Insulin-like growth factor signalling and its significance as a biomarker in fish and shellfish research.

The insulin-like growth factor signalling system comprises insulin-like growth factors, insulin-like growth factor receptors and insulin-like growth factor-binding proteins. Along with the growth hormones, insulin-like growth factor signalling is very pivotal in the growth and development of all vertebrates. In fishes, insulin-like growth factors play an important role in osmoregulation, besides the neuroendocrine regulation of growth. Insulin-like growth factor concentration in plasma can assess the growth in fishes and shellfishes and therefore widely applied in nutritional research as an indicator to evaluate the performance of selected nutrients. The present review summarizes the role of insulin-like growth factor signalling in fishes and shellfishes, its significance in aquaculture and in evaluating growth, reproduction and development, and discusses the utility of this system as biomarkers for early indication of growth in aquaculture.

Rivaroxaban vs warfarin in high-risk patients with antiphospholipid syndrome.

Rivaroxaban is an effective and safe alternative to warfarin in patients with atrial fibrillation and venous thromboembolism. We tested the efficacy and safety of rivaroxaban compared with warfarin in high-risk patients with thrombotic antiphospholipid syndrome. This is a randomized open-label multicenter noninferiority study with blinded end point adjudication. Rivaroxaban, 20 mg once daily (15 mg once daily based on kidney function) was compared with warfarin (international normalized ratio target 2.5) for the prevention of thromboembolic events, major bleeding, and vascular death in patients with antiphospholipid syndrome. Only high-risk patients triple positive for lupus anticoagulant, anti-cardiolipin, and anti-ß2-glycoprotein I antibodies of the same isotype (triple positivity) were included in the study. The trial was terminated prematurely after the enrollment of 120 patients (59 randomized to rivaroxaban and 61 to warfarin) because of an excess of events among patients in the rivaroxaban arm. Mean follow-up was 569 days. There were 11 (19%) events in the rivaroxaban group, and 2 (3%) events in the warfarin group. Thromboembolic events occurred in 7 (12%) patients randomized to rivaroxaban (4 ischemic stroke and 3 myocardial infarction), whereas no event was recorded in those randomized to warfarin. Major bleeding occurred in 6 patients: 4 (7%) in the rivaroxaban group and 2 (3%) in the warfarin group. No death was reported. The use of rivaroxaban in high-risk patients with antiphospholipid syndrome was associated with an increased rate of events compared with warfarin, thus showing no benefit and excess risk. This trial was registered at www.clinicaltrials.gov as #NCT02157272.

Risk factors for intracranial hemorrhage during vitamin K antagonist therapy in patients with nonvalvular atrial fibrillation: A case-control study.

BACKGROUND: Intracranial hemorrhage (ICH) is the most fearful side effect of oral anticoagulant therapy. It is still unclear which risk factor is involved in ICH during vitamin K antagonists (VKAs) treatment and if commonly used bleeding risk scores are able to predict ICH. PURPOSE: Search for individual risk factors and bleeding risk scores (HAS-BLED, ATRIA and ORBIT) associated with ICHs in patients with atrial fibrillation treated with VKAs. METHODS: This is a retrospective case-control study in a single Thrombosis Center. During a 7-year period, patients with nonvalvular atrial fibrillation (NVAF) who developed ICH during VKAs were identified as cases. Four control patients matched for gender, age and length of VKAs were assigned to each case. The association between considered risk factors and ICHs was evaluated using a linear logistic regression method and expressed as odds ratio. Receiver operator characteristic (ROC) curves to assess the predictive ability of bleeding risk scores HAS-BLED, ATRIA and ORBIT were also evaluated. RESULTS: Fifty-one cases of ICH, most of whom were 80 years of age or older (72.5%), were retrieved from the Thrombosis Center's database. Compared to 204 controls, no individual risk factors were associated with ICH. Poor ability to predict ICH was also found using ROC curves (C-statistic for HAS-BLED, ATRIA, and ORBIT were 0.55, 0.53 and 0.54, respectively). CONCLUSIONS: ICHs during VKA therapy preferentially occur in very elderly patients. The risk of ICH is not predicted by the commonly used risk scores.

Laboratory Diagnostics of Antiphospholipid Syndrome.

Diagnosis of antiphospholipid syndrome (APS) lies in the recognition of antiphospholipid antibodies (aPL). As standardization of tests for the detection of aPL is far from being optimal and reference material is not available, inappropriate diagnoses of APS are not unusual. In the last few years, the concept of triple test positivity has emerged as a useful tool to identify patients with APS. Clinical studies on patients and carriers of triple positivity clearly show that these individuals are at high risk of thromboembolic events and pregnancy loss. Moreover, triple positivity arises from a single (probably pathogenic) antibody directed to domain 1 of ß2-glycoprotein I, a protein whose function is still unknown. Studies on homogenous group of patients with single or double positivity are scant, and uncertainties arise on their association with clinical events. Promising but undetermined results come also from the determination of antibodies directed to phosphatidylserine/prothrombin complex. Interpretation of laboratory profile in APS is challenging, and the collaboration between clinical pathologists and clinicians is highly desirable.

Warfarin prescription in patients with nonvalvular atrial fibrillation and one non-gender-related risk factor (CHA2 DS2 VASc 1 or 2): A treatment dilemma.

INTRODUCTION: The issue of anticoagulation in individuals with nonvalvular atrial fibrillation (NVAF) and 1 non-gender-related (NGR) risk factor is subject to debate. The reported risk of stroke in untreated individuals is not uniform, and the rate of hemorrhage associated with anticoagulation in this group of individuals is not well defined. To this end, we assessed the rate of stroke and major hemorrhage in individuals treated with warfarin. MATERIALS AND METHODS: individuals were extracted from the START register, an observational, multicenter, dynamic inception cohort study that collects data on NVAF individuals starting anticoagulation therapy. Risk of stroke is stratified using the CHA2 DS2 VASc score upon entry into the registry. RESULTS: Overall, 431 individuals with 1 NGR risk factor were followed up for 604 person-years. One nonfatal ischemic stroke was recorded (0.17 per 100 person-years) during follow-up. On the other hand, there were 9 major bleeding events (1.49 per 100 person-years), with 4 being intracranial hemorrhage (0.66 per 100 person-years), 1 of which was fatal. No difference in patient characteristics, bleeding risk factors, and quality of treatment were found between individuals who bled versus those who did not. However, a trend toward more bleeding events was observed in individuals <65 years old. CONCLUSION: We found an elevated risk of major bleeding and intracranial hemorrhage in NVAF individuals treated with warfarin with 1 NGR risk factor for stroke. These data call for caution when treating with warfarin these individuals.

Effectiveness and safety of oral anticoagulation with non-vitamin K antagonists compared to well-managed vitamin K antagonists in naïve patients with non-valvular atrial fibrillation: Propensity score matched cohort study.

BACKGROUND: The global real-life impact of non-vitamin K antagonist oral anticoagulants (NOACs) introduction in the healthcare system in a setting of well-managed vitamin K antagonist (VKA) therapy has not been specifically addressed. METHODS: We did a population-based retrospective cohort study in naïve patients initiating oral anticoagulants for stroke prevention in atrial fibrillation in a region with a well-managed VKA therapy. NOAC and VKA cohorts were identified using Anatomical Therapeutic Chemical (ATC) codes, while excluding other indications for anticoagulation therapy using ICD-9CM codes. Propensity score was conducted using two different approaches: stratification and 1:1 matching. Event-rates were assessed using both an intention to treat (ITT) and as treated analyses. RESULTS: Of the 137,800 selected patients, 40,411 (6923 treated with NOACs and 33,488 with VKAs) were identified (June 2013-December 2015). Overall ischaemic stroke and major bleeding risk did not significantly differ between the groups both in the ITT and as treated analyses. Noteworthy, intracranial bleeding risk was lower with NOACs (stratified model HR=0.69; 95%CI 0.48-0.99; 1:1 matched model HR=0.73; 95%CI 0.47-1.13) reaching statistical significance in the as treated analysis in both stratified and 1:1 matched models (HR=0.51; 95%CI 0.32-0.80 and HR=0.52; 95%CI 0.30-0.90, respectively). CONCLUSION: Despite well-managed anticoagulation with VKAs, NOACs' introduction has a positive global impact in the public healthcare system in terms of effectiveness and safety especially by lowering intracranial bleeding.

Lupus Anticoagulant Testing: Diluted Russell Viper Venom Time (dRVVT).

Diluted Russell Viper Venom Time (dRVVT) has become the most popular test to detect Lupus Anticoagulant (LA). dRVVT is more sensitive than other global tests employed to detect LA and is not affected by inhibitors of factor VIII or IX. The test is most successfully implemented if you observe three steps in its execution: screening, mixing, and confirmatory studies. Interference due to the presence of heparin in tested plasma must be excluded by means of thrombin time (TT). The prior use of Vitamin K Antagonists (VKAs) or Non-vitamin K Oral Anticoagulants (NOACs) must also be evaluated by means of International Normalized Ratio, or specific tests, respectively.

Regulating gonad inhibition and vitellogenin/vitellin induction in Penaeus monodon using mature GIH fusion protein and polyclonal antisera.

Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed.

A Randomized Trial of Pharmacogenetic Warfarin Dosing in Naïve Patients with Non-Valvular Atrial Fibrillation.

UNLABELLED: Genotype-guided warfarin dosing have been proposed to improve patient's management. This study is aimed to determine whether a CYP2C9- VKORC1- CYP4F2-based pharmacogenetic algorithm is superior to a standard, clinically adopted, pharmacodynamic method. Two-hundred naïve patients with non-valvular atrial fibrillation were randomized to trial arms and 180 completed the study. No significant differences were found in the number of out-of-range INRs (INR<2.0 or >3.0) (p = 0.79) and in the mean percentage of time spent in the therapeutic range (TTR) after 19 days in the pharmacogenetic (51.9%) and in the control arm (53.2%, p = 0.71). The percentage of time spent at INR>4.0 was significantly lower in the pharmacogenetic (0.7%) than in the control arm (1.8%) (p = 0.02). Genotype-guided warfarin dosing is not superior in overall anticoagulation control when compared to accurate clinical standard of care. TRIAL REGISTRATION: ClinicalTrials.gov NCT01178034.

Cellular and molecular markers in monitoring the fate of lymphoid cell culture from Penaeus monodon Fabricius (1798).

Lymphoid cell culture from penaeid shrimps has gained much acceptance as an in vitro platform to facilitate research on the development of prophylaxis, and therapeutic strategies against viruses and for cell line development. However, lymphoid cells can be used as platform for in vitro research, only if they are in metabolically and mitotically active state in vitro with unaltered cell surface receptors. Through this study, we addressed the response of lymphoid cells to a new microenvironment at cellular and molecular levels; including the study of mitotic events, DNA synthesis, expression profile of cell cycle genes, cytoskeleton organization, metabolic activity and viral susceptibility. The S-phase entry and synthesis of new DNA was recorded by immunoflourescent technique. Cdc2, CycA, CycB, EF-1α and BUB3 genes involved in cell cycle were studied in both the cells and tissue, of which EF-1α showed an elevated expression in cells in vitro (∼ 19.7%). Cytoskeleton network of the cell was examined by studying the organization of actin filaments. As the markers for metabolic status, mitochondrial dehydrogenase, protein synthesis and glucose assimilation by the cells were also assessed. Viral susceptibility of the cell was determined using WSSV to confirm the preservation of cellular receptors. This study envisages to strengthen the shrimp cell line research and to bring forth lymphoid cell culture system as a 'model' in vitro system for shrimp and crustaceans altogether.

Minimizing the risk of hemorrhagic stroke during anticoagulant therapy for atrial fibrillation.

INTRODUCTION: Oral anticoagulation (OAC) is given for ischemic stroke prevention in patients with nonvalvular atrial fibrillation. OAC's most serious complications are major bleeding and, in particular, hemorrhagic stroke. Together with vitamin K antagonists (VKAs), direct oral anticoagulants (DOAC) are now available which have a more rapid onset/offset of action and more predictable anticoagulant effect. The advent of DOAC has given to the clinician an opportunity to tailor OAC therapy in order to maximize advantages and minimize complications. AREAS COVERED: This review covers data published in literature regarding the risk of hemorrhagic stroke in patients taking OAC. Bleeding risk assessment is discussed and different bleeding risk factors are presented. The paper will also review clinical studies comparing DOAC against standard anticoagulation, in regard to the risk of hemorrhagic stroke. EXPERT OPINION: Bleeding assessment is mandatory in order to select patients at high hemorrhagic risk who will benefit the most from close monitoring. Blood pressure, alcohol intake, concomitant medication and comorbidities should be constantly evaluated and treated accordingly. During VKA therapy, adherence and intensity of anticoagulation must be strictly monitored. DOAC are associated with lower risk of hemorrhagic stroke than VKA. However, periodic hepatic and renal checks as well as careful evaluation of time adherence are necessary to reduce the risk of bleeding.

Incidence of a first thromboembolic event in carriers of isolated lupus anticoagulant.

Among the so called antiphospholipid (aPL) antibodies Lupus Anticoagulant (LAC) is considered the strongest risk factor for thromboembolic events. In individuals without a previous thromboembolic event (carriers), LAC is a risk factor when associated with the presence of anticardiolipin (aCL) and aß2-Glycoprotein I (aß2GPI) antibodies. On the other hand, data on carriers of isolated LAC positivity are sparse and inconclusive. The aim of this study was to prospectively determine the incidence of thrombosis in a cohort of carriers of isolated LAC positivity. One-hundred seventy-nine carriers of LAC confirmed twelve weeks apart and in a reference laboratory were studied. During a total follow up of 552 person-years, there were seven thromboembolic events (1.3% person-y). All the seven patients had at least one adjunctive major risk factor for thrombosis. The cumulative incidence of thromboembolic events was 3.1% (95% CI 0.6-5.6) after 2years, and 5.9% (95% CI 1.2-10.6) after 5 and 10years. On a multivariate regression analysis considering age, sex, autoimmune disease, risk factors for arterial and venous thrombosis, use of aspirin, only age was found to be an independent predictor of thromboembolic events (HR=1.1, 95% CI 1.0-1.2, p=0.02). These data might be relevant in clinical practice and underline the importance of differentiating LAC carriers in terms of isolated positivity or positivity associated with the presence of antibodies to aCL and ß2-glycoprotein I.

The paradox of the lupus anticoagulant: history and perspectives.

A unique coagulation inhibitor prolonging whole-blood clotting time was described more than 50 years ago in two patients with systemic lupus erythematosus (SLE). The immunoglobulin nature of the inhibitor and its interaction with antiphospholipid antibodies was later demonstrated and the term "lupus anticoagulant (LA)" was coined to describe this laboratory finding. It soon became apparent that LA was a misnomer as it is often found in plasma from patients with clinical conditions other than SLE and is associated with thromboembolic events that may occur in otherwise healthy individuals. Individuals with LA have circulating autoantibodies that inhibits blood coagulation. These are mostly of IgG or IgM class and mainly directed against a phospholipid (PL)-binding plasma protein, ß2-glycoprotein I (ß2GPI). The presence of ß2GPI-dependent LA represents a well-recognized risk factor for venous and arterial thromboembolism, as well as pregnancy loss and morbidity. ß2GPI-dependent LA in the presence of documented previous thromboembolism, or history of pregnancy loss/morbidity, identifies definite anti-PL syndrome. Laboratory diagnosis of LA is thus of particular importance, as it may assign patients with a common event (thrombosis) to a group with a high risk for recurrence, which is a prerequisite for long-term oral antithrombotic treatment.

Left ventricular myocardial strain by three-dimensional speckle-tracking echocardiography in healthy subjects: reference values and analysis of their physiologic and technical determinants.

BACKGROUND: Despite growing interest in applying three-dimensional (3D) speckle-tracking echocardiography (STE) to measure left ventricular (LV) myocardial deformation in various diseases, normative values for 3D speckle-tracking echocardiographic parameters and the effects of demographic, hemodynamic, and technical factors on these values are unknown. METHODS: In 265 healthy volunteers (age range, 18-76; 57% women), longitudinal strain (3DLε), circumferential strain (3DCε), radial strain (3DRε), and area strain (3DAε) were measured by using vendor-specific (Vsp) 3D speckle-tracking echocardiographic equipment. LV strain was also measured by using Vsp two-dimensional (2D) and vendor-independent 3D speckle-tracking echocardiographic software packages, for comparison. RESULTS: Reference values (lower limit of normality) for Vsp 3D STE were -17% to -21% (-15%) for 3DLε, -17% to -20% (-14%) for 3DCε, -31% to -36% (-26%) for 3DAε, and 47% to 59% (38%) for 3DRε. Three-dimensional longitudinal strain decreased, whereas 3DCε increased, with aging (P < .003), with different trends in men and women. Men had lower 3DLε, 3DRε, 3DAε, and 2D longitudinal strain than women (P < .02). LV 3D strain parameters were also influenced by LV volumes and mass, image quality, and temporal resolution (P < .02). Reference values obtained by Vsp 2D STE were -20% to -23% (-18%) for 2D longitudinal strain, -20% to -24% (-17%) for 2D circumferential strain, and 39% to 54% (28%) for 2D radial strain (P < .001 vs Vsp 3D STE). Significantly different 3DCε and 3DRε values were obtained with vendor-independent versus Vsp 3D STE (P < .001). CONCLUSIONS: In healthy subjects, reference values of LV 3D strain parameters were significantly influenced by demographic, cardiac, and technical factors. Limits of normality of LV strain by Vsp 3D STE should not be used interchangeably with Vsp 2D STE or with Vin 3D STE software.

Normal left ventricular mechanics by two-dimensional speckle-tracking echocardiography. Reference values in healthy adults.

INTRODUCTION AND OBJECTIVES: Two-dimensional speckle-tracking echocardiography is a novel tool to assess myocardial function. The purpose of this study was to evaluate left ventricular myocardial strain and rotation parameters by two-dimensional speckle-tracking echocardiography in a large group of healthy adults across a wide age range to establish their reference values and to assess the influence of age, sex, and hemodynamic factors. METHODS: Transthoracic echocardiograms were acquired in 247 healthy volunteers (139 women, 44 years [standard deviation, 16 years old] (range, 18-80 years). We measured longitudinal, circumferential, and radial peak systolic strain values, and left ventricular rotation and twist. RESULTS: Average values of global longitudinal, radial, and circumferential strain were -21.5% (standard deviation, 2.0%), 40.1% (standard deviation, 11.8%) and -22.2% (standard deviation, 3.4%), respectively. Longitudinal strain was significantly more negative in women, whereas radial and circumferential strain and rotational parameters were similar in both sexes. Accordingly, lower limits of normality for the strain components were -16.9% in men and -18.5% in women for longitudinal strain, and -15.4% for circumferential and 24.6% for radial strain, irrespective of sex. Longitudinal strain values were more negative at the base than at apical segments. Mean rotational values were -6.9° (standard deviation, 3.5°) for the base, 13.0° (standard deviation, 6.5°) for apical rotation, and 20.0° (standard deviation, 7.3°) for net twist. CONCLUSIONS: We report the comprehensive assessment of normal myocardial deformation and rotational mechanics in a large cohort of healthy volunteers. We found that women have more negative longitudinal strain, accounting for their higher left ventricular ejection fraction. Availability of reference values for these parameters may foster their implementation in the clinical routine.

Ascending aorta diameters measured by echocardiography using both leading edge-to-leading edge and inner edge-to-inner edge conventions in healthy volunteers.

AIMS: Reference ranges of ascending aorta diameters (AAoD) for two-dimensional echocardiography (2DE) using inner edge (IE) convention are lacking, preventing the comparison of AAoD measurements by 2DE with those obtained by other imaging modalities. METHODS AND RESULTS: We used harmonic imaging 2DE to prospectively study 218 healthy volunteers (56% women, 42 ± 15 years, 18-80 years). Measurements were performed at the level of aortic root (AoR), sinotubular junction (STJ), and proximal tubular portion (TAo, 1 cm from the STJ) using both leading edge (LE) and IE conventions at end-diastole and end-systole. Feasibility of AAoD measurements between end-diastole and end-systole was similar at AoR and STJ levels, but it was significantly different at TAo level (82 vs. 96%, respectively, P < 0.0001). Ascending aorta diameters indexed to height were larger in men than in women (P < 0.0001). After adjusting for the effect of gender, only age and body surface area (BSA) were independent predictors of AAoD at multivariable analysis. Average end-diastolic AoR, STJ, and TAo diameters measured using IE convention were similar between genders (17 ± 2, 15 ± 2, and 15 ± 2 mm/m(2), respectively). Corresponding AAoD measured using the LE convention were 18 ± 2, 16 ± 2, and 17 ± 4 mm/m(2), respectively. On average, the end-systolic AAoD measured using LE were 2 mm larger than those performed using IE or at end-diastole. Mean aortic wall thickness was 2.4 ± 0.8 mm. CONCLUSION: End-diastolic AAoD measured using IE were significantly smaller than those obtained either using LE convention or at end-systole. Gender-specific reference values for AAoD indexed for BSA should be used to identify ascending aorta pathology.

A novel medium for the development of in vitro cell culture system from Penaeus monodon.

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8-7.2. Osmolality was adjusted to 720 ± 10 mOsm kg(-1) and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace's insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation "cocktail".