Endocan: A biomarker for endothelial dysfunction and inflammation, linking maternal obesity and pediatric obesity in a cohort of preterm neonates.

OBJECTIVES: Numerous animal and epidemiologic studies have demonstrated a positive association between maternal obesity in pregnancy and obesity in offspring. The biologic mechanisms of this association remain under investigation. One proposed mechanism includes fetoplacental endothelial dysfunction secondary to inflammation. Endocan is a relatively new biomarker for endothelial dysfunction and inflammation. Our objectives were to examine (1) the association between maternal obesity and neonatal serum endocan at birth, and (2) the association between neonatal serum endocan at birth and pediatric obesity at 24-36 months of age. STUDY DESIGN: This was a secondary analysis of a prospective cohort of neonates born < 33 weeks gestation. Serum endocan was collected within 48 hours of birth. Serum endocan levels were compared in neonates born to obese mothers vs. those born to non-obese mothers. BMI data were retrospectively collected from cohort neonates between 24 and 36 months of age. RESULTS: The analysis included 120 mother/neonate dyads. Neonates born to obese mothers had higher median serum endocan at birth compared to neonates born to non-obese mothers (299 ng/L [205-586] vs. 251 ng/L [164-339], p = 0.045). In a linear regression modeled on neonatal serum endocan level, maternal obesity had a statistically significant positive association (p = 0.021). Higher mean serum endocan level at birth was associated with pediatric obesity between 24 and 36 months (obese vs. non-obese offspring; 574 ng/L (222) vs. 321 ng/L (166), p = 0.005). CONCLUSIONS: In our cohort of preterm neonates, elevated serum endocan at birth was associated with both maternal obesity and downstream pediatric obesity. More research is needed to understand intergenerational transmission of obesity. A large focus has been on epigenetic modification. Endothelial dysfunction and inflammation may play important roles in these pathways. Effective biomarkers, including endocan, may also serve as intermediate outcomes in future pregnancy research.

Exposure to the Amino Acids Histidine, Lysine, and Threonine Reduces mTOR Activity and Affects Neurodevelopment in a Human Cerebral Organoid Model.

Evidence of the impact of nutrition on human brain development is compelling. Previous in vitro and in vivo results show that three specific amino acids, histidine, lysine, and threonine, synergistically inhibit mTOR activity and behavior. Therefore, the prenatal availability of these amino acids could be important for human neurodevelopment. However, methods to study the underlying mechanisms in a human model of neurodevelopment are limited. Here, we pioneer the use of human cerebral organoids to investigate the impact of amino acid supplementation on neurodevelopment. In this study, cerebral organoids were exposed to 10 mM and 50 mM of the amino acids threonine, histidine, and lysine. The impact was determined by measuring mTOR activity using Western blots, general cerebral organoid size, and gene expression by RNA sequencing. Exposure to threonine, histidine, and lysine led to decreased mTOR activity and markedly reduced organoid size, supporting findings in rodent studies. RNA sequencing identified comprehensive changes in gene expression, with enrichment in genes related to specific biological processes (among which are mTOR signaling and immune function) and to specific cell types, including proliferative precursor cells, microglia, and astrocytes. Altogether, cerebral organoids are responsive to nutritional exposure by increasing specific amino acid concentrations and reflect findings from previous rodent studies. Threonine, histidine, and lysine exposure impacts the early development of human cerebral organoids, illustrated by the inhibition of mTOR activity, reduced size, and altered gene expression.

The association between AB blood group and neonatal disease.

BACKGROUND: Numerous studies have examined the association between ABO blood groups and adult disease states, but very few have studied the neonatal population. The objective of this study was to determine the relationship between AB blood group and the occurrence of common neonatal disorders such as neutropenia at birth, sepsis, respiratory distress syndrome (RDS), intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP), and patent ductus arteriosus (PDA) compared to all other blood groups. METHODS: We performed a retrospective review on 3,981 infants born at 22 0/7 to 42 6/7 weeks' gestational age and compared the relative risk of neonatal diseases in infants with AB blood group to that of infants with all other blood groups (A, B, and O). RESULTS: When compared to all other blood groups, AB infants demonstrated an increased risk for developing negative clinical outcomes. AB blood group was significantly associated with a 14-89% increased risk of neutropenia at birth, sepsis, RDS, and ROP. Risks for IVH and PDA were not significant. CONCLUSION: We hypothesize that the phenotypic expression of A and B antigens, rather than the antigens themselves, in the AB group may reveal an enhanced susceptibility to injury at the endothelial level resulting in an increased risk for disease development.

Thromboelastographic evaluation of blood coagulation in the presence of branded and generic enoxaparins.

AIM: Enoxaparin is the most widely used low-molecular-weight heparin (LMWH) in the USA and has been approved for clinical use in multiple indications. Enoxaparin is a complex biological product with multiple known activities relevant to its antithrombotic effects, and variations in different forms of enoxaparin may have important clinical implications. This study aimed to compare the physiological anticoagulant activity of branded and a generic enoxaparin, using thromboelastography (TEG) to evaluate their effect on the dynamic formation of the blood clot as quantitated by interactions between coagulation factors and inhibitors, fibrinogen, platelets and the fibrinolytic system. METHODS: Whole native (no preservative) blood was obtained from 7 healthy volunteers. Samples were immediately mixed with various concentrations of branded or generic enoxaparin and TEG was performed to assess anticoagulant activity. Five different batches of each enoxaparin (branded and generic) were tested. RESULTS: Generic enoxaparin showed more variation in anticoagulation response with a less predictable concentration-dependent and linear response compared with branded enoxaparin. There was also an apparent batch-to-batch variation for generic enoxaparin. The results demonstrated a lower overall anticoagulant effect (P=0.05; no overlap of 95% confidence intervals) with a wider inter-individual variation for generic enoxaparin in comparison with branded enoxaparin. Some individuals responded with a higher than expected anticoagulant response to the given concentration of the generic enoxaparin. CONCLUSION: The findings of this study suggest that other pre-clinical and clinical studies should be done to validate the clinical interchangeability between branded and generic enoxaparin.

Leptin and ghrelin concentrations and weight loss in Parkinson's disease.

OBJECTIVES: To investigate the role of leptin, ghrelin, GH and IGF-1 in energy balance disturbances in Parkinson's disease (PD). MATERIALS AND METHODS: Thirty-nine patients were included: 11 PD patients with unintentional weight loss, 16 PD patients without weight loss and 12 controls. UPDRS, MMSE, MADRS, appetite scale, BMI, adipose tissue content, plasma leptin and active ghrelin concentrations and serum GH, IGF-1, TSH, T3 and T4, concentrations were evaluated. RESULTS: A lower plasma leptin concentration and a higher serum IGF-1 concentration were found in PD patients with weight loss. BMI and the content of adipose tissue were positively correlated with leptin concentration in all PD patients. Paradoxically, the lower BMI was, the lower plasma active ghrelin concentration was in PD patients with the weight loss. CONCLUSION: These findings confirm that changes of plasma leptin concentration occur in PD patients with loss of weight.

Molecular profiling of heparinase-I resistant glycosaminoglycans in contaminated heparins. Comparative studies with uncontaminated heparin and porcine oversulfated chondroitin sulfate.

AIM: Heparin is a widely used anticoagulant which is usually obtained from porcine mucosal tissue. The structure of heparin is comparable to other naturally occurring glycosaminoglycans such as chondroitin sulfate and dermatan sulfate. The commercially available heparin preparations may contain small amounts of dermatan sulfate as a carry-over impurity. More recently (November 2007 to April 2008), an increased incidence of adverse events and deaths associated with the use of heparin alerted regulatory agencies to investigate the composition of heparin. As a result, oversulfated chondroitin sulfate was found to be the main determinant of the observed adverse reactions. This glycosaminoglycan is not usually found in the mammalian tissues. METHODS: This investigation reports on the comparison of contaminant free and contaminated heparins and their digestion by heparinase-I. It also describes the molecular profile of the contaminant isolated from the recalled heparin preparations in comparison to oversulfated chondroitin sulfate. The anticoagulant and anti-Xa activities are also reported. RESULTS: The contaminant is found to be comparable to the synthesized OSCS as both were resistant to heparinase-I digestion. The contaminant and OSCS exhibited weaker anticoagulant activities than heparin and did not have any anti-Xa effects. CONCLUSION: This data strongly suggests that such glycosaminoglycans as chondroitin sulfate can be structurally modified to exhibit anticoagulant activities and their molecular weight can be adjusted to mimic heparin.

Incidentally discovered adrenal tumors: a lesson from observation of 1,444 patients.

This study was aimed at summarizing our experience in the management of 1,444 patients with incidentally found adrenal tumors observed at a single endocrinological centre. Hormonal determinations were performed in all patients at the beginning of the observation period to detect subclinical adrenal hyperfunction. The imaging phenotype on CT and MRI was analyzed for defining the malignant potential of the tumors. Based on the results of these examinations we diagnosed among our cohort probably benign masses in 87%, malignant tumors in 10% (adrenal carcinoma - 9%), and metastases in 3%. Subclinical hyperfunction was diagnosed in 8%; the most frequent was the pre-Cushing's syndrome. A subgroup of 480 patients (33%) was submitted to surgery because of oncological or endocrinological indications. The patients not qualified for surgery were carefully controlled by imaging and hormonal examinations. Malignancy is the most serious risk in the group of patients with incidentally discovered adrenal tumors.

1,161 patients with adrenal incidentalomas: indications for surgery.

BACKGROUND AND AIMS: The aim of this study is to analyze the clinical data and criteria for surgery in a group of over 1,100 patients with adrenal incidentalomas (AI) observed at the Department of Endocrinology. PATIENTS AND METHODS: The material consisted of 1,161 patients (842 women and 319 men, 10-87 years old) with AI ranging in size from 1.0 to 23.0 cm. The methods included clinical examination, imaging studies, hormonal determinations in the blood and in the urine as well as histological and immunocytochemical investigations in 390 patients treated by surgery. RESULTS: Basing on these studies, we diagnosed 112 patients with primary malignant adrenal tumors (100 with carcinoma), 45 with metastatic infiltrations, and 1,004 with probable benign AI. Imaging phenotypes (especially high density on computed tomography, CT) were characteristic of malignant and chromaffin tumors. Subclinical adrenal hyperactivity was found in 8% of the patients with pre-Cushing's syndrome as the most frequent form (6.5%). Chromaffin tumors were detected in 3%. CONCLUSIONS: (1) Indications for surgery include malignant tumors (both primary and metastatic), tumors with subclinical hyperfunction, and chromaffin tumors. High density on CT, >20 HU, appeared to be an important indication for surgery. (2) A slight prevalence of oncological indications over endocrinological indications (14 vs. 11%) was found.

Nelson's syndrome -- 46 years later: clinical experience with 37 patients.

OBJECTIVE: Pituitary tumours occurring after bilateral adrenalectomy for Cushing's disease (Nelson's syndrome) are frequently aggressive, so an early diagnosis and careful management are of prime importance. For a new insight into this entity it is necessary to analyse the factors predisposing to its development and the course of the disease, as well as the methods of diagnosis and modalities of treatment. PATIENTS AND METHODS: Thirty-seven patients with Nelson's syndrome were observed, 32 women and 5 men, aged 16 to 61 years at the time of pituitary tumour detection (at present, 27 to 82 years old). The diagnostic methods included clinical observation, imaging examinations (X-ray studies, CT, MRI), hormonal evaluation (especially ACTH and cortisol levels during replacement therapy) and ophthalmologic investigations. Neurosurgery was the main method of treatment. RESULTS: The clinical analysis indicated that young age at the time of adrenalectomy, pregnancy, insufficient replacement therapy and fulminant course of Cushing's disease were the main factors predisposing to Nelson's syndrome. MRI appeared to be the most valuable imaging method, as this detected Nelson's tumours in the microadenoma stage in 7 patients. Plasma ACTH levels varied between 32.6 pmol/l in an early phase to 2 000 pmol/l in the full-blown syndrome. Absolute temporal scotomas found in ophthalmologic examinations were an early abnormality. The best results after therapy were obtained in patients treated by neurosurgery using a transsphenoidal approach in an early stage. CONCLUSIONS: MRI, ophthalmologic examination and plasma ACTH determination were the most valuable investigations for early diagnosis of Nelson's syndrome. Early neurosurgery offered the best outcome in our group of patients.

Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin.

The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.

Comparative tissue factor pathway inhibitor release potential of heparins.

Tissue factor pathway inhibitor (TFPI) is released following the administration of unfractionated heparin, low-molecular-weight heparins, defibrotide and PI-88. In this study, the comparative effects of heparin, a low-molecular-weight heparin-gammaparin and a heparin-derived oligosaccharide mixture-subeparin (C3) were studied on functional and immunologic tissue factor pathway inhibitor activity levels in a non-human primate (Macaca mulatta) model. The dose-dependent effect was studied following intravenous and subcutaneous administration. Following the administration of 1 mg/kg of heparin, gammaparin, and C3, the functional levels of TFPI at 5 minutes were 2.40, 2.56, and 1.08 U/mL and the corresponding TFPI immunologic levels were 4.3-, 4.0-, and 2.1-fold, increased, respectively, over the baseline value. From these results, it can be concluded that heparin and gammaparin produced similar levels of TFPI release. Hence, gammaparin and heparin have similar TFPI release potential despite their differences in molecular weight. The influence of molecular weight, charge density, and interactions with heparin cofactor II on TFPI release are also discussed.

Statin drugs and dietary isoprenoids downregulate protein prenylation in signal transduction and are antithrombotic and prothrombolytic agents.

Statins and various isoprenoids of dietary origins inhibit L-mevalonic acid synthesis, which in turn downregulates cholesterol and various other dependent substances, including farnesyl- and geranylgeranyl-conjugated proteins involved in cell signaling processes. Such signaling processes are stimulated by protease-activated receptor-1 (PAR-1), which upon activation, causes the expression of various substances including tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1). Tissue factor promotes thrombin generation, where thrombin stimulates a variety of cellular processes, as well as activating PAR-1 to produce more thrombin. Statins downregulate TF mitigating thrombin generation and also downregulate PAI-1, which normally consumes tissue plasminogen activator (tPA). In the absence of PAI-1, tPA activates plasminogen to generate plasmin. Thus, statins behave as antithrombotic agents and prothrombolytic agents.

Downregulation of clusterin expression in testicular germ cell tumours.

OBJECTIVES: Clusterin is implicated in many biological processes including cell adhesion, apoptosis and transformation. Clusterin expression was demonstrated during sperm maturation and is overexpressed in different malignancies including breast and prostate carcinomas and anaplastic large-cell lymphomas. METHODS: The aim of this study was to determine the expression of clusterin in a series of different germ cell tumours by immunohistochemistry. Twenty-two seminomas, 27 embryonal carcinomas, 22 mature and immature teratomas, 8 yolk sac tumours and 1 chorion carcinoma as well as 30 normal testes were analysed using a monoclonal antibody. RESULTS: In normal testes strong signals were seen in the maturing germ cells and in Leydig cells. In most tumours examined no clusterin expression was detected (84%). Only a focal weak expression was seen in some teratomas (32%), embryonal carcinomas (15%) and yolk sac tumours (25%). CONCLUSIONS: Our results suggest that clusterin is associated with normal germ cell development and that the loss of clusterin expression might play a role in the malignant transformation of germ cells.

Singlet oxygen inhibits agonist-induced P-selectin expression and formation of platelet aggregates.

Major mediators of activated polymorphonuclear leukocytes (PMN) are the oxidants HOCl and chloramine, which are a source for the nonradical photon-emitting oxidant singlet oxygen (1O2). We were interested in a possible platelet-modulating activity of 1O2. As a stable 1O2 source we chose the mild oxidant chloramine T (CT), which mimics the natural chloramine N-chloro-taurine. Freshly drawn native whole blood from donors (n = 5) was incubated at 0 to 3 mM CT for 1 minute at 37 degrees C. Then saline. 10 microM adenosine diphosphate (ADP), 5 microg/mL collagen, or 6.25 microM thrombin receptor activator peptide (TRAP) were added and the mixtures were allowed to incubate for 3 minutes at 37 degrees C. Aliquots of activated blood were fixed in 1% para-formaldehyde. After removal of the fixative, platelets were labeled with anti-CD61-FITC and anti-CD62P-PE antibodies and analyzed by flow cytometry. An oxidant concentration-dependent decrease in the expression of P-selectin appeared (at 3 mM CT to 39, 23, and 20% of the 100% saline control level for ADP, collagen, and TRAP, respectively). There was also an oxidant concentration-dependent decrease in the formation of platelet aggregates (at 3 mM CT to 8, 12, and 13% of the 100% saline control level for ADP, collagen, and TRAP, respectively; the 50% effective dose was 1.0 to 1.5 mM chloramine). In ADP- and TRAP-stimulated platelets, an oxidant-mediated increase in platelet fragments appeared (at 3 mM CT: three- to fourfold of the initial value). The addition to the blood of 30 mM of the oxyradical scavenger mannitol in contrast to excess methionine did not antagonize these oxidative modulations of platelet activation. The results were confirmed using equimolar concentrations of NaOCI and N-chloro-taurine. This study shows that 1O2 inhibits platelets, decreasing the expression of CD62P and the formation of platelet aggregates. Activated PMN might modulate hemostasis, shifting it into an antithrombotic state. The physiologic signal action and the direct anticoagulant action of 1O2 (released by chloramines such as vancomycin) might be a new principle for pharmacologic intervention in atherothrombosis.

Evaluation of a new point of care heparin test for cardiopulmonary bypass: the TAS heparin management test.

Patients undergoing cardiopulmonary bypass (CPB) require anticoagulation with heparin to avoid thrombosis within the bypass circuit. The common method used to monitor the degree of anticoagulation is the activated clotting time (ACT). We evaluated a novel point of care device, the TAS (Pharmanetics, Raleigh, NC, USA) heparin management test (HMT), for its suitability in monitoring anticoagulation during CPB. In vitro analysis showed a dose-response (r2=0.988) of the HMT from 0.078-10.0 U/ml heparin, covering the range of heparin used during cardiac surgery (2-5 U/ml). Fifty randomly selected patients undergoing CPB were studied. Preheparin clotting times for these patients were 143+/-32 s for the HMT and 146+/-18 s for the ACT; 435+/-60 s HMT and 438+/-39 s ACT during CPB; 145+/-50 s HMT and 128+/-14 s ACT post-protamine (r2=0.797). epsilon-Aminocaproic acid treatment for inhibition of fibrinolysis did not affect the HMT. We conclude that the HMT correlates well with the ACT and may be useful for monitoring heparin during CPB. Advantages of the HMT are small sample volume and good sensitivity to heparin.

Inhibition of tissue factor-activated platelets by low-molecular-weight heparins and glycoprotein IIb/IIIa receptor antagonist.

Thrombotic disorders can lead to vascular distress and platelet activation eventually resulting in the rupture of the lesions where a sizable amount of tissue factor (TF) is generated during the pathogenesis of arterial diseases. Since low-molecular-weight heparins (LMWHs) and platelet glycoprotein (GP) IIb/IIIa inhibitors are clinically used for the management of acute coronary syndrome (ACS), studies were taken to determine the effects of these agents on TF-mediated activation of platelets. Freshly drawn native whole blood (WB) from normal healthy volunteers (n = 6) supplemented with a predetermined amount of TF was incubated with equivalent anti-Xa adjusted amounts of various LMWHs at 0.01-1.0 U/ml and tirofiban from 10 to 100 ng/ml. Platelet activation was assessed by measuring the expression of P-selectin (CD62) and the generation of platelet aggregates. At 0.01 U/ml, enoxaparin exhibited a stronger inhibition of TF-induced platelet activation compared to ardeparin and dalteparin. At 0.1 U/ml, these LMWHs produced a comparable inhibition of total P-selectin expression, and at 1.0 U/ml, a marked inhibition was noted. Since enoxaparin produced the best concentration-dependent inhibition of P-selectin expression (saline: 76 +/- 10% vs. 1.0 U/ml enoxaparin: 18 +/- 7%; P < .02) and platelet aggregate formation (saline: 63 +/- 7% vs. 1.0 U/ml enoxaparin: 35 +/- 6%, P < .035), this agent was used for additional studies. Unlike enoxaparin, tirofiban produced a weak concentration-dependent inhibition of platelet activation. At 100 ng/ml, tirofiban produced a 40% inhibition of P-selectin expression and about 60% inhibition of platelet aggregate formation. To elucidate the potential interaction between tirofiban and enoxaparin, the effect of 10 and 100 ng/ml tirofiban was studied with enoxaparin-supplemented WB in a 0.01-1.0 U/ml range. Additive effects between these two agents were noted only at lower concentrations. Thus, at therapeutic concentrations (0.8-1.2 U/ml), enoxaparin itself was capable of inhibiting TF-mediated activation of platelets to > 70%; whereas tirofiban failed to produce such concentration-dependent inhibition. This suggests that the simultaneous administration of GPIIb/IIIa receptor antagonist with LMWH may not have any added benefit in the clinical management of patients with ACS.

The effect of a novel glycoprotein IIb/IIIa antagonist, SR 121566A, on platelet aggregation and activation in rhesus monkeys.

SR 121566A represents a peptidomimetic glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitor 3-[N- 4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl ) -N-(1-carboxymethylpiperid-4-yl) amino] propionic acid, trihydrochloride. To investigate the intravenous and subcutaneous pharmacodynamics of this agent, a primate model ( Macaca mulatta) was used. The IC50 for adenosine diphosphate (ADP) (10 micromol/L)-induced platelet aggregation in this primate platelet system was found to be 45 +/- 6 nmol/L. Comparatively in the human platelet rich plasma system, SR 121566A demonstrated an IC50 of 39 +/- 4 nmol/L. Graded doses of SR 121566A in the range of 25-400 microg/kg were administered intravenously. Blood samples were drawn from individual groups of primates (n = 4-6) at varying periods of time up to 24 hours after administration of SR 121566A. The pharmacodynamic effects were measured by platelet aggregation using ADP (10 micromol/L) as an agonist. In addition, flow cytometric methods were used to measure thrombin receptor-activating peptide (TRAP) (6.25 micromol/L)-induced platelet activation. In the subcutaneous studies, 50, 100, 250, and 400 microg/kg of SR 121566A was administered with an identical blood-drawing schedule and analysis as with the intravenous studies. In the intravenous studies, all doses of SR 121566A produced > 80% inhibition of platelet aggregation 5 minutes after the administration of the drug. The duration of the inhibitory effect is proportional to the dose administered and the 50% recovery time ranged from 2 to 15 hours. By flow cytometry, TRAP-induced P-selectin expression was also blocked for a varying duration of time in a dose-dependent fashion. The subcutaneous studies showed > 90% inhibition of platelet aggregation, which was observed at 15 minutes after administration of both 50 and 100 microg/kg of the drug. The recovery time after the subcutaneously administered doses was found to be shorter than the intravenously administered doses. These studies demonstrate that SR 121566A is an effective platelet inhibitor with predictable pharmacokinetic and pharmacodynamic characteristics.