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 role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum

P.M. Hastie and W. Haresign (2006). A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum. Animal Reproduction Science, 96(1-2), 196-209. Sponsorship: DEFRA RAE2008

Individual variation in the milk yield response to bovine somatotropin in dairy cows

M. T. Rose, T. E. C. Weekes and P. Rowlinson (2004). Individual variation in the milk yield response to bovine somatotropin in dairy cows. Journal of Dairy Science, 87(7), 2024-2031. Sponsorship: industry 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.

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.

Interleukin 10 reduces the incidence of pancreatitis after therapeutic endoscopic retrograde cholangiopancreatography.

BACKGROUND & AIMS: Prophylactic administration of interleukin (IL)-10 decreases the severity of experimental pancreatitis. Prevention of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis in humans is a unique model to study the potential role of IL-10 in this setting. METHODS: In a single-center, double-blind, randomized, placebo-controlled study, the effect of a single injection of 4 microg/kg (group 1) or 20 microg/kg (group 2) IL-10 was compared with that of placebo (group 0), all administered 30 minutes before therapeutic ERCP. The primary endpoint was the effect of IL-10 on serum levels of amylases and lipases measured 4, 24, and 48 hours after ERCP. The secondary objective was to evaluate changes in plasma cytokines (IL-6, IL-8, tumor necrosis factor) at the same time points and the incidence of acute pancreatitis in the 3 groups. Subjects undergoing a first therapeutic ERCP were eligible for inclusion. RESULTS: A total of 144 patients were included. Seven were excluded based on intention to treat (n = 1) or per protocol (n = 6). Forty-five, 48, and 44 patients remained in groups 0, 1, and 2, respectively. The 3 groups were comparable for age, sex, underlying disease, indication for treatment, type of treatment, and plasma levels of C-reactive protein (CRP), cytokines, and hydrolases at baseline. No significant difference was observed in CRP, cytokine, and hydrolase plasma levels after ERCP. Forty-three patients developed hyperhydrolasemia (18 in group 0, 14 in group 1, and 11 in group 2; P = 0.297), and 19 patients developed acute clinical pancreatitis (11 in group 0, 5 in group 1, 3 in group 2; P = 0.038). Two severe cases were observed in the placebo group. No mortality related to ERCP was observed. Logistic regression identified 3 independent risk factors for post-therapeutic ERCP pancreatitis: IL-10 administration (odds ratio [OR], 0.46; 95% confidence interval [95% CI], 0.22-0.96; P = 0.039), pancreatic sphincterotomy (OR, 5.04; 95% CI, 1.53-16.61; P = 0.008), and acinarization (OR, 8.19; 95% CI, 1.83-36.57; P = 0.006). CONCLUSIONS: A single intravenous dose of IL-10, given 30 minutes before the start of the procedure, independently reduces the incidence of post-therapeutic ERCP pancreatitis.

A binding site for the cyclic adenosine 3',5'-monophosphate-response element-binding protein as a regulatory element in the grp78 promoter.

The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.

Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.

Attenuation of inflammatory events in human intervertebral disc cells with a tumor necrosis factor antagonist.

STUDY DESIGN: The inflammatory responses of primary human intervertebral disc (IVD) cells to tumor necrosis factor α (TNF-α) and an antagonist were evaluated in vitro. OBJECTIVE: To investigate an ability for soluble TNF receptor type II (sTNFRII) to antagonize TNF-α-induced inflammatory events in primary human IVD cells in vitro. SUMMARY OF BACKGROUND DATA: TNF-α is a known mediator of inflammation and pain associated with radiculopathy and IVD degeneration. sTNFRs and their analogues are of interest for the clinical treatment of these IVD pathologies, although information on the effects of sTNFR on human IVD cells remains unknown. METHODS: IVD cells were isolated from surgical tissues procured from 15 patients and cultured with or without 1.4 nmol/L TNF-α (25 ng/mL). Treatment groups were coincubated with varying doses of sTNFRII (12.5-100 nmol/L). Nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin-6 (IL6) levels in media were quantified to characterize the inflammatory phenotype of the IVD cells. RESULTS: Across all patients, TNF-α induced large, statistically significant increases in NO, PGE₂, and IL6 secretion from IVD cells compared with controls (60-, 112-, and 4-fold increases, respectively; P < 0.0001). Coincubation of TNF-α with nanomolar doses of sTNFRII significantly attenuated the secretion of NO and PGE₂ in a dose-dependent manner, whereas IL6 levels were unchanged. Mean IC₅₀ values for NO and PGE₂ were found to be 35.1 and 20.5 nmol/L, respectively. CONCLUSION: Nanomolar concentrations of sTNFRII were able to significantly attenuate the effects of TNF-α on primary human IVD cells in vitro. These results suggest this sTNFR to be a potent TNF antagonist with potential to attenuate inflammation in IVD pathology.

The implementation of a translational study involving a primary care based behavioral program to improve blood pressure control: The HTN-IMPROVE study protocol (01295).

BACKGROUND: Despite the impact of hypertension and widely accepted target values for blood pressure (BP), interventions to improve BP control have had limited success. OBJECTIVES: We describe the design of a 'translational' study that examines the implementation, impact, sustainability, and cost of an evidence-based nurse-delivered tailored behavioral self-management intervention to improve BP control as it moves from a research context to healthcare delivery. The study addresses four specific aims: assess the implementation of an evidence-based behavioral self-management intervention to improve BP levels; evaluate the clinical impact of the intervention as it is implemented; assess organizational factors associated with the sustainability of the intervention; and assess the cost of implementing and sustaining the intervention. METHODS: The project involves three geographically diverse VA intervention facilities and nine control sites. We first conduct an evaluation of barriers and facilitators for implementing the intervention at intervention sites. We examine the impact of the intervention by comparing 12-month pre/post changes in BP control between patients in intervention sites versus patients in the matched control sites. Next, we examine the sustainability of the intervention and organizational factors facilitating or hindering the sustained implementation. Finally, we examine the costs of intervention implementation. Key outcomes are acceptability and costs of the program, as well as changes in BP. Outcomes will be assessed using mixed methods (e.g., qualitative analyses--pattern matching; quantitative methods--linear mixed models). DISCUSSION: The study results will provide information about the challenges and costs to implement and sustain the intervention, and what clinical impact can be expected.

Diagnosis of partial body radiation exposure in mice using peripheral blood gene expression profiles.

In the event of a terrorist-mediated attack in the United States using radiological or improvised nuclear weapons, it is expected that hundreds of thousands of people could be exposed to life-threatening levels of ionizing radiation. We have recently shown that genome-wide expression analysis of the peripheral blood (PB) can generate gene expression profiles that can predict radiation exposure and distinguish the dose level of exposure following total body irradiation (TBI). However, in the event a radiation-mass casualty scenario, many victims will have heterogeneous exposure due to partial shielding and it is unknown whether PB gene expression profiles would be useful in predicting the status of partially irradiated individuals. Here, we identified gene expression profiles in the PB that were characteristic of anterior hemibody-, posterior hemibody- and single limb-irradiation at 0.5 Gy, 2 Gy and 10 Gy in C57Bl6 mice. These PB signatures predicted the radiation status of partially irradiated mice with a high level of accuracy (range 79-100%) compared to non-irradiated mice. Interestingly, PB signatures of partial body irradiation were poorly predictive of radiation status by site of injury (range 16-43%), suggesting that the PB molecular response to partial body irradiation was anatomic site specific. Importantly, PB gene signatures generated from TBI-treated mice failed completely to predict the radiation status of partially irradiated animals or non-irradiated controls. These data demonstrate that partial body irradiation, even to a single limb, generates a characteristic PB signature of radiation injury and thus may necessitate the use of multiple signatures, both partial body and total body, to accurately assess the status of an individual exposed to radiation.

The effects of sleep hypoxia on coagulant factors and hepatic inflammation in emphysematous rats.

OBJECTIVES: To develop a sleep hypoxia (SH) in emphysema (SHE) rat model and to explore whether SHE results in more severe hepatic inflammation than emphysema alone and whether the inflammation changes levels of coagulant/anticoagulant factors synthesized in the liver. METHODS: Seventy-five rats were put into 5 groups: SH control (SHCtrl), treated with sham smoke exposure (16 weeks) and SH exposure (12.5% O(2), 3 h/d, latter 8 weeks); emphysema control (ECtrl), smoke exposure and sham SH exposure (21% O(2)); short SHE (SHEShort), smoke exposure and short SH exposure (1.5 h/d); mild SHE (SHEMild), smoke exposure and mild SH exposure (15% O(2)); standard SHE (SHEStand), smoke exposure and SH exposure. Therefore, ECtrl, SHEShort, SHEMild and SHEStand group were among emphysematous groups. Arterial blood gas (ABG) data was obtained during preliminary tests. After exposure, hepatic inflammation (interleukin -6 [IL-6] mRNA and protein, tumor necrosis factor α [TNFα] mRNA and protein) and liver coagulant/anticoagulant factors (antithrombin [AT], fibrinogen [FIB] and Factor VIII [F VIII]) were evaluated. SPSS 11.5 software was used for statistical analysis. RESULTS: Characteristics of emphysema were obvious in emphysematous groups and ABGs reached SH criteria on hypoxia exposure. Hepatic inflammation parameters and coagulant factors are the lowest in SHCtrl and the highest in SHEStand while AT is the highest in SHCtrl and the lowest in SHEStand. Inflammatory cytokines of liver correlate well with coagulant factors positively and with AT negatively. CONCLUSIONS: When SH is combined with emphysema, hepatic inflammation and coagulability enhance each other synergistically and produce a more significant liver-derivative inflammatory and prothrombotic status.

Association of a peripheral blood metabolic profile with coronary artery disease and risk of subsequent cardiovascular events.

BACKGROUND: Molecular tools may provide insight into cardiovascular risk. We assessed whether metabolites discriminate coronary artery disease (CAD) and predict risk of cardiovascular events. METHODS AND RESULTS: We performed mass-spectrometry-based profiling of 69 metabolites in subjects from the CATHGEN biorepository. To evaluate discriminative capabilities of metabolites for CAD, 2 groups were profiled: 174 CAD cases and 174 sex/race-matched controls ("initial"), and 140 CAD cases and 140 controls ("replication"). To evaluate the capability of metabolites to predict cardiovascular events, cases were combined ("event" group); of these, 74 experienced death/myocardial infarction during follow-up. A third independent group was profiled ("event-replication" group; n=63 cases with cardiovascular events, 66 controls). Analysis included principal-components analysis, linear regression, and Cox proportional hazards. Two principal components analysis-derived factors were associated with CAD: 1 comprising branched-chain amino acid metabolites (factor 4, initial P=0.002, replication P=0.01), and 1 comprising urea cycle metabolites (factor 9, initial P=0.0004, replication P=0.01). In multivariable regression, these factors were independently associated with CAD in initial (factor 4, odds ratio [OR], 1.36; 95% CI, 1.06 to 1.74; P=0.02; factor 9, OR, 0.67; 95% CI, 0.52 to 0.87; P=0.003) and replication (factor 4, OR, 1.43; 95% CI, 1.07 to 1.91; P=0.02; factor 9, OR, 0.66; 95% CI, 0.48 to 0.91; P=0.01) groups. A factor composed of dicarboxylacylcarnitines predicted death/myocardial infarction (event group hazard ratio 2.17; 95% CI, 1.23 to 3.84; P=0.007) and was associated with cardiovascular events in the event-replication group (OR, 1.52; 95% CI, 1.08 to 2.14; P=0.01). CONCLUSIONS: Metabolite profiles are associated with CAD and subsequent cardiovascular events.

Genetic variants in IGF-I, IGF-II, IGFBP-3, and adiponectin genes and colon cancer risk in African Americans and Whites.

PURPOSE: Evaluating genetic susceptibility may clarify effects of known environmental factors and also identify individuals at high risk. We evaluated the association of four insulin-related pathway gene polymorphisms in insulin-like growth factor-1 (IGF-I) (CA)( n ) repeat, insulin-like growth factor-2 (IGF-II) (rs680), insulin-like growth factor-binding protein-3 (IGFBP-3) (rs2854744), and adiponectin (APM1 rs1501299) with colon cancer risk, as well as relationships with circulating IGF-I, IGF-II, IGFBP-3, and C-peptide in a population-based study. METHODS: Participants were African Americans (231 cases and 306 controls) and Whites (297 cases, 530 controls). Consenting subjects provided blood specimens and lifestyle/diet information. Genotyping for all genes except IGF-I was performed by the 5'-exonuclease (Taqman) assay. The IGF-I (CA)(n) repeat was assayed by PCR and fragment analysis. Circulating proteins were measured by enzyme immunoassays. Odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated by logistic regression. RESULTS: The IGF-I (CA)( 19 ) repeat was higher in White controls (50 %) than African American controls (31 %). Whites homozygous for the IGF-I (CA)(19) repeat had a nearly twofold increase in risk of colon cancer (OR = 1.77; 95 % CI = 1.15-2.73), but not African Americans (OR = 0.73, 95 % CI 0.50-1.51). We observed an inverse association between the IGF-II Apa1 A-variant and colon cancer risk (OR = 0.49, 95 % CI 0.28-0.88) in Whites only. Carrying the IGFBP-3 variant alleles was associated with lower IGFBP-3 protein levels, a difference most pronounced in Whites (p-trend <0.05). CONCLUSIONS: These results support an association between insulin pathway-related genes and elevated colon cancer risk in Whites but not in African Americans.