Determination of fatty acid ethyl esters in dried blood spots by LC-MS/MS as markers for ethanol intake: application in a drinking study.

The forensic utility of fatty acid ethyl esters (FAEEs) in dried blood spots (DBS) as short-term confirmatory markers for ethanol intake was examined. An LC-MS/MS method for the determination of FAEEs in DBS was developed and validated to investigate FAEE formation and elimination in a drinking study, whereby eight subjects ingested 0.66-0.84 g/kg alcohol to reach blood alcohol concentrations (BAC) of 0.8 g/kg. Blood was taken every 1.5-2 h, BAC was determined, and dried blood spots were prepared, with 50 μL of blood, for the determination of FAEEs. Lower limits of quantitation (LLOQ) were between 15 and 37 ng/mL for the four major FAEEs. Validation data are presented in detail. In the drinking study, ethyl palmitate and ethyl oleate proved to be the two most suitable markers for FAEE determination. Maximum FAEE concentrations were reached in samples taken 2 or 4 h after the start of drinking. The following mean peak concentrations (c̅ max) were reached: ethyl myristate 14 ± 4 ng/mL, ethyl palmitate 144 ± 35 ng/mL, ethyl oleate 125 ± 55 ng/mL, ethyl stearate 71 ± 21 ng/mL, total FAEEs 344 ± 91 ng/mL. Detectability of FAEEs was found to be on the same time scale as BAC. In liquid blood samples containing ethanol, FAEE concentrations increase post-sampling. This study shows that the use of DBS fixation prevents additional FAEE formation in blood samples containing ethanol. Positive FAEE results obtained by DBS analysis can be used as evidence for the presence of ethanol in the original blood sample. Graphical Abstract Time courses for fatty acid ethyl ester (FAEE) concentrations in DBS and ethanol concentrations for subject 1 over a period of 7 h. Ethanol ingestion occured during the first hour of the time course.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Blood glucose and prognosis in children with presumed severe malaria: is there a threshold for 'hypoglycaemia'?

OBJECTIVES: Hypoglycaemia (glucose <2.2 mmol/l) is a defining feature of severe malaria, but the significance of other levels of blood glucose has not previously been studied in children with severe malaria. METHODS: A prospective study of 437 consecutive children with presumed severe malaria was conducted in Mali. We defined hypoglycaemia as <2.2 mmol/l, low glycaemia as 2.2-4.4 mmol/l and hyperglycaemia as >8.3 mmol/l. Associations between glycaemia and case fatality were analysed for 418 children using logistic regression models and a receiver operator curve (ROC). RESULTS: There was a significant difference between blood glucose levels in children who died (median 4.6 mmol/l) and survivors (median 7.6 mmol/l, P < 0.001). Case fatality declined from 61.5% of the hypoglycaemic children to 46.2% of those with low glycaemia, 13.4% of those with normal glycaemia and 7.6% of those with hyperglycaemia (P < 0.001). Logistic regression showed an adjusted odds ratio (AOR) of 0.75 (0.64-0.88) for case fatality per 1 mmol/l increase in baseline blood glucose. Compared to a normal blood glucose, hypoglycaemia and low glycaemia both significantly increased the odds of death (AOR 11.87, 2.10-67.00; and 5.21, 1.86-14.63, respectively), whereas hyperglycaemia reduced the odds of death (AOR 0.34, 0.13-0.91). The ROC [area under the curve at 0.753 (95% CI 0.684-0.820)] indicated that glycaemia had a moderate predictive value for death and identified an optimal threshold at glycaemia <6.1 mmol/l, (sensitivity 64.5% and specificity 75.1%). CONCLUSIONS: If there is a threshold of blood glucose which defines a worse prognosis, it is at a higher level than the current definition of 2.2 mmol/l.

A case of Maffucci syndrome

The Maffucci syndrome consists of a combination of multiple enchondromas and haemangiomas. It appears in the first two decades of life, with no family history. In this case we are reporting about a 26-year-old female who had suffered from multiple enchondromas since the age of two. At the age of nine, the patient presented with additional haemangiomas, which facilitated making proper diagnosis. She now presents with a massive lesion of her left upper extremity. The patient had initially rejected operative treatment when the disease was at early stages. At later stages, a more complex reconstruction of the hand would have been necessary to secure hand function. This procedure that sometimes induces a risk related to potential necessity of blood transfusion was rejected by the patient for religious reasons. Amputation of the extremity was therefore the last resort procedure.

Pulse or hyaline ring granuloma. Review of the literature on etiopathogenesis of oral and extraoral lesions

Since the late 1950s, reports on an unusual giant-cell granulomatous lesion affecting the jaws, lungs, stomach and intestines have been published. Histopathologically, the lesions showed the presence of structureless hyaline rings with multinucleated giant cells. The aim of this review was to summarize the literature on the etiopathogenesis of the so-called oral and extraoral pulse or hyaline ring granuloma. Literature was searched using PubMed and Medline. In addition, hand search was performed. Search words were oral and extraoral hyaline ring granuloma, giant-cell hyaline angiopathy, pulse granuloma and chronic periostitis. Numerous terms for hyaline ring granuloma have been introduced over time (1971-2008). One hundred seventy-three cases of oral hyaline ring granuloma have been retrieved from the literature. In the mandible, 72.3% occurred . Two theories for etiopathogenesis have been proposed: (1) the origin of the hyaline rings is due to a foreign material (pulse and legumes) having penetrated the oral mucosa or gastrointestinal tract and lungs (exogenous theory) and (2) the rings are due to hyaline degenerative changes in walls of blood vessels (endogenous theory). Experimental production of oral and extraoral hyaline ring granulomas is consistent with the exogenous origin. Particles or remains of leguminous cells having been implanted or aspirated into human tissues whether located to the oral cavity or throughout the entire digestive tract and respiratory system are thought to be causative. Pulse or hyaline ring granulomas are rare but are well-defined oral and extraoral lesions due to implantation of the cellulose moiety of plant foods in contrast to the starch components.

Development and in-vitro characterization of an implantable flow sensing transducer for hydrocephalus

An implantable transducer for monitoring the flow of Cerebrospinal fluid (CSF) for the treatment of hydrocephalus has been developed which is based on measuring the heat dissipation of a local thermal source. The transducer uses passive telemetry at 13.56 MHz for power supply and read out of the measured flow rate. The in vitro performance of the transducer has been characterized using artificial Cerebrospinal Fluid (CSF) with increased protein concentration and artificial CSF with 10\% fresh blood. After fresh blood was added to the artificial CSF a reduction of flow rate has been observed in case that the sensitive surface of the flow sensor is close to the sedimented erythrocytes. An increase of flow rate has been observed in case that the sensitive surface is in contact with the remaining plasma/artificial CSF mix above the sediment which can be explained by an asymmetric flow profile caused by the sedimentation of erythrocythes having increased viscosity compared to artificial CSF. After removal of blood from artificial CSF, no drift could be observed in the transducer measurement which could be associated to a deposition of proteins at the sensitive surface walls of the packaged flow transducer. The flow sensor specification requirement of +-10\% for a flow range between 2 ml/h and 40 ml/h. could be confirmed at test conditions of 37 degrees C.

Cardiovascular changes after PMMA vertebroplasty in sheep: the effect of bone marrow removal using pulsed jet-lavage

Clinically, the displacement of intravertebral fat into the circulation during vertebroplasty is reported to lead to problems in elderly patients and can represent a serious complication, especially when multiple levels have to be treated. An in vitro study has shown the feasibility of removing intravertebral fat by pulsed jet-lavage prior to vertebroplasty, potentially reducing the embolization of bone marrow fat from the vertebral bodies and alleviating the cardiovascular changes elicited by pulmonary fat embolism. In this in vivo study, percutaneous vertebroplasty using polymethylmethacrylate (PMMA) was performed in three lumbar vertebrae of 11 sheep. In six sheep (lavage group), pulsed jet-lavage was performed prior to injection of PMMA compared to the control group of five sheep receiving only PMMA vertebroplasty. Invasive recording of blood pressures was performed continuously until 60 min after the last injection. Cardiac output and arterial blood gas parameters were measured at selected time points. Post mortem, the injected cement volume was measured using CT and lung biopsies were processed for assessment of intravascular fat. Pulsed jet-lavage was feasible in the in vivo setting. In the control group, the injection of PMMA resulted in pulmonary fat embolism and a sudden and significant increase in mean pulmonary arterial pressure. Pulsed jet-lavage prevented any cardiovascular changes and significantly reduced the severity of bone marrow fat embolization. Even though significantly more cement had been injected into the lavaged vertebral bodies, significantly fewer intravascular fat emboli were identified in the lung tissue. Pulsed jet-lavage prevented the cardiovascular complications after PMMA vertebroplasty in sheep and alleviated the severity of pulmonary fat embolism.

Pros and cons of post-mortem CT imaging on aspiration diagnosis

Recently, the field of forensics has experienced a rapid increase in the use of modern cross-sectional imaging in forensic investigations. We examined the value of post-mortem computed tomography (CT) imaging relative to autopsy for distinguishing aspiration into the lungs from airways, from lung alterations due to other causes, and for identifying the aspirated material. We selected 54 bodies submitted to whole-body CT scanning prior to autopsy. All cases had autopsy findings of blood (31 cases), fresh water (12 cases), or gastric content (11 cases) aspiration. The radiological images were retrospectively analyzed for airway and lung aspiration. In all cases, CT imaging detected pulmonary abnormalities suggestive of aspiration. Nevertheless, analysis of the CT images alone was not able to identify the aspirated material or to distinguish pulmonary findings of aspiration from lung changes due to other causes, except for a few cases of hemo-aspiration. However, due to its ability to visualize the entire parenchyma, CT imaging was superior to autopsy in providing additional data about the distribution and severity of the aspiration as well as in detecting small abnormalities. Post-mortem CT imaging should be considered as a superior tool for forensic investigations of aspiration due to its ability to document diagnostic conclusions and to guide the forensic pathologist during lung tissue examination.

Blood aspiration as a vital sign detected by postmortem computed tomography imaging

Blood aspiration is a significant forensic finding. In this study, we examined the value of postmortem computed tomography (CT) imaging in evaluating findings of blood aspiration. We selected 37 cases with autopsy evidence of blood in the lungs and/or in the airways previously submitted to total-body CT scanning. The CT-images were retrospectively analyzed. In one case with pulmonary blood aspiration, biopsy specimens were obtained under CT guide for histological examination. In six cases, CT detected pulmonary abnormalities suggestive of blood aspiration, not mentioned in the autopsy reports. CT reconstructions provided additional data about the distribution and extent of aspiration. In one needle-biopsied case, the pulmonary specimens showed blood in the alveoli. We suggest the use of CT imaging as a tool complementary to traditional techniques in cases of blood aspiration to avoid misdiagnosis, to guide the investigation of lung tissue, and to allow for more evidence-based inferences on the cause of death.

Differential roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium

Endothelial ICAM-1 and ICAM-2 were shown to be essential for T cell diapedesis across the blood-brain barrier (BBB) in vitro under static conditions. Crawling of T cells prior to diapedesis was only recently revealed to occur preferentially against the direction of blood flow on the endothelial surface of inflamed brain microvessels in vivo. Using live cell-imaging techniques, we prove that Th1 memory/effector T cells predominantly crawl against the direction of flow on the surface of BBB endothelium in vitro. Analysis of T cell interaction with wild-type, ICAM-1-deficient, ICAM-2-deficient, or ICAM-1 and ICAM-2 double-deficient primary mouse brain microvascular endothelial cells under physiological flow conditions allowed us to dissect the individual contributions of endothelial ICAM-1, ICAM-2, and VCAM-1 to shear-resistant T cell arrest, polarization, and crawling. Although T cell arrest was mediated by endothelial ICAM-1 and VCAM-1, T cell polarization and crawling were mediated by endothelial ICAM-1 and ICAM-2 but not by endothelial VCAM-1. Therefore, our data delineate a sequential involvement of endothelial ICAM-1 and VCAM-1 in mediating shear-resistant T cell arrest, followed by endothelial ICAM-1 and ICAM-2 in mediating T cell crawling to sites permissive for diapedesis across BBB endothelium.

Regulation of GPCR signaling in hypertension

Hypertension represents a complex, multifactorial disease and contributes to the major causes of morbidity and mortality in industrialized countries: ischemic and hypertensive heart disease, stroke, peripheral atherosclerosis and renal failure. Current pharmacological therapy of essential hypertension focuses on the regulation of vascular resistance by inhibition of hormones such as catecholamines and angiotensin II, blocking them from receptor activation. Interaction of G-protein coupled receptor kinases (GRKs) and regulator of G-protein signaling (RGS) proteins with activated G-protein coupled receptors (GPCRs) effect the phosphorylation state of the receptor leading to desensitization and can profoundly impair signaling. Defects in GPCR regulation via these modulators have severe consequences affecting GPCR-stimulated biological responses in pathological situations such as hypertension, since they fine-tune and balance the major transmitters of vessel constriction versus dilatation, thus representing valuable new targets for anti-hypertensive therapeutic strategies. Elevated levels of GRKs are associated with human hypertensive disease and are relevant modulators of blood pressure in animal models of hypertension. This implies therapeutic perspective in a disease that has a prevalence of 65million in the United States while being directly correlated with occurrence of major adverse cardiac and vascular events. Therefore, therapeutic approaches using the inhibition of GRKs to regulate GPCRs are intriguing novel targets for treatment of hypertension and heart failure.

Mobile phone technologies and advanced data analysis towards the enhancement of diabetes self-management

Advances in the area of mobile and wireless communication for healthcare (m-Health) along with the improvements in information science allow the design and development of new patient-centric models for the provision of personalised healthcare services, increase of patient independence and improvement of patient's self-control and self-management capabilities. This paper comprises a brief overview of the m-Health applications towards the self-management of individuals with diabetes mellitus and the enhancement of their quality of life. Furthermore, the design and development of a mobile phone application for Type 1 Diabetes Mellitus (T1DM) self-management is presented. The technical evaluation of the application, which permits the management of blood glucose measurements, blood pressure measurements, insulin dosage, food/drink intake and physical activity, has shown that the use of the mobile phone technologies along with data analysis methods might improve the self-management of T1DM.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A? followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Adsorption of Enamel Matrix Proteins to a Bovine Derived Bone Grafting Material and its Regulation of Cell Adhesion, Proliferation and Differentiation

The use of various combinations of enamel matrix derivative (EMD) and grafting materials has been shown to promote periodontal wound healing/regeneration. However, the downstream cellular behavior of periodontal ligament (PDL) cells and osteoblasts has not yet been studied. Furthermore, it is unknown to what extent the bleeding during regenerative surgery may influence the adsorption of exogenous proteins to the surface of bone grafting materials and the subsequent cellular behavior. In the present study, the aim is to test EMD adsorption to the surface of natural bone mineral (NBM) particles in the presence of blood and determine the effect of EMD coating to NBM particles on downstream cellular pathways, such as adhesion, proliferation, and differentiation of primary human osteoblasts and PDL cells.

Formation of Phosphatidylethanol and Its Subsequent Elimination During an Extensive Drinking Experiment Over 5 Days

BACKGROUND: For almost 30 years, phosphatidylethanol (PEth) has been known as a direct marker of alcohol consumption. This marker stands for consumption in high amounts and for a longer time period, but it has been also detected after 1 high single intake of ethanol (EtOH). The aim of this study was to obtain further information about the formation and elimination of PEth 16:0/18:1 by simulating extensive drinking. METHODS: After 3 weeks of alcohol abstinence, 11 test persons drank an amount of EtOH leading to an estimated blood ethanol concentration of 1 g/kg on each of 5 successive days. After the drinking episode, they stayed abstinent for 16 days with regular blood sampling. PEth 16:0/18:1 analysis was performed using liquid chromatography-tandem mass spectrometry (high-performance liquid chromatography 1100 system and QTrap 2000 triple quadrupole linear ion trap mass spectrometer. Values of blood alcohol were obtained using a standardized method with headspace gas chromatography flame ionization detector. RESULTS: Maximum measured concentrations of EtOH were 0.99 to 1.83 g/kg (mean 1.32 g/kg). These values were reached 1 to 3 hours after the start of drinking (mean 1.9 hours). For comparison, 10 of 11 volunteers had detectable PEth 16:0/18:1 values 1 hour after the start of drinking, ranging from 45 to 138 ng/ml PEth 16:0/18:1. Over the following days, concentrations of PEth 16:0/18:1 increased continuously and reached the maximum concentrations of 74 to 237 ng/ml between days 3 and 6. CONCLUSIONS: This drinking experiment led to measurable PEth concentrations. However, PEth 16:0/18:1 concentrations stayed rather low compared with those of alcohol abusers from previous studies.