Current practices in pediatric hospital-acquired thromboembolism: Survey of the Children's Hospital Acquired Thrombosis (CHAT) Consortium.

Background: A rise in hospital-acquired venous thromboembolism (HA-VTE) in children has led to increased awareness regarding VTE prophylaxis and risk assessment. Despite no consensus exists regarding these practices in pediatrics. Objective: To describe common practices in VTE prophylaxis, VTE risk assessment models, and anticoagulation dosing strategies in pediatric hospitals that are members of the Children's Hospital Acquired Thrombosis (CHAT) Consortium. Methods: An electronic survey of 44 questions evaluating practices surrounding pediatric HA-VTE risk assessment and prevention was distributed between August 9, 2021, and August 30, 2021, to the primary investigators from the 32 institutions within the CHAT Consortium. Results: The survey response rate was 100% (n = 32). In total, 85% (n = 27) of the institutions assess HA-VTE, but only 63% (n = 20) have formal hospital guidelines. Within the institutions with formal guidelines, 100% (n = 20) include acute systemic inflammation or infection and presence of a central venous catheter (CVC) as risk factors for VTE. Pharmacologic prophylaxis is prescribed at 87% (28) of institutions, with enoxaparin being the most frequent (96%, n = 27). Variability in responses persisted regarding risk factors, risk assessment, thromboprophylaxis, dosing of prophylactic anticoagulation or anticoagulant drug monitoring. A majority of providers were comfortable providing thromboprophylaxis across all age groups. In addition, the global coronavirus disease 2019 increased the providers' use of prophylactic anticoagulation 78% (n = 25). Conclusion: Practices among institutions are variable in regard to use of HA-VTE prophylaxis, risk assessment, or guideline implementation, highlighting the need for further research and a validated risk assessment model through groups like the CHAT Consortium.

Hepatocellular malignant neoplasm, NOS: a clinicopathological study of 11 cases from a single institution.

AIMS: The primary aim of this study is to characterize hepatocellular malignant neoplasm, NOS (HEMNOS), a new provisional entity describing a subset of paediatric hepatocellular tumours, which have histological features of neither typical hepatoblastoma (HB) nor hepatocellular carcinoma (HCC). METHODS AND RESULTS: The clinicopathological features of 11 patients with HEMNOS were analysed retrospectively. The median age and serum alpha-fetoprotein level at diagnosis was 7 years and 182 000 ng/ml, respectively. Ten patients presented with pretreatment extent of disease (PRETEXT) stages III/IV multifocal tumours, eight with major vascular involvement, three with lung metastases and three with extrahepatic extension. The original pathology diagnoses were: HB in seven patients, HCC in two and HEMNOS in two. Our pathology review of pre-chemotherapy specimens showed that six tumours had equivocal/overlapping histological features of HB and HCC, four had predominant HB histology along with focal HCC-like histology and one had HB histology. Seven of nine post-chemotherapy resection specimens showed predominant HCC-like histology. Beta-catenin, glypican 3 and spalt-like transcription factor 4 immunostaining showed that all the tumours had a mixed HB/HCC immunophenotype. Telomerase reverse transcriptase immunostaining showed nuclear staining in nine of the 11 tumours. All patients received chemotherapy and achieved gross total primary tumour resection. Nine of the 11 patients were treated with established HB chemotherapy regimens. After a median follow-up of 6.1 years (range: 1.2-11.8 years), all patients were in remission. CONCLUSIONS: HEMNOS is a subtype of HB with focal HCC-like histology, a high-risk clinical profile but favourable outcome following chemotherapy and complete tumour resection.

Thromboelastographic characterization of the activated clotting system in children with sickle cell trait or sickle cell disease.

BACKGROUND: Recent epidemiological evidence suggests sickle cell disease (SCD) and sickle cell trait (SCT) is a risk factor for venous thromboembolism. The increased in-vivo markers of thrombin generation support the notion that such patients are in a chronic hypercoagulable state. In an attempt to better understand the underlying mechanism, global hemostatic assays including thrombin generation assay (TGA) and thromboelastography (TEG) have been utilized by several groups, but thus far, have shown inconsistent results either due to small sample size or technical differences. OBJECTIVES: Global hemostatic characterization of children with SCD or SCT by using TGA and modified TEG methods. MATERIALS AND METHODS: In this pilot study, we obtained TGA, TEG and other hemostatic data on specimens from 13 patients with SCD, 14 with SCT and 12 race-matched healthy controls (NC). RESULTS: R time and K time with modified TEG methods were significantly shorter in SCD when compared to SCT and NC. Alpha and MA did not show any significant differences between the groups. There was no difference seen between SCT and NC. TGA profiles did not show any difference between the three groups. As expected the in-vivo markers of thrombin generation and activation of fibrinolysis including D dimer and thrombin-antithrombin complexes were significantly higher in SCD subjects as compared to SCT and NC. CONCLUSION: The modified TEG methods are able to detect the activated coagulation system for the SCD population but a larger and more homogenous SCT cohort needs to be studied for more conclusive results.

Anti-inflammatory potency of nano-formulated puerarin and curcumin in rats subjected to the lipopolysaccharide-induced inflammation.

Puerarin (PU) and curcumin (CU), used commonly in traditional Chinese medicine and Ayurveda, have been shown to possess potent anti-inflammatory, anti-oxidation, and neuro-protective properties. Despite the experimental success of CU and PU in in vitro and animal models, their effectiveness has not yet been demonstrated in clinical trials, possibly because of their poor bioavailability. We hypothesized that gold nanoparticle (AuNP)-formulated PU (PU-AuNP), CU (CU-AuNP), or a combination of PU and CU (PU-CU-AuNP) were a more effective and nontoxic alternative to their bulk (nonformulated) counterparts. To test the hypothesis, bioavailability, therapeutic potency, and toxicity of bulk CU and/or PU were compared with those of their nanotized counterparts in rats subjected to the lipopolysaccharide (LPS)-induced inflammation. This study showed that a 20-mg/kg dose of bulk PU or a mixture of PU and CU did not, while their nanotized counterparts, PU-AuNP, CU-AuNP, or PU-CU-AuNP, effectively suppressed the LPS-induced inflammation and cytotoxicity in rats. In addition, PU-CU-AuNP was more potent than PU-AuNP or CU-AuNP alone. The blank AuNP (bAuNP) at ≤40 mg/kg dose did not cause any adverse effects (blood and brain lactic acid concentrations, kidney function, and neuronal apoptosis were measured) in animals. Therefore, the present observations suggest that a bi-functional AuNP loaded with CU and PU may effectively suppress the LPS-induced inflammation and cytotoxicity provided the following conditions are met: (1) The AuNP dose is at or below the no-effect dose; (2) the nanoparticles release a therapeutic dose of CU and PU in vivo; and (3) the active ingredients are released into the intracellular component of the brain.

Quantitative analysis of conjugated and free estrogens in swine manure: solutions to overcome analytical problems due to matrix effects.

Although animal manure is an important source for environmental estrogens, quantitative analysis of estrogens in manure is complicated due to matrix interference. In the present study, chromatographic methods have been developed for quantification of conjugated and free estrogens in manure samples collected from pig farms. The whole manure samples, immediately after collection, were stored at 4°C, acidified (pH≈2.0) and spiked with (i) (13)C-labeled internal standards to account for possible storage related degradation and (ii) deuterium labeled internal standards for calibration and quantitative analysis. The liquid samples were extracted with ethyl acetate for separating conjugated and free estrogens. The solid samples were eluted with water for desorbing conjugated hormones followed by methanol for desorbing free hormones. The water and extracts were further purified using hydrophilic-lipophilic balance and/or aminopropyl cartridges. The conjugated estrogens were analyzed using high-performance liquid chromatograph-mass spectrometer, while the free estrogens were analyzed using gas chromatograph-mass spectrometer. The extraction and calibration methods used in the present study yielded excellent sensitivity, reproducibility and >85% recovery of both free and conjugated estrogens that was independent of the manure matrix. In general, the total estrogen loads in liquid and solid samples were 5.1mg/l and 4.93mg/kg, respectively. This may represent the hormonal load of approximately 2.3tons estrogen per day.

In vitro neurogenesis from neural progenitor cells isolated from the hippocampus region of the brain of adult rats exposed to ethanol during early development through their alcohol-drinking mothers.

AIMS: This study was aimed to determine whether ethanol exposure during early development altered neurogenesis in the brain of adult rats. METHODS: Pregnant rats were given either ethanol-mixed or mannose-mixed (for control) rodent liquid diet ad libitum. Ethanol drinking continued during pregnancy and nursing. After weaning, the pups (AC(o): pups from control mothers, AE(o): pups from ethanol exposed mothers) received normal diet and water ad libitum for 11 weeks. Then the rats were anesthetized, their brains were collected and the hippocampal samples were processed for isolation of neural progenitor cells (NPCs). AC(o) NPCs and AE(o) NPCs were sequentially grown in media containing different growth factors that induced proliferation and differentiation. RESULTS AND CONCLUSIONS: Neuronal maturation was significantly delayed in ethanol-exposed rats. AC(o) NPCs, up to day 7 of culture, exhibited high beta-catenin-probe binding, an increase in Ca(2+) when exposed to gamma-amino butyric acid (GABA) and lack of response to glutamate (Glu) exposure. beta-Catenin-probe binding and the stimulatory effects of GABA declined thereafter. AC(o) NPCs, at culture day 29, exhibited high beta-catenin-probe binding, lack of response to GABA and elevated Glu-induced increase in Ca(2+i). Cultures of AE(o) NPCs showed an amplified stimulatory effects of GABA, attenuated stimulatory effects of Glu and attenuated the delayed (culture day 29) increase in the expression of Wnt proteins and beta-catenin-probe binding. This suggests a significant alteration in neurogenesis and synapse formation in adult rats exposed to ethanol at early development through their alcohol-drinking mothers.

Oxidative stress and protein oxidation in the brain of water drinking and alcohol drinking rats administered the HIV envelope protein, gp120.

Possible roles of oxidative stress and protein oxidation on alcohol-induced augmentation of cerebral neuropathy in gp120 administered alcohol preferring rats drinking either pure water (W rats) or a free-choice ethanol and water (E rats) for 90 days. This study showed that peripherally administered gp120 accumulated into the brain, liver, and RBCs samples from water drinking - gp120 administered rats (Wg rats) and ethanol drinking - gp120 administered rats (Eg rats), although gp120 levels in samples from Eg rats were significantly greater than the levels in samples from Wg rats. The brain samples from ethanol drinking-saline administered (EC) and Wg rats exhibited comparable levels of free radicals that were significantly lower than the levels in Eg rats. Peroxiredoxin-I (PrxI) activity in the brain samples exhibited the following pattern: Wg >> >> WC >> EC > Eg. Total protein-carbonyl and carbonylated hippocampal cholinergic neurostimulating peptide precursor protein levels, but not N-acetylaspartate or N-acetyl aspartylglutamate or total protein-thiol levels, paralleled the free radical levels in the brain of all four groups. This suggests PrxI inhibition may be more sensitive indicator of oxidative stress than measuring free radicals or metabolites. As PrxI oxidation in WC, Wg, and EC rats was reversible, while PrxI oxidation in Eg rats was not, we suggest that alcohol drinking and gp120 together hyperoxidized and inactivated PrxI that suppressed free radical neutralization in the brain of Eg rats. In conclusion, chronic alcohol drinking, by carbonylating and hyperoxidizing free radical neutralization proteins, augmented the gp120-induced oxidative stress that may be associated with an increase in severity of the brain neuropathy.

Effects of chronic alcohol drinking on receptor-binding, internalization, and degradation of human immunodeficiency virus 1 envelope protein gp120 in hepatocytes.

Although alcohol drinking increases susceptibility to human immunodeficiency virus (HIV) infection, possible mechanisms underlying the effects of alcohol are not yet known. Since the HIV envelope protein gp120 plays a key role in progression of HIV infection, the aim of the present study was to evaluate the toxicity and degradation of gp120 in hepatocytes isolated from liver of alcohol-preferring rats drinking either 15% ethanol in water or pure water for 70 days. The hypothesis was that alcohol drinking augmented the toxicity, but suppressed degradation of gp120. Hepatocytes from water-drinking rats (C-cells) or ethanol-drinking rats (Et-cells) were treated with laptacystin, anti-CD4 antibodies, CCR5 antagonist, or mannose, followed by [(125)I]gp120 or native gp120. At predetermined intervals, control (C) and ethanol exposed (Et) cells were analyzed for toxicity and degradation of gp120. In C-cells, [(125)I]gp120 binding and internalization peaked within 5-45 min and remained elevated for up to 10h and then decreased gradually. In Et-cells, [(125)I]gp120 binding peaked comparably to C-cells, but the binding remained to the peak level throughout the experimental period. C-cells exhibited the lysosomal/ubiquitin-mediated degradation of intracellular gp120, resulting in released gp120 fragments into the incubation medium that suppressed gp120-CD4 binding, improved cell viability, and inhibited gp120-induced apoptosis. Ethanol drinking suppressed gp120 degradation in and release of gp120 fragments from hepatocytes. The incubation medium of Et-cells did not suppress gp120-CD4 binding or the gp120-mediated apoptosis in hepatocytes. Thus, chronic alcohol drinking augmented the adverse effects of gp120 possibly by suppressing its degradation in hepatocytes. The present observation also suggests that a number of CCR5 or ubiquitin-based therapeutic drugs may not be effective in suppressing HIV infection in alcohol-drinking subjects.

Herbal mixtures consisting of puerarin and either polyenylphosphatidylcholine or curcumin provide comprehensive protection against alcohol-related disorders in P rats receiving free choice water and 15% ethanol in pure water.

Chronic alcohol drinking has been associated with the development of a number of abnormalities, including neuron-behavioral disorders, liver, pancreas, and heart-related diseases and inflammation and immune disorders. Because diverse mechanisms are involved in the development of these disorders, the commonly used receptor- or enzyme-specific drugs do not provide comprehensive protection against the adverse effects of alcoholism. This study describes possible therapeutic potency of puerarin (PU) from kudzu root, polyenylphosphatidylcholine from soy (SPCh), and curcumin (CU) from turmeric against alcohol's addiction-related and inflammatory-related abnormalities in alcohol-preferring P rats receiving free choice water and 15% ethanol in water. P-rats were fed once daily either the vehicle (for control) or different doses of PU, SPCh, CU, PU + SPCh, or PU + CU. The rats were divided in two groups: one received water alone, and the other free choice water and ethanol. Four rats from each group were fitted with electroencephalogram (EEG) electrodes for EEG recording. After 70 days of alcohol drinking, alcohol was withdrawn for 2 weeks, and the withdrawal symptoms were assessed. This study showed that alcohol drinking for 70 days (1) caused liver inflammation characterized by elevated tumor necrosis factor-alpha, interleukin-1beta, and matrix metalloproteinase-9 expression and (2) dysregulated lipopolysaccharide (LPS)-induced pleurisy. Alcohol withdrawal after 70 days of drinking generated severe withdrawal symptoms including seizure-type EEG activity. PU suppressed the addiction-mediated abnormalities but did not affect the inflammation-related abnormalities, while SPCh or CU suppressed only the inflammation-related abnormalities in alcohol-drinking rats subjected to LPS-induced pleurisy. A combination of PU with SPCh or CU suppressed both the addiction-related and inflammation-related abnormalities of alcohol drinking. Therefore, a mixture consisting of PU and either SPCh or CU may provide alternative therapy for alcohol-related disorders.

Effects of chronic ethanol drinking on the blood brain barrier and ensuing neuronal toxicity in alcohol-preferring rats subjected to intraperitoneal LPS injection.

AIMS: Although alcohol drinking impairs the blood-brain barrier (BBB), the underlying mechanism is not fully understood. Thus, the effects of chronic ethanol drinking on the BBB were studied in vivo. METHODS: Alcohol-preferring rats were given for 70 days free choice water and 15% ethanol. Then, they received LPS by i.p. injection. Efflux of [(14)C]sucrose or [(14)C]dextran was measured by their microinjection into the brain. Endothelial cells and neurons were isolated from the brain and analysed for mitogen-activated protein kinase (MAPK) and the tight-junction (TJ) protein phosphorylation, NFkappaB activation, mRNA levels of TJ proteins, inducible nitric oxide synthase, tumour necrosis factor alpha, interleukin-1 beta (IL-1beta), IL-10, CASPASE-8, and DNA damage. RESULTS: LPS transiently increased [(14)C]sucrose efflux in water drinking, while it caused a lasting increase in [(14)C]sucrose and [(14)C]dextran efflux in ethanol-drinking rats. The time-course of changes in the TJ correlated with (i) an increase in extracellular signal-regulated kinase (ERK), p38(mapk) Jun-N-terminal Kinase (JNK), and TJ protein phosphorylation, (ii) RelA-p50 and p50-p50 activation, and (iii) a decrease in the TJ proteins' mRNA levels in endothelial cells and neurons. Apoptotic cells were detected in water drinking and LPS (WC-LPS) neurons at 24 h after LPS exposure. Neurons from Et-LPS rats did not exhibit apoptosis. CONCLUSIONS: LPS injection in WC-LPS rats transiently disrupted the BBB. Lack of JNK activation and CASPASE-8 may be responsible for lack of apoptosis in endothelial cells and vice versa in neurons. Chronic alcohol drinking in ethanol drinking and LPS (Et-LPS) rats augmented and dysregulated the LPS-induced BBB abnormalities but suppressed apoptosis in neurons.

Effects of bacterial toxins on endothelial tight junction in vitro: a mechanism-based investigation.

ABSTRACT Lipopolysaccharide (LPS) and lipoteichoic acid (LTA), principal cell wall components of Gram-negative and Gram-positive bacteria, respectively, play a central role in altering the blood-brain barrier and facilitate bacterial infection of the host brain. Despite the significance of bacterial toxins in disease pathogenesis, mechanisms by which toxins impair the barrier are not yet known. This study, using an in vitro cell culture model, showed that LPS and LTA interacted with the endothelial cells and disrupted the tight junction between the cells that increased the barrier's permeability. Both toxins increased inducible nitric oxide synthase (iNOS) mRNA that is indicative of an increase in intracellular NO release, disrupted architecture of the tight junction proteins, suppressed zonula occludens-1 (ZO-1) and occludin (OCL) and junctional adhesive molecules (JAM) mRNA levels, and increased tumor necrosis factor alpha (TNFalpha) and interleukin-1 beta (IL-1beta) mRNA levels. Anti-CD14 antibodies blocked the increase in TNFalpha and IL-1beta mRNA levels but did not affect either changes in the tight junction or iNOS, ZO-1, OCL, and JAM mRNA levels in endothelial cells and astrocytes. Although both toxins did not cross the endothelial barrier, the abluminal neurons exhibited high inflammatory activity characterized by a sequential increase in TNFalpha, IL-1beta, external receptor kinase (ERK), and RelA-p50 that induced inflammation, followed by an increase in anti-inflammatory/apoptotic factors including IL-10 and cysteine-aspartic acid protease-8 (CASPASE-8), which resolve inflammation and induce apoptosis. Anti-CD14 antibodies in luminal buffer blocked the pro- and anti-inflammatory effects of the toxins in neurons. Thus, the CD14-TLR cascade that participates in the inflammatory effects of toxins may not participate in the toxin-induced barrier disruption in vitro. Since the toxins did not cross the endothelial barrier, induction of inflammation in neurons was due to a release of proinflammatory cytokines in the abluminal fluid.

Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma.

Darbepoietin (DAR) and recombinant human erythropoietin (rhEPO) stimulate erythropoiesis, leading to an increase in red blood cells. Along with their legitimate clinical use, rhEPO and DAR are also misused in racing horses for performance enhancement. To control the illegal use of DAR and rhEPO, it is important to develop analytical methods for the detection and confirmation of these proteins in plasma. Analysis of rhEPO and DAR in plasma is challenging due to the presence of a number of high abundance proteins including albumin that interferes with their extraction. The present study showed that the extraction of rhEPO or DAR from plasma using anti-EPO-antibody coupled immunoaffinity (IA) extraction yielded low (25-40%) recovery. Albumin-depletion using antialbumin-antibody coupled IA columns also depleted the target proteins and further reduced their recovery. Pre-extraction of spiked plasma using hydroxyapatite (HTP)-ProGel or ConA columns followed by the IA column yielded 65 to 70% recovery. The extracted samples were (i) analyzed directly with or without SDS-PAGE for intact proteins and (ii) analyzed after trypsin hydrolysis, with or without SDS-PAGE, for peptide fingerprinting using MALDI-TOF. Trypsin and enolase were used as internal calibrators for intact protein analysis and a peptide EYEATLEECCAK was used as internal calibrator for fragment analysis. Analysis of extracted sample without SDS-PAGE yielded, along with the target proteins (rhEPO and DAR), albumin and other related proteins. SDS-PAGE separated the target proteins with albumin and yielded clean samples. Inclusion of internal calibrators resulted in a linear dose-response relationship for both intact protein and digested fragments and allowed quantification of the target peptides. Thus, extraction of plasma using a combination of ConA and IA extractions yielded approximately 70% recovery of target proteins with a small amount of albumin and other proteins. SDS-PAGE improved the quality of the MALDI-TOF results. Minimum detection limits for digested fragments were lower than those for intact proteins.