A polymorphic variant inside the osteopontin gene shows association with disease course in oligoarticular juvenile idiopathic arthritis.

BACKGROUND: Oligoarticular onset juvenile idiopathic arthritis (JIA) has a variable disease course. In some patients the disease remains confined to a few joints (persistent oligoarticular) while in others it extends to affect more joints (oligoarticular extended). Osteopontin is thought to play a role in the pathogenesis. OBJECTIVE: To investigate whether a polymorphic variant in the human osteopontin gene, which is in linkage disequilibrium with recently characterised promoter variants, is associated with the disease course in oligoarticular JIA. METHODS: Genotyping of the two base pair insertion/deletion variant at +245 in the first intron was undertaken by polymerase chain reaction (PCR) amplification of DNA fragments, using a fluorescently labelled primer, followed by allele detection after rapid separation of PCR products on an automated DNA sequencer. RESULTS: Allele 2 of the polymorphic variant in the osteopontin first intron was significantly associated with the persistent oligoarticular form rather than the extended form of JIA. This was verified at the level of genotype and allele frequencies. CONCLUSIONS: The results suggest that osteopontin gene polymorphism is associated with the disease course in oligoarticular JIA and might therefore represent a useful genetic marker to characterise patients with oligoarticular JIA who are at risk of a worse outcome.

Synovial expression of osteopontin correlates with angiogenesis in juvenile idiopathic arthritis.

OBJECTIVE: To evaluate the synovial expression of osteopontin (OPN) and its possible correlation with the degree of synovial angiogenesis in human chronic idiopathic arthritis. METHODS: Forty-five patients with active juvenile idiopathic arthritis (JIA) were studied. All patients underwent SF aspiration before steroid injection. A paired plasma sample was collected from 22 JIA patients. Plasma from 15 age-matched healthy subjects was used as control. Plasma and SF were tested by ELISA for OPN and vascular endothelial growth factor (VEGF). Synovial tissue was obtained at synovectomy from 10 JIA patients. Immunohistochemistry was performed according to a standard technique with anti-OPN, anti-CD68, anti-CD31 anti-VEGF and anti-alpha(v)beta(3) antibodies. RESULTS: OPN levels were significantly higher in SF than in paired plasma samples (P<0.001). The same pattern was observed for VEGF (P<0.001). A positive correlation between OPN and VEGF concentrations was found in SF (r = 0.6, P = 0.001). In synovial tissue, OPN was expressed at the level of the lining and sublining layers with a distribution similar to that observed for VEGF. OPN expression in the lining layer correlated with the number of vessels present in the areas underlying the sublining layer. CONCLUSIONS: Synovial expression of OPN correlates with parameters of angiogenesis in JIA. These data support, in human disease, the possible role of OPN in the vascularization of inflamed synovial tissue, as previously shown in OPN-deficient animal models of arthritis.

Outcome of primary antiphospholipid syndrome in childhood.

The objective of this paper is to investigate the long-term outcome of primary antiphospholipid syndrome (APS) in the paediatric age. The features of unselected patients with primary APS who had disease onset before the age of 16 years were retrospectively analysed in three Italian referralcentres. Clinical and laboratory manifestations were assessed to establish whether, at the end of follow-up, the final diagnosis was still primary APS or whether they had developed definite SLE or lupus-like syndrome. Fourteen patients, nine boys and five girls, who had the presenting clinical manifestation of APS between three and 13 years of age (median nine years) and were followed for two to 16 years (median six years). Six patients presented with deep vein thrombosis, five with cerebral stroke, two with peripheral artery occlusion and onewith myocardial infarction. During follow-up, four patients had one or more recurrences of vascular thrombosis. At last observation, 10 patients could still be classified as having primary APS, two had developed SLE, one lupus-like syndrome and one Hodgkin's lymphoma. In conclusion; our analysis suggests that some children who present with the features of primary APS may progress to develop SLE or lupus-like syndrome.

MICA gene polymorphisms in an Italian paediatric series of juvenile Behçet disease.

The objective of this study was to investigate MICA (major histocompatibility complex MHC class I chain-related genes) polymorphisms in an Italian series of patients with juvenile Behcet disease (jBD) and to compare these genetic findings with the high prevalence of inflammatory mucosal disease, which occurs in Western populations. Ten families which included at least 1 affected patient were studied. We genotyped 18 patients (13 children and 5 adults) affected with the complete or incomplete form of jBD comparing the results to those found in a population of 20 apparently healthy individuals. The MICA transmembrane polymorphism was analysed by PCR and polyacrylamide gel electrophoresis. HLA typing was assessed by SSP-PCR technique. Statistical analysis was performed using chi2 based methods. In our series the prevalence of gastrointestinal disease was high (41%). Seven of 10 patients were HLA-B51 positive. MICA A6 allele was present in 70% of probands as compared to 25% of an ethnically matched control population. On the other hand, MICA A5.1 was present in 20% of probands as compared to 60% in controls. Out of 5 A6 homozygotes, 2 probands and 2 affected relatives developed a severe gut inflammatory disease. The study of MICA gene polymorphisms disclosed an independent association with genetic risk for jBD. The combination of MICA A6 and HLA-B51 is the strongest genetic marker for this disease. Homozygous A6 patients seem to develop more severe mucosal gut involvement. This finding sheds light on the role of a receptor for MICA, named NKG2D, presented by natural killer cells, and CD8+, alphabetaT cells and gammadeltaT cells, usually localised in gut mucosa.

Kawasaki's disease in northern Italy: a multicenter retrospective study of 250 patients.

OBJECTIVE: To evaluate (i) the clinical course and outcome in a cohort of 250 children affected with Kawasaki disease (KD) in northern Italy; (ii) the prevalence of coronary aneurysms and their correlation with treatment; and (iii) the prevalence of incomplete and atypical KD in the series as a whole. RESULTS: The male to female ratio was 1.8 to 1 and the median age at diagnosis 37 months. The majority of cases occurred in children under five years, with the youngest patient aged 51 days. Median duration offever was 8 days; median erythrocyte sedimentation rate (ESR) and C Reactive protein (CRP) values, performed before any treatment, were 77 mm/1st h and 10.4 mg/dl respectively. Median haemoglobin (Hb) levels was 9.2 g/dl and median platelet (PTL) count 408,000/mm3. Out of 250 patients, 209 (83.6%) fulfilled all criteria for the diagnosis of KD whilst 41/250 (16.4%) had an incomplete disease and 11/250 (4.4%) had an atypical onset. Fifty-four children (21.6%) were treated with aspirin alone (50-80 mg/kg/day, during the acute phase), while 196/250 (78.4%) were given aspirin (50-80 mg/kg day, during the acute phase and then 3-5 mg/Kg/day) and high-dose intravenous gammaglobulin (IVIG, 400 mg/Kg/day or 2 g/Kg/day). Coronary artery abnormalities, including aneurysms and dilatation, were reported in 7/54 patients (5 males and 2 females) who had not received IVIG and 53/196 patients (27 males and 26 females) who were treated with the IVIG in addition to aspirin. Other clinical manifestations included abdominal pain/diarrhoea (11), arthritis (10), hydrops of the gallbladder (8), disseminated intravascular coagulation (3), and hemiparesis (1). All patients except one infant are still alive. In most of the patients coronary alterations normalised, while in 20 patients they persisted for more than one year; in 12/20 patients they were permanent. CONCLUSION: Pediatricians should be aware of the increasing number of children with atypical and incomplete KD, in order to diagnose and treat the disease even in the absence of all typical manifestations and to prevent coronary complications.

Involvement of the hypothalamic-pituitary-adrenal axis in children with oligoarticular-onset idiopathic arthritis.

Adult patients with rheumatic arthritis and other rheumatic disorders show inappropriate cortisol secretion and peculiar CRH promoter gene polymorphisms. So far, no data are available about this topic in children with juvenile idiopathic arthritis (JIA). We have studied a series of 13 prepubertal patients (10 female, 3 male) affected with oligoarticular JIA (o-JIA) without clinical and biological signs of disease activity (ESR and IL-6). ACTH plasma concentrations were significantly increased at 8 a.m. in o-JIA patients, whereas no differences were found in cortisol plasma concentrations. The ACTH/cortisol ratio was significantly increased in o-JIA patients with respect to the normal population both at 8 a.m. and at noon. DHEAS and testosterone plasma concentration did not statistically differ in the two populations. The genetic study was aimed at defining the prevalence of polymorphisms A1 and A2 in o-JIA patients, but we failed to find allelic or genotypic differences. Our study suggests the presence of a partial resistance to ACTH with a dysregulated pattern of secretion also in inactive o-JIA patients. These preliminary data need further confirmation in larger pediatric studies.

Serum and synovial fluid concentration of vascular endothelial growth factor in juvenile idiopathic arthritides.

OBJECTIVE: To evaluate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of local joint inflammation in juvenile idiopathic arthritis (JIA). METHODS: Sera from 50 patients affected with JIA and 10 age-matched healthy controls were tested with a commercial ELISA for VEGF. Corresponding synovial fluid (SF) concentrations of VEGF and p75 soluble tumour necrosis factor receptor (sTNFR) were evaluated in 20 active JIA patients. RESULTS: Serum concentrations of VEGF were significantly higher in patients with active polyarticular disease than in patients with active and inactive oligoarticular disease and healthy controls. In JIA patients, serum concentrations of VEGF displayed a significant correlation with a number of clinical and laboratory parameters of disease activity. VEGF concentrations in SF were significantly higher than those detected in corresponding sera. Moreover, a clear correlation was found between corresponding SF and serum VEGF concentrations. In SF, VEGF showed a strong positive correlation with p75 sTNFR. CONCLUSIONS: Concentrations of VEGF in SF in patients with JIA are higher than corresponding serum concentrations, suggesting that this pro-angiogenic factor may have a major role in the outgrowth of hyperplastic pannus and tissue damage at the site of tissue inflammation.

Levels of soluble CD27 in sera and synovial fluid and its expression on memory T cells in patients with juvenile idiopathic arthritides.

OBJECTIVE: CD27 is a member of tumour necrosis factor receptor family. Its expression is predominantly confined to mature lymphocytes and is strongly enhanced after cell activation. Shedding of the CD27 from the surface of activated cells is related to their effector phase. The aim of the present study was to evaluate the levels of soluble CD27 in sera and synovial fluids, together with its expression on circulating and synovial fluid (SF) memory T cells, in children with JIA. METHODS: Sera from 40 patients with active JIA were studied for soluble CD27. Paired SF samples were available in 20 patients. Sera from 12 age-matched patients affected with various acute infectious diseases and 12 age-matched healthy subjects were used as controls. In 8 JIA patients freshly isolated circulating and SF lymphocytes were stained for CD27 in CD4+CD45 RO+ T cell subpopulation and analyzed by cytometry. RESULTS: Soluble CD27 serum levels were significantly higher in patients with polyarticular JIA and acute systemic infectious diseases than in patients with active oligoarticular or healthy controls. Both polyarticular and oligoarticular JIA patients showed increased levels of soluble CD27 in SF when compared with paired serum samples (p = 0.01). In all the patients tested a significant enrichment of CD27- T cells was seen in the SF (median 39.5%, range 18-56%) when compared to paired CD4+CD45RO+ peripheral lymphocytes (median 19.5%, range 5-43%; p = 0.01). CONCLUSIONS: A clear enrichment of CD4+ memory SF T cells with a CD27-phenotype is observed when compared to correspondent circulating T lymphocytes. This issue is conceivably related to re-activation and recruitment of memory T cells to the site of inflammation, and to the subsequent expansion of a subpopulation of "effector" memory T cells.

Brain perfusion spect in juvenile neuro-Behçet's disease.

UNLABELLED: Regional cerebral blood flow was evaluated by (99m)Tc-hexamethylpropyleneamine oxime SPECT in 7 patients (age range, 7--18 y; mean age, 9.1 y) affected with Behçet's disease and signs or symptoms of central nervous system involvement at different times of their clinical history. METHODS: Three patients suffered from seizures, 3 patients were affected with severe persistent headache that was refractory to common analgesic and nonsteroidal antiinflammatory drugs, and 1 patient had recurrent episodes of acute intracranial hypertension. Electroencephalography was performed on all patients, MRI on 5 patients, and CT on 1 patient. Brain SPECT was performed using a high-resolution, brain-dedicated camera. After conventional visual analysis by 2 expert readers, 2 transaxial sections were drawn parallel to the bicommissural line: the first across the thalami and the second across the temporal lobe at the level of the mesiotemporal structures. Cortical regions of interest were drawn automatically on the cortical ribbon on the 2 sections, whereas other regions of interest were drawn by hand around the basal ganglia, the thalami, and the mesiotemporal structures. Asymmetry analysis was then applied, and hypoperfusion was considered when the asymmetry value was >10%. RESULTS: Hypoperfusion was observed in all patients by visual and asymmetry analyses; this finding was localized mainly in the basal ganglia, the thalami, and the temporal cortex, including its mesial portion. Temporal hypoperfusion was found primarily in patients with seizures, and hypoperfusion of deep gray nuclei was found mainly in the other patients. Electroencephalography disclosed brain functional impairment in 5 of 6 patients, where- as MRI showed multiple bilateral white matter lesions in 1 patient suffering from persistent headache. CONCLUSION: As in adults, perfusion SPECT seems to be very sensitive in disclosing brain abnormalities in children and adolescents with Behçet's disease and signs or symptoms of central nervous system involvement, even with negative findings on brain MRI.

Efficacy and safety of alendronate for the treatment of osteoporosis in diffuse connective tissue diseases in children: a prospective multicenter study.

OBJECTIVE: Osteopenia/osteoporosis is being increasingly reported as a complication of many chronic diseases, even in children. In this preliminary study, we evaluated the effect of an oral bisphosphonate (alendronate) on bone mass in children with diffuse connective tissue diseases. METHODS: Thirty-eight children with low bone mass were treated with alendronate for 1 year; 38 children who had the same primary disorders as the study patients but in a less severe form served as untreated control patients. We were also able to evaluate changes in bone mass (before and after alendronate) in 16 of the treated patients whose bone mineral density (BMD) had been routinely measured before the present study was initiated. RESULTS: BMD increased by a mean +/- SD of 14.9 +/- 9.8% (P < 0.002 versus baseline) in the treated patients (reaching the normal range in 13 patients), while the BMD was 2.6 +/- 5% (not significant versus baseline) in the control group (15 had a decrease). Most interestingly, there was a large increase in BMD (15.3 +/-9.9%) after alendronate therapy in the 16 children who had their BMD followed up in the year before the study, during which time they had shown little increase in BMD (1.03 +/- 6.3%), and often a decrease. Considering their condition, increases in the height of all patients was satisfactory. No new fractures were observed after alendronate therapy was initiated. CONCLUSION: Bisphosphonates can be considered essential components of the treatment of secondary osteoporosis, not only in adults, but also in pediatric patients. Alendronate has a positive effect on secondary osteopenia/osteoporosis in children with connective tissue diseases.

MEFV mutations in Behçet's disease.

Familial Mediterranean fever (FMF) and Behçet's disease (BD), both inflammatory diseases, are highly prevalent in the Middle Eastern and Mediterranean populations. FMF is a Mendelian autosomic recessive disease linked to MEFV, a gene of unknown function. BD in contrast is a polyfactorial disease associated with the major histocompatibility complex. Because FMF and BD have epidemiological similarities, we asked whether the FMF gene was implicated in BD. We screened for the common MEFV mutations a cohort of 114 chromosomes from definite BD patients [meeting the criteria of the International study group] and probable cases [meeting at least two of these criteria]. We screened in parallel an ethnically matched cohort of FMF and control chromosomes. The M694V, V726A and E148Q mutations tended to be more frequent in definite BD (2.6%, 2.6%, and 5.2%, respectively) than in controls (0%, 0%, and 2.2%). The P706 polymorphism was found in 10.5% of the probable BD chromosomes, but in only 1.6% of the controls (p=0.01). Because some MEFV mutations were more frequent in BD than in controls, we suggest that they may act as additional susceptibility factors in BD.

Tumor necrosis factor induced adhesion molecule serum concentrations in Henoch-Schönlein purpura and pediatric systemic lupus erythematosus.

OBJECTIVE: Animal models of immune complex mediated tissue injury have shown that tumor necrosis factor (TNF) and TNF induced adhesion molecules play an important role in the pathogenesis of tissue damage mediated by IgG, but not in that mediated by IgA, immune complexes. We compared possible differences in the behavior of 2 TNF induced adhesion molecules (VCAM-1 and ICAM-1) in Henoch-Schönlein purpura (HSP), which is characterized by the formation of IgA immune complexes, versus systemic lupus erythematosus (SLE), which is mostly associated with the vascular deposition of IgG immune complexes. METHODS: Serum concentrations of soluble (s)VCAM-1 and ICAM-1 were determined by ELISA methods in 20 patients with pediatric SLE showing variably active disease, 20 active patients with active HSP, and 19 healthy controls. TNF-alpha as well as p55 and p75 soluble receptors (sTNF-R) were simultaneously tested by enzyme amplified sensitivity immunoassay in 22 patients (12 SLE, 10 HSP). RESULTS: Serum sVCAM-1 concentration was significantly higher in patients with SLE (mean +/- SD, 608 +/- 76 ng/ml), than in patients with HSP (501.9 +/- 63.3 ng/ml) and controls (446.8 +/- 139.2 ng/ml) (p < 0.001). In SLE patients, sVCAM-1 correlated positively with ESR (r = 0.45, p = 0.02) and negatively with C4 serum levels (r = -0.57, p = 0.004), platelets (r = -0.38, p = 0.03), and lymphocyte count (r = -0.42, p = 0.03). No differences in sICAM-1 serum concentrations were detected among SLE, HSP, or control groups. Soluble VCAM, but not sICAM-1, showed a positive correlation with TNF-alpha (r = 0.71, p = 0.01), p55 (r = 0.63, p = 0.02), and p75 (r = 0.7, p = 0.01) sTNF-R serum concentrations in SLE, but not in patients with HSP. CONCLUSION: Our study provides additional evidence of a possible differential involvement of TNF and TNF induced adhesion molecules in the pathogenesis of tissue damage between pediatric SLE and HSP.

Recurrent antiphospholipid-related deep vein thrombosis as presenting manifestation of systemic lupus erythematosus.

UNLABELLED: Antiphospholipid antibodies (aPL) are frequently associated with thrombotic disorders in the so-called antiphospholipid syndrome. Together with anticardiolipin antibodies (aCL), lupus anticoagulant (LA) is the main diagnostic tool for aPL detection. Since LA determination is based on the finding of prolonged clotting time in vitro, concomitant anticoagulant therapy may significantly interfere with its detection. We report a case of a boy in whom recurrent aPL-related thrombosis heralded for several months the onset of systemic lupus erythematosus (SLE). Abnormally increased in vitro clotting times at the time of the second thrombotic event led to the suspicion of the presence of LA activity. However, this latter finding was difficult to interpret since the patient was already on heparin treatment at the time of our first observation. Thus, LA was assayed using a commercial kit in which a heparin neutralizer is included (Staclot LA). Two consecutive samples from the patient were compared with eight patients on anticoagulant therapy for non-aPL-related thrombotic events and 20 healthy controls. The study showed that, taking into account the concomitant anticoagulant treatment, Staclot LA was positive only in the propositus, raising the suspicion of a possible aPL-related origin of the thrombotic event. This issue was definitively confirmed in a subsequent follow-up. CONCLUSION: The present report shows that aPL-related deep vein thrombosis can be the earliest clinical manifestation of pediatric SLE, and that Staclot LA may have a role in LA detection during the course of anticoagulant treatment.

Increased gut permeability in juvenile chronic arthritides. A multivariate analysis of the diagnostic parameters.

OBJECTIVE: This study was aimed at evaluating intestinal permeability (IP) in patients with oligoarticular juvenile idiopathic arthritis (o-JIA), spondyloarthropathy (SpA) associated with inflammatory bowel disease (IBD) and other forms of juvenile-onset chronic arthritiis (OIA) using the lactulose/mannitol (L/M) test in comparison with other non-invasive parameters of gut involvement. METHODS: A series of 26 children affected with o-JIA and 14 with either SpA/IBD or OIA were assessed for IP. The urinary L/M ratio was measured by gas chromatography. The erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and faecal alpha 1 antitrypsin concentrations were also evaluated. Ten o-JIA patients displayed active arthritis while in 16 the disease was under control. Among the OIA patients, 11 were affected with psoriatic arthritis and the remaining 3 with chronic reactive arthritis. 14 patients with SpA-IBD had active synovitis or spine inflammation. 14 eo-pJCA and 22 OIA and SpA-IBD patients, respectively, were receiving NSAID therapy. RESULTS: The mean L/M ratios for the Spa-IBD (0.07 +/- 0.02, mean +/- SD), OIA (0.05 +/- 0.02) and o-JIA (0.04 +/- 0.02) patients were significantly higher (p < 0.001, p = 0.022 and p = 0.01, respectively) than those found in controls (0.02 +/- 0.01). Logistic regression analysis disclosed a positive correlation between the L/M ratio and the presence of gastrointestinal manifestations (p = 0.011). The type of disease (p = 0.28), the disease activity in the JCA patient group (p = 0.24) and NSAID administration (p = 0.210) did not seem to significantly influence the L/M ratio. CONCLUSIONS: All of the subtypes of juvenile chronic arthritides that we studied displayed an increased IP. Hence, gut wall inflammation (albeit asymptomatic) may also be present in o-JIA patients. The SpA-IBD patients with gastrointestinal symptoms displayed the highest mean L/M ratio values. The L/M test seemed to correlate with histopathological features of the gut mucosa. The L/M ratio was shown to be a highly sensitive but poorly specific test for predicting gut inflammatory disease compared to other non-invasive screening tests.