Hypoxia targeted gene therapy to increase the efficacy of tirapazamine as an adjuvant to radiotherapy: reversing tumor radioresistance and effecting cure

Solid tumors are characterized by regions of hypoxia that are inherently resistant to both radiotherapy and some chemotherapy. To target this resistant population, bioreductive drugs that are preferentially toxic to tumor cells in a hypoxic environment are being evaluated in clinical trials; the lead compound, tirapazamine (TPZ), is being used in combination with cisplatin and/or with radiotherapy. Crucially, tumor response to TPZ is also dependent on the cellular complement of reductases. In particular, NADPH:cytochrome P450 reductase (P450R) plays a major role in the metabolic activation of TPZ. In a gene-directed enzyme prodrug therapy (GDEPT) approach using adenoviral delivery, we have overexpressed human P450R specifically within hypoxic cells in tumors, with the aim of harnessing hypoxia as a trigger for both enzyme expression and drug metabolism. The adenovirus used incorporates the hypoxia-responsive element (HRE) from the lactate dehydrogenase gene in a minimal SV40 promoter context upstream of the cDNA for P450R. In a human tumor model in which TPZ alone does not potentiate radiotherapeutic outcome (HT1080 fibrosarcoma), we witnessed complete tumor regression when tumors were virally transduced before treatment.     

Tyrosine-free amino acid mixture attenuates amphetamine-induced displacement of [11C]raclopride in striatum in vivo: a rat PET study

Previous neurochemical and behavioural studies show that tyrosine depletion using a nutritionally balanced tyrosine-free amino acid mixture attenuates the dopamine-releasing and psychostimulant properties of amphetamine. Here we investigate the effect of a tyrosine-free amino acid mixture on striatal binding of [(11)C]raclopride, and amphetamine-induced [(11)C]raclopride displacement, using positron emission tomography in the rat. Rats were scanned for 60 min after an i.v. injection of approximately 11 MBq [(11)C]raclopride using a quad-HIDAC system. Amphetamine (2 mg/kg i.p., 30 min prior to scan) caused a 12% reduction in [(11)C]raclopride distribution volume ratio (DVR) compared to saline-injected controls. The tyrosine-free amino acid mixture (1 g/kg i.p.) caused a small (+7%) but statistically insignificant increase in [(11)C]raclopride DVR and attenuated, although it did not fully block, the amphetamine-induced reduction. These data are in keeping with previous neurochemical, immunocytochemical, and behavioural studies showing that tyrosine-free amino acid mixtures reduce dopamine function and offer promise for future PET studies testing the effect of tyrosine-depleting paradigms on dopamine release in humans.     

Fos immunocytochemical studies on the neuroanatomical sites of action of acute tyrosine depletion in the rat brain

Rationale Acute depletion of brain tyrosine using a tyrosine-free amino acid mixture offers a nutritional approach to reduce central catecholamine function. Recent preclinical data suggest that tyrosine-free amino acid mixtures may have region-specific effects through targeting dopamine neurones.Objectives Here we used fos immunocytochemistry to examine the neuroanatomical sites of action of a tyrosine-free amino acid mixture administered either alone or combined with amphetamine.Methods Rats (male, Sprague Dawley, 240–260 g) were administered (IP) either a tyrosine-free amino acid mixture (1 g/kg), or the same mixture supplemented with tyrosine and phenylalanine (1 g/kg). Mixtures were injected twice (1 h apart) followed 1 h later by amphetamine (2 mg/kg SC). Two hours later, cardiac perfusion was performed and brains were processed for fos immunocytochemistry. Fos positive cells were counted using computer imaging software.Results The tyrosine-free amino acid mixture alone did not alter fos expression in ten regions of the rat forebrain compared to saline controls. However, the mixture reduced the increase in fos expression evoked by amphetamine. This effect was region-specific and was greatest in caudate putamen, nucleus accumbens, bed nucleus stria terminalis and lateral habenula, and lacking in other areas including cingulate and insular cortices, lateral septum and central amygdaloid nucleus. Moreover, in most regions the effect of the tyrosine-free mixture was less after tyrosine and phenylalanine supplementation.Conclusions In summary, a tyrosine-free amino acid mixture reduced amphetamine-induced fos expression but this effect was region-specific and included dopamine-rich regions. These data further support the idea that tyrosine depletion strategies have potential as treatments for mania and other hyperdopaminergic states.     

The pattern of astigmatism in a Canadian preschool population

PURPOSE. To measure magnitude, type, and central tendency of astigmatism found in a county-wide population of Canadian preschool children (mean age, 48.1 months). METHODS. Noncycloplegic autorefractive measures were taken in 1179 children attending a preschool health fair operated by their county board of health. Spherocylinder measures were transformed into three independent components. RESULTS. The equivalent sphere showed considerable variation between retinoscopy and autorefraction that was attributed to the variable overaccommodation induced by the autorefractor. Astigmatic components were not affected. Small discrepancies between the two techniques were similar to those in adults and were not of sufficient magnitude to affect validity. With-the-rule (WTR) astigmatism of at least 0.25 D was the most frequent form (45%) followed by against-the-rule (ATR; 40%) and oblique (15%). The 95th percentile for cylinder magnitude was found at 1.25 D. Astigmatisms beyond this value were predominately WTR. The mean (negative) cylinder magnitude was 0.08 Dx 015 degrees. CONCLUSIONS. When spherocylinder values are transformed into a mathematical continuum rather than WTR and ATR classifications, the true central tendency of the population is better defined and is close to zero. Astigmatisms of more than 1.25 D in the preschool child exceed the 95th percentile in this population and were more frequently WTR.     

Impairments in working memory associated with naturalistic dieting in women: no relationship between task performance and urinary 5-HIAA levels

The present study investigated the question of whether the previously observed impairments of working memory characteristic of dieting to lose weight can be explained in terms of preoccupying cognitions relating to body shape or to alterations in serotonergic function resulting from a low dietary intake of tryptophan. The population comprised female non-dieting, lower restrained eaters (N=23), non-dieting higher restrained eaters (N=11) and current dieters (N=19). Each participant completed three tasks, each of which selectively loaded on to a different sub-component of working memory. The tasks comprised the Tower of London task, a letter string recall task and a mental rotation task. In addition, all participants completed self-report measures of body shape concern and affective state. Serotonin turnover was assessed by means of 24 h urine sample collection for each participant on their day of testing. This was analysed (via HPLC) for levels of the main serotonin metabolite 5-HIAA.The results of the present study broadly replicated previous findings of a Central Executive and Phonological Loop (but not Visuo-Spatial Sketchpad) deficit in those subjects who reported themselves to be currently dieting. Tower of London task performance also significantly correlated with self-reported feelings of fatness and body shape disparagement. There were no group differences in 5-HIAA levels nor did 5-HIAA levels correlate with task performance. However, there was a significant negative correlation between 5-HIAA levels and self-reported depression. These results support the hypothesis that the variables mediating this deficit are preoccupying cognitions concerning body shape. They do not support the hypothesis that the serotonergic function of dieters is compromised, although this conclusion is tentative.     

Acute reboxetine administration increases plasma and salivary cortisol

We investigated the effect of a single oral dose of the selective noradrenaline reuptake inhibitor, reboxetine (4 mg), on plasma and salivary cortisol in 24 healthy volunteers in a randomized, placebo-controlled, parallel-group design. Reboxetine significantly increased both plasma and salivary cortisol, although the correlation between the responses in plasma and saliva was modest. Our results are consistent with previous neuroendocrine challenge studies showing that potentiation of brain noradrenaline function stimulates the hypothalamic-pituitary-adrenal axis. Reboxetine-induced salivary cortisol release appears to be a simple and relatively non-invasive test of hypothalamic noradrenaline function. However, placebo-controlled, within-subject designs are likely to yield a more valid measure of noradrenaline-mediated cortisol release.     

Facial reanimation surgery utilizing the Mitek anchor system: A case report

A flaccid hemi-face is frequently the most noticeable and cosmetically unacceptable consequence of facial nerve palsy, whether due to trauma, Bell's palsy or other etiologies. A variety of face-lift and reanimation techniques have been utilized in the past, but with time, these frequently require further surgery. We describe the use of Mitek (Norwood, MA) suture anchors for cheek resuspension in a patient with facial palsy. This system is composed of a drill guide, drill, inserter, and anchor. Although the titanium alloy anchors come in multiple sizes, the Mini GII Anchor is typically most appropriate for use in facial procedures. The actual size of the Mini GII Anchor is 1.8 mm in diameter and 5.4 mm in length. Two small arched prongs extend from the body of the anchor, and an eyelet at the superior surface is used for suture placement. When placed into a pre-drilled hole with the insertion tool, the prongs extend, effectively fixing the anchor in place. The drill guide protects adjacent soft tissues during the drilling process and allows drilling to a predetermined fixed depth. Sutures attached to the anchor may then be used for soft tissue fixation to bone.     

Subcellular post-transcriptional targeting: delivery of an intracellular protein to the extracellular leaflet of the plasma membrane using a glycosyl-phosphatidylinositol (GPI) membrane anchor in neurons and polarised epithelial cells

The effectiveness of viral vector-mediated gene transfer depends on the expression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribution of expressed transgenes. Targeting individual transgenes to particular subcellular compartments will provide various advantages in increasing the safety, efficacy, and specificity of viral vector-mediated gene delivery. Viruses normally hijack the cellular protein synthesis machinery for their own advantages. It is thus unknown whether cells infected with viral vectors will be able to target proteins to the correct subcellular organelles, or whether the subcellular targeting machinery would be selectively disrupted by viral infection. In this article we explored whether a herpes simplex virus type 1-derived vector could be used to deliver a transgene engineered to be targeted to the extracellular membrane of target cells. To do so we constructed a temperature-sensitive mutant HSV-1 vector, tsK-TT21 expressing a recombinant marker protein, tissue inhibitor of metalloproteinases (TIMP), linked to sequence encoding a signal for the addition of a glycosyl-phosphatidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results demonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, without disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell membrane to modify cell-cell interactions, the function of specific plasma membrane proteins, or their interactions with other membrane proteins, and also to target a prodrug converting enzyme to the plasma membrane of target cells, therefore enhancing its cell killing effects.