Quantification of beta-galactosidase activity after non-viral transfection in vivo.

The limited efficacy of non-viral gene delivery systems currently hampers their wider therapeutic use. In order to further develop novel gene delivery systems, it is important to quantify their efficacy. Many reporter gene assays have limitations when being used to quantify expression in vivo. We have developed a simple assay which allows the quantification of beta-galactosidase transgene activity in vivo. The assay is based on beta-galactosidase cleavage of the DDAO-galactopyranoside substrate to DDAO, which shifts the fluorescence towards longer wavelengths. Reaction conditions were optimised to minimise degradation, activity of endogenous beta-galactosidase, and non-specific background fluorescence. The spectrofluorimetric quantification of the reaction product DDAO in the red part of the spectrum avoided interference from haemoglobin or other bio-molecules which hamper many in vivo assays. Routinely, amounts of less than 1 ng of beta-galactosidase (1 mU) per gram tissue could be detected and quantified. After intravenous administration of beta-galactosidase complexed with linear polyethylenimine (PEI, 22 kD) in mice, 134 mU g(-1) beta-galactosidase were detected in the lung, but only 2.9 mU g(-1) were found in the liver.     

The release of model macromolecules may be controlled by the hydrophobicity of palmitoyl glycol chitosan hydrogels

A non-covalently cross-linked palmitoyl glycol chitosan (GCP) hydrogel has been evaluated as an erodible controlled release system for the delivery of hydrophilic macromolecules. Samples of GCP with hydrophobicity decreasing in the order GCP12>GCP11>GCP21 were synthesised and characterised by ¹H NMR. Hydrogels were prepared by freeze-drying an aqueous dispersion of the polymer in the presence or absence of either a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran, MW 4400), and/or amphiphilic derivatives Gelucire 50/13 or vitamin E d--tocopherol polyethylene glycol succinate. Gels were analysed for aqueous hydration, FITC-dextran release, and bioadhesion, and imaged by scanning electron microscopy. The gels were highly porous and could be hydrated to up to 95× their original weight without an appreciable volume change and most gels eventually eroded. Hydration and erosion were governed by the hydrophobicity of the gel and the presence of the amphiphilic additives. GCP gels could be loaded with up to 27.5% (w/w) of FITC-dextran by freeze-drying a dispersion of GCP in a solution of FITC-dextran. The controlled release of FITC-dextran was governed by the hydrophobicity of the gel following the trend GCP21>GCP11>GCP12. GCP gels were bioadhesive but less so than hydroxypropylmethylcellulose, Carbopol 974NF (7:3) tablets.     

Sustained buccal delivery of the hydrophobic drug denbufylline using physically cross-linked palmitoyl glycol chitosan hydrogels.

A physically cross-linked palmitoyl glycol chitosan hydrogel has been evaluated as a controlled release system for the delivery of hydrophobic drugs via the buccal route. Samples of palmitoyl glycol chitosan (GCP) with diminishing hydrophobicity (GCP12>GCP11>GCP21) were synthesized, characterized by (1)H nuclear magnetic resonance and hydrogels prepared by freeze-drying an aqueous dispersion of the polymer in the presence of a model hydrophobic drug denbufylline and in some cases the soluble detergent sodium glycodeoxycholate (GDC). GDC was employed as a penetration enhancer. Gels were analysed for hydration, erosion, mucoadhesion and imaged by scanning electron microscopy. The buccal absorption of denbufylline from GCP12, denbufylline, GDC (20:12:1.5) formulations was also investigated in the rabbit model with Carbopol 974NF (CP), denbufylline, GDC (60:36:4) tablets used as controls. Denbufylline reduced the porosity, erosion and hydration of the gels while GDC increased the hydration and erosion. All gels were mucoadhesive but less so than the control CP tablets. Denbufylline was detected 0.5 h after dosing with the GCP12 formulation and delivery was sustained for at least 5 h after dosing. In comparison delivery from the CP tablets was not sustained and was first detected 1 h after dosing.     

The effect of <Emphasis Type="Italic">Iresine herbstii</Emphasis> Hook on some haematological parameters of experimentally induced anaemic rats

Iresine herbstii is fed to livestock in South-eastern Nigeria in the belief that it boosts their blood supply. The aim of this work is to study the effect of the methanolic extract of I. herbstii Hook on some blood parameters of experimentally induced anaemic albino rats. For acute toxicity study, graded doses of the methanolic leaf extract of I. herbstii at 10, 100, 1000, 1600, 2900 and 5000 mg/kg were administered orally to rats randomly allotted to six groups of three animals each and signs of toxicity were observed for 24 h. To evaluate its haematological effects, 100, 200, and 400 mg/kg of extract was administered orally to three out of five groups of six rats each for 14 days after the induction of acute blood loss anaemia. Parameters monitored were packed cell volume (PCV), haemoglobin concentration (Hb), weight gain and red blood cell counts (RBC). Phytochemical, proximate and nutritional analysis of the plant was done. I. herbstii had an LD₅₀?>?5000 mg/kg. Mean group weight gain and RBC were significantly (p?<?0.05) higher in treated than untreated groups. PCV and Hb did not differ significantly between the treated and untreated groups. Plant contained flavonoids; phenols; alkaloids; 22.85 % crude protein; 18.58 % ash; 9.62 % crude fibre; 1.5 % ether extract; 12.05 % moisture; vitamins A, C, E, B1, B3, B5, B6 and B12; zinc; iron; phosphorus; calcium; and magnesium. Methanolic extract of I. herbstii significantly improved red blood cell count of anaemic rats at the dose of 400 mg/kg bw and the body weights of anaemic rats at all treatment doses (100, 200 and 400 mg/kg bw).     

Converting partial hospitals to community integrated recovery centers

This paper describes the conversion of partial hospitals into recovery-oriented programs as part of system transformation. Steps included: participatory planning with stakeholders; strength based assessment of resources and needs; technical assistance; and changing funding strategies. Over a period of 8 years, use of partial hospitals decreased as persons with serious mental illnesses were transitioned to community integrated recovery centers. Preliminary outcomes suggest that these programs are more effective in engaging people in the community activities of their choice, confirming previous findings that showed that partial hospitals can be converted to recovery-oriented programs that focus more directly on promoting community inclusion.     

Gene Transfer with Three Amphiphilic Glycol Chitosans—the Degree of Polymerisation is the Main Controller of Transfection Efficiency

A number of studies have examined the possibility of delivering genes for the treatment of genetic diseases using various polymers and lipids. We have previously demonstrated the gene transfer ability of amphiphilic polymers (a soluble amine polymer covalently bound to lipid pendant groups). In the current communication we explore the gene transfer activity of amphiphilic glycol chitosans. Glycol chitosan was acid depolymerised to give polymers of various molecular weights. Palmitoyl or hexadecyl and in some cases additional N-methyl quaternary ammonium groups were attached to the polymers. DNA binding was studied by measuring the reduced fluorescence of ethidium bromide and the polyplex particle size and zeta potential. Biological characterisation of the polyplexes involved haemolysis, cytotoxicity and gene transfer assays. For the 22 polymers tested, DNA binding was optimum at a nitrogen to phosphate ratio of 2:1 and above. Polyplexes were 200–500 nm in diameter with a neutral or positive zeta potential. The haemolytic activity of the N-methyl polymers was studied and no haemolysis was detected up to a concentration of 10 mg ml^?1. Cytotoxicity studies showed that the biocompatibility of glycol chitosan was adversely affected by a combination of a palmitoyl group and depolymerisation and that biocompatibility was subsequently restored with the introduction of N-methyl groups. In vitro transfection efficiency superior to the cationic lipid formulation N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP) was seen with depolymerised glycol chitosan in the A431 cell line only and with the depolymerised N-methyl quaternary ammonium amphiphilic derivatives in both the A431 and A549 cell lines. Degree of polymerisation (DP) was the most important controller of transfection efficiency and transfection resided within polymers with a DP of 73–171. High DP polymers diminished DNA–cell association, the first step in the cellular gene transfer process, thus apparently diminishing cell uptake. In vivo transfection with the N-methyl quaternary ammonium amphiphile was best at a DP of 86 and this glycol chitosan amphiphile gave superior liver and heart gene expression levels when compared to both Exgen 500 (linear polyethylenimine) and Superfect (a polyamidoamine dendrimer).     

Intervention for stress management among skilled construction workers

Objective:Previous studies have demonstrated the need for a stress management intervention among construction workers. Construction workers, despite their degree of stress, are expected to contribute to the accomplishment of projects on construction sites. This study aimed to ascertain the effect of a group rational emotive behavior therapy (group REBT) on stress management among a select sample of skilled construction workers in construction industry in Nigeria.Methods:With a randomized controlled trial design, 160 skilled construction workers completed the study and responded to the perceived stress scale-14 and work-related irrational beliefs questionnaire. Participants were randomly allocated to either the intervention group (n = 80) or the control group (n = 80). The main method of data analysis used in the study was repeated measures within-between subjects analysis of variance statistic.Results:Results show that group REBT significantly improved stress and work-related irrational beliefs scores of the skilled construction workers after they were exposed to the intervention and compared with their colleagues in the control group. The significant reduction in stress and work-related irrational beliefs scores of the treatment group were also sustained at follow-up.Conclusion:Group REBT was significant in reducing stress and work-related irrational beliefs among the skilled construction workers.     

The identification and characterization of Clostridium perfringens by real-time PCR, location of enterotoxin gene, and heat resistance

Clostridium perfringens carrying the enterotoxin gene is an important cause of both foodborne and non-foodborne diarrheal disease. Rapid identification of isolates carrying the enterotoxin gene is invaluable for outbreak investigation whilst information on the genomic location of the enterotoxin (cpe) gene can improve our understanding of disease transmission. This paper describes the validation of a real-time polymerase chain reaction (PCR) assay for the identification of C. perfringens and assessment of the potential to cause diarrhea, together with an investigation into the genomic location of the cpe genes in isolates from confirmed incidents of C. perfringens diarrhea. The real-time assay was shown to be specific for the identification of 253 C. perfringens cultures and gave results concordant with those from motility nitrate and lactose gelatine media, the Nagler reaction, and a conventional block-based PCR assay. The cpe gene was detected in 223 of 253 C. perfringens cultures isolated in association with human gastrointestinal disease. A subset of cpe-positive C. perfringens isolates associated with separate incidents of diarrheal disease were investigated further for plasmid or chromosomal location of the cpe gene using a multiplex PCR assay. The cpe gene was plasmid encoded in two isolates from cases of sporadic diarrhea and six isolates from cases of food poisoning. The cpe gene from the remaining 11 isolates from different food poisoning outbreaks was found to be chromosomally encoded. One of the C. perfringens strains with a plasmid encoded cpe gene formed spores of high heat resistance and five formed spores that were sensitive to heating. Eight of the isolates with a chromosomal cpe gene formed heat-resistant spores, and two formed spores with an intermediate heat resistance.     

HIV/AIDS-related stigma and discrimination: accounts of HIV-positive Caribbean people in the United Kingdom

This paper explores the effects of HIV/AIDS-related stigma and discrimination (HASD) on HIV-positive Caribbean people in the Caribbean and the UK. In-depth, semi-structured interviews were held with a purposively selected group of 25 HIV-positive people of Caribbean origin, using primary selection criteria of sex, age, sexuality and country of birth. Interviews with respondents revealed that they are keenly aware of the stigma surrounding HIV/AIDS, which some attribute to a particularly Caribbean combination of fear of contamination, homophobia, and ignorance, reinforced by religious beliefs. In fact, religion serves a double role: underpinning stigma and assisting in coping with HIV. HASD has usually occurred where respondents have lost or do not have control over disclosure. Compared to UK-born respondents, the accounts of Caribbean-born respondents, most of whom were born in Jamaica, include more reports of severe HASD, particularly violence and employment discrimination. All respondents mobilise a variety of strategies in order to avoid HASD, which have implications for their social interactions and emotional well being. While some manage to avoid the "spoiled identity" of the stigmatised, thereby creating their own understandings of HIV infection, these may remain individual-level negotiations. HASD affects HIV-positive Caribbean people at home and in the diaspora in a variety of ways: emotionally, mentally, financially, socially and physically. Interventions specifically addressing stigma and discrimination must be formulated for the UK's Caribbean population. Tackling stigma and discrimination requires more than education; it requires "cultural work" to address deeply entrenched notions of sexuality.