Expression of two major isoforms of MYO7A in the retina: Considerations for gene therapy of Usher syndrome type 1B.

Usher syndrome type 1B (USH1B) is a deaf-blindness disorder, caused by mutations in the MYO7A gene, which encodes the heavy chain of an unconventional actin-based motor protein. Here, we examined the two retinal isoforms of MYO7A, IF1 and IF2. We compared 3D models of the two isoforms and noted that the 38-amino acid region that is present in IF1 but absent from IF2 affects the C lobe of the FERM1 domain and the opening of a cleft in this potentially important protein binding domain. Expression of each of the two isoforms of human MYO7A and pig and mouse Myo7a was detected in the RPE and neural retina. Quantification by qPCR showed that the expression of IF2 was typically âˆ¼ 7-fold greater than that of IF1. We discuss the implications of these findings for any USH1B gene therapy strategy. Given the current incomplete knowledge of the functions of each isoform, both isoforms should be considered for targeting both the RPE and the neural retina in gene augmentation therapies.

Deriving National Continued Competency Priorities for Emergency Medical Services Clinicians.

OBJECTIVE: Continued competency is poorly defined in emergency medical services (EMS), with no established method for verifying continued competency at a national level. The objective of this project was to refine understanding of continued competency for EMS clinicians in the U.S. and establish priorities for developing competency assessments. METHODS: A panel of EMS managers, educators, medical directors, and experts in competency assessment, simulation, and certification used a modified Delphi technique to address two questions: "What is the content for continued competency in EMS that should be assessed or verified?" (content) and "How should continued competency of EMS clinicians be demonstrated?" (process). The Delphi process was conducted through electronic conferencing and survey software over a 6-month period. In round one, panelists responded to open-ended prompts and their contributions were analyzed and categorized into themes by independent reviewers. In round two, the panel rated theme importance using five-point Likert-type scales. In round three, the panel ranked their top 10 themes, and in round four, the panel selected the most important themes for each of the two questions through consensus-building discussions. Descriptive statistics and thematic analyses were performed with Excel and STATA 16. RESULTS: Fourteen invited experts participated in all Delphi activities. The panel contributed 70 content and 35 process items from the original prompts. Following thematic analysis, these contributions were reduced to 21 and 14 unique themes, respectively. The final top five prioritized themes for content important for continued competency included (1) airway, respiration, and ventilation, (2) patient assessment, (3) pharmacology, (4) pediatrics, and (5) management of time critical disease progressions. The final top five prioritized themes for the processes for continued competency assessment included (1) assessments of evidence-based practice, (2) performance-based assessments, (3) combined knowledge and skill assessments, (4) performance improvement over time, and (5) frequent, short knowledge assessments. CONCLUSION: This modified Delphi process identified priorities for content and assessment, laying the groundwork for EMS continued competency at a national level. These findings can be leveraged by national task forces to develop transparent and consistent guidelines for systems that verify continued competency related to certification, licensure, and local credentialing.

Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies.

Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

The relationship between gender and age with monocyte tissue factor expression.

BACKGROUND: In normal healthy individuals, the level of tissue factor (TF) expression on monocytes is low. However, studies have shown that patients with cardiovascular disease (CVD) have elevated levels of TF. As the risk of CVD increases with age and is more prominent in the male population, it is postulated that TF expression may be positively correlated with these factors. However, very few studies have examined the relationship between age and gender on TF expression. METHODS: This study evaluated the influence of age and gender on TF expression using data obtained from female (n = 44) and male (n = 27) subjects. We also examined the influence of BMI and total fat intake on TF expression in the same subjects. RESULTS: The results of our study found no significant difference in TF expression between the male and female subgroups. No correlation was found between TF and age, BMI or total fat intake in the male or female groupings. CONCLUSION: It may be postulated that the risk of CVD development in such populations may not be due to increases in TF expression with increasing age or gender differences.

Effect of testicular steroids on catalytic activities of cytochrome P450 enzymes in porcine liver microsomes.

The testicular steroids androstenone (A), 17beta-oestradiol (E2) and testosterone (T) were tested for their ability to alter CYP2E1 and CYP2A activity in porcine liver microsomes from male and female pigs. This is the first in vitro study indicating that sex steroids have a potential to modify microsomal CYP2E1 activity, the main skatole-metabolising enzyme. A and E2 exerted an inhibitory effect on CYP2E1 mediated hydroxylation of p-nitrophenol to p-nitrocatechol although the mechanism of this inhibition differed for these steroids. The inhibitory effect of A on CYP2E1, as determined by kinetic analysis, might be due to the competitive binding of A and p-nitrophenol to the same site of CYP2E1. Including E2 into the incubations resulted in decreased activities of CYP2E1 in male microsomes through a mixed mode of inhibition. Including pre-incubation steps eliminated this inhibition in male microsomes, and resulted in increased CYP2E1 activities in the microsomes from female pigs. Testosterone was ineffective as an inhibitor of either CYP2E1 or CYP2A activities. Overall, our findings indicate that A and E2 have the potential to modify the catalytic activities of porcine CYP2E1 in vitro. However, the significance of this modification for skatole metabolism in vivo is questionable.

Molecular cloning and regulation of porcine SULT2A1: relationship between SULT2A1 expression and sulfoconjugation of androstenone.

Hydroxysteroid sulfotransferase (SULT2A1) is a key enzyme in the testicular and hepatic metabolism of 5alpha-androstenone, which is a major component of the off-odor and off-flavor in pork known as boar taint. The goals of this study were to determine the role of testicular and hepatic SULT2A1 activity on plasma 5alpha-androstenone sulfate levels, the accumulation of 5alpha-androstenone in adipose tissue, and to gain insight into the regulatory control of SULT2A1. Testicular SULT2A1 activity was negatively correlated (r = -0.57; P < 0.01) with 5alpha-androstenone concentrations in fat. The differences observed in SULT2A1 activity warranted investigation into potential genetic variation within porcine SULT2A1. The cDNA sequence of porcine Sult2A1 was determined to be > 82% homologous to the human, mouse, and rat Sult2A1 genes. A single nucleotide polymorphism was detected within the coding region of the Sult2A1 from individual testes and liver samples; however, this did not affect the amino acid sequence of the enzyme. Western blot analysis determined that animals with high concentrations of 5alpha-androstenone in fat and low SULT2A1 activity had corresponding low levels of SULT2A1 protein compared with animals with low levels of 5alpha-androstenone in fat. Real-time PCR analysis indicated that Sult2A1 mRNA was increased 2.8-fold in animals with high levels of the protein relative to animals with low levels of the protein. Furthermore, we demonstrated the positive role of the nuclear receptors constitutive androstane receptor and pregnane X receptor, as well as the possible role of farnesoid X receptor in the regulation of testicular SULT2A1 activity. Together, the results of this study suggest that differences in SULT2A1 expression can influence 5alpha-androstenone accumulation in fat.

The role of CYP2A and CYP2E1 in the metabolism of 3-methylindole in primary cultured porcine hepatocytes.

The accumulation of 3-methylindole (3MI) in uncastrated male pigs (boars) is a major cause of boar taint, which negatively affects the quality of meat from the animal. Previously, CYP2E1 and CYP2A have been identified as cytochrome P450 (P450) isoforms involved in the metabolism of 3MI using porcine liver microsomes. This study further examines the role of these isoforms in the metabolism of 3MI using a primary porcine hepatocyte model by examining metabolic profiles of 3MI after incubation with P450 inhibitors. Incubation of hepatocytes with 4-methylpyrazole resulted in a selective inhibition of CYP2E1 activity as determined by p-nitrophenol hydroxylase activity and an associated significant decrease in the production of the 3MI metabolites 3-hydroxy-3-methyloxindole and 3-methyloxindole. Furthermore, inhibition of CYP2A, as assayed by coumarin 7-hydroxylase activity, using 8-methoxypsoralen and diethyldithiocarbamate was not associated with any further significant inhibition of the production of 3MI metabolites. Treatment with general P450 inhibitors resulted in further decreases in CYP2E1 activity and a more dramatic decrease in the production of 3MI metabolites, suggesting that additional P450s may be involved in the phase 1 metabolism of 3-methylindole. In conclusion, CYP2E1 activity levels are more important than CYP2A activity levels for the metabolism of 3-methylindole in isolated pig hepatocytes.

Zinc status and age-related changes in peripheral blood leukocyte subpopulations in healthy men and women aged 55-70 y: the ZENITH study.

OBJECTIVE: To determine zinc status and age-related changes in the immune function of healthy late-middle-aged men and women (aged 55-70 y). DESIGN: Observational study. SETTING: Population of Northern Ireland. SUBJECTS: Apparently healthy, free-living individuals (45 men, 48 women) aged 55-70 y. INTERVENTION: Zinc status markers were analysed by flame atomic absorption spectrometry and commercially available kits. Immune function was assessed by flow cytometry. RESULTS: Serum and erythrocyte zinc concentrations were 13.0 (s.d. 1.40) micromol/l and 222 (s.d. 48.2) micromol/l, respectively. Serum alkaline phosphatase (ALP) concentrations were 76.8 (s.d. 16.1) U/l; women showed significantly higher concentrations of ALP (P = 0.011). Women demonstrated (1) a significant inverse correlation in naive T lymphocytes, specifically naive T-helper lymphocytes (% expression, r = -0.364, P = 0.007 and absolute count, r = -0.275, P = 0.036) with age and (2) a significant positive correlation between late activation of T lymphocytes (% expression, r = 0.299, P = 0.019 and absolute count, r = 0.260, P = 0.039) with advancing age. Men demonstrated a significant positive correlation in the % expression of (CD3-/CD16+/CD56+) natural killer (NK) cells with age (r = 0.316, P = 0.017). CONCLUSIONS: Between the ages of 55 and 70 y, healthy individuals experience significant alterations in immune function; however, such changes appear largely sex specific. Given the reported importance of adequate zinc status in maintaining optimal immune function, further studies are required to explore the effect of enhanced zinc status on emerging immune deficiencies in cell-mediated immunity in healthy 55-70 y olds.

T cell expansion is regulated by activated Gr-1+ splenocytes.

CD4+ T cell proliferation depends on the balance between NO and extra-cellular superoxide (O2-). By reducing NO bio-availability, O2- promotes splenic T cell proliferation and immune response intensity. Here, we show that spleen cells from naïve mice produced neither NO nor O2- during T cell activation, but Gr-1+ splenocytes from primed mice regulated Ag-specific T cell expansion via production of both molecules. Purified splenic Gr-1+ cells included mostly granulocytes at various stages of maturation, as well as monocytes. Activation or recruitment of regulatory Gr-1+ cells was dependent on immunization with CFA. Importantly, these regulatory cells were not detected in draining lymph nodes. These data suggest that innate Gr-1+ splenic cells regulate adaptive immunity.

Metabolism of the 16-androstene steroids in primary cultured porcine hepatocytes.

The hepatic metabolism of the 16-androstene steroids was investigated using isolated porcine hepatocytes. This study demonstrated that the liver is capable of producing both phase I and phase II steroid metabolites from 16-androstene steroid precursors. 16-Androstene metabolites were recovered by solid-phase extraction and identified by gas chromatography-mass spectrometry (GC-MS). When 5alpha-androstenone was provided as a substrate, both 3beta- and 3alpha-androstenol were produced as well as a metabolite that showed evidence of hydroxylation. Incubations with the various 16-androstene steroids produced metabolic profiles which suggested that the major role of the liver is phase II conjugation. Sulfoconjugated 16-androstene steroids included androstadienol, 5alpha-androstenone, 3beta-, 3alpha-androstenol, and possibly the hydroxylated metabolite of 5alpha-androstenone. It was determined that hydroxysteroid sulfotransferase (HST) is the likely candidate for the sulfoconjugation of the 16-androstene steroids within the liver. Despite the capacity of the hepatocytes to sulfoconjugate the 16-androstene steroids, the principle metabolites produced from incubations with 5alpha-androstenone, 3beta-, and 3alpha-androstenol were glucuronide conjugates, accounting for approximately 68% of all phase II metabolism. These findings underline the importance of steroid conjugation and suggest that hepatic metabolism of the 16-androstene steroids may influence the levels of 5alpha-androstenone present in the circulation, and thus, capable of accumulating in fat.

Annexin A2 expression during cellular differentiation in myeloid cell lines.

Annexin A2 is a calcium-dependent, phospholipid-binding protein found on many cell types. It consists of a short hydrophobic tail (Ser(2)-Asn(32)), which dictates its function, and a core domain (Phe(33)-Asp(339)), which is involved in phospholipid binding. Annexin A2 has been implicated in a number of biochemical processes, including cell proliferation, foetal immune tolerance, ion-channel activation, cell-cell interactions and the bridging of membranes. Annexin A2 is reported to be a powerful activator of plasminogen and, therefore, is implicated in many normal and pathological processes such as haemostasis and metastasis. Myeloid cell lines are used, extensively, to study many aspects of cellular proliferation, differentiation and function. In the present study, we have used flow cytometry and real-time PCR to investigate the role of annexin A2 expression in the proliferation and differentiation of a number of myeloid cell lines. The results demonstrated that annexin A2 expression was affected when the cells were induced to differentiate by stimulation with all-trans-retinoic acid. Annexin A2 may, therefore, be an important player in cellular differentiation and its disorders.

Endoplasmic reticulum stress due to altered cellular redox status positively regulates murine hepatic CYP2A5 expression.

Murine hepatic cytochrome P450 2A5 (CYP2A5) is uniquely induced by a variety of agents that cause liver injury and inflammation, conditions that are typically associated with downregulation of P450s. We hypothesized that induction of CYP2A5 occurs in response to hepatocellular damage resulting in endoplasmic reticulum (ER) stress. Treatment of mice in vivo and mouse hepatocytes in primary culture with the CYP2A5 inducer pyrazole resulted in overexpression of the ER stress biomarker glucose-regulated protein (GRP) 78. Treatment of primary hepatocytes with ER stress activators thapsigargin, tunicamycin, and trans-4,5-dihydroxy-1,2-dithiane (DTT(ox)) and the calcium ionophore A23187 (calcimycin) resulted in elevated GRP78 mRNA levels; however, only the reducing agent DTT(ox) induced levels of CYP2A5 mRNA, protein, and coumarin 7-hydroxylase activity. To test the hypothesis that CYP2A5 induction is due to liver injury resulting from altered cellular redox status, we demonstrated that CYP2A5 induction, elevated serum alanine aminotransferase, and oxidative protein damage occur concurrently in pyrazole-treated mice. Pyrazole also induced the expression of cytosolic alpha and mu class glutathione S-transferase expression both in vivo and in primary mouse hepatocytes. Moreover, treatment of hepatocytes with the redox cycling quinone menadione resulted in overexpression of CYP2A5 and GSTM1 mRNA. Finally, pretreatment of hepatocytes with the antioxidants N-acetylcysteine and vitamin E attenuated pyrazole-mediated increases in CYP2A5 mRNA levels. These findings clearly indicate that induction of mouse hepatic CYP2A5 during liver injury occurs via a novel mechanism involving ER stress due to altered cellular redox status.

Plasma levels of the immunomodulatory cytokine interleukin-10 during normal human pregnancy: a longitudinal study.

Pregnancy is proposed to be a Th2 phenomenon, where Th2 cytokines inhibit Th1 responses to improve foetal survival. The importance of interleukin-10 (IL-10), an immunomodulatory cytokine produced by Th2 cells, in the maintenance of normal pregnancy is becoming increasingly apparent. In a longitudinal case-control study, the physiological effect of pregnancy on plasma IL-10 was investigated. The plasma concentration of IL-10 was determined using an ELISA technique in 99 pregnant women sampled at 12, 20 and 35 weeks of gestation, 38 non-pregnant control subjects sampled in parallel and in a subgroup of women sampled at 3 days post-partum (n, pregnant 21, non-pregnant 21). Plasma IL-10 was significantly higher in pregnant women at 12, 20 and 35 weeks of gestation (p<0.05, p<0.01 and p<0.0001, respectively), and in mothers post-delivery (p<0.01) when compared to non-pregnant control subjects. Furthermore, there was no significant effect of gestational time on IL-10 concentration. Results from the current study suggest that elevated IL-10 is a physiological consequence of normal healthy pregnancy. These findings help clarify previous conflicting results and establish a range for plasma levels of IL-10 in normal healthy pregnancy.

Copper supplementation has no effect on markers of DNA damage and liver function in healthy adults (FOODCUE project).

BACKGROUND/AIMS: Copper is routinely used in the laboratory to promote oxidation in vitro. However, copper concentrations are million-fold higher than physiological concentrations and, in contrast, accumulating evidence suggests that copper may have an antioxidant role in vivo. The aim of this study was to provide data on how increased intake of copper affected mononuclear leukocyte DNA damage and liver function in healthy young free-living men and women. METHODS: The study design was a double-blind repeated crossover trial with treatment and intervening placebo periods, each of 6 weeks' duration. The following supplementations were given orally in sequence: CuSO(4) at a dose of 3 mg copper/day and copper amino acid chelates at doses of 3 and 6 mg copper/day. Oxidative DNA damage was assessed using a modification of the alkaline Comet assay incorporating an endonuclease III digestion step. The assessment of liver function was by measurement of the liver enzymes, alanine aminotransferase and L-gamma-glutamyltransferase. RESULTS: There was no significant alteration in mononuclear leukocyte DNA damage or on liver function after 6 weeks of copper supplementation at two doses (3 and 6 mg/day). CONCLUSIONS: Copper supplementation (giving total copper intake at the highest level of 7 mg/day) did not induce DNA damage or adversely affect liver function in healthy adults.

Effects of lipopolysaccharide-stimulated inflammation and pyrazole-mediated hepatocellular injury on mouse hepatic Cyp2a5 expression.

Murine hepatic cytochrome P450 2a5 (Cyp2a5) is induced during hepatotoxicity and hepatitis, however, the specific regulatory mechanisms have not been determined. We compared the influence of acute inflammation elicited in vivo by bacterial endotoxin lipopolysaccharide (LPS) and liver injury caused by the hepatotoxin pyrazole on hepatic Cyp2a5 expression in mice. Pyrazole treatment resulted in statistically significant increases in levels of Cyp2a5 mRNA, protein and catalytic activity by 540, 273 and 711%, respectively (P<0.05). In LPS-treated livers Cyp2a5 expression was significantly reduced compared to controls at the mRNA (46%) protein (35%), and activity (23%) levels (P<0.05). Treatment of mice with recombinant murine interleukin-1 beta and interleukin-6 had no significant effect on Cyp2a5 mRNA and protein levels. Liver injury, as assessed by serum alanine aminotransferase, was greater with pyrazole than with LPS treatment (609 vs 354% of control levels respectively). ER stress, determined by hepatic glucose regulated protein 78 (grp78) levels, was greater with pyrazole (185% of controls) than with LPS (128% of controls). In pyrazole-treated liver, overexpression of immunoreactive grp78 protein revealed that ER stress was localized to pericentral hepatocytes in which Cyp2a5 was induced. Evidence of glycogen loss and membrane damage in these cells was suggestive of oxidative damage. Moreover, vitamin E attenuated Cyp2a5 induction by pyrazole in vivo. These results suggest that induction of Cyp2a5 that has been observed in mouse models of hepatitis and hepatoxicity may be related to oxidative injury to the endoplasmic reticulum of pericentral hepatocytes rather than exposure to pro-inflammatory cytokines.

Boron supplementation and activated factor VII in healthy men.

OBJECTIVE: The aim of the present study was to determine whether postprandial concentrations of the active component of serine protease coagulation factor VII (VIIa) were lowered by acute boron supplementation in vivo. DESIGN: An acute, randomized, placebo-controlled, double blind, cross-over study. SETTING: Free-living population. SUBJECTS: Fifteen apparently healthy men, aged 45-65 y. INTERVENTIONS: Subjects visited the centre on two occasions, with the study days separated by a minimum of 2 weeks. Following collection of a fasting blood sample, subjects received either placebo or acute bolus of 11.6 mg boron (given as 102.6 mg sodium tetraborate decahydrate) together with a standard fat-rich meal. Blood samples were obtained at 1, 2, 4 and 6 h after the administration of the test meal, during which time subjects were at liberty to consume deionized water only. Blood samples were assayed for concentrations of insulin, glucose, lipids and boron. Measurement of the concentration of activated factor VIIa and of factor VII antigen, and of the activity of coagulation factors VII, IX and X was also carried out. RESULTS: Plasma boron concentrations were significantly higher following consumption of the boron supplement compared with placebo (0.124+/-0.02 vs 0.008+/-0.01 mg/l; P< or =0.001). There was no significant effect of acute boron supplementation on plasma insulin and glucose concentration or on blood lipid or coagulation factor profile. Factor VIIa rose significantly following consumption of the high fat meal (1.05+/-0.07 vs 1.26+/-0.07; P< or =0.001), but this increase was not altered by boron supplementation. CONCLUSIONS: Results from this study suggest that acute boron supplementation (at 11.6 mg boron) does not alter the activity of factor VIIa following consumption of a high-fat meal. SPONSORSHIP: This work was funded by Borax Europe Ltd.

Soluble P-selectin levels during normal pregnancy: a longitudinal study.

OBJECTIVE: To investigate soluble P-selectin (sP-selectin) levels and platelet parameters in normal pregnant women compared with non-pregnant control subjects. DESIGN: A longitudinal case-control study. SETTING: Obstetric outpatient clinic in the Jubilee Maternity Hospital, Belfast. POPULATION: One hundred and twenty normal pregnant women and 41 non-pregnant age-matched control subjects. METHODS: The plasma concentration of sP-selectin in pregnant women sampled at 12, 20 and 35 weeks of gestation, and, in a subgroup at three days postpartum, and non-pregnant controls sampled in parallel, was determined using a commercial quantitative sandwich immunoassay kit. Platelet parameters on each blood sample were also recorded using a SYSMEX SE 9500 analyser. MAIN OUTCOME MEASURES: Plasma sP-selectin as a measure of platelet activation in normal pregnancy. RESULTS: Soluble P-selectin was significantly higher in pregnant women than in non-pregnant control subjects at 20 and 35 weeks of gestation, (P < 0.01, and P < 0.001, respectively). Correlation analyses showed positive correlation between sP-selectin and platelet count in pregnant women at 20 and 35 weeks of gestation (r = 0.247, P < 0.05 and r = 0.360, P < 0.001, respectively). Soluble P-selectin concentration per platelet was also significantly higher in pregnant women than in non-pregnant control subjects at 20 and 35 weeks of gestation (P < 0.001). CONCLUSIONS: Our results show that sP-selectin concentration is significantly higher in the second and third trimester of pregnancy when compared with non-pregnant control subjects sampled in parallel. This finding clarifies previous conflicting results on platelet activation in normal pregnancy, and is in agreement with those earlier studies which reported, using other methods, increased platelet activation in normal pregnancy.

Tissue factor expression on monocyte subpopulations during normal pregnancy.

Pregnancy is often referred to as a hypercoagulable state due to changes in the haemostatic system. Tissue factor (TF) is the initiator of blood clotting in vivo. The effect of pregnancy on monocyte TF expression was determined in a longitudinal case control study (89 pregnant, 39 non-pregnant). Using whole blood flow cytometry and CD14 as a monocyte marker, TF expression was measured on all CD14 positive, CD14Bright and CD14Dim cells. TF expression was significantly lower in pregnant women than in non-pregnant control subjects, on all CD14 positive cells at 20 and 35 weeks, on CD14Bright cells at 12 and 35 weeks and on CD14Dim cells at 20 weeks. Additionally, we report that a higher percentage of CD14Dim than CD14Bright cells express TF. These results suggest that, in order to maintain homeostasis in haemostasis in an otherwise hypercoagulable state, monocyte TF expression is reduced during normal pregnancy.