Expression of a Constitutively Active Human Insulin Receptor in Hippocampal Neurons Does Not Alter VGCC Currents.

Memory and cognitive decline are the product of numerous physiological changes within the aging brain. Multiple theories have focused on the oxidative, calcium, cholinergic, vascular, and inflammation hypotheses of brain aging, with recent evidence suggesting that reductions in insulin signaling may also contribute. Specifically, a reduction in insulin receptor density and mRNA levels has been implicated, however, overcoming these changes remains a challenge. While increasing insulin receptor occupation has been successful in offsetting cognitive decline, alternative molecular approaches should be considered as they could bypass the need for brain insulin delivery. Moreover, this approach may be favorable to test the impact of continued insulin receptor signaling on neuronal function. Here we used hippocampal cultures infected with lentivirus with or without IRß, a constitutively active, truncated form of the human insulin receptor, to characterize the impact continued insulin receptor signaling on voltage-gated calcium channels. Infected cultures were harvested between DIV 13 and 17 (48 h after infection) for Western blot analysis on pAKT and AKT. These results were complemented with whole-cell patch-clamp recordings of individual pyramidal neurons starting 96 h post-infection. Results indicate that while a significant increase in neuronal pAKT/AKT ratio was seen at the time point tested, effects on voltage-gated calcium channels were not detected. These results suggest that there is a significant difference between constitutively active insulin receptors and the actions of insulin on an intact receptor, highlighting potential alternate mechanisms of neuronal insulin resistance and mode of activation.

Modeling Temperature Regime and Physical Habitat Impacts from Restored Streamflow.

Water infrastructure updates at Grand Canyon National Park (GRCA) provide an opportunity to restore natural flow to Bright Angel Creek, adding an additional ~20% to baseflow. This creek provides habitat for endangered humpback chub (Gila cypha) and invasive brown trout (Salmo trutta). We assess how increased flow may alter habitat and how that change may impact native and nonnative species using physical habitat modeling and statistical analysis of stream temperature data. We used System for Environmental Flow Analysis to calculate the change in habitat area for both species in the lower 2.1 km of the creek before and after the increased flow. Results indicate a slight increase in available habitat for juveniles of both species and a slight decrease for spawning brown trout. We used regression modeling to relate daily average air temperature to stream temperature and periods of increased discharge during water system maintenance were used to model the temperatures during likely future conditions. Both high and low stream temperature were dampened due to the added water resulting in fewer days with suitable spawning temperature and more days with suitable growth temperature for humpback chub. Fewer suitable days for growth upstream but more suitable days downstream, were predicted for brown trout. Compared to other streams that sustain populations of humpback chub, flow conditions for Bright Angel Creek provide fewer days throughout the year with suitable temperatures, particularly during the winter months. Juvenile humpback chub rearing may improve through the restoration of flow however the presence of predatory brown trout complicates the net beneficial impact.

Efficacy and safety of Nuwiq® (human-cl rhFVIII) in patients with severe haemophilia A undergoing surgical procedures.

INTRODUCTION: Haemophilia A patients are at a high risk of excess bleeding during surgeries. The aim of haemostatic therapy during the perioperative period is to normalize FVIII level perioperatively and postoperatively to maintain normal haemostasis until wound healing is complete. AIMS/METHODS: To examine the efficacy of Nuwiq® (simoctocog alfa, human-cl rhFVIII), a 4th generation recombinant FVIII produced in a human cell line, for surgical prophylaxis in patients with severe haemophilia A. This analysis assessed the efficacy of Nuwiq® during surgical procedures and in the postoperative period in seven clinical studies of previously treated patients (PTPs) with severe haemophilia A. RESULTS: Thirty-six patients, aged 3-55 years, received surgical prophylaxis with Nuwiq® for 60 surgeries (28 major and 32 minor). Efficacy was evaluated for 52 surgeries (25 major and 27 minor). The success rate of Nuwiq® treatment was 98.1% (51 of 52 evaluated surgeries); haemostatic efficacy was assessed as "excellent" or "good" in all but one major surgery (assessed as "moderate"). The number of infusions ranged from 1 to 19 for minor surgeries and from 3 to 76 for major surgeries. The median (range) daily doses were 42.0 (28.2-100.9) IU kg-1 for minor surgeries and 69.3 (43.3-135.6) IU kg-1 for major surgeries. There were no serious treatment-related adverse events, and none of the patients developed FVIII inhibitors. CONCLUSIONS: The results of this pooled analysis show that Nuwiq® was efficacious in maintaining haemostasis during and after major and minor surgical procedures in PTPs with severe haemophilia A.

First report on the safety and efficacy of an extended half-life glycoPEGylated recombinant FVIII for major surgery in severe haemophilia A.

BACKGROUND: N8-GP (turoctocog alfa pegol) is an extended half-life glycoPEGylated recombinant factor VIII (FVIII) product developed for the prevention and treatment of bleeds in haemophilia A patients. AIM: This is a planned interim analysis of pathfinder™3, an international, open-label, Phase 3 trial evaluating the efficacy and safety (including immunogenicity) of N8-GP administered before, during and after major surgery in severe haemophilia A patients aged ≥12 years. METHODS: Sixteen patients who underwent 18 major surgical procedures (including synovectomy, joint replacement and ankle arthrodesis) were included here. Postoperative assessments were conducted daily for days 1-6, and once for days 7-14. Primary endpoint was N8-GP haemostatic efficacy, assessed after completion of surgery using a four-point scale ('excellent', 'good', 'moderate', 'none'). RESULTS: Haemostasis was successful (rated 'excellent' or 'good') on completion of surgery in 17 (94.4%) procedures and rated as 'moderate' (5.6%) for one surgery in a patient with multiple comorbidities who needed an intraoperative N8-GP dose (20.7 IU kg-1 ). In the postoperative period, three bleeds occurred (one during days 1-6; two during days 7-14); all were successfully treated with N8-GP. Mean N8-GP consumption on day of surgery was 80.0 IU kg-1 ; patients received a mean of 1.7 doses (median: 2, range: 1-3). No safety concerns were identified. CONCLUSION: The data showed that N8-GP was effective and well tolerated for the prevention and treatment of bleeds during major surgery; such FVIII products with extended half-lives may modify current treatment schedules, enabling fewer infusions and earlier patient discharge.

Safety and efficacy of BAY 81-8973 for surgery in previously treated patients with haemophilia A: results of the LEOPOLD clinical trial programme.

INTRODUCTION: BAY 81-8973 is a recombinant factor VIII (rFVIII) with the same amino acid sequence as Bayer's sucrose-formulated rFVIII (rFVIII-FS) but manufactured with certain more advanced technologies. AIM: To describe surgery outcomes with BAY 81-8973 in the LEOPOLD trials. METHODS: Male patients with severe haemophilia A and no inhibitors aged 12-65 years with ≥150 exposure days (EDs) to FVIII (LEOPOLD I and II), or aged ≤12 years with ≥50 EDs to FVIII (LEOPOLD Kids), received BAY 81-8973 based on dosing recommendations for rFVIII-FS according to surgical requirements. Haemostasis-related complications, investigator/surgeon assessment of haemostasis, blood loss, need for transfusion and use of BAY 81-8973 were determined. RESULTS: In LEOPOLD I and II, 11 patients (mean age, 35.3 years) underwent 13 major surgeries. In LEOPOLD Kids, one patient (aged 6 years) underwent one major surgery. Thirty-two adult and paediatric patients underwent 46 minor surgeries. Haemostasis was rated good or excellent in all major and minor surgeries. Blood loss during surgery did not exceed expected amounts; blood transfusions were required in three of the 14 major surgeries. For major surgeries in LEOPOLD I and II, patients received a presurgical 50-IU kg(-1) dose of BAY 81-8973; median nominal dose on day of surgery was 7000 IU (107.5 IU kg(-1) ). Total BAY 81-8973 dose was 2500 IU (108.7 IU kg(-1) ) on the day of the only major surgery in LEOPOLD Kids. No haemostasis-related complications were reported. CONCLUSIONS: Haemostatic control with BAY 81-8973 during all surgeries in the LEOPOLD trials was good or excellent, with no haemostasis-related complications.

Novel, human cell line-derived recombinant factor VIII (human-cl rhFVIII; Nuwiq® ) in adults with severe haemophilia A: efficacy and safety.

INTRODUCTION: Nuwiq® [human cell line-derived recombinant factor VIII (human-cl rhFVIII)] is a new generation rFVIII protein, without chemical modification or fusion to any other protein, produced in a human cell line. AIM/METHODS: This prospective, open-label, multinational phase III study assessed the efficacy and safety of human-cl rhFVIII in 32 adult previously treated patients (PTPs) with severe haemophilia A during standard prophylaxis for ≥6 months and ≥50 exposure days. Efficacy in treating bleeds and during surgical prophylaxis was also assessed. RESULTS: Prophylactic efficacy, based on mean monthly bleeding rate, was rated as 'excellent' or 'good' in 97% of patients for all bleeds and in 100% of patients for spontaneous bleeds. Mean (SD) annualized bleeding rate was 2.28 (3.73) [median = 0.9] for all bleeds, 1.16 (2.57) [median = 0] for spontaneous bleeds and 1.00 (1.79) [median = 0] for traumatic bleeds. There were no bleeds in 50% of patients and there were no major, life-threatening bleeds. Efficacy was 'excellent' or 'good' in treating 28 (100%) of 28 bleeds. Overall efficacy was rated as 'excellent' during four surgical procedures (three major, one minor) and 'moderate' during one major surgery. Incremental in vivo recovery (IVR) data were comparable with the one-stage and chromogenic assays. IVR was >2.0% per IU kg-1 for all measurements and stable over 6 months. No patients developed FVIII inhibitors and there were no treatment-related serious or severe adverse events. CONCLUSION: These results in adult PTPs indicate that human-cl rhFVIII is effective for the prevention and treatment of bleeds in adults with severe haemophilia A.

Major surgery in severe haemophilia A with inhibitors using a recombinant factor VIIa and activated prothrombin complex concentrate hybrid regimen.

Major surgery in persons with haemophilia A and inhibitors is increasingly being performed. Both recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (APCC) are used to cover surgery but it remains unclear what the optimal dosing schedules are. We describe the use of a hybrid regimen in four inhibitor patients undergoing eight major surgical procedures using rFVIIa in the initial 2-6 postoperative days followed by FEIBA for the remaining period. All patients were also treated with tranexamic acid while receiving rFVIIa. We performed six major orthopaedic procedures, one emergency orchidectomy and one open appendectomy. The dosing schedules were at the higher end of those described in the literature but within the recommendations of the summary of product characteristics. Despite this, we encountered non-surgical bleeding in four of eight episodes. Three of these occurred in one individual suggesting a patient factor. The overall outcome was good for all episodes. The hybrid regimen combines flexibility of dose and dosing frequency of rFVIIa in the immediate postoperative setting with the advantage of a reduced dosing frequency with FEIBA in the subsequent days. This study also emphasizes that surgical procedures in this patient group remain a challenge.

Understanding attraction stimuli of the cat flea, Ctenocephalides felis, in non-chemical control methods.

Comparisons were conducted of flea catches of four commercially available flea traps in the laboratory and under field conditions, in both rural and urban locations. The results clearly showed the My Flea Trap™, which utilizes an intermittent light to attract fleas, to be far superior in trapping ability to the three continuous light traps; it caught up to 23 times as many fleas as the other traps. Altering the lighting mechanism to provide continuous rather than intermittent light significantly decreased the number of fleas captured. In addition, the use of a green filter significantly increased trapping efficiency, whereas the addition of a heat source had no apparent effect.

Survivors of tobacco-related diseases and advocacy for tobacco control.

Survivors and victims of tobacco-related diseases can and should play significant roles in tobacco control advocacy efforts. This article describes one example of how to successfully incorporate their talents into a statewide programme.

Best practice in primary care pathology: review 8.

This eighth best practice review examines four series of common primary care questions in laboratory medicine: (i) sodium abnormalities; (ii) faecal occult blood testing; (iii) warfarin management; and (iv) sputum cytology in diagnosis of bronchopulmonary malignancy. The review is presented in question-answer format, referenced for each question series. The recommendations represent a précis of guidance found using a standardised literature search of national and international guidance notes, consensus statements, health policy documents and evidence-based medicine reviews, supplemented by Medline Embase searches to identify relevant primary research documents. They are not standards but form a guide to be set in the clinical context. Most are consensus rather than evidence-based. They will be updated periodically to take account of new information.

The effects of general anaesthetics on glucose and phosphate transport across the membrane of the human erythrocyte.

A study has been carried out into the effects of clinically important general anaesthetics, althesin, thiopentone and propanidid, on the transport of glucose and phosphate across the membrane of the human erythrocyte. In general these three substances all inhibit both transport processes but with characteristic inhibition profiles and varying degrees of efficacy. Glucose transport was more sensitive to the hydrophobic steroids and phosphate transport to propanidid. Some hydrophobic agents, e.g., iodobenzene and its azide, were not inhibitory. Removal of cholesterol to some extent augmented the inhibitory effects of most of these compounds (not propanidid). It is argued that these effects are due to the penetration of the anaesthetics into the lipid bilayer and either subsequent disruption of the lipid annuli surrounding the integral membrane proteins and/or direct anaesthetic-protein interaction.

Novel platelet membrane glycoprotein VI dimorphism is a risk factor for myocardial infarction.

BACKGROUND: Glycoprotein (GP) VI plays a crucial role in platelet activation and aggregation. We investigated whether polymorphic variation at the GP VI locus confers an increased risk of myocardial infarction (MI). METHODS AND RESULTS: Coding and 5' and 3' non-coding regions of the GP VI gene were analyzed by polymerase chain reaction and conformation sensitive gel electrophoresis in 21 healthy subjects. Ten dimorphisms, 5 of which predicted amino acid substitutions (T13254C, A19871G, A21908G, A22630T, C22644A), were identified. Two core haplotypes involving 7 dimorphisms (C10781A and G10873A and all those predicting amino acid substitutions) were apparent. The contribution of the T13254C dimorphism, which predicted the substitution of serine 219 by proline, to risk of MI was assessed in 525 patients with acute MI and 474 controls, all aged <75 years. The allelic odds ratio (OR) for MI associated with the 13254C allele was 1.16 (95% CI, 0.91 to 1.46; P=0.23). Compared with corresponding control subgroups, the 13254CC genotype was more common among cases who were female (OR, 4.52; 95% CI, 1.23 to 16.64; P=0.029), nonsmokers (OR, 2.50; 95% CI, 0.98 to 6.38; P=0.048), aged >/=60 years (OR, 6.48; 95% CI, 1.47 to 28.45; P=0.009) or carried the beta-fibrinogen -148T allele associated with increased fibrinogen levels (OR, 10.49; 95% CI, 1.32 to 83.42; P=0.02). In logistic regression analysis that took other cardiovascular risk factors into account, the interactions of GP VI genotype with age (P=0.005) and beta-fibrinogen genotype (P=0.035) remained significant. CONCLUSIONS: The GP VI 13254CC genotype increases the risk of MI, particularly in older individuals, and the interaction of the GP VI 13254C allele with other candidate risk alleles may accentuate this risk.

A genetic screen for modifiers of E2F in Drosophila melanogaster.

The activity of the E2F transcription factor is regulated in part by pRB, the protein product of the retinoblastoma tumor suppressor gene. Studies of tumor cells show that the p16(ink4a)/cdk4/cyclin D/pRB pathway is mutated in most forms of cancer, suggesting that the deregulation of E2F, and hence the cell cycle, is a common event in tumorigenesis. Extragenic mutations that enhance or suppress E2F activity are likely to alter cell-cycle control and may play a role in tumorigenesis. We used an E2F overexpression phenotype in the Drosophila eye to screen for modifiers of E2F activity. Coexpression of dE2F and its heterodimeric partner dDP in the fly eye induces S phases and cell death. We isolated 33 enhancer mutations of this phenotype by EMS and X-ray mutagenesis and by screening a deficiency library collection. The majority of these mutations sorted into six complementation groups, five of which have been identified as alleles of brahma (brm), moira (mor) osa, pointed (pnt), and polycephalon (poc). osa, brm, and mor encode proteins with homology to SWI1, SWI2, and SWI3, respectively, suggesting that the activity of a SWI/SNF chromatin-remodeling complex has an important impact on E2F-dependent phenotypes. Mutations in poc also suppress phenotypes caused by p21(CIP1) expression, indicating an important role for polycephalon in cell-cycle control.

Successful ketoconazole treatment of protothecosis with ketoconazole-associated hepatotoxicity.

A 46-year-old woman had a chronic, unresponsive wrist infection that was proved to be due to the algaelike organism Prototheca wickerhamii. Treatment with ketoconazole resulted in prompt improvement and ultimate healing. Therapy was complicated by hepatitis that was ketoconazole-related. Ketoconazole may be effective and easily administered therapy for this generally unresponsive infection.

The impact of Xpert(®) MTB/RIF in sparsely populated rural settings.

BACKGROUND: The impact of implementing Xpert(®) MTB/RIF and the choice of instrument placement on patient care in sparsely populated areas with poor access to laboratory and radiology services have not yet been elucidated. METHODS: Prospective evaluation of three diagnostic approaches in the Central Karoo, South Africa: smear microscopy as the initial diagnostic, with sputum processing at centralised laboratories, and Xpert as the initial diagnostic with instrument placement at facility level or centralised laboratory. RESULTS: Of 1449 individuals, 196 were diagnosed with TB. The proportion positive on initial testing was respectively 8%, 20% and 8% during the smear microscopy, decentralised Xpert and centralised Xpert periods. The proportion of bacteriologically confirmed cases was respectively 88%, 99% and 91% during the smear microscopy, decentralised Xpert and centralised Xpert periods. The median time to treatment was respectively 11.5 (interquartile range [IQR] 6-24), 1 (IQR 0-2) and 6 days (IQR 2-9) during the smear microscopy, decentralised Xpert and centralised Xpert periods. CONCLUSION: Introducing Xpert as the initial diagnostic in areas with poor access to TB diagnostics increased the proportion of cases with bacteriological confirmation and reduced time to treatment initiation; however, point-of-care placement may have resulted in fewer people being evaluated for TB.

Coagulation and fibrinolytic activity in Behçet's disease.

Coagulation and fibrinolytic activities were studied in 18 subjects with Behçet's disease and compared with results from 14 matched control patients suffering from sero-negative arthritis. Significantly higher plasma concentrations (median and range) were found in Behçet's patients for the following variables: fibrinogen 3.7 (1.7-6.9) vs 3.0 (2.0-5.1) g/l, p less than 0.05; von Willebrand factor antigen, 115 (72-344) vs 74 (60-119)%, p less than 0.002; plasminogen activator activity (10(6)/ECLT2) 219 (94-329) vs 137 (78-197) units, p less than 0.002; tissue plasminogen activator inhibitor (t-PA-I) activity, 9.1 (5.5-19.3) vs 5.1 (1.8-12.0) IU/ml, p less than 0.002; and PAI-1 antigen, 13.9 (4.5-20.9) vs 6.4 (2.4-11.1) ng/ml, p less than 0.002. Protein C antigen was significiantly lower: 97 (70-183) vs 126 (96-220)%, p less than 0.02. No differences were observed in antithrombin III activity or antigen, factor VIII coagulant activity, fibrinopeptides A and B beta 15-42, plasminogen, alpha-2-antiplasmin, functional and immunological tissue-plasminogen activator, thrombin-antithrombin complexes and D-dimer. Levels of tissue plasminogen activator inhibitor (activity and antigen) correlated with disease activity while fibrinogen and von Willebrand factor concentrations did not. Seven of the 18 subjects with Behçet's disease had suffered thrombotic events but it was not possible to distinguish these from the 11 patients without thrombosis using the assays performed. The results suggest the abnormal fibrinolytic activity in Behçet's disease is due to increased inhibition of tissue plasminogen activator. No abnormality of coagulation or fibrinolytic activity specific to Behçet's disease was detected.

A common splice site mutation is shared by two families with different type 2N von Willebrand disease mutations.

Using an ELISA-based method to detect type 2N von Willebrand disease (VWD), we found two individuals with absent FVIII binding. Direct sequencing of the FVIII binding region of the von Willebrand factor (VWF) gene showed that one individual had an R854Q substitution whilst the other had a T791M substitution. The very low FVIII binding and the VWF:Ag levels in both individuals suggested a second defect on the other VWF allele. Conformation sensitive gel electrophoresis of polymerase chain reaction amplified DNA was used to detect an additional change in the VWF gene of each patient. Direct sequencing confirmed a previously unreported G to A transition in the donor splice site in intron 25 of both individuals which should result in a null allele. This was confirmed by mRNA analysis. These two individuals therefore have compound heterozygous VWD in which the only expressed allele has a type 2N mutation. In our population, such compound heterozygosity appears to be a significant cause of type 2N VWD.

Co-inheritance of the 20210A allele of the prothrombin gene increases the risk of thrombosis in subjects with familial thrombophilia.

The presence of the 20210A allele of the prothrombin (PT) gene has recently been shown to be a risk factor for venous thromboembolism. This is probably mediated through increased plasma prothrombin levels. The aim of this study was to compare the prevalence of the prothrombin 20210A allele in control subjects and in subjects with recognised thrombophilia and to establish whether the additional inheritance of the PT 20210A allele is associated with an increased risk of venous thromboembolism. 101 subjects with a history of venous thromboembolism and diagnosed as having either factor V Leiden (R506Q) or heritable deficiencies of protein C, protein S or antithrombin were studied. The prevalence of the PT 20210A allele in this group was compared with the results obtained for 150 control subjects. In addition, the relationships were examined between genetic status and the number of documented thromboembolic episodes, and between plasma prothrombin levels and possession of the PT 20210A allele. 8 (7.9%) of the 101 patients were also heterozygous for the PT 20210A allele. This compares with 0.7% in the control subjects (p = 0.005). After exclusion of patients on warfarin, the mean plasma prothrombin of 113 subjects without 20210A was 1.09 U/ml, as compared with 1.32 U/ml in 8 with the allele (p = 0.0002). Among the 101 patients with either factor V Leiden, protein S deficiency, protein C deficiency or antithrombin deficiency, the age adjusted mean (SD) number of venous thromboembolic episodes at diagnosis was 3.7 (1.5) in those with the PT 20210A allele, as compared with 1.9 (1.1) in those without (p = 0.0001). We have demonstrated that the prevalence of the PT 20210A allele is significantly greater in subjects with venous thrombosis and characterised heritable thrombophilia than in normal control subjects and that the additional inheritance of PT 20210A is associated with an increased risk of venous thromboembolism. We have also confirmed that plasma prothrombin levels are significantly greater in subjects possessing the PT 20210A compared with those who do not.

The prothrombin 20210A allele and its association with myocardial infarction.

The relationship between the prothrombin (PT) 20210A allele and arterial disease is controversial. We conducted a case-control study to assess its contribution to risk of myocardial infarction (MI). Five hundred and thirty-nine acute MI patients and 498 control subjects aged <75 years were studied. Two percent of cases carried the PT20210A allele compared to 2.8% of controls. The odds ratio for MI was 0.72 (95% CI 0.32-1.60) indicating that the PT20210A allele confers no increased risk for MI. Subgroup analysis showed no association between the PT20210A allele and either premature MI or MI in females. We conclude the PT20210A allele is not a risk factor for MI and suggest that discrepancies in studies relating the PT20210A allele to MI may be due to difficulties in estimating its low allelic frequency in the general population and thus random differences in the observed frequencies in the control populations studied.

Coagulation, fibrinolytic and platelet function in patients on long-term therapy with aspirin 300 mg or 1,200 mg daily compared with placebo.

Aspirin has been shown to be beneficial in the prophylaxis of arterial thromboembolic disease. The rationale for its use as an antithrombotic drug lies in its inhibition of thromboxane A2-dependent platelet function. However, the effect of aspirin on coagulation and fibrinolysis during chronic therapy has not been studied. We have measured a range of haemostatic and platelet functions in 49 patients with transient ischaemic attacks randomly allocated to aspirin 300 mg a day, aspirin 1,200 mg a day or placebo. All had been taking their allocated treatment for between 9 months and 4 years prior to investigation. Bleeding time was prolonged, serum thromboxane diminished and platelet aggregation to arachidonic acid but not ADP was abolished by both 300 mg and 1,200 mg aspirin, in a non-dose dependent fashion. Serum salicylate increased with the dose of aspirin ingested. No effect was seen with either dose of aspirin on urinary thromboxane and 6-keto-PGF1 alpha excretion, or on coagulation. Patients taking 1,200 mg aspirin a day had a lower haemoglobin and packed cell volume, lower resting fibrinopeptide A concentration and lower basal plasminogen activator activity than those on placebo. Response to venous occlusion was normal in all groups. The results suggest 300 mg and 1,200 mg aspirin have an equivalent platelet inhibitory effect but 1,200 mg aspirin causes greater gastro-intestinal blood loss.