Vasiliki Tzimouli

Comparative study of dual energy X‐ray absorptiometry and quantitative ultrasonography with the use of biochemical markers of bone turnover in boys with haemophilia

Summary.  Our aim was to evaluate bone status in boys with haemophilia using dual energy X‐ray absorptiometry (DXA) and quantitative ultraSonography (QUS), and in addition, to compare these two methods with the use of biochemical markers of bone turnover. Twenty‐six boys with a mean decimal age of 12.08 ± 4.44 years were included in the study which included a DXA scan at lumbar spine and radial, as well as tibial QUS. Serum levels of soluble receptor activator of nuclear factor κB ligand (sRANK‐L), osteoprotegerin (OPG) and osteocalcin (OC) were measured and joint evaluation was performed using the Hemophilia Joint Health Score (HJHS). With regard to the study results, only 2 of 26 patients (7.7%) had bone mineral density (BMD) Z‐scores <−2, and 4 patients (15.4%) had BMD Z‐scores between −1 and −2. Only one patient had radial and other two had tibial QUS Z‐scores <−2. No agreement was recorded between QUS and DXA in identifying patients at risk for osteoporosis (k = 0.275, P = 0.063). Haemophiliacs had significantly higher serum levels of sRANK‐L (21.04 ± 4.78 vs. 18.58 ± 2.28 ng mL−1, P = 0.038) and of OC (5.35 ± 2.29 vs. 3.09 ± 0.61 ng mL−1, P = 0.002) and significantly decreased levels of OPG (15.78 ± 2.53 vs. 23.79 ± 4.39 pg mL−1, P < 0.001) compared with controls. QUS Z‐scores at tibia significantly correlated with HJH Scores (r = −0.450, P = 0.040), whereas lumbar BMD Z‐scores significantly correlated with body mass index Z‐scores (r = 0.500, P = 0.009). More studies are warranted to identify the most accurate densitometric method for assessing bone status in haemophiliacs.

The significance of persistent newly developed autoantibodies in JIA patients under long-term anti-TNF treatment

OBJECTIVE To study the significance of persistent (12 months) new autoantibodies, in Juvenile Idiopathic Arthritis (JIA) patients treated with either Infliximab (INFL) or Etanercept (ET) for 2 years. PATIENTS-METHODS 26 children under INFL (n=12) or ET (n=14) were prospectively studied. A large panel of autoantibodies was tested using indirect immunofluorescence (ANA, anti-dsDNA, anti-ENA, SMA, LKM, AMA, PCA, anti-R1, ATA), ELISA (ANA, anti-ENA, anti-cardiolipin, ANCA), immunoblotting assay (anti-ENA: anti-Ro, anti-La, anti-Sm, anti-URNP, anti-Jo, anti-Scl70, anti-centromere, anti-ribosomal and anti-histone) and rate nephelometry (RF). RESULTS Apart from the positive patients for ANA (13/26) and RF (2/26) prior to anti-TNF treatment, 6/26 patients (23%) developed new autoantibodies (SMA, anti-R1, ATA) which persisted for 12-50 months. None developed antibodies to nuclear antigens. In only one case, ATA was associated with the development of Hashimotos thyroiditis. CONCLUSIONS These findings indicate that in JIA patients in contrast to adult RA patients, development of new autoantibodies to various nuclear antigens is rare. Other non relevant to rheumatic diseases autoantibodies, may appear and persist for >12 months, but very rarely they may be related to clinical entities, especially in the presence of a positive family history of autoimmunity.

Fok-I polymorphism of vitamin D receptor gene and the presence of renal dysfunction in patients with β-thalassemia major.

Recent evidence supports the presence of renal dysfunction even among young patients with β-thalassemia major. However, the possible genetic contribution has never been investigated. The aim of this study was to correlate the presence of Fok-I polymorphism of the vitamin D receptor gene with abnormal levels of early markers of renal impairment in children and young adults with thalassemia. Thirty-four patients (19 male and 15 female) with β-thalassemia major on conventional treatment, with a mean decimal age of 14.62 ± 5.47 years (range: 5–22 years), were included in the study. Markers of renal function were determined in serum and in urine and patients were genotyped for Fok-I gene polymorphism. Genotype frequencies were similar to those previously reported for other populations: 47.06% of the patients were homozygous for the F allele, 41.18% were heterozygous, and 11.76% were homozygous for the f allele. A considerable number of patients demonstrated impaired renal function with increased serum cystatin C levels (29.41%), glomerular dysfunction with proteinuria (68%), as well as significant tubulopathy with hypercalciuria (73.08%), and increased levels of urinary β2-microglobulin (29.41%). When patients were stratified according to Fok-I polymorphism, a significantly higher prevalence of abnormally increased serum levels of cystatin C was observed in patients being homozygous for the f allele (75%) compared with those being heterozygous (Ff) or homozygous for the F allele (14.29% and 31.25%, respectively, P = .02). Further studies are needed to confirm these preliminary results and elucidate the possible mechanisms involved.

Increased osteoclastic activity as shown by increased sRANK-L/OPG ratio in boys with hemophilia

Dear Editor, We read with interest the report by Katsarou et al. that originally assessed biochemical bone markers in patients with hemophilia and correlated these results to clinical and laboratory parameters [1]. As we have recently measured, bone markers in our hemophilic patients and our results differ significantly with those already released; we would like to report our experience. In 27 hemophilic boys with a mean decimal age of 11.63±4.66 years (range: 4-17 years) and 25 age-matched male controls (mean decimal age: 11.45±5.26 years), levels of soluble receptor activator of nuclear factor κB ligand (sRANK-L), osteoprotegerin (OPG) and osteocalcin (OC) were measured in serum. All samples were deep frozen after immediate centrifugation until the day of measurement. Enzyme-linked immunosorbent assays were used for the determination of bone markers: sRANK-L (total), Biovendor Laboratorni Medicina, Modrice, Czech Republic; OPG, Biomedica Medizinprodukte GmbH & Go, Vienna, Austria; OC, Bender MedSystems GmbH, Vienna, Austria. Our results demonstrate that, compared to controls, patients with hemophia have significant higher serum levels of sRANK-L (395.86±90.60 vs. 297.25±36.46 pmol/l, p=0.001) and of OC (5.31±2.26 vs. 3.09±0.61 ng/ml, p=0.001) and significantly decreased levels of OPG (0.78±0.13 vs. 1.19±0.22 pmol/l, p<0.001). These result to an even more striking difference of the ratio sRANK-L/OPG between patients and controls (527.27± 181.51 vs. 256.24±45.01, p<0.001, Fig. 1). Our results indicate an increased osteoclastic activity followed by a compensatory up-regulated osteoblastic function and further substantiate the common pathogenetic mechanism that both hemophilic and rheumatoid arthritis share [2, 3]. In the study by Katsarou et al. [1], serum sRAKL-L and OPG levels did not differ significantly between patients and controls, whereas OC levels were significantly decreased in hemophiliacs, in contrast to our results. In that study, an up-regulated osteoclastic activity in hemophiliacs was shown by the increased levels of Nterminal and C-terminal cross-linking telopeptide of collagen type I (NTX and CTX); the latter was also found in the recent study by Jansen et al. [4]. Further studies are warranted to elucidate the exact mechanism of the hemophilic arthropathy and translate this knowledge into potential therapeutic options.

Antenatal Betamethasone Does Not Influence Lymphocyte Apoptosis in Preterm Neonates

Despite the widespread use of antenatal glucocorticosteroids (GCs), the possibility of adverse effects on the immune response in preterm neonates remains a major concern. GCs stimulate lymphocyte apoptosis, resulting in lymphopenia and functional disorders, which have been associated with sepsis-related death in critically ill neonates. We sought to assess the effect of antenatal betamethasone (BM) on lymphocyte apoptosis in preterm neonates. Fifty preterm neonates exposed to antenatal BM and 50 controls were studied prospectively. Lymphocyte apoptosis was assessed using the annexin-V/propidium iodide (PI) assay, analysis of cell cycle after staining with PI, and intracellular caspase-3 activity. The two groups did not differ significantly as regards absolute lymphocyte counts and the percentage of lymphocytes being annexin-V (+)/PI (-) (early apoptotic) or lymphocytes in the subG1 peak after staining with PI and those with intracellular caspase-3 activation. The lymphocyte number and apoptosis were not associated with the time elapsed between antenatal BM administration and delivery. A single course of antenatal BM does not influence apoptosis of neonatal lymphocytes. This is of significant importance with respect to the preservation of lymphocyte-associated immune response in preterm neonates.

AB1011 Synovial Fluid Biomarkers, Prognostic for the Course of Juvenile Idiopathic Arthritis: A Decade's Follow-Up Study

Background The course and outcome of oligo-JIA is more or less unpredictable. So far, no internationally acceptable prognostic biomarkers, regarding the progression of oligo- to the extended disease type have been defined. Objectives The aim of this study was to assess the immunophenotype plus cytokine profile in synovial fluid (SF) of patients (pts) with JIA and to evaluate the findings in respect to the severity of the disease course in order to define those pts who need early aggressive treatment. Methods 73 SF samples from the knees of 57 JIA patients (F: 48, median age 11.49 yrs) were studied. 20/57 pts had oligo-persistent (O-per), 27 oligo-extended (O-ext) and 10 polyarthritis (poly). 25 age-matched children (25 SF samples) with recent traumatic knee arthritis served as controls. The SF immunophenotype and cytokine levels were assessed by flow cytometry and ELISA respectively. Results The median CD4:CD8 T cell ratio was found to be significantly lower in 19/57 pts (24/73 samples) from all 3 JIA pt groups as compared to the controls (0.75 vs 1.55, p<0.01). Also, the median CD4:CD8 ratio was significantly lower in the O-ext disease type (0.70) as compared to O-per and poly (1.54 and 1.57 respectively p<0.01). 23/24 SF samples with a low (<0.8) ratio had a polyarticular disease course (21 O-ext, 3/10 poly). Tregs were significantly lower and Th17 significantly higher in all 3 JIA pt groups as compared to the controls (p<0.001), especially in O-ext. Cytokines IL-17, IL-23 and IL-6 were significantly higher in all JIA pt groups as compared to the controls. A positive correlation between low CD4:CD8 ratio and the disease duration as well as, values of Th17 cells, IL-17 and IL-23 levels were found regardless of the JIA subtype. Conclusions A low ratio of CD4:CD8 T cells in combination with high levels of IL-17 and IL-23 in SF of JIA pts can predict a prolonged, polyarticular course of the disease and may be used as “the window of opportunity” for a more targeted treatment. References Hunter PJ, et al. Biologic predictors of extension of oligoarticular juvenile idiopathic arthritis as determined from synovial fluid cellular composition and gene expression. Arthritis Rheum 2010;62: 896-907. Disclosure of Interest None declared

B lymphocyte stimulator, interferon-α and HMGB 1 interrelation in childhood onset systemic lupus erythematosus: associations with disease activity and severity

The role of B lymphocyte stimulator (Blys) in Childhood onset Systemic Lupus Erythematosus (cSLE) has not been elucidated.

PReS-FINAL-2299: Novel biomarkers for the assessment of pediatric systemic lupus erythematosus nephritis (preliminary report)

Results The pSLE nephritis patients had significantly higher serum levels of anti-NCS [median: 48.89 (IQR: 31.4880.81) U/ml versus 12.5 (11.5-27.8) U/ml, p < 0.001], anti-C1q [22.75 (12.77-56.4) U/ml versus 12.5 (12.512.5) U/ml, p < 0.001], anti-GBM [3.88 (2.25-6.94) U/ml versus 2.2 (2.2-2.4) U/ml, p = 0.002] and HMGB1 [9.9 (5.7-32.23) ng/ml versus 2.5 (2.5-2.5) ng/ml, p < 0,001], than the patients with nephritis of other causality. Serum anti-GBM levels were significantly higher in the pSLE nephritis patients compared to the pSLE patients without nephritis [3.88 (2.25-6.94) U/ml versus 2.25 (2.2-2.83) U/ml, p = 0.014], while this was not true for the rest of the biomarkers. In the pSLE nephritis patients no correlation was found between serum antiGBM levels and pSLE nephritis disease activity. Serum anti-NCS and anti-C1q levels were positively correlated with the ECLAM score in the pSLE patients as a whole (p = 0.002, rho = 0.492 and p = 0.007, rho = 0.461, respectively). Conclusion In this pure Caucasian Northern Greek pSLE population, high serum anti-GBM levels were found to be associated with the presence of nephritis, but not with the nephritis disease activity. Serum anti-GBM, anti-NCS, anti-C1q and HMGB1 may be used to differentiate patients with pSLE nephritis from patients with nephritis of other causality. Furthermore, serum anti-NCS and anti-C1q may be useful for the estimation of pSLE disease activity.