An Amphiphilic Nanocarrier Based on Guar Gum-graft-Poly(ε-caprolactone) for Potential Drug-Delivery Applications

Amphiphilic guar gum grafted with poly(ε-caprolactone) (GG-g-PCL) was fabricated as a drug-delivery carrier using microwave irradiation. The structure of the GG-g-PCL co-polymer was characterized by ¹H-NMR spectroscopy. By microwave irradiation, GG-g-PCL with high grafting percentage (>200%) was obtained in a short reaction time. The GG-g-PCL co-polymer is capable of self-assembling into nanosized spherical micelles in aqueous solution with the diameter of around 75–135 nm and 60–100 nm, as determined by DLS and TEM, respectively. The critical micelle concentration (CMC) of GG-g-PCL was found to be approx. 0.56 mg/l in a phosphate buffer solution. The drug-release profile showed that the GG-g-PCL micelles provided an initial burst release followed by a sustained release of the entrapped hydrophobic model drug, ketoprofen, over a period of 10–68 h. Under physiological conditions, the GG-g-PCL co-polymer hydrolytically degraded into lower-molecular-weight fragments within a 7-week period. These results suggest that the GG-g-PCL micelles could be used as a nanocarrier for in vitro controlled drug delivery.     

PEG- and PDMAEG-Graft-Modified Branched PEI as Novel Gene Vector: Synthesis,Characterization and Gene Transfection

The cytotoxicity of polyethylenimine (PEI) was a dominating obstacle to its application. Introduction of poly(ethylene glycol) (PEG) blocks to PEI is one of the strategies to alleviate the cytotoxocity of PEI. However, it is well known that the transfection efficiency of PEGylated PEI is decreased to some extent compared to the corresponding PEI. Thus, the aim of our study was to enhance the transfection efficiency of PEGylated PEI. A series of tri-block co-polymers, PEG-g-PEI-g-poly(dimethylaminoethyl L-glutamine) (PEG-g-PEI-g-PDMAEG), as novel vectors for gene therapy was synthesized and evaluated. PEG-g-PEI was first obtained by linking PEG and PEI using isophorone diisocyanate (IPDI) as coupling reagent. The anionic co-polymerization of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) using PEG-g-PEI as a macro-initiator was carried out, followed by aminolysis with 2-dimethylaminoethylamine to obtain the target water-soluble tri-block co-polymer. The structures of the polymers were confirmed by FT-IR and ¹H-NMR. The influence of the molecular weight of PEI and the length of the PDMAEG chain on the physicochemical properties and transfection activity of polymer/DNA was evaluated. All PEI derivates were revealed to compact plasmid DNA effectively to give polyplexes with suitable size (approx. 100 nm) and moderate zeta potentials (10–15 mV) at N/P ratios over 10. The PEG-g-PEI-g-PDMAEG tri-block co-polymers displayed particularly low cytotoxicity, even at high concentration, reflecting an improved safety profile compared to PEI 25k. Gene transfection efficiency of PEG-g-PEI-g-PDMAEG on HeLa in the presence and absence of serum was determined. Remarkably, the transfection activity of PEG-g-PEI (10k)-g-PDMAEG (PPP-4)/DNA polyplex formulations was nearly twofold higher than PEI 25k/DNA formulations in vitro, and the transfection efficiency was less affected by the presence of serum. These results indicated that the synthesized PEG-g-PEI-g-PDMAEG tri-block co-polymers are promising candidates as carriers for gene delivery.     

Insoluble ionic complexes of polyacrylic acid with a cationic drug for use as a mucoadhesive,ophthalmic drug delivery system

We have developed a new mucoadhesive drug delivery formulation based on an ionic complex of partially neutralized poly(acrylic acid) (PAA) and a highly potent beta blocker drug, levobetaxolol · hydrochloride (LB · HCl), for use in the treatment of glaucoma. PAA was neutralized with sodium hydroxide to varying degrees of neutralization. Aqueous solutions containing concentrations of LB · HCl equivalent to the degree of PAA neutralization were added to the PAA solutions and formed insoluble complexes, which were isolated. The complex formation was followed by turbidimetric titration, and the complexes were characterized by IR and ¹H NMR spectroscopy. Complexes were prepared with varying degrees of drug loading, such that the same PAA chain would have free COOH groups for mucoadhesion along with ionic complexes of LB · H⁺ with COO^- groups. Thin films of the complexes dissociated to release the drug by ion exchange with synthetic tear fluid. The films shrunk continuously during release of the drug and dissolved completely in 1 h. Solid inserts of these films could be useful as a mucoadhesive ophthalmic drug delivery system.     

A polymeric micelle system with a hydrolysable segment for drug delivery

A potential anti-cancer drug-delivery polymeric micelle system with an in vitro degradation half-life of about 48 h that releases its drug upon application of ultrasound was synthesized. This vehicle was composed of an amphiphilic co-polymer, poly(ethylene oxide)-b-poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate-lactate _n ). The degree of polymerization of the lactate side group, n, was 0, 3 or 5. The molar ratio of NIPAAm to HEMA-lactate _n to PEO in polymerization was optimized to produce an in vitro polymeric micelle half-life of about 48 h at 40°C. 1,6-Diphenyl-1,3,5-hexatriene (DPH) was used as a fluorescent probe to study the hydrophobicity of the cores of the polymeric micelles. The results showed that the cores of the polymeric micelles were hydrophobic enough to sequester DPH and the anti-cancer drug doxorubicin (Dox). Dox was encapsulated into the polymeric micelles having a molar feed ratio of NIPAAm to HEMA-lactate₃ to PEO equal to 20 : 5 : 1; this drug was released upon the application of low-frequency ultrasound. The Dox release was about 2% at room temperature and 4% at body temperature, and the drug returned to the polymeric micelles when insonation ceased.     

Apatite nano-crystalline surface modification of poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering: implications for protein adsorption

A number of bone tissue engineering approaches are aimed at (i) increasing the osteconductivity and osteoinductivity of matrices, and (ii) incorporating bioactive molecules within the scaffolds. In this study we examined the growth of a nano-crystalline mineral layer on poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for tissue engineering. In addition, the influence of the mineral precipitate layer on protein adsorption on the scaffolds was studied. Scaffolds were mineralized by incubation in simulated body fluid (SBF). Scanning electron microscopy (SEM) analysis revealed that mineralized scaffolds possess a rough surface with a plate-like nanostructure covering the surface of microspheres. The results of protein adsorption and release studies showed that while the protein release pattern was similar for PLAGA and mineralized PLAGA scaffolds, precipitation of the mineral layer on PLAGA led to enhanced protein adsorption and slower protein release. Mineralization of tissue-engineered surfaces provides a method for both imparting bioactivity and controlling levels of protein adsorption and release.     

A novel construct as a cell carrier for tissue engineering

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.     

A New Injectable Thermogelling Material: Methoxy Poly(ethylene glycol)–Poly(sebacic acid-D,L-lactic acid)–Methoxy Poly(ethylene glycol) Triblock Co-polymer

A novel injectable thermogelling poly(ester-anhydride) co-polymer, methoxy poly(ethylene glycol)–poly(sebacic acid-D,L-lactic acid)–methoxy poly(ethylene glycol) (mPEG–poly(SA-LA)–mPEG) triblock co-polymer, was prepared by melt-condensation polymerization. The synthesized triblock co-polymer was characterized by FT-IR and ¹H-NMR. The aqueous solutions of mPEG–poly(SA-LA)–mPEG underwent sol–gel precipitation transition when the temperature was increased from 20 to 70°C, depending on the concentration of the polymer. 5-FU, as the model drug, was mixed into the gel in a low-viscous sol state at room temperature. About 63 wt% of the loaded 5-FU could be released in vitro from the gel over 72 h at 37°C. Subcutaneous injection of 25 wt% mPEG–poly(SA-LA)–mPEG aqueous solution resulted in the formation of a in situ gel depot in a rat model, which sustained for longer time than that of Pluronic F-127 aqueous solution. The biodegradable thermogelling mPEG–poly(SA-LA)–mPEG triblock co-polymer is believed to be a promising candidate for drug-delivery applications.     

Novel Poly(L-lactide) PLLA/SWNTs Nanocomposites for Biomedical Applications: Material Characterization and Biocompatibility Evaluation

Poly(L-lactide) (PLLA)/single-walled carbon nanotubes (SWNTs) nanocomposite films were produced using the solvent casting method, and morphological, thermal and mechanical properties were investigated. Biocompatibility was evaluated by using human bone cells, performing adhesion and proliferation studies. The role of single-walled nanotube incorporation and functionalization on PLLA bio-polymers was investigated. Pristine (SWNTs) and carboxylated (SWNTs–COOH) carbon nanotubes were considered in order to control the interaction between PLLA and nanotubes. SWNTs and SWNTs–COOH showed a good dispersion in the polymer matrix and improved the PLLA crystallinity. Thermal, morphological and dynamic-mechanical analyses revealed that carboxylic groups on the tube sidewalls increased compatibility between PLLA and nanostructures. Mechanical properties demonstrated an enhancement related to introduction and functionalization of carbon nanotubes. Biological investigations showed osteoblasts cultured on PLLA/SWNTs–COOH nanocomposites has higher cell adhesion and proliferation than osteoblasts cultured on PLLA and PLLA/SWNTs nanocomposites. These studies suggest that combination of biodegradable polymers and SWNTs opens a new perspective in the self-assembly of nanomaterials and nanodevices for biomedical applications with tunable properties.     

Poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine], a potential non-viral vector for DNA delivery

A potential non-viral gene-transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] (PSL), has been developed by thermal polycondensation of aspartic acid and lysine under reduced pressure. Low-molecular-mass branch poly(ethylenimine) (PEI600) was conjugated to the backbone. The chemical structure of the resulting co-polymer was identified by H-NMR, FT-IR, TGA and X-ray diffraction. The results of the MTT assay showed that at concentration up to 4000 nmol/l of the vector cell viability was over 80% and showed low toxicity. Electrophoretic retardation and ethidum bromide assay showed that at N/P ratios 12–15 (w/w) the DNA could be condensed and neutralized. Using the zeta potential assay we discovered that it had a high positive charge on its surface of the particle (over 30 mV). The particle sizes of the co-polymer/DNA complexes were 150–170 nm, as measured by DLS and AFM. Compared with PEI600, co-polymer/DNA complexes showed a significant enhancement of transfection activity in the absence and presence of serum in NT2 and COS7 cell lines. This means that the PEI600-PSL co-polymer is a promising candidate for gene delivery.     

Differential degradation rates in vivo and in vitro of biocompatible poly(lactic acid) and poly(glycolic acid) homo- and co-polymers for a polymeric drug-delivery microchip

The biocompatibility and biodegradation rate of component materials are critical when designing a drug-delivery device. The degradation products and rate of degradation may play important roles in determining the local cellular response to the implanted material. In this study, we investigated the biocompatibility and relative biodegradation rates of PLA, PGA and two poly(lactic-co-glycolic acid) (PLGA) polymers of 50 : 50 mol ratio, thin-film component materials of a drug-delivery microchip developed in our laboratory. The in vivo biocompatibility and both in vivo and in vitro degradation of these materials were characterized using several techniques. Total leukocyte concentration measurements showed normal acute and chronic inflammatory responses to the PGA and low-molecular-weight PLGA that resolved by 21 days, while the normal inflammatory responses to the PLA and high-molecular-weight PLGA were resolved but at slower rates up to 21 days. These results were paralleled by thickness measurements of fibrous capsules surrounding the implants, which showed greater maturation of the capsules for the more rapidly degrading materials after 21 days, but less mature capsules of sustained thicknesses for the PLA and high-molecular-weight PLGA up to 49 days. Gel-permeation chromatography of residual polymer samples confirmed classification of the materials as rapidly or slowly degrading. These materials showed thinner fibrous capsules than have been reported for other materials by our laboratory and have suitable biocompatibility and biodegradation rates for an implantable drug-delivery device.     

Preparation of poly(ethylene glycol)-introduced cationized gelatin as a non-viral gene carrier

The objective of this study was to prepare cationized gelatins grafted with poly(ethylene glycol) (PEG) (PEG-cationized gelatin) and evaluate the in vivo efficiency as a non-viral gene carrier. Cationized gelatin was prepared by chemical introduction of ethylenediamine to the carboxyl groups of gelatin. PEG with one terminal of active ester group was coupled to the amino groups of cationized gelatin to prepare PEG-cationized gelatins. Electrophoretic experiments revealed that the PEG-cationized gelatin with low PEGylation degrees was complexed with a plasmid DNA of luciferase, in remarked contrast to that with high PEGylation degrees. When the plasmid DNA complexed with the cationized gelatin or PEG-cationized gelatin was mixed with deoxyribonuclease I (DNase I) in solution to evaluate the resistance to enzymatic degradation, stronger protection effect of the PEG-cationized gelatin was observed than that of the cationized gelatin. The complex of plasmid DNA and PEG-cationized gelatin had an apparent molecular size of about 300 nm and almost zero surface charge. These findings indicate that the PEG-cationized gelatin–plasmid DNA complex has a nano-order structure where the plasmid DNA is covered with PEG molecules. When the PEG-cationized gelatin–plasmid DNA complex was intramuscularly injected, the level of gene expression was significantly increased compared with the injection of plasmid DNA solution. It is concluded that the PEG-cationized gelatin was a promising non-viral gene carrier to enhance gene expression in vivo.     

Pilocarpine-loaded chitosan-PAA nanosuspension for ophthalmic delivery

Chitosan-poly(acrylic acid) (CS-PAA) nanoparticles, to be used as ophthalmic drug carrier, were successfully prepared using template polymerization of acrylic acid (AA) in a chitosan solution. When the polymerization was done at 70°C for 45 min with a CS/AA weight ratio of 1:1, the surface structure of the prepared nanoparticles was most stable with the smallest mean diameter (92.0±7.5 nm) and a stable zeta potential (25.5±2.6 mV) in a buffer solution (pH 4.5). The size of the nanoparticles dramatically increased with the pH value of the medium. Both in vitro and in vivo studies revealed that the prepared nanoparticle suspension was better at sustaining the release of pilocarpine than either simulated tear fluid or commercial eye drops.     

Hydrophilic interpenetrating polymer networks of poly(dimethyl siloxane) (PDMS) as biomaterial for cochlear implants

Poly(dimethyl siloxane) (PDMS) was bulk-modified to develop a new intra-cochlear electrode that can closely hug the inner wall of scala tympani (ST). The hydrophilicity of bulk and surface of PDMS was changed using a sequential method for preparation of interpenetrating polymer networks (IPNs). A series of IPNs, based on PDMS and poly(acrylic acid) (PAAc), was synthesized and characterized by means of attenuated total reflectance Fourier transform infrared spectroscopy, water contact-angle measurement, dynamic mechanical thermal analysis and peel strength tests. The performances of actual-sized fabricated electrodes were assessed inside a transparent model of ST, which was filled with saline. The cell behavior of L929 fibroblasts on materials was studied in vitro.     

A Novel In-Situ-Gelling Liquid Suppository for Site-Targeting Delivery of Anti-Colorectal Cancer Drugs

In order to avoid anti-cancer drugs undergoing a first-pass effect and reduce their toxicity, and to solve conventional suppositories defects, we developed an in-situ-gelling and injectable Pluronic–poly(acrylic acid) (Pluronic–PAA) liquid suppository, which could gel fast in the physiological state and had suitable gel strength and bioadhesive force. The liquid suppositories were inserted into the rectum of rabbits without difficulty and leakage, and retained in the rectum for at least 6 h and while releasing the drug. The toxicity and cytotoxic tests indicated that Pluronic and PAA were non-toxic materials and could inhibit colon cancer cells when oxaliplatin was incorporated. C _max and AUC_0→12h values of oxaliplatin after rectal administration of a oxaliplatin suppository were higher than those for an oxaliplatin solution administered orally. These results suggest that an in-situ-gelling and injectable liquid suppository for humans can be further developed as a more convenient and effective rectal dosage form.     

Novel Castor Oil‐Derived Block Copolymers as Promising Candidates for Biological Applications: Biorelevant and Biocompatible

The castor oil‐derived fatty acid (thioether‐containing ω‐hydroxyacid) is obtained using the UV‐catalyzed thiol‐Michael addition and is used for the first time as a monomer, for the development of biorelevant copolymers based on ε‐caprolactone, methoxy‐poly(ethylene glycol), and poly(ethylene glycol). Proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and size exclusion chromatography, confirm the successful synthesis of diblock and triblock copolymers with very narrow molecular weight distributions. X‐ray diffractometry, polarizing light microscopy, and differential scanning calorimetry, suggest that crystallization of the crystalline block chains is restricted within the microphase‐separated morphology and that it is strongly affected by the competition between the two semicrystalline blocks. Moreover, thermogravimetric analysis reveals that the thermal properties of the copolymers are intermediate between those of their parent homopolymers. Regardless of their block composition, the biorelevant copolyesters do not exhibit apparent cytotoxicity, according to the (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) (MTT) and fluorescence microscopy assays.     

Use of real-time polymerase chain reaction (rtPCR) as a diagnostic tool for influenza infection in a vaccine efficacy trial

Background

Conventional techniques for diagnosing influenza based on viral cell culture or disease serology have limitations, and molecular assays, such as real-time polymerase chain reaction (rtPCR) are increasingly used.

Objectives

To evaluate the use of rtPCR as a diagnostic tool for the determination of influenza virus infection.

Study design

This prospective, double-blind, placebo-controlled, randomised efficacy study was conducted in persons aged 18-64 years. Cases of influenza-like-illness (ILI), defined as at least one systemic symptom [fever ≥37.8 °C and/or myalgia] and at least one respiratory symptom [cough and/or sore throat] were identified by active and passive surveillance. For each case of suspected ILI, nasal and throat swabs were collected and analysed by viral culture and rtPCR.

Results

227 ILI cases were positive by rtPCR while 64% (145/227) were positive by both rtPCR and culture. For both assays, the maximum percentage of swabs that tested positive was on Day 0, thereafter positive samples by rtPCR remained constant until Day 5 but decreased progressively by culture. All rtPCR positive cases with a viral load of below 4.5 log₁₀ copies/sample were negative by culture. There were however culture negative cases with high viral loads. Vaccine efficacy for influenza was estimated as 54.7% by rtPCR (culture positive or negative) and 61.6% by culture irrespective of match to vaccine strain. Clinical severity was not significantly different between culture positive cases and culture negative but rtPCR positive cases.

Conclusions

rtPCR is a sensitive and specific diagnostic tool for influenza vaccine efficacy studies.     

Use of Lipoprotein(a) in clinical practice: A biomarker whose time has come. A scientific statement from the National Lipid Association

Lipoprotein(a) [Lp(a)] is a well-recognized, independent risk factor for atherosclerotic cardiovascular disease, with elevated levels estimated to be prevalent in 20% of the population. Observational and genetic evidence strongly support a causal relationship between high plasma concentrations of Lp(a) and increased risk of atherosclerotic cardiovascular disease–related events, such as myocardial infarction and stroke, and valvular aortic stenosis. In this scientific statement, we review an array of evidence-based considerations for testing of Lp(a) in clinical practice and the utilization of Lp(a) levels to inform treatment strategies in primary and secondary prevention.     

A Poly(vinyl alcohol)‐graft‐Copolyester: Synthesis of a Novel Graft Copolymer Containing Adamantane Moieties as Guest for Cyclodextrin

A novel graft copolymer is synthesized from commercially available poly(vinyl alcohol) using ring‐opening polymerization. For the polymerization reaction of novel brush‐like poly(vinyl alcohol)‐graft‐poly(?‐caprolactone‐co‐(3‐/7‐(prop‐2‐ynyl)oxepan‐2‐one) 5 Sn(Oct)₂ is used as a catalyst. The formation of the graft copolymer is confirmed by ¹H NMR, ¹³C NMR, and Fourier transform infrared (FTIR) spectroscopy. Furthermore, the modification of the novel synthesized graft copolymer via a “click” reaction to implement adamantane groups is described. The success of the “click” reaction is proven by ¹H NMR spectroscopy and visualized by decomplexation of cyclodextrin with included phenolphthalein.

     

Allergic rhinitis as a key factor for the development of gastroesophageal reflux disease in children

BackgroundGastroesophageal reflux disease (GERD) may cause airway symptoms and some airway diseases exacerbate GERD symptoms. Asthma and allergic rhinitis (AR) have been identified as united airway disease because of their similar epidemiology and pathophysiology. Asthma has been considered a risk factor to develop GERD. However, the association between AR and GERD is not clear. We tried to investigate whether AR could increase the development of GERD.MethodsChildren diagnosed as AR without a prior history of GERD were conducted from the National Health Insurance Research Database between 2000 and 2005. After propensity score matching, we enrolled 36,588 children with AR and 36,588 non-AR children as the controls. Cox regression models were adopted to calculate the hazard ratio (HR) of GERD.ResultsAR children had a significantly increased risk of GERD than non-AR children (adjusted HR 1.91, 95% CI = 1.73–2.11, p < 0.001), especially in the age less than 6 years old (adjusted HR 2.68, 95% CI = 1.64–4.38, p < 0.001). The risk factor related to increased risk of GERD including age, gender, and chronic sinusitis.ConclusionAR is a risk factor associated with the development of GERD in children.     

Lymphocytopenia as a marker for pandemic influenza A/H1N1 2009 virus infection in children

Lymphocytopenia has been reported in adults with pandemic influenza A/H1N1 2009 infection, but data in children are inconclusive. Data from 76 children presented with flu‐like symptoms between July and November 2009 and tested for pandemic influenza A/H1N1 2009 virus and white blood cell (WBC) counts were analyzed. Samples from 37 (48.7%) children resulted in a positive PCR assay for pandemic influenza A/H1N1 2009 virus. When comparing data from these children with data from 39 (51.3%) children with uncomplicated flu‐like illness and negative PCR assay for pandemic influenza A/H1N1 2009 virus, no difference in disease duration, median age, red blood cell count, hemoglobin concentration, C reactive protein concentration, and absolute neutrophil count was observed, whereas significant differences were apparent when considering WBC count, relative and absolute lymphocyte count, absolute lymphocyte count z‐score, and platelet count. Receiver operating characteristic curve analysis revealed that the best absolute lymphocyte count and absolute lymphocyte count z‐score cut‐points that simultaneously maximized sensitivity and specificity were 2,256 cells/µl and ?0.89, respectively, sensitivity being 0.81 (95% CI: 0.68–0.94), specificity 0.87 (95% CI: 0.77–0.98), positive predictive value 0.85 (95% CI: 0.74–0.97), and negative predictive value 0.83 (95% CI: 0.71–0.94). In conclusion, lymphocytopenia is a marker for influenza A/H1N1 2009 virus infection in children. Absolute lymphocyte count <2,556 cells/µl or absolute lymphocyte count z‐score < ?0.89 may be useful cut‐offs to discriminate against children at higher risk of infection during epidemics. Considering that the pandemic virus is highly likely to continue to circulate in the coming winter season, these findings provide direct and practical implications for the near future. J. Med. Virol. 83:1–4, 2011. © 2010 Wiley‐Liss, Inc.