Cognitive health after menopause: Does menopausal hormone therapy affect it?

Dementia is a pandemic chronic non-communicable disease. 10 in 100 women above age 65 will be diagnosed with dementia, primarily Alzheimer's disease (AD). Apart from age and hereditary risk factors, there are multiple acquired risk and protective factors. So far, Menopausal Hormone Therapy (MHT) is not recommended as preventive measure. A systematic literature search on MHT and dementia risk and MHT and cognitive performance in demented women, respectively, was performed. Two recent meta-analyses identified 18 and 16 studies analyzing the impact of MHT on dementia/AD risk. Our systematic literature search identified eight additional original articles. The majority of studies found a risk reducing impact of MHT by 11-33%. However, results may vary depending on MHT type, age at initiation and study design. For example, the Women's Health Initiative Memory Study (WHIMS) reported an approximately 2-fold increased risk of dementia/Alzheimer's disease if MHT comprising conjugated equine estrogens and medroxyprogesterone acetate was initiated in predominantly comorbid postmenopausal women above age 65. In general, MHT displays a beneficial effect on several dementia risk factors and also augments some protective factors. Accordingly, clinicians can be reassured that MHT can be safely prescribed in the context of cognition in women free of dementia. However, MHT is not indicated for cognitive improvement in demented women. International scientific guidelines on MHT and dementia should consider incorporating most recent data.

E3 ubiquitin ligase Smurf2: a prognostic factor in microsatellite stable colorectal cancer.

PURPOSE: Smurf2 is a member of the homologous to E6-AP carboxyl terminus family of E3 ubiquitin ligases. Changes in their expression pattern are known to contribute to tumorigenesis. Smurf2 plays a decisive role in cell differentiation, proliferation, and migration and exhibits a dual role in cancer - functioning as both oncogene and tumor suppressor. Dysregulation of Smurf2 in different cancer types has been described, besides colorectal cancer (CRC). We therefore examined the expression and oncogenic potential of Smurf2 in human CRC patients. MATERIALS AND METHODS: Expression levels of Smurf2 were analyzed via qRT-PCR in CRC specimens and healthy mucosa from 98 patients who had undergone surgery due to CRC. Spatial expression of Smurf2 was additionally studied by immunohistochemistry. siRNA-mediated knockdown of Smurf2 was applied for migration and invasion assays in DLD-1 and SW-480 cells. RESULTS: Smurf2 was significantly overexpressed in CRC tissue compared to corresponding healthy colon mucosa. Smurf2 expression levels differed significantly between microsatellite instable (MSI) and microsatellite stable (MSS) CRC. In patients suffering from MSS CRC, high tumoral expression of Smurf2 was significantly associated with impaired overall survival. Consistently, in vitro analysis revealed that knockdown of Smurf2 reduced the invasive and migratory potential of MSS CRC cells. CONCLUSION: Smurf2 expression is upregulated in CRC specimens and affects survival dependent on patients' MSI status. Moreover, Smurf2 supports cancer cell migration and invasion, collectively suggesting an oncogenic function in CRC.

Determination of direct oral anticoagulants from human serum samples.

Dabigatran, rivaroxaban, and apixaban are direct oral anticoagulants (DOAC) inhibiting thrombin or factor Xa and effectively preventing thromboembolic complications using fixed doses without need for laboratory-guided dose adjustment. Plasma samples are needed to determine the actual concentration or activity of DOACs, which may be required for special patient populations such as those with acute deterioration of renal function due to any disease, before surgical interventions, during bleeding or thrombotic episodes while on therapy with DOACs, the elderly and youngest populations, unexpected pregnancy, suspicion of overdose and toxication, and to control adherence to therapy. Serum samples have several advantages over plasma samples such as no need of sampling with a specific coagulation tube, reduced pre-analytical errors, and longer storage stability. Determination of rivaroxaban and apixaban from serum samples of patients on treatment performed well and better than samples of patients treated with dabigatran compared with plasma samples. Specific adaption to automated coagulation platforms may improve the performance of the assays from serum samples.

Novel methods for assessing oral direct factor Xa and thrombin inhibitors: use of point-of-care testing and urine samples.

Rivaroxaban and dabigatran are new oral anticoagulants (NOACs) that inhibit directly factor Xa and thrombin, respectively. These NOACs effectively prevent thromboembolic complications using fixed doses without the need for dose adjustment according to laboratory results. About 60% of rivaroxaban is cleared from circulation by glomerular filtration, 30% of which is excreted as active drug. About 80% of dabigatran is excreted into urine as active compound. Accordingly, both NOACs can be determined in urine by means of chromatographic methods. Only a few laboratories are able to perform such methods, and results are not available within short time frames. New methods have to be developed to obtain results within minutes and possibly as point-of-care (POC) techniques. This testing may be useful for special patient populations such as those with acute deterioration of renal function due to any disease, before surgical interventions, during unexpected bleeding or thrombotic episodes while on therapy with NOACs, the oldest and youngest populations, pregnancy, suspicion of overdose and intoxication, and to determine adherence to therapy. Here we describe results of a POC qualitative assay using urine samples from patients on treatment with dabigatran and rivaroxaban.

The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity.

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.

Determination of dabigatran in human plasma samples.

The oral direct thrombin inhibitor dabigatran effectively prevents arterial and venous thromboembolism using fixed doses without the need for adjustment according to laboratory results. Dabigatran is eliminated from the circulation by ∼80% through the kidneys. However, the in vitro anticoagulant effect of dabigatran may be necessary to determine in special patient populations such as in the elderly, for renal impairment, before operations, bleeding or thrombotic episodes, and to monitor self-compliance. Several clotting and thrombin-specific chromogenic substrate assays are available to analyze the biological activity of dabigatran. All of them are prolonged in the presence of dabigatran. This article reports the effects of dabigatran on clinical routine assays and the potential usefulness for determination in special risk groups of patients when overdose or lack of compliance are suspected.

Determination of an international sensitivity index of thromboplastin reagents using a WHO thromboplastin as calibrator for plasma spiked with rivaroxaban.

Rivaroxaban and other direct factor Xa inhibitors are used at fixed doses without drug monitoring and dose adjustment. Patients may require determination of the anticoagulant effect during treatment. The aim of this study was to develop a method to reduce the differences between thromboplastin reagents and coagulation analysers for determination of the anticoagulant effect of rivaroxaban in human plasma. Purity of rivaroxaban extracted from commercially available drug was confirmed by mass spectrometry, elemental analysis and 1H-NMR spectroscopy. Coagulation times of pooled human plasma spiked with 50-900  ng/ml rivaroxaban were analysed. Thromboplastin reagents, WHO RBT/90, Innovin, RecombiPlasTin 2G, STA Neoplastin Plus, Technoclot PT Plus and Thromborel S, the manual Kolle-Hook method and the KC10 analyser were used. An international sensitivity index (ISI) was determined for each reagent and coagulation method using the RBT/90 thromboplastin reagent as reference. The orthogonal, used for the determination of the ISI of coumarin plasmas, and ordinary regression analyses were compared. The results showed than increasing concentrations of rivaroxaban prolonged coagulation values of all thromboplastin assays linearly (r (2)= 0.96 and r(2) = 0.99, respectively). The coefficient of variation between the slopes of the dilution curves and the ratios of the thromboplastin reagents were reduced using the international normalized ratio (INR) and ISI calculated for rivaroxaban. The ISIs of the thromboplastin reagents ranged from 0.73 to 1.67 as compared with the WHO reagent using the manual technique. The coefficient of variations between the thromboplastin reagents comparing the orthogonal and the ordinary regression analysis were 6.8 versus 3.7% (Kolle-Hook method, P = 0.0011) and 8.5 versus 4.8% (KC10 method, P < 0.0001). Using ISI for vitamin-K antagonist and rivaroxaban, the INRs for the rivaroxaban-containing samples were significantly different for one of five commercial thromboplastin reagents. In conclusion, the determination of an ISI for rivaroxaban using a WHO thromboplastin reagent is required for commercial thromboplastin reagents. The manual Kolle-Hook method and an ordinary linear regression analysis should be adopted.

Determination of rivaroxaban by different factor Xa specific chromogenic substrate assays: reduction of interassay variability.

Rivaroxaban and other oral direct factor Xa inhibitors (ODiXa) are currently developed for prophylaxis and treatment of thromboembolic diseases using fixed doses. Although routine monitoring is not required, assessing the intensity of anticoagulation may be useful under certain clinical conditions. ODiXa prolong coagulation times of several clotting assays and, thus, their concentration may be determined in factor Xa specific chromogenic substrate assays. So far, no standardized and validated assay is commercially available. Here, five methods (A through E) are studied and optimized to reduce interassay variability. Human pooled plasma was spiked by a serial dilution of rivaroxaban (25-900 ng/ml). The release of para-nitroaniline from the chromogenic substrates was measured by the optical density (OD) at 405 nm. Method B was identified to yield the lowest sum of deviations from the mean value of the OD concentration curve calculated from all assays. Spline functions were developed for OD versus concentration curves for all methods. The calculated OD versus concentration curves overlapped for all methods. The coefficient of variation for all assays and concentrations of rivaroxaban decreased from 25.3 ± 11.4% using the original data to 3.8 ± 2.2% using the calculated data (P < 0.0001). The robustness of the chromogenic assay (method B) remains to be corroborated in interlaboratory comparisons.

Determination of antithrombin-dependent factor Xa inhibitors by prothrombin-induced clotting time.

Prothrombinase-induced clotting time (PiCT) determines the anticoagulant effects of heparins, low molecular weight heparins (LMWHs), and direct thrombin inhibitors. At present, this is the only method that measures the effects of all of these inhibitors, in contrast to the prothrombin time, activated partial thromboplastin time (aPTT), Heptest, ecarin clotting time, and the chromogenic assays. The antithrombin-dependent direct factor (F) Xa inhibitors fondaparinux and idraparinux were compared with the LMWH dalteparin on PiCT, aPTT, Heptest, and chromogenic anti-FXa assays in pooled human normal plasma samples. Fondaparinux and idraparinux prolonged the coagulation times in the PiCT, Heptest, and chromogenic FXa assays in a dose-dependent manner, in contrast to the aPTT. We conclude that PiCT is a suitable assay to determine the anticoagulant effects of these two new FXa inhibitors in patients receiving treatment with these compounds.