Safety and immunogenicity of whole-virus,alum-adjuvanted whole-virus,virosomal, and whole-virus intradermal influenza A/H9N2 vaccine formulations

Avian influenza H9N2 viruses are considered as a pandemic threat. We assessed the safety and immunogenicity of fourteen H9N2 vaccine formulations. A randomized, phase I trial was done in 353 adults, aged 18–82 years. Subjects received two doses of A/Hong Kong/1073/99 (H9N2) whole-virus, alum-adjuvanted whole-virus, virosomal, or intradermal whole-virus vaccine at four doses (1.7, 5, 15 or 45 μg haemagglutinin). Sera were obtained before and three weeks after each vaccination (days 0, 21, and 42) for haemagglutination–inhibition (HAI) and neutralization assays. All formulations were well tolerated. Pre-vaccination sera from subjects aged below or above 40 years had baseline antibody to H9N2 in 1% and 16% of samples. Compared to intramuscular whole-virus vaccine, alum-adjuvanted vaccine was more immunogenic, intradermal vaccine was comparable, and virosomal vaccine less immunogenic. Among subjects under 40 years, two doses (45, 15, and 5 μg) of alum-adjuvanted vaccine achieved seroprotective HAI titres in 50%, 41%, and 39% respectively, and neutralization seroconversions in 83%, 82%, and 78% of recipients. Among subjects over 40 years, one dose (45, 15, and 5 μg) of alum-adjuvanted vaccine achieved seroprotective HAI titres in 50%, 25% and 0% respectively, and neutralization seroconversions in 88%, 63% and 63% of recipients. Among immunologically naive subjects under 40 years, two doses of vaccine are required and alum-adjuvanted vaccines were most immunogenic. Among immunologically primed subjects over 40 years, one dose of whole-virus or alum-adjuvanted vaccine induced immune responses; the second dose provided less additional benefit. However, no vaccine formulation satisfied all European regulatory criteria for pandemic vaccines.     

Evaluation of circulating tumor necrosis factor-alpha in patients with gynecological malignancies

TNF-alpha levels in sera from patients with gynecological cancers were evaluated by ELISA and compared with those of patients with benign ovarian cysts or of anonymous healthy donors. Patients with cervical and endometrial carcinoma and with benign ovarian cysts showed levels of TNF-alpha similar to those of healthy donors. In contrast, significantly increased levels of TNF-alpha were found in patients with ovarian carcinoma, regardless of the stage of disease.     

Therapeutic antibody gene transfer: an active approach to passive immunity.

Advances in gene transfer approaches are enabling the possibility of applying therapeutic antibodies using DNA. In particular gene transfer in combination with electroporation is promising and can result in generating in vivo antibody concentrations in the low therapeutic range. However, several important problems need to be dealt with before antibody gene transfer can become a valuable supplement to the current therapies. As antibody production following gene transfer is difficult to control, the danger of inducing autoimmune conditions or uncontrollable side effects occurs in cases in which autologous antigens are targeted. It is suggested that the most promising area of application therefore appears to be infectious disease in which heterologous antigens are targeted and concerns for long-term antibody exposure are minimal. Finally, genes encoding fully human antibodies will enhance long-term expression and decrease problems linked to immunogenicity.     

Does a Gender Difference in Response to Cardiac Resynchronization Therapy Exist?

BACKGROUND: Cardiac resynchronization therapy (CRT) has a beneficial effect on clinical symptoms, exercise capacity, and systolic left ventricular (LV) performance in patients with heart failure. The aim of the current study was to evaluate whether a gender difference exists in response to CRT. METHODS: Consecutive patients with end-stage heart failure (New York Heart Association, NYHA, class III-IV), LV ejection fraction (LVEF) < or =35%, QRS duration >120 ms, and left bundle branch block configuration underwent CRT. At baseline and 6 months post-CRT, clinical and echocardiographic parameters were evaluated; follow-up was obtained up to 5 years. The effects of CRT were compared between women and men. RESULTS: The study population comprised 137 men and 36 women (mean age 66 +/- 11 years). No differences in baseline characteristics were observed except that nonischemic cardiomyopathy was more frequent in women than men (67% vs 38%, P < 0.05). In all patients, clinical and echocardiographic parameters improved significantly at 6-month follow-up. The magnitude of improvement in different parameters was similar between women and men, e.g., the improvement in NYHA Class was 0.9 +/- 0.6 in women and 1.0 +/- 0.7 in men (NS) and the increase in LVEF was 8 +/- 8% in women as compared to 7 +/- 9% in men (NS). The percentage of individual responders was not different between women and men (76% vs 80%, NS) and 2-year survival was comparable for women and men (84% vs 80%, NS). CONCLUSION: No gender differences were observed in response to CRT and long-term survival after CRT.     

Evaluation of the Novel PET Tracer [11C]HACH242 for Imaging the GluN2B NMDA Receptor in Non-Human Primates

Purpose

There are currently no positron emission tomography (PET) radiotracers for the GluN2B (NR2B) binding sites of brain N-methyl-d-aspartate (NMDA) receptors. In rats, the GluN2B antagonist Ro25-6981 reduced the binding of N-((5-(4-fluoro-2-[¹¹C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamin ([¹¹C]HACH242). This paper reports the evaluation of [¹¹C]HACH242 PET in non-human primates at baseline and following administration of the GluN2B negative allosteric modulator radiprodil.

Procedures

Eight 90-min dynamic [¹¹C]HACH242 PET scans were acquired in three male anaesthetised rhesus monkeys, including a retest session of subject 1, at baseline and 10 min after intravenous 10 mg/kg radiprodil. Standardised uptake values (SUV) were calculated for 9 brain regions. Arterial blood samples were taken at six timepoints to characterise pharmacokinetics in blood and plasma. Reliable input functions for kinetic modelling could not be generated due to variability in the whole-blood radioactivity measurements.

Results

[¹¹C]HACH242 entered the brain and displayed fairly uniform uptake. The mean (±?standard deviation, SD) T_max was 17?±?7 min in baseline scans and 24?±?15 min in radiprodil scans. The rate of radioligand metabolism in plasma (primarily to polar metabolites) was high, with mean parent fractions of 26?±?10 % at 20 min and 8?±?5 % at 85 min. Radiprodil increased [¹¹C]HACH242 whole-brain SUV in the last PET frame by 25 %, 1 %, 3 and 17 % for subjects 1, 2, 3 and retest of subject 1, respectively. The mean brain to plasma ratio was 5.4?±?2.6, and increased by 39 to 110 % in the radiprodil condition, partly due to lower parent plasma radioactivity of ?11 to ?56 %.

Conclusions

The present results show that [¹¹C]HACH242 has a suitable kinetic profile in the brain and low accumulation of lipophilic radiometabolites. Radiprodil did not consistently change [¹¹C]HACH242 brain uptake. These findings may be explained by variations in cerebral blood flow, a low fraction of specifically bound tracer, or interactions with endogenous NMDA receptor ligands at the binding site. Further experiments of ligand interactions are necessary to facilitate the development of radiotracers for in vivo imaging of the ionotropic NMDA receptor.

     

Preanalytical,analytical, and biological variation of blood plasma submicron particle levels measured with nanoparticle tracking analysis and tunable resistive pulse sensing

Background: Nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) enable measurement of extracellular vesicles (EVs) in blood plasma but also measure other particles present in plasma. Complete isolation of EVs from similarly sized particles with full EV recovery is currently not possible due to limitations in existing isolation techniques.

Aim: This study aimed to evaluate preanalytical, analytical, and biological variation of particle measurements with NTA and TRPS on blood plasma.

Methods: Blood from 20 healthy subjects was sampled in the fasting and postprandial state. Platelet free plasma (PFP) was analyzed immediately and after a freeze-thaw cycle. Additionally, the effect of prandial state and a freeze-thaw cycle on EV-enriched particle fractions obtained via size-exclusion chromatography (SEC) was examined.

Results: We observed analytical linearity in the range of 1.0–10.0?×?10⁸ particles/mL for NTA and 1.0?×?10⁸–1.8?×?10⁹ particles/mL for TRPS. The analytical variation was generally below 10%. A considerable intra- and inter-individual variation was demonstrated with estimated reference intervals of 1.4?×?10¹¹–1.2?×?10¹² particles/mL for NTA and 1.8?×?10⁸–1.6?×?10⁹ particles/mL for TRPS. Food intake and to a lesser extent a freeze-thaw cycle affected particle populations in PFP and, similarly, in EV-enriched fractions.

Conclusion: In this study NTA and TRPS enabled acceptably precise concentration and size measurement of submicron particles in PFP. An appreciable intra- and inter-individual biological variation was observed. In studies on particle populations in PFP or EV-enriched fractions, we recommend analysis of fresh, fasting samples.     

Molecular profiling of the “plexinome” in melanoma and pancreatic cancer

Plexins are transmembrane high‐affinity receptors for semaphorins, regulating cell guidance, motility, and invasion. Functional evidences implicate semaphorin signals in cancer progression and metastasis. Yet, it is largely unknown whether plexin genes are genetically altered in human tumors. We performed a comprehensive gene copy analysis and mutational profiling of all nine members of the plexin gene family (plexinome), in melanomas and pancreatic ductal adenocarcinomas (PDACs), which are characterized by high metastatic potential and poor prognosis. Gene copy analysis detected amplification of PLXNA4 in melanomas, whereas copy number losses of multiple plexin genes were seen in PDACs. Somatic mutations were detected in PLXNA4, PLXNB3, and PLXNC1; providing the first evidence that these plexins are mutated in human cancer. Functional assays in cellular models revealed that some of these missense mutations result in loss of plexin function. For instance, c.1613G>A, p.R538H mutation in the extracellular domain of PLXNB3 prevented binding of the ligand Sema5A. Moreover, although PLXNA4 signaling can inhibit tumor cell migration, the mutated c.5206C>T, p.H1736Y allele had lost this activity. Our study is the first systematic analysis of the “plexinome” in human tumors, and indicates that multiple mutated plexins may be involved in cancer progression. Hum Mutat 30,1–8, 2009. © 2009 Wiley‐Liss, Inc.     

Reverse ventricular remodelling after cardiac resynchronization therapy is associated with a reduction in serum tenascin-C and plasma matrix metalloproteinase-9 levels

BACKGROUND: In heart failure patients, cardiac resynchronization therapy (CRT) leads to reverse ventricular remodelling. AIM: The aim of this study was to evaluate whether changes in levels of circulating biomarkers of extracellular matrix metabolism correlate with the response to CRT. METHODS AND RESULTS: Clinical parameters, left ventricular (LV) volumes, and circulating levels of tenascin-C (TNC), matrix metalloproteinase-2 (MMP-2), MMP-9, and amino-terminal propeptide of brain natriuretic peptide (NT-proBNP) were assessed in 64 patients at baseline and 6 months follow-up. The majority of patients (72%) showed a >10% reduction in LV end-systolic volume at follow-up, and were classified as responders to CRT. The remaining patients were classified as non-responders. In responders, a significant decrease in circulating levels of TNC (from 60+/-40 ng/mL to 47+/-30 ng/mL, p<0.01), MMP-9 (from 55+/-30 AU to 44+/-27 AU, p<0.01), and NT-proBNP (from 2106+/-1805 pg/mL to 1132+/-1289 pg/mL, p<0.001) were observed at follow-up; MMP-2 levels were unchanged. In non-responders TNC, NT-proBNP, MMP-9 and MMP-2 levels remained unchanged. CONCLUSION: At 6 months follow-up, CRT was associated with reverse LV remodelling, and a significant decrease in TNC, MMP-9, and NT-proBNP levels. This suggests an important role of ECM modulation in the process of reverse ventricular remodelling in patients responding to CRT.     

Synthesis of methoxypoly(ethylene glycol) carbonate prodrugs of zidovudine and penetration through human skin in vitro

Objectives The aim of this study was to synthesise a series of novel methoxypoly (ethylene glycol) carbonate prodrugs of the antiretroviral drug zidovudine (azidothymidine, AZT) in an attempt to enhance the physicochemical properties for transdermal delivery, which may reduce the severe side‐effects and toxicity associated with high oral doses of AZT. Methods Methoxypoly(ethylene glycol) carbonates of AZT were synthesised in two steps: activation of the relevant methoxypoly(ethylene glycol) with p‐nitrophenyl chloroformate, followed by reaction with AZT. Analysis of the hydrolytic stability in phosphate buffer at pH 5.0 and 7.4 revealed that all the carbonates were markedly more stable at pH 5.0 than at pH 7.4 (0.01 m), with half‐lives ranging from 15 to 44 days at pH 5.0 and from 6 to 24 days at pH 7.4. The potential of the series to penetrate the skin was evaluated in vitro by measuring diffusion through excised abdominal female human skin at pH 5.0. Key findings Prodrugs with 1–3 or 8 oxyethylene units in the methoxypoly(ethylene glycol) moiety were found to permeate the skin whereas those with 12 or 17 units did not. The prodrug with eight oxyethylene units was the most effective penetrant, permeating the skin with a mean flux of 53.3 ± 46.5 nmol/cm² per h, which is 2.4–10.1 times that of AZT (8.55 ± 5.3 nmol/cm² per h). Conclusions The bioreversible conjugation of the methoxypoly(ethylene glycol) promoiety to AZT appears to be a promising strategy for the transdermal delivery of AZT at a therapeutic dose.