Herpes Virus Infections During Treatment With Etanercept in Juvenile Idiopathic Arthritis.

Incidence rates for varicella and herpes zoster were similar in patients with juvenile idiopathic arthritis receiving etanercept/methotrexate (n = 85, 184.9 patient-years [PY]) or methotrexate alone (n = 71, 199.4 PY); no complicated varicella or herpes zoster cases were reported; herpes labialis incidence was higher in patients receiving etanercept/methotrexate versus methotrexate alone (0.38 vs. 0.24 PY).

An experimental therapy to improve skeletal growth and prevent bone loss in a mouse model overexpressing IL-6.

UNLABELLED: Premature osteoporosis and stunted growth are common complications of childhood chronic inflammatory disease. Presently, no treatment regimens are available for these defects in juvenile diseases. We identified the sequential Fc-OPG/hPTH treatment as an experimental therapy that improves the skeletal growth and prevents the bone loss in a mouse model overexpressing IL-6. INTRODUCTION: Premature osteoporosis and stunted growth are common complications of childhood chronic inflammatory diseases and have a significant impact on patients' quality of life. Presently, no treatment regimens are available for these defects in juvenile diseases. To test a new therapeutic approach, we used growing mice overexpressing the pro-inflammatory cytokine IL-6 (TG), which show a generalized bone loss and stunted growth. METHODS: Since TG mice present increased bone resorption and impaired bone formation, we tested a combined therapy with the antiresorptive modified osteoprotegerin, Fc-OPG, and the anabolic PTH. We injected TG mice with Fc-OPG once at the 4th day of life and with hPTH(1-34) everyday from the 16th to the 30th day of age. RESULTS: A complete prevention of growth and bone defects was observed in treated mice due to normalization of osteoclast and osteoblast parameters. Re-establishment of normal bone turnover was confirmed by RT-PCR analysis and by in vitro experiments that revealed the full rescue of osteoclast and osteoblast functions. The phenotypic recovery of TG mice was due to the sequential treatment, because TG mice treated with Fc-OPG or hPTH alone showed an increase of body weight, tibia length, and bone volume to intermediate levels between those observed in vehicle-treated WT and TG mice. CONCLUSIONS: Our results identified the sequential Fc-OPG/hPTH treatment as an experimental therapy that improves the skeletal growth and prevents the bone loss in IL-6 overexpressing mice, thus providing the proof of principle for a therapeutic approach to correct these defects in juvenile inflammatory diseases.

Increased muscle expression of interleukin-17 in Duchenne muscular dystrophy.

OBJECTIVES: Duchenne muscular dystrophy (DMD) is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. Dystrophic muscle is characterized by chronic inflammation, and inflammatory mediators could be promising targets for innovative therapeutic interventions. We analyzed muscle biopsy samples of DMD-affected children to characterize interleukin (IL)-17 and Forkhead box P3 (Foxp3) expression levels and to identify possible correlations with clinical status. METHODS: Expression levels of IL-17, Foxp3, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), IL-6, and transforming growth factor-ß (TGF-ß) were analyzed by real-time PCR in muscle biopsy samples from patients with DMD (n = 27) and juvenile dermatomyositis (JDM) (n = 8). Motor outcome of patients with DMD was evaluated by North Star Ambulatory Assessment score. RESULTS: In DMD, we found higher levels of IL-17 and lower levels of Foxp3 mRNA compared with those for a typical inflammatory myopathy, JDM. Moreover, the IL-17/Foxp3 ratio was higher in DMD than in JDM biopsy samples. IL-17 mRNA levels appeared to be related to the expression levels of other proinflammatory cytokines (TNF-α and MCP-1) and significantly associated with clinical outcome of patients. CONCLUSIONS: The association of IL-17 expression with levels of other inflammatory cytokines and with the clinical course of DMD suggests a possible pathogenic role of IL-17.

Performance of a component-based allergen-microarray in the diagnosis of cow's milk and hen's egg allergy.

BACKGROUND: The food challenge test (FCT) is the gold standard for the diagnosis of food allergy. This procedure is time consuming, costly and can induce potentially severe symptoms. An ideal in vitro test should allow to avoid the FCT. Objective To assess the clinical performance of microarray for specific IgE (sIgE) detection in children with challenge-proven/excluded cow's milk (CM) or hen's egg (HE) allergy. METHODS: One-hundred and four children with suspected IgE-mediated hypersensitivity to CM or HE were studied. In all patients, skin prick test, ImmunoCAP, microarray and FCT were performed. RESULTS: The microarray components Bos d 8 for CM (27/58 patients) and Gal d 1 (20/46 patients) and Gal d 2 (24/46) for HE were the most frequently recognized allergens. Using the FCT results as the reference parameter, sIgE to Bos d 8 and Gal d 1 had the highest area under the curves. These were not significantly different from those obtained using the ImmunoCAP. Use of 95% clinical decision points (CDP) for sIgE to Bos d 8 and Gal d 1 resulted in higher negative predictive values (78% and 79%, respectively) than those obtained with the ImmunoCAP (57% and 59%). CONCLUSIONS: Our results show that in children with suspected CM or HE allergy, the microarray has a good ability to predict the FCT results. In a clinical application perspective, the microarray could be used as a second-level assay, if the ImmunoCAP sIgE is <95% CDP. This approach would lead to a decrease in the number of the FCT to be performed, as well as of positive FCTs with a subsequent decrease in severe reaction risk.

The impact of chronic inflammation on the growing skeleton: lessons from interleukin-6 transgenic mice.

BACKGROUND: Chronic inflammatory diseases in children are associated with impairment of linear growth and bone mineral accrual. In addition to poor nutrition, reduced mobility and glucocorticoid treatment, several observations in patients suggest that inflammation itself may have a direct detrimental effect on the growing skeletal system. Among the various inflammatory cytokines produced during the inflammatory response, data in animals suggest that interleukin-6 (IL-6) may mediate the effects of inflammation on the developing skeleton. Mice overexpressing IL-6 during the prepubertal stage show stunted growth, abnormalities of the insulin-like growth factor I system, defective growth plates, delayed development of ossification centres, uncoupling of osteoblast and osteoclast activity and defective ossification. These changes appear to mirror the features observed in children with chronic inflammatory diseases. CONCLUSIONS: The tissue, cellular and molecular abnormalities reviewed provide a rationale for therapeutic approaches aimed at correcting the detrimental effects of inflammation on the developing skeletal system.

Macrophage migration inhibitory factor (MIF) and oligoarticular juvenile idiopathic arthritis (o-JIA): association of MIF promoter polymorphisms with response to intra-articular glucocorticoids.

OBJECTIVES: To address the clinical relevance of macrophage migration inhibitory factor (MIF) promoter polymorphisms in oligoarticular juvenile idiopathic arthritis (o-JIA) by evaluating their associations with serum and SF MIF levels, with response to intra-articular glucocorticoid injections and with outcome of the disease. METHODS: Seventy-five Caucasian patients with o-JIA were studied. Alleles of the -794 CATT variable number of tandem repeats (VNTR) and of the -173 G/C single nucleotide polymorphism (SNP) were identified by capillary electrophoresis following fluorescently labelled PCR and by allelic discrimination assay, respectively. MIF levels were measured by ELISA. The association of MIF promoter polymorphisms with polyarticular extension, Childhood Health Assessment Questionnaire (CHAQ) score at the last follow-up visit and occurrence of chronic anterior uveitis was evaluated only in patients with a follow up > 5 years. RESULTS: Neither of the MIF promoter polymorphisms was associated with serum MIF levels, nor with the long-term outcome of o-JIA. The -173 G/C SNP was significantly associated with both SF MIF levels and duration of response to intra-articular glucocorticoid injection. Carriers of a MIF -173 C allele were 4 times more likely to relapse within 3 months. No association was found between the different MIF CATT alleles and both SF MIF levels and duration of response to intra-articular glucocorticoids. CONCLUSION: Our study shows the clinical relevance of the MIF -173 G/C SNP in o-JIA and suggests that the -173 C allele may represent a predictor of poor response to intra-articular glucocorticoid treatment.

High circulating levels of biologically inactive IL-6/SIL-6 receptor complexes in systemic juvenile idiopathic arthritis: evidence for serum factors interfering with the binding to gp130.

We previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.

Role of interleukin-6 in growth failure: an animal model.

Indirect evidence suggests a link between factors produced during the inflammatory response and stunted growth. The demonstration of this link was provided by the observation that mice transgenic for the inflammatory cytokine interleukin-6 (IL-6), expressing high circulating levels of IL-6 since birth, show a marked decrease in growth rate leading to adult mice 50-70% the size of wild-type littermates. The growth defect is completely abolished by neutralization of IL-6. In these mice the production of GH is normal, while circulating levels of IGF-I are markedly decreased. Administration of IL-6 to wild-type mice results in a marked decrease in IGF-I levels. These observations show that in vivo high levels of IL-6 are associated with low levels of IGF-I. However, IL-6 does not directly affect IGF-I production both in vitro and in vivo. In contrast, markedly decreased levels of IGFBP-3 are present in the IL-6 transgenic mice and administration of IL-6 to wild-type mice results in a marked decrease in IGFBP-3 levels. In these mice the decrease in IGFBP-3 levels is associated with impaired formation of the 150 kD ternary complex, even in the presence of normally functional ALS. As a consequence, IL-6 transgenic mice show increased clearance of circulating IGF-I, suggesting that IL-6 decreases IGF-I levels by increased clearance. Proteolytic degradation of IGFBP-3 occurs in the IL-6 transgenic mice, suggesting that the decrease in IGFBP-3 could be at least in part due to proteolysis. The abnormalities of the IGF-I system observed in the IL-6 transgenic mice are similar to those found in patients with systemic juvenile idiopathic arthritis, one of the chronic inflammatory diseases characterized by stunted growth and prominent production of IL-6. The IL-6 transgenic mice represent a faithful animal model of the growth impairment associated with chronic inflammation and may therefore provide information relevant to the understanding and treatment of this complication of inflammatory diseases.

Effect of IL-6 on IGF binding protein-3: a study in IL-6 transgenic mice and in patients with systemic juvenile idiopathic arthritis.

Stunted growth is a common complication of childhood diseases characterized by chronic inflammation or infections. We previously demonstrated that NSE/hIL-6 transgenic mice, overexpressing the inflammatory cytokine IL-6 since early phase of life, showed a marked growth defect associated with decreased IGF-I levels, suggesting that IL-6 is one of the factors involved in stunted growth complicating chronic inflammation in childhood. Here we show that NSE/hIL-6 mice have normal liver IGF-I production, decreased levels of IGF binding protein-3 (IGFBP-3) and increased serum IGFBP-3 proteolysis. Reduced IGFBP-3 levels results in a marked decrease in the circulating 150-kDa ternary complex, even in the presence of normally functional acid labile subunit. Pharmacokinetic studies showed that NSE/hIL-6 mice have accelerated IGF-I clearance. Patients with systemic juvenile idiopathic arthritis (s-JIA), a chronic inflammatory disease characterized by prominent IL-6 production and complicated by stunted growth associated with low IGF-I levels, have markedly decreased IGFBP-3 levels, increased serum IGFBP-3 proteolysis and normal acid labile subunit levels. Our data show that chronic overproduction of IL-6 causes decreased IGFBP-3 levels, resulting in a decreased association of IGF-I in the 150-kDa complex. Decreased levels of IGF-I appear to be secondary to increased clearance.

Abnormal regulation of interleukin 6 in systemic juvenile idiopathic arthritis.

OBJECTIVE: To evaluate the in vitro production of interleukin 6 (IL-6) by peripheral blood mononuclear cells (PBMC) of patients with systemic juvenile idiopathic arthritis (JIA), its regulation by IL-10, and the association of abnormal regulation of IL-6 production with presence of polymorphisms in the regulatory sequence of the IL-6 gene. METHODS: PBMC were cultured in the absence or presence of lipopolysaccharide (LPS). IL-6 and IL-10 levels were measured by ELISA. Polymorphisms in the regulatory sequences of the IL-6 gene were assessed by restriction fragment length polymorphism analysis and sequencing of amplified regions. RESULTS: Patients' PBMC produced high amounts of IL-6 compared to controls in unstimulated conditions. The IL-10 50% inhibitory dose of LPS stimulated IL-6 production was significantly higher in patients than controls. IL-10 levels produced in the absence or presence of LPS were comparable between patients and controls. The -174 G/C polymorphism in the IL-6 gene does not appear to be correlated with the high unstimulated IL-6 production or with the reduced inhibition by IL-10 observed in patients with JIA. No differences in patients compared to controls and with respect to the published sequence were found in the 3' untranslated region (UTR) of the IL-6 gene. CONCLUSION: Most patients with JIA have increased unstimulated production of IL-6 and reduced inhibition of IL-6 production by IL-10. This abnormal regulation of IL-6 production is not secondary to a defect in IL-10 production, and is not associated with polymorphism of alleles at position -174 of the 5' flanking region or with mutations in the 3' untranslated region of the IL-6 gene.

Activation-induced cell death and Fas-induced apoptosis in patients with systemic or pauciarticular juvenile idiopathic arthritis.

OBJECTIVE: To investigate the functionality of the Fas-induced apoptotic pathway in peripheral blood mononuclear cells (PBMC) from patients with systemic or pauciarticular juvenile idiopathic arthritis (JIA). METHODS: PBMC from 12 patients with systemic and 6 with pauciarticular JIA were activated with anti-CD3 and rhIL-2 and then incubated in the presence or absence of the anti-Fas MoAb CH11 inducing activation of the Fas apoptotic pathway. Apoptosis was evaluated by flow cytometry and fluorescence microscopy. RESULTS: The percentage of apoptotic cells following triggering of Fas did not differ between patients with systemic JIA (12.5 +/- 9.5%) or pauciarticular JIA (18.7 +/- 8.9%) and controls (16.1 +/- 6.8%). Evaluation of activation-induced cell death (AICD) in the absence of exogenous triggering of Fas showed that 44% (8/18) of the patients with JIA, compared to none of the controls (0/16), had a percentage of apoptotic cells higher than the mean + 2 SD of controls. The increased AICD was neutralized by the addition of an anti-TNF-alpha antibody. CONCLUSION: Patients with systemic or pauciarticular JIA do not show a defect in the Fas-dependent apoptotic pathway of T cells. The increased AICD present in some patients with JIA appears to be at least in part related to the inflammatory cytokine TNF-alpha.

In vivo neutralization of human IL-6 (hIL-6) achieved by immunization of hIL-6-transgenic mice with a hIL-6 receptor antagonist.

Neutralization of IL-6 represents an attractive therapeutic option in several diseases, including B cell neoplasia, osteoporosis, and autoimmunity. Therapeutic attempts in humans have shown that administration of injectable doses of a mAb to IL-6 does not provide efficient neutralization of the cytokine in vivo. Therefore, alternative approaches are needed. In this study, we evaluated whether the Ab response to human IL-6 (hIL-6) elicited by vaccination with Sant1 (a hIL-6 variant with seven amino acid substitutions) was able to fully correct in vivo the clinical and biological effects of a chronic endogenous overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice. Because of the overexpression of hIL-6, occurring since birth, with circulating levels in the nanogram per milliliter range, NSE/hIL-6 mice have a marked decrease in growth rate, associated with decrease in insulin-like growth factor I levels, and represent an animal model of the growth impairment associated with human chronic inflammatory diseases. Following immunization with Sant1, but not with hIL-6, NSE/hIL-6 mice developed high titers of polyclonal Abs to hIL-6. The Abs, acquired by transplacental transfer, effectively neutralized IL-6 activities in vivo as shown by the complete correction of the growth defect and normalization of insulin-like growth factor levels in the hIL-6-transgenic offspring. Immunization with Sant1 could therefore represent a novel and simple therapeutic approach for the specific neutralization of IL-6 in humans.