The use of 19F spectroscopy and diffusion-weighted MRI to evaluate differences in gene-dependent enzyme prodrug therapies.

To evaluate noninvasive measures of gene expression and tumor response in a gene-dependent enzyme prodrug therapy (GDEPT), a bifunctional fusion gene between Saccharomyces cerevisiae cytosine deaminase (CD) and Haemophilus influenzae uracil phosphoribosyltransferase (UPRT) was constructed. CD deaminates 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), and UPRT subsequently converts 5FU to fluorouridine monophosphate, and both of these reactions can be monitored noninvasively in vitro and in vivo using 19F magnetic resonance spectroscopy (MRS). Following transient transfection the CD-UPRT fusion protein exhibited both UPRT and CD enzymatic activities as documented by 19F MRS. In addition, an increase in CD activity and thermal stability was witnessed for the fusion protein compared to native CD. Stable expression of CD-UPRT in 9L glioma cells increased both 5FC and 5FU sensitivity in vitro compared to CD-expressing and wild-type 9L cells. Noninvasive 19F MRS of both CD and UPRT gene function in vivo demonstrated that in animals bearing CD-expressing tumors there was limited conversion of 5FC to 5FU with no measurable accumulation of cytotoxic fluorinated nucleotides (F-nucs). In contrast, CD-UPRT-expressing tumors had increased CD gene activity with a threefold higher intratumoral accumulation of 5FU and significant generation of F-nucs. Finally, CD-UPRT yielded increased efficacy in an orthotopic animal model of high-grade glioma. More importantly, early changes in cellular water mobility, which are felt to reflect cellular death, as measured by diffusion-weighted MRI, were predictive of both durable response and increased animal survival. These results demonstrate the increased efficacy of the CD-UPRT GDEPT compared to CD alone both biochemically and in a preclinical model and validate both 19F MRS and diffusion-weighted MRI as tools to assess gene function and therapeutic efficacy.     

N-acetylcysteine does not modify nitroglycerin-induced tolerance in canine vascular rings

The mechanism whereby nitroglycerin initiates relaxation in vascular smooth muscle remains unclear. One hypothesis states that nitroglycerin oxidizes critical sulfhydryl groups in smooth muscle to initiate relaxation, and that repeated exposure to nitroglycerin results in tolerance. In the current study, N-acetylcysteine, a sulfhydryl-reducing agent, was used to explore the sulfhydryl hypothesis by assessing whether or not tolerance to nitroglycerin was reversed by N-acetylcysteine in canine dorsal pedal artery rings. Two nitroglycerin dose-response curves were performed (n = 18)--one before (1st dose-response curve, from 10(-9) to 1.1 X 10(-5) M nitroglycerin) and one after (2nd dose-response curve, from 10(-9) to 5 X 10(-7) M nitroglycerin) incubation with 10(-5) M nitroglycerin for 105 min. At 5 X 10(-7) M nitroglycerin there was 50.7 +/- 10.0% relaxation during the first dose-response curve. During the second dose-response curve, tolerance to nitroglycerin was evident, as demonstrated by a 6.8 +/- 4.8% relaxation (p less than 0.001) at 5 X 10(-7) M nitroglycerin. A 10-min treatment with 10(-3) M N-acetylcysteine (n = 10) during the second nitroglycerin dose-response curve was performed after the 5 X 10(-7) M concentration of nitroglycerin; the second dose-response curve was then completed up to 1.1 X 10(-5) M nitroglycerin. The dose of 10(-3) M N-acetylcysteine was chosen since higher concentrations (i.e., 1.3 X 10(-2) and 1.2 X 10(-1) M N-acetylcysteine) produced 20.3 +/- 8.4 and 43.6 +/- 11.6% relaxation in vascular rings (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of percutaneous ureteral stent to indwelling pigtail stent over guidewire

A ureteral stent placed percutaneously through a nephrostomy can be readily exchanged endoscopically for a ureteral indwelling pigtail stent over a percutaneous guide wire. Percutaneous antegrade stent placement can sometimes be accomplished when retrograde placement cannot. We report 10 successful conversions to indwelling stent in 11 cases. In 1 case the percutaneous guide wire could not be retrieved endoscopically because of a bleeding tumor in the bladder. No serious difficulties or complications were encountered. The percutaneous approach offers an alternative method of providing internal urinary diversion if retrograde ureteral indwelling stent placement has failed.     

Reconstructive options after temporal bone resection for squamous cell carcinoma.

Squamous cell carcinoma (SCC) of the temporal bone is a rare, aggressive and highly malignant tumour that requires specialised, multidisciplinary surgery for its treatment. Reconstruction of the defect is as crucial as the tumour ablation in terms of mortality and postoperative morbidity. METHODS: The experience of the East Anglian Skull Base Surgery Service from 1982 to 2004 in managing 42 consecutive patients (22 females; age range 37-80 years) undergoing extended and lateral temporal bone resection for SCC is presented. RESULTS: The overall 5-year survival was 31.0% (median follow up: 97 months) when both curative and palliative procedures were included. On multivariate analysis, the favourable prognostic factors were male sex, well-differentiated tumours and stage N(0) neck. The reconstructions in 38 patients were analysed. The majority (24/38) had free tissue transfer reconstructions and most of these were radial forearm free flaps. Later in the series, other free flap types such as anterolateral thigh flaps and lateral arm flaps were employed. Pedicled myocutaneous flaps and local flaps were also used. CONCLUSIONS: This paper analyses the outcomes of the various reconstructions and discusses the evolution of their care in our unit. The information obtained from the review of our data is extrapolated to offer guidance on the choice of reconstructive option in these patients.     

Experimental intrarenal reflux and blood pressure.

The effect on the blood pressure of experimental vesico-ureteric reflux was investigated in adult female Wistar rats. In 6 rats, reflux with isotonic saline produced a transient rise in systemic blood pressure followed by a fall, with return to normal within 2 min (mean BP readings: 121-130-93 mmHg). In 6 rats during water diuresis, reflux with distilled water produced similar changes (114-120-79 mmHg). In 6 rats in which the ureters were divided before reflux, no rise in blood pressure occurred although in 2 of these the pressure showed a marked fall. The pattern of blood pressure changes which occurred as a result of reflux was similar to that produced by a rapid i.v. injection of a corresponding volume of saline and it was concluded that the changes accompanying reflux are due to pyelovenous backflow. This was confirmed by producing reflux with 5% lissamine green which appeared in the peripheral capillaries within 4 s of the reflux in 3 rats. Slow dilatation of the pelvis with saline in 9 rats showed that rupture of the pelvic epithelium occurred at a mean pressure of 99 mmHg.     

Age-related changes in the kinetics of water transport in normal human lenses

The aim of this work was to study blink frequency changes and levels of ocular discomfort during work at a video display terminal, and the effects on these parameters of augmented or reduced humidification of the ocular surface.Blink rate was measured from recordings of the electrical signal evoked by the contraction of the orbicularis oculi muscle. Blink rate and interblink intervals were analyzed at rest and during performance of a task with a computer (playing a card game) for 10 or 30 min in steady environmental conditions and during application of a continuous stream of air to the face. In two separate sessions, the effect of pretreatment with humidifying ocular solutions of different elastoviscosity (balanced salt solution or elastoviscous 0.1% Hylan A solution) was assayed. At the end of each experimental period, the subjects marked the level of ocular discomfort experienced on a 0-10 cm visual analogue scale.The blink frequency at rest (12.4+/-1.2 blinks min-1) was reduced significantly (to 10.3+/-1.1 blinks min-1) by pretreatment with elastoviscous eyedrops both with and without air applied to the face. This effect was not obtained with balanced salt solution. During performance of the visual task for 10 or 30 min, basal blink rate decreased significantly, to about 40% of the control value. Neither application of an air jet on the face nor application of eye solutions of different viscosity modified this reduced blink rate.A low degree of ocular discomfort developed after performance of the visual task that was enhanced by air application to the face. This discomfort was reduced by pretreatment with ocular solutions, the elastoviscous eye solution being more efficient than the balanced salt solution. Interblink interval duration was also more regular after treatment with the elastoviscous solution.These data suggest that blink rate at rest is maintained in part by activation of sensory receptors of the cornea and conjunctiva, which are stimulated by desiccation of the ocular surface. Reduction of eye blink frequency elicited by the performance of a visual task with a computer appears to depend on central neural mechanisms that are quite independent of peripheral sensory inputs. The reduction of blink frequency consecutive to computer use was associated with a sensation of discomfort that was attenuated more effectively by elastoviscous eyedrops than by regular balanced salt solution.