CD226 (DNAM-1) is associated with susceptibility to juvenile idiopathic arthritis.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is considered a complex genetic autoimmune disease. We investigated the association of genetic variants previously implicated in JIA, autoimmunity and/or immunoregulation, with susceptibility to JIA. METHODS: A genetic association study was performed in 639 JIA patients and 1613 healthy controls of northwest European descent. Ninety-three single nucleotide polymorphisms (SNP) were genotyped in a candidate gene approach. Results of the entire JIA patient group (all subtypes) were compared with results obtained, alternatively, with a clinically homogeneous patient group including only oligoarticular and rheumatoid factor (RF) negative polyarticular JIA patients (n=493). Meta-analyses were performed for all SNPs that have been typed in other Caucasian JIA cohorts before. RESULTS: SNPs in or near PTPN22, VTCN1, the IL2-IL21 region, ANKRD55 and TNFA were confirmed to be associated with JIA (p<0.05), strengthening the evidence for involvement of these genes in JIA. In the majority of these replicated SNPs, effect sizes were larger when analysing a homogeneous patient cohort than when analysing all subtypes. We identified two novel associations with oligoarticular and RF-negative polyarticular JIA: CD226 rs763361 (OR 1.30, 95% CI 1.12 to 1.51, p=0.0006) and CD28 rs1980422 (OR 1.29, 95% CI 1.07 to 1.55, p=0.008). Meta-analyses including reported studies confirmed the association of both SNPs with susceptibility to JIA (OR 1.16, p=0.001 and OR 1.18, p=0.001, for rs763361 and rs1980422, respectively). CONCLUSIONS: The CD226 gene has been identified as novel association with JIA, and a SNP near CD28 as a suggestive association. Both genes are probable candidate risk factors, since they are involved in costimulation of T cells.

Genetic variation in VTCN1 (B7-H4) is associated with course of disease in juvenile idiopathic arthritis.

OBJECTIVE: The course of disease in juvenile idiopathic arthritis (JIA) is unpredictable with episodes of activity and remission. In order to identify predictive factors, 93 SNPs, JIA subtype, age at onset and ANA status were studied in relation to disease course. METHODS: Genetic and clinical parameters were analysed in a cohort of 272 Caucasian patients with persistent oligoarthritis (n=129), extended oligoarthritis (n=57) and rheumatoid factor negative polyarthritis (n=86). Categories of disease course (remitting (n=65), intermediate (n=96) and unremitting (n=111)) were designed based on the cumulative time spent in active disease in the first 2 years. RESULTS: Univariate analysis revealed association of the course of disease with JIA subtype (p=5.7*10(-5)) and three SNPs; VTCN1 rs10 923 223 (p=4.4*10(-5)), VTCN1 rs12 046 117 (p=0.017) and CDK6 rs42 041 (p=0.038). In a subsequent multivariate ordinal logistic regression analysis, VTCN1 rs10 923 223 (OR 0.41, 95%-CI 0.26 to 0.63) and JIA subtype (OR 3.8, 95%-CI 2.0 to 7.2; OR 2.5, 95%-CI 1.4 to 4.2, for extended oligoarthritis and RF-negative polyarthritis vs persistent oligoarthritis, respectively) were the strongest independent factors for course of disease. CONCLUSIONS: This study provides evidence that VTCN1, encoding B7-H4, is associated with course of disease in selected subtypes of JIA. VTCN1 might be useful in predicting the course of disease.

Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.

Previously, hepatic iron overload resembling that in hereditary hemachromatosis (HH) has been found in beta 2-microglobulin knockout (beta 2m-/-) mice. We have now characterized iron metabolism in beta 2m-/- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transferrin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that (a) iron metabolism is defective in the gut mucosa as well as the liver of beta 2m-/- mice; and (b) a beta 2m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the major histocompatibility complex class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH.

The TRAF1/C5 region is a risk factor for polyarthritis in juvenile idiopathic arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is a chronic disorder in which both genetic and environmental factors are involved. Recently, we identified the TRAF1/C5 region (located on chromosome 9q33-34) as a risk factor for rheumatoid arthritis (RA) (p(combined) = 1.4 x 10(-8)). In the present study the association of the TRAF1/C5 region with the susceptibility to JIA was investigated. METHODS: A case-control association study was performed in 338 Caucasian patients with JIA and 511 healthy individuals. We genotyped the single nucleotide polymorphism rs10818488 as a marker for the TRAF1/C5 region. RESULTS: The A allele was associated with the susceptibility to rheumatoid factor-negative polyarthritis with an 11% increase in allele frequency (OR 1.54, 95% CI 1.09 to 2.18; p = 0.012). This association was stronger when combining subtypes with a polyarticular phenotype (OR 1.46, 95% CI 1.12 to 1.90; p = 0.004). In addition, we observed a trend towards an increase in A allele frequency in patients with extended oligoarthritis versus persistent oligoarthritis (49%, 38% respectively); p = 0.055. CONCLUSIONS: Apart from being a well replicated risk factor for RA, TRAF1/C5 also appears to be a risk factor for the rheumatoid factor-negative polyarthritis subtype of JIA and, more generally, seems to be associated with subtypes of JIA characterised by a polyarticular course.

Uncoupling of S phase and mitosis in cardiomyocytes and hepatocytes lacking the winged-helix transcription factor Trident.

In order to maintain a stable karyotype, the eukaryotic cell cycle is coordinated such that only one round of S phase precedes each mitosis, and mitosis is not initiated until DNA replication is completed. Several checkpoints and regulatory proteins have been defined in lower eukaryotes that govern this coordination, but little is known about the proteins that are involved in mammalian cells. Previously, we have shown that the winged-helix transcription factor Trident - also known as HFH-11, FKL16 and WIN [1] [2] [3] - is exclusively expressed in cycling cells and is phosphorylated during mitosis [1] [4]. The cellular function of Trident has yet to be described, however. Here, we have shown that disruption of the Trident gene in mice resulted in postnatal death, most probably because of circulatory failure. Histological analysis of Trident -/- embryos from embryonic day 10 (E10) onwards revealed a specific, characteristic defect in the developing myocardium. The orientation of the myocytes was highly irregular and the nuclei of these disorganized cardiomyocytes were clearly polyploid with up to a 50-fold increase in DNA content. Polyploidy was also observed in embryonic hepatocytes. Our results indicate that expression of Trident is required to prevent multiple rounds of S phase in the heart and the liver. Trident therefore appears to have a role in preventing DNA re-replication during the G2 and M phases.

Is there a role for minimal residual disease levels in the treatment of ALL patients who receive allogeneic stem cells?

Relapse is the major complication after allogeneic stem cell transplantation (SCT) for acute lymphoblastic leukemia (ALL) in children. Since it has been possible to measure minimal residual disease (MRD) by real-time quantitative polymerase chain reaction, this parameter is used more frequently in the treatment of ALL. In this article, the role of MRD and chimerism in the treatment and monitoring of pediatric transplantation recipients is described. Pre-SCT MRD levels can predict the risk of relapse and can thus be used to adjust treatment. Post-SCT MRD levels and changes in chimerism can predict relapses as well, although not many treatment options are available today, except relying on a graft-versus-leukemia effect mediated by graft-versus-host disease. Finding new treatments will be the challenge for the near future.

Adenovirus infection in children after allogeneic stem cell transplantation: diagnosis, treatment and immunity.

Human adenoviruses (HAdV) are a frequent cause of potentially fatal infections in patients after allogeneic stem cell transplantation, especially in children. Monitoring of serum/plasma by real-time quantitative PCR is a sensitive tool for the recognition of patients at risk of a potentially fatal infection and for the evaluation of the efficacy of treatment. Data from a retrospective study and from a prospective study demonstrate that recovery of immunity after transplantation is essential for the elimination of HAdV infection. The feasibility of several approaches for the manipulation of immunity in the immunocompromised host to prevent a fatal course of the infection is discussed.

Animal models of congenital defects in the ventriculoarterial connection of the heart.

The embryonic heart functions as a pump without one-way valves. To accomplish this, a long, slowly conducting myocardial structure, the outflow tract, functions as a sphincter at the arterial pole of the heart. During subsequent development tissue remodeling in the outflow tract and immigrating cells of the neural crest are responsible for connecting the right ventricle with the pulmonary trunk and the left ventricle with the aorta, that is, for the developmental formation of the ventriculoarterial junction. Most congenital malformations of the ventriculoarterial junction stem from disturbances that result in developmental arrest or in abnormal pattern formation ("real" teratology). Abnormal pattern formation can in turn originate from problems with laterality or from aberrant or incomplete formation of structural elements. Genetically modified animals with well-defined gene deficiencies are beginning to provide insight in the signal-transduction pathways and structural elements that are responsible for normal development.

Effect of ribavirin on the plasma viral DNA load in patients with disseminating adenovirus infection.

Adenovirus (AdV) infections are an increasingly frequent and potentially fatal complication in allogeneic stem cell transplant recipients. To determine the antiviral potential of ribavirin in an unbiased way, 4 patients without immune recovery were prospectively analyzed by quantitative measurement of plasma AdV DNA load. Administration of ribavirin at the first signs of AdV dissemination was not accompanied by a decrease in the plasma AdV DNA load in any of these patients, and an increase in the AdV load was even documented in 3. These observations question the potential of ribavirin to improve the outcome for patients with disseminating AdV infection and support a critical evaluation of antiviral treatments for AdV infection that involves the kinetics of virus DNA load as an objective parameter of viral replication.

In vivo mucosal uptake, mucosal transfer and retention of iron in mice.

An improved and sensitive method for studying iron absorption in mice with alterations in body iron stores is described. Mice with varying iron status were given a double isotope-labelled test dose containing 59Fe and 51Cr as a non-absorbable indicator, via an oroesophageal needle. Using a whole-body counter it was possible to measure in vivo the initial mucosal iron uptake and long-term iron retention and to calculate mucosal iron transfer. A significant difference was demonstrated between normal and both anaemic and dietary iron-loaded mice with regard to the various steps of iron absorption. When mice were tested twice for iron absorption, the results were highly reproducible. In conjunction with other parameters, the method described is useful in studying the mechanism and the regulation of iron absorption in mice.

Clinical course and prognostic value of disease activity in the first two years in different subtypes of juvenile idiopathic arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is a heterogeneous disease involving chronic arthritis. The clinical course is characterized by a fluctuating pattern of active and inactive disease. We have described in detail the clinical course in different JIA subtypes during the first 2 years after diagnosis and studied its relationship to disease activity in the following years. METHODS: Detailed clinical data on different parameters describing the disease activity in sequential time periods covering the first 2 years after diagnosis were retrieved from the charts of 311 patients with JIA and compared between subtypes. In a cohort of 146 patients, the relation of these different clinical variables to the course of disease in the following 3 years was evaluated. RESULTS: The percentage of time with active disease in the first 2 years differed significantly between subtypes. In all subtypes, a broad spectrum of activity was observed. The time with active disease in the first 2 years was the most significant factor associated with the duration of active disease in the following years. CONCLUSION: Different percentages of time with active disease have been observed between JIA subtypes in the first 2 years. The cumulative duration of activity varied widely within each subtype. Regarding the prognosis of the individual patient, the clinical course in the first 2 years appears to be predictive of the clinical course in the following years. Patients that have less time with active disease in the first 2 years are not likely to develop an unremitting clinical course later on.

Preemptive alloimmune intervention in high-risk pediatric acute lymphoblastic leukemia patients guided by minimal residual disease level before stem cell transplantation.

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level >or=1 x 10(-4) were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n=1-4). The intervention was associated with graft versus host disease >or=grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0-30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells.

Time to treatment as an important factor for the response to methotrexate in juvenile idiopathic arthritis.

OBJECTIVE: Methotrexate (MTX) is the most commonly used disease-modifying antirheumatic drug in juvenile idiopathic arthritis (JIA). Currently, individual response to MTX cannot be reliably predicted. Identification of clinical and genetic factors that influence the response to MTX could be helpful in realizing the optimal treatment for individual patients. METHODS: A cohort of 128 JIA patients treated with MTX were studied retrospectively. Eleven clinical parameters and genotypes of 6 single nucleotide polymorphisms in 5 genes related to the mechanism of action of MTX were compared between MTX responders and nonresponders using a multivariate regression analysis. RESULTS: The time from diagnosis to start of MTX treatment, physician's global assessment at baseline, and the starting dose were significantly associated with the response to MTX at 6 months after initiation. Patients with a shorter time from diagnosis to start of MTX and a higher disease activity according to the physician but with a lower MTX dose showed an increased response. The effect of the starting dose on MTX response seemed to be mainly due to the influence of the systemic JIA subtype. The time from diagnosis to start of MTX treatment and physician's global assessment at baseline were highly correlated. Therefore, the precise effect size of each independent variable could not be determined. CONCLUSION: In children with JIA, the time from diagnosis to start of MTX appears to be an important factor for MTX response. Our results suggest that an earlier start of MTX treatment will lead to an increased response.

Association of the autoimmunity locus 4q27 with juvenile idiopathic arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is characterized by chronic arthritis and an autoimmune etiology. In several autoimmune diseases, including rheumatoid arthritis (RA), an association with the 4q27 locus has been reported. We undertook this study to investigate the possible role of the 4q27 locus in JIA. METHODS: A case-control association study was conducted, with a total of 655 Caucasian JIA patients and 791 healthy controls divided into 2 independent sample sets. The rs6822844 marker in the 4q27 locus was genotyped. RESULTS: In the first and larger sample set, a 5% decrease in T allele frequency was observed in patients compared with controls (allelic odds ratio [OR] 0.72 [95% confidence interval 0.55-0.95], P = 0.019), and in the second set, a 3% decrease was observed (allelic OR 0.81 [95% confidence interval 0.61-1.09], P = 0.169). The combined data set generated an OR of 0.76 (95% confidence interval 0.62-0.93, P = 7.08 x 10(-3)). When the different JIA subtypes were analyzed individually, significant decreases were seen in the subtypes with a polyarticular course of disease (extended oligoarthritis [P = 0.019] and rheumatoid factor-negative polyarthritis [P = 0.038]). CONCLUSION: Our findings suggest that the 4q27 locus, previously reported to be associated with RA, type 1 diabetes mellitus, celiac disease, and psoriatic arthritis, is also associated with susceptibility to JIA.

HMG box containing transcription factors in lymphocyte differentiation.

The identification of the mammalian sex-determining gene Sry has led to the discovery of a large family of related ('HMG box') transcription factors that control developmental events in yeast, C. elegans, Drosophila and vertebrates. In lymphocyte differentiation, several HMG box proteins play a decisive role. Sox-4 is important for very early B-cell differentiation, while TCF-1/LEF-1 play a crucial role in early thymocyte development. TCF/LEF proteins have recently been found to constitute a downstream component of the Wingless/Wnt signal transduction pathway. In flies, this pathway controls segment polarity; in Xenopus it controls the definition of the body axis. Deregulation of the pathway occurs in several human tumors. These insights in the molecular events that are involved in TCF/LEF function in these organisms may eventually lead to the understanding of the function of these HMG box proteins in lymphoid development.

T cell repertoire and clonal deletion of Mtv superantigen-reactive T cells in mice lacking CD4 and CD8 molecules.

CD4-CD8- double-negative T cells constitute a lymphocyte subpopulation within the thymus and peripheral lymphatic organs that express a unique T cell receptor (TCR) repertoire and do not undergo negative selection. To test whether these cells develop as a distinct lineage or due to altered selection in the absence of CD4 and CD8 expression, we analyzed the TCR repertoire in mice lacking both CD4 and CD8 accessory molecules after homologous recombination (CD40/0CD80/0). We show that mature T cells of CD40/0CD80/0 mice express an unbiased diverse TCR V beta repertoire comparable to wild type mice. In addition, clonal deletion of mouse mammary tumor virus superantigen-reactive T cells did occur in CD40/0CD80/0 mice. These data show that the intrinsic lack of CD4 and CD8 expression has no effect on the mature TCR repertoire and that clonal deletion of superantigen-reactive cells is independent of CD4 and CD8 co-receptors.

Characterization of an Lyt-2+ alloreactive cytotoxic T cell clone specific for H-2Db that cross-reacts with I-Ek.

An alloreactive cytotoxic T cell clone (433) possessing the L3T4-, Lyt-2+ phenotype is described that shows a double specificity. It has been derived from unprimed BALB/c (H-2d) spleen cells by repetitive in vitro restimulation with C57BL/6(H-2b) cells. The specificity of clone 433 was determined in cytotoxicity and proliferation experiments. One specificity was for the class I antigen H-2Db and the second was for the class II antigen I-Ek. Inhibition of cytotoxicity with monoclonal antibodies confirmed these results. Cold target competition experiments demonstrated that the two specificities were mediated by the same cell population. Anti-Lyt-2 antibodies inhibited only the H-2Db- but not the I-Ek-specific lysis, suggesting a higher affinity of the antigen receptor for I-Ek than for Db. To our knowledge, this is the first description of a T cell clone that is specific for a class I antigen and cross-reacts heteroclitically with a class II antigen.

Sox-4 facilitates thymocyte differentiation.

The mouse Sry-like transcription factor Sox-4 is expressed in thymus, bone marrow, and gonads of adult mice. Sox-4-deficient mice die at embryonic day E14 due to cardiac malformation. In transfer experiments to irradiated recipients, B cell development was shown to be severely impaired in Sox-4-deficient progenitor cells. However, no drastic effects on T lymphocyte development were noted, despite the high level expression of the Sox-4 gene in the thymus of normal mice. Here, we report a detailed analysis of T cell development from Sox-4-deficient progenitors. Explanted fetal thymic organ cultures (FTOC) of Sox-4-deficient thymi yielded 10-50-fold fewer CD4 CD8 double-positive and single-positive cells than FTOC of littermates. This effect was T cell-autonomous, since similar observations were made when FTOC were performed by culturing of Sox-4-deficient progenitors in wild-type thymus lobes. When Sox-4-deficient fetal liver cells were injected together with normal cells intrathymically, they did not compete efficiently for reconstitution. It is concluded that Sox-4 facilitates thymocyte development.