SUMMARY In this study, Anginex lipoplexes (AxL) were developed for intracellular delivery of non-coding RNA (ncRNA) to endothelial cells of tumor vasculature. Physicochemical characteristics, stability, cell binding and cell uptake of this system were investigated in HUVEC. Upon transfection of HUVEC with AxL containing siVEGFR2, considerable gene silencing was achieved. INTRODUCTION Since the discovery of RNA interference (RNAi) as a gene regulation process, successful delivery of ncRNAs appears as the major challenge for therapeutic activity. ncRNAs are mediators of the RNAi phenomenon and are involved in various processes in cells among which cell proliferation and cell migration play a central role in angiogenesis. Prevention of angiogenesis inhibits tumor growth as it restricts the access of tumor cells to oxygen and nutrients. Endothelial cell (EC) activation is the first step in angiogenesis and is influenced by various effectors. Vascular endothelial growth factor (VEGF) is the major trigger in EC activation. Therefore, siRNA silencing VEGF receptor 2 (VEGFR2) gene of EC seems an effective tool to inhibit tumor progression. Although different polymeric and lipidic delivery systems have already been investigated, efficient delivery to EC has remained a challenge. Here, we developed lipoplexes targeted to tumor EC for delivering siRNA against VEGFR2. Anginex, a 33-mer peptide, was used to target galectin-1 receptors the expression of which is upregulated upon EC activation. EXPERIMENTAL METHODS Lipoplexes were formed by complexation of siRNA with protamine in 20mM Hepes Buffer containing 5% glucose at pH:7.4 and consequent hydration of a lipid film (solvent evaporation-lipid film hydration method). The lipid film was composed of DOPE:CHEMS:PEG2000-DSPE:MaleimidePEG2000-DSPE (6:4:0.3:0.3). The prepared lipoplexes with a total lipid concentration of 10 mM and RNA concentration of 1 uM were extruded repeatedly through polycarbonate membranes with a final pore size of 100 nm by a high-pressure extruder. A solution of 0.5 M Hepes, 0.5 M Hydroxylamine, 0.25 mM EDTA at pH:7 was added to N-succinimidyl S-acetylthioacetate (SATA) modified Anginex at volume ratio of 1/10 and incubated for 45 minutes at room temperature to deprotect the SATA groups. Unprotected peptides were then added to lipoplexes at a concentration of 10 ug peptide per 1 umol phospholipid and left at 4°C overnight. Uncoupled peptides were separated by ultracentrifuge for 60 minutes at 200,000XG (Brandwijk et al., 2007). Hydrodynamic diameter of lipoplexes was measured by dynamic light scattering (DLS) on an ALV CGS-3 system (Malvern Instruments Ltd., Worcestershire, UK) and Nanosight LM-10SH (NanoSight Ltd. Wiltshire, UK). ζpotential was determined by Zetasizer Nano-Z (Malvern Instruments Ltd., Worcestershire, UK). Complex stability was evaluated by gel retardation and encapsulation efficiency was determined by Ribogreen assay after treatment of samples with Triton X-100 5% and dextran sodium sulfate (DSS)1%. Cytotoxicity was studied by Lactate Dehydrogenase Activity Assay (LDH) on HUVEC. Cell binding and uptake were investigated by flow cytometry after treatment of HUVEC with AxL containing Alexa488-labeled siRNA. Gene knockdown was determined by measuring expression of VEGFR2 in HUVEC using Western blot. RESULTS AND DISCUSSION Monodisperse AxL with an average size of 126 nm and a net negative charge were prepared and were shown to protect the encapsulated siRNA when treated with Triton X-100 5% and dextran sodium sulfate (DSS) 1% as demonstrated in Fig. 1.

Summary. This is a descriptive study, which aims to report adult carriers’ and their husbands/partners’ experiences of carrier diagnosis and their views as to how these issues should be handled for the next generation. Following an initial pilot, 105 carriers and husbands/partners responded to a postal questionnaire. Most of the adult carriers had been tested because either they or their parents wanted to know their carrier status or they had a son diagnosed with haemophilia. The respondents agreed that the main reasons for testing young potential carriers should be either a family history of severe haemophilia or that the young person or her parents wanted to know her status. Forty per cent (35/87) believed the earliest age for carrier testing should be 0–9 years, 44% (38/87) 10–15 years and 16% (14/87) ≥16 years. Respondents aged 18–39 years were more likely to be in favour of testing <2 years. If parents and teenagers disagreed, the majority of parents thought that a test should not be forced, consent refused or results withheld. Genetic counselling provides an important opportunity for parents, who want a very early genetic test, to explore their motivations and balance their desire to prepare and protect their daughter with her right to decide as a teenager.

BACKGROUND:Hepatitis C is a major co-morbidity in patients with hemophilia. However, there is little information on the efficacy of antiviral therapy and long-term follow-up after treatment. OBJECTIVES:To assess the effect of interferon-based (IFN-based) therapy on hepatitis C virus (HCV) eradication, to identify determinants associated with treatment response, and to assess the occurrence of end-stage liver disease (ESLD) after completing antiviral therapy. PATIENTS AND METHODS:In a multicenter cohort study, 295 treatment-naïve hemophilia patients chronically infected with HCV were included. The effect of therapy was expressed as sustained virological response (SVR). Determinants associated with treatment response were expressed as odds ratios (ORs). Cumulative incidence of ESLD was assessed using a Kaplan-Meier survival table. RESULTS:Among human immunodeficiency virus (HIV) negative patients (n = 235), SVR was 29% (29/101) for IFN monotherapy, 44% (32/72) for IFN with ribavirin, and 63% (39/62) for pegylated IFN (PegIFN) with ribavirin. In patients co-infected with HIV (n = 60), IFN monotherapy, IFN with ribavirin, and PegIFN with ribavirin eradicated HCV in 7/35 (20%), 1/2 (50%), and 11/23 (48%), respectively. SVR increased with genotype 2 and 3 [OR 11.0, 95% CI: 5.8-20.5], and combination therapy (IFN and ribavirin OR 3.7, 95% CI: 1.7-8.4), PegIFN and ribavirin (OR 4.2, 95% CI: 1.8-9.5). Up to 15 years after antiviral treatment, none of the patients with a SVR relapsed and none developed ESLD. In contrast, among unsuccessfully treated patients the cumulative incidence of ESLD after 15 years was 13.0%. CONCLUSIONS:Successful antiviral therapy appears to have a durable effect and reduces the risk of ESLD considerably.

a twofold higher mortality by 65 than those who continued working (1.89, 1.58 to 2.27).The mortality by 65 of employees who retired at 60 was similar to those who continued working at 60 (1.04, 0.82 to 1.31).

HaemophiliaVolume 9, Issue 6 p. 745-745 Comparative study of full-length and B-domain deleted factor VIII concentrates S. A. Brown, S. A. Brown The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, UKSearch for more papers by this authorT. T. Yee, T. T. Yee The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, UKSearch for more papers by this authorA. Griffioen, A. Griffioen The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, UKSearch for more papers by this authorC. A. Lee, C. A. Lee The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, UKSearch for more papers by this author S. A. Brown, S. A. Brown The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, UKSearch for more papers by this authorT. T. Yee, T. T. Yee The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, UKSearch for more papers by this authorA. Griffioen, A. Griffioen The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, UKSearch for more papers by this authorC. A. Lee, C. A. Lee The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, UKSearch for more papers by this author First published: 21 November 2003 https://doi.org/10.1046/j.1365-2516.2003.00805.xCitations: 5 Dr Simon A Brown, The Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, Pond Street, London NW3 2QG, UK. Tel.: 020 7830 2068; fax: 020 7472 6759; e-mail: [email protected]. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Gruppo RA, Brown D, Wilkes MM, Navickis RJ. Comparative effectiveness of full-length and B-domain deleted factor VIII for prophylaxis – a meta-analysis. Haemophilia 2003; 9: 251 – 60. 2 Yee TT, Beeton K, Griffioen A et al. Experience of prophylaxis treatment in children with severe haemophilia. Haemophilia 2002; 8: 76 – 82. Citing Literature Volume9, Issue6November 2003Pages 745-745 ReferencesRelatedInformation

BACKGROUND:Low serum albumin concentration is associated with short-term survival in individuals with HIV-1. However, few investigators have assessed whether individuals with a low serum albumin concentration have delayed progression to AIDS, or survive in the long term. We aimed to assess the relation between markers of liver function and progression to AIDS and death in individuals with haemophilia infected with HIV-1 and hepatitis C virus. METHODS:We measured markers of liver function and took CD4 counts every 3 months in 111 patients registered at the Royal Free Hospital Haemophilia Centre, London, UK. HIV RNA concentrations were measured yearly and then every 3-6 months from 1996. We used Cox's regression models to assess the independent prognostic value of these markers for AIDS and death. FINDINGS:As a fixed covariate, albumin concentrations measured shortly after HIV-1 seroconversion were associated with risk of AIDS (relative hazard 0.91 [95% CI 0.84-1.00], p=0.04) and death (0.89 [0.82-0.96], p=0.004) over a 15-year period. These findings were independent of the CD4 count and HIV-1 RNA concentration. As a time-updated covariate, after adjustment for CD4 count and HIV-1 RNA concentrations, albumin was not associated with progression to AIDS (0.96 [0.90-1.01], p=0.13), but was strongly associated with death (0.88 [0.84-0.93], p<0.0001) in the short term. INTERPRETATION:Low concentrations of albumin in individuals infected with HIV-1 could indicate a poor outlook and should therefore prompt concern at any stage of infection.

Hepatitis C virus (HCV) RNA loads are measured sporadically in HCV-positive individuals. However, the prognostic value of these isolated measurements for predicting progression to acquired immune deficiency syndrome (AIDS) and all-cause mortality in coinfected individuals remains unclear. In this study, the prognostic value of a single HCV RNA load measurement taken early after human immunodeficiency virus (HIV) seroconversion was investigated in a cohort of 96 male patients with inherited bleeding disorders. Dates of HIV seroconversion had been estimated for all patients, and at least 4 HCV RNA load measurements per patient were done retrospectively after HIV seroconversion. HCV RNA load stabilized at 4 years after HIV seroconversion, and this point was used for analysis. There was a significant correlation between increased age and early HCV RNA load (r=0.25; P=.01). Adjusting for HIV RNA levels, CD4 cell counts, and the age effect, HCV RNA load >5.90 log10 copies/mL was predictive of progression to AIDS and all-cause mortality over a period of at least 15 years

The practice of prophylactic treatment of boys with severe haemophilia has been evaluated in our centre. Prophylaxis was started at the median age of 3.7 years (range 0.4-12.7 years) in 38/41 children (93%) under 17 years of age. Median follow-up was 4.1 years (range 0.4-12.7 years). The criteria of primary prophylaxis according to the definition by the European Paediatric Network of Haemophilia Management was fulfilled by 9/38 (24%). Although a majority [76%, 29/38] of the children started prophylaxis after a median number of joint bleeds of 3.5, 70% of the children in this group had clinical joint scores of 0. Intravenous catheter insertion was required at a median age of 15.5 months (range 5-36 months) in 21% of the children, resulting in a catheter infection rate of 1.74 per 1000 catheter days. None developed an inhibitor on prophylaxis and three patients who had low-titre inhibitors (< 5 Bethesda units) prior to prophylaxis had undetectable inhibitors after prophylaxis. The home-treatment training programme required considerable time and cost. As a result, 87% of the children used peripheral venous access and hospital visits declined as prophylaxis became established. Parents' incentives for prophylaxis were that the children undertook many physical activities and sports previously not recommended, there was less parental anxiety and an overall improvement in the quality of life for the whole family.

Limited data are available regarding optimal treatment with desmopressin (DDAVP) or intermediate‐purity FVIII concentrates rich in VWF (CFCs) in patients with von Willebrand disease (VWD) who undergo planned surgery. We undertook a retrospective review over 10 years (1988–1997) and identified 27 patients treated with DDAVP for 35 surgical events and 38 patients who received CFCs for 68 elective surgical events. Tranexamic acid was usually added for mucosal surgery. The FVIII:C levels and the severity of surgery were used to determine the frequency and the doses of postoperative treatment. For major surgery the median pre‐ and post‐operative doses of CFCs were 54 and 43 IU/kg, respectively, and for minor surgery the median doses varied between 34 and 52 IU/kg preoperatively and between 23 and 37 IU/kg postoperatively. The effectiveness of haemostasis was excellent in 32 events (91%) treated with DDAVP and in 56 events (82%) treated with CFCs. It is concluded that patients with VWD do not carry an increased operative risk if appropriate therapy is given. Am. J. Hematol. 66:280–284, 2001. © 2001 Wiley‐Liss, Inc.

This clinical retrospective study investigated the difficulties in diagnosing type 1 von Willebrand disease (VWD). A total of 246 patients previously diagnosed with type 1 VWD were reclassified into 'possible' type 1 VWD (patients with low levels of VWF adjusted for the blood group and either a significant bleeding history or family history) and 'definite' type 1 VWD, requiring low levels of von Willebrand factor (VWF), a bleeding history and inheritance. On reclassification, only 144/246 (59%) patients had low VWF levels adjusted for blood group, 88/246 (36%) patients met all the criteria for 'definite' type 1 VWD and 51/246 (21%) patients were 'possible' type 1 VWD. A significant proportion of patients, 102/246 (42%), remained an indeterminate group with blood type O, VWF levels between 35 and 50 U/dl and personal and/or family bleeding history. This subgroup might require reclassification as 'not VWD'. However, a similar bleeding tendency was found in two matched groups of patients of blood groups O and non-O and VWF levels between 35 and 50 U/dl. These results suggest that the use of ABO adjusted ranges for VWF levels might not be essential for diagnosis, because bleeding symptoms may depend on the VWF level regardless of the ABO type. Of the diagnostic criteria, the bleeding history was of prime importance in the clinical decision to diagnose and treat type 1 VWD. These observations could help in the reconsideration of how the criteria for diagnosing type 1 VWD could be adjusted in order to maximize their clinical relevance.