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.     

P300 and contingent negative variation in migraine

We reviewed P300 and contingent negative variation (CNV) studies performed in migraine in order to identify their relevance in migraine and the role of neurophysiology in migraine. Publications available to us were completed by a Medline search. There is experimental and clinical evidence for loss of cognitive habituation in migraine which may serve as a specific diagnostic tool; therefore, we reviewed studies on migraine that analyzed habituation and lack of habituation by P300 and CNV, performing short-term habituation (STH) and long-term habituation (LTH). Finally, we described the two components of P300 (a and b) and of CNV (early and late wave) and the two abnormalities reported from the majority of studies on event-related potential in migraine: increased amplitude of average event-related potential and lack of habituation. These abnormalities are especially related to the early component characterizing orienting activity.     

A rapid and sensitive method for measuring monooxygenase activities in hepatocytes cultured in 96-well plates

Summary Measurement of biotransformation activities in cells is of great importance for drug metabolism and toxicologic studies. It is currently done by measuring the enzymatic activities in partially purified microsomes. In the present work we report on a rapid, easy, sensitive, and reproducible fluorimetric assay for quantifying cytochrome P450-dependent monooxygenase activities (P450IA1, P450IIB1) in hepatocytes cultured in 96-well plates. The procedure involves the direct determination of enzymatic activities in intact hepatocytes while avoiding cell homogenization, thereby permitting use of a the reduced number of cells and allowing cultured cells to be used in later experiments. Substrates (7-ethoxyresorufin, 7-pentoxyresorufin) are added to culture medium and metabolized by hepatocytes. After enzymatic deconjugation, the fluorescent resorufin present in culture medium is quantified by means of a microplate fluorimetric reader. Major advantages of this technique, as compared to other available methods, are: a) no cell disruption is required; b) activity can be measured with a very small number of cells; c) rapid processing time; and d) possibility of performing repeated assays with the same cell monolayer.     

Management of intraabdominal testicle during laparoscopic hernia repair

A 22-year-old man who had previously undergone work-up for right cryptorchidism and been diagnosed as having right testicular absence was referred for repair of a right inguinal hernia. A laparoscopic approach was chosen for this patient, and at the time of laparoscopic herniorrhaphy, a small intraabdominal testicle was visualized and removed laparoscopically.Laparoscopy has served as an excellent procedure for locating intraabdominal testes and planning the most appropriate treatment for many cryptorchid patients. Until recently, atrophic testes located within the abdominal cavity through use of a laparoscope were removed via an open technique. Advances in laparoscopy now enable detection and definitive treatment for these patients without the need for large, more painful incisions.The laparoscopic approach to this patient enabled diagnosis and management of his cryptorchidism and provided a means for repair of his hernia.     

Palliation of cardiac malformations associated with right isomerism (asplenia syndrome) in infancy

Right isomerism is characterized by the combination of an obstructed pulmonary outflow tract and a total anomalous pulmonary venous connection (TAPVC), which is obstructed in nearly half of the patients. Fourteen patients less than 2 years of age with right isomerism have been seen in our unit. Thirteen of them underwent palliation consisting of a systemic-pulmonary shunt (10 patients) or a combination of shunt and TAPVC repair (3). There were 7 hospital deaths (54%). The presence of obstructed pulmonary venous drainage was the major risk factor in the surgical treatment of these complex cardiac anomalies (p less than .02). Guidelines for the palliative management of right isomerism are suggested.