Microparticles from stored red blood cells induce thrombin generation

Background: Microparticles are small phospholipid vesicles of <1 lm shed in blood flow by various cell types including red blood cells. Erythrocyte-derived microparticles (EMPs) accumulate in erythrocyte concentrates (ECs) during their storage time. EMPs are considered as part of storage lesion and as their exact role is not elucidated, they could be involved in these clinical outcomes. Aims: The aim of this study is to evaluate the impact and implication of EMPs isolate from ECs on coagulation. Methods: EMPs were first isolated from erythrocyte concentrates by centrifugation and counted by flow cytometry. Using a calibrated automated thrombogram, EMPs were then added to different type of plasmas in order to evaluate the potential of thrombin generation. Results: We demonstrate that EMPs isolated from ECs are capable to accelerate and amplify thrombin generation in presence of a low exogenous tissue factor concentration, thanks to their negatively charged membrane necessary for the assembly of coagulation complexes. Interestingly, in the absence of exogenous tissue factor, EMPs are also able to trigger thrombin generation. In addition, thrombin generation induced by EMPs is not affected by the presence of anti-TF antibodies. Finally, thrombin generation induced by EMPs is not affected by using plasma samples deficient in factor VII, XI or XII. However, thrombin generation is reduced in plasma deficient in factor VIII or IX and is completely abolished in plasma deficient in factor X, V or II. No thrombin generation was observed in plasma samples without EMPs. Summary/conclusion: Several studies have shown a link between storage time of blood products and post transfusion complications. We provide evidence that EMPs accumulated during storage of erythrocyte concentrates were not only able to accelerate and support thrombin generation in plasma in presence of a low exogenous tissue-factor concentration, but also to trigger thrombin generation in absence of exogenous TF. The impact of those transfused EMs is unknown on recipients, nevertheless it could be hypothesized that under certain circumstances, transfused EMPs could be involved in thrombin generation and could be linked to adverse clinical outcome. Further work is needed to determine whether procoagulant EMPs transfused with erythrocyte concentrate may account for some of the complications occurring after red blood cell transfusion, and more particularly after transfusion of ''older''stored blood, rich in EMPs.

CRTC2 polymorphism as a risk factor for the incidence of metabolic syndrome in patients with solid organ transplantation.

NlmCategory="UNASSIGNED">Metabolic syndrome after transplantation is a major concern following solid organ transplantation (SOT). The CREB-regulated transcription co-activator 2 (CRTC2) regulates glucose metabolism. The effect of CRTC2 polymorphisms on new-onset diabetes after transplantation (NODAT) was investigated in a discovery sample of SOT recipients (n1=197). Positive results were tested for replication in two samples from the Swiss Transplant Cohort Study (STCS, n2=1294 and n3=759). Obesity and other metabolic traits were also tested. Associations with metabolic traits in population-based samples (n4=46'186, n5=123'865, n6>100,000) were finally analyzed. In the discovery sample, CRTC2 rs8450-AA genotype was associated with NODAT, fasting blood glucose and body mass index (Pcorrected<0.05). CRTC2 rs8450-AA genotype was associated with NODAT in the second STCS replication sample (odd ratio (OR)=2.01, P=0.04). In the combined STCS replication samples, the effect of rs8450-AA genotype on NODAT was observed in patients having received SOT from a deceased donor and treated with tacrolimus (n=395, OR=2.08, P=0.02) and in non-kidney transplant recipients (OR=2.09, P=0.02). Moreover, rs8450-AA genotype was associated with overweight or obesity (n=1215, OR=1.56, P=0.02), new-onset hyperlipidemia (n=1007, OR=1.76, P=0.007), and lower high-density lipoprotein-cholesterol (n=1214, β=-0.08, P=0.001). In the population-based samples, a proxy of rs8450G>A was significantly associated with several metabolic abnormalities. CRTC2 rs8450G>A appears to have an important role in the high prevalence of metabolic traits observed in patients with SOT. A weak association with metabolic traits was also observed in the population-based samples.The Pharmacogenomics Journal advance online publication, 8 December 2015; doi:10.1038/tpj.2015.82.

Establishing International Blood Pressure References Among Nonoverweight Children and Adolescents Aged 6 to 17 Years.

BACKGROUND: Several distributions of country-specific blood pressure (BP) percentiles by sex, age, and height for children and adolescents have been established worldwide. However, there are no globally unified BP references for defining elevated BP in children and adolescents, which limits international comparisons of the prevalence of pediatric elevated BP. We aimed to establish international BP references for children and adolescents by using 7 nationally representative data sets (China, India, Iran, Korea, Poland, Tunisia, and the United States). METHODS AND RESULTS: Data on BP for 52 636 nonoverweight children and adolescents aged 6 to 19 years were obtained from 7 large nationally representative cross-sectional surveys in China, India, Iran, Korea, Poland, Tunisia, and the United States. BP values were obtained with certified mercury sphygmomanometers in all 7 countries by using standard procedures for BP measurement. Smoothed BP percentiles (50th, 90th, 95th, and 99th) by age and height were estimated by using the Generalized Additive Model for Location Scale and Shape model. BP values were similar between males and females until the age of 13 years and were higher in males than females thereafter. In comparison with the BP levels of the 90th and 95th percentiles of the US Fourth Report at median height, systolic BP of the corresponding percentiles of these international references was lower, whereas diastolic BP was similar. CONCLUSIONS: These international BP references will be a useful tool for international comparison of the prevalence of elevated BP in children and adolescents and may help to identify hypertensive youths in diverse populations.

Fibroblast growth factor-23 (FGF-23) levels differ across populations by degree of industrialization.

CONTEXT: Compensatory increases in FGF23 with increasing phosphate intake may adversely impact health. However, population and clinical studies examining the link between phosphate intake and FGF23 levels have focused mainly on populations living in highly industrialized societies in which phosphate exposure may be homogenous. OBJECTIVE: Contrast dietary phosphate intake, urinary measures of phosphate excretion and FGF23 levels across populations that differ by level of industrialization. DESIGN: Cross-sectional analysis of three populations Setting: Maywood, IL, U.S., Mah|fe Island, Seychelles, and Kumasi, Ghana Participants: Adults with African ancestry aged 25-45 years Main Outcome: Fibroblast growth factor 23 (FGF23) levels Results: The mean age was 35.1 (6.3) years and 47.9% were male. Mean phosphate intake and fractional excretion of phosphate were significantly higher in the U.S. vs. Ghana while no significant difference in phosphate intake or fractional excretion of phosphate was noted between U.S. and Seychelles for men or women. Overall, median FGF23 values were 57.41 RU/ml (IQR 43.42, 75.09) in U.S., 42.49 RU/ml (IQR 33.06, 55.39) in Seychelles and 33.32 RU/ml (IQR 24.83, 47.36) in Ghana. In the pooled sample, FGF23 levels were significantly and positively correlated with dietary phosphate intake (r=0.11; P < 0.001), and the fractional excretion of phosphate (r=0.13; P < 0.001) but not with plasma phosphate levels (-0.001; P = 0.8). Dietary phosphate intake was significantly and positively associated with the fractional excretion of phosphate (r=0.23; P < 0.001). CONCLUSION: The distribution of FGF23 levels in a given population may be influenced by the level of industrialization, likely due to differences in access to foods preserved with phosphate additives.

A Headspace needle-trap method for the analysis of volatile organic compounds in whole blood

Needle trap devices (NTDs) are a relatively new and promising tool for headspace (HS) analysis. In this study, a dynamic HS sampling procedure is evaluated for the determination of volatile organic compounds (VOCs) in whole blood samples. A full factorial design was used to evaluate the influence of the number of cycles and incubation time and it is demonstrated that the controlling factor in the process is the number of cycles. A mathematical model can be used to determine the most appropriate number of cycles required to adsorb a prefixed amount of VOCs present in the HS phase whenever quantitative adsorption is reached in each cycle. Matrix effect is of great importance when complex biological samples, such as blood, are analyzed. The evaluation of the salting out effect showed a significant improvement in the volatilization of VOCs to the HS in this type of matrices. Moreover, a 1:4 (blood:water) dilution is required to obtain quantitative recoveries of the target analytes when external calibration is used. The method developed gives detection limits in the 0.020–0.080 μg L−1 range (0.1–0.4 μg L−1 range for undiluted blood samples) with appropriate repeatability values (RSD < 15% at high level and <23% at LOQ level). Figure of merits of the method can be improved by using a smaller phase ratio (i.e., an increase in the blood volume and a decrease in the HS volume), which lead to lower detection limits, better repeatability values and greater sensibility. Twenty-eight blood samples have been evaluated with the proposed method and the results agree with those indicated in other studies. Benzene was the only target compound that gave significant differences between blood levels detected in volunteer non-smokers and smokers

Effect of meal ingestion on liver stiffness in patients with cirrhosis and portal hypertension.

BACKGROUND AND AIMS: Liver stiffness is increasingly used in the non-invasive evaluation of chronic liver diseases. Liver stiffness correlates with hepatic venous pressure gradient (HVPG) in patients with cirrhosis and holds prognostic value in this population. Hence, accuracy in its measurement is needed. Several factors independent of fibrosis influence liver stiffness, but there is insufficient information on whether meal ingestion modifies liver stiffness in cirrhosis. We investigated the changes in liver stiffness occurring after the ingestion of a liquid standard test meal in this population. METHODS: In 19 patients with cirrhosis and esophageal varices (9 alcoholic, 9 HCV-related, 1 NASH; Child score 6.9±1.8), liver stiffness (transient elastography), portal blood flow (PBF) and hepatic artery blood flow (HABF) (Doppler-Ultrasound) were measured before and 30 minutes after receiving a standard mixed liquid meal. In 10 the HVPG changes were also measured. RESULTS: Post-prandial hyperemia was accompanied by a marked increase in liver stiffness (+27±33%; p<0.0001). Changes in liver stiffness did not correlate with PBF changes, but directly correlated with HABF changes (r = 0.658; p = 0.002). After the meal, those patients showing a decrease in HABF (n = 13) had a less marked increase of liver stiffness as compared to patients in whom HABF increased (n = 6; +12±21% vs. +62±29%,p<0.0001). As expected, post-prandial hyperemia was associated with an increase in HVPG (n = 10; +26±13%, p = 0.003), but changes in liver stiffness did not correlate with HVPG changes. CONCLUSIONS: Liver stiffness increases markedly after a liquid test meal in patients with cirrhosis, suggesting that its measurement should be performed in standardized fasting conditions. The hepatic artery buffer response appears an important factor modulating postprandial changes of liver stiffness. The post-prandial increase in HVPG cannot be predicted by changes in liver stiffness.

The Regulation of Skeletal and Cardiac Muscle Blood Flow in Humans. Studies by positron emission tomography with special reference to exercise, adenosine and nitric oxide

Virtually every cell and organ in the human body is dependent on a proper oxygen supply. This is taken care of by the cardiovascular system that supplies tissues with oxygen precisely according to their metabolic needs. Physical exercise is one of the most demanding challenges the human circulatory system can face. During exercise skeletal muscle blood flow can easily increase some 20-fold and its proper distribution to and within muscles is of importance for optimal oxygen delivery. The local regulation of skeletal muscle blood flow during exercise remains little understood, but adenosine and nitric oxide may take part in this process. In addition to acute exercise, long-term vigorous physical conditioning also induces changes in the cardiovasculature, which leads to improved maximal physical performance. The changes are largely central, such as structural and functional changes in the heart. The function and reserve of the heart’s own vasculature can be studied by adenosine infusion, which according to animal studies evokes vasodilation via it’s a_2A receptors. This has, however, never been addressed in humans in vivo and also studies in endurance athletes have shown inconsistent results regarding the effects of sport training on myocardial blood flow. This study was performed on healthy young adults and endurance athletes and local skeletal and cardiac muscle blod flow was measured by positron emission tomography. In the heart, myocardial blood flow reserve and adenosine A_2A receptor density, and in skeletal muscle, oxygen extraction and consumption was also measured. The role of adenosine in the control of skeletal muscle blood flow during exercise, and its vasodilator effects, were addressed by infusing competitive inhibitors and adenosine into the femoral artery. The formation of skeletal muscle nitric oxide was also inhibited by a drug, with and without prostanoid blockade. As a result and conclusion, it can be said that skeletal muscle blood flow heterogeneity decreases with increasing exercise intensity most likely due to increased vascular unit recruitment, but exercise hyperemia is a very complex phenomenon that cannot be mimicked by pharmacological infusions, and no single regulator factor (e.g. adenosine or nitric oxide) accounts for a significant part of exercise-induced muscle hyperemia. However, in the present study it was observed for the first time in humans that nitric oxide is not only important regulator of the basal level of muscle blood flow, but also oxygen consumption, and together with prostanoids affects muscle blood flow and oxygen consumption during exercise. Finally, even vigorous endurance training does not seem to lead to supranormal myocardial blood flow reserve, and also other receptors than A_2A mediate the vasodilator effects of adenosine. In respect to cardiac work, atheletes heart seems to be luxuriously perfused at rest, which may result from reduced oxygen extraction or impaired efficiency due to pronouncedly enhanced myocardial mass developed to excel in strenuous exercise.

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC

Transoperative refusion: a simple and safe method in emergency surgery

The objective is to reinforce the importance of blood reinfusion as a cheap, safe and simple method, which can be used in small hospitals, especially those in which there is no blood bank. Moreover, even with the use of devices that perform the collection and filtration of blood, more recent studies show that the cost-benefit ratio is much better when autologous transfusion is compared with blood transfusions, even when there is injury to hollow viscera and blood contamination. It is known that the allogeneic blood transfusion carries a number of risks to patients, among them are the coagulation disorders mediated by excess enzymes in the conserved blood, and deficiency in clotting factors, mainly the Factor V, the proacelerin. Another factor would be the risk of contamination with still unknown pathogens or that are not investigated during screening for selection of donors, such as the West Nile Fever and Creutzfeldt-Jacob, better known as "Mad Cow" disease. Comparing both methods, we conclude that blood autotransfusion has numerous advantages over heterologous transfusion, even in large hospitals. We are not against blood transfusions, just do not agree that the patient's own blood is discarded without making sure there will be enough blood in stock to get him out of the hemorrhagic shock.

Blood-gas and electrolyte values for Amazon parrots (Amazona aestiva)

The aim was to provide reference data for blood gas/acid-base status and electrolytes for non-anesthetized Amazon parrots (Amazona aestiva). Thirty-five adult parrots from Tietê ecologic park were utilized. Arterial blood (0.3ml) samples were anaerobically collected from the superficial ulnar artery in heparinized (sodium heparin) 1-ml plastic syringes. The samples were immediately analyzed through a portable analyzer (i-STAT*, Abbot, Illinois, USA) with cartridges (EG7+). These data were grouped in such a way as to present both mean and standard deviation: body weight (360±37g), respiratory rate (82±33 b/m), temperature (41.8±0.6°C), hydrogen potential (7.452±0.048), carbon dioxide partial pressure (22.1±4.0mmHg), oxygen partial pressure (98.1±7.6mmHg), base excess (-7.9±3.1), plasma concentration of bicarbonate ions (14.8±2.8mmol/L), oxygen saturation (96.2±1.1%), plasma concentration of sodium (147.4±2.2mmol/L), plasma concentration of potassium (3.5±0.53mmol/L), plasma concentration of calcium (0.8±0.28mmol/L), hematocrit (38.7±6.2%) and concentration of hemoglobin (13.2±2.1g/dl). This study led us to conclude that, although the results obtained showed hypocapnia and low values of bicarbonate and base excess, when compared to other avian species, these data are very similar. Besides, in spite of the equipment being approved only for human beings, it was considered simple and very useful in the analysis of avian blood samples. By using this equipment we were able to provide references data for non-anaesthetized Amazon parrots.

Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro?

Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)¹ - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (P<0.05). There was no difference between UBP and CBP effects. In conclusion, treatment of stimulated equine synovial cells with either UBP or CBP efficiently restored their redox equilibrium.

Porcine follicular fluid concentration of free insulin-like growth factor-I collected from different diameter ovarian follicles

The study aimed to quantify the concentrations of free IGF-I in serum and fluid of ovarian follicles in pre-pubertal gilts and describe the ovarian morphology by measuring the size of the ovaries and counting the number of surface follicles. Ovaries (n=1,000) from pre-pubertal gilts were obtained immediately after slaughter. A total of 10 samplings were performed, with ovaries obtained from 50 females for each collection. The follicles situated on the surface of each ovary were classified as small (SFs, 2 to 5mm in diameter) or large (LFs 6 to 10mm in diameter) and the follicular fluid was obtained by follicle aspiration. The collection of serum samples was performed after the gilts exsanguination using sterile tubes. From the pool of serum and follicular fluid obtained from 50 females, the concentration of free IGF-I was determined in each sample using an enzyme immunoassay kit (ELISA). The description of ovarian morphometry was performed in 100 ovaries from randomly selected gilts. The larger and smaller lengths of ovaries were measured, and the total number of SFs and LFs present on the surface of each ovary were also counted. The IGF-I concentration was greater (P<0.05) in LFs (170.92±88.29 ng/mL) compared with SFs (67.39±49.90ng/mL) and serum (73.48±34.63ng/mL). The largest and smallest length of the ovaries was 26.0±3.0 and 19.0mm ±2.0mm, respectively. The number of SFs (70.86±25.76) was greater (P<0.01) than LFs (6.54±5.26). The study concluded that LFs present greater levels of IGF-I when compared with SFs and blood, which is related to increased activity of the LFs and its differentiation to ovulation. In addition, ovaries of pre-pubertal gilts have a higher number of SFs compared to LFs. Therefore, our study demonstrated unique data regarding the physiological concentration of free IGF-I in ovarian follicles, that can be used in future research to evaluate the addition of this hormone in the in vitro production media of porcine embryos with the goal to improve the technique efficiency.

Long term dietary deficiency in steers: vital functions and T3 and IGF-1 relationships

To evaluate the influence of diets with different degrees of energy deficiency on the hormonal profile and vital functions, 12 steers were randomly distributed into 3 groups of 4 animals. For 140 days, each group received (G1) a diet to promote a weight gain of 900gr/day (17.7 Mcal/d DE and 13% CP), (G2) 80% of the maintenance requirements (5.8 Mcal/d DE and 7% CP), or (G3) 60% of the maintenance requirements (4.7 Mcal/d DE and 5% CP). In G2 and G3, the energy deficit caused a marked decrease in the heart rate and respiratory rate and a reduction in the blood levels of Insulin like growth factor-1 (IGF-1) and triiodothyronine (T3). The decrease in heart rate, respiratory movement and, to a lesser extent, reduction of the rectal temperature, reflected the low status of energy and was negatively impacted by the low levels of T3. There was a strong correlation between the hormones T3 and IGF-1 (r=0.833). There were also strong correlations between T3 and HR (r=0.701), T3 and RR (r=0.632), IGF-1 and HR (r=0.731), and IGF-1 and RR (r=0.679). There were intermediate correlations between T3 and TºC (r=0.484), T3 and insulin (r=0.506), IGF-1 and insulin (r=0.517), and IGF-1 and TºC (r=0.548). This study showed the influence of a long period of providing an energy-deficient diet on animal performance, correlating hormonal status and vital functions in growing cattle. The results indicated that the evaluated parameters represent an important tool for the early detection of dietary deficiency.

Interoperability specifications and an automatic validation tool as a key factor for the support of a repository ecosystem

Presentation at Open Repositories 2014, Helsinki, Finland, June 9-13, 2014