Evaluation of EC50 of factor VIII as predictor of prophylaxis efficacy in patients with severe haemophilia A.

Trough factor (F) VIII level is a not reliable bleeding risk indicator to predict prophylaxis efficacy in severe haemophilia A (SHA), therefore, accurate biomarkers are much needed. Thrombelastography (TEG) monitors both thrombin and clot formation addressing the global haemostatic status but its usefulness to tailor prophylaxis in haemophilia has been poorly evaluated. In this study, correspondence between individual pharmacodynamic/pharmacokinetic profile of FVIII and joint condition, physical activity and bleeding phenotype of SHA patients under prophylactic treatment was assessed. Nineteen SHA patients < 18 years old on long-term prophylaxis treatment with FVIII were studied in an observational cross-sectional study. Whole blood was withdrawn before FVIII administration and at five time-points after infusion for a TEG-based pharmacodynamic- and pharmacokinetic-study. Type of prophylaxis and joint condition at inclusion and physical activity as well as onset of treated spontaneous bleeding events in the previous two years were retrospectively assessed. Six patients had suffered at least one treated spontaneous bleeding event and were named as "bleeders". The rest were named as "non-bleeders". Only the half maximal effective concentration of FVIII (FVIII-EC50) for TEG parameters R-time, K-time and α-angle correlated with the bleeding phenotype being significantly higher in bleeders suggestive of a poorer response to FVIII. Poorer joint condition, trough FVIII levels or type of prophylaxis were not definitive predicting variables of bleeding phenotype. In conclusion, this study reveals FVIII-EC50 for the first time as a valuable biomarker to anticipate individual efficacy of prophylaxis in SHA.

Preclinical pharmacokinetics and biodistribution of subcutaneously administered glycoPEGylated recombinant factor VIII (N8-GP) and development of a human pharmacokinetic prediction model.

Essentials N8-GP is an extended half-life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous (SC) FVIII dosing might reduce the treatment burden of prophylaxis. SC N8-GP has a favorable PK profile in animal models and disappears from skin injection sites. Combined animal (SC) and clinical (IV) data suggest that daily SC dosing may provide prophylaxis. SUMMARY: Background N8-GP is an extended half-life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous administration of FVIII may reduce the treatment burden of prophylaxis; however, standard FVIII products have low bioavailability after subcutaneous dosing in animals. Objective To evaluate the pharmacokinetics, effectiveness and local distribution of subcutaneously administered N8-GP in preclinical models and predict the human pharmacokinetic (PK) profile. Methods The pharmacokinetics of subcutaneously administered N8-GP were evaluated in FVIII knockout (F8-KO) mice and cynomolgus monkeys; a human PK prediction model in hemophilia A patients was developed. The hemostatic effect was evaluated in a tail vein bleeding model in F8-KO mice. The injection-site distribution and absorption of subcutaneously administered N8-GP were assessed in F8-KO mice by the use of temporal fluorescence imaging and immunohistochemistry. Results Subcutaneously administered N8-GP had a bioavailability, a first-order absorption rate and a half-life, respectively, of 24%, 0.094 h-1 and 14 h in F8-KO mice, and 26%, 0.33 h-1 and 15 h in cynomolgus monkeys. A dose-dependent effect of subcutaneously administered N8-GP on blood loss was observed in mice. A minimal amount of N8-GP was detected at the injection site 48-72 h after single or multiple dose(s) in F8-KO mice. Subcutaneously administered N8-GP was localized to the skin around the injection site, with time-dependent disappearance from the depot. PK modeling predicted that subcutaneously administered N8-GP at a daily dose of 12.5 IU kg-1 will provide FVIII trough levels of 2.5-10% in 95% of patients with severe hemophilia A. Conclusions Subcutaneously administered N8-GP may provide effective hemophilia A prophylaxis. A phase I clinical trial is underway to investigate this possibility.

A comparative study on basophil activation test, histamine release assay, and passive sensitization histamine release assay in the diagnosis of peanut allergy.

BACKGROUND: Allergy can be diagnosed using basophil tests. Several methods measuring basophil activation are available. This study aimed at comparing basophil activation test (BAT), histamine release assay (HR), and passive sensitization histamine release assay (passive HR) in the diagnosis of peanut allergy. METHODS: BAT, HR, and passive HR were performed on 11 peanut-allergic and 14 nonallergic subjects. Blood was incubated with peanut extract or anti-IgE and tests were performed as follows: BAT-CD63 upregulation was assessed by flow cytometry; HR-released histamine was quantified by a glass fiber-based fluorometric method; passive HR-IgE-stripped donor basophils were incubated with participants' serum and histamine release was quantified as HR. RESULTS: CDsens, a measure of basophil allergen sensitivity, was significantly higher for BAT (80.1±17.4) compared to HR (23.4±10.31) and passive HR (11.1±2.0). BAT, HR, and passive HR had a clinical sensitivity of 100%, 100%, and 82% and specificity of 100%, 100%, and 100%, respectively, when excluding inconclusive results. BAT identified 11 of 11 allergic patients, HR 10, and passive HR 9. Likewise, BAT recognized 12 of 14 nonallergic subjects, HR 10, and passive HR 13. However, the tests' diagnostic performances were not statistically different. Interestingly, nonreleasers in HR but not in BAT had lower basophil count compared to releasers (249 vs 630 counts/min). CONCLUSION: BAT displayed a significantly higher CDsens compared to HR and passive HR. The basophil tests' diagnostic performances were not significantly different. Still, BAT could diagnose subjects with low basophil number in contrast to HR.

The immunoglobulin superfamily member CD200R identifies cells involved in type 2 immune responses.

BACKGROUND: The pathology of allergic diseases involves type 2 immune cells, such as Th2, ILC2, and basophils exerting their effect by production of IL-4, IL-5, and IL-13. However, surface receptors that are specifically expressed on type 2 immune cells are less well documented. The aim of this investigation was to identify surface markers associated with type 2 inflammation. METHODS: Naïve human CD4+ T cells were short-term activated in the presence or absence of IL-4 and analyzed for expression of >300 cell-surface proteins. Ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from peanut-allergic (PA) and nonallergic subjects were stimulated (14-16 h) with peanut extract to detect peanut-specific CD4+ CD154+ T cells. Biopsies were obtained for transcriptomic analysis from healthy controls and patients with extrinsic or intrinsic atopic dermatitis (AD) and psoriasis. RESULTS: Expression analysis of >300 surface proteins enabled identification of IL-4-upregulated surface proteins, such as CD90, CD108, CD109, and CD200R (CD200R1). Additional analysis of in vitro-differentiated Th0, Th1, and Th2 cultures identified CD200R as upregulated on Th2 cells. From ex vivo-isolated PBMCs, we found high expression of CD200R on Th2 and ILC2 cells and basophils. In PA subjects, the peanut-specific Th2 (CD154+ CRTh2+ ) cells expressed more CD200R than the non-allergen-specific Th2 (CD154- CRTh2+ ) cells. Moreover, costaining of CD161 and CD200R identified peanut-specific highly differentiated IL-4+ IL-5+ Th2 cells. Finally, transcriptomic analysis revealed upregulation of CD200R in lesional skin from subjects with an extrinsic AD phenotype compared to healthy skin. CONCLUSION: These results indicate that CD200R expression strongly correlates with Th2 pathology; though, the mechanism is as yet elusive.

Adipose tissue lipin expression levels distinguish HIV patients with and without lipodystrophy.

OBJECTIVE: Lipodystrophy is the major complication of antiretroviral therapy in HIV-infected patients. Its pathophysiology is not well understood, but has been linked to antiadipogenic effects of antiretroviral drugs. Lipin represents a newly characterized protein that is critical for adipocyte differentiation, and lipin deficiency leads to lipodystrophy in the mouse. The objective of this study was to determine whether altered lipin gene expression is associated with HIV lipodystrophy in humans. DESIGN: We measured lipin mRNA levels in subcutaneous abdominal and femoral-gluteal adipose tissue biopsies from HIV-infected patients with or without lipodystrophy, and in healthy controls. Real-time reverse transcription-PCR was performed to quantitate total lipin expression levels, and expression of two lipin isoforms (lipin-alpha and -beta) that are generated by alternative mRNA splicing. RESULTS: As predicted from studies with mice, lipin mRNA levels were correlated with limb fat mass in HIV patients, with lower lipin levels in patients with lipodystrophy than those without lipodystrophy. Unexpectedly, however, this was explained by an increase in lipin-beta expression in HIV patients without lipodystrophy compared to patients with lipodystrophy and control subjects. In addition, lipin expression levels were inversely correlated with adipose tissue expression of inflammatory cytokines interleukin (IL)-6, IL-8 and IL-18, which typically increase in HIV-associated lipoatrophy. CONCLUSIONS: Elevated lipin expression levels are associated both with the maintenance of greater fat mass and lower cytokine expression in HIV-infected patients. Based on the demonstrated role for lipin in promoting lipogenic gene expression, these observations raise the possibility that variations in lipin levels may contribute to variations in adipose tissue mass and function that distinguish HIV patients with and without lipodystrophy.

Fish oil feeding is associated with an increased accumulation of dietary lipids in enterocytes: results from an in vivo study in rats.

BACKGROUND: Chronic fish oil consumption is associated with reduced postprandial lipaemia, but the mechanism behind this effect is not fully understood. We studied whether lipid absorption might be altered in rats fed fish oil. METHODS: Male Wistar rats were fed fish oil enriched chow (n = 6) or control oil enriched chow (n = 6). After 4 weeks, 61 mg 3H-triolein was instilled into duodenal tied-off loops. Intestinal segments were removed after 15, 30, 45, 60 and 90 min. Enterocytes were then isolated by calcium chelation and quantified by DNA determination. Non-absorbed 3H-lipid and 3H-lipid contents of enterocytes were determined by liquid scintillation counting. Two other groups of rats (2 x 6) fed the experimental diets were given an oral fat load and fasting and postprandial blood samples were taken. RESULTS: The accumulation of 3H-lipids in enterocytes was higher in rats fed fish oil than in controls (area under the 3H-lipid time curve: 1041.3 versus 670.3 nmol oleic acid x min/microg DNA, P < 0.05). Separation of lipids showed that an accumulation of triglycerides and free fatty acids occurred in rats fed fish oil. The amount of non-absorbed 3H-lipid tended to be higher in the fish oil fed rats (P > 0.1). It was confirmed that the fish oil enriched chow caused lower postprandial lipaemia (34% reduction in serum triglyceride concentrations, P < 0.05). CONCLUSION: Attenuated postprandial lipaemia following fish oil feeding is explained, at least partly, by a transient lipid accumulation in enterocytes which may result in a delayed triglyceride efflux from the enterocytes into the circulation.

The Göttingen minipig as a model for postprandial hyperlipidaemia in man: experimental observations.

Postprandial hyperlipidaemia is believed to be atherogenic. This study aimed to establish a minipig model to investigate determinants of postprandial lipid metabolism. In a randomized cross-over design seven minipigs were subjected to six different feeding regimens: intragastric fat loads of 1, 2, and 4 g fat (Intralipid, 20%) kg(-1) in two fractions 1.5 h apart (1/3 first, 2/3 second), 2 g fat (Intralipid kg(-1) in one fraction, and 2 g olive oil kg(-1) in two fractions, all after pre-feeding with standard diet, and finally 2 g fat (Intralipid kg(-1) in two fractions without pre-feeding. Blood was sampled before and hourly for 7 h after gavaging, and plasma triglycerides were measured. Triglycerides increased significantly in all the feeding regimens (P < 0.001), except when olive oil was used as the fat source. A borderline significant dose-response effect of the Intralipid dose on the triglyceride response was observed. We found no significant differences in triglyceride response whether 2 g fat (Intralipid kg(-1) was given in one or two fractions, with or without pre-feeding. We conclude that postprandial hyperlipidaemia in minipigs can be induced by gavaging an emulgated lipid solution (1-4 g fat/kg, Intralipid, while olive oil is not applicable. There is no need to administer the fat fractionated or to withhold food prior to administration.

A high-fat meal does not activate blood coagulation factor VII in minipigs.

It is a matter of debate whether postprandial activation of blood coagulation factor VII (FVII) is associated with an increased risk of thrombosis. To clarify this question, an animal model in which consequences of dietary FVII activation can be studied in a more detailed way would be an important tool. We studied postprandial FVII activation in seven non-fasting Göttingen minipigs. Intralipid (4 g/kg) was administered through a gastric tube in two fractions at 9.00 a.m. (one-third of total dose) and 10.30 a.m. (two-thirds of total dose). Blood samples were drawn 0.5 h before (baseline) and 2, 3, 3.5, 4, 5, and 6 h after the first fat load. Triglycerides, activated FVII (FVIIa), FVII coagulant activity (FVIIc), FVII amidolytic activity (FVIIam) and prothrombin fragment I + 2 (F1 + 2) were analysed in plasma samples. Median plasma triglycerides were significantly raised from 0.67 mmol/l (baseline) to 2.56 mmol/l 5 h postprandially (P < 0.001). There were no significant changes in FVIIa (9.6 U/l at baseline), FVIIam (142% at baseline) and F1 + 2 (0.13 nmol/l at baseline). FVIIc decreased from 141% at baseline to 114% 6 h postprandially (P < 0.001). As a high-fat meal does not seem to activate blood coagulation FVII in minipigs, the pig is apparently not a relevant model for the study of dietary FVII activation and thrombin generation.

The link between high-fat meals and postprandial activation of blood coagulation factor VII possibly involves kallikrein.

Contrary to low-fat meals, high-fat meals are known to cause postprandial factor VII (FVII) activation, but the mechanism is unknown. To study the postprandial FVII activation in detail, 18 young men consumed in randomized order high-fat or low-fat test meals. Fasting and non-fasting blood samples were collected. The high-fat test was associated with an increase in plasma triglyceride and kallikrein concentrations and postprandial FVII activation (p<0.001). Plasma kallikrein was strongly associated with triglycerides in fasting and non-fasting samples (r2=0.74-0.87, p<0.0001), suggesting that triglyceride-rich lipoproteins may activate prokallikrein. Neither plasma triglycerides nor kallikrein and activated FVII were statistically associated. This may suggest that additional factors are involved in the postprandial FVII activation. No clear evidence for a role of tissue factor expression by monocytes, factor XII or insulin in postprandial FVII activation was observed. Tissue factor pathway inhibitor and prothrombin fragment 1+2, a marker of thrombin generation, were not affected postprandially after either the high-fat or the low-fat meals. Our findings indicate that triglyceride-rich lipoproteins activate prokallikrein postprandially, which might form an important initial event in FVII activation after consumption of high-fat meals.

In vitro effects of heparin and tissue factor pathway inhibitor on factor VII assays. possible implications for measurements in vivo after heparin therapy.

The coagulant activity of blood coagulation factor VII (FVII:C) can be lowered by changes in lifestyle and by therapeutic intervention, e.g. heparin infusion. The question is, however, whether FVII:C determined ex vivo is a valid measure of the FVII activity in vivo. We measured plasma FVII:C, activated FVII (FVIIa), FVII protein (FVII:Ag), tissue factor pathway inhibitor (TFPI), triglycerides, and free fatty acids (FFA) before and 15 min after infusion of a bolus of unfractionated heparin (50 IU/kg body weight) in 12 healthy subjects. Additionally, we conducted in vitro experiments to investigate the effect of unfractionated heparin and TFPI, which is released from the endothelium by heparin, on FVII:C, FVIIa, and FVII:Ag. Heparin infusion decreased triglycerides and increased FFA and TFPI. This was accompanied by significant reductions in FVIIa, FVII:C and FVII:Ag. In vitro, anti-TFPI antibodies increased FVIIa and FVII:C, and heparin reduced FVIIa. The heparinase Hepzyme was unable to abolish the effect of heparin. There were no in vitro effects on FVII:Ag. We conclude that, due to interference by TFPI and heparin in post-heparin plasma, it is impossible to measure the in vivo FVII activity by means of FVII clotting assays. These assays should therefore not be used to measure the coagulation status of patients in heparin therapy, unless extraordinary precautions are taken to eliminate TFPI and heparin effects ex vivo. The observed effect of heparin on FVII:Ag should be investigated further.

Are olive oil diets antithrombotic? Diets enriched with olive, rapeseed, or sunflower oil affect postprandial factor VII differently.

BACKGROUND: The incidence of ischemic heart disease (IHD) in Crete was lower than expected on the basis of blood lipid concentrations of participants in the Seven Countries Study. A favorable effect of a high intake of olive oil on thrombogenesis may have contributed to this finding. OBJECTIVE: We compared the effects of virgin olive oil with those of rapeseed and sunflower oils on blood coagulation factor VII (FVII), a key factor in thrombogenesis. DESIGN: In a randomized and strictly controlled crossover study, 18 healthy young men consumed diets enriched with 5 g/MJ (19% of total energy) olive oil, sunflower oil, or rapeseed oil for periods of 3 wk. On the final day of each period, participants consumed standardized high-fat meals (42% of energy as fat). Fasting and nonfasting blood samples were collected after each period. RESULTS: Mean (+/-SEM) nonfasting peak concentrations of activated FVII (FVIIa) were 11.3 +/- 5.1 U/L lower after olive oil than after sunflower oil, an 18% reduction (P < 0.05). Olive oil also tended to cause lower FVIIa peak concentrations than did rapeseed oil (mean difference: 8.6 U/L, a 15% reduction; P = 0.09). There were no significant differences between diets with respect to nonfasting factor VII coagulant activity (FVII:c), prothrombin fragment 1+2 (F1+2), and tissue factor pathway inhibitor (TFPI) concentrations, or with respect to fasting plasma values of FVII protein, FVII:c, FVIIa, F1+2, or TFPI. CONCLUSION: A background diet rich in olive oil may attenuate the acute procoagulant effects of fatty meals, which might contribute to the low incidence of IHD in Mediterranean areas.

Effects of dietary fat quality and quantity on postprandial activation of blood coagulation factor VII.

Acute elevation of the coagulant activity of blood coagulation factor VII (FVIIc) is observed after consumption of high-fat meals. This elevation is caused by an increase in the concentration of activated FVII (FVIIa). In a randomized crossover study, we investigated whether saturated, monounsaturated, or polyunsaturated fats differed regarding postprandial activation of FVII. Eighteen healthy young men participated in the study. On 6 separate days each participant consumed two meals (times, 0 and 1 3/4 hours) enriched with 70 g (15 and 55 g) of either rapeseed oil, olive oil, sunflower oil, palm oil, or butter (42% of energy from fat) or isoenergetic low-fat meals (6% of energy from fat). Fasting and series of nonfasting blood samples (the last at time 8 1/2 hours) were collected. Plasma triglycerides, FVIIc, FVIIa, and free fatty acids were analyzed. There were marked effects of the fat quantity on postprandial responses of plasma triglycerides, FVII, and free fatty acids. The high-fat meals caused, in contrast to the low-fat meals, considerable increases in plasma triglycerides. Plasma levels of FVIIc and FVIIa peaks were 7% and 60% higher after consumption of high-fat meals than after consumption of low-fat meals. The five different fat qualities caused similar postprandial increases in plasma triglycerides, FVIIc, and FVIIa. These findings indicate that high-fat meals may be prothrombotic, irrespective of their fatty acid composition. The postprandial FVII activation was not associated with the plasma triglyceride or free fatty acid responses.

Stimulation of glioma-cell migration by laminin and inhibition by anti-alpha3 and anti-beta1 integrin antibodies.

An induction of laminin in the confrontation zone between tumor cells and normal brain tissue has been observed in our model systems in vivo and in vitro. In order to study the effects of ECM components on glioma-cell migration and invasion, we have used 2 lacZ-transfected glioma cell lines, AN1/lacZ and U-251 /lacZ. Cell migration from multicellular spheroids was studied using different types of media: DMEM with 10% serum, Ultra Culture medium, and filtrated DMEM with serum in which the protein fraction > 100 kDa had been removed by ultrafiltration. Laminin, fibronectin and collagen type-IV were individually added to the different media, and cell migration from the spheroids was studied. The results show that cell migration in both cell lines, was stimulated by laminin and fibronectin. Collagen type-IV stimulated only cell migration of U-251/lacZ cells. Scanning electron microscopy revealed an extensive change in cell shape as a result of laminin stimulation. Flowcytometric studies showed that both AN1/lacZ and U-251/lacZ strongly express the alpha3 beta1 integrin receptor, which can bind to several ECM components (laminin, fibronectin, collagen). Immunofluorescence microscopy demonstrated that the same integrin sub-units were expressed in multicellular spheroids. When monoclonal antibodies to alpha3 and beta1 were added to the laminin-stimulated cultures, cell migration was significantly reduced. This indicates that the alpha3 beta1 integrin receptor plays an important role during glioma-cell migration.

Variation in risk indicators of cardiovascular disease during the menstrual cycle: an investigation of within-subject variations in glutathione peroxidase, haemostatic variables, lipids and lipoproteins in healthy young women.

Variations in erythrocyte glutathione peroxidase activity, serum concentrations of lipids and lipoproteins and in blood coagulation and fibrinolysis during the menstrual cycle were studied in healthy young women. Blood samples were drawn twice a week for 9 weeks. A group of males was used for estimation of the influence on the results of factors which were not related to the menstrual cycle. Variations during the menstrual cycle were demonstrated in several of the factors analysed. The activity of glutathione peroxidase was lowest at ovulation. The clotting activity of factor II+ VII+X and the concentration of fibrinogen were lowest during mid-cycle, and the number of platelets increased in the follicular phase (days 5-9). Statistically significant variations in the fibrinolytic factors analysed (tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were not observed during the menstrual cycle. The serum concentrations of cholesterol and LDL cholesterol were significantly higher at the start of the menstrual cycle (days 3-8) than later in the cycle (days 19-24). The concentration of HDL cholesterol was lowest in the late luteal phase (days 23-28).