Cerebral glucose metabolism and cognition in newly diagnosed Parkinson's disease: ICICLE-PD study.

OBJECTIVE: To assess reductions of cerebral glucose metabolism in Parkinson's disease (PD) with 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET), and their associations with cognitive decline. METHODS: FDG-PET was performed on a cohort of 79 patients with newly diagnosed PD (mean disease duration 8 months) and 20 unrelated controls. PD participants were scanned while on their usual dopaminergic medication. Cognitive testing was performed at baseline, and after 18 months using the Cognitive Drug Research (CDR) and Cambridge Neuropsychological Test Automated Battery (CANTAB) computerised batteries, the Mini-Mental State Examination (MMSE), and the Montreal Cognitive Assessment (MoCA). We used statistical parametric mapping (SPM V.12) software to compare groups and investigate voxelwise correlations between FDG metabolism and cognitive score at baseline. Linear regression was used to evaluate how levels of cortical FDG metabolism were predictive of subsequent cognitive decline rated with the MMSE and MoCA. RESULTS: PD participants showed reduced glucose metabolism in the occipital and inferior parietal lobes relative to controls. Low performance on memory-based tasks was associated with reduced FDG metabolism in posterior parietal and temporal regions, while attentional performance was associated with more frontal deficits. Baseline parietal to cerebellum FDG metabolism ratios predicted MMSE (ß=0.38, p=0.001) and MoCA (ß=0.3, p=0.002) at 18 months controlling for baseline score. CONCLUSIONS: Reductions in cortical FDG metabolism were present in newly diagnosed PD, and correlated with performance on neuropsychological tests. A reduced baseline parietal metabolism is associated with risk of cognitive decline and may represent a potential biomarker for this state and the development of PD dementia.

The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition.

BACKGROUND: Combined targeting of MAPK and PI3K signalling pathways may be necessary for optimal therapeutic activity in cancer. This study evaluated the MEK inhibitors AZD6244 and PD0325901, alone and in combination with the dual mTOR/PI3K inhibitor NVP-BEZ235 or the PI3K inhibitor GDC-0941, in three colorectal cancer cell lines. METHODS: Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively, in HCT116, HT29 and DLD1 cell lines. RESULTS: All MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition; however, GDC-0941 displayed greater synergy in combination with either MEK inhibitor. NVP-BEZ235 exhibited stronger inhibition of 4EBP1 phosphorylation, and similar inhibition of S6 and AKT phosphorylation, compared with GDC-0941. Both PD0325901 and AZD6244 inhibited ERK phosphorylation, and with MEK/PI3K inhibitor combinations inhibition of S6 phosphorylation was increased. The reduced synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared with GDC-0941, may be due to inhibition of mTOR, and the addition of the mTORC1/2 inhibitor KU0063794 compromised the synergy of GDC-0941:PD0325901 combinations. CONCLUSION: These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the combination of specific PI3K inhibitors, rather than dual mTOR/PI3K inhibitors, with MEK inhibitors results in greater synergy.

Carbogen breathing increases prostate cancer oxygenation: a translational MRI study in murine xenografts and humans.

Hypoxia has been associated with poor local tumour control and relapse in many cancer sites, including carcinoma of the prostate. This translational study tests whether breathing carbogen gas improves the oxygenation of human prostate carcinoma xenografts in mice and in human patients with prostate cancer. A total of 23 DU145 tumour-bearing mice, 17 PC3 tumour-bearing mice and 17 human patients with prostate cancer were investigated. Intrinsic susceptibility-weighted MRI was performed before and during a period of carbogen gas breathing. Quantitative R(2)* pixel maps were produced for each tumour and at each time point and changes in R(2)* induced by carbogen were determined. There was a mean reduction in R(2)* of 6.4% (P=0.003) for DU145 xenografts and 5.8% (P=0.007) for PC3 xenografts. In all, 14 human subjects were evaluable; 64% had reductions in tumour R(2)* during carbogen inhalation with a mean reduction of 21.6% (P=0.0005). Decreases in prostate tumour R(2)* in both animal models and human patients as a result of carbogen inhalation suggests the presence of significant hypoxia. The finding that carbogen gas breathing improves prostate tumour oxygenation provides a rationale for testing the radiosensitising effects of combining carbogen gas breathing with radiotherapy in prostate cancer patients.

Rationale for cancer prevention strategies in high-risk ulcerative colitis.

Colorectal cancer (CRC) is the most feared long-term complication in patients with ulcerative colitis (UC) and Crohn's colitis. Surveillance by colonoscopy and serial biopsy is conducted to identify patients most likely to benefit from potentially curative surgery. Within this paradigm, patients with high grade dysplasia or early stage CRC typically undergo colectomy, while patients free of dysplasia continue within surveillance programmes. However, detection of dysplasia in colitis may be difficult. Underdiagnosis and undertreatment of dysplasia may be accompanied by 'interval cancers' after apparently negative colonoscopy, frustrating the goal of cancer prevention. In the absence of a best practice model, surgical decisions for effective cancer prevention and control can be aided by greater understanding of cancer biology, in particular the close relationship between processes of inflammation and neoplastic change. This review will summarise recent knowledge in this area and consider clinical variables of disease duration, severity and anti-inflammatory therapy against stepwise events of neoplastic transformation. Against this background, indications for surveillance and prophylactic colectomy in specific clinical situations will be discussed.

Preclinical evaluation of the fluorinated 2-nitroimidazole N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) as a probe for the measurement of tumor hypoxia.

A novel probe, N-(2-hydroxy-3,3,3,-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554), has been used to detect tumor hypoxia noninvasively by 19F magnetic resonance spectroscopy (19F MRS). The compound was designed to undergo a hypoxia-dependent, one-electron reduction to metabolites that are selectively retained in tumors and has attractive pharmacokinetic, toxicological, and detection sensitivity properties. As a prelude to clinical studies, we report here for the first time on the ability to detect a MR signal following SR-4554 administration in various transplantable tumors and describe validation studies, consisting of a correlation between signal retention and radiobiological hypoxic fraction, and the effects of modulating the degree of hypoxia by hydralazine and carbogen breathing. SR-4554 was absorbed and then eliminated from EMT6 tumors with a half-life of 51 min following an injection of 180 mg/kg i.p. of SR-4554. Using a quantitative 19F MRS technique, the 19F retention index (19FRI; 19F signal level at 6 h/45 min) was determined for four commonly used murine tumors (EMT6, SCCVII, KHT, and RIF-1). The retention of high tumor concentrations of fluorinated probe at 6 h, despite the much lower (20-fold) concentration of parent SR-4554 detected by high-performance liquid chromatography, was consistent with the involvement of one or more nitroreduced metabolites and suggested that 19F MRS might give a quantitative measure of tumor hypoxia. In these murine tumors, 19FRI correlated with the reported radiobiological hypoxic fraction of the tumors (r = 0.988; P = 0.01). In addition, changes in tumor microenvironment were detected by 19F MRS. An increase in hypoxia induced by hydralazine treatment of RIF-1 tumor-bearing mice was associated with a 2.4-fold increase in 19FRI compared to untreated controls. In contrast, carbogen breathing by C3H mammary tumor-bearing mice produced a 6-fold decrease in the 19FRI compared to air-breathing mice. The data presented support the preclinical and clinical development of SR-4554 as a noninvasive probe for tumor hypoxia.

Effect of changing tumor oxygenation on glycolytic metabolism in a murine C3H mammary carcinoma assessed by in vivo nuclear magnetic resonance spectroscopy.

The rate of conversion of D-[1-(13)C]glucose into [3-(13)C]lactate (apparent glycolytic rate) has been determined in C3H murine mammary carcinomas in vivo using tumor-selective (13)C nuclear magnetic resonance spectroscopy with (1)H-(13)C cross-polarization. Under conditions of acute hypoxia induced by breathing carbon monoxide at 660 ppm, the apparent glycolytic rate was 0.0239 +/- 0.0019 min(-1). The proportion of (13)C label incorporated into [4-(13)C]glutamate (measured in tumor extracts) was 25-fold lower than that incorporated into [3-(13)C]lactate, reflecting a very limited oxidative metabolism during this hypoxic episode. For animals breathing air or carbogen (95% O(2) + 5% CO(2)), the calculated glycolytic rates were correspondingly lower (0.0160 +/- 0.0021 min(-1) and 0.0050 +/- 0.0011 min(-1), respectively). Although (13)C labeling of glutamate at C4 was still an order of magnitude lower than that for lactate at C3 (11-fold for air and 9-fold for carbogen), these ratios did show a greater degree of oxidative metabolism than that seen in animals breathing carbon monoxide at 660 ppm. The marked difference in apparent glycolytic rate for this tumor model between well-oxygenated and hypoxic conditions demonstrates a substantial Pasteur effect (inhibition of glycolysis by oxygen). Dynamic (13)C nuclear magnetic resonance spectroscopy provides a noninvasive estimate of tumor glycolysis that can be used to evaluate the relationship between oxygenation and energy metabolism, and this has potential consequences for the sensitivity of hypoxic cells to treatment and their ability to promote angiogenesis.

Vasoactive intestinal peptide stimulates glycolysis in pituitary tumours; 1H-NMR detection of lactate in vivo.

1H- and 31P-NMR spectroscopy has been applied to rats carrying implanted tumours in vivo, and used to observe simultaneous changes in intracellular pH (pHi) and lactate concentration during the stimulatory action of vasoactive intestinal polypeptide (VIP). A maximal decrease in pHi to a mean of 0.29 units below basal values was recorded. At the same time, 11 min after VIP, a maximal increase in tumour lactate was found, with a mean value of 150% of the basal concentration. The magnitude of these changes was compatible with in vitro measurements of basal lactate concentration and buffering capacity made on the same tumour line. It is concluded that VIP stimulates glycolysis by the tumour cells, resulting in an accumulation of lactate and a consequent fall in pHi.

Applications of magnetic resonance in model systems: cancer therapeutics.

The lack of information regarding the metabolism and pathophysiology of individual tumors limits, in part, both the development of new anti-cancer therapies and the optimal implementation of currently available treatments. Magnetic resonance [MR, including magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), and electron paramagnetic resonance (EPR)] provides a powerful tool to assess many aspects of tumor metabolism and pathophysiology. Moreover, since this information can be obtained nondestructively, pre-clinical results from cellular or animal models are often easily translated into the clinic. This review presents selected examples of how MR has been used to identify metabolic changes associated with apoptosis, detect therapeutic response prior to a change in tumor volume, optimize the combination of metabolic inhibitors with chemotherapy and/or radiation, characterize and exploit the influence of tumor pH on the effectiveness of chemotherapy, characterize tumor reoxygenation and the effects of modifiers of tumor oxygenation in individual tumors, image transgene expression and assess the efficacy of gene therapy. These examples provide an overview of several of the areas in which cellular and animal model studies using MR have contributed to our understanding of the effects of treatment on tumor metabolism and pathophysiology and the importance of tumor metabolism and pathophysiology as determinants of therapeutic response.

Multiple mitochondrial DNA deletions in an elderly human individual.

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69-year-old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.

Identification of a 66 KDa protein associated with yeast mitochondrial ATP synthase as heat shock protein hsp60.

A 66 kDa protein, denoted P66, not hitherto classified as an integral component of yeast mitochondrial ATPase, is often observed in preparations of this enzyme complex. A physical association exists between P66 and the assembled ATPase complex since both components are coimmunoprecipitated by anti-F1 beta monoclonal antibody. Two recombinant clones expressing proteins immunologically similar to P66 were isolated from a yeast genomic library in lambda gt11 by screening with a polyclonal anti-holo-ATPase antibody. Based on restriction site mapping and partial nucleotide sequence analysis, both clones encompass the gene encoding the yeast heat shock protein hsp60. The identification of P66 with hsp60, taken together with its demonstrated association with the mitochondrial ATPase complex, is consistent with recent suggestions that hsp60 is involved in assembly of the ATP synthase complex.

Demonstration of tumor-selective retention of fluorinated nitroimidazole probes by 19F magnetic resonance spectroscopy in vivo.

We have evaluated two fluorinated misonidazole analogues, Ro 07-0741 and CCI-103F, as potential probes for the non-invasive identification of hypoxic tumor cells by 19F magnetic resonance spectroscopy (MRS) in vivo. The equipment used was a 1.9 T Oxford Research Systems TMR-32 spectrometer, fitted with a 15 mm diameter surface coil. Signal was readily detectable, with similar intensity from EMT6 tumor, liver, and brain at early times (1-2 hr) after i.v. injection in BALB/c mice, indicative of an initial uniform biodistribution of parent probes. At later times (5-10 hr) there was a progressive reduction in signal intensity from brain and liver, but tumor levels remained constant or declined more slowly. This is illustrated by tumor/brain ratios at 6-7 hr of 2.9 (Ro 07-0741) and 4.2 (CCI-103F). In 4/5 mice analyzed at 20-24 hr after Ro 07-0741, and 1/2 following CCI-103F, tumor signal remained detectable. This occurred in the absence of parent probe as measured by HPLC, suggesting the involvement of a product of nitroreductive bioactivation. Studies with KHT and RIF-1 tumors in C3H/He mice showed a similar trend but retention in RIF-1 was less dramatic, and this was consistent with the known hypoxic fractions and comparative in vivo nitroreductase activities. These promising results support the continuing development of 19F nitroimidazole probes for non-invasive identification of hypoxic cells in vivo.

Effects of combretastatin on murine tumours monitored by 31P MRS, 1H MRS and 1H MRI.

PURPOSE: Combretastatins have tubulin-binding activity and are being investigated for their toxicity against tumour vasculature. We report the use of 31P and 'H magnetic resonance (MR) spectroscopy and 1H MR imaging for monitoring the effects of combretastatin A-4 prodrug (100mg/kg, i.p.) on energy metabolism and necrosis, respectively, in the C3H murine mammary tumour. MATERIALS AND METHODS: The tumours (volume ca. 200mm3) were grown in the hind foot of mice. MR examinations were performed without anaesthesia within a 7.1 Tesla magnet. 31P MRS (TR = 6 s) was performed before treatment and at 1-, 2-, 3-, and 24-h after injection of drug or saline via an i.p. line. 1H MRS (PRESS; 24microl voxel; TR = 2 s; TE = 135 ms) and both T1-weighted (TR = 0.2 s; TE = 0.02 s) and T2-weighted (TR = 2 s; TE = 0.20 s) 1H MRI were performed before treatment and 2.5 and 24 h afterwards. RESULTS: The ratio beta-nucleotide triphosphate/inorganic phosphate fell by 33% within 1 h of treatment and remained constant for a further 2 h. A small but significant fall in pH (by 0.11 units) was observed at 1 h. Although an increase in the 1H MR spectroscopy signal at about 1.32 ppm (predominantly from lactate) was observed in some tumours following combretastatin treatment, this effect was not seen consistently. No changes in the intensity of T2-weighted 1H MR images or in tumour necrosis (measured histologically) were detected within 3 h of treatment. CONCLUSIONS: The reduction in tumour energetics and pH was consistent with a reduction in tumour blood flow but this occurred before any significant incidence of haemorrhagic necrosis was detected. The combretastatin dose used to achieve these effects was less than one tenth of the maximum tolerated dose in mice.

Phosphorus-31 magnetic resonance spectroscopy and blood perfusion of the RIF-1 tumor following X-irradiation.

Phosphorus-31 magnetic resonance spectra were obtained from the RIF-1 tumor in C3H mice before and up to 2 days after various doses of X rays. Parallel studies were performed to measure relative changes in tumor blood perfusion using [14C]iodo-antipyrine and changes in % tumor necrosis using Chalkley's method. Tumor ratios of phosphocreatine to inorganic phosphate (PCr/Pi) and nucleotide triphosphates to inorganic phosphate (NTP/Pi) as well as pH as measured by 31P-MRS increased significantly at most time points after irradiation with doses of 5, 10, and 20 Gy. Tumor blood perfusion was found to significantly improve after a dose of 20 Gy but not after a dose of 2 Gy. Percent tumor necrosis increased to about 3 times its control level at 1 day after a dose of 20 Gy and then declined to about twice its control value at 2 days. The magnitude of the changes in the 31P-MRS parameters makes it unlikely that any of them are entirely due to radiation-induced changes in the radiobiologically hypoxic fraction of these tumors. Changes in the necrotic fraction did not appear to influence the tumor spectra. However, the observed improvement in tumor blood perfusion may have resulted in an increase in oxidative phosphorylation of the whole tumor population as well as a clearance of inorganic phosphate and acid metabolites, so that 31P-MRS changes may indirectly reflect changes in tumor blood perfusion.

The energy metabolism of RIF-1 tumours following hydralazine.

Phosphorus-31 Magnetic Resonance Spectroscopy (MRS) was used to observe the effect of two doses of the vasodilator hydralazine on the energy status of RIF-1 tumours. An intravenous dose of 5 mg/kg hydralazine reduced the high energy phosphate metabolites PCr and ATP, lowered pHMRS and raised the levels of inorganic phosphate of tumours within 20 min of administering the drug. The levels of high energy metabolites continued to decrease for at least 24 h. Normal muscle spectra obtained up to 1 h after drug administration remained unchanged. An intravenous dose of 0.5 mg/kg hydralazine also reduced NTP/Pi and PCr/Pi levels of tumours up to at least 5 h after drug administration, but the effect was smaller than for the higher dose. Blood flow measurements and measurements of systemic blood pressure demonstrated that 5 mg/kg of hydralazine produced a reduction in both systemic blood pressure and tumour blood flow relative to most normal tissues investigated. It is concluded that the changes in the P-31 MRS spectra of tumours were due to a reduction in tumour vascular perfusion following administration of hydralazine.

In vivo 19F NMR spectroscopy of the antimetabolite 5-fluorouracil and its analogues. An assessment of drug metabolism.

The metabolism of 5-fluorouracil (5FU) and its analogues 2'-deoxy-5-fluorouridine (2'FdURD), 5'-deoxy-5-fluorouridine (5'FdURD) and R,S-1-(tetrahydro-2-furyl)-5-fluorouracil (Ftorafur) has been studied by 19F NMR in rat liver and the rat S. G. Prolactinoma. In experiments using i.v. bolus injections of 0.46 mmol/kg 5FU was cleared more rapidly from the liver than 5'FdURD (t1/2 of 4.7 +/- 0.6 vs 15.8 +/- 0.8 min, P less than 0.001). Alphafluoro-beta-alanine (FBALA) production was almost identical after 5FU or 2'FdURD but slower and more sustained after 5'FdURD and still slower after Ftorafur. Both 5FU and 2'FdURD caused formation of toxic fluoronucleotides in S.G. Prolactinomas when administered i.v. (0.92 mmol/kg bolus). After i.v. infusion (0.23 mmol/kg/hr for 4 hr) 5FU produced fluoronucleotides whereas 2'FdURD did not; however, both 5FU and 2'FdURD (0.19 mmol/kg daily i.p. for 7 days) suppressed tumour growth. FBALA was observed in tumors following 5FU and 2'FdURD. Infusion of FBALA itself (0.17 mmol/kg/hr for 4 hr i.v.) led to signal in the tumour, so this compound could have been formed in the liver. These data demonstrate that 19F NMR can monitor drug metabolism in vivo.

Systemic vasculitis and aneurysm formation in the Wiskott-Aldrich syndrome.

A 24 year old male who suffered from the Wiskott-Aldrich syndrome developed intra-abdominal bleeding on two occasions. Radiological investigations showed aneurysmal dilatation of branches of the hepatic and superior mesenteric arteries. The second abdominal bleed necessitated laparotomy and the bleeding was localised to the kidneys. Right nephrectomy was performed and histological examination showed a necrotising vasculitis, mainly involving medium and small sized renal blood vessels. Steroids, immunosuppressive treatment, and control of blood pressure resulted in resolution of the vasculitic process and prevented further haemorrhage. Vasculitis and aneurysm formation are rarely described complications of Wiskott-Aldrich syndrome and may account for the life threatening haemorrhage which occurs in this condition.

Mitochondrial DNA mutation associated with aging and degenerative disease.

Previous theories of aging based on somatic mutation neglected mtDNA, which has a high propensity for mutational error. Knowledge of yeast mtDNA mutations and their functional effects, and of human mtDNA mutations identified in the mitochondrial cytopathies, provides for a concept of aging based on the cumulative effect of mutations affecting human mtDNA. An essential feature of this concept is heteroplasmy, representing mixtures of normal and mutant mtDNA at the cellular and mitochondrial level, resulting in a "tissue mosaic" of focal bioenergetic deficits. Direct evidence for the concept is provided by (i) focal loss of staining for mitochondrially encoded enzymes, such as cytochrome c oxidase, in tissues of aged individuals (humans and rats) and (ii) an age-related increase in deletional mutations in mtDNA demonstrable by application of the polymerase chain reaction to DNA templates from individuals of different ages.

Effects of pulmonary embolism on survival of patients with Greenfield vena caval filters.

The late mortality in 167 patients who had Greenfield vena caval filters inserted after episodes of acute pulmonary embolism is reported. The mean follow-up period of the survivors was 42 months; the mean survival time of the 48% who died was 9.3 months. The mortality rate in 50 comparable patients who did not have pulmonary embolism but had prophylactic filter insertion was 52% with a mean survival time of 9.4 months. The mean follow-up period of the survivors was 28 months. Age, mean pulmonary artery pressure, and severity of the initial embolic episode did not predict the increased rate of late mortality. We conclude that for patients who survive acute pulmonary embolism and have treatment to prevent recurrence, the embolic history of thromboembolism has little impact on late mortality, which is determined by their preexisting serious medical conditions.

Motilin's induction of phasic contractility in canine jejunum is mediated by the luminal release of serotonin.

To determine whether mucosal serotonin (5-HT) is involved in the intestinal motility response to motilin, we studied the effects of intra-arterial infusions of motilin (5 X 10(-10) mol/L) in isolated, vascularly perfused segments of canine jejunum. Intraluminal pressures were continuously recorded through a perfused manometric catheter, and venous and luminal 5-HT levels were measured by radioimmunoassay. Luminal 5-HT output rose from a basal of 54 +/- 12 to a peak of 151 +/- 30 ng/min X 100 gm (p less than 0.01) with the motilin infusion. The earliest significant rise in luminal (5-HT), which occurred at 1 minute after the start of the motilin infusion, preceded the onset of phasic contractility. The rise in the motility index induced by motilin demonstrated a strong correlation (r = 0.72, p less than 0.01) with rises in luminal 5-HT output. 5-HT tachyphylaxis abolished the motility response to motilin; prior treatment with atropine abolished, while tetrodotoxin inhibited the luminal release of 5-HT. An infusion of exogenous 5-HT giving equivalent luminal 5-HT levels as induced by motilin led to similar phasic contractility and rise in the motility index. These findings suggest that motilin initiates phasic contractility in canine jejunum through the cholinergically mediated release of mucosal serotonin.

Hyperparathyroid crisis and posterior mediastinal parathyroid adenoma: a case for preoperative localization.

Hyperparathyroid crisis is a rare, life-threatening condition that requires prompt removal of the involved parathyroid gland(s). This report describes a patient with hyperparathyroid crisis caused by a mediastinal parathyroid adenoma, which was localized before operation and removed at the initial operation, without neck exploration. Surgical approaches to hyperparathyroid crisis and the importance of preoperative localization studies are discussed.