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.     

Effects of a monoclonal antibody raised against nerve growth factor on skeletal pain and bone healing after fracture of the C57BL/6J mouse femur.

A closed femur fracture pain model was developed in the C57BL/6J mouse. One day after fracture, a monoclonal antibody raised against nerve growth factor (anti-NGF) was delivered intraperitoneally and resulted in a reduction in fracture pain-related behaviors of approximately 50%. Anti-NGF therapy did not interfere with bone healing as assessed by mechanical testing and histomorphometric analysis. INTRODUCTION: Current therapies to treat skeletal fracture pain are limited. This is because of the side effect profile of available analgesics and the scarcity of animal models that can be used to understand the mechanisms that drive this pain. Whereas previous studies have shown that mineralized bone, marrow, and periosteum are innervated by sensory and sympathetic fibers, it is not understood how skeletal pain is generated and maintained even in common conditions such as osteoarthritis, low back pain, or fracture. MATERIALS AND METHODS: In this study, we characterized the pain-related behaviors after a closed femur fracture in the C57BL/6J mouse. Additionally, we assessed the effect of a monoclonal antibody that binds to and sequesters nerve growth factor (anti-NGF) on pain-related behaviors and bone healing (mechanical properties and histomorphometric analysis) after fracture. RESULTS: Administration of anti-NGF therapy (10 mg/kg, days 1, 6, and 11 after fracture) resulted in a reduction of fracture pain-related behaviors of approximately 50%. Attenuation of fracture pain was evident as early as 24 h after the initial dosing and remained efficacious throughout the course of fracture pain. Anti-NGF therapy did not modify biomechanical properties of the femur or histomorphometric indices of bone healing. CONCLUSIONS: These findings suggest that therapies that target NGF or its cognate receptor(s) may be effective in attenuating nonmalignant fracture pain without interfering with bone healing.     

Changes to osteoporosis prevalence according to method of risk assessment.

The impact of clinical risk factor-based absolute risk methods on the prevalence of high risk for osteoporotic fracture is unknown. We applied absolute risk methods to 6646 subjects and found that the prevalence of elderly women deemed to be at high risk increased substantially, whereas the overall prevalence was highly dependent on the threshold used to designate high risk. INTRODUCTION: Many groups have advocated using absolute risk methods that incorporate clinical risk factors to target patients for osteoporosis therapy. We examined how the application of such absolute risk classification systems influences the prevalence of those considered to be at high risk for osteoporotic fracture and compared these systems to one based solely on BMD. MATERIALS AND METHODS: Using 6646 subjects from the Canadian Multicentre Osteoporosis Study (CaMos), a prospective, randomly selected, population-based cohort, we assessed three different systems for determining prevalence of high risk for osteoporotic fracture: a BMD-based system; a simplified risk factor system incorporating age, sex, BMD, and two clinical risk factors; and a comprehensive system, incorporating age, sex, BMD, and seven clinical risk factors. The 10-year absolute risks of incident fragility fracture were compared across systems using three different high-risk thresholds. RESULTS: The prevalence of a T score < or = -2.5 was 18.8% (95% CI: 17.7-19.9%) in women and 3.9% (95% CI: 3.0-4.7%) in men. Using a 15% 10-year risk of fracture threshold, the prevalence of women at high risk increased to 46.9% (95% CI: 45.4-48.4) and 42.5% (95% CI: 41.1-43.9) when the comprehensive and simplified risk factor classification systems were used, respectively. Using a 25% 10-year absolute risk threshold, the prevalence of high risk was similar to that of the BMD-based system, whereas the 20% threshold gave intermediate rates. All thresholds analyzed resulted in an increased prevalence of older women at high risk for fracture, whereas only the 15% 10-year risk of fracture threshold resulted in an increase in the prevalence of men at high risk. CONCLUSIONS: The application of risk factor-based systems results in an increased prevalence of older women at high risk. The prevalence of individuals at high risk may increase with changes to the methods used to determine those who are eligible for therapy. These data have important implications for the pattern of care and costs of treating osteoporotic fractures.     

18F-FDG PET can replace conventional work-up in primary M staging of nonkeratinizing nasopharyngeal carcinoma.

Conventional work-up (CWU) with chest radiography, abdominal ultrasonography, and skeletal scintigraphy has limited value in M staging of nonkeratinizing nasopharyngeal carcinoma (NPC). Our aim was to evaluate whether (18)F-FDG PET could replace CWU by comparing their diagnostic efficacies. METHODS: Patients with histologically proven nonkeratinizing NPC and no prior treatment were prospectively enrolled. All study participants underwent CWU and (18)F-FDG PET for primary M staging. Distant metastasis was considered to be present if there was any reliable evidence identified within 1 y after diagnosis. The comparative diagnostic efficacies of (18)F-FDG PET, CWU, and the combination of (18)F-FDG PET and CWU (PET+CWU) were evaluated using the areas under the receiver-operating-characteristic (ROC) curves. RESULTS: Sixty-one (20.3%) of 300 eligible patients were found to have distant metastases. On a patient-based analysis, (18)F-FDG PET was found to be more effective than CWU (P < 0.001), whereas it was equally effective with PET+CWU (P = 0.130). On region-based analyses, (18)F-FDG PET was more effective than skeletal scintigraphy and chest radiography for detecting bone metastases (P < 0.001) and chest metastases (P < 0.001), respectively. (18)F-FDG PET and abdominal ultrasound were equally effective for detecting hepatic metastases (P = 0.127). On region-based analyses, the combination of (18)F-FDG PET and CWU did not yield any noticeable increase in diagnostic efficacy. CONCLUSION: (18)F-FDG PET can replace CWU in primary M staging of nonkeratinizing NPC.     

Corpus callosotomy in refractory idiopathic generalized epilepsy

RATIONALE: A small percentage of patients with idiopathic generalized epilepsy (IGE) do not respond to medical therapy. Generalized tonic-clonic (GTC) seizures are especially debilitating and can be associated with severe injuries. The benefit, safety and effect of corpus callosotomy (CC) in patients with IGE have not been studied. METHODS: We reviewed patients with presumed IGE who underwent CC between 1991 and 2000. Criteria for selection included history, examination, brain imagining, interictal and ictal EEG. All patients had refractory and debilitating tonic-clonic seizures (GTCS) and had failed four or more antiepileptic drugs. Seizure frequency was calculated per month over the last year and pre-operative baseline was compared to last follow-up using paired t-tests. IQ, executive function, language and verbal, non-verbal memory and quality of life (QOL) was compared before and after surgery. Serial EEGs after surgery were reviewed. RESULTS: There were nine patients (seven men), mean age 37.9 (range: 22-49), mean IQ 87.3 (range: 75-107). All had anterior CC. Mean follow-up time was 5.4 years (range: 0.6-10.3 years). One patient died from sudden death in epilepsy 9 months after surgery. There was a significant reduction of GTC seizures from 6.3 to 1.1 (p<0.005). Four patients had more than 80% and eight more than 50% reduction. Of five patients with absence seizures, two became seizure free and one had more than 80% reduction and two worsened slightly, and of three with myoclonic seizures one had more than 90% reduction. One patient had completion of the CC with improvement of myoclonus and absence seizures, but not of GTC seizures and suffered a disconnection syndrome. Another had right frontal focal resection without improvement after new seizures of focal onset. Cognitive testing showed a good outcome (improved or no change) in all cognitive domains. Post-surgical EEG showed new focal slowing and sharp waves. There was no change in QOL. CONCLUSION: CC can be effective in reducing GTC, absence and myoclonic seizures in patients with refractory IGE. These findings suggest that interhemispheric communication of the cerebral cortices plays an important role in the generation of seizures in IGE. Anterior CC appears safe while complete callosotomy has a risk of disconnection syndrome.