Encapsulation of viral vectors for gene therapy applications

In gene therapy, a number of viruses are currently being used as vectors to provide transient expression of therapeutic proteins. A drawback of using free virus is that it gives a potent immune response, which reduces gene transfer and limits re-administration. An alternative delivery system is to encapsulate the virus in poly(lactide-co-glycolide) (PLG) microspheres prior to administration. A recombinant adenovirus (Ad) expressing green fluorescent protein (GFP) was used to test the transduction efficiency of Ad encapsulated in microspheres on target cells. The number of infected cells that expressed GFP was measured by flow cytometry. It was demonstrated that encapsulated viral vectors could successfully transduce target cells with encapsulation efficiencies up to 23% and that the level of transduction could be controlled by varying both the quantity of microspheres and the amount of Ad in the microspheres. High transduction efficiencies and its recognized biocompatibility make PLG-encapsulated Ad an attractive alternative to the use of free virus in gene therapy applications. The infectivity of Ad was found to be significantly influenced by the processing conditions and changes in environmental factors. Free Ad and encapsulated Ad were able to infect both E1 complimenting cells (HEK 293) and non-complimenting cells (A549), with the viral expression in HEK 293 cells being 2.1 times greater than for A549 cells.     

Spatial and temporal analysis of the microbial community in slow sand filters used for treating horticultural irrigation water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.     

Deleterious effect of soil‐applied metalaxyl and mancozeb on the mycoparasite Pythium oligandrum

Pythium oligandrum was recovered, identified and quantified from air‐dried soil plated on 1.5% water agar containing 0.1% glucose. Isolations of P. oligandrum over 2 years from soils treated with single applications of metalaxyl plus mancozeb were consistently lower than those from untreated soil from the same fields. In three fields in the first year P. oligandrum was reduced from a range of 43.3–115.0 to 17.0–43.2 isolates g^‐1 soil. In the second year, results from 11 fields showed reductions from 27.8–141.8 to 2.8–44.5 isolates g^‐1 soil. P. oligandrum was sensitive to both metalaxyl and mancozeb, with median effective dose (ED₅₀) values of 0.13 ± 0.02 μg m^‐1 and 3.33 ± 0.12 μg ml^‐1, respectively. In a pot test with three soils treated with metalaxyl, mancozeb or the combination of fungicides, levels of P. oligandrum declined over 3 months, with effects first recorded 2 weeks after treatment. Levels of P. oligandrum were reducd by differing degrees in the three soils. Isolate counts from untreated soils declined from a mean of 58.0 g^‐1 soil at the start of the experiment to 27.5 g^‐1 after 3 months, whereas fungicide treatments caused further reductions to 11.1 (metalaxyl), 9.7 (mancozeb) and 4.8 isolates g^‐1 (metalaxyl plus mancozeb).     

Venous Graft Surgery in Treatment of Coronary Heart Disease

Sixty-seven patients have had aortocoronary venous graft bypass surgery by one surgeon for the relief of symptoms of severe coronary heart disease, including eight emergency operations. The overall operative, hospital, and late mortality was low in patients with favourable myocardial function and no previous myocardial infarction. There was a 7% mortality in patients with a normal preoperative chest radiogram, 8% mortality when the left ventricular end-diastolic pressure was normal preoperatively, and a 5% mortality in patients who had normal left ventricular angiograms. The overall mortality in all elective operations for cardiac pain resistant to medical treatment was 15·8%. 89% of survivors improved; 67% are pain-free. Exercise tolerance in survivors is increased by 135%, atrial pacing results are improved by 10%. Left ventricular end-diastolic pressure is unchanged. Left ventricular function on angiography is improved. The improvement in left ventricular function assessed objectively correlates positively with vein-graft patency, as does freedom from angina pectoris.