Identification of phospholipid structures in human blood by direct-injection quadrupole-linear ion-trap mass spectrometry

Direct-injection electrospray ionization mass spectrometry in combination with information-dependent data acquisition (IDA), using a triple-quadrupole/linear ion trap combination, allows high-throughput qualitative analysis of complex phospholipid species from child whole blood. In the IDA experiments, scans to detect specific head groups (precursor ion or neutral loss scans) were used as survey scans to detect phospholipid classes. An enhanced resolution scan was then used to confirm the mass assignments, and the enhanced product ion scan was implemented as a dependent scan to determine the composition of each phospholipid class. These survey and dependent scans were performed sequentially and repeated for the entire duration of analysis, thus providing the maximum information from a single injection. In this way, 50 different phospholipids belonging to the phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin classes were identified in child whole blood. Copyright (C) 2005 John Wiley & Sons, Ltd.

Altered of apoptotic markers of both extrinsic and intrinsic pathways induced by hepatitis C virus infection in peripheral blood mononuclear cells

Background: Chronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U. S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection.Methods: the present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined.Results: HCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). in group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05).Conclusions: HCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways.

Mixed methods feasibility study for a trial of blood pressure telemonitoring for people who have had stroke/transient ischaemic attack (TIA).

Background Good blood pressure (BP) control reduces the risk of recurrence of stroke/transient ischaemic attack (TIA). Although there is strong evidence that BP telemonitoring helps achieve good control, none of the major trials have considered the effectiveness in stroke/TIA survivors. We therefore conducted a feasibility study for a trial of BP telemonitoring for stroke/ TIA survivors with uncontrolled BP in primary care. Method Phase 1 was a pilot trial involving 55 patients stratified by stroke/TIA randomised 3:1 to BP telemonitoring for 6 months or usual care. Phase 2 was a qualitative evaluation and comprised semi-structured interviews with 16 trial participants who received telemonitoring and 3 focus groups with 23 members of stroke support groups and 7 carers. Results Overall, 125 patients (60 stroke patients, 65 TIA patients) were approached and 55 (44%) patients were randomised including 27 stroke patients and 28 TIA patients. Fifty-two participants (95%) attended the 6-month follow-up appointment, but one declined the second daytime ambulatory blood pressure monitoring (ABPM) measurement resulting in a 93% completion rate for ABPM − the proposed primary outcome measure for a full trial. Adherence to telemonitoring was good; of the 40 participants who were telemonitoring, 38 continued to provide readings throughout the 6 months. There was a mean reduction of 10.1 mmHg in systolic ABPM in the telemonitoring group compared with 3.8 mmHg in the control group, which suggested the potential for a substantial effect from telemonitoring. Our qualitative analysis found that many stroke patients were concerned about their BP and telemonitoring increased their engagement, was easy, convenient and reassuring Conclusions A full-scale trial is feasible, likely to recruit well and have good rates of compliance and follow-up.

Blood and urinary abnormalities induced during and after 24-hour continuous running: A case report

In this reported clinical case, a healthy and well-trained male subject [aged 37 years, maximal oxygen uptake (V[Combining Dot Above]O2max) 64 mL·kg·min] ran for 23 hours and 35 minutes covering 160 km (6.7 km/h average running speed). The analysis of hematological and biochemical parameters 3 days before the event, just after termination of exercise, and after 24 and 48 hours of recovery revealed important changes on muscle and liver function, and hemolysis. The analysis of urine sediments showed an increment of red and white blood cells filtrations, compatible with transient nephritis. After 48 hours, most of these alterations were recovered. Physicians and health professionals who monitor such athletic events should be aware that these athletes could exhibit transient symptoms compatible with severe pathologies and diseases, although the genesis of these blood and urinary abnormalities are attributable to transient physiological adaptations rather to pathological status.

Comparison of the Lactate Pro and Analox GM7 blood lactate analysers

van Someren KA, Howatson G, Nunan D, Thatcher R, Shave R., Comparison of the Lactate Pro and Analox GM7 blood lactate analysers, Int J Sports Med. 2005 Oct;26(8):657-61. RAE2008

Influence of blood donation on O-2 uptake on-kinetics, peak O-2 uptake and time to exhaustion during severe-intensity cycle exercise in humans

Thatcher, Rhys, et al., 'Influence of blood donation on O-2 uptake on-kinetics, peak O-2 uptake and time to exhaustion during severe-intensity cycle exercise in humans', Experimental Physiology (2006) 91(3) pp.499-509 RAE2008

A genome-wide association study for blood lipid phenotypes in the Framingham Heart Study

BACKGROUND:Blood lipid levels including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) are highly heritable. Genome-wide association is a promising approach to map genetic loci related to these heritable phenotypes.METHODS:In 1087 Framingham Heart Study Offspring cohort participants (mean age 47 years, 52% women), we conducted genome-wide analyses (Affymetrix 100K GeneChip) for fasting blood lipid traits. Total cholesterol, HDL-C, and TG were measured by standard enzymatic methods and LDL-C was calculated using the Friedewald formula. The long-term averages of up to seven measurements of LDL-C, HDL-C, and TG over a ~30 year span were the primary phenotypes. We used generalized estimating equations (GEE), family-based association tests (FBAT) and variance components linkage to investigate the relationships between SNPs (on autosomes, with minor allele frequency [greater than or equal to]10%, genotypic call rate [greater than or equal to]80%, and Hardy-Weinberg equilibrium p [greater than or equal to] 0.001) and multivariable-adjusted residuals. We pursued a three-stage replication strategy of the GEE association results with 287 SNPs (P < 0.001 in Stage I) tested in Stage II (n ~1450 individuals) and 40 SNPs (P < 0.001 in joint analysis of Stages I and II) tested in Stage III (n~6650 individuals).RESULTS:Long-term averages of LDL-C, HDL-C, and TG were highly heritable (h2 = 0.66, 0.69, 0.58, respectively; each P < 0.0001). Of 70,987 tests for each of the phenotypes, two SNPs had p < 10-5 in GEE results for LDL-C, four for HDL-C, and one for TG. For each multivariable-adjusted phenotype, the number of SNPs with association p < 10-4 ranged from 13 to 18 and with p < 10-3, from 94 to 149. Some results confirmed previously reported associations with candidate genes including variation in the lipoprotein lipase gene (LPL) and HDL-C and TG (rs7007797; P = 0.0005 for HDL-C and 0.002 for TG). The full set of GEE, FBAT and linkage results are posted at the database of Genotype and Phenotype (dbGaP). After three stages of replication, there was no convincing statistical evidence for association (i.e., combined P < 10-5 across all three stages) between any of the tested SNPs and lipid phenotypes.CONCLUSION:Using a 100K genome-wide scan, we have generated a set of putative associations for common sequence variants and lipid phenotypes. Validation of selected hypotheses in additional samples did not identify any new loci underlying variability in blood lipids. Lack of replication may be due to inadequate statistical power to detect modest quantitative trait locus effects (i.e., < 1% of trait variance explained) or reduced genomic coverage of the 100K array. GWAS in FHS using a denser genome-wide genotyping platform and a better-powered replication strategy may identify novel loci underlying blood lipids.

PARAMETERS AFFECTING THE IMAGING AND DETECTION OF MICROBUBBLES IN BLOOD

Stabilized micron-sized bubbles, known as contrast agents, are often injected into the body to enhance ultrasound imaging of blood flow. The ability to detect such bubbles in blood depends on the relative magnitude of the acoustic power backscattered from the microbubbles (‘signal’) to the power backscattered from the red blood cells (‘noise’). Erythrocytes are acoustically small (Rayleigh regime), weak scatterers, and therefore the backscatter coefficient (BSC) of blood increases as the fourth power of frequency throughout the diagnostic frequency range. Microbubbles, on the other hand, are either resonant or super-resonant in the range 5-30 MHz. Above resonance, their total scattering cross-section remains constant with increasing frequency. In the present thesis, a theoretical model of the BSC of a suspension of red blood cells is presented and compared to the BSC of Optison® contrast agent microbubbles. It is predicted that, as the frequency increases, the BSC of red blood cell suspensions eventually exceeds the BSC of the strong scattering microbubbles, leading to a dramatic reduction in signal-to-noise ratio (SNR). This decrease in SNR with increasing frequency was also confirmed experimentally by use of an active cavitation detector for different concentrations of Optison® microbubbles in erythrocyte suspensions of different hematocrits. The magnitude of the observed decrease in SNR correlated well with theoretical predictions in most cases, except for very dense suspensions of red blood cells, where it is hypothesized that the close proximity of erythrocytes inhibits the acoustic response of the microbubbles.

P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants

Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms

The use of different blood sample preparations in the development of biomarkers for the environmental monitoring of domestic farm animals

The application of biological effect monitoring for the detection of environmental chemical exposure in domestic animals is still in its infancy. This study investigated blood sample preparations in vitro for their use in biological effect monitoring. When peripheral blood mononuclear cells (PBMCs), isolated following the collection of multiple blood samples from sheep in the field, were cryopreserved and subsequently cultured for 24 hours a reduction in cell viability (<80%) was attributed to delays in the processing following collection. Alternative blood sample preparations using rat and sheep blood demonstrated that 3 to 5 hour incubations can be undertaken without significant alterations in the viability of the lymphocytes; however, a substantial reduction in viability was observed after 24 hours in frozen blood. Detectable levels of early and late apoptosis as well as increased levels of ROS were detectable in frozen sheep blood samples. The addition of ascorbic acid partly reversed this effect and reduced the loss in cell viability. The response of the rat and sheep blood sample preparations to genotoxic compounds ex vivo showed that EMS caused comparable dose-dependent genotoxic effects in all sample preparations (fresh and frozen) as detected by the Comet assay. In contrast, the effects of CdCl2 were dependent on the duration of exposure as well as the sample preparation. The analysis of leukocyte subsets in frozen sheep blood showed no alterations in the percentages of T and B lymphocytes but led to a major decrease in the percentage of granulocytes compared to those in the fresh samples. The percentages of IFN-γ and IL-4 but not IL-6 positive cells were comparable between fresh and frozen sheep blood after 4 hour stimulation with phorbol 12-myrisate 13-acetate and ionomycin (PMA+I). These results show that frozen blood gives comparable responses to fresh blood samples in the toxicological and immune assays used.

IL-12 inhibits in vitro immunoglobulin production by human lupus peripheral blood mononuclear cells (PBMC).

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by polyclonal B cell activation and by the production of anti-double-stranded (ds) DNA antibodies. Given the inhibitory effects of IL-12 on humoral immune responses, we investigated whether IL-12 displayed such an activity on in vitro immunoglobulin production by SLE PBMC. Spontaneous IgG, IgG1, IgG2, IgG3 and IgM antibody production was dramatically reduced by addition of IL-12. These results were confirmed by Elispot assays detecting IgG- and anti-dsDNA-secreting cells. While IL-6 and TNF titres measured in PBMC supernatants were not modified by addition of IL-12, interferon-gamma (IFN-gamma) titres were up-regulated and IL-10 production down-regulated. Since addition of IFN-gamma did not down-regulate immunoglobulin production and since the inhibitory activity of IL-12 on immunoglobulin synthesis was not suppressed by anti-IFN-gamma antibody, we concluded that the effect of IL-12 on immunoglobulin production was not mediated through IFN-gamma. Our data also argue against the possibility that down-regulation of endogenous IL-10 production was responsible for the effect of IL-12. Thus, inhibition of IL-10 production by IFN-gamma was not accompanied by inhibition of immunoglobulin production, and conversely, restoration of IL-10 production by anti-IFN-gamma antibody did not suppress the inhibitory activity exerted by IL-12 on immunoglobulin production. Taken together, our data indicate that reduction of excessive immunoglobulin and anti-dsDNA antibody production by lupus PBMC can be achieved in vitro by IL-12, independently of IFN-gamma and IL-10 modulation.

Development of a new blood test based on the helper lymphocytes Th-17 response for the diagnosis of allergies to dental metals: Preliminary results

info:eu-repo/semantics/nonPublished

Development of a new blood test for the diagnosis of the allergies to dental metals

Congrès du GIRSO, Lille, avril 2011

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.