THE ROLE OF TUMOR-SEEKING RADIOPHARMACEUTICALS IN THE DIAGNOSIS AND MANAGEMENT OF ADRENAL TUMORS.

CONTEXT: The variety of tumor-seeking radiopharmaceuticals, which are currently in clinical use, may have a potential role as imaging agents for adrenal gland tumors, due to physiological characteristics of this organ. OBJECTIVE: The purpose of this study was to evaluate the diagnostic potential of 99mTc-HYNIC-TOC, 99mTc(V)-DMSA, and 99mTc-MIBI in the assessment of adrenal tumors, by correlating with imaging findings and histopathologic results. DESIGN: The research is designed as a cross-sectional prospective study. PATIENTS AND METHOD: The study included 50 patients with adrenal tumors (19 hormone-secreting and 31 nonfunctioning) and 23 controls without adrenal involvement. In all patients, single-photon emission computed tomography (SPECT) was performed, using qualitative and semiquantitative analysis. The tumor to non-tumor tracer uptake was conducted by using a region-of-interest technique. Adrenal to background (A/B) ratio was calculated in all cases. RESULTS: 99mTc-HYNIC-TOC scintigraphy showed a high statistical significance between A/B ratios, while other two tracers resulted in a lower sensitivity, specificity and accuracy. Futhermore, 99mTc-HYNIC-TOC could have a high diagnostic yield to detect adrenal tumors (the receiver-operating-characteristic curve analysis, A/B ratio cut-off value of 8.40). CONCLUSION: A semiquantitative SPECT analysis showed that 99mTc-HYNIC-TOC is a highly sensitive tumor-seeking agent for the accurate localization of adrenal tumors.

Glucose and lipid abnormalities in patients with adrenal incidentalomas.

Adrenal incidentalomas (AI), defined as masses detected during imaging procedures of non-adrenal disorders, have become a common clinical problem that appear to have impairment of glucose and lipid metabolism. PATIENTS AND METHODS: One hundred and ten patients (mean 53.5 age; 24-72), who were diagnosed with functioning and non-functioning AI, were assessed. Patients with hormone-secreting AI underwent biochemical evaluation regarding metabolic disorders. Data about hormone status (cortisol profile and DEX screening test), lipid profile, glycemia, insulinemia were evaluated. RESULTS: This prospective study included 41 (37.28%) patients with non-functional and 69 (62.72%) with functional AI. Tumors associated with (sub)clinical Cushing's syndrome (functional AI) are considered to have higher cortisol concentration at 8h (p=0.027), 16h (p=0.025) and after DEX screening (p<0.010), compared to the controls. Patients with cortisol-secreting AI have significantly higher concentrations of cholesterol (p=0.040), triglycerides (p=0.027) and insulin (p<0.01) than controls. The patients with metabolic disorders have a significantly higher total cholesterol, triglyceride and insulin concentration (p<0.001) compared to controls. There was significant positive correlation between cortisol concentration after DEX screening and total cholesterol (r=0.727, p=0.007), triglycerides (r=0.564, p=0.041) and insulin (r=0.957, p=0.043) in the group with metabolic disorders. CONCLUSION: The present study demonstrates the patients with functional AI have significantly higher lipid, glucose and insulin concentration than controls. There was a significant positive correlation between metabolic parameters and cortisol concentration.

Cardiopulmonary bypass changes the plasma proteome in children undergoing tetralogy of Fallot repair.

INTRODUCTION: Cardiopulmonary bypass (CPB) can be associated with deleterious clinical effects. However, the impact of CPB on inflammatory, immunological and other homeostatic pathways remains poorly understood. We investigated the impact of CPB on the plasma proteome in children undergoing tetralogy of Fallot repair. METHODS: Blood samples were taken from 20 children prior to and at the end of CPB and 6h, 12h and 24h after CPB. Plasma was analysed by liquid chromatography-mass spectrometry (LC-MS) in a label-free, untargeted approach. Data were analysed using Genedata software to identify peptides that were differentially expressed (p<0.01 above a false discovery rate). Proteins were identified from peptides that demonstrated differential expression. RESULTS: The proteins that were found to be differentially expressed were haptoglobin isoform 1 preproprotein, isoform 2 of semaphorin-6C, vitamin D-binding protein, inter-alpha-trypsin inhibitor, ceruloplasmin, apolipoprotein B100 and fibrinogen alpha. CONCLUSION: CPB alters the plasma proteome with differences most apparent at 6h and 12h post CPB. There was a return to baseline with no proteins differentially regulated by 24h.

The expression of placental proteoglycans in pre-eclampsia.

BACKGROUND/AIMS: Pre-eclampsia (PE) is one of the leading causes of maternal and perinatal morbidity and mortality. PE is defined clinically as the onset of maternal hypertension and proteinuria following 20 weeks of gestation. It is associated with altered maternal uterine decidual spiral artery remodelling, which may lead to reduced blood flow and increased thrombosis within the uteroplacental vasculature. Proteoglycans (PGs) are macromolecules which have (in combination with glycosaminoglycans) important anticoagulant roles in vascular endothelial environments, including the uteroplacental circulation. The hypothesis under consideration in this study was that differential expression of placental PGs may be associated with PE. METHODS: PE and control placental samples were collected with ethics approval and patient consent. RNA and protein were extracted and real-time PCR and Western immunoblotting were performed to determine the expression of the PGs in the samples. RESULTS: Of the nine PGs investigated, none showed increased expression, whereas the mRNA and protein expression of five of them was significantly decreased in the placentae of pre-eclamptic women compared to gestation-matched controls. CONCLUSION: Therefore, the results of this study support the hypothesis that a placental PG deficiency may contribute to the placental thrombotic lesions characteristic of PE.

Decorin expression is decreased in human idiopathic fetal growth restriction.

Fetal growth restriction (FGR) is a clinically significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. Most cases of FGR are idiopathic and are associated with placental thrombosis. Previous studies suggest that proteoglycans, such as decorin, that contain the glycosaminoglycan dermatan sulfate are the principal anticoagulants in the normal placenta. The present study investigated decorin expression in placentas from pregnancies complicated by idiopathic FGR (n = 26) and gestation-matched controls (n = 27). Real-time polymerase chain reaction demonstrated significantly reduced decorin mRNA expression in FGR compared with control (1.52 +/- 0.14 v. 2.21 +/- 0.22, respectively; P < 0.01). Immunoblotting revealed decreased decorin protein (40 kDa) expression in FGR compared with controls (420.8 +/- 39.0 v. 690.1 +/- 42.2, respectively; n = 12 in each group; P = 0.0007). Immunohistochemistry demonstrated the presence of immunoreactive decorin protein in the placental villous stroma surrounding the fetal capillaries and a significant decrease in decorin protein presence in FGR compared with control (1.75 +/- 0.66 v. 2.98 +/- 1.12, respectively; n = 6 in each group; P < 0.01, t-test). This is the first study to demonstrate reduced decorin in idiopathic FGR, indicating a potentially significant role for decorin in the aetiology of placental thrombosis in idiopathic FGR.

Decreased placental glypican expression is associated with human fetal growth restriction.

Placental mediated fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. Heparan sulphate proteoglycans (HSPG) are highly expressed in placentae and regulate haemostasis. We hypothesise that altered expression of HSPGs, glypicans (GPC) may contribute to the development of FGR and small-for-gestational-age (SGA). GPC expression was determined in first-trimester chorionic villous samples collected from women with later SGA pregnancies and in placentae from third-trimester FGR and gestation-matched uncomplicated pregnancies. The expression of both GPC1 and GPC3 were significantly reduced in first-trimester SGA as well as in the third-trimester FGR placentae compared to controls. This is the first study to report a relationship between altered placental GPC expression and subsequent development of SGA/FGR.

The differences of platelet response to snake venoms: a comparative study of children and adults.

Platelets play a vital role in the coagulation, yet the potential for differences in platelet function, between adults and children, remains underexplored. This is despite the age-related variation in haemostatic proteins, that is encompassed by the term Developmental Haemostasis. Hemotoxins found in the venoms of Australian snakes mimic human blood coagulation factors. The effects of Australian snake venoms on platelets, as well as the possible differential response in adults and children were subject of this study.

The molecular basis of cross-reactivity in the Australian Snake Venom Detection Kit (SVDK).

The Snake Venom Detection Kit (SVDK) is of major medical importance in Australia, yet it has never been rigorously characterised in terms of its sensitivity and specificity, especially when it comes to reports of false-negative and false-positive results. This study investigates reactions and cross-reactions of five venoms the SVDK is directed against and a number of purified toxins. Snakes showing the closest evolutionary relationships demonstrated the lowest level of cross-reactivity between groups. This was, instead, far more evident between snakes that are extraordinarily evolutionary separated. These snakes: Pseudechis australis, Acanthophis antarcticus and Notechis scutatus, in fact displayed more false-positive results. Examination of individual toxin groups showed that phospholipase A(2)s (PLA(2)s) tends to react strongly and display considerable cross-reactivity across groups while the three-finger toxins (3FTx) reacted poorly in all but the Acanthophis well. The hook effect was evident for all venoms, particularly Oxyuranus scutellatus. The results of this study show considerable variation in toxin detection, with implications in further development of venom detection, both in Australia and other countries.

Cognitive impairment and functional ability in the acute phase of ischemic stroke.

OBJECTIVE: Acute ischemic stroke significantly affects cognitive efficiency and functional ability, primarily physical ability. Decreased cognitive efficiency is often in correlation with decreased functional ability following an ischemic stroke. The aim of the study was to determine whether there is cognitive impairment in patients with preserved physical ability in acute ischemic stroke, and if so, determine whether there is a significant correlation with the functional, i.e. physical status. PATIENTS AND METHODS: The study included a total of 80 subjects: 40 subjects (26 male and 14 female) in the acute phase of ischemic stroke and 40 healthy subjects (20 male and 20 female) with no history of neurological disease. Both groups were matched with regard to basic sociodemographic characteristics. The cognitive status was evaluated using a comprehensive neuropsychological battery, while physical status was assessed using the modified Rankin scale. Cognitive performance was presented using the following seven cognitive domains: executive function, immediate recall, delayed recall, speech, divergent thinking, attention and concentration, and visual-constructive performance. RESULTS: The two groups differed in all the studied cognitive domains, with acute ischemic stroke subjects achieving poorer results. There was no correlation between the cognitive status and the functional, i.e.physical ability in the acute ischemic stroke group. CONCLUSIONS: The results showed significant impairment in all cognitive domains in the acute phase of ischemic stroke, regardless of the preserved functional, i.e. physical ability of these patients.

Inhalational use of antithrombotics in humans: Review of the literature.

INTRODUCTION: Off label use of anticoagulants is common. The association between fibrin deposition in the lungs and primary lung disease, injury or prematurity affords a strong theoretical basis for the potential benefit of antithrombotic therapies administered directly to the lung tissue. This review offers a critical appraisal of current evidence related to the inhalational administration of antithrombotic therapy in humans. MATERIALS AND METHODS: An interrogation of 2 databases across a 13 year period of time was undertaken using key words selected a priori. Identified publications were categorized according to the following themes: 1. Inhaled antithrombotic therapy in healthy subjects 2. Inhaled antithrombotic therapy for vascular thromboprophylaxis 3. Inhaled antithrombotic therapy in smoke inhalation and lung injury 4. Inhaled antithrombotic therapy in asthma or allergy 5. Inhaled antithrombotic therapy for plastic bronchitis post-Fontan surgery 6. Inhaled antithrombotic therapy for other indications. RESULTS: 33 articles were identified consistent with the inclusion criteria developed for this review. Unfractionated heparin, LMWH, activated protein C and thrombolytic agents have been administered via the respiratory track, with asthma and smoke inhalation/lung injury being the most frequently investigated clinical scenarios described. All studies reported had significant methodological limitations. CONCLUSIONS: The safety and clinical utility of inhaled antithrombotic therapies have not been adequately investigated to support the generation of any firm evidence. This review highlights where inhaled antithrombotic therapies have shown promise and importantly, the further research required to confirm mechanism of action and a definitive risk: benefit profile.

Lack of anti-factor Xa assay standardization results in significant low molecular weight heparin (enoxaparin) dose variation in neonates and children.

INTRODUCTION: Enoxaparin is a frequently used anticoagulant in children. Unlike in adults, consensus guidelines recommend therapeutic monitoring to a target anti-factor Xa level of 0.5-1 U mL(-1) . Therapeutic ranges are not well correlated with clinical outcomes (e.g. thrombosis or hemorrhage), and assays are not standardized. Owing to limited reagent supplies, our clinical laboratory conducted a validation process and switched anti-FXa assays. Although the assays correlated well with each other, anti-FXa values were, on average, 33% higher with the new assay. The target anti-FXa range was not altered. We evaluated how this change in anti-FXa assays influenced enoxaparin dosing (mg kg(-1) ). METHODS: Enoxaparin dosing and anti-FXa values for all patients started on enoxaparin for the 6 months before and after assay change were retrospectively compiled and analyzed with a Student's t-test. RESULTS: One hundred and nine children were started on enoxaparin before assay change, and 104 after assay change. The mean therapeutic enoxaparin dose (mg kg(-1) ) was significantly lower in subjects aged < 3 months (P = 0.01) and 3 months to 2 years (P < 0.0001), but not in subjects aged > 2 years (P = 0.18), after assay change. The median number of enoxaparin dose changes required to achieve the target range was significantly reduced after assay change, from 1 to 0 (P = 0.004). CONCLUSIONS: The current pediatric practice of dose adjustment to achieve and maintain a target anti-FXa range is vulnerable to assay determination, which may provide false reassurance of efficacy and safety and represent misappropriation of time and resources. These data support a pediatric randomized controlled clinical trial comparing the safety and efficacy of enoxaparin weight-based dosing with or without dose titration based on anti-FXa.

Beta (ß)-antithrombin activity in children and adults: implications for heparin therapy in infants and children.

BACKGROUND: Antithrombin, a hemostatic protein and naturally occurring anticoagulant, is a major thrombin inhibitor. The capacity of antithrombin to inhibit thrombin is known to increase a 1000-fold whilst in the presence of unfractionated heparin. ß-antithrombin is an isoform of antithrombin with a high affinity for unfractionated heparin. This study aimed to determine the differences in the anticoagulant activity of the ß-antithrombin isoform in children compared with adults. METHODS: Plasma samples were obtained from 105 healthy individuals from the following age groups: neonates (day 1 and day 3), 28 days to 1 year, 1-5 years, 6-10 years, 11-16 years and adults. The method utilized to measure the activity of ß-antithrombin in plasma is a modified version of the total antithrombin assay routinely used in diagnostic laboratories. The modified version of this assay allows for the specific quantification of the ß-antithrombin glycoform anticoagulant activity alone, as the ß-antithrombin molecule is activated under a high salt concentration, which in turn does not allow activation of other antithrombin isoforms. CONCLUSIONS: This study demonstrated that there are no age-specific differences in the activity of ß-antithrombin. However, considering that the total AT activity is significantly reduced in neonates, our results suggest that in this population ß-antithrombin activity is a major contributor to the overall activity of AT.

Altered decorin leads to disrupted endothelial cell function: a possible mechanism in the pathogenesis of fetal growth restriction?

OBJECTIVE: Fetal growth restriction (FGR) is a key cause of adverse pregnancy outcome where maternal and fetal factors are identified as contributing to this condition. Idiopathic FGR is associated with altered vascular endothelial cell functions. Decorin (DCN) has important roles in the regulation of endothelial cell functions in vascular environments. DCN expression is reduced in FGR. The objectives were to determine the functional consequences of reduced DCN in a human microvascular endothelial cell line model (HMVEC), and to determine downstream targets of DCN and their expression in primary placental microvascular endothelial cells (PLECs) from control and FGR-affected placentae. APPROACH: Short-interference RNA was used to reduce DCN expression in HMVECs and the effect on proliferation, angiogenesis and thrombin generation was determined. A Growth Factor PCR Array was used to identify downstream targets of DCN. The expression of target genes in control and FGR PLECs was performed. RESULTS: DCN reduction decreased proliferation and angiogenesis but increased thrombin generation with no effect on apoptosis. The array identified three targets of DCN: FGF17, IL18 and MSTN. Validation of target genes confirmed decreased expression of VEGFA, MMP9, EGFR1, IGFR1 and PLGF in HMVECs and PLECs from control and FGR pregnancies. CONCLUSIONS: Reduction of DCN in vascular endothelial cells leads to disrupted cell functions. The targets of DCN include genes that play important roles in angiogenesis and cellular growth. Therefore, differential expression of these may contribute to the pathogenesis of FGR and disease states in other microvascular circulations.

Developmental hemostasis: age-specific differences in the levels of hemostatic proteins.

INTRODUCTION: Developmental hemostasis recognizes the physiologic differences between the hemostatic system of neonates and children and that of adults. As compared with the knowledge of hemostatic system physiology in adults, our understanding in neonates and children remains inadequate. Routine clinical coagulation testing most commonly measures functional parameters of the hemostatic system. Very few studies have measured age-specific levels of hemostatic proteins. An understanding of the normal fluctuations in the levels of hemostatic proteins is vital in the prevention, diagnosis and treatment of hemostatic problems during infancy and childhood. This study was designed as the first comprehensive study of the age-specific changes in the levels of important hemostatic proteins in healthy neonates, children, and adults. METHODS: Plasma samples were obtained from 120 healthy individuals from the following age groups: neonates (day 1 and day 3), 28 days to 1 year, 1-5 years, 6-10 years, 11-16 years, and adults. Factor II, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, plasminogen, protein C and total and free protein S were quantified with commercially available ELISA kits. RESULTS: The levels of 10 proteins were significantly different between neonates and adults, and these differences persisted throughout childhood for most of these proteins. CONCLUSION: The results of this study confirm that the levels of the majority of coagulation proteins vary significantly with age. Future studies should investigate how hemostatic protein level relates to functional changes with age.

The in vitro anticoagulant effect of rivaroxaban in children.

INTRODUCTION: Current anticoagulation therapy in children is less than ideal, requiring regular venous monitoring and dosing adjustments. Limitations associated with conventional anticoagulants have prompted the development of novel drugs that specifically target key proteins in the coagulation system. Rivaroxaban is the first oral, direct Factor Xa inhibitor available for the prevention of venous thromboembolism in adults. Its predictable pharmacokinetic profile, high oral bioavailability and once-daily dosing make rivaroxaban an optimal anticoagulant that warrants investigation in children. The aim of this study was to investigate the age-related anticoagulant effect of rivaroxaban in vitro. MATERIALS AND METHODS: Age-specific plasma pools were created (i.e. 28 days-23 months, 2-6, 7-11, 12-16 years and adults) and spiked with increasing concentrations of rivaroxaban (0-500 ng/ml). Commercially available PT, APTT and anti-Factor Xa assays, as well as sub-sampling thrombin generation assays, were used to measure rivaroxaban effect. RESULTS: The results of this study indicate that there are no significant differences in rivaroxaban effect across the age groups in vitro. CONCLUSION: In vivo studies are required to confirm the consistency of dose-response across the paediatric age groups.

The effect of different snake venoms and anti-venoms on thrombin clotting time in human plasma.

BACKGROUND: Crotaline snake species, or pit vipers, are distributed throughout Asia and America. While much is known about the clinical effect of these snake venoms, there is a lack of evidence related to the various anti-venoms available and their effectiveness in reversing the effect of different venoms. AIM: This study aimed to determine the interaction of the venoms of the following species: Crotalus unicolor, Crotalus adamanteus, Crotalus vegrandis, Trimeresurus spp, Calloselasma rhodostoma, Bothriechis schlegelii and Agkistrodon and the following anti-venoms: Anticrotalico, Antivipmyn, Antibotropico, Antifidico and SAIMR by evaluating their effect on the thrombin clotting time in human plasma. METHOD: The interactions of venoms and anti-venoms were evaluated using thrombin clotting time in human plasma. RESULTS: The results demonstrate that Anticrotalico anti-venom was most effective for the Crotalid species (Crotalus unicolor, Crotalus adamanteus, Crotalus vegrandis). Anticrotalico extended the time to clot formation 2.7 fold for Crotalus Unicolor, 3 fold for Crotalus Adamanteus and 4.6 fold for Crotalus Vegrandis. The anti-venoms most efficient in reversing the effect of the Trimeresurus spp venom, were Anticrotalico, Antivipmyn, Antibotropico and Antifidico anti-venoms, which all completely reversed the effect of clot formation as evident by no clot formation within the 999 seconds measurement limit. Bothriechis schlegelii venom was neutralized by all anti-venoms tested. Calloselasma rhodostoma venom was neutralized by Antifidico as well as Anticrotalico. The most efficient anti-venoms against the Agkistrodon venom were Anticrotalico and Antibotropico. In general, monovalent anti-venoms had improved efficiency for their corresponding snake species, depending highly on the composition of the snake venom. This study confirms the importance of considering the choice of anti-venom in a clinical setting, to reverse the effect of specific snake venoms. In addition, this study suggests that some anti-venoms can be considered for use against a variety of snake-venoms.

Age-specific differences in binding of heparin to plasma proteins.

BACKGROUND: Clinically significant age-related differences in the anticoagulation effect of heparin have previously been established in vitro as well as in different clinical settings in vivo. These differences were hypothesized to be due to the age-specific differences in binding of heparin to plasma proteins. OBJECTIVES: The aim of this project was to investigate global age-related differences in heparin binding to plasma proteins. PATIENTS/METHODS: Heparin-binding proteins were identified by incubating heparin-coated magnetic beads with plasma samples from neonates, children and adults, and purifying the proteins that were bound to the beads in this reaction system. RESULTS: These results provide the first preliminary evidence of age-related differences in the total number and concentration of proteins bound to heparin. The results also suggest, for the first time, that there are age-related differences of heparin binding to antithrombin and thrombin. CONCLUSIONS: The results of this study, although preliminary, support and contribute to the explanation of the mechanism of age-related differences in the effect of heparin observed previously in vitro and in vivo.