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.     

Therapeutic Consequences of Variation in Intraarterial Pressure Measurements After Iliac Angioplasty

Purpose: To assess the accuracy of intraarterial measurement of transstenotic pressure gradients for the detection of hemodynamically suboptimal iliac angioplasty. Methods: In 14 patients, referred for diagnostic angiography, mean pressure gradients in the aorta and iliac artery were obtained twice, using a double-sensor pressure catheter. Additional iliac measurements were performed during pharmacologically induced flow augmentation. Repeatability was assessed by calculation of the mean difference plus standard deviation (MD ± SD) and repeatability coefficient (2 × SD). These results were extrapolated to 137 iliac angioplasty procedures with secondary stenting where there was a residual pressure gradient > 10 mmHg. Results: MD ± SD for repeated measurements at rest and during flow augmentation were 0 ± 2 mmHg and 1 ± 3 mmHg, respectively. Repeatability coefficients were 3 and 6 mmHg. Mean pressure gradients after hemodynamically insufficient angioplasty were 8 ± 7 mmHg at rest and 17 ± 5 mmHg following vasodilatation. Inaccurate pressure recordings may have led to inappropriate stent placement in less than 2.5%, and inappropriate denial of stent placement in less than 5% of the lesions. Conclusion: Variability of intraarterial pressure measurements has little consequence in the detection of hemodynamically significant stenosis after angioplasty. Received: 0/00/00/Accepted: 0/00/00     

Studies on Propafenone-type Modulators of Multidrug-Resistance IV: Synthesis and Pharmacological Activity of 5-Hydroxy and 5-Benzyloxy Derivatives

A series of 5-hydroxy and 5-benzyloxy analogs of the antiarrhythmic and multidrug resistance (MDR) modulating drug propafenone was synthesized and the MDR-modulating activity of the compounds was evaluated using a daunomycin efflux assay system. The key step of the synthesis is the selective reduction of the double bond in 1 without cleavage of the benzyl group thus leading to the phenol 3 . Alkylation with epichlorohydrine followed by nucleophilic epoxide ring opening gave the benzylated target compounds 5a–d . Subsequent cleavage of the benzyl group gave the 5-hydroxy analogs 6a–d . Structure activity relationship studies showed, that the 5-hydroxy derivates 6a–d fit the log P/log potency correlation line previously established for a series of propafenone analogs. In contrast, all four 5-benzyloxy analogs 5a–d showed almost identical EC₅₀ values, independent of their log P value.     

Electro-encephalographic disturbances due to chronic toxin abuse in young people, with special reference to glue-sniffing

A study was carried out in order to document any abnormalities in the electro-encephalogram (EEG) that might appear in young adolescents who have deliberately inhaled the range of volatile substances loosely referred to as 'glue'. The EEGs of a group of 'street children' being assisted in a Johannesburg shelter were examined. The records were analysed for any clinical abnormalities and also subjected to spectral analysis in order to examine the overall characteristics of frequency, power and spatial distribution. The EEGs clearly revealed that, although at the time of the examination the subjects were ostensibly abstinent, both clinical and normative evidence of continuing brain disturbance was present. It was concluded that glue sniffing is likely to have long term electrocerebral sequelae.     

5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel

The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 microM and 5 microM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 microM and 100 microM in the pipette, the half-maximal activation voltage ( V1/2) was shifted by +28.5+/-1.7 mV. The activation time constant (tau(a)) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. tau(a) changed from 1106+/-60 ms at 0 microM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 microM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 microM forskolin induced an elevation of the cAMP level by about 1.6+/-0.2 microM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4a- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4a-receptors by 0.1 microM 5-HT induced an increase of cAMP of about 4.6+/-1.5 microM, whereas cAMP levels decreased from a control value of 1+/-0.2 microM to 0.41+/-0.1 microM after stimulation of the m5-HT1A-receptors.     

Diagnostik des Synovialsarkoms Simultane t(X;18) FISH- und SYT/SSX-RT-PCR-Analyse

Synovialsarkome sind mitunter schwierig von anderen Spindelzellsarkomen zu unterscheiden. In diesen Fällen kann der Nachweis einer t(X;18) Translokation unter Verwendung der FISH und RT-PCR helfen. Die vorliegende Arbeit wurde unter der Fragestellung durchgeführt, ob bei Synovialsarkom-verdächtigen Tumoren der simultane Einsatz beider Methoden zum Translokationsnachweis erforderlich ist oder ob eine der Methoden ausreicht.In die Studie wurde Paraffin-eingebettetes Tumorgewebe von 53 Patienten einbezogen, bei denen nach Lichtmikroskopie und Immunhistochemie der Verdacht auf das Vorliegen eines Synovialsarkoms bestand. Es erfolgte der Nachweis von t(X;18) mittels FISH und RT-PCR.     

Dioxin treatment of rats results in increased in vitro induction of sister chromatid exchanges by alpha-naphthoflavone: an animal model for human exposure to halogenated aromatics

Recent reports have shown that alpha-naphthoflavone (alpha-NF) in vivo enhances the sister chromatid exchange (SCE) frequency in lymphocytes from human populations exposed to cigarette smoke or polychlorinated biphenyls and dibenzofurans. In this study, female Sprague-Dawley rats (9-11 weeks old) were administered a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and killed 6 days after treatment. Blood cultures were established with or without alpha-NF. The baseline and alpha-NF-induced SCE frequencies were assessed in lymphocytes after a 72-hr culture period. No effect on the SCE baseline frequency (cultures without alpha-NF) was detected in rats exposed to 0-30 micrograms TCDD/kg. However, the SCE frequencies from cultures incubated in the presence of alpha-NF were significantly higher in lymphocytes from rats treated with TCDD. Moreover, delta SCE values (SCE alpha-NF minus SCE baseline) were significantly higher in lymphocytes from rats treated with TCDD than in controls. A dose-dependent increase in delta SCE values was observed between 0 and 3 micrograms TCDD/kg, followed by a plateau at higher doses. This induction pattern closely resembled the induction of the liver microsomal aryl hydrocarbon hydroxylase activity by TCDD. In contrast to TCDD, phenobarbital treatment of rats (75 mg/kg/day) had no effect on alpha-NF-induced SCE frequencies in lymphocytes. Liver microsomes from TCDD-treated rats metabolized alpha-NF at a rate much faster than that of control microsomes. These studies indicate that TCDD-exposed rats provide a useful model to investigate the mechanism of enhanced in vitro induction of SCE frequency in lymphocytes from humans exposed to toxic halogenated aromatics or cigarette smoke.     

Bioreductive activation of quinones: A mixed blessing

Quinones can be metabolized by various routes: substitution or reductive addition with nucleophilic compounds (mainly glutathione and protein thiol groups), one-electron reduction (mainly by NADPH: cytochrome P-450 reductase) and two-electron reduction (by D,T-diaphorase). During reduction semiquinone radicals and hydroquinones are formed, which can transfer electrons to molecular oxygen, resulting in the formation of reactive oxygen intermediates and back-formation of the parent quinone (redox cycling). Reaction of semiquinones and reactive oxygen intermediates with DNA and other macromolecules can lead to acute cytotoxicity and/or to mutagenicity and carcinogenicity. The enhanced DNA-alkylating properties of certain hydroquinones are exploited in the bioreductive alkylating quinones. Acute cytotoxicity of quinones appears to be related to glutathione depletion and to interaction with mitochondria and subsequent disturbance of cellular energy homoeostasis and calcium homoeostasis. These effects can to a certain extent be predicted from the electron-withdrawing and electron-donating effects of the substituents on the quinone nucleus of the molecule. Prediction of cytostatic potential remains much more complicated, because reduction of the quinones and the reactivity of the reduction products with DNA are modulated by the prevailing oxygen tension and by the prevalence of reducing enzymes in tumour cells.This article is based on a lecture given at the 16th LOF Symposium, 27 October 1989, Utrecht, the Netherlands.     

Activation-induced changes in evoked and slow brain potentials: effect of cocaine in rabbits previously subchronically treated by cocaine

To study the type of adaptive modification (tolerance vs. sensitization) in different organism's indices following intermittent cocaine (COC) administrations, acute COC (2 mg/kg, SC)-induced changes in heart and breathing rate, response to increasing noxious stimulation, spontaneous EEG and event-related evoked and slow brain potentials (ERP) registered from the occipital cortex and hippocampal CA1 region were investigated in naive rabbits and one subchronically treated with COC (6 injections at a same dose). Tolerance developed for bradycardia, depression of noxious responsiveness, cortical desynchronization and increase of main components of cortical ERP, typical to acute COC, while the changes in breathing and hippocampal ERP were stable. These changes as well as a significant increase due to COC administrations of the basal values of an animal's noxious responsiveness and increase in relative changes in main component of cortical ERP (N205) are considered as a consequence of adaptive changes in neurophysiological/neurochemical substrate accepting COC action and mediating phenomena tolerance-sensitization and dependence typical to the drug.