Bioreducible liposomes for gene delivery: from the formulation to the mechanism of action

Background

A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined.

Methodology/Principal Findings

With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25∶50∶25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro.

Conclusions/Significance

The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors.     

Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy

Background

Formulation of DNA/cationic lipid complexes (lipoplexes) designed for nucleic acid delivery mostly results in positively charged particles which are thought to enter cells by endocytosis. We recently developed a lipoplex formulation called Neutraplex that allows preparation of both cationic and anionic stable complexes with similar lipid content and ultrastructure.

Methodology/Principal Findings

To assess whether the global net charge could influence cell uptake and activity of the transported oligonucleotides (ON), we prepared lipoplexes with positive and negative charges and compared: (i) their physicochemical properties by zeta potential analysis and dynamic light scattering, (ii) their cell uptake by fluorescence microscopy and flow cytometry, and (iii) the biological activity of the transported ON using a splicing correction assay. We show that positively or negatively charged lipoplexes enter cells cells using both temperature-dependent and -independent uptake mechanisms. Specifically, positively charged lipoplexes predominantly use a temperature-dependent transport when cells are incubated OptiMEM medium. Anionic lipoplexes favour an energy-independent transport and show higher ON activity than cationic lipoplexes in presence of serum. However, lipoplexes with high positive global net charge and OptiMEM medium give the highest uptake and ON activity levels.

Conclusions

These findings suggest that, in addition to endocytosis, lipoplexes may enter cell via a temperature-independent mechanism, which could be mediated by lipid mixing. Such characteristics might arise from the specific lipoplex ultrastructure and should be taken into consideration when developing lipoplexes designed for in vivo or ex vivo nucleic acid transfer.     

Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium

Background

Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF) via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy.

Methodology

The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN) of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI). We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo.

Results

RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm–1.4 µm cut off (NGI stages 3–6) compatible with deposition in the central and lower airways.

Conclusions

RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.     

Skin vaccination against cervical cancer associated human papillomavirus with a novel micro-projection array in a mouse model

Background

Better delivery systems are needed for routinely used vaccines, to improve vaccine uptake. Many vaccines contain alum or alum based adjuvants. Here we investigate a novel dry-coated densely-packed micro-projection array skin patch (Nanopatch™) as an alternate delivery system to intramuscular injection for delivering an alum adjuvanted human papillomavirus (HPV) vaccine (Gardasil®) commonly used as a prophylactic vaccine against cervical cancer.

Methodology/Principal Findings

Micro-projection arrays dry-coated with vaccine material (Gardasil®) delivered to C57BL/6 mouse ear skin released vaccine within 5 minutes. To assess vaccine immunogenicity, doses of corresponding to HPV-16 component of the vaccine between 0.43±0.084 ng and 300±120 ng (mean ± SD) were administered to mice at day 0 and day 14. A dose of 55±6.0 ng delivered intracutaneously by micro-projection array was sufficient to produce a maximal virus neutralizing serum antibody response at day 28 post vaccination. Neutralizing antibody titres were sustained out to 16 weeks post vaccination, and, for comparable doses of vaccine, somewhat higher titres were observed with intracutaneous patch delivery than with intramuscular delivery with the needle and syringe at this time point.

Conclusions/Significance

Use of dry micro-projection arrays (Nanopatch™) has the potential to overcome the need for a vaccine cold chain for common vaccines currently delivered by needle and syringe, and to reduce risk of needle-stick injury and vaccine avoidance due to the fear of the needle especially among children.     

Chitosan-graft-branched polyethylenimine copolymers: influence of degree of grafting on transfection behavior

Background

Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI_x) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked.

Methodology/Principal Findings

With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI_x derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI_x series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI_x copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI_2.7% was as effective as and less cytotoxic than the gold standard 25 kDa bPEI.

Conclusions/Significance

This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI_x copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity.     

A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

Background

The gene delivery vector for DNA-based therapy should ensure its transfection efficiency and safety for clinical application. The Micro-Linear vector (MiLV) was developed to improve the limitations of traditional vectors such as viral vectors and plasmids.

Methods

The MiLV which contained only the gene expression cassette was amplified by polymerase chain reaction (PCR). Its cytotoxicity, transfection efficiency in vitro and in vivo, duration of expression, pro-inflammatory responses and potential application for Epstein-Barr virus (EBV) positive tumors were evaluated.

Results

Transfection efficiency for exogenous genes transferred by MiLV was at least comparable with or even greater than their corresponding plasmids in eukaryotic cell lines. MiLV elevated the expression and prolonged the duration of genes in vitro and in vivo when compared with that of the plasmid. The in vivo pro-inflammatory response of MiLV group was lower than that of the plasmid group. The MEKK1 gene transferred by MiLV significantly elevated the sensitivity of B95-8 cells and transplanted tumor to the treatment of Ganciclovir (GCV) and sodium butyrate (NaB).

Conclusions

The present study provides a safer, more efficient and stable MiLV gene delivery vector than plasmid. These advantages encourage further development and the preferential use of this novel vector type for clinical gene therapy studies.     

Peroral Amphotericin B Polymer Nanoparticles Lead to Comparable or Superior In Vivo Antifungal Activity to That of Intravenous Ambisome? or Fungizone?

Background

Despite advances in the treatment, the morbidity and mortality rate associated with invasive aspergillosis remains unacceptably high (70–90%) in immunocompromised patients. Amphotericin B (AMB), a polyene antibiotic with broad spectrum antifungal activity appears to be a choice of treatment but is available only as an intravenous formulation; development of an oral formulation would be beneficial as well as economical.

Methodology

Poly(lactide-co-glycolode) (PLGA) nanoparticles encapsulating AMB (AMB-NPs) were developed for oral administration. The AMB-NPs were 113±20 nm in size with ∼70% entrapment efficiency at 30% AMB w/w of polymer. The in vivo therapeutic efficacy of oral AMB-NPs was evaluated in neutropenic murine models of disseminated and invasive pulmonary aspergillosis. AMB-NPs exhibited comparable or superior efficacy to that of Ambisome® or Fungizone™ administered parenterally indicating potential of NPs as carrier for oral delivery.

Conclusions

The present investigation describes an efficient way of producing AMB-NPs with higher AMB pay-load and entrapment efficiency employing DMSO as solvent and ethanol as non-solvent. The developed oral formulation was highly efficacious in murine models of disseminated aspergillosis as well as an invasive pulmonary aspergillosis, which is refractory to treatment with IP Fungizone™and responds only modestly to AmBisome®.     

Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems

Objective

In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry.

Results

The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p?<?0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~?83.94 and ~?77.01% for HEK-293T), and cancer cells (~?71.69 and ~?67.19% for TC-1, and ~?73.86 and ~?67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~?60–80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~?63.66%) compared to TurboFect (~?32.95%); however, the efficiency level of hPP10 was only ~?17.51 and ~?16.36% in TC-1 and A549 cells.

Conclusions

Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.

     

Evaluation of cationic assemblies constructed with amino acid based lipids for plasmid DNA delivery

We synthesized cationic lipids bearing lysine, histidine, or arginine as a cationic headgroup for use in gene transfer studies. The cationic assemblies formed from lysine- or arginine-type lipids gave unilamellar vesicles (approximately 100 nm diameter), whereas the morphology of the histidine-type lipids was tube-like. The competences of the cationic assemblies were sufficient to form lipoplexes, and the resulting lipoplexes were evaluated in terms of gene expression efficiencies with COS-7 cells. The lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, a conventional transgenic reagent, indicating that stable lipoplexes could be prepared between spherical cationic assemblies and plasmid DNA. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine > or = arginine > histidine. In addition, gene expression efficiency was enhanced by decreasing the length of the alkyl chain of the hydrophobic moiety. Unlike Lipofectamine2000, no reduction in transfection efficiency in the presence of fetal bovine serum was observed for the lipoplexes formed using synthetic cationic lipids. Moreover, the synthetic cationic lipids revealed remarkably low cytotoxicity compared with Lipofectamine2000. In conclusion, cationic assemblies formed from 1,5-ditetradecyl-N-lysyl-L-glutamate or 1,5-ditetradecyl-N-arginyl-L-glutamate can be used as an effective plasmid DNA delivery system.     

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Background

Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH₂CH₂OH) and oligo-oxyethylene -(CH₂CH₂O)_n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media.

Methodology/Principal Findings

To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH₂-CH₂-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features.

Conclusions/Significance

-OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.     

Ab-externo AAV-mediated gene delivery to the suprachoroidal space using a 250 micron flexible microcatheter

Background

The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV) vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter.

Methods

6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP.

Results

Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically.

Conclusions

The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.     

Non-invasive measurement of hemoglobin: assessment of two different point-of-care technologies

Background

Measurement of blood hemoglobin (Hb) concentration is a routine procedure. Using a non-invasive point-of-care device reduces pain and discomfort for the patient and allows time saving in patient care. The aims of the present study were to assess the concordance of Hb levels obtained non-invasively with the Pronto-7 monitor (version 2.1.9, Masimo Corporation, Irvine, USA) or with the NBM-200MP monitor (Orsense, Nes Ziona, Israel) and the values obtained from the usual colorimetric method using blood samples and to determine the source of discordance.

Methods and Findings

We conducted two consecutive prospective open trials enrolling patients presenting in the emergency department of a university hospital. The first was designed to assess Pronto-7™ and the second NBM-200MP™. In each study, the main outcome measure was the agreement between both methods. Independent factors associated with the bias were determined using multiple linear regression. Three hundred patients were prospectively enrolled in each study. For Pronto-7™, the absolute mean difference was 0.56 g.L⁻¹ (95% confidence interval [CI] 0.41 to 0.69) with an upper agreement limit at 2.94 g.L⁻¹ (95% CI [2.70;3.19]), a lower agreement limit at -1.84 g.L⁻¹ (95% CI [-2.08;-1.58]) and an intra-class correlation coefficient at 0.80 (95% CI [0.74;0.84]). The corresponding values for the NBM-200MP™ were 0.21 [0.02;0.39], 3.42 [3.10;3.74], -3.01 [-3.32;-2.69] and 0.69 [0.62;0.75]. Multivariate analysis showed that age and laboratory values of hemoglobin were independently associated with the bias when using Pronto-7™, while perfusion index and laboratory value of hemoglobin were independently associated with the bias when using NBM-200MP™.

Conclusion

Despite a relatively limited bias in both cases, the large limits of agreement found in both cases render the clinical usefulness of such devices debatable. For both devices, the bias is independently and inversely associated with the true value of hemoglobin.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01321580","term_id":"NCT01321580"}}NCT01321580 {"type":"clinical-trial","attrs":{"text":"NCT01321593","term_id":"NCT01321593"}}NCT01321593     

Quality control and performance of HIV rapid tests in a microbicide clinical trial in rural KwaZulu-Natal

Background

Quality control (QC) and evaluation of HIV rapid test procedures are an important aspect of HIV prevention trials. We describe QC and performance of two rapid tests, Determine™ and Uni-Gold™ used in a microbicide clinical trial in rural KwaZulu-Natal, South Africa.

Methods/Results

Internal QC of both HIV rapid tests was conducted at the trial site using a Uni-Gold control kit (Uni-Gold™Recombigen® HIV). Both assays produced the expected results for a total of 4637 QC tests. Study participants were tested for HIV at screening and, if enrolled, at regular time points throughout the study. Positive or discordant results were confirmed by a double HIV immunoassay testing strategy at a local laboratory. Overall, 15292 HIV rapid test were performed. Sensitivity and specificity of Determine was 98.95% (95% CI: 97.72–99.61) and 99.83% (95% CI: 99.70–99.91) respectively [positive predictive value (PPV) 97.91% (95% CI: 96.38–98.92)], for Uni-Gold it was 99.30% (95% CI: 98.21–99.81) and 99.96% (95% CI: 99.88–99.99) respectively [PPV 99.47% (95% CI: 98.46–99.89)].

Conclusions

The results suggest that a Uni-Gold control kit can be used for internal QC of both Uni-Gold and the HIV-1 component of the Determine rapid tests. Both rapid tests performed proficiently in the trial population.     

Treatment of Colorectal Cancer Using a Combination of Liposomal Irinotecan (Irinophore C?) and 5-Fluorouracil

Purpose

To investigate the use of liposomal irinotecan (Irinophore C™) plus or minus 5-fluorouracil (5-FU) for the treatment of colorectal cancer.

Experimental Design

The effect of irinotecan (IRI) and/or 5-FU exposure times on cytotoxicity was assessed in vitro against HT-29 or LS174T human colon carcinoma cells. The pharmacokinetics and biodistribution of Irinophore C™ (IrC™) and 5-FU, administered alone or in combination, were compared in vivo. A subcutaneous model of HT-29 human colorectal cancer in Rag2-M mice was utilized to assess the efficacy of IrC™ alone, and in combination with 5-FU.

Results

The cytotoxicity of IRI and 5-FU were strongly dependent on exposure time. Synergistic interactions were observed following prolonged exposure to IRI/5-FU combinations. Pharmacokinetics/biodistribution studies demonstrated that the 5-FU elimination rate was decreased significantly when 5-FU was co-administered intravenously with IrC™, versus alone. Significant decreases in 5-FU elimination were also observed in plasma, with an associated increase of 5-FU in some tissues when 5-FU was given by intraperitoneal injection and IrC™ was given intravenously. The elimination of IrC™ was not significantly different when administered alone or in combination with 5-FU. Therapeutic studies demonstrated that single agent IrC™ was significantly more effective than the combination of IRI/5-FU; surprisingly, IrC™/5-FU combinations were no more effective than IrC™ alone. The administration of combinations of 5-FU (16 mg/kg) and IrC™ (60 mg IRI/kg) showed increased toxicity when compared to IrC™ alone. Treatment with IrC™ alone (60 mg IRI/kg) delayed the time required for a 5-fold increase in initial tumor volume to day 49, compared to day 23 for controls. When IrC™ (40 mg IRI/kg) was used in combination with 5-FU (16 mg/kg), the time to increase tumor volume 5-fold was 43 days, which was comparable to that achieved when using IrC™ alone (40 mg IRI/kg).

Conclusions

Single agent IrC™ was well tolerated and has significant therapeutic potential. IrC™ may be a suitable replacement for IRI treatment, but its use with free 5-FU is complicated by IrC™-engendered changes in 5-FU pharmacokinetics/biodistribution which are associated with increased toxicity when using the combination.     

Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells

Background

To demonstrate the involvement of tobacco smoking in the pathophysiology of lung disease, the responses of pulmonary epithelial cells to cigarette smoke condensate (CSC) — the particulate fraction of tobacco smoke — were examined.

Methods

The human alveolar epithelial cell line A549 and normal human bronchial epithelial cells (NHBEs) were exposed to 0.4 μg/ml CSC, a concentration that resulted in >90% cell survival and <5% apoptosis. Changes in gene expression and signaling responses were determined by RT-PCR, western blotting and immunocytofluorescence.

Results

NHBEs exposed to CSC showed increased expression of the inflammatory mediators sICAM-1, IL-1β, IL-8 and GM-CSF, as determined by RT-PCR. CSC-induced IL-1β expression was reduced by PD98059, a blocker of mitogen-actived protein kinase (MAPK) kinase (MEK), and by PDTC, a NFκB inhibitor. Analysis of intracellular signaling pathways, using antibodies specific for phosphorylated MAPKs (extracellular signal-regulated kinase [ERK]-1/2), demonstrated an increased level of phosphorylated ERK1/2 with increasing CSC concentration. Nuclear localization of phosphorylated ERK1/2 was seen within 30 min of CSC exposure and was inhibited by PD98059. Increased phosphorylation and nuclear translocation of IκB was also seen after CSC exposure. A549 cells transfected with a luciferase reporter plasmid containing a NFκB-inducible promoter sequence and exposed to CSC (0.4 μg/ml) or TNF-α (50 ng/ml) had an increased reporter activity of approximately 2-fold for CSC and 3.5-fold for TNF-α relative to untreated controls.

Conclusion

The acute phase response of NHBEs to cigarette smoke involves activation of both MAPK and NFκB.     

CCL3L1 copy number variation and susceptibility to HIV-1 infection: a meta-analysis

Background

Although several studies have investigated whether CCL3L1 copy number variation (CNV) influences the risk of HIV-1 infection, there are still no clear conclusions. Therefore, we performed a meta-analysis using two models to generate a more robust estimate of the association between CCL3L1 CNV and susceptibility to HIV-1 infection.

Methods

We divided the cases and controls into two parts as individuals with CCL3L1 gene copy number (GCN) above the population specific median copy number (PMN) and individuals with CCL3L1 GCN below PMN, respectively. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were given for the main analysis. We also conducted stratified analyses by ethnicity, age group and sample size. Relevant literatures were searched through PubMed and ISI Web of Knowledge up to March 2010.

Results

In total, 9 studies with 2434 cases and 4029 controls were included. ORs for the main analysis were 1.35 (95% CI, 1.02–1.78, model: GCN ≤ PMN Vs. GCN > PMN) and 1.70 (95% CI, 1.30–2.23, model: GCN < PMN Vs. GCN ≥ PMN), respectively. Either in stratified analysis, statistically significant results can be detected in some subgroups.

Conclusions

Our analyses indicate that CCL3L1 CNV is associated with susceptibility to HIV-1 infection. A lower copy number is associated with an increased risk of HIV-1 infection, while a higher copy number is associated with reduced risk for acquiring HIV-1.     

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo

Background

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.

Methods and results

We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino‐sugar‐based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin‐cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.

Conclusions

These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Sour taste responses in mice lacking PKD channels

Background

The polycystic kidney disease-like ion channel PKD2L1 and its associated partner PKD1L3 are potential candidates for sour taste receptors. PKD2L1 is expressed in type III taste cells that respond to sour stimuli and genetic elimination of cells expressing PKD2L1 substantially reduces chorda tympani nerve responses to sour taste stimuli. However, the contribution of PKD2L1 and PKD1L3 to sour taste responses remains unclear.

Methodology/Principal Findings

We made mice lacking PKD2L1 and/or PKD1L3 gene and investigated whole nerve responses to taste stimuli in the chorda tympani or the glossopharyngeal nerve and taste responses in type III taste cells. In mice lacking PKD2L1 gene, chorda tympani nerve responses to sour, but not sweet, salty, bitter, and umami tastants were reduced by 25–45% compared with those in wild type mice. In contrast, chorda tympani nerve responses in PKD1L3 knock-out mice and glossopharyngeal nerve responses in single- and double-knock-out mice were similar to those in wild type mice. Sour taste responses of type III fungiform taste cells (GAD67-expressing taste cells) were also reduced by 25–45% by elimination of PKD2L1.

Conclusions/Significance

These findings suggest that PKD2L1 partly contributes to sour taste responses in mice and that receptors other than PKDs would be involved in sour detection.     

Immunogenicity is not improved by increased antigen dose or booster dosing of seasonal influenza vaccine in a randomized trial of HIV infected adults

Introduction

The risk of poor vaccine immunogenicity and more severe influenza disease in HIV necessitate strategies to improve vaccine efficacy.

Methods

A randomized, multi-centered, controlled, vaccine trial with three parallel groups was conducted at 12 CIHR Canadian HIV Trials Network sites. Three dosing strategies were used in HIV infected adults (18 to 60 years): two standard doses over 28 days, two double doses over 28 days and a single standard dose of influenza vaccine, administered prior to the 2008 influenza season. A trivalent killed split non-adjuvanted influenza vaccine (Fluviral™) was used. Serum hemagglutinin inhibition (HAI) activity for the three influenza strains in the vaccine was measured to assess immunogenicity.

Results

297 of 298 participants received at least one injection. Baseline CD4 (median 470 cells/µL) and HIV RNA (76% of patients with viral load <50 copies/mL) were similar between groups. 89% were on HAART. The overall immunogenicity of influenza vaccine across time points and the three influenza strains assessed was poor (Range HAI ≥40 = 31–58%). Double dose plus double dose booster slightly increased the proportion achieving HAI titre doubling from baseline for A/Brisbane and B/Florida at weeks 4, 8 and 20 compared to standard vaccine dose. Increased immunogenicity with increased antigen dose and booster dosing was most apparent in participants with unsuppressed HIV RNA at baseline. None of 8 serious adverse events were thought to be immunization-related.

Conclusion

Even with increased antigen dose and booster dosing, non-adjuvanted influenza vaccine immunogenicity is poor in HIV infected individuals. Alternative influenza vaccines are required in this hyporesponsive population.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00764998","term_id":"NCT00764998"}}NCT00764998