 1.4 (1.1-1.7), p = 0.17] slightly improved and beta cell function [133% (115-151) to 118% (109-130), p <0.05] significantly improved. Improvement in IR and beta cell function was most pronounced in RA patients with highest decrease in CRP and ESR. Our observations indicate that IR and increased beta cell function are more common in RA patients with active disease. Anti-TNF reduced IR and beta cell function especially in RA patients with highest decrease in systemic inflammation and this effect was not explained by changes in body composition.

Comparison of drug survival and clinical outcome in patients with ankylosing spondylitis treated with etanercept or adalimumab.

OBJECTIVE: To compare rates of drug survival and clinical response during 2 years of follow-up in ankylosing spondylitis (AS) patients treated with etanercept or adalimumab in routine care. METHOD: Biological-naïve consecutive AS patients treated with etanercept (n = 163) or adalimumab (n = 82) were followed. Treatment discontinuation was due to inefficacy, adverse events, loss to follow-up, planning a pregnancy, or uveitis. Disease activity was assessed by the Ankylosing Spondylitis Disease Activity Score using C-reactive protein (ASDAS-CRP). Moderate disease activity was defined as an ASDAS-CRP < 2.1. RESULTS: Twenty-seven patients (32.9%) treated with adalimumab and 30 (18.4%) with etanercept discontinued treatment. Cox regression analysis demonstrated a significant difference in survival rate between discontinuation of the drug in adalimumab patients compared with etanercept patients [hazard ratio (HR) 2.1, 95% confidence interval (CI) 1.3-4.5, p = 0.005; corrected for confounding factors: HR 2.5, 95% CI 1.3-4.5, p = 0.006]. There was no significant difference at 2 years of follow-up between the adalimumab- and the etanercept-treated patients in mean ± sd ASDAS-CRP (1.9 ± 1.1 and 2.0 ± 0.9, respectively, p = 0.624), and 23 out of 34 (67.6%) compared to 71 out of 117 (60.7%) reached ASDAS-CRP moderate disease activity (odds ratio 0.738, 95% CI 0.329-1.657, p = 0.530). CONCLUSION: No significant difference was found between AS patients treated with etanercept and those treated with adalimumab in mean ASDAS-CRP and reaching ASDAS-CRP minimal disease activity at 2 year follow-up. Drug survival rate was higher in etanercept- compared to adalimumab-treated patients. However, this should be interpreted cautiously as the risk of allocation bias cannot be excluded.

What do we miss? ASAS non-responders on anti-TNF therapy show improvement in performance-based physical function.

OBJECTIVE: A prospective study was conducted in order to establish whether AS patients, who are defined as non-responders after 3 months of anti-TNF therapy, show improvement on performance-based tests of physical functioning. METHODS: At baseline and 3 months after the start of anti-TNF therapy, AS patients completed seven performance-based tests of physical functioning, questionnaires on self-reported physical functioning (BASFI) and disease activity (BASDAI), and a pain and a global patient assessment. The concordance between ≥ 20% intra-individual improvement on the performance-based test of physical functioning and (i) response to anti-TNF therapy [Assessment of SpondyloArthritis International Society 20% (ASAS20) response] and (ii) ≥ 20% intra-individual improvement on self-reported physical functioning (BASFI) was assessed. RESULTS: One hundred AS patients were included, of which 82 patients completed all tests at both time points. After 3 months of anti-TNF therapy, 27 (32.9%) patients were categorized as non-responders according to the ASAS20 response criteria. Improvement in performance-based physical functioning was seen in 13 of the 27 non-responders (48.1%) (i.e. n = 13/82 = 15.9% of the total group). Furthermore, 30 (36.6%) patients showed no improvement on self-reported physical functioning (BASFI). However, 17 of the 30 (56.7%) patients did improve on the performance-based tests of physical functioning (i.e. n = 17/82 = 20.7% of the total group). CONCLUSION: After 3 months of anti-TNF therapy, performance-based tests of physical functioning showed improvement in 48.1% of the ASAS20 non-responders. With these performance-based tests, new information on outcome after anti-TNF therapy can be generated. Using performance-based tests alongside the BASFI could have additional value in the evaluation of outcomes for patients receiving anti-TNF therapy.

Prevalence and Radiographic Progression of Hip Involvement in Patients With Ankylosing Spondylitis Treated With Tumor Necrosis Factor Inhibitors.

OBJECTIVE: To examine the prevalence of hip involvement between sexes in patients with ankylosing spondylitis (AS) treated with tumor necrosis factor inhibitors (TNFi) and to estimate the effect of TNFi on radiographic progression of hip involvement compared to the spine. METHODS: Two hundred ninety-nine patients with AS treated with TNFi (215 men; median age: 43 yrs [IQR 36-52], median disease duration: 7.6 yrs [IQR 2-15]) were evaluated for hip involvement, defined radiographically as Bath AS Radiological Hip Index (BASRI-hip) score ≥ 2. Those who received TNFi for ≥ 2 years (263/299) were assessed for radiographic progression. Radiographs of the pelvis and spine, obtained at baseline (ie, before TNFi initiation), were compared retrospectively to those obtained after 2.5 (SD 0.7) years and 7.0 (SD 2.3) years of TNFi treatment. Both hips were scored by BASRI-hip score and mean joint space width (MJSW). Spinal radiographs were scored by modified Stoke AS Spinal Score (mSASSS). RESULTS: The prevalence of hip involvement at baseline was 113/299 (38%) patients, of whom 87/215 (41%) were male and 26/84 (31%) were female (P = 0.10). In both sexes with hip involvement at baseline, BASRI-hip score and MJSW did not change significantly during follow-up. In males and females without baseline hip involvement, the BASRI-hip score remained unchanged after 2.5 (SD 0.7) years but increased significantly after 7.0 (SD 2.3) years, without reaching the cut-off of 2. In contrast, the MJSW slightly decreased at the 2 follow-up timepoints (ie, after 2.5 and 7.0 yrs). The mSASSS increased significantly during the follow-up in both sexes, regardless of hip involvement. CONCLUSION: In our study, approximately one-third of patients with AS had hip involvement, which seemed to stabilize with TNFi treatment. No sex differences in the prevalence or progression of this manifestation were found.

High frequency of vertebral fractures in early spondylarthropathies.

UNLABELLED: We demonstrated that vertebral fractures (VF) are commonly found in early SpA. Patients with VF had lower lumbar BMD than patients without VF. VF remained frequently 'unrecognized' and untreated. VF have been associated with more back pain, reduced Qol, and increased risk of future fractures which stresses the importance of recognition also in early stage SpA. INTRODUCTION: VF are a common complication of long-standing ankylosing spondylitis (AS). However, data of VF in early AS patients and in other spondylarthropathies (SpA) are scarce. Therefore we examined the prevalence of VF in early SpA patients and investigated the associations between VF and demographic and disease-related variables. METHODS: SpA patients were included consecutively and radiographs of the spine were made. VF were assessed according to the method of Genant et al.: fractures were defined as reduction of ≥20% of the vertebrae. Descriptive statistics, t-tests and logistic regression analyses were used to study the relationship between VF and demographic and disease-related variables, radiographic damage and BMD. RESULTS: A total of 113 early SpA patients were included with a disease duration of 7 months, a mean age of 37 years. Seventeen patients (15%) had at least one VF. Fourteen patients had one VF, three patients had two VF. Most VF were located at Th6-Th8. In patients with VF, bone mineral density (BMD) of lumbar spine was lower than BMD of patients without VF (t-test: p = 0.043). Axial Psoriatic Arthritis (PsA) was significantly associated with a higher risk for VF (odds ratio [OR]: 4.62, 95% confidence interval [CI] 1.15-18.58, p = 0.031). No significant associations were found with disease activity variables nor with radiographic severity. CONCLUSION: In a group of 113 early, young SpA patients, 15% already had at least one VF. Most VF were asymptomatic, undetected by routine diagnostic procedures and located at the mid-thoracic spine. The VF were associated with low BMD of the lumbar spine and with axial PsA.

The effect of anti-TNF on renal function in patients with ankylosing spondylitis: a prospective cohort study.

BACKGROUND: Biologicals, such as anti-tumor necrosis factor (anti-TNF), reduce cardiovascular disease (CVD) in patients with inflammatory rheumatic diseases. Impaired renal function is a known predictor of CVD and elevated in ankylosing spondylitis (AS). OBJECTIVE: To assess the effect of anti-TNF on renal function in patients with AS and whether anti-TNF use is safe in AS patients with pre-existing risk factors for renal decline. METHOD: Biological-naïve consecutive AS patients treated with etanercept or adalimumab were prospectively followed from 2005 to 2014. Renal function was determined by calculation of the estimated glomerular filtration rate (eGFR), estimated with the abbreviated modification of diet in renal disease (MDRD) formula. The effect of anti-TNF on eGFR was analyzed using mixed model analysis. RESULTS: 211 AS patients were followed for a median of 156 (36-286) weeks. Overall mixed model analyses showed a significant decrease of eGFR over time (ß = - 0.040, p = 0.000), although this association did not remain significant after adjustment for responding to anti-TNF, alcohol use, disease duration, body mass index (BMI), C-reactive protein (CRP), and disease activity (ß = - 0.018, p = 0.094). However, patients with pre-existing risk factors for renal decline did have a significant change in eGFR over time (ß = - 0.029, p = 0.006). CONCLUSIONS: We found a significant change in eGFR over time, although this small decrease was not clinically relevant. This study further demonstrates that anti-TNF does not affect renal function in AS patients with and without existing risk factors for renal decline, which means that use of anti-TNF is safe concerning renal function in patients with AS. Key Points • Previous studies showed that biologicals, such as anti-tumor necrosis factor (anti-TNF), reduce cardiovascular disease (CVD) in patients with inflammatory rheumatic diseases, such as ankylosing spondylitis (AS). • Impaired renal function is a known predictor of CVD, and also a known concern for many AS patients. • Use of anti-TNF is safe with regard to renal function in patients with AS. • The effect of anti-TNF on CVD in AS patients does not seem to be mediated by changes in renal function.

Antibody recognition of the tumor-specific bcr-abl joining region in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by the presence of a 210-kD protein (P210bcr-abl) in the cytoplasm of leukemic cells, generated by the reciprocal translocation between chromosome 9 and chromosome 22. Due to this translocation, the abl oncogene is coupled to the bcr gene, forming a new determinant in this protein encoded by the bcr-abl joining region. In the joining region itself, either the bcr exon 2 is coupled to the abl exon 2 (b2-a2), or the bcr exon 3 is coupled to the abl exon 2 (b3-a2). Thus, these joining regions form by definition new tumor-specific determinants in the respective chimeric P210-bcr-abl molecules. This paper addresses the question as to whether these tumor-specific joining regions are exposed on the P210bcr-abl molecule in such a way that antibodies can be generated to detect these sites. To test this possibility a polyclonal antiserum, termed BP-1, was raised against a synthetic peptide representative for the b2-a2 joining region. The reactivity of BP-1 was analyzed in an ELISA system on various synthetic peptides. Peptide inhibition studies showed the presence of antibodies to different parts of the b2-a2 peptide in the polyvalent antiserum. The reactivity of BP-1 was then tested with native P210bcr-abl molecules in various CML cell lines (K562, LAMA-84, and BV173) using a protein kinase assay. In this context, the bcr-abl junctions were first analyzed at the DNA and RNA level. The present study indicates that BP-1 specifically recognizes the b2-a2 junction in native P210bcr-abl. Furthermore, BP-1 clearly discriminates between b2-a2 P210bcr-abl and b3-a2 P210bcr-abl. We conclude that the tumor-specific b2-a2 joining region is antigenically exposed on the native P210bcr-abl molecule.

Survival, comorbidities and joint damage 11 years after the COBRA combination therapy trial in early rheumatoid arthritis.

BACKGROUND: COBRA (for 'COmbinatie therapie Bij Rheumatoide Artritis') combination therapy is effective for the treatment of rheumatoid arthritis (RA), but long-term safety is unknown. This study evaluates survival, comorbidities and joint damage in the original COBRA trial cohort. METHODS: In the COBRA trial, 155 patients with early RA were treated with sulfasalazine (SSZ) monotherapy (SSZ group) or a combination of step-down prednisolone, methotrexate (MTX) and SSZ (COBRA group). The current 11-year follow-up study of the COBRA trial invited all original patients and performed protocollised scrutiny of clinical records, questionnaires, physical examination, laboratory and imaging tests. RESULTS: In all, 152 out of 155 patients yielded at least partial data. After a mean of 11 years follow-up, 18 (12%) patients had died, 6 COBRA patients and 12 SSZ patients, HR 0.57 (95% CI 0.21 to 1.52). Treatment for hypertension was significantly more prevalent in the COBRA group (p=0.02) with similar trends for diabetes and cataract. Conversely, hypercholesterolaemia, cancer and infection showed a trend in favour of COBRA. Other comorbidities such as cardiovascular disease and fractures appeared in similar frequency. Radiographic findings suggest as a minimum sustained benefit for COBRA therapy, that is, difference in joint damage but similar subsequent progression rates after 5 years. Imputation to compensate for selective dropout suggests increasing benefit for COBRA, that is, difference in yearly progression rates similar to that seen in the first 5 years of follow-up. CONCLUSIONS: After 11 years, initial COBRA combination therapy resulted in numerically lower mortality and similar prevalence of comorbidity compared with initial SSZ monotherapy. In addition, lower progression of joint damage suggests long-term disease modification.

Immunogenicity does not influence treatment with etanercept in patients with ankylosing spondylitis.

BACKGROUND: Immunogenicity, specifically the onset of antibodies against tumour necrosis factor (TNF) blocking agents, seems to play an important role in non-response to treatment with these drugs. OBJECTIVES: To assess the relation of clinical response of ankylosing spondylitis (AS) to etanercept with etanercept levels, and the presence of antibodies to etanercept. METHODS: Patients with AS were treated with etanercept 25 mg twice weekly, according to the international Assessment in Ankylosing Spondylitis (ASAS) working group consensus statement. Sera were collected at baseline and after 3 and 6 months of treatment. Clinical response was defined as a 50% improvement or as an absolute improvement of 2 points on a (0-10 scale) Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score. Functional etanercept levels were measured by a newly developed ELISA, measuring the binding of etanercept to TNF. Antibodies against etanercept were measured with a two-site assay and antigen binding test. Clinical data were used to correlate disease activity with serum etanercept levels. RESULTS: In all, 53 consecutive patients were included. After 3 months of treatment 40 patients (76%) fulfilled the response criteria. Mean etanercept levels were 2.7 mg/litre and 3.0 mg/litre after 3 and 6 months respectively. Characteristics and etanercept levels of responders and non-responders were similar. No antibodies to etanercept were detected with any of the assays. CONCLUSION: Etanercept levels of responders and non-responders were similar and no antibodies to etanercept were detected with any of the assays. This study indicates that etanercept is much less immunogenic compared with the other TNF-blocking agents.

Fat Mass Lowers the Response to Tumor Necrosis Factor-α Blockers in Patients with Ankylosing Spondylitis.

OBJECTIVE: Our main objective was to assess the relationship between body composition (BC) and response to tumor necrosis factor-α (TNF-α) blocker treatment in patients with ankylosing spondylitis (AS). Our secondary objective was to evaluate the change of BC after treatment, accounting for sex and age. METHODS: All included patients fulfilled the modified New York criteria for AS and were naive to TNF-α blocker. They were followed for at least 6 months after the start of etanercept or adalimumab. The Ankylosing Spondylitis Disease Activity Score containing C-reactive protein (ASDAS-CRP) and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) were reported. BC was assessed by whole body dual-energy X-ray absorptiometry. Body fat percentage (BF%), fat mass index (FMI), and fat free mass index (FFMI) were reported as absolute values and as percentiles. RESULTS: Forty-one patients were included (61% men). The median followup was 14.3 months (interquartile range 8.4-19.4). After multivariate regression analysis, more fat at baseline (BF%, FMI, or FMI percentile) was significantly related with a lower chance of achieving a clinically important improvement of the ASDAS-CRP or BASDAI after treatment. The body composition did not change significantly after treatment, but there was a trend toward muscle recovery in men (FFMI change from 34.0th to 37.4th percentile). CONCLUSION: Higher body fat content at baseline was independently associated with a worse response to treatment with TNF-α blockers, measured by ASDAS-CRP and BASDAI change, and might contribute to the lower response rates in female patients. Also, there is a trend toward muscle mass recovery in male patients after treatment.

Etanercept Increases Bone Mineral Density in Ankylosing Spondylitis, but Does Not Prevent Vertebral Fractures: Results of a Prospective Observational Cohort Study.

OBJECTIVE: Ankylosing spondylitis (AS) is characterized by chronic inflammation leading to ankylosis, but also to low bone mineral density (BMD) and vertebral fractures (VFx). Treatment with tumor necrosis factor-α blockers decreases inflammation and has shown to be effective in increasing BMD. We studied the effects of etanercept (ETN) on BMD and VFx in patients with AS after 2 years of treatment. Further, we studied changes in bone turnover markers and radiological damage. METHODS: Patients with active AS, treated with ETN for 2 years, were included. BMD lumbar spine and hip were measured at baseline and after 2 years, as well as radiological damage (modified Stoke Ankylosing Spondylitis Spinal Score with the addition of the thoracic spine), VFx (Genant method), and change in bone turnover markers. RESULTS: Forty-nine patients with AS were included. After 2 years of ETN, hip BMD increased by 2.2% (p = 0.014) and lumbar spine BMD by 7.0% (p < 0.001). The Bath Ankylosing Spondylitis Disease Activity Index decreased significantly (p < 0.001), as well as C-reactive protein and erythrocyte sedimentation rate (p < 0.001). Despite ETN therapy, the number of patients with VFx more than doubled (from 6 to 15 patients, p = 0.003). Also, the radiological damage increased significantly over time (from 12.1 to 18.5, p < 0.001); however, no significant change in bone turnover markers was found. CONCLUSION: This prospective longitudinal observational cohort study showed that after 2 years of ETN, BMD of the hip and spine increased significantly, but the number of patients with VFx and the severity of VFx increased as well. Besides that, radiological progression, including the thoracic spine, increased significantly. Thus, the favorable bone-preserving effect is accompanied by unfavorable outcomes on VFx and radiological damage.

Specific immunologic recognition of the tumor-specific E2A-PBX1 fusion-point antigen in t(1;19)-positive pre-B cells.

The t(1;19) translocation is the most commonly observed chromosomal translocation in childhood acute lymphoblastic leukemia (ALL). Its presence among pre-B cell ALL cases, has been associated with a poor prognosis. Two genes, E2A and PBX1, are involved in this t(1;19) translocation. As a consequence, parts of the E2A and PBX1 genes are fused, resulting in a chimeric E2A-PBX1 gene, encoding chimeric E2A-PBX1 proteins. As such, the amino acid sequence at the fusion site represents a unique tumor-specific determinant. We report on the generation of a polyclonal antiserum, termed BP 1/19, raised against the tumor-specific E2A-PBX1 junction of E2A-PBX1 proteins. The specificity of antiserum BP 1/19 for the E2A-PBX1 fusion-point is demonstrated at the peptide and at the protein level. Furthermore, specific binding of antiserum BP 1/19 to t(1;19) positive cells was shown using immunofluorescence techniques. The study shows that: (1) the tumor-specific fusion-point epitope on E2A-PBX1 proteins is presented in an antigenic fashion, and (2) this particular fusion-point epitope can be used in immunological marker analysis using fluorescence microscopy.

A novel variant of the bcr-abl fusion product in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Two patients with Philadelphia chromosome-positive acute lymphoblastic leukemia showed novel variants of the chimeric bcr-abl mRNA. The bcr-abl breakpoint region on cDNA derived from the chimeric mRNA was amplified using the polymerase chain reaction (PCR). Sequence analysis of the breakpoint-containing fragment showed that in both patients exon a2 of the abl gene was deleted, giving rise to an in-frame joining at the mRNA level of 5' bcr sequences to the abl exon a3. These findings were confirmed by Southern blot analysis and cloning of chromosomal DNA. Protein studies showed a bcr-abl protein with heightened tyrosine kinase activity in blast cells of both patients: one of the P190 type, the other of the P210 type. The significance of these findings and the role of this new type of translocation in the disregulation of the abl gene are discussed.

Antibody recognition of the tumor-specific b3-a2 junction of bcr-abl chimeric proteins in Philadelphia-chromosome-positive leukemias.

The reciprocal translocation between chromosome 9 and chromosome 22, as observed in chronic myeloid leukemia (CML) as well as in acute lymphoblastic leukemia (ALL), results in a 22q- chromosome, the so-called Philadelphia chromosome. The translocation event creates on the Philadelphia chromosome a fusion between two genes: bcr and abl. Depending on the localization of the breakpoint in the bcr gene different chimeric bcr-abl genes are generated, each encoding their own tumor-specific protein: e1-a2P190bcr-abl, b2-a2p210bcr-abl, or b3-a2P210bcr-abl. Especially in ALL, the presence of such a tumor-specific protein is highly associated with a poor prognosis. Detection of these proteins therefore has a strong clinical significance. In this study a polyclonal antiserum, termed BP-2, was raised against a synthetic peptide, corresponding to the tumor-specific 'fusion-point' epitope of the b3-a2P210bcr-abl protein. The specificity of BP-2 for the bcr-abl joining region in b3-a2P210bcr-abl is demonstrated by means of peptide inhibition studies in combination with immunoprecipitation. In addition we show the reactivity of BP-2 with bcr-abl proteins in leukemic cells of a Philadelphia-chromosome-positive ALL patient.

Recognition of the ALL-specific BCR-ABL junction in P190bcr-abl by monoclonal antibody ER-FP1.

The pH chromosome, resulting from the t(9;22) translocation, is the most frequently observed cytogenetic aberration in acute lymphoblastic leukemia (ALL). Two genes, bcr and abl, are involved in this translocation. As a consequence, parts of the bcr and abl genes are fused, resulting in chimeric bcr-abl genes encoding chimeric BCR-ABL proteins. Three bcr-abl genes and proteins have been identified: e1-a2 P190bcr-abl, b2-a2 P210bcr-abl, and b3-a2 P210bcr-abl. Since these chimeric proteins only occur in Ph-chromosome-positive leukemic cells, they are by definition tumor-specific markers. Ph-chromosome-positive ALL is correlated with a bad prognosis, therefore the detection of chimeric BCR-ABL proteins is of prime importance in ALL diagnosis. In the present study, we report on the generation of a monoclonal antibody termed ER-FP1, raised against the tumor-specific e1-a2 BCR-ABL junction in P190bcr-abl. We show that ER-FP1 reacts highly specifically with e1-a2 P190bcr-abl in different assays. The reactivity of ER-FP1 with e1-a2 P190bcr-abl in soluble form was analyzed in an immunoprecipitation assay; specificity was confirmed by peptide inhibition studies. Binding of ER-FP1 to e1-a2 P190bcr-abl at the single cell level was detected by using immunofluorescence techniques. Immunological double-staining experiments using ER-FP1 and a monoclonal antibody recognizing all BCR-ABL proteins confirmed the specificity of ER-FP1 for the e1-a2 fusion point.

Objective evaluation of physical functioning after tumor necrosis factor inhibitory therapy in patients with ankylosing spondylitis: a selection of 3 feasible performance-based tests.

OBJECTIVE: (1) To select a limited number of performance-based tests that are reliable, show improvement in physical functioning after tumor necrosis factor inhibitor (TNFi) therapy in patients with ankylosing spondylitis (AS), and generate information equivalent to the full set of tests, and (2) are feasible for use in daily clinical practice. METHODS: Eight performance-based tests were evaluated. To eliminate redundant testing, the tests that showed adequate reliability, the highest standardized response mean (SRM), and the largest proportion of patients with an improved performance-based physical functioning were selected. The selected tests were combined into a new criterion for improvement in physical functioning (AS Performance-based Improvement; ASPI). The number and percentage of improved patients identified with the ASPI and identified with the full set of performance tests were compared. RESULTS: Reliability for all tests was adequate to excellent (ICC 0.73-0.96). The tests for bending, putting on socks, and getting up from the floor had the highest SRM (0.52-0.74) and showed the largest proportion of improved patients after TNFi therapy. The combination of these 3 tests was feasible in daily clinical practice and showed improved physical functioning after TNFi therapy in 67% of the patients. CONCLUSION: The 3 selected tests are recommended for use in daily practice because they generate information comparable to the full set. They are reliable and feasible, and the combination of these tests showed improved physical functioning after TNFi therapy in 67% of the patients. Evaluation of physical functioning might be improved by adding these tests to other AS outcome measures.

Purification of bovine thyroid-stimulating hormone by a monoclonal antibody.

A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by [3H]thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH.

Calretinin in the entorhinal cortex of the rat: distribution, morphology, ultrastructure of neurons, and co-localization with gamma-aminobutyric acid and parvalbumin.

Calretinin is a marker that differentially labels neurons in the central nervous system. We used this marker to distinguish subtypes of neurons within the general population of neurons in the entorhinal cortex of the rat. The distribution, morphology, and ultrastructure of calretinin-immunopositive neurons in this cortical area were documented. We further analyzed the co-localization of the marker with gamma-aminobutyric acid (GABA) and studied whether calretinin-positive neurons project to the hippocampal formation. Methods used included single-label immunocytochemistry at the light and electron microscopic level, retrograde tracing combined with immunocytochemistry, and double-label confocal laser scanning microscopy (CLSM). The entorhinal cortex contained calretinin-positive cells in a scattered fashion, in all layers except layer IV (lamina dissecans). Bipolar and multipolar dendritic configurations were present, displaying smooth dendrites. Bipolar cells had a uniform morphology whereas the multipolar calretinin cell population consisted of large neurons, cells with long ascending dendrites, horizontally oriented neurons, and small spherical cells. Retrograde tracing combined with immunocytochemistry showed that calretinin is not present in cells projecting to the hippocampus. Few synapic contacts between calretinin-positive axon terminals and immunopositive cell bodies and dendrites were seen. Most axon terminals of calretinin fibers formed asymmetrical synapses, and immunopositive axons were always unmyelinated. Results obtained in the CLSM indicate that calretinin co-exists in only 18-20% of the GABAergic cell population (mostly small spherical and bipolar cells). Thus, the entorhinal cortex contains two classes of calretinin interneurons: GABA positive and GABA negative. The first class is presumably a classical, GABAergic inhibitory interneuron. The finding of calretinin-immunoreactive axon terminals with asymmetrical synapses suggests that the second class of calretinin neuron is a novel type of a (presumably excitatory) interneuron.