OXR1A, a Coactivator of PRMT5 Regulating Histone Arginine Methylation

Oxidation resistance gene 1 (OXR1) protects cells against oxidative stress. We find that male mice with brain-specific isoform A knockout (Oxr1A−/−) develop fatty liver. RNA sequencing of male Oxr1A−/− liver indicates decreased growth hormone (GH) signaling, which is known to affect liver metabolism. Indeed, Gh expression is reduced in male mice Oxr1A−/− pituitary gland and in rat Oxr1A−/− pituitary adenoma cell-line GH3. Oxr1A−/− male mice show reduced fasting-blood GH levels. Pull-down and proximity ligation assays reveal that OXR1A is associated with arginine methyl transferase PRMT5. OXR1A-depleted GH3 cells show reduced symmetrical dimethylation of histone H3 arginine 2 (H3R2me2s), a product of PRMT5 catalyzed methylation, and chromatin immunoprecipitation (ChIP) of H3R2me2s shows reduced Gh promoter enrichment. Finally, we demonstrate with purified proteins that OXR1A stimulates PRMT5/MEP50-catalyzed H3R2me2s. Our data suggest that OXR1A is a coactivator of PRMT5, regulating histone arginine methylation and thereby GH production within the pituitary gland.publishedVersio

Exploratory Analysis of CA125-MGL and –STn Glycoforms in the Differential Diagnostics of Pelvic Masses

BackgroundThe cancer antigen 125 (CA125) immunoassay (IA) does not distinguish epithelial ovarian cancer (EOC) from benign disease with the sensitivity needed in clinical practice. In recent studies, glycoforms of CA125 have shown potential as biomarkers in EOC. Here, we assessed the diagnostic abilities of two recently developed CA125 glycoform assays for patients with a pelvic mass. Detailed analysis was further conducted for postmenopausal patients with marginally elevated conventionally measured CA125 levels, as this subgroup presents a diagnostic challenge in the clinical setting.MethodsOur study population contained 549 patients diagnosed with EOC, benign ovarian tumors, and endometriosis. Of these, 288 patients were postmenopausal, and 98 of them presented with marginally elevated serum levels of conventionally measured CA125 at diagnosis. Preoperative serum levels of conventionally measured CA125 and its glycoforms (CA125-MGL and CA125-STn) were determined.ResultsThe CA125-STn assay identified EOC significantly better than the conventional CA125-IA in postmenopausal patients (85% vs. 74% sensitivity at a fixed specificity of 90%, P = 0.0009). Further, both glycoform assays had superior AUCs compared to the conventional CA125-IA in postmenopausal patients with marginally elevated CA125. Importantly, the glycoform assays reduced the false positive rate of the conventional CA125-IA.ConclusionsThe results indicate that the CA125 glycoform assays markedly improve the performance of the conventional CA125-IA in the differential diagnosis of pelvic masses. This result is especially valuable when CA125 is marginally elevated.</div

Human epididymis protein 4 reference limits and natural variation in a Nordic reference population

The objectives of this study are to establish reference limits for human epididymis protein 4, HE4, and investigate factors influencing HE4 levels in healthy subjects. HE4 was measured in 1,591 samples from the Nordic Reference Interval Project Bio-bank and Database biobank, using the manual HE4 EIA (Fujirebio) for 802 samples and the Architect HE4 (Abbott) for 792 samples. Reference limits were calculated using the statistical software R. The influence of donor characteristics such as age, sex, body mass index, smoking habits, and creatinine on HE4 levels was investigated using a multivariate model. The study showed that age is the main determinant of HE4 in healthy subjects, corresponding to 2% higher HE4 levels at 30 years (compared to 20 years), 9% at 40 years, 20% at 50 years, 37% at 60 years, 63% at 70 years, and 101% at 80 years. HE4 levels are 29% higher in smokers than in nonsmokers. In conclusion, HE4 levels in healthy subjects are associated with age and smoking status. Age-dependent reference limits are suggested

Heterophilic Antibody Interference in Immunometric Assays

Monoclonal and polyclonal antibodies from animals are used in immunological assays to measure concentrations of many clinically relevant proteins, peptides and drugs in blood samples. Heterophilic antibodies are immunoglobulins (present in some blood samples) that bind to the animal antibodies used in immunoassays. This type of assay interference is usually called heterophilic antibody interference. In some cases, false results caused by such interference may go undetected and lead to unnecessary diagnostic and potentially harmful therapeutic interventions, and immunoassay companies invest substantial resources to make their assays resilient to antibody interference. Heterophilic antibodies are found in the blood of both patients and healthy individuals, and are only rarely associated with prior exposure to animal antibodies. While some patient groups have an increased risk of antibody-mediated interference, particularly patients with seropositive rheumatoid arthritis, it is difficult to suspect assay interference before it appears. Bolstad and colleagues tested how resistant 170 commercially available immunoassay kits were to heterophilic antibody interference, and found that 21 assays lacked adequate protection against Fc-reactive heterophilic antibodies, which is the most common reactivity associated with heterophilic antibodies. Since the results were published, several of these vulnerable assays have been relaunched with improved protection against heterophilic antibody interference. Continued focus was put on improving execution and interpretation of tests to detect interference, but also to develop strategies to limit the damage from heterophilic antibodies relevant to both clinical laboratories and immunoassay developers. Finally, the prevalence of heterophilic antibodies in patient samples was examined by screening more than 5000 blood samples in an in house assay designed to detect reactivities to eight different animal antibodies used in immunoassays. Candidate samples identified in the screening assay were subsequently tested in a range of individual assays to identify the individual reactivities in each sample. Heterophilic antibodies with reactivity to rabbit IgG (5.5 %) and murine IgG1 (4.8 %) were the most common, while reactivity to murine IgG2a (0.1 %), murine IgG2b (0.6 %), goat and sheep IgG (both 0.2 %) was rare. When specific blocking of the commonly occurring anti-bovine antibodies was lifted, vast cross-reactivity to sheep and goat IgG was evident. These findings should enable immunoassay developers to further improve protection against heterophilic antibodies in immunoassays, thereby reducing the risk of unnecessary harm to patients

Expression of Ogg1 and detection of 8-oxoG DNA glycosylase activity in neural progenitors

ABSTRACT Normal cellular metabolism generates reactive oxygen species which interact with DNA, lipids and proteins in cells. Cellular damage due to oxidative stress is proposed to contribute to the pathophysiology of cancer, neurodegenerative diseases including Alzheimer s and Parkinson s and to the process of aging. The central nervous system is thought to be particularly susceptible to oxidative stress due to the high rate of oxygen consumption. Base excision DNA repair (BER) is the major pathway that removes oxidative DNA base lesions. Among hundreds of lesions, 7,8-dihydro-8-oxoguanine (8-oxoG) is believed to be one of the most important oxidized lesions due to their relatively high incidence and their miscoding properties.OGG1 (8-oxoguanine DNA glycosylase-1) is one of the main DNA glycosylases present in mammalian cells that removes 7,8-dihydro-8-oxoguanine (8-oxoG) lesions. In this study we have investigated 8-oxoG repair capacity of neurospheres derived from newborn and adult mice. We show that Ogg1 is the major glycosylase initiating 8-oxoG repair. Moreover 8-oxoG activity decrases with age and upon cell differentiation

Determination of lower cut-off levels of adalimumab associated with biochemical remission in Crohn's disease

Background and Aim: Adalimumab is administered and dosed using a standardized treatment regimen. Although therapeutic drug monitoring (TDM) may help optimize treatment efficacy, the lower cut-off concentration of adalimumab needed to retain disease remission has not been established. This cross-sectional study of patients with Crohn’s disease on stable medication aimed to determine a lower therapeutic drug concentration threshold of adalimumab associated with biochemical disease remission. Methods: C-reactive protein (CRP) and fecal calprotectin were used as established markers and albumin as an explorative marker of disease activity. Time since introduction, treatment interval, drug dosage, serum drug concentration and antidrug antibodies, disease duration, age, and sex were recorded. Results: The study included 101 patients who were divided into “active disease” and “remission” groups for inflammatory markers based on cut-off levels of 5 mg/L for CRP and 50 mg/kg for fecal calprotectin. Cut-off levels for albumin of 36.5 and 41.5 g/L were also added as further indicatives of remission. Receiver operating characteristic analysis found optimal thresholds for adalimumab associated with remission at 6.8–7.0 mg/L for the combination of CRP and fecal calprotectin and when combining CRP, fecal calprotectin, and albumin. Conclusions: In patients with Crohn’s disease, serum adalimumab of at least 6.8 mg/L was associated with biochemical disease remission based on CRP and fecal calprotectin, supporting the use of TDM to ensure disease control. Albumin should be further tested in this setting.publishedVersio

Serum etanercept concentrations in relation to disease activity and treatment response assessed by ultrasound, biomarkers and clinical disease activity scores: results from a prospective observational study of patients with rheumatoid arthritis.

Objectives: To identify the therapeutic range for etanercept and to assess the incidence of anti-etanercept antibody formation. Methods: Associations between etanercept serum concentration and disease activity as well as treatment response were examined in a longitudinal observational study of rheumatoid arthritis patients starting etanercept. Disease activity was assessed by ultrasound (grey scale and power Doppler), 28-joint Disease Activity Score (DAS28), Simplified Disease Activity Index, plasma calprotectin and C reactive protein. Etanercept concentration and anti-etanercept antibodies were analysed using automated in-house fluorescence assays. Results: A total of 89 patients were included, whereof 66% were biological disease-modifying antirheumatic drug (DMARD) naïve and 91% used concomitant synthetic DMARD. At 3 months, the median etanercept concentration was 1.8 (IQR 1.1-2.5) mg/L. Longitudinal associations were found between etanercept concentration and disease activity assessed by plasma calprotectin, C reactive protein and DAS28, but not between etanercept concentration and improvement in disease activity by any of the parameters at 3, 6 or 12 months of treatment. Etanercept concentrations were not significantly different among patients who achieved response or remission, compared with non-response or non-remission. Hence, no therapeutic range could be identified. None of the patients developed anti-etanercept antibodies. Conclusion: Despite the use of sensitive and objective markers of inflammation, a therapeutic range could not be identified for etanercept. Hence, this study suggests that proactive therapeutic drug monitoring is unlikely to benefit rheumatoid arthritis patients treated with etanercept, but a potential benefit in certain clinical situations cannot be excluded