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Aims: Nrf2 is an essential transcription factor for protection against oxidant disorders. However, its role in organ development and neonatal disease has received little attention. Therapeutically administered oxygen has been considered to contribute to bronchopulmonary dysplasia (BPD) in prematurity. The current study was performed to determine Nrf2-mediated molecular events during saccular-to-alveolar lung maturation, and the role of Nrf2 in the pathogenesis of hyperoxic lung injury using newborn Nrf2-deficient (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mice., Results: Pulmonary basal expression of cell cycle, redox balance, and lipid/carbohydrate metabolism genes was lower while lymphocyte immunity genes were more highly expressed in Nrf2(-/-) neonates than in Nrf2(+/+) neonates. Hyperoxia-induced phenotypes, including mortality, arrest of saccular-to-alveolar transition, and lung edema, and inflammation accompanying DNA damage and tissue oxidation were significantly more severe in Nrf2(-/-) neonates than in Nrf2(+/+) neonates. During lung injury pathogenesis, Nrf2 orchestrated expression of lung genes involved in organ injury and morphology, cellular growth/proliferation, vasculature development, immune response, and cell-cell interaction. Bioinformatic identification of Nrf2 binding motifs and augmented hyperoxia-induced inflammation in genetically deficient neonates supported Gpx2 and Marco as Nrf2 effectors., Innovation: This investigation used lung transcriptomics and gene targeted mice to identify novel molecular events during saccular-to-alveolar stage transition and to elucidate Nrf2 downstream mechanisms in protection from hyperoxia-induced injury in neonate mouse lungs., Conclusion: Nrf2 deficiency augmented lung injury and arrest of alveolarization caused by hyperoxia during the newborn period. Results suggest a therapeutic potential of specific Nrf2 activators for oxidative stress-associated neonatal disorders including BPD.

Objective: To determine the spectrum of mutations associated with familial hypercholesterolaemia (FH) and their detection rate in the FH Western Australia (FHWA) Program., Methods: Mutation testing of the LDLR gene, plus select regions in APOB and PCSK9, was performed in the first 343 patients considered to be phenotypic index cases of FH and classified on the basis of the Dutch Lipid Clinic Network Criteria (DLCNC) score as "possible", "probable", or "definite" FH., Results: Overall, 86 different pathogenic (or likely pathogenic) mutations were identified in 129 patients, including four compound heterozygotes manifesting a more severe clinical phenotype. Fourteen of these mutations were novel and twelve (9.6%) were large deletions/duplications of the LDLR. The most common mutations were the familial defective apoB-100 mutation APOB p.Arg3527Gln (7.2%) and an LDLR intron 3 splice site mutation c.313 + 1G > A (4.8%). While 70% of 'definite' FH patients were found to carry a mutation, only 29% of 'probable' and 11% of 'possible' FH patients were mutation-positive., Conclusion: This information provides a useful DNA database on which to base ongoing cascade screening for FH and future research into the genetic aetiology of FH in Western Australia. These findings suggest genetic testing should be prioritised to those with high DLCNC scores and offers a cost-effective family screening method from FH index cases, leading to detection of other previously undiagnosed and younger family members, enabling early instigation of intervention and preventative measures for premature coronary heart disease., (Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Epigenetic modifications, such as DNA methylation, due to in utero exposures may play a critical role in early programming for childhood and adult illness. Maternal smoking is a major risk factor for multiple adverse health outcomes in children, but the underlying mechanisms are unclear., Objective: We investigated epigenome-wide methylation in cord blood of newborns in relation to maternal smoking during pregnancy., Methods: We examined maternal plasma cotinine (an objective biomarker of smoking) measured during pregnancy in relation to DNA methylation at 473,844 CpG sites (CpGs) in 1,062 newborn cord blood samples from the Norwegian Mother and Child Cohort Study (MoBa) using the Infinium HumanMethylation450 BeadChip (450K)., Results: We found differential DNA methylation at epigenome-wide statistical significance (p-value < 1.06 × 10-7) for 26 CpGs mapped to 10 genes. We replicated findings for CpGs in AHRR, CYP1A1, and GFI1 at strict Bonferroni-corrected statistical significance in a U.S. birth cohort. AHRR and CYP1A1 play a key role in the aryl hydrocarbon receptor signaling pathway, which mediates the detoxification of the components of tobacco smoke. GFI1 is involved in diverse developmental processes but has not previously been implicated in responses to tobacco smoke., Conclusions: We identified a set of genes with methylation changes present at birth in children whose mothers smoked during pregnancy. This is the first study of differential methylation across the genome in relation to maternal smoking during pregnancy using the 450K platform. Our findings implicate epigenetic mechanisms in the pathogenesis of the adverse health outcomes associated with this important in utero exposure.

Introduction: Adrenal insufficiency is a rare life threatening condition with non-specific clinical signs and symptoms. This necessitates a high level of reliance on laboratory results for making the diagnosis., Methods: We report a case involving an interference in cortisol measurement leading to spuriously low-cortisol measurements and the incorrect diagnosis and treatment of adrenal insufficiency. We also discuss the testing used to confirm the presence and nature of the interferent., Results: Interference with an IgG antibody was demonstrated, and the patient was found to have a normally functioning adrenal axis., Conclusion: Interference in immunoassay is well described but often difficult to detect in clinical practice. Negative interference in cortisol measurement can occur and may lead to inappropriate diagnosis and treatment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells' SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis.

We studied the influence of glacial oscillations on the genetic structure of seven species of white-headed gull that breed at high latitudes (Larus argentatus, L. canus, L. glaucescens, L. glaucoides, L. hyperboreus, L. schistisagus, and L. thayeri). We evaluated localities hypothesized as ice-free areas or glacial refugia in other Arctic vertebrates using molecular data from 11 microsatellite loci, mitochondrial DNA (mtDNA) control region, and six nuclear introns for 32 populations across the Holarctic. Moderate levels of genetic structure were observed for microsatellites (F(ST)= 0.129), introns (Φ(ST)= 0.185), and mtDNA control region (Φ(ST)= 0.461), with among-group variation maximized when populations were grouped based on subspecific classification. Two haplotype and at least two allele groups were observed across all loci. However, no haplotype/allele group was composed solely of individuals of a single species, a pattern consistent with recent divergence. Furthermore, northernmost populations were not well differentiated and among-group variation was maximized when L. argentatus and L. hyberboreus populations were grouped by locality rather than species, indicating recent hybridization. Four populations are located in putative Pleistocene glacial refugia and had larger τ estimates than the other 28 populations. However, we were unable to substantiate these putative refugia using coalescent theory, as all populations had genetic signatures of stability based on mtDNA. The extent of haplotype and allele sharing among Arctic white-headed gull species is noteworthy. Studies of other Arctic taxa have generally revealed species-specific clusters as well as genetic structure within species, usually correlated with geography. Aspects of white-headed gull behavioral biology, such as colonization ability and propensity to hybridize, as well as their recent evolutionary history, have likely played a large role in the limited genetic structure observed.

Context: Inflammatory myofibroblastic tumor is a predominantly benign, spindle cell, mesenchymal neoplasm with myxoid areas that occurs rarely in the female genital tract and may be confused with other spindle cell lesions, particularly leiomyosarcoma., Objective: To investigate the utility of detecting anaplastic lymphoma kinase-1 protein expression and ALK gene rearrangements in the diagnosis of inflammatory myofibroblastic tumors in the female genital tract., Design: Eight inflammatory myofibroblastic tumors arising in the female genital tract and seen in consultation (from 2004 to 2011) were reviewed. Immunohistochemistry for anaplastic lymphoma kinase-1 and fluorescence in-situ hybridization studies for ALK gene rearrangements were performed., Results: The anatomic sites included myometrium (4 cases) and endometrium, fallopian tube, cervix, and a cervical polyp (1 each), with a patient age range from 25 to 52 years. Histologic features ranged from bland spindle cells to striking cytologic atypia, embedded in a prominent myxoid background. Anaplastic lymphoma kinase-1 immunohistochemistry was positive in 7 cases. Fluorescence in-situ hybridization studies detected ALK gene rearrangements in 5 cases. Five cases had both immunopositivity and fluorescence in-situ hybridization abnormalities, 2 cases had immunopositivity only, and 1 case was negative by both methods., Conclusions: This is the first report, to our knowledge, of ALK gene rearrangements in inflammatory myofibroblastic tumors in the female genital tract. If a myxoid background is appreciated in a spindle cell lesion of the female genital tract, especially if inflammatory cells are present, anaplastic lymphoma kinase-1 staining along with fluorescence in situ hybridization studies, for ALK gene rearrangements, may aid in distinguishing inflammatory myofibroblastic tumors from their malignant mimics.

Background: Prolactin has multiple forms and macroprolactin, which is thought not to be bioavailable, can cause a raised serum prolactin concentration. Gel filtration chromatography (GFC) is currently the gold standard method for separating macroprolactin, but is labour-intensive. Polyethylene glycol (PEG) precipitation is suitable for routine use but may not always be accurate. We developed a high pressure liquid chromatography (HPLC) assay for macroprolactin measurement., Methods: Chromatography was carried out using an Agilent Zorbax GF-250 (9.4 × 250 mm, 4 μm) size exclusion column and 50 mmol/L Tris buffer with 0.15 mmol/L NaCl at pH 7.2 as mobile phase, with a flow rate of 1 mL/min. Serum or plasma was diluted 1:1 with mobile phase and filtered and 100 μL injected. Fractions of 155 μL were collected for prolactin measurement and elution profile plotted. The area under the curve of each prolactin peak was calculated to quantify each prolactin form, and compared with GFC., Results: Clear separation of monomeric-, big- and macroprolactin forms was achieved. Quantification was comparable to GFC and precision was acceptable. Total time from injection to collection of the final fraction was 16 min., Conclusions: We have developed an HPLC method for quantification of macroprolactin, which is rapid and easy to perform and therefore can be used for routine measurement.

Aims/hypothesis: To compare the effectiveness of low-fat high-protein and low-fat high-carbohydrate dietary advice on weight loss, using group-based interventions, among overweight people with type 2 diabetes. Study design Multicentre parallel (1:1) design, blinded randomised controlled trial., Methods: Individuals with type 2 diabetes aged 30–75 years and a BMI >27 kg/m2 were randomised, by an independent statistician using sequentially numbered sealed envelopes, to be prescribed either a low-fat high-protein (30% of energy as protein, 40% as carbohydrate, 30% as fat) or a low-fat high carbohydrate(15% of energy as protein, 55%as carbohydrate,30% as fat) diet. Participants attended 18 group sessions over 12 months. Primary outcomes were change in weight and waist circumference assessed at baseline, 6 and 12 months.Secondary outcomes were body fatness, glycaemic control,lipid profile, blood pressure and renal function. A further assessment was undertaken 12 months after the intervention.Research assessors remained blinded to group allocation throughout. Intention-to-treat analysis was performed., Results: A total of 419 participants were enrolled (mean±SDage 58±9.5 years,BMI 36.6±6.5 kg/m2 and HbA1c 8.1±1.2%(65 mmol/mol)). The study was completed by 70%(294/419).No differences between groups were found in change in weight or waist circumference during the intervention phase or the 12-month follow-up. Both groups had lost weight (2–3 kg, p<0.001) and reduced their waist circumference (2–3 cm, p<0.001) by 12 months and largely maintained this weight loss for the following 12 months. By 6 months, the difference in self-reported dietary protein between groups was small (1.1%total energy; p<0.001). No significant differences between groups were found in secondary outcomes: body fatness, HbA1c, lipids, blood pressure and renal function.There were no important adverse effects., Conclusions/interpretation: In a 'real-world' setting, prescription of an energy-reduced low-fat diet, with either increased protein or carbohydrate, results in similar modest losses in weight and waist circumference over 2 years

Familial hypercholesterolaemia (FH) is an autosomal dominant disorder characterised by increased plasma concentrations of low density lipoprotein (LDL) cholesterol leading to atherosclerosis and premature coronary heart disease (CHD) and death. The clinical diagnosis of FH is based on a personal and family history, physical examination findings and LDL-cholesterol concentrations. FH is primarily caused by mutations in the LDL-receptor gene (LDLR), and less frequently by mutations in genes for APOB and the more recently identified PCSK9. Lifestyle modification and pharmacotherapy can delay or prevent the onset of CHD in FH. It is estimated that only 20% of cases have been diagnosed in Australia and that the majority are inadequately treated. Screening options for FH include population screening (of children or adults), targeted screening of patients with premature CHD and their relatives, or opportunistic screening such as flagging laboratory lipid reports. Cascade screening, a form of targeted screening, is an ethically acceptable, cost-effective strategy for the identification of FH. However, for screening to be successful, medical practitioners need to be aware of the signs and diagnosis of FH and the benefits of early treatment.

Lipid disorders, also known as dyslipidaemias, are abnormalities of lipoprotein metabolism and include elevations of total cholesterol, low density lipoprotein (LDL) cholesterol, and triglyceride, and reductions in high density lipoprotein (HDL) cholesterol, and can be acquired or familial in nature. Dyslipidaemia is a major risk factor for coronary heart disease and cardiovascular disease (CVD), which is the leading cause of morbidity and mortality in Australia. Dyslipidaemia is defined by laboratory testing and using statistically determined criteria. Although the benefits of detecting and treating dyslipidaemia in patients with known CVD is clear, controversy remains regarding screening asymptomatic individuals who are not known to be at increased cardiovascular risk. This review examines the role of screening in the detection and treatment of individuals with lipid disorders.

Objective: To examine the hypothesis that the subset of rheumatoid arthritis (RA) characterized by antibodies to citrullinated α-enolase is mediated by Porphyromonas gingivalis enolase in the context of DR4 alleles., Methods: Recombinant human α-enolase and P gingivalis enolase, either citrullinated or uncitrullinated, were used to immunize DR4-IE-transgenic mice and control mice (class II major histocompatibility complex-deficient [class II MHC(-/-)] and C57BL/6 wild-type mice). Arthritis was quantified by measurement of ankle swelling in the hind paws and histologic examination. Serum IgG reactivity with α-enolase and citrullinated α-enolase was assayed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Antibodies to peptide 1 of citrullinated α-enolase (CEP-1) and its arginine-bearing control peptide, REP-1, were also assessed by ELISA., Results: Significant hind-ankle swelling (≥0.3 mm) occurred in DR4-IE-transgenic mice immunized with citrullinated human α-enolase (9 of 12 mice), uncitrullinated human α-enolase (9 of 12 mice), citrullinated P gingivalis enolase (6 of 6 mice), and uncitrullinated P gingivalis enolase (6 of 6 mice). Swelling peaked on day 24. None of the control groups developed arthritis. The arthritic joints showed synovial hyperplasia and erosions, but there was a paucity of leukocyte infiltration. Antibodies to human α-enolase, both citrullinated and unmodified, and to CEP-1 and REP-1 were detectable in all immunized mice except the class II MHC(-/-) control mice., Conclusion: This is the first animal model that links an immune response to P gingivalis enolase to an important subset of RA, defined by antibodies to citrullinated α-enolase in the context of DR4. The fact that arthritis and anti-CEP-1 antibodies were induced independent of citrullination of the immunizing antigen suggests that the unmodified form of α-enolase may be important in initiating the corresponding subset of human RA., (Copyright © 2011 by the American College of Rheumatology.)

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein-DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process.

Introduction: mipomersen is a second-generation antisense oligonucleotide (ASO) targeted to human apolipoprotein (apo) B-100, a large protein synthesized by the liver that plays a fundamental role in human lipoprotein metabolism. Mipomersen predominantly distributes to the liver and decreases the production of apoB-100, the primary structural protein of the atherogenic lipoproteins including low density lipoprotein (LDL), thereby reducing plasma LDL-cholesterol and apoB-100 concentrations., Areas Covered: the mode of action, preclinical development and clinical trials of mipomersen, an antisense apoB synthesis inhibitor. The paper provides an understanding of the pharmacokinetic and pharmacodynamic characteristics of mipomersen and insight into its clinical efficacy and safety. In clinical trials, mipomersen produced dose-dependent and prolonged reductions in LDL-cholesterol and other apoB-containing lipoproteins, including lipoprotein (a) [Lp(a)] in healthy volunteers and in patients with mild to moderate hypercholesterolemia. Mipomersen has been shown to decrease apoB, LDL-cholesterol and Lp(a) in patients with heterozygous and homozygous familial hypercholesterolemia on maximally tolerated lipid-lowering therapy., Expert Opinion: mipomersen shows promise as an adjunctive agent by reducing apoB-containing lipoproteins in patients at high risk of atherosclerotic cardiovascular disease who are not at target or are intolerant of statins. Although the short-term efficacy and safety of mipomersen has been established, concern exists regarding the long-term potential for hepatic steatosis with this ASO.

p53 coordinates the expression of an intricate network of genes in response to stress signals. Sequence-specific DNA binding is essential for p53-mediated tumor suppression. We evaluated the impact of single-nucleotide polymorphisms (SNPs) in p53 response elements (p53RE) on DNA binding and gene expression in response to DNA damage. Using a bioinformatics approach based on incorporating p53 binding strength into a position weight matrix, we selected 32 SNPs in putative and validated p53REs. The microsphere assay for protein-DNA binding (MAPD) and allele-specific expression analysis was employed to assess the impact of SNPs on p53-DNA binding and gene expression, respectively. Comparing activated p53 binding in nuclear extracts from doxorubicin- or ionizing radiation (IR)-treated human cells, we observed little difference in binding profiles. Significant p53 binding was observed for most polymorphic REs and several displayed binding comparable to the p21 RE. SNP alleles predicted to lower p53 binding indeed reduced binding in 25 of the 32 sequences. Chromatin immunoprecipitation-sequencing in lymphoblastoid cells confirmed p53 binding to seven polymorphic p53 REs in response to doxorubicin. In addition, five polymorphisms were associated with altered gene expression following doxorubicin treatment. Our findings demonstrate an effective strategy to identify and evaluate SNPs that may alter p53-mediated stress responses.

Objective: To assess the role of T cells in the mouse model of citrullinated human fibrinogen-induced rheumatoid arthritis (RA) using CTLA-4Ig, an agent that blocks T cell costimulation, which is required for T cell activation., Methods: Humanized HLA-DRβ1*0401-transgenic (DR4-Tg) mice were immunized with Cit-human fibrinogen to induce arthritis. Prior to, and at the onset or peak of, arthritis, the DR4-Tg mice were treated with CTLA-4Ig or control human IgG1 or were left untreated. Arthritis development and progression were monitored by measuring ankle swelling with calipers and by assessing histopathologic changes. The immune responses to the citrullinated antigens and the corresponding unmodified antigens, as well as the arthritogenicity of lymphocytes from these mice, were examined. The latter was performed using lymphocyte transfers from CTLA-4Ig-treated or control mice via intraperitoneal injection into naive DR4-Tg mice. Recipient mice also received an intraarticular injection of Cit-human fibrinogen, unmodified human fibrinogen, or vehicle., Results: CTLA-4Ig-treated, but not human IgG1-treated, arthritic mice had significantly reduced ankle swelling and pathologic joint damage. Treatment with CTLA-4Ig, but not human IgG1, suppressed Cit-human fibrinogen-induced T cell activation, including citrulline-specific T cell activation, when given prior to disease onset. Transfer of splenic lymphocytes from untreated or human IgG1-treated arthritic mice caused arthritis in recipients, and this occurred when Cit-human fibrinogen, but not unmodified fibrinogen, was deposited into the joint. Splenocytes from CTLA-4Ig-treated mice were unable to transfer arthritis., Conclusion: Activated citrulline-specific T cells play a direct role in the development and progression of arthritis in this model of Cit-human fibrinogen-induced RA.

Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. As a first step in applying functional genomic analysis to population studies, we have examined the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer (SC, n = 20), smokers without lung cancer (SNC, n = 24), and never smokers (NS, n = 8). Functional enrichment analysis showed that the NRF2-mediated, antioxidant response element (ARE)-regulated genes, were significantly lower in SC, when compared with expression levels in SNC. Importantly, we found that the expression of MAFG (a binding partner of NRF2) was correlated with the expression of ARE genes, suggesting MAFG levels may limit target gene induction. Bioinformatically we identified single nucleotide polymorphisms (SNPs) in putative ARE genes and to test the impact of genetic variation, we genotyped these putative regulatory SNPs and other tag SNPs in selected NRF2 pathway genes. Sequencing MAFG locus, we identified 30 novel SNPs and two were associated with either gene expression or lung cancer status among smokers. This work demonstrates an analysis approach that integrates bioinformatics pathway and transcription factor binding site analysis with genotype, gene expression and disease status to identify SNPs that may be associated with individual differences in gene expression and/or cancer status in smokers. These polymorphisms might ultimately contribute to lung cancer risk via their effect on the airway gene expression response to tobacco-smoke exposure.

Objective: To examine the association between aberrant IgG galactosylation and disease parameters in rheumatoid arthritis (RA)., Methods: Analysis of N-glycan in serum samples from multiple cohorts was performed. The IgG N-glycan content and the timing of N-glycan aberrancy relative to disease onset were compared in healthy subjects and in patients with RA. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, sex, age, anti-cyclic citrullinated peptide (anti-CCP) antibody titer, disease duration, and C-reactive protein level on aberrant galactosylation was determined using multivariate analysis. The N-glycan content was also compared between epitope affinity-purified autoantibodies and the remaining IgG repertoire in RA patients., Results: Our results confirm the aberrant galactosylation of IgG in RA patients as compared with healthy controls (mean +/- SD 1.36 +/- 0.43 versus 1.01 +/- 0.23; P < 0.0001). We observed a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman's rho = 0.37, P < 0.0001). This correlation was higher in women (Spearman's rho = 0.60, P < 0.0001) than in men (Spearman's rho = 0.16, P = 0.10). Further, aberrant IgG galactosylation substantially predated the onset of arthritis and the diagnosis of RA (3.5 years) and resided selectively in the anticitrullinated antigen fraction., Conclusion: Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, associates with disease activity in a sex-specific manner, and resides preferentially in autoantibodies.

Endometriosis is a common gynecologic disorder characterized by ectopic endometrium associated with pelvic pain and infertility. The pathogenesis of endometriosis is unclear, and several genetic, endocrine, immune, and environmental agents have been studied as putative causative factors. However, consistent somatic genetic alterations have not been identified. Rarely, endometriosis presents as a mass lesion with an infiltrative pattern reminiscent of malignancy. We describe cytogenetic and molecular cytogenetic findings of mass-forming endometriosis. The index case of pulmonary endometriosis underwent conventional and molecular cytogenetics analysis. In addition, 16 cases of mass-forming endometriosis, 11 cases of usual endometriosis, and six endometriomas were investigated by fluorescence in situ hybridization (FISH) for HMGA1 and HMGA2 loci, performed on paraffin-embedded thin tissue sections with custom-designed probes. The index patient had an endometriotic lung nodule, with a 46,XX, t(5;6)(q13;p21) karyotype and HMGA1 rearrangement by FISH. A second patient had decidualized endometriosis forming a large abdominal mass and HMGA1 rearrangement by FISH. Of the 15 other cases of mass-forming endometriosis, one had HMGA1 rearrangement and two had HMGA2 rearrangement. The rearrangements were found in the stromal component exclusively. None of the usual endometriosis cases or endometriomas had HMGA1 or HMGA2 rearrangements. In conclusion, mass-forming endometriosis is an uncommon subset of endometriosis that harbors HMGA1 or HMGA2 rearrangements in up to 29% of cases. The present findings support the concept that endometriosis is clonal and that rearrangement of HMGA genes likely contributes to its pathogenesis., ((c) 2010 Wiley-Liss, Inc.)

A patient with bilateral ovarian adenocarcinomas composed predominantly of ciliated cells incidentally found at autopsy is reported. Although obviously malignant, a majority of the cells expressed well-differentiated cilia with terminal bar formation. In one of the masses, the neoplastic cells seemed to arise from a serous adenofibroma. The tumor was confined to the ovaries without evidence of metastatic spread. Although morphologically resembling an endometrioid-type neoplasm, immunohistochemical and molecular studies were more consistent with a serous phenotype, especially in light of its apparent origination from a serous adenofibroma. We agree with previous observations suggesting that although the neoplasm seems morphologically worrisome, it may actually portend a more benign clinical course.

Objective: Despite recent advances in the conceptual understanding of the pathogenesis of ovarian cancer, it remains the foremost cause of death from gynecologic malignancies in developed countries. The main reason for such a high rate of mortality is the lack of sensitive and specific biomarkers and imaging techniques for early detection of ovarian cancer. Additional biological insights into early-stage ovarian carcinogenesis are needed to help speed the development of markers for early detection of ovarian cancer. The objective of this study was to characterize differentially expressed genes in high-grade stage I serous carcinoma of the ovary., Methods: We analyzed gene expression in macrodissected formalin-fixed, paraffin-embedded samples from 5 high-grade stage I serous carcinomas and 5 stage I borderline tumors of the ovary using the Illumina Whole Genome DASL assay (cDNA-mediated annealing, selection, extension, and ligation) corresponding to 24,000 genes. Significance Analysis of Microarrays was performed to determine differentially expressed genes in stage I serous carcinoma, and class prediction analysis was performed to determine the predictive value of differentially expressed gene sets to correctly classify serous carcinoma from borderline tumors in 3 independent data sets. Altered transcription factor pathways and biological pathways unique to stage I serous carcinoma were identified through class comparison of differentially expressed genes., Results: Unsupervised cluster analysis of gene expression correctly classified stage I serous carcinomas from serous borderline tumors. Supervised analysis identified several known, as well as novel, genes differentially expressed in stage I ovarian cancer. Using a differentially expressed gene set, class comparison prediction analysis correctly identified serous carcinomas from serous borderline tumors in 3 independent data sets at over 80% accuracy, sensitivity, and specificity. Pathway analysis demonstrated the significance of p53 and E2F pathways in serous carcinogenesis and significant involvements of cell cycle and immune response pathways in stage I serous epithelial ovarian cancer., Conclusion: We have identified differentially expressed genes associated with the carcinogenesis of high-grade stage I serous EOC. Furthermore, integrative analysis of biological and transcription pathway data contributed to the confirmation of important biological pathways and discovery of additional unique genes and pathways that may have potential importance in ovarian pathogenesis and biomarker development.

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks., Competing Interests: The authors have declared that no competing interests exist.

We investigated the role of selection in generating and maintaining species distinctness in spite of ongoing gene flow, using two zones of secondary contact between large gull species in Europe (Larus argentatus and Larus cachinnans) and North America (Larus glaucescens and Larus occidentalis). We used the pattern of neutral genetic differentiation at nine microsatellite loci (F(ST)) as an indicator of expected changes under neutral processes and compared it with phenotypic differentiation (P(ST)) for a large number of traits (size, plumage melanism and coloration of bare parts). Even assuming very low heritability, interspecific divergence between L. glaucescens and L. occidentalis in plumage melanism and orbital ring colour clearly exceeded neutral differentiation. Similarly, melanism of the central primaries was highly divergent between L. argentatus and L. cachinnans. Such divergence is unlikely to have arisen randomly and is therefore attributed to spatially varying selection. Variation in plumage melanism in both transects agrees with Gloger's rule, which suggests that latitude (and associated sun and humidity gradients) could be the selective pressure shaping differentiation in plumage melanism. We suggest that strong species differentiation in orbital ring colour results from sexual selection. We conclude that these large gull species, along with other recently diverged species that hybridize after coming into secondary contact, may differ only in restricted regions of the genome that are undergoing strong disruptive selection because of their phenotypic effects.

The study of zones of secondary contact provides insight into the maintenance of reproductive isolation. Tension zone theory supplies powerful tools for assessing how dispersal and selection shape hybrid zones. We present a multimodal analysis of phenotypic clines in conjunction with clines at molecular markers in a hybrid zone between Larus glaucescens and Larus occidentalis. We developed a new method to analyze simultaneously clines of quantitative traits and molecular data. Low linkage disequilibrium and the lack of coincidence between clines at six microsatellites, a mitochondrial DNA region, and two phenotypic traits indicated introgression. However, the hypothesis of neutral diffusion was rejected based on evidence that all of the clines were concordant and narrower than expected for neutral clines, indicating some indirect selection. The analysis of phenotypic variance gave evidence of restricted phenotypic introgression and together with the bimodal distribution of phenotypes suggested that disruptive selection is acting across the hybrid zone, especially on the coloration of bare parts. Multimodal analysis of phenotypic clines also highlighted a shift between the peak of intermediates and the cline center, left behind by hybrid zone motion. High-resolution analysis of phenotypes distribution thus proved useful for detecting hybrid zone movement even without temporal data.

The most common form of genetic variation, single nucleotide polymorphisms or SNPs, can affect the way an individual responds to the environment and modify disease risk. Although most of the millions of SNPs have little or no effect on gene regulation and protein activity, there are many circumstances where base changes can have deleterious effects. Non-synonymous SNPs that result in amino acid changes in proteins have been studied because of their obvious impact on protein activity. It is well known that SNPs within regulatory regions of the genome can result in disregulation of gene transcription. However, the impact of SNPs located in putative regulatory regions, or rSNPs, is harder to predict for two primary reasons. First, the mechanistic roles of non-coding genomic sequence remain poorly defined. Second, experimental validation of the functional consequences of rSNPs is often slow and laborious. In this review, we summarize traditional and novel methodologies for candidate rSNPs selection, in particular in silico techniques that aid in candidate rSNP selection. Additionally we will discuss molecular biological techniques that assess the impact of rSNPs on binding of regulatory machinery, as well as functional consequences on transcription. Standard techniques such as EMSA and luciferase reporter constructs are still widely used to assess effects of rSNPs on binding and gene transcription; however, these protocols are often bottlenecks in the discovery process. Therefore, we highlight novel and developing high-throughput protocols that promise to aid in shortening the process of rSNP validation. Given the large amount of genomic information generated from a multitude of re-sequencing and genome-wide SNP array efforts, future focus should be to develop validation techniques that will allow greater understanding of the impact these polymorphisms have on human health and disease.

Genetically determined factors that alter the metabolism of tobacco carcinogens can influence an individual's susceptibility to bladder cancer. The associations between the genotypes of glutathione S-transferase (GST) M1, GSTP1, GSTT1 and N-acetyltransferase (NAT) 1 and the phenotypes of NAT2 and cytochrome P450 (CYP) 1A2 and bladder cancer risk were examined in a case-control study involving 731 bladder cancer patients and 740 control subjects in Los Angeles County, California. Individual null/low-activity genotypes of GSTM1, GSTT1 and GSTP1 were associated with a 19-48% increase in odds ratio (OR) of bladder cancer. The strongest association was noted for GSTM1 [OR for the null genotype = 1.48, 95% confidence interval (CI) = 1.19-1.83]. When the three GST genes were examined together, there was a monotonic, statistically significant association between increasing number of null/low-activity genotypes and risk (P for trend = 0.002). OR (95% CI) for one and two or more null/low-activity GST genotypes was 1.42 (1.12-1.81) and 1.71 (1.25-2.34), respectively, relative to the absence of null/low-activity GST genotype. NAT2 slow acetylation was associated with doubled risk of bladder cancer among individuals with known high exposures to carcinogenic arylamines (OR = 2.03, 95% CI = 1.12-3.69, P = 0.02). The effect of NAT2 slow acetylation was even stronger in the presence of two or more null/low-activity GST genotypes. There were no associations between bladder cancer risk and NAT1 genotype or CYP1A2 phenotype.

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0-13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0-13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi-in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical (1/2)-(a single decamer) and (3/4)-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of (1/2)- and (3/4)-site REs greatly expands the p53 master regulatory network., Competing Interests: The authors have declared that no competing interests exist.

Rheumatoid arthritis (RA) is a common autoimmune disease that afflicts the synovium of diarthrodial joints. The pathogenic mechanisms inciting this disease are not fully characterized, but may involve the loss of tolerance to posttranslationally modified (citrullinated) antigens. We have demonstrated that this modification leads to a selective increase in antigenic peptide affinity for major histocompatibility complex (MHC) class II molecules that carry the RA-associated shared epitope, such as HLA-DRB1*0401 (DR4). We describe the induction of arthritis in DR4-IE transgenic (tg) mice with citrullinated fibrinogen, a protein commonly found in inflamed synovial tissue and a frequent target of autoantibodies in RA patients. The disease induced in these mice was characterized by synovial hyperplasia followed by ankylosis, but lacked a conspicuous polymorphonuclear cell infiltrate. Immunological analysis of these mice through T cell epitope scanning and antibody microarray analysis identified a unique profile of citrulline-specific reactivity that was not found in DR4-IE tg mice immunized with unmodified fibrinogen or in wild-type C57BL/6 mice immunized with citrullinated fibrinogen, two conditions where arthritis was not observed. These observations directly implicate citrullinated fibrinogen as arthritogenic in the context of RA-associated MHC class II molecules.

Ovarian cancer, the most aggressive gynecologic cancer, is the foremost cause of death from gynecologic malignancies in the developed world. Two primary reasons explain its aggressive behavior: most patients present with advanced disease at diagnosis, and die of recurrences from disease that has become resistant to conventional chemotherapies. In this paper on epithelial ovarian cancer (EOC), we will review molecular alterations associated with the few precursor lesions identified to date, followed by the more commonly recognized processes of de novo carcinogenesis, metastasis, and the development of chemoresistance. We will propose a unifying model of ovarian epithelial tumorigenesis that takes into account various hypotheses. We will also review novel approaches to overcome the major problem of chemoresistance in ovarian cancer. Finally, we will discuss advances and new challenges in the development of mouse model systems to investigate EOC precursor lesions, progression, metastasis, and chemoresistance., ((c) 2007 Wiley-Liss, Inc.)

The p53 tumor suppressor is a sequence-specific pleiotropic transcription factor that coordinates cellular responses to DNA damage and stress, initiating cell-cycle arrest or triggering apoptosis. Although the human p53 binding site sequence (or response element [RE]) is well characterized, some genes have consensus-poor REs that are nevertheless both necessary and sufficient for transactivation by p53. Identification of new functional gene regulatory elements under these conditions is problematic, and evolutionary conservation is often employed. We evaluated the comparative genomics approach for assessing evolutionary conservation of putative binding sites by examining conservation of 83 experimentally validated human p53 REs against mouse, rat, rabbit, and dog genomes and detected pronounced conservation differences among p53 REs and p53-regulated pathways. Bona fide NRF2 (nuclear factor [erythroid-derived 2]-like 2 nuclear factor) and NFkappaB (nuclear factor of kappa light chain gene enhancer in B cells) binding sites, which direct oxidative stress and innate immunity responses, were used as controls, and both exhibited high interspecific conservation. Surprisingly, the average p53 RE was not significantly more conserved than background genomic sequence, and p53 REs in apoptosis genes as a group showed very little conservation. The common bioinformatics practice of filtering RE predictions by 80% rodent sequence identity would not only give a false positive rate of approximately 19%, but miss up to 57% of true p53 REs. Examination of interspecific DNA base substitutions as a function of position in the p53 consensus sequence reveals an unexpected excess of diversity in apoptosis-regulating REs versus cell-cycle controlling REs (rodent comparisons: p < 1.0 e-12). While some p53 REs show relatively high levels of conservation, REs in many genes such as BAX, FAS, PCNA, CASP6, SIVA1, and P53AIP1 show little if any homology to rodent sequences. This difference suggests that among mammalian species, evolutionary conservation differs among p53 REs, with some having ancient ancestry and others of more recent origin. Overall our results reveal divergent evolutionary pressure among the binding targets of p53 and emphasize that comparative genomics methods must be used judiciously and tailored to the evolutionary history of the targeted functional regulatory regions., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Single nucleotide polymorphisms (SNPs) in transcription factor binding sites (TFBSs) may affect the binding of transcription factors, lead to differences in gene expression and phenotypes and therefore affect susceptibility to environmental exposure. We developed an integrated computational system for discovering functional SNPs in TFBSs in the human genome and predicting their impact on the expression of target genes. In this system, we (i) construct a position weight matrix (PWM) from a collection of experimentally discovered TFBSs; (ii) predict TFBSs in SNP sequences using the PWM and map SNPs to the upstream regions of genes; (iii) examine the evolutionary conservation of putative TFBSs by phylogenetic footprinting; (iv) prioritize candidate SNPs based on microarray expression profiles from tissues in which the transcription factor of interest is either deleted or over-expressed and (v) finally, analyze association of SNP genotypes with gene expression phenotypes. The application of our system has been tested to identify functional polymorphisms in the antioxidant response element (ARE), a cis-acting enhancer sequence found in the promoter region of many genes that encode antioxidant and Phase II detoxification enzymes/proteins. In response to oxidative stress, the transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) binds to AREs, mediating transcriptional activation of its responsive genes and modulating in vivo defense mechanisms against oxidative damage. Using our novel computational tools, we have identified a set of polymorphic AREs with functional evidence, showing the utility of our system to direct further experimental validation of genomic sequence variations that could be useful for identifying high-risk individuals.

Aggrecan has been implied as an autoantigen in rheumatoid arthritis (RA). Immunization with aggrecan induces arthritis in BALB/c (H-2(d)) mice but not in other strains of mice [e.g. C57BL/6 (H-2(b))]. In humans, the strongest genetic association with RA is to the shared epitope (SE), and aggrecan peptides are predicted to bind to the SE. Therefore, we hypothesized that C57BL/6 mice transgenic (tg) for the RA SE (DR4 tg mice) may be susceptible to aggrecan-induced arthritis. C57BL/6 and DR4 tg mice were immunized with a mixture of SE-binding aggrecan peptides and tested for immune responses to the corresponding peptides as well as aggrecan. Sustained T- and B-cell immune responses to aggrecan and several of its peptides were detected in DR4 tg mice. C57BL/6 mice showed only transient T-cell responses to different immunizing peptides and little B-cell response. Therefore, an immune response to peptides of aggrecan can be induced experimentally in DR4 tg mice as anticipated from the predicted and actual binding affinities of these peptides for the RA SE. Failure to induce arthritis in these DR4 tg mice may be due to a lack of appropriate non-MHC genes.

Unlabelled: The indications for magnetic resonance imaging (MRI) in suspected cauda equina syndrome, and the urgency for this investigation are regularly disputed. In this study we assess the ability of neurosurgical residents to predict on clinical grounds in which patients with cauda equina syndrome (CES) this was due to prolapsed intervertebral disc thereby justifying a request for urgent MR imaging., Design: Prospective cohort study of all adult patients with a suspected diagnosis of cauda equina syndrome., Setting: A single tertiary referral neurosurgical centre., Participants: All patients referred over a four month period with a suspected diagnosis of cauda equina syndrome., Results: MRI was normal in 10 (43%) patients. A disc prolapse causing cauda equina distortion was present in 5 (22%) patients. The diagnostic accuracy of urinary retention, urinary frequency, urinary incontinence, altered urinary sensation and altered perineal sensation were 0.57, 0.65, 0.61 ,0.65 and 0.60 respectively., Conclusions: Because it is impossible in a significant proportion of patients to exclude the diagnosis of prolapsed intervertebral disc in the context of referral with suspected cauda equina compromise the authors recommend urgent MRI assessment in all patients who present with new onset urinary symptoms in the context of lumbar back pain or sciatica.

Background: In vitro, human isoenzymes encoded by genes homozygous for the ADH1C*1 or ADH1B*2 alleles metabolize ethanol to acetaldehyde at a faster rate than those homozygous for the ADH1C*2 or ADH1B*1 allele. Because alcohol is a known risk factor for breast cancer, we evaluated the joint association of genetic variants in ADH and alcohol consumption in relation to breast cancer., Methods: A nested case-control study of 321 cases and matched controls was conducted. Five single nucleotide polymorphisms (SNPs) in the ADH1C and ADH1B genes were genotyped. Logistic regression was used to assess odds ratios (ORs) and 95% confidence limits (CIs) for each SNP. Haplotype analysis of all 5 SNPs was also undertaken., Results: Among drinkers, the median intake of total alcohol was 13 g/wk (10th-90th percentiles; 4.5-135.9) in cases and 18 g/wk (10th-90th percentiles; 4.5-104.1) in controls. Women who drank alcohol tended to be at an increased risk of developing breast cancer compared with those who did not drink (OR=1.40%, 95% CI 0.97-2.03), particularly those who were premenopausal at the time of breast cancer diagnosis (OR=2.69%, 95% CI: 1.00-7.26). Of the known functional alleles, breast cancer risk was not significantly increased among carriers of at least 1 ADH1C*1 or ADH1B*2 allele, when compared with those homozygous for the genotype at each locus. However, breast cancer risk tended to be lower among women who inherited the G allele at ADH1B IVS1+896A>G (OR=0.62, 95% CI 0.37-1.04). Overall haplotype frequencies were not significantly different between cases and controls., Conclusions: In this study low levels of alcohol are associated with a modest increase in breast cancer risk that is not altered by known functional allelic variants of the ADH1B and 1C gene. The protective association conferred by the G allele at ADH1B IVS1+896A>G needs further evaluation.

Objective: Cyclooxygenase-1 (COX-1, PTGS1) catalyzes the conversion of arachidonic acid to prostaglandin H2, which is subsequently metabolized to various biologically active prostaglandins. We sought to identify and characterize the functional relevance of genetic polymorphisms in PTGS1., Methods: Sequence variations in human PTGS1 were identified by resequencing 92 healthy individuals (24 African, 24 Asian, 24 European/Caucasian, and 20 anonymous). Using site-directed mutagenesis and a baculovirus/insect cell expression system, recombinant wild-type COX-1 and the R8W, P17L, R53H, R78W, K185T, G230S, L237M, and V481I variant proteins were expressed. COX-1 metabolic activity was evaluated in vitro using an oxygen consumption assay under basal conditions and in the presence of indomethacin., Results: Forty-five variants were identified, including seven nonsynonymous polymorphisms encoding amino acid substitutions in the COX-1 protein. The R53H (35+/-5%), R78W (36+/-4%), K185T (59+/-6%), G230S (57+/-4%), and L237M (51+/-3%) variant proteins had significantly lower metabolic activity relative to wild-type (100+/-7%), while no significant differences were observed with the R8W (104+/-10%), P17L (113+/-7%), and V481I (121+/-10%) variants. Inhibition studies with indomethacin demonstrated that the P17L and G230S variants had significantly lower IC50 values compared to wild-type, suggesting these variants significantly increase COX-1 sensitivity to indomethacin inhibition. Consistent with the metabolic activity data, protein modeling suggested the G230S variant may disrupt the active conformation of COX-1., Conclusions: Our findings demonstrate that several genetic variants in human COX-1 significantly alter basal COX-1-mediated arachidonic acid metabolism and indomethacin-mediated inhibition of COX-1 activity in vitro. Future studies characterizing the functional impact of these variants in vivo are warranted.

Objective: Autoantibodies that bind citrullinated antigens are a sensitive and specific marker for rheumatoid arthritis (RA). While synthetic cyclic citrullinated peptides (CCP) are typically used to identify these antibodies, little is known about antibody reactivity to the predominant citrullinated protein found in the inflamed synovium, citrullinated fibrinogen (CitFib). We assessed the prevalence of anti-CitFib antibodies in patients with various rheumatic diseases., Methods: In total, 65 patients with established RA and 63 patients with other rheumatic diseases were tested for serum IgM rheumatoid factor (RF), IgG anti-CCP2, and IgG anti-CitFib antibodies. This cohort was used to determine optimal positive cutoff values for antibody reactivity to CitFib through receiver operating characteristic curve analysis. The specificity of these assays was confirmed with sera from 49 patients with psoriatic arthritis., Results: Antibodies to both citrullinated antigens were identified in the majority of RA patients tested. The overall sensitivity and specificity of the assays were: CCP 82%, 96%, CitFib 75%, 98%, and IgM RF 80%, 64%, respectively. All but one patient that was positive for CitFib was also positive for CCP2, and close to half the RF-negative RA patients were positive for CitFib and CCP2., Conclusion: These results suggest that autoimmunity to CitFib is common in patients with RA and may play a role in disease pathogenesis.

Background: Psammoma bodies in cervicovaginal cytology specimens are associated with malignant and benign conditions. Few studies have evaluated which features distinguish patients with underlying malignancy from those with benign conditions., Methods: Pathology files were searched for cervicovaginal specimens having psammoma bodies. The cytology specimen was assessed for the background, glandular atypia, squamous atypia, and presence of non-psammomatous calcifications. Clinical data was obtained from chart review., Results: Nineteen women (mean age 42.7 years) had benign outcomes. None had signs or symptoms suggesting malignancy. None had highly atypical or malignant appearing glandular cells. Twelve women had malignant neoplasms (mean age 56 years), including 6 with recurrent disease. Four women without prior malignancy had worrisome signs including bleeding or mass. All six women with prior malignancy had signs of recurrent disease. All specimens contained highly atypical or malignant glandular cells., Conclusions: The only cytologic feature predictive of outcome was the presence of highly atypical glandular cells in the specimen (P = 0.001), but these cells may be few. Women with underlying malignancy were older than those with benign outcome (P = 0.014) and more likely to be postmenopausal (P = 0.05). Women with malignancy had signs that warranted additional investigation whereas those with benign outcome were usually asymptomatic (P = 0.001).

Background: DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have been developed for evaluating the significance of the observed differences in gene expression. However, until now little attention has been given to the characterization of dispersion of DNA microarray data., Results: Here we examine the expression data obtained from 682 Affymetrix GeneChips with 22 different types and we demonstrate that the Gaussian (normal) frequency distribution is characteristic for the variability of gene expression values. However, typically 5 to 15% of the samples deviate from normality. Furthermore, it is shown that the frequency distributions of the difference of expression in subsets of ordered, consecutive pairs of genes (consecutive samples) in pair-wise comparisons of replicate experiments are also normal. We describe a consecutive sampling method, which is employed to calculate the characteristic function approximating standard deviation and show that the standard deviation derived from the consecutive samples is equivalent to the standard deviation obtained from individual genes. Finally, we determine the boundaries of probability intervals and demonstrate that the coefficients defining the intervals are independent of sample characteristics, variability of data, laboratory conditions and type of chips. These coefficients are very closely correlated with Student's t-distribution., Conclusion: In this study we ascertained that the non-systematic variations possess Gaussian distribution, determined the probability intervals and demonstrated that the K(alpha) coefficients defining these intervals are invariant; these coefficients offer a convenient universal measure of dispersion of data. The fact that the K(alpha) distributions are so close to t-distribution and independent of conditions and type of arrays suggests that the quantitative data provided by Affymetrix technology give "true" representation of physical processes, involved in measurement of RNA abundance., Reviewers: This article was reviewed by Yoav Gilad (nominated by Doron Lancet), Sach Mukherjee (nominated by Sandrine Dudoit) and Amir Niknejad and Shmuel Friedland (nominated by Neil Smalheiser).

Purpose: One of the major obstacles in the treatment of ovarian cancer is the development of multidrug resistance. Recent evidence shows that high-grade ovarian cancer often shows activation of the signal transducers and activators of transcription 3 (Stat3) pathway with subsequent transcription of genes that support tumor growth and survival. Less studied is the role of the Stat3 pathway in acquired drug resistance. There is no information on Stat3 expression in chemotherapy naïve ovarian cancer as compared with tumors collected later in the natural history of the disease. To further clarify the significance of Stat3 activation in ovarian cancer, here we investigated the Stat3 expression and activation in ovarian cancer and ovarian cancer multidrug resistance cell lines., Experimental Design: Western blotting, electrophoretic mobility shift assay, luciferase assays, ELISA assay, and real-time reverse transcription-PCR determined interleukin-6 and Stat3 pathway expression and activation in cell lines. Stat3 expression in ovarian cancer tissue microarray was evaluated by immunohistochemistry., Results: Activated (phosphorylated) Stat3 is overexpressed in most paclitaxel-resistant ovarian cancer cells. Inhibition of Stat3 activation results in significant decreases in paclitaxel resistance and enhanced apoptosis. Drug-resistant recurrent tumors have significantly greater phosphorylated Stat3 (pStat3) expression as compared with matched primary tumors. Tumors with associated inflammatory cell infiltrates also have a higher proportion of cells staining intensely for nuclear phosphorylated Stat3 as compared with tumors without inflammatory infiltrates, consistent with paracrine activation of the Stat3 pathway by immune-mediated cytokines., Conclusions: These data support the hypothesis that interruption of Stat3 signaling could reverse resistance to paclitaxel and perhaps other chemotherapy agents in human cancer.

Background: Gene expression microarray technology continues to evolve and its use has expanded into all areas of biology. However, the high dimensionality of the data makes analysis a difficult challenge. Evaluating measurements and estimating the significance of the observed differences among samples remain important issues that must be addressed for each technology platform. In this work we use a consecutive sampling method to characterize the dispersion patterns of data generated from Illumina fiberoptic bead-based oligonucleotide arrays., Results: To describe general properties of the dispersion we used a linear function SD = a + bY(mean), approximating the standard deviation across arrays (Y(mean) is the mean expression of a given consecutive sample). First we examined three levels of variability: 1) same cell culture, same reverse transcription, duplicate hybridizations; 2) same cell culture, reverse transcription replicates; 3) parallel cultures. Each higher level is expected to introduce a new source of variability. We observed minor differences in the constant term: the mean values are 3.5, 3.1 and 3.5, respectively. However, the mean coefficient b increased from 0.045 to 0.147 and 0.133. We compared the coefficients derived from the consecutive sampling to those obtained from the standard deviation of individual gene expressions and found them in good agreement. In the second experiment samples we detected 11 genes with systematically different expressions between the experiment samples treated with glucose oxidase and controls and corroborated the selection using the Mann-Whitney and other tests. We also compared the consecutive sampling and coincidence method to t-test: the average percentage of consistency was above 80 for the former and below 50 for the latter., Conclusion: Our results indicate that the consecutive sampling method and standard deviation function provide a convenient description of the overall dispersion of Illumina arrays. We observed that the constant term of the standard deviation function is at average approximately the same for duplicate hybridization as for the assays with additional sources of variability. Furthermore, among the genes affected by glucose oxidase treatment we identified 6 genes in oxidative stress pathways and 5 genes involved in DNA repair. Finally, we noted that the consecutive sampling and coincidence test provide, under given conditions, more consistent results than the t-test., Reviewers: This article was reviewed by Alexander Karpikov (nominated by Mark Gerstein), Jordan King and Eugene V. Koonin.

The clinical and pathologic features of serous borderline tumors (SBTs) with autoimplants (AIs) were studied with emphasis on their relation to survival, ovarian SBT with a micropapillary pattern, and their distinction from serous adenocarcinoma. The 30 patients ranged in age from 17 to 70 years (mean, 35.8 years). Two had stage I disease, 10 had stage II disease, and 18 had stage III disease. Twenty-five patients had bilateral ovarian tumors. In 28 cases, AIs were present on the surface of the neoplasm or between exophytic surface tumor papillae; in 2 cases, AIs were between papillae within intracystic tumor. The AIs consisted of single cells or glands and clusters of cells with mild to moderate nuclear atypia within a fibroblastic stroma; the stroma dominated over the epithelium in extent within the AI. In many cases, the epithelial cells in the AI had abundant eosinophilic cytoplasm. All but 2 of the patients had coexisting noninvasive peritoneal implants; 3 of them also had invasive peritoneal implants. Six of the SBTs had a micropapillary pattern; 3 of these were stage III and 3 stage II tumors. Clinical follow-up was available for 11 patients. Eight were alive and well after 4 to 7 years (mean, 5 years); 3 of these patients had stage II disease and 5 had stage III disease. Three were dead of disease after 7 to 12 years (mean, 9 years). Two patients who died of disease had stage III disease, and 1 of them had invasive implants. The third patient had stage II disease, invasive implants, and the tumor was of the micropapillary type. Our study indicates that the majority of patients with SBT with AI have stage II or III disease and abundant exophytic tumor. SBTs with AI had a micropapillary pattern in 20% of the cases. AIs do not appear to have an adverse impact on survival when controlled for peritoneal implant type and for this reason must be distinguished from true stromal invasion in serous carcinoma, a misdiagnosis sometimes made, or seriously entertained, initially in these cases. Features that favor AI over carcinoma arising in an SBT are 1) a location between tumor papillae or on the ovarian surface 2) a predominance of stroma over epithelial cells, and 3) the "borderline" cytologic appearance of epithelial cells.

Genes involved in phase I and phase II regulation of aromatic hydrocarbon-induced effects exhibit sequence variability that may mediate the risk of adult brain tumors. We evaluated associations between gene variants in CYP1A1, CYP1B1, GSTM3, EPHX1, and NQO1 and adult brain tumor incidence. Cases were patients with glioma (n = 489), meningioma (n = 197), or acoustic neuroma (n = 96) diagnosed from 1994 to 1998 at three U.S. hospitals. Controls were 799 patients admitted to the same hospitals for nonmalignant conditions. DNA was extracted from blood samples collected from 1277 subjects, and genotyping was conducted for CYP1A1 I462V, CYP1B1 V432L, EPHX1 Y113H, GSTM3 *A/*B (intron 6 deletion), and NQO1 P187S. The CYP1B1 V432L homozygous variant was associated with decreased risk of meningioma (odds ratio [OR] = 0.6; 95% CI, 0.3-1.0) but not the other tumor types. The GSTM3 *B/*B genotype was associated with increased risk of glioma (OR = 2.3; 95% CI, 1.0-5.2) and meningioma (OR = 3.6; 95% CI, 1.3-9.8). Increased risks associated with GSTM3 *B/*B were observed in younger subjects (age < 50) and older subjects (age > or = 50), in men and women, and within each study site. The magnitude of association for GSTM3 with glioma and meningioma was greater among ever-smokers than among those who had never smoked. None of the other genotypes showed consistent associations with any tumor type. The association with the GSTM3 *B allele, while intriguing, requires replication, and additional research is needed to clarify the function of the GSTM3 alleles studied here.

This study expands a previous study of NAT2 polymorphisms and bladder cancer in male subjects occupationally exposed only to benzidine. The combined analysis of 68 cases and 107 controls from a cohort of production workers in China exposed to benzidine included 30 new cases and 67 controls not previously studied. NAT2 enzymatic activity phenotype was characterized by measuring urinary caffeine metabolite ratios. PCR-based methods identified genotypes for NAT2, NAT1 and GSTM1. NAT2 phenotype and genotype data were consistent. A protective association was observed for the slow NAT2 genotype (bladder cancer OR = 0.3; 95% CI = 0.1 = 1.0) after adjustment for cumulative benzidine exposure and lifetime smoking. Individuals carrying NAT1wt/*10 and NAT1*10/*10 showed higher relative risks of bladder cancer (OR = 2.8, 95% CI = 0.8-10.1 and OR = 2.2, 95% CI = 0.6-8.3, respectively). No association was found between GSTM1 null and bladder cancer. A metaanalysis risk estimate of case-control studies of NAT2 acetylation and bladder cancer in Asian populations without occupational arylamine exposures showed an increased risk for slow acetylators. The lower limit of the confidence interval (OR = 1.4; 95% CI = 1.0-2.0) approximated the upper confidence interval for the estimate obtained in our analysis. These results support the earlier finding of a protective association between slow acetylation and bladder cancer in benzidine-exposed workers, in contrast to its established link as a risk factor for bladder cancer in people exposed to 2-naphthylamine and 4-aminobiphenyl. Study findings suggest the existence of key differences in the metabolism of mono- and diarylamines., (Published 2005 Wiley-Liss, Inc.)