Accuracy profile validation of a new method for carbon monoxide measurement in the human blood using headspace-gas chromatography-mass spectrometry (HS-GC-MS).

The aim of our study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable for the routine determination of blood CO concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature for CO measurement is the absence of a specific CO internal standard necessary for performing quantification. Even if stable isotope of CO is commercially available in the gaseous state, it is essential to develop a safer method to limit the manipulation of gaseous CO and to precisely control the injected amount of CO for spiking and calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in a vial in situ, an internal labeled standard gas ((13)CO) formed by the reaction of labeled formic acid formic acid (H(13)COOH) with sulfuric acid. As sulfuric acid can also be employed to liberate the CO reagent from whole blood, the procedure allows for the liberation of CO simultaneously with the generation of (13)CO. This method allows for precise measurement of blood CO concentrations from a small amount of blood (10 μL). Finally, this method was applied to measure the CO concentration of intoxicated human blood samples from autopsies.

Repeated exposure to Lutzomyia intermedia sand fly saliva induces local expression of interferon-inducible genes both at the site of injection in mice and in human blood.

During a blood meal, Lutzomyia intermedia sand flies transmit Leishmania braziliensis, a parasite causing tegumentary leishmaniasis. In experimental leishmaniasis, pre-exposure to saliva of most blood-feeding sand flies results in parasite establishment in absence of any skin damages in mice challenged with dermotropic Leishmania species together with saliva. In contrast, pre-immunization with Lu. intermedia salivary gland sonicate (SGS) results in enhanced skin inflammatory exacerbation upon co-inoculation of Lu. intermedia SGS and L. braziliensis. These data highlight potential unique features of both L. braziliensis and Lu. intermedia. In this study, we investigated the genes modulated by Lu. intermedia SGS immunization to understand their potential impact on the subsequent cutaneous immune response following inoculation of both SGS and L. braziliensis. The cellular recruitment and global gene expression profile was analyzed in mice repeatedly inoculated or not with Lu. intermedia. Microarray gene analysis revealed the upregulation of a distinct set of IFN-inducible genes, an immune signature not seen to the same extent in control animals. Of note this INF-inducible gene set was not induced in SGS pre-immunized mice subsequently co-inoculated with SGS and L. braziliensis. These data suggest the parasite prevented the upregulation of this Lu. intermedia saliva-related immune signature. The presence of these IFN-inducible genes was further analyzed in peripheral blood mononuclear cells (PBMCs) sampled from uninfected human individuals living in a L. braziliensis-endemic region of Brazil thus regularly exposed to Lu. intermedia bites. PBMCs were cultured in presence or absence of Lu. intermedia SGS. Using qRT-PCR we established that the IFN-inducible genes induced in the skin of SGS pre-immunized mice, were also upregulated by SGS in PBMCs from human individuals regularly exposed to Lu. intermedia bites, but not in PBMCs of control subjects. These data demonstrate that repeated exposure to Lu. intermedia SGS induces the expression of potentially host-protective IFN-inducible genes.

Measurement of immunoreactive angiotensin-(1-7) heptapeptide in human blood.

BACKGROUND: The renal enzyme renin cleaves from the hepatic alpha(2)-globulin angiotensinogen angiotensin-(1-10) decapeptide [Ang-(1-10)], which is further metabolized to smaller peptides that help maintain cardiovascular homeostasis. The Ang-(1-7) heptapeptide has been reported to have several physiological effects, including natriuresis, diuresis, vasodilation, and release of vasopressin and prostaglandins. METHODS: To investigate Ang-(1-7) in clinical settings, we developed a method to measure immunoreactive (ir-) Ang-(1-7) in 2 mL of human blood and to estimate plasma concentrations by correcting for the hematocrit. A sensitive and specific antiserum against Ang-(1-7) was raised in a rabbit. Human blood was collected in the presence of an inhibitor mixture including a renin inhibitor to prevent peptide generation in vitro. Ang-(1-7) was extracted into ethanol and purified on phenylsilylsilica. The peptide was quantified by radioimmunoassay. Increasing doses of Ang-(1-7) were infused into volunteers, and plasma concentrations of the peptide were measured. RESULTS: The detection limit for plasma ir-Ang-(1-7) was 1 pmol/L. CVs for high and low blood concentrations were 4% and 20%, respectively, and between-assay CVs were 8% and 13%, respectively. Reference values for human plasma concentrations of ir-Ang-(1-7) were 1.0-9.5 pmol/L (median, 4.7 pmol/L) and increased linearly during infusion of increasing doses of Ang-(1-7). CONCLUSIONS: Reliable measurement of plasma ir-Ang-(1-7) is achieved with efficient inhibition of enzymes that generate or metabolize Ang-(1-7) after blood sampling, extraction in ethanol, and purification on phenylsilylsilica, and by use of a specific antiserum.

A New Approach for the Carbon Monoxide (CO) Exposure Diagnosis: Measurement of Total CO in Human Blood Versus Carboxyhemoglobin (HbCO).

The aim of the study is to present the application of a headspace-gas chromatography-mass spectrometry (HS-GC-MS) method for the determination of the carbon monoxide (CO) blood concentration and to compare it with carboxyhemoglobin (HbCO) saturation. In postmortem cases, the HbCO measured by spectrophotometry frequently leads to inaccurate results due to inadequate samples or analyses. The true role of CO intoxication in the death of a person could be misclassified. The estimation of HbCO from HS-GC-MS CO measurements provides helpful information by determining the total CO levels (CO linked to hemoglobin (HbCO) and CO dissociated from hemoglobin). The CO concentrations were converted in HbCO saturation levels to define cutoff blood CO values. CO limits were defined as less than 1 μmol/mL for living persons, less than 1.5 μmol/mL for dead persons without CO exposure, and greater than 3 μmol/mL for dead persons with clear CO poisoning.

Transfer of ultrasmall iron oxide nanoparticles from human brain-derived endothelial cells to human glioblastoma cells.

Nanoparticles (NPs) are being used or explored for the development of biomedical applications in diagnosis and therapy, including imaging and drug delivery. Therefore, reliable tools are needed to study the behavior of NPs in biological environment, in particular the transport of NPs across biological barriers, including the blood-brain tumor barrier (BBTB), a challenging question. Previous studies have addressed the translocation of NPs of various compositions across cell layers, mostly using only one type of cells. Using a coculture model of the human BBTB, consisting in human cerebral endothelial cells preloaded with ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) and unloaded human glioblastoma cells grown on each side of newly developed ultrathin permeable silicon nitride supports as a model of the human BBTB, we demonstrate for the first time the transfer of USPIO NPs from human brain-derived endothelial cells to glioblastoma cells. The reduced thickness of the permeable mechanical support compares better than commercially available polymeric supports to the thickness of the basement membrane of the cerebral vascular system. These results are the first report supporting the possibility that USPIO NPs could be directly transferred from endothelial cells to glioblastoma cells across a BBTB. Thus, the use of such ultrathin porous supports provides a new in vitro approach to study the delivery of nanotherapeutics to brain cancers. Our results also suggest a novel possibility for nanoparticles to deliver therapeutics to the brain using endothelial to neural cells transfer.

Heterogeneity of human monocytes: an optimized four-color flow cytometry protocol for analysis of monocyte subsets.

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5-conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14(+)CD16(-) monocytes (here termed "Mo1" subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX(3)CR1, whereas "nonclassical" CD14(lo)CD16(+) monocytes (Mo3) essentially showed the inverse expression pattern. CD14(+)CD16(+) monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas "nonclassical" monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX(3)CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition.

A phenotypical study of vascular smooth muscle cells in human arterial and venous stenosis

Abstract Introduction The primary function of the contractile vascular smooth muscle cells (cVSMCs) is the regulation of the vascular contractility which means the adaptation of the vascular tonus in response to the modulation of the blood pressure and blood flow. The cVSMCs are essentially quiescent, and therefore their synthesis rate is very limited. They are characterized by the expression of contractile proteins specific to the muscular tissue including myosin, h-­‐caldesmon and <-­‐smooth muscle actin (〈-­‐SMA). These contractile cells are strongly represented in the media layer of the arterial wall and, in a smaller proportion, of the vein wall. Their typical stretched-­‐out morphology allows recognizing them by a histological analysis. They do not produce any extracellular matrix (ECM), and do not migrate through the different layers of the vessel wall, and are not directly involved in the development of intimal hyperplasia (IH). Neointimal formation occurs after endothelial disruption leading to complex molecular and biological mechanisms. The de-­‐differentiation of cVSMCs into synthetic VSMCs (sVSMCs) is mentioned as a key element. These non mature cells are able to proliferate and produce ECM. The characterization of the vascular smooth muscle cells (VSMCs) from healthy and stenosed vascular tissues will contribue to the understanding of the different biological processes leading to IH and will be useful for the development of new therapies to interfere with the cVSMCs growth and migration. The aim of our research was to quantify the proportion of cVSMCs and sVSMCs into the healthy and pathologic human blood vessel wall and to characterize their phenotype. Methods We selected 23 specimens of arterial and venous segments from 18 patients. All these specimens were stored in the biobank from the thoracic and vascular surgery departement. 4 groups were designed (group 1 :arteries without lesions (n=3) ;group 2 : veins without lesions (n=1); group 3: arteries with stenosis (n=9); group 4: veins with stenosis (n=10)). Histology: 5µm-­‐sections were made from each sample embedded in paraffin wax and further stained with hematoxylin & eosin (HE), Van Gieson's stain (VGEL) and Masson's Trichrome (TMB). Pathologic tissues were defined using the label that was given to the macroscopic samples by the surgeon and also, based on the histological analysis with HE and VGEL evaluating the presence of a thickened intima. The same was done to the control samples evaluating the absence of thickening. Immunohistochemistry : The primary antibodies were used :〈-­‐SMA, vimentin, h-­‐ caldesmon, calponin, smooth muscle-myosin heavy chain (SM-­‐MHC), tropomyosin-­‐4, retinol binding protein-­‐1 (RBP-­‐1), nonmuscle-­‐myosin heavy chain-­‐B (NM-­‐MHC-­‐B), Von Willebrand factor (VWF). A semi-­‐quantitative assessment of the intensity of each sample stained was performed. Western Blot : Segments of arteries and veins were analyzed using the following primary antibodies :〈-­‐SMA, Calponin, SM-­‐MHC, NM-­‐MHC-­‐B. The given results were then normalized with tubulin. Results Our data showed that, when using immunohistochemistry analysis we found that〈-­‐SMA was mostly expressed in control arteries, whereas NM-­‐MHC-­‐B in the pathologic ones. Using SM-­‐MHC, calponin, vimentin and caldesmon we found no significative differences in the expression of these proteins in the control and in the pathologic samples. Western Blot analysis showed an inverse correlation between healthy and pathological samples as <-­‐ SMA was more expressed in the pathological samples, while NM-­‐MHC-­‐B in the control group; SM-­‐MHC and calponin were mostly expressed in the pathologic samples. Conclusion Our study showed no clear differences between stenotic and control arterial and venous segments using semi-­‐quantitative assessement by immunohistochemistry. Western Blot showed a significant increased expression of 〈-­‐SMA, calponin and SM-­‐MHC in the arteries with stenosis, while NM-­‐MHC-­‐B was mostly expressed in the arteries without lesions. Further studies are needed to track the lineage of VSMCs to understand the mechanisms leading toIH.

Blood plasma lipidomic signature of epicardial fat in healthy obese women.

OBJECTIVES: A lipidomic approach was employed in a clinically well-defined cohort of healthy obese women to explore blood lipidome phenotype ascribed to body fat deposition, with emphasis on epicardial adipose tissue (EAT). METHODS: The present investigation delivered a lipidomics signature of epicardial adiposity under healthy clinical conditions using a cohort of 40 obese females (age: 25-45 years, BMI: 28-40 kg/m(2) ) not showing any metabolic disease traits. Lipidomics analysis of blood plasma was employed in combination with in vivo quantitation of mediastinal fat depots by computerized tomography. RESULTS: All cardiac fat depots correlated to indicators of hepatic dysfunctions (ALAT and ASAT), which describe physiological connections between hepatic and cardiac steatosis. Plasma lipidomics encompassed overall levels of lipid classes, fatty acid profiles, and individual lipid species. EAT and visceral fat associated with diacylglycerols (DAG), triglycerides, and distinct phospholipid and sphingolipid species. A pattern of DAG and phosphoglycerols was specific to EAT. CONCLUSIONS: Human blood plasma lipidomics appears to be a promising clinical and potentially diagnostic readout for patient stratification and monitoring. Association of blood lipidomics signature to regio-specific mediastinal and visceral adiposity under healthy clinical conditions may help provide more biological insights into obese patient stratification for cardiovascular disease risks.

RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise.

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.

Evaluation of uptake and transport of ultrasmall superparamagnetic iron oxide nanoparticles by human brain-derived endothelial cells.

Aim: Ultrasmall superparamagnetic iron oxide nanoparticles (USPIO-NPs) are under development for imaging and drug delivery; however, their interaction with human blood-brain barrier models is not known. Materials & Methods: The uptake, reactive oxygen species production and transport of USPIO-NPs across human brain-derived endothelial cells as models of the blood-brain tumor barrier were evaluated for either uncoated, oleic acid-coated or polyvinylamine-coated USPIO-NPs. Results: Reactive oxygen species production was observed for oleic acid-coated and polyvinylamine-coated USPIO-NPs. The uptake and intracellular localization of the iron oxide core of the USPIO-NPs was confirmed by transmission electron microscopy. However, while the uptake of these USPIO-NPs by cells was observed, they were neither released by nor transported across these cells even in the presence of an external dynamic magnetic field. Conclusion: USPIO-NP-loaded filopodia were observed to invade the polyester membrane, suggesting that they can be transported by migrating angiogenic brain-derived endothelial cells.

Urotensin-II System in Genetic Control of Blood Pressure and Renal Function.

Urotensin-II controls ion/water homeostasis in fish and vascular tone in rodents. We hypothesised that common genetic variants in urotensin-II pathway genes are associated with human blood pressure or renal function. We performed family-based analysis of association between blood pressure, glomerular filtration and genes of the urotensin-II pathway (urotensin-II, urotensin-II related peptide, urotensin-II receptor) saturated with 28 tagging single nucleotide polymorphisms in 2024 individuals from 520 families; followed by an independent replication in 420 families and 7545 unrelated subjects. The expression studies of the urotensin-II pathway were carried out in 97 human kidneys. Phylogenetic evolutionary analysis was conducted in 17 vertebrate species. One single nucleotide polymorphism (rs531485 in urotensin-II gene) was associated with adjusted estimated glomerular filtration rate in the discovery cohort (p = 0.0005). It showed no association with estimated glomerular filtration rate in the combined replication resource of 8724 subjects from 6 populations. Expression of urotensin-II and its receptor showed strong linear correlation (r = 0.86, p<0.0001). There was no difference in renal expression of urotensin-II system between hypertensive and normotensive subjects. Evolutionary analysis revealed accumulation of mutations in urotensin-II since the divergence of primates and weaker conservation of urotensin-II receptor in primates than in lower vertebrates. Our data suggest that urotensin-II system genes are unlikely to play a major role in genetic control of human blood pressure or renal function. The signatures of evolutionary forces acting on urotensin-II system indicate that it may have evolved towards loss of function since the divergence of primates.

BDNF promoter I methylation correlates between post-mortem human peripheral and brain tissues.

Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n = 98, Pearson, r = 0.48, p = 4.5 × 10(-7)). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n = 37, r = 0.74, p = 1.4 × 10(-7)). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood.

Higher memory responses in HIV-infected and kidney transplanted patients than in healthy subjects following priming with the pandemic vaccine.

BACKGROUND: Memory responses require immune competence. We assessed the influence of priming with AS03-adjuvanted pandemic vaccine (Pandemrix®) on memory responses of HIV patients, kidney recipients (SOT) and healthy controls (HC). METHOD: Participants (HIV: 197, SOT: 53; HC: 156) were enrolled in a prospective study and 390/406 (96%) completed it. All had been primed in 2009/2010 with 1 (HC) or 2 (patients) doses of Pandemrix®, and were boosted with the 2010/2011 seasonal influenza vaccine. Geometric mean titres and seroprotection rates were measured 12 months after priming and 4 weeks after boosting. Primary and memory responses were directly compared in 191 participants (HCW: 69, HIV: 71, SOT: 51) followed during 2 consecutive seasons. RESULTS: Most participants (HC: 77.8%, HIV: 77.6%, SOT: 66%) remained seroprotected at 12 months post-priming. Persisting A/09/H1N1 titers were high in HIV (100.2) and HC (120.1), but lower in SOT (61.4) patients. Memory responses reached higher titers in HIV (507.8) than in HC (253.5) and SOT (136.9) patients. Increasing age and lack of HAART reduced persisting and memory responses, mainly influenced by residual antibody titers. Comparing 2009/2010 and 2010/2011 titers in 191 participants followed for 2 seasons indicated lower post-2010/2011 titers in HC (240.2 vs 313.9), but higher titers in HIV (435.7 vs 338.0) and SOT (136 vs 90.3) patients. CONCLUSIONS: Priming with 2 doses of Pandemrix® elicited persistent antibody responses and even stronger memory responses to non-adjuvanted seasonal vaccine in HIV patients than 1 dose in healthy subjects. Adjuvanted influenza vaccines may improve memory responses of immunocompromised patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT01022905.

Malaria haplotype frequency estimation.

We present a Bayesian approach for estimating the relative frequencies of multi-single nucleotide polymorphism (SNP) haplotypes in populations of the malaria parasite Plasmodium falciparum by using microarray SNP data from human blood samples. Each sample comes from a malaria patient and contains one or several parasite clones that may genetically differ. Samples containing multiple parasite clones with different genetic markers pose a special challenge. The situation is comparable with a polyploid organism. The data from each blood sample indicates whether the parasites in the blood carry a mutant or a wildtype allele at various selected genomic positions. If both mutant and wildtype alleles are detected at a given position in a multiply infected sample, the data indicates the presence of both alleles, but the ratio is unknown. Thus, the data only partially reveals which specific combinations of genetic markers (i.e. haplotypes across the examined SNPs) occur in distinct parasite clones. In addition, SNP data may contain errors at non-negligible rates. We use a multinomial mixture model with partially missing observations to represent this data and a Markov chain Monte Carlo method to estimate the haplotype frequencies in a population. Our approach addresses both challenges, multiple infections and data errors.