Strategies to enhance transductional efficiency of adenoviral-based gene transfer to primary human fibroblasts and keratinocytes as a platform in dermal wounds

Genetically modified keratinocytes and fibroblasts are suitable for delivery of therapeutic genes capable of modifying the wound healing process. However, efficient gene delivery is a prerequisite for successful gene therapy of wounds. Whereas adenoviral vectors (Ads) exhibit superior levels of in vivo gene transfer, their transductional efficiency to cells resident within wounds may nonetheless be suboptimal, due to deficiency of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR). We explored CAR-independent transduction to fibroblasts and keratinocytes using a panel of CAR-independent fiber-modified Ads to determine enhancement of infectivity. These fiber-modified adenoviral vectors included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7). To evaluate whether transduction efficiencies of the fiber-modified adenoviral vectors correlated with the expression of their putative receptors on keratinocytes and fibroblasts, we analyzed the mRNA levels of CAR, alpha upsilon integrin, syndecan-1, and glypican-1 using quantitative polymerase chain reaction. Analysis of luciferase and green fluorescent protein transgene expression showed superior transduction efficiency of Ad5.pK7 in keratinocytes and Ad5.RGD.pK7 in fibroblasts. mRNA expression of alpha upsilon integrin, syndecan-1 and glypican-1 was significantly higher in primary fibroblasts than CAR. In keratinocytes, syndecan-1 expression was significantly higher than all the other receptors tested. Significant infectivity enhancement was achieved in keratinocytes and fibroblasts using fiber-modified adenoviral vectors. These strategies to enhance infectivity may help to achieve higher clinical efficacy of wound gene therapy.     

Preparation and bioevaluation of 99mTc-carbonyl complex of 5-hydroxy tryptamine derivative.

Studies on the development of imaging agents for targeting neuroreceptors is an area of considerable interest owing to the limited availability of specific as well as selective radiolabeled agents. Therefore, with an aim of developing a receptor-specific agent, iminodiacetic acid (IDA) derivative of 5-hydroxy tryptamine viz., HTIDA has been synthesized. HTIDA could be radiolabeled with the synthon [(99m)Tc(CO)(3)(H(2)O)(3)](+) in >98% yield. The biodistribution studies in normal Swiss mice showed that the (99m)Tc(CO)(3)-HTIDA crosses the blood-brain barrier successfully with a brain uptake of 0.5%ID/g at 5min post injection. The other relevant observations from biodistribution studies included no significant uptake in any other organ and fast clearance from blood, lungs and liver.     

Corrosive oesophagitis: an analysis of 50 cases

A total of 50 consecutive patients who were treated in JIPMER Hospital between 1970 and 1981 for corrosive injuries of the oesophagus and stomach were analysed. There were 23 males and 27 females. All but seven presented with dysphagia due to an established stricture. In addition seven of them had associated stricture of the stomach. They were treated with either repeated dilatations or, in selected cases, oesophageal replacement. Perforation of the oesophagus is an important complication associated with oesophageal dilatation indicating the need for oesophageal replacement in multiple or long dense strictures. Results are quite satisfactory with both modalities of treatment. However, oesophageal replacement surgery, performed properly in selected cases, offers a permanent solution to these unfortunate victims.     

Epidemic measles in an isolated unvaccinated population, India

Between June and September, 1986, an outbreak of measles occurred in Pilkhi Primary Health Centre area (population 56,000) in Tehri Garhwal district, Uttar Pradesh, India. Overall, 1092 cases were identified and 62 died; case-fatality ratio was 5.7%. Illness was restricted primarily to children below 15 years of age; 38% cases were in children under 5 and 58% between 5-14 years of age. To better characterize the outbreak, a survey was conducted in 13 affected villages. The age of the cases ranged from 5 months to 19 years (median = 7.0 years). The age-specific attack rates were 22.4%, 54.5%, 46.2% and 35.3% for children under 1, 1-4, 5-9, 10-14 years of age respectively. In as many as four villages, the attack rate in children below ten was 80% or more. Secondary attack rate among family members was 70%. Overall, 82% of children with measles developed complications which consisted mainly of pneumonia, diarrhoea and dysentery. The age-specific case-fatality ratios in infants and children 1-4 years of age were 23.1% and 11.5% respectively; thereafter the rates tended to decline with increasing age and was higher in females than in males (less than 0.05). Pneumonia which was a complication in 39% of measles cases contributed to 56% of deaths. Traditional beliefs and customs in the area were strong and did not encourage treatment of measles cases. Although a measles vaccination programme has been launched in India since 1985, only 30 districts could be covered during the first year and another 90 during 1986.(ABSTRACT TRUNCATED AT 250 WORDS)     

Return of fertility following discontinuation of an injectable contraceptive — Norethisterone oenanthate (NET EN) 200mg dose

The return of fertility following discontinuation of norethisterone oenanthate (NET EN) 200 mg injectable contraceptive after use for a minimum period of six months or more was studied in 69 women who discontinued the method for planning pregnancy. Former users of copper intra-uterine device (CuT 200) were enrolled as a control group. Another 161 women who had discontinued NET EN due to other reasons (e.g. amenorrhoea, excessive bleeding or personal reasons) were also studied for return of fertility after ensuring that they were not using any other method of contraception and were exposed to the risk of pregnancy. The subjects from both groups were followed for a period of one year. The cumulative conception rates at one year were 72.5 and 83.6 per 100 subjects for ex-NET EN and ex-CuT 200 users who had discontinued the method for planning pregnancy and this difference was not statistically significant (P > 0.05). The median time for conception for ex-NET EN users was 7.8 months as compared to 3.7 months in ex-CuT 200 users but the cumulative conception rates at the end of one year show that future return of fertility in NET EN users does not appear to be adversely affected.

In 51 subjects who had discontinued NET EN due to amenorrhoea, the return of fertility was predictably slower and less. The return of fertility in subjects who discontinued NET EN for other reasons (e.g. excessive bleeding and other personal reasons) was similar to ex-NET EN and ex-CuT 200 users.     

Inhibition of the replication of hepatitis B virus by the carbocyclic analogue of 2''-deoxyguanosine.

We report that treatment of 2.2.15, a human hepatoblastoma-derived cell line in which hepatitis B virus is actively replicating, with the carbocyclic analogue of 2'-deoxyguanosine [Shealy, Y. F., O'Dell, C. A., Shannon, W. M. & Arnett, G. (1984) J. Med. Chem. 27, 1416-1421] resulted in the nearly complete cessation of viral replication, as monitored by the absence of both intracellular episomal and secreted viral DNAs and by the absence of viral DNA polymerase activity. The drug was nontoxic in concentrations up to 200 times the minimum effective inhibitory concentration.     

Effects of periodic weight support in a simulated weightless environment in preventing bone demineralisation.

Anti Orthostatic Hypokinetic posture in rats by tail suspension for 15 days (d) simulates the deconditioning effects of weightlessness on the weight bearing bones. The present study evaluates the effects of daily 4 hour (h) weight support (WS) during simulated weightlessness (S-W) in preventing these changes. Adult male albino rats were divided into three groups as (i) Control (CON, n = 12), (ii) Hind limb unweighing by tail suspension for 15 d (HU, n = 18), (iii) HU with daily 4 h WS (4 HRWS, n = 11). After 15 d tibia from all the animals were removed and subsequently dried, ashed and then calcium content of the bones were determined. HU showed reductions in the water content by 35.8%, organic matrix by 12.2% and calcium content by 33.4% of tibia. 4 h WS during S-W resulted in complete prevention of water loss and organic matrix loss and partial prevention of the loss of calcium content. Calcium content of tibia in 4 HRWS remained 15.2% less as compared to CON. These findings indicate that 4 h WS is partially successful in preventing the demineralisation effects of S-W on weight bearing bone tibia.     

Poisoning with oxybutynin.

A case of poisoning with 100 mg of oxybutynin in a 34-year-old female is reported. The main features were anticholinergic effects, including stupor, followed by disorientation and agitation on awakening, dilated pupils, dry skin and retention of urine. She had a sinus tachycardia which resolved 3 h after admission, and in addition ventricular ectopics and bigeminy which continued for a further 30 h. She recovered fully on symptomatic treatment alone.