Search

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose‐1‐phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno‐associated virus (AAV)‐mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV‐mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFβRII protein levels and that these effectors of cellular homeostasis could be restored following AAV‐mediated expression of GALT. Finally, we show initial in vivo proof‐of‐concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV‐GALT. [ABSTRACT FROM AUTHOR]

Most lysosomal storage diseases (LSDs) have a significant neurological component, including types 2 and 3 Gaucher disease (neuronal forms of Gaucher disease; nGD). No therapies are currently available for nGD since the recombinant enzymes used in the systemic form of Gaucher disease do not cross the blood–brain barrier (BBB). However, a number of promising approaches are currently being tested, including substrate reduction therapy (SRT), in which partial inhibition of the synthesis of the glycosphingolipids (GSLs) that accumulate in nGD lowers their accumulation. We now induce nGD in mice by injection with conduritol B‐epoxide (CBE), an irreversible inhibitor of acid beta‐glucosidase (GCase), the enzyme defective in nGD, with or without co‐injection with Genz‐667161, a prototype for SRT which crosses the BBB. Significant neuropathology, and a reduction in lifespan, was observed upon CBE injection, and this was largely reversed by co‐injection with Genz‐667161, along with a reduction in glucosylceramide and glucosylsphingosine levels. Analysis of gene expression by RNAseq revealed that Genz‐667161 largely reversed the changes in genes and pathways that were differentially expressed upon CBE injection, specifically pathways of GSL metabolism, lipoproteins and other lipid metabolic pathways, lipid droplets, astrocyte activation, neuronal function, and to some extent, neuroinflammation. Together, this demonstrates the efficacy of SRT to reverse the effects of substrate accumulation on pathological components and pathways in nGD brain. [ABSTRACT FROM AUTHOR]

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α- galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy. [ABSTRACT FROM AUTHOR]

TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%( v/ v) ethylene glycol and 5%( v/ v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å. [ABSTRACT FROM AUTHOR]

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent intracellular accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb). Vascular endothelial cells may play important roles in disease pathogenesis, and are one of the main target cell types in therapeutic interventions. In this study, we generated immortalized aortic endothelial cell lines from a mouse model of Fabry disease. These cells retained endothelial cell-specific markers and functions. Gb expression level in one of these clones (referred to as FMEC2) was highly susceptible to culture media, and appeared to be regulated by glucosylceramide synthase. Results also showed that Gb could be upregulated by hydrocortisone. FMEC2 express the mannose 6-phosphate receptor and sortilin but not the mannose receptor. Uptake studies suggested that sortilin plays a role in the binding and internalization of mammalian cell-produced α-galactosidase A. Moss-aGal (a plant-made enzyme) was endocytosed by FMEC2 via a receptor other than the aforementioned receptors. In conclusion, this study suggests that glucosylceramide synthase and hydrocortisone may play important roles in modulating Gb levels in Fabry mouse aortic endothelial cells, and that endocytosis of recombinant α-galactosidase A involves a combination of multiple receptors depending on the properties of the enzyme. [ABSTRACT FROM AUTHOR]

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age-related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin-1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin-1 (Fbn1Prx1-/- mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1Prx1-/- mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1Prx1-/- mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1-silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARg levels. Consonant with fibrillin-1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1Prx1-/- limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin-1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches. [ABSTRACT FROM AUTHOR]

Background The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. Methods We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug). Results TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls. Conclusions Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron cell loss, muscular atrophy, and a shortened life span. Survival is highly variable, as some patients die within months, while others live for many years. Exposure to stress or the development of a nonoptimal stress response to disease might account for some of this variability. We show in the SODIG93A mouse model of ALS that recurrent exposure to restraint stress led to an earlier onset of astrogliosis and microglial activation within the spinal cord, accelerated muscular weakness, and a significant decrease in median survival (105 vs. 122 d) when compared to non-stressed animals. Moreover, during normal disease course, ALS mice display a cacostatic stress response by developing an aberrant serum corticosterone circadian rhythm. Interestingly, we also found that higher corticosterone levels were significantly correlated with both an earlier onset of paralysis (males: r²=0.746; females: r²=0.707) and shorter survival times (males: r²=0.680; females: r²=0.552) in ALS mice. These results suggest that stress is capable of accelerating disease progression and that strategies that modulate glucocorticoid metabolism might be a viable treatment approach for ALS. [ABSTRACT FROM AUTHOR]

Several diseases of the nervous system are characterized by neurodegeneration and death in childhood. Conventional medicine is ineffective, but fetal or neonatal gene therapy may provide an alternative route to treatment. We evaluated the ability of single-stranded and self-complementary adeno-associated virus pseudotype 2/9 (AAV2/9) to transduce the nervous system and target gene expression to specific neural cell types following intravenous injection into fetal and neonatal mice, using control uninjected age-matched mice. Fetal and neonatal administration produced global delivery to the central (brain, spinal cord, and all layers of the retina) and peripheral (myenteric plexus and innervating nerves) nervous system but with different expression profiles within the brain; fetal and neonatal administration resulted in expression in neurons and protoplasmic astrocytes, respectively. Neither single-stranded nor self-complementary AAV2/9 triggered a microglia-mediated immune response following either administration. In summary, intravenous AAV2/9 targets gene expression to specific neural cell types dependent on developmental stage. This represents a powerful tool for studying nervous system development and disease. Furthermore, it may provide a therapeutic strategy for treatment of early lethal genetic diseases, such as Gaucher disease, and for disabling neuropathies, such as preterm brain injury. [ABSTRACT FROM AUTHOR]

Background The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis. Methods Apolipoprotein E deficient (ApoE [ABSTRACT FROM AUTHOR]

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid β-glucosidase), with consequent cellular accumulation of glucosylceramide (GL-1). The disease is managed by intravenous administrations of recombinant glucocerebrosidase (imiglucerase), although symptomatic patients with mild to moderate type 1 Gaucher disease for whom enzyme replacement therapy (ERT) is not an option may also be treated by substrate reduction therapy (SRT) with miglustat. To determine whether the sequential use of both ERT and SRT may provide additional benefits, we compared the relative pharmacodynamic efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V/null). As expected, ERT with recombinant glucocerebrosidase was effective in reducing the burden of GL-1 storage in the liver, spleen, and lung of 3-month-old Gaucher mice. SRT using a novel inhibitor of glucosylceramide synthase (Genz-112638) was also effective, albeit to a lesser degree than ERT. Animals administered recombinant glucocerebrosidase and then Genz-112638 showed the lowest levels of GL-1 in all the visceral organs and a reduced number of Gaucher cells in the liver. This was likely because the additional deployment of SRT following enzyme therapy slowed the rate of reaccumulation of GL-1 in the affected organs. Hence, in patients whose disease has been stabilized by intravenously administered recombinant glucocerebrosidase, orally administered SRT with Genz-112638 could potentially be used as a convenient maintenance therapy. In patients naïve to treatment, ERT followed by SRT could potentially accelerate clearance of the offending substrate. [ABSTRACT FROM AUTHOR]

Background When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs). Methods We have compared laser microdissection, pronase digestion and nasal brushing for: (i) the ability to enrich RECs from the wild-type mouse nose and (ii) the length of time to perform the procedure. Using TaqMan, we subsequently assessed gene transfer in enriched RECs after nasal perfusion of GL67A/pCF1-CFTR complexes in a CF mouse model. Results Laser microdissection successfully isolated RECs; however, time-consuming sample preparation made this technique unsuitable for high-throughput studies. Pronase digestion was sufficiently rapid but only yielded 19% (range = 13%) RECs ( n = 6). The nasal brushing method was superior, yielding 92% (range = 15%) RECs ( n = 8) and was equally effective in CF knockout mice (91%, range = 14%, n = 10). Importantly, gene transfer was detectable in brushed RECs from 70% of perfused mice and the number of vector-specific transcripts was comparable to 3.5% of endogenous wild-type Cftr levels. Conclusions Isolation of RECs by brushing allows accurate assessment of GTA transfection efficiency in an experimental system that is relevant for CF gene therapy. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background Diabetes mellitus is a common comorbidity of atherosclerosis. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of the angiogenic response to hypoxia. Methods We studied the effects of adenoviral vectors expressing a constitutively active HIF-1α hybrid (Ad2/HIF-1α/VP16) or vascular endothelial growth factor (Ad2/VEGF) on collateral development and vascular leakiness in a diabetic rat model of hindlimb ischemia. Results After the removal of the right femoral artery, the mRNA levels of VEGF, angiopoietin-1 and angiopietin-4 in the calf muscles, as measured by Taqman reverse transcriptase-polymerase chain reaction, were transiently elevated in Zucker lean (ZL) but not Zucker diabetic fatty (ZDF) rats. The angiographic score, as determined by post-mortem angiography, was significantly lower in ZDF animals 35 days after surgery compared to their ZL counterparts. In separate animals, intramuscular injection of Ad2/HIF-1a/VP16 and Ad/2VEGF into the thigh muscles significantly increased the angiographic score and capillary density 21 and 35 days after the injection compared to Ad2/CMVEV (a vector expressing no transgene) or vehicle. After the injection of Ad2/CMVEV or vehicle, the Evans-blue dye content in the thigh muscles was significantly higher in ZDF rats than their ZL counterparts. Ad2/HIF-1α/VP16 but not Ad2/VEGF reduced tissue Evans blue dye content. Conclusions The endogenous angiogenic response to ischemia was impaired in ZDF rats, possibly due to down-regulation of angiogenic factors. Ad2/HIF-1α/VP16 enhanced collateral development and reduced vascular leakage in the ischemic hindlimb of ZDF rats indicating that hybrid HIF-1α angiogenic therapy may be efficacious for peripheral vascular disease with a diabetic comorbidity. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Affected individuals can experience episodic, life-threatening, acute neurological attacks that are precipitated by various drugs, dieting, and hormonal changes. Intravenous hematin is used to treat the attacks, but a more effective, preventive therapy is needed, especially for patients with frequent attacks. Since the disease is a hepatic encephalopathy, efforts were focused towards evaluating four different combinations of liver-specific enhancers and promoters for maximal hepatic HMB-synthase expression. Methods Four different mammalian expression vectors, each carrying a unique combination of liver-specific enhancers and promoters driving murine HMB-synthase cDNA expression, were transiently transfected into HepG2 cells. The vectors included: HMBS-1; human α1-microglobulin enhancer/α1-antityrpsin promoter (α1Me/α1ATp), HMBS-2; α1Me/human serum albumin promoter (α1Me/SAp), HMBS-3; human prothrombin enhancer/SAp (PTe/SAp), and HMBS-4; (PTe/α1ATp). Each HMB-synthase construct and a luciferase reporter construct were hydrodynamically coinjected into mice with HMB-synthase deficiency and evaluated for hepatic expression 24 h post-injection, the time-point of peak hepatic HMB-synthase expression. Results Following transient transfection into HepG2 cells, HMBS-1 (α1Me/α1ATp) had the highest HMB-synthase expression level, with an ∼8-fold increase over endogenous cellular activities. Construct HMBS-1 also had the highest hepatic HMB-synthase activity following hydrodynamic delivery into HMB-synthase deficient mice, with a ∼6-fold increase over saline-treated mice. Conclusions These studies support the use of a gene therapy vector containing the α1Me/α1ATp combination for preclinical studies of the efficacy and safety of liver-targeted gene therapy for AIP. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. Methods Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h. Results Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone. Conclusions These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background Identification of the cell types transfected following gene transfer is an important factor in the selection of appropriate gene transfer agents (GTAs). Due to the relatively low gene expression mediated by non-viral GTAs, current methodologies for the detection and identification of transfected cells in the lung have proven insensitive and unreliable. We have investigated the use of the green fluorescent protein (GFP) to identify transfected cells in a mouse lung model. Methods Direct visualisation of GFP fluorescence in frozen histological sections was used in conjunction with a panel of cell type specific antibodies to investigate the distribution and level of gene expression in mouse lungs following instillation of non-viral GTAs. Results Despite considerable tissue autofluorescence, dose-dependent expression of GFP was detected following instillation of as little as 25 µg naked plasmid DNA (pDNA). Naked pDNA and pDNA complexed with polyethylenimine appeared to transfect mainly ciliated cells and Clara cells of the conducting airway, whereas expression mediated by pDNA complexed with the cationic lipid GL67 was found predominantly in type I pneumocytes. Conclusions Direct visualisation of GFP expression was used to detect transfected cell types in the mouse lung. In contrast with observations made using ß-galactosidase as a reporter, gene expression from several non-viral GTAs was readily demonstrated and no false GFP-positive cells were ever detected in untreated lung tissues. Lung delivery of different GTAs resulted in GFP expression in different cell types, confirming the importance of identification of transfected cells when screening and selecting GTAs for disease targets. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy. Methods A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs. Results Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme. Conclusions These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A, leading to an accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in most tissues of the body. The goal of this study was to determine if systemic delivery of a nonviral vector could correct the enzyme deficiency and reduce the levels of GL-3 in different tissues of a transgenic knockout mouse model of the disease. Methods Cationic lipid was complexed with a CpG-depleted plasmid DNA vector and then injected intravenously into Fabry mice. The levels of α-galactosidase A and GL-3 in different tissues were assayed at various time points after injection. Results Expression of α-galactosidase A was detected in the different tissues of Fabry mice for up to 3 months after complex administration, but resulted in minimal reductions in GL-3 levels. However, the use of the anti-inflammatory drug dexamethasone and multiple dosing increased α-galactosidase A expression and resulted in significant reductions of GL-3 in all the organs with the exception of the kidney. In addition, injecting complex into young Fabry mice partially prevented the normal accumulation of GL-3 in the heart, lung, and liver. Conclusions Systemic delivery of a cationic lipid-pDNA complex partially corrected the enzyme deficiency and reduced glycolipid storage in a mouse model of Fabry disease. The results are one of the few demonstrations of long-term efficacy in a genetic disease model using nonviral vectors. However, substantial improvements in expression, especially in critical organs such as the kidney, are required before these vectors can become a viable approach to treat Fabry disease and other lysosomal storage disorders. Copyright © 2003 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]