Lysosomal localization and mechanism of membrane penetration influence nonenveloped virus activation of the NLRP3 inflammasome.

Adenovirus (Ad) endosomal membrane penetration activates the NLRP3 inflammasome by releasing lysosomal cathepsin B (catB) into the cytoplasm. We therefore examined the extent to which inflammasome activation correlates with Ad colocalization with catB-enriched lysosomes. Inflammasome activation, is greater during infections with Ad5 possessing an Ad16 fiber (Ad5F16gfp), or Ad5gfp neutralized by human serum, than Ad5gfp alone. Enhanced IL-1ß release by Ad5F16gfp is partially due to increased TLR9 signaling but also correlates with greater release of catB into the cytoplasm. This increased TLR9 signaling and catB release correlates with a greater localization of Ad5F16gfp to lysosomes prior to endosomal escape. Another nonenveloped virus, reovirus, requires catB to penetrate cell membranes. However, reovirus did not release catB into the cytoplasm despite significantly greater colocalization with lysosomes compared to Ad5gfp and efficient membrane penetration. Thus, not only lysosomal localization, but the mechanism of membrane penetration influences viral activation of the NLRP3 inflammasome.

Adenovirus membrane penetration activates the NLRP3 inflammasome.

Adenovirus type 5 (Ad5) infection of macrophages results in rapid secretion of interleukin-1ß (IL-1ß) and is dependent on the inflammasome components NLRP3 and ASC and the catalytic activity of caspase-1. Using lentivirus-expressed short hairpin RNA (shRNA) and competitive inhibitors, we show that Ad-induced IL-1ß release is dependent upon Toll-like receptor 9 (TLR9) sensing of the Ad5 double-stranded DNA (dsDNA) genome in human cell lines and primary monocyte-derived macrophages but not in mouse macrophages. Additionally, a temperature-sensitive mutant of Ad5 unable to penetrate endosomal membranes, ts1, is unable to induce IL-1ß release in TLR2-primed THP-1 cells, suggesting that penetration of endosomal membranes is required for IL-1ß release. Disruption of lysosomal membranes and the release of cathepsin B into the cytoplasm are required for Ad-induced NLRP3 activation. Ad5 cell entry also induces reactive oxygen species (ROS) production, and inhibitors of ROS prevent Ad-induced IL-1ß release. Ad5 activation of NLRP3 also induces necrotic cell death, resulting in the release of the proinflammatory molecule HMGB1. This work further defines the mechanisms of virally induced inflammasome activation.

The right ventricle in Fabry disease.

AIM: Left ventricular (LV) hypertrophy is a common feature in Fabry disease-related progressive infiltrative hypertrophic cardiomyopathy and affects both men and women, but at different ages. To date, however, little is known about the role of right ventricular (RV) function in Fabry disease. Therefore, this study aimed to investigate the extent of RV involvement in patients with Fabry disease. METHODS: Echocardiographic examination of the right and left ventricle was carried out in 129 patients (80 women and 49 men) with Fabry disease. RESULTS: RV hypertrophy was present in 46 patients (35.7%). Of these patients, 13 showed signs of severely depressed right systolic function (tricuspid annulus movement < 10 mm and a prolonged RV pre-ejection period/pulmonary ejection time ratio) and six patients showed additional severe depression of parameters of diastolic function (pseudo-normal or restrictive RV filling pattems). Those patients with RV hypertrophy and severely compromised systolic and diastolic function had the highest LV masses (92 +/- 11.7 g/m(2.7)). CONCLUSION: RV involvement is common in Fabry disease and ultimately progresses to severe systolic and diastolic RV dysfunction. These findings might explain why patients with preserved LV function can develop clinical features such as reduced exercise capacity, organomegaly and lymphoedema.

Enzyme replacement therapy in heterozygous females with Fabry disease: results of a phase IIIB study.

Fabry disease is an X-linked glycosphingolipid storage disorder caused by a deficiency of alpha-galactosidase A. Affected patients experience debilitating neuropathic pain and have premature mortality due to renal failure, cardiovascular disease or cerebrovascular complications. The disease may be X-linked dominant, since most females heterozygous for Fabry disease are affected clinically. We evaluated the safety, efficacy and pharmacokinetics of agalsidase alfa (Replagal) administered intravenously to female patients with Fabry disease in an open-label, single-centre study. Fifteen severely affected patients received agalsidase alfa at 0.2 mg/kg every other week for up to 55 weeks. Agalsidase alfa was safe and well-tolerated in female patients. None of the patients developed antibodies or experienced an infusion reaction to agalsidase alfa. The pharmacokinetic profile of agalsidase alfa in female patients is comparable to the pharmacokinetics of agalsidase alfa in male patients. Mean urine sediment and plasma Gb3 levels decreased from baseline at 13, 27 and 41 weeks. A significant decrease in left ventricular mass from baseline was seen at weeks 27 (p = 0.003) and 41 (p = 0.039), and a significant reduction in QRS durations was seen at week 27 (p = 0.007). Furthermore, there was a significant improvement in quality of life. Renal function did not deteriorate in these 15 female patients over the 13- to 41-week period of observation. We conclude that enzyme replacement therapy with agalsidase alfa was safe and effective in female patients heterozygous for Fabry disease.

Electrocardiographic signs of hypertrophy in fabry disease-associated hypertrophic cardiomyopathy.

AIMS: Fabry disease results from deficient activity of the lysosomal enzyme alpha-galactosidase A. Progressive accumulation of the major substrates leads, in both men and women, to progressive hypertrophic cardiomyopathy. We aimed to evaluate the utility of different electrocardiographic (ECG) parameters for assessing the degree and severity of hypertrophic cardiomyopathy in patients with Fabry disease. METHODS: A total of 166 ECGs of 94 hemi- and heterozygous patients with Fabry disease were analysed and compared with echocardiographic-estimated left ventricular mass (LVM). RESULTS: There was a significant (p < 0.0001) correlation between QRS duration (R2 = 0.59), 12-lead amplitude/duration product (R2 = 0.61), Sokolow-Lyon voltage/duration product (R2 = 0.52) and LVM. Analysis of receiver operating characteristics revealed that the 12-lead amplitude/duration product had the highest sensitivity-specificity relationship (p < 0.01 compared with the Cornell index). CONCLUSION: In general, ECG signs of left ventricular hypertrophy correlated well with LVM as revealed by echocardiography. Of the parameters studied, the 12-lead amplitude/duration product was the most successful at describing the severity of cardiac involvement in Fabry disease. These data suggest that ECG parameters have potential for use as a simple and cost-effective means of screening for hypertrophic cardiomyopathy in patients with Fabry disease.

The potential role of proteoglycans in cationic lipid-mediated gene delivery. Studies of the interaction of cationic lipid-DNA complexes with model glycosaminoglycans.

Recent evidence supports a role for proteoglycans in polycation-mediated gene delivery. Therefore, the interaction of glycosaminoglycans with cationic lipid-DNA complexes (CLDCs) has been characterized using a combination of biophysical approaches. At low ionic strength, CLDCs bind to heparin-derivatized Sepharose particles, with the ratio of cationic lipid to DNA controlling the binding. Incorporation of the helper lipids cholesterol or 1,2-dioleoyl-phosphatidylethanolamine increases the amount of bound CLDC. Heparin also induces the aggregation of CLDCs, with cholesterol reducing this effect. Isothermal titration calorimetry demonstrates an endothermic heat for the binding of heparin to CLDCs at low ionic strength, whereas circular dichroism studies suggest a heparin-stimulated release of DNA from CLDCs at a greater than 20-fold charge excess. Increasing the ionic strength to 0.11 reduces CLDC binding to heparin beads, and greatly enhances the release of DNA from CLDCs by heparin. The ability of the cell surface glycosaminoglycan heparan sulfate to release DNA from CLDCs is more sensitive than heparin to the incorporation of the cholesterol or 1,2-dioleoyl-phosphatidylethanolamine. Titration calorimetry reveals an exothermic heat for the interaction glycosaminoglycans with CLDCs at higher ionic strength. These results are consistent with the direct involvement of proteoglycans in transfection.

Infrared spectroscopic characterization of the interaction of cationic lipids with plasmid DNA.

Fourier transform infrared spectroscopy was used to characterize the interaction of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dioctadecyldimethylammonium bromide with plasmid DNA. The effect of incorporating the neutral colipids cholesterol and dioleoylphosphatidylethanolamine on this interaction was also examined. Additionally, dynamic and phase analysis light scattering were used to monitor the size and zeta potential of the resulting complexes under conditions similar to the Fourier transform infrared measurements. Results suggest that upon interaction of cationic lipids with DNA, the DNA remains in the B form. Distinct changes in the frequency of several infrared bands arising from the DNA bases, however, suggest perturbation of their hydration upon interaction with cationic lipids. A direct interaction of the lipid ammonium headgroup with and dehydration of the DNA phosphate is observed when DNA is complexed with these lipids. Changes in the apolar regions of the lipid bilayer are minimal, whereas the interfacial regions of the membrane show changes in hydration or molecular packing. Incorporation of helper lipids into the cationic membranes results in increased conformational disorder of the apolar region and further dehydration of the interfacial region. Changes in the hydration of the DNA bases were also observed as the molar ratio of helper lipid in the membranes was increased.

Characterization of synthetic gene delivery vectors by infrared spectroscopy.

For many decades, infrared (IR) spectroscopy has been used to characterize the structure of molecules. In IR spectroscopy, absorption of light, corresponding to vibrational and rotational transitions of a molecule, is measured. For a transition to be IR-active, a change in the dipole moment of a particular bond must occur upon excitation. This vibrational energy is not only dependent on the chemical nature of the particular covalent bonds, but also on the environment of these coupled atoms and bonds. IR spectroscopy has been previously employed in the study of the structure of nucleic acids, producing not only information about the individual bases, sugars, and phosphate backbone, but also providing information about the helical conformation of polynucleotides (1-3). IR spectroscopy has also been successfully applied to the analysis of lipids, as well as to numerous other polymers (4). Thus, IR spectroscopy potentially possesses the ability to obtain structural information about all of the components of most synthetic gene delivery complexes, as well as changes in the structure of polymeric or lipid components upon complex formation. In addition to the ability to gather detailed structural information, there are also some practical advantages to the use of IR spectroscopy for the study of plas-mid DNA and DNA complexes compared to other techniques, including the availability of a variety of sampling techniques, permitting the analysis of samples in a wide variety of physical states including solutions, solids, and gels. There is also no upper limit to the size of the sample molecule examined, allowing both short oligonucleotides and higher molecular weight DNA to be studied. IR spectroscopy is not a destructive technique, and requires only small amounts of material, making it ideal for the analysis of valuable samples.

Characterization of cationic vector-based gene delivery vehicles using isothermal titration and differential scanning calorimetry.

Within the past 10 years, major advances in the design and development of differential scanning calorimeters (DSC) (1) and isothermal titration calorimeters (ITC) (2) have resulted in an unparalleled level of sensitivity, stability, and reproducibility in calorimetric measurements of large molecules. These improvements have allowed the thermal stability and ligand binding processes of biological macromolecules to be thermodynamically characterized with speed, accuracy, and convenience. With their increasing commercial availability, experiments that were previously limited to specialist calorimetry laboratories can now be routinely performed by most investigators.

Light-scattering techniques for characterization of synthetic gene therapy vectors.

The colloidal properties of delivery systems currently being developed for nonviral gene therapy are extremely important. The physical stability of these systems on the shelf, as well as in the biological milieu, is mostly based on their size and interfacial properties. The size and surface charge of these systems can also have dramatic effects on their biological activity (1-4). With these facts in mind, it is apparent that adequate characterization of these properties is necessary for the development of any synthetic gene delivery system.

Normal values of M mode echocardiographic measurements of more than 2000 healthy infants and children in central Europe.

OBJECTIVE: To obtain normal M mode (one dimensional) echocardiographic values in a substantial sample of normal infants and children. DESIGN: Data were obtained over three years from a single centre in central Europe. PATIENTS: 2036 healthy infants and children aged one day to 18 years. METHODS: In line with recommendations for standardising measurements from M mode echocardiograms, and using digital echocardiographic equipment, measurements were obtained of the following: right ventricular anterior wall thickness at end diastole, right ventricular end diastolic dimension, thickness of interventricular septum at end diastole and end systole, thickness of posterior wall of the left ventricle at end diastole and end systole, left ventricular dimension at end diastole and end systole, pulmonary and aortic valve diameter, and left atrial dimension. RESULTS: Measurements are presented graphically on centile charts with respect to body surface area, and as tables with mean and 2 SD values for newborns in relation to body weight, and for infants and children in relation to body surface area. Best fitting regression equations are given for each measured variable, using the 50th centile values. CONCLUSION: In comparison with previously published normal values, the presented charts and tables make it possible to judge echocardiographic measurements of a particular patient as normal or abnormal.