Correlation analyses of clinical and molecular findings identify candidate biological pathways in systemic juvenile idiopathic arthritis.

BACKGROUND: Clinicians have long appreciated the distinct phenotype of systemic juvenile idiopathic arthritis (SJIA) compared to polyarticular juvenile idiopathic arthritis (POLY). We hypothesized that gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with each disease would reveal distinct biological pathways when analyzed for significant associations with elevations in two markers of JIA activity, erythrocyte sedimentation rate (ESR) and number of affected joints (joint count, JC). METHODS: PBMC RNA from SJIA and POLY patients was profiled by kinetic PCR to analyze expression of 181 genes, selected for relevance to immune response pathways. Pearson correlation and Student's t-test analyses were performed to identify transcripts significantly associated with clinical parameters (ESR and JC) in SJIA or POLY samples. These transcripts were used to find related biological pathways. RESULTS: Combining Pearson and t-test analyses, we found 91 ESR-related and 92 JC-related genes in SJIA. For POLY, 20 ESR-related and 0 JC-related genes were found. Using Ingenuity Systems Pathways Analysis, we identified SJIA ESR-related and JC-related pathways. The two sets of pathways are strongly correlated. In contrast, there is a weaker correlation between SJIA and POLY ESR-related pathways. Notably, distinct biological processes were found to correlate with JC in samples from the earlier systemic plus arthritic phase (SAF) of SJIA compared to samples from the later arthritis-predominant phase (AF). Within the SJIA SAF group, IL-10 expression was related to JC, whereas lack of IL-4 appeared to characterize the chronic arthritis (AF) subgroup. CONCLUSIONS: The strong correlation between pathways implicated in elevations of both ESR and JC in SJIA argues that the systemic and arthritic components of the disease are related mechanistically. Inflammatory pathways in SJIA are distinct from those in POLY course JIA, consistent with differences in clinically appreciated target organs. The limited number of ESR-related SJIA genes that also are associated with elevations of ESR in POLY implies that the SJIA associations are specific for SJIA, at least to some degree. The distinct pathways associated with arthritis in early and late SJIA raise the possibility that different immunobiology underlies arthritis over the course of SJIA.

Early gene expression profiles of patients with chronic hepatitis C treated with pegylated interferon-alfa and ribavirin.

UNLABELLED: Responsiveness to hepatitis C virus (HCV) therapy depends on viral and host factors. Our aim was to assess sustained virologic response (SVR)-associated early gene expression in patients with HCV receiving pegylated interferon-alpha2a (PEG-IFN-alpha2a) or PEG-IFN-alpha2b and ribavirin with the duration based on genotypes. Blood samples were collected into PAXgene tubes prior to treatment as well as 1, 7, 28, and 56 days after treatment. From the peripheral blood cells, total RNA was extracted, quantified, and used for one-step reverse transcription polymerase chain reaction to profile 154 messenger RNAs. Expression levels of messenger RNAs were normalized with six "housekeeping" genes and a reference RNA. Multiple regression and stepwise selection were performed to assess differences in gene expression at different time points, and predictive performance was evaluated for each model. A total of 68 patients were enrolled in the study and treated with combination therapy. The results of gene expression showed that SVR could be predicted by the gene expression of signal transducer and activator of transcription-6 (STAT-6) and suppressor of cytokine signaling-1 in the pretreatment samples. After 24 hours, SVR was predicted by the expression of interferon-dependent genes, and this dependence continued to be prominent throughout the treatment. CONCLUSION: Early gene expression during anti-HCV therapy may elucidate important molecular pathways that may be influencing the probability of achieving virologic response.

A single-tube quantitative assay for mRNA levels of hormonal and growth factor receptors in breast cancer specimens.

Knowledge of estrogen receptor (ER) and progesterone receptor (PR) status has been critical in the evolution of modern targeted therapy of breast cancer and remains essential for making informed therapeutic decisions. Recently, growth factor receptor HER2/neu (ERBB2) status has made it possible to provide another form of targeted therapy linked to the overexpression of this protein. Presently, pathologists determine the receptor status in formalin-fixed, paraffin-embedded sections using subjective, semiquantitative immunohistochemistry (IHC) assays and quantitative fluorescence in situ hybridization for HER2. We developed a single-tube multiplex TaqMan (mERPR+HER2) assay to quantitate mRNA levels of ER, PR, HER2, and two housekeeping genes for breast cancer formalin-fixed, paraffin-embedded sections. Using data from the discovery sample sets, we evaluated IHC-status-dependent cutoff-point and IHC-status-independent clustering methods for the classification of receptor status and then validated these results with independent sample sets. Compared with IHC-status, the accuracies of the mERPR+HER2 assay with the cutoff-point classification method were 0.98 (95% CI: 0.97-1.00), 0.92 (95% CI: 0.88-0.95), and 0.97 (95% CI: 0.95-0.99) for ER, PR, and HER2, respectively, for the validation sets. Furthermore, the areas under the receiver operating-characteristic curves were 0.997 (95% CI: 0.994-1.000), 0.967 (95% CI: 0.949-0.985), and 0.968 (95% CI: 0.915-1.000) for ER, PR, and HER2, respectively. This multiplex assay provides a sensitive and reliable method to quantitate hormonal and growth factor receptors.

Increased expression of alpha(1,3)-fucosyltransferase-VII and P-selectin binding of synovial fluid T cells in juvenile idiopathic arthritis.

OBJECTIVE: The mechanisms controlling the recruitment of T helper type 1 (Th1) cells to the inflamed synovium are not fully understood. Here, we focus on alpha(1,3)-fucosyltransferase-VII (FucT-VII), an enzyme responsible for the generation of functional P- and E-selectin ligands that is upregulated in Th1 cells. METHODS: Expression of transcripts encoding FucT-VII, interferon-gamma (IFN-gamma), and interleukin 12Rbeta2 (IL-12Rbeta2) were analyzed in T cells purified from synovial fluid (SF) and from peripheral blood (PB) of children with juvenile idiopathic arthritis (JIA) using kinetic reverse transcriptase polymerase chain reaction analysis. Binding of SF and PB T cells to P-selectin was determined by flow cytometry using a soluble P-selectin/IgG1 fusion molecule. Recruitment of T cells to synovial tissue in vivo was studied by analyzing the migration of FucT-VII transfected Jurkat T cells into human rheumatoid synovial tissue grafted into SCID mice. RESULTS: In patients with JIA, the mRNA levels of FucT-VII, as well as of IFN-gamma and IL-12Rbeta2, were up-regulated in SF T cells compared to paired PB T cells. A higher expression of FucT-VII mRNA in SF T cells was associated with increased binding of T cells to P-selectin. Moreover, FucT-VII expression and increased P-selectin binding capacity of T cells were associated with a polyarticular course of oligoarticular JIA. Expression of FucT-VII in Jurkat T cells resulted in an increased accumulation of these cells in human rheumatoid synovial tissue grafted into SCID mice. CONCLUSION: Our data indicate an important role of FucT-VII in the enhanced homing of T cells to the inflamed synovium.

A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most common systemic autoimmune disease, affecting approximately 1% of the adult population worldwide, with an estimated heritability of 60%. To identify genes involved in RA susceptibility, we investigated the association between putative functional single-nucleotide polymorphisms (SNPs) and RA among white individuals by use of a case-control study design; a second sample was tested for replication. Here we report the association of RA susceptibility with the minor allele of a missense SNP in PTPN22 (discovery-study allelic P=6.6 x 10(-4); replication-study allelic P=5.6 x 10(-8)), which encodes a hematopoietic-specific protein tyrosine phosphatase also known as "Lyp." We show that the risk allele, which is present in approximately 17% of white individuals from the general population and in approximately 28% of white individuals with RA, disrupts the P1 proline-rich motif that is important for interaction with Csk, potentially altering these proteins' normal function as negative regulators of T-cell activation. The minor allele of this SNP recently was implicated in type 1 diabetes, suggesting that the variant phosphatase may increase overall reactivity of the immune system and may heighten an individual carrier's risk for autoimmune disease.