Role of L-Arginine on the Expression of Coagulation Factor VIII Gene.

Objective: To explore the key sites in which L-arginine affects the expression of human coagulation factor VIII gene, and to create new drug targets for the treatment of hemophilia. Methods: A total of 5 human FVIII genes (A1, A2, A3, C1 and C2) with B domain deletion were transfected into human umbilical vein endothelial cells (HUVECs) as promoters. Run-on assay and ELISA analysis were performed to observe the driving effect of each domain gene on chloramphenicol acetyl transferase (CAT) gene transcription and expression, and the effect of L-arginine on each promoter. Results: In co-culture with L-arginine, transcriptional expression of the CAT gene was not detected in the PCAT3-Basic group (negative control without promoters), PA3-CAT3-Enhancer group or PC1-CAT3-Enhancer group. The transcriptional expression of CAT gene in the PCAT3-Control group (positive control with promoters) and PA1-CAT3-Enhancer group was unchanged compared with the non-L-arginine intervention, while the transcriptional expression of CAT gene in the PA2-CAT3-Enhancer group was significantly enhanced. Conclusions: A1 and A2 domain genes had promoter function and could initiate the transcription and expression of CAT gene, but A3, C1 and C2 domain genes could not. Moreover, L-arginine can significantly enhance transcription and expression of human coagulation factor VIII via A2 domain.

The full-length Auxin Response Factor 8 isoform ARF8.1 controls pollen cell wall formation and directly regulates TDF1, AMS and MS188 expression.

Auxin Response Factor 8 plays a key role in late stamen development: its splice variants ARF8.4 and ARF8.2 control stamen elongation and anther dehiscence. Here, we characterized the role of ARF8 isoforms in pollen fertility. By phenotypic and ultrastructural analysis of arf8-7 mutant stamens, we found defects in pollen germination and viability caused by alterations in exine structure and pollen coat deposition. Furthermore, tapetum degeneration, a prerequisite for proper pollen wall formation, is delayed in arf8-7 anthers. In agreement, the genes encoding the transcription factors TDF1, AMS, MS188 and MS1, required for exine and pollen coat formation, and tapetum development, are downregulated in arf8-7 stamens. Consistently, the sporopollenin content is decreased, and the expression of sporopollenin synthesis/transport and pollen coat protein biosynthetic genes, regulated by AMS and MS188, is reduced. Inducible expression of the full-length isoform ARF8.1 in arf8-7 inflorescences complements the pollen (and tapetum) phenotype and restores the expression of the above transcription factors. Chromatin immunoprecipitation-quantitative polymerase chain reaction assay revealed that ARF8.1 directly targets the promoters of TDF1, AMS and MS188. In conclusion, the ARF8.1 isoform controls pollen and tapetum development acting directly on the expression of TDF1, AMS and MS188, which belong to the pollen/tapetum genetic pathway.

Efficacy and safety of prophylaxis with BAY 81-8973 in Chinese patients with severe haemophilia A enrolled in the LEOPOLD II trial.

INTRODUCTION: BAY 81-8973 (Kovaltry® ) is a full-length, unmodified recombinant human factor VIII approved in China for prophylaxis and on-demand treatment in patients with haemophilia A. Limited access to FVIII prophylaxis in China has historically led to this population being undertreated. This subanalysis of LEOPOLD II investigated whether the efficacy and safety of BAY 81-8973 varied between Chinese and non-Chinese patients. AIM: To evaluate BAY 81-8973 efficacy and safety in Chinese patients. METHODS: LEOPOLD II enrolled males aged 12-65 years with severe haemophilia A who were receiving on-demand treatment. Patients were randomly assigned to receive BAY 81-8973 as low-dose prophylaxis (20-30 IU/kg twice-weekly), high-dose prophylaxis (30-40 IU/kg 3 times weekly) or on-demand for 1 year. RESULTS: Data were available from 23 Chinese and 57 non-Chinese patients; Chinese patients had a higher prestudy bleeding rate and were more likely to have target joints than non-Chinese patients. 74% of patients were assigned to prophylaxis. Annualized bleeding rates (ABRs) in Chinese and non-Chinese patients receiving prophylaxis were significantly lower compared to patients treated on-demand. Median ABRs for all bleeds in the last 6 months of the study were 2.0 and 1.0 for Chinese and non-Chinese patients, respectively, in the combined prophylaxis groups, and 61.3 and 58.5 in the on-demand group. A treatment-related adverse event occurred in 1 Chinese patient; no patients developed FVIII inhibitors. CONCLUSION: BAY 81-8973 prophylaxis was efficacious and well tolerated in Chinese patients with severe haemophilia A, with ABRs comparable to those in non-Chinese patients receiving prophylaxis.

European principles of inhibitor management in patients with haemophilia.

BACKGROUND: In spite of recent major advances in the understanding and treatment of inhibitor development in patients with haemophilia, multidisciplinary management of many of these patients remains suboptimal and highly heterogenous across Europe. METHODS: Following a series of multidisciplinary meetings and a review of the literature, the European haemophilia community of health professionals and patients jointly defined practical optimum standards for ensuring and harmonizing treatment and care for patients with an inhibitor. RESULTS: Ten complementary principles for the management of inhibitors in haemophilia have been developed, emphasizing the importance and benefits of a centralized, multidisciplinary, expert and holistic approach. CONCLUSIONS: This document will serve as a benchmark to improve the multidisciplinary and practical management of patients with inhibitor. Implementation and adherence to each of these principles should have a major positive impact on the management and outcomes of patients developing an inhibitor.

Manual therapy in the treatment of patients with hemophilia B and inhibitor.

BACKGROUND: The main clinical manifestations of hemophilia are muscle and joint bleeding. Recurrent bleeding leads to a degenerative process known as hemophilic arthropathy. The development of inhibitors (antibodies against FVIII/FIX concentrates) is the main complication in the treatment of hemophilia. The objective was to assess the safety and efficacy of manual therapy treatment in a patient with hemophilia and inhibitor. CASE PRESENTATION: A 26-year-old patient with hemophilia B and inhibitor received physiotherapy treatment based on manual therapy for 3 months, with a frequency of 2 sessions per week. The joint status was evaluated using the Hemophilia Joint Health Score; pain was assessed with the Visual Analog Scale; and the range of movement was evaluated using a universal goniometer. The patient developed no joint bleeding in the knees or ankles as a result of the physiotherapy treatment. Following treatment, improvements were noted in the range of movement of knees and ankles, the perception of pain in both knees, and ankle functionality. CONCLUSIONS: Until now, manual therapy using joint traction was contraindicated in patients with hemophilia and inhibitor, as it was feared to cause possible joint bleeding. This is the first case study to address the safety and efficacy of manual therapy in a patient with hemophilia and an inhibitor. The results of this study may help to establish which manual therapy treatments are indicated in patients with hemophilic arthropathy and inhibitors. Thus, a physiotherapy program based on manual therapy may be safe in patients with hemophilia and inhibitor and such therapy may improve joint condition, pain, and joint range of motion in patients with hemophilia and inhibitor. Randomized clinical trials are needed to confirm the results of this case study.

Expression and characterization of a codon-optimized blood coagulation factor VIII.

Essentials Recombinant factor VIII (FVIII) is known to be expressed at a low level in cell culture. To increase expression, we used codon-optimization of a B-domain deleted FVIII (BDD-FVIII). This resulted in 7-fold increase of the expression level in cell culture. The biochemical properties of codon-optimized BDD-FVIII were similar to the wild-type protein. SUMMARY: Background Production of recombinant factor VIII (FVIII) is challenging because of its low expression. It was previously shown that codon-optimization of a B-domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well recognized that synonymous mutations may affect the protein structure and function. Objectives To compare biochemical properties of a BDD-FVIII variants expressed from codon-optimized and wild-type cDNAs (CO and WT, respectively). Methods Each variant of the BDD-FVIII was expressed in several independent Chinese hamster ovary (CHO) cell lines, generated using a lentiviral platform. The proteins were purified by two-step affinity chromatography and analyzed in parallel by PAGE-western blot, mass spectrometry, circular dichroism, surface plasmon resonance, and chromogenic, clotting and thrombin generation assays. Results and conclusion The average yield of the CO was 7-fold higher than WT, whereas both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low-density lipoprotein receptor-related protein 1. The CO preparations had on average 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure as a result of considerably higher protein concentrations during the production. We concluded that the codon-optimization of the BDD-FVIII resulted in significant increase of its expression and did not affect the structure-function properties.

Effects of Danhong Injection (丹红注射液) and its main components on anticoagulation and fibrinolysis in cultured vein endothelial cells.

OBJECTIVE: To observe the effects of Danhong Injection (丹红注射液) and its main components, including daiclzein and hydroxysafflor yellow A (HSYA), on the anticoagulation, fibrinolysis, anti-apoptosis in hypoxia model of vein endothelial cells (VECs). METHODS: VECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N and 5% CO mixed gas, when reached confluent culture. Five groups used different treatments, including normal control group, hypoxia group, daiclzein group, HSYA group and Danhong Injection group. The VECs were identified by fluorescence double labeling methods. The morphology was observed by a phase contrast microscopy. The effects of Danhong Injection, daiclzein and HSYA on 6 keto prostaglandin F1α (6-keto-PGF1α) level was measured by the method of radioimmunoassay (RIA). Superoxide dismutase (SOD) activity was tested by water soluble tetrazolium salt. The content of malondialdehyde (MDA) was measured by thiobarbituric acid. The activities of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were measured by the method of chromogenic substrate. The contents of endothelin (ET) and nitric oxide (NO) were detected by non-equilibrium RIA and enzymelinked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry. RESULTS: Compared with the normal control group, the floating cells number, PAI activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1α and NO contents, and t-PA and SOD activities were decreased significantly (P<0.01). Compared with the hypoxia group, Danhong Injection markedly increased the 6-keto-PGF1α content and SOD activity, regulated PAI and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate (P<0.05 or P<0.01). CONCLUSIONS: Danhong Injection and its main components played an important role in protecting primary VECs from hypoxic damage by regulating the secretion and vasomotor function of VECs. The function of Danhong Injection was most remarkable.

Effects of a Low Dose of Fish Oil on Inflammatory Markers of Brazilian HIV-Infected Adults on Antiretroviral Therapy: A Randomized, Parallel, Placebo-Controlled Trial.

BACKGROUND: The benefits of antiretroviral therapy for HIV-infected subjects have been limited by an increased risk of metabolic and cardiovascular diseases. The objective of this study was to assess the effects of a low dose of marine omega-3 fatty acids on inflammatory marker concentrations in HIV-infected subjects under antiretroviral therapy (ART). METHODS: This was a randomized, parallel, placebo-controlled trial that investigated the effects of 3 g fish oil/day (540 mg of eicosapentaenoic acid-EPA plus 360 mg of docosahexaenoic acid-DHA) or 3 g soy oil/day (placebo) for 24 weeks in 83 male and non-pregnant female HIV-infected adults on ART. RESULTS: There were no differences between groups for the measures at baseline. Multilevel analyses revealed no statistically significant relationship between the longitudinal changes in high sensitivity-C reactive protein (hs-CRP) (Wald Chi2 = 0.17, p = 0.918), fibrinogen (Wald Chi2 = 3.82, p = 0.148), and factor VIII (Wald Chi2 = 5.25, p = 0.073) with fish oil. No significant changes in interleukin-6 (IL6), interleukin-1 beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha) serum concentrations were observed with fish oil supplements for 12 weeks. CONCLUSIONS: Compared to placebo, a low dose of 900 mg omega-3 fatty acids (EPA plus DHA) in fish oil capsules did not change hs-CRP, fibrinogen, factor VIII, IL6, IL1-beta and TNF-alpha serum concentrations in HIV-infected subjects on ART. Further investigations should consider the assessment of more sensitive inflammatory markers or higher doses to evaluate the effects of marine omega-3 fatty acids in this population. Registered at the Nederlands Trial Register, Identifier no. NTR1798.

Current management of the hemophilic child: a demanding interlocutor. Quality of life and adequate cost-efficacy analysis.

Hemophilias are the most known inherited bleeding disorders. The challenges in the management of hemophilic children are different from those in adults: prophylaxis regimen removed the hallmark of crippling disease with lifelong disabilities; individualized regimens are being implemented in order to overcome venous access problems. Presently, at least in high-income countries, advances in treatment of hemophilia resulted in continuous improvement of the patients' quality of life and life expectancy. Inhibitors remain the most severe complication of hemophilia therapy. The treatment' compliance is the key to achieve a successful management. The patient, his family, the medical and psychological team are the players of a comprehensive care system. The current management of hemophilic children is the example of huge resource investments enabling long-term benefits in particular quality of life as a primary objective of the healthcare process.

Continuous infusion in haemophilia: current practice in Europe.

Summary. Continuous infusion (CI) of factor VIII (FVIII) is an effective method for replacement therapy in haemophilia. Recently, concerns have been raised regarding association of CI with the development of inhibitors. The aim of this study was to gain information on the current practices in Europe regarding CI and the true inhibitor incidence after this mode of therapy. In a cross sectional study performed in 22 Comprehensive Care Centres (CCCs), we evaluated CI techniques, treatment protocols, efficacy, safety and complications of CI including inhibitors. Thirteen (59%) CCCs reported a total of 1079 CI treatments, given peri-operatively or for major bleeds, in 742 patients. Most centres used 'adjusted dose' CI aimed at median target FVIII level of 0.8 IU mL(-1). CI was haemostatically very effective with a low incidence of complications: median incidence of postoperative bleeding was 1.8%, six centres observed phlebitis in 2-11% of CI treatments. Only nine (1.2%) patients developed inhibitors (0.45% of 659 severe and 7.2% of 83 mild haemophilia patients). Additional analysis of inhibitor patients revealed several confounding factors (low number of prior FVIII exposure days, high steady-state factor levels during CI, high-risk genotype). In this unprecedentedly large cohort, CI treatment appears to be an effective and safe treatment that does not increase the risk of inhibitor development in patients with severe haemophilia. Thus, previous small case series reports suggesting that CI may increase inhibitors cannot be confirmed. Inhibitor risk in mild haemophilia could not be evaluated as the influence of other, potentially confounding, risk factors could not be excluded.

Enhancement of gastric ulcer healing and angiogenesis by cochinchina Momordica seed extract in rats.

Cochinchina momordica seed is the dried ripe seed of Momordica cochinchinensis, a perennial vine. The antiulcer effect of an extract from cochinchina momordica seeds (SK-MS10) was evaluated in a rat model of acetic acid-induced gastric ulcers. Gastric ulcers were produced by subserosal injection of acetic acid. SK-MS10 (200 mg/kg) or vehicle was administered orally once per day for 14 days after the acetic acid injection. The stomach was removed and the ulcer size measured at day 7 and 14 of the treatment. Expression of vascular endothelial growth factor (VEGF) was assessed by real-time RT-PCR and Western blot analysis. In addition, the microvasculature density (MVD) adjacent to the ulcer margin was examined by immunohistochemistry. The treatment with SK-MS10 for 7 and 14 days significantly accelerated ulcer healing and increased the expression of mRNA (at day 7) as well as VEGF protein (at day 14) compared to the vehicle-treated rats. The MVD for factor VIII was also higher in the SK-MS10 treatment group compared to the vehicle-treated rats; however, these differences were not statistically significant. These results suggest that SK-MS10 treatment accelerates the healing of gastric ulcers via upregulation of VEGF and angiogenesis in an acetic acid rat model.

Thalidomide-dexamethasone as up-front therapy for patients with newly diagnosed multiple myeloma: thrombophilic alterations, thrombotic complications, and thromboprophylaxis with low-dose warfarin.

BACKGROUND: Venous thromboembolism (VTE) is a major complication of myeloma therapy recently observed with the increasing use of up-front thalidomide and dexamethasone (thal-dex). The pathogenesis of thal-induced VTE is not well recognized, and the role of prothrombotic factors, especially of thrombophilic abnormalities, is not yet determined. MATERIAL AND METHODS: Two hundred and sixty-six patients with newly diagnosed multiple myeloma (MM) were primarily treated with thal-dex in preparation for subsequent high-dose therapy and autologous stem-cell transplantation. Out of these 266 patients, 190 were evaluated for thrombophilic alterations at baseline, and 125 of them were also re-assessed after thal-dex therapy. RESULTS: The presence of genetic thrombophilic polymorphisms among patients with MM was superimposable to that of normal controls and was associated with a twofold increase in the relative risk of VTE. aAPCR and elevated factor VIII levels were frequent, albeit transient, alterations and were not associated with a significant increase in the risk of VTE. Two hundred and forty-six patients received a thromboprophylaxis with fixed low-dose warfarin (1.25 mg/day) during thal-dex therapy. Of these patients (or 10.6%), 26 had symptomatic VTE events. Their patients-years rate of VTE (35.5%) was significantly lower in comparison with the 86.2% rate recorded among the first 19 patients who initially entered the study and did not receive any kind of thromboprophylaxis (P = 0.043). CONCLUSIONS: On the basis of these data, a baseline thrombophilic work up is not recommended in patients with receiving up-front thal-dex. For these patients, fixed low-dose warfarin may be a valuable prophylaxis against VTE.

Enhanced efficacy of recombinant FVIII in noncovalent complex with PEGylated liposome in hemophilia A mice.

Recombinant FVIII formulated in PEG-ylated liposomes (rFVIII-PEG-Lip) was reported to increase the bleed-free days from 7 to 13 days (at 35 IU/kg rFVIII) in severe hemophilia A patients. To understand the underlying mechanism, we sought to recapitulate its efficacy in hemophilia A mice. Animals treated with rFVIII-PEG-Lip achieved approximately 30% higher survival relative to rFVIII after tail vein transection inflicted 24 hours after dosing. The efficacy of rFVIII-PEG-Lip represents an approximately 2.5-fold higher "apparent" FVIII activity, which is not accounted for by its modestly increased (13%) half-life. The enhanced efficacy requires complex formation between rFVIII and PEG-Lip before the administration. Furthermore, PEG-Lip associates with the majority of platelets and monocytes in vivo, and results in increased P-selectin surface expression on platelets in response to collagen. Rotational thromboelastometry (ROTEM) analysis of whole blood from rFVIII-PEG-Lip-treated animals at 5 minutes up to 72 hours after dosing recapitulated the 2- to 3-fold higher apparent FVIII activity. The enhanced procoagulant activity is fully retained in plasma unless microparticles are removed by ultracentrifugation. Taken together, the efficacy of rFVIII-PEG-Lip is mediated mainly by its sensitization of platelets and the generation of procoagulant microparticles that may express sustained high-affinity receptors for FVIII.

Cyclooxygenase-2 and vascular endothelial growth factor expression in 5-fluorouracil-induced oral mucositis in hamsters: evaluation of two low-intensity laser protocols.

GOAL OF WORK: The aim of this study was to investigate the mechanisms whereby low-intensity laser therapy may affect the severity of oral mucositis. MATERIALS AND METHODS: A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil followed by surface irritation. Animals were randomly allocated into three groups and treated with a 35 mW laser, 100 mW laser, or no laser. Clinical severity of mucositis was assessed at four time-points by a blinded examiner. Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. This tissue was used for immunohistochemistry for cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and factor VIII (marker of microvessel density) and the resulting staining was quantified. MAIN RESULTS: Peak severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW laser and control groups. This reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a significantly lower level of COX-2 staining. The 100 mW laser did not have an effect on the severity of clinical mucositis, but was associated with a decrease in VEGF levels at the later time-points, as compared to the other groups. There was no clear relationship of VEGF levels or microvessel density to clinical mucositis severity. CONCLUSION: The tissue response to laser therapy appears to vary by dose. Low-intensity laser therapy appears to reduce the severity of mucositis, at least in part, by reducing COX-2 levels and associated inhibition of the inflammatory response.

Efficacy and safety of a new-class hemostatic drug candidate, AV513, in dogs with hemophilia A.

AV513 is a select fucoidan, a sulfated polysaccharide of botanical origin. It inhibits tissue factor pathway inhibitor (TFPI) activity and accelerates clotting of human hemophilia A and B plasma. In prior work, subcutaneous administration of AV513 to mice with hemophilia A improved hemostasis. The current studies were designed to evaluate potential efficacy and safety in dogs with hemophilia A (hemophilia A dogs) with minimally increased hemostasis after adenoassociated viral-FVIII gene transfer and in treatment-naive severe hemophilia A dogs. AV513 administered subcutaneously to low-FVIII dogs for multiple weeks improved hemostasis as exhibited in thromboelastography (TEG) and cuticle bleeding time (CBT) tests. Moreover, AV513 administered orally to AAV-FVIII dogs and treatment-naive severe hemophilia A dogs for a multiweek dose-escalating period yielded correction to normal ranges in both TEG and CBT end points at 5 to 15 mg/kg and 15 to 20 mg/kg dose levels, respectively. In all 3 separate studies, throughout their duration, AV513 was well tolerated by the dogs without any adverse events. Additional pharmacologic characterization of AV513 included intravenous pharmacokinetic analysis in rats. In summary, the combination of safety and efficacy in 2 global tests of hemostasis in the hemophilia A dog model indicate that further evaluation of AV513 as a hemostatic agent in hemophilia A patients is warranted.

Sinomenine suppresses TNF-alpha-induced VCAM-1 expression in human umbilical vein endothelial cells.

Sinomenine (SN), an alkaloid prepared from the root of Sinomenium acutum Rehd. Et wils, is used to alleviate the symptoms of rheumatism in Chinese medicine. In the present study, the potential inhibition of TNF-alpha-induced VCAM-1 expression on human umbilical vein endothelial cells (HUVECs) was evaluated in vitro. HUVECs were isolated from freshly collected umbilical cords. Positive controls were stimulated with TNF-alpha, omitting SN. Negative controls were cultured omitting TNF-alpha and SN. Experimental groups were co-cultured with TNF-alpha and SN at different concentrations (0.25, 0.5, and 1.0 mol/L), or TNF-alpha and Dexamethasone (Dex) at a concentration of 1.0 x 10(-6) mol/L. Cells were harvested after culturing with the above drugs for 12 h. VCAM-1 mRNA expression was detected by real-time quantitative PCR, and VCAM-1 expression was detected by flow cytometry. The experimental data indicated that VCAM-1 mRNA and VCAM-1 were induced by TNF-alpha. The relative VCAM-1 mRNA expression decreased in the experimental groups (p<0.05). Concentrations of SN at 0.5 and 1.0 mol/L inhibited expression of VCAM-1 (p<0.05). SN at concentration of 0.25 mol/L and Dex at concentration of 1.0 x 10(-6) mol/L did not show an inhibitory effect on VCAM-1 expression in TNF-alpha-induced HUVECs. Our preliminary data indicates that SN has an inhibitory effect in vitro on TNF-alpha-induced VCAM-1 expression at both mRNA level and protein level in HUVECs, and suggests that SN may be a novel method of immunotherapy for rheumatic carditis or rheumatic heart disease.

A solvent/detergent-treated and 15-nm filtered factor VIII: a new safety standard for plasma-derived coagulation factor concentrates.

BACKGROUND: Since the early 1990 s the Committee for Proprietary Medicinal Products has set the mandatory requirement that all manufacturing processes for blood products include two virus removal/inactivation steps that are complementary in their action. OBJECTIVES: The objective was to develop a manufacturing process for factor VIII (FVIII) including two complementary steps of viral inactivation/elimination. METHODS: A 35-15 nm nanofiltration step was added to a former FVIII manufacturing process that included solvent/detergent (S/D) treatment to generate a new FVIII concentrate called Factane. The impact of nanofiltration on the structural and functional characteristics of FVIII, as well as virus/transmissible spongiform encephalopathy reduction factors were assessed. RESULTS: Using an innovative approach, FVIII was successfully nanofiltered at 35-15 nm, while the biological properties of the active substance were unmodified. FVIII coagulant and antigen content for Factane and previous S/D-treated FVIII (FVIII-LFB, commercialized as Facteur VIII-LFB) were comparable. The FVIII one-stage chromogenic and coagulant/antigen ratios confirmed that nanofiltered FVIII was not activated. After nanofiltration, the copurified von Willebrand factor (vWF) was reduced but vWF/FVIII binding properties were unaffected. Phospholipid binding and thrombin proteolysis studies displayed no differences between Factane and FVIII-LFB. The rate of factor Xa generation was slightly lower for Factane when compared to FVIII-LFB. Viral validation studies with different viruses showed no detectable virus in the filtrate. CONCLUSIONS: Nanofiltration of FVIII at 15 nm is feasible despite the large molecular weight of FVIII and vWF. Nanofiltration has been proven to be highly effective at removing infectious agents while preserving the structural and functional integrity of FVIII.

Celecoxib inhibits meningioma tumor growth in a mouse xenograft model.

BACKGROUND: Treatments for recurrent meningiomas are limited. We previously demonstrated universal expression of COX-2 in meningiomas and dose-dependent growth inhibition in vitro with celecoxib, a COX-2 inhibitor. We therefore tested the effects of celecoxib on meningioma growth in a mouse xenograft model. METHODS: Meningioma cell lines (IOMM-Lee, CH157-MN, WHO grade I primary cultured tumor) were transplanted into flanks of nude mice fed mouse chow with celecoxib at varying concentrations (0, 500, 1000, 1500 ppm) ad libitum. Tumors were measured biweekly and processed for MIB-1, Factor VIII, COX-2, and VEGF, and assayed with transferase-mediated dUTP-biotin nick-end labeling (TUNEL). RESULTS: Celecoxib reduced growth of mean tumor volume by 66% (P < .05), 25% (P > .05), and 65% (P < .05) compared with untreated controls in IOMM-Lee, CH157-MN, and benign tumors, respectively. IOMM-Lee tumors removed from celecoxib treatment regained a growth rate similar to the control. Blood vessel density decreased and apoptotic cells increased in treated flank tumors. Diminished COX-2 expression and VEGF were observed in treated IOMM-Lee tumors. Mean plasma celecoxib levels were 845, 1540, and 2869 ng/mL, for low-, medium-, and high-dose celecoxib, respectively. CONCLUSIONS: Celecoxib inhibits meningioma growth in vivo at plasma levels achievable in humans. Celecoxib-treated tumors were less vascular with increased apoptosis. IOMM-Lee tumors treated with celecoxib showed decreased COX-2 and VEGF expression. COX-2 inhibitors may have a role in the treatment of recurrent meningiomas.

Inhibition of tumor angiogenesis by Brahma Rasayana (BR).

The current therapy for prostate cancer includes radical prostatectomy, radiation therapy and hormonal ablation. Chemotherapy also provides beneficial results for some patients with advanced prostate cancer but with several harmful side effects. Hence there is a need to identify and develop alternate therapies, which can reduce the disease progression with minimal or few side effects. Earlier studies from our laboratory have shown that a Polyherbal mixture, Brahma Rasayna (BR) rich in anti-oxidant principles has a potential to be an anti-tumor agent. BR treatment of MAT-LyLu cell inoculated Copenhagen rats resulted in a decrease of palpable tumor incidence, delay in tumor occurrence and lower mean tumor volumes. Also, a significant reduction in tumor weight and lung metastasis was observed in BR treated animals in comparison to untreated controls. In the present study, we focused to examine the effect of BR on angiogenesis and regulation of molecular markers involved in angiogenesis using in-vivo and in-vitro models. BR treatment showed a significant reduction in Factor VIII expression compared to control indicating reduced angiogenesis. BR treated tumor specimens showed a decrease in the pro-angiogenic factors like VEGF, MMP-9 and MMP-2. Methanolic extract of BR was found to inhibit the proliferation, tube formation, cell migration and attachment of HUVEC on matrigel in a dose dependant manner. These findings suggest the possible mechanism(s) of action of BR in the reduction of tumor growth and metastatic spread.

Distinctive effects of red wine and diet on haemostatic cardiovascular risk factors.

The aim of this study was to compare the effects of Mediterranean-type diet (MD), high-fat diet (HFD), and red wine supplementation on plasma concentration of emergent haemostatic cardiovascular risk factors (HCVRF) and on variables of primary haemostasis (bleeding time, plasma von Willebrand factor and platelet aggregation/secretion). In a controlled prospective intervention study, two groups (21 healthy males each) received either MD or HFD during 90 days. Between days 30-60, both diets were supplemented with 240 ml/ day of red wine. After adjusting by baseline values, MD was associated with: lower plasma fibrinogen (p =0.03), factor VIIc (p=0.034) and factor VIIIc (p=0.0057); higher levels of protein S (p=0.013); longer bleeding time (p=0.017); and marginal increases in platelet serotonin aggregation and secretion after stimulation with epinephrine. Red wine supplementation, in both diets, resulted in decreased plasma fibrinogen (p=0.001) and factor VIIc (p=0.05), and in increased t-PA (p=0.01) and PAI-1 (p=0.0003). The effects of wine on antithrombin III (p=0.01) were divergent: there was a decrease in the HFD group but it increased slightly in the MD group. No effects of diet or wine were detected in plasma protein C, C-reactive protein or von Willebrand factor. BT did not change significantly with wine supplementation. Wine intake resulted in a significant increase in ex vivo platelet aggregation and secretion after stimulation with collagen (1 and 2 microg/ml, p < or = 0.01). MD and moderate consumption of red wine have complementary, mostly beneficial effects on haemostatic CV risk factors. The longer BT in individuals on MD, obtained independently of red wine, denotes less interaction of platelets with the vascular wall, which could be beneficial from the point of view of CV risk.