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.     

Experimental arthritis induced by a clinical Mycoplasma fermentans isolate

Background  

Mycoplasma fermentans has been associated with rheumatoid arthritis. Recently, it was detected in the joints and blood of patients with rheumatoid arthritis, but it is not clear yet how the bacteria enter the body and reach the joints. The purpose of this study was to determine the ability of M. fermentans to induce experimental arthritis in rabbits following inoculation of the bacteria in the trachea and knee joints.     

Predicting Stricture in Morbidly Obese Patients Undergoing Laparoscopic Roux-en-Y Gastric Bypass: A Logistic Regression Analysis

Gastrojejunostomy stricture after Roux-en-Y gastric bypass occurs in 3 to 27% of morbidly obese patients in the USA. We questioned whether preoperative patient characteristics, including demographic attributes and comorbid disease, might be significant factors in the etiology of stricture. In this study from November 2001 to February 2006 (51 months), at a high-volume bariatric center, of the 1,351 patients who underwent laparoscopic gastric bypass, 92 developed stricture (6.8%). All but two were treated successfully by endoscopic dilation. All patients stopped nonsteroidal anti-inflammatory medications 2 weeks prior to surgery and did not restart them. The operative procedure included the use of a 21-mm transoral circular stapler to create the gastrojejunostomy; the Roux limb was brought retrogastric, retrocolic. In an effort to reduce our center’s stricture rate, late in the study, U-clips used at the gastrojejunostomy were replaced by absorbable sutures, and postoperative H₂ antagonists were added to the treatment protocol. The change to absorbable polyglactin suture proved to be significant, resulting in a lower stricture rate. The addition of H₂ antagonists showed no significant effect. Following the retrospective review of the prospective database, univariate and multivariate logistic regression analyses identified factors associated with the development of stricture. Gastroesophageal reflux disease and age were each shown to be statistically significant independent predictors of stricture following laparoscopic gastric bypass. Presented at the 2006 Annual Meeting of the Society for Surgery of the Alimentary Tract, May 20–24, Los Angeles, CA (poster presentation).     

Microbiology of adult cellulitis

Needle aspiration of cellulitis sites is commonly advocated to assist in the identification of causative organisms. Twenty-five nondiabetic, adult patients with a clinical diagnosis of cellulitis had site aspirations and blood cultures obtained before antibiotic therapy was initiated. Site cultures were positive in 6 of 25 patients. Blood cultures were positive in 4 of 25 patients. All organisms except one (Enterobacter agglomerans) were staphylococci or streptococci. The gram-negative bacilli were not believed to be a pathogen based on the patient's prompt response to nafcillin. In adult patients who do not have complications, the use of needle aspiration was not supported. Empiric treatment of cellulitis aimed at gram-positive cocci appears to be sufficient.     

Distribution and function of cholinergic receptors in the sheep detrusor muscle

The distribution of cholinergic nerve fibres, as well as the characterization of the muscarinic receptors responsible for the contraction, were determined in the detrusor smooth muscle of the sheep. The results obtained demonstrated a rich presence of acetylcholinesterase (AChE)-positive fibres distributed throughout the bladder body forming dense neuromuscular, subepithelial and perivascular plexuses. Furthermore, intramural ganglia containing AChE-positive cell bodies were identified. However, acetylcholine and carbachol induced a dose-dependent contraction of detrusor smooth muscle. The effect observed with carbachol was competitively antagonized by atropine (pA2: 8.94), pirenzepine (pA2: 7.38), AF-DX 116 (pA2: 7.35), 4-DAMP (pA2: 9.26) and hexahydroxiladifenidol (HHSiD) (pA2: 8.49). The pA2 value for pirenzepine is intermediate between M1- and M2-receptors which suggests that this antagonist does not act on M1- or M2-receptors, but that it does on M3-receptors. The pA2 value for AF-DX 116 is consistent with the presence of M2-receptors in this tissue. Moreover, the pA2 values obtained for both 4-DAMP and HHSiD are in agreement with the presence of M3-receptors, due to the lack of effect of pirenzepine on M1-muscarinic receptors. These results indicate the existence of a rich parasympathetic innervation in the sheep detrusor muscle and suggest that its contraction could be mediated by the stimulation of muscarinic receptors belonging to both M3- and M2-subtypes.     

Adenocarcinoma at an ileostomy site after colectomy for ulcerative colitis

In this article we present the tenth case, according to the literature, of adenocarcinoma occurring at a long standing ileostomy originally performed for ulcerative colitis. Study of this and previous cases demonstrates common factors such as a previous history of ulcerative colitis, a long interval until the appearance of symptoms, together with a similar clinical and pathological characterisation with invasion of neighbouring skin layers close to the ileostomy. Several pathogenic hypotheses are considered. We believe that local excision in oder to obtain the diagnosis is a better option than a sampling biopsy and that surgery should include a wide resection of the abdominal wall and intestine in order to comply with therapeutic criteria in malignant disease.     

The cardioprotective action of nifedipine during short-term transient ischemia

To determine the therapeutic activity on regional myocardial function of nifedipine intravenous administration (0.16 mg/kg) during short term myocardial induced ischemia, we used 6 mongrel dogs anesthetized and intubated those who we have implanted 3 pairs of ultrasonic crystals in the subendocardial layer. Utilizing an open chest canine model we performed total 1 minute and partial 2 minutes 30 seconds alternative obstructions, controlled with electromagnetic flowmeters in the anterior descending (AD) and circumflex (Cx) coronary arteries before and after drug administration. During the 70-80% obstructions we have shown a significant improvement in segment shortening, reduced by ischemia, in the ischemic and peri-ischemic zone. The ischemic markers, per cent increment of end diastolic length (% IEDL) and per cent increment of end systolic length (% IESL), have shown significant improvement during partial obstructions in the ischemic and peri-ischemic zone. After total obstructions the improvement on regional myocardial function is less marked and limited to ischemic zone.