Identification of imaging biomarkers for the assessment of tumour response to different treatments in a preclinical glioma model.

PURPOSE: Hypoxia-inducible factor 1α (HIF-1α) activity is one of the major players in hypoxia-mediated glioma progression and resistance to therapies, and therefore the focus of this study was the evaluation of HIF-1α modulation in relation to tumour response with the purpose of identifying imaging biomarkers able to document tumour response to treatment in a murine glioma model. METHODS: U251-HRE-mCherry cells expressing Luciferase under the control of a hypoxia responsive element (HRE) and mCherry under the control of a constitutive promoter were used to assess HIF-1α activity and cell survival after treatment, both in vitro and in vivo, by optical, MRI and positron emission tomography imaging. RESULTS: This cell model can be used to monitor HIF-1α activity after treatment with different drugs modulating transduction pathways involved in its regulation. After temozolomide (TMZ) treatment, HIF-1α activity is early reduced, preceding cell cytotoxicity. Optical imaging allowed monitoring of this process in vivo, and carbonic anhydrase IX (CAIX) expression was identified as a translatable non-invasive biomarker with potential clinical significance. A preliminary in vitro evaluation showed that reduction of HIF-1α activity after TMZ treatment was comparable to the effect of an Hsp90 inhibitor, opening the way for further elucidation of its mechanism of action. CONCLUSION: The results of this study suggest that the U251-HRE-mCherry cell model can be used for the monitoring of HIF-1α activity through luciferase and CAIX expression. These cells can become a useful tool for the assessment and improvement of new targeted tracers for potential theranostic procedures.

Validation of an engineered cell model for in vitro and in vivo HIF-1α evaluation by different imaging modalities.

PURPOSE: The aim of this study was to characterize a cell-based model for the molecular study of hypoxia-inducible factor (HIF)-1α activity, in the context of hypoxia, by means of different imaging techniques. PROCEDURES: Engineered U251-HRE glioma cells were used to analyze the molecular mechanisms underlying HIF-1α activity in vitro in relation to luciferase expression. The same cells were orthotopically implanted in mice to evaluate tumor progression and hypoxia induction by bioluminescence imaging, fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI). RESULTS: In vitro analyses highlighted the relationship between HIF-1α and luciferase activity in hypoxic conditions and after pharmacological treatments in U251-HRE cells. Through in vivo studies, it was possible to assess hypoxia establishment in relation to tumor growth by optical imaging, PET and MRI. CONCLUSIONS: The findings of this study indicate that the U251-HRE orthotopic murine model can be used to reliably evaluate processes modulating HIF-1α activity, using both molecular and preclinical non-invasive imaging techniques.

The DRAGO gamma camera.

In this work, we present the results of the experimental characterization of the DRAGO (DRift detector Array-based Gamma camera for Oncology), a detection system developed for high-spatial resolution gamma-ray imaging. This camera is based on a monolithic array of 77 silicon drift detectors (SDDs), with a total active area of 6.7 cm(2), coupled to a single 5-mm-thick CsI(Tl) scintillator crystal. The use of an array of SDDs provides a high quantum efficiency for the detection of the scintillation light together with a very low electronics noise. A very compact detection module based on the use of integrated readout circuits was developed. The performances achieved in gamma-ray imaging using this camera are reported here. When imaging a 0.2 mm collimated (57)Co source (122 keV) over different points of the active area, a spatial resolution ranging from 0.25 to 0.5 mm was measured. The depth-of-interaction capability of the detector, thanks to the use of a Maximum Likelihood reconstruction algorithm, was also investigated by imaging a collimated beam tilted to an angle of 45 degrees with respect to the scintillator surface. Finally, the imager was characterized with in vivo measurements on mice, in a real preclinical environment.

Quinolinic acid induced neurodegeneration in the striatum: a combined in vivo and in vitro analysis of receptor changes and microglia activation.

PURPOSE: Huntington's disease (HD) is a progressive neurodegenerative disorder, which is characterised by prominent neuronal cell loss in the basal ganglia with motor and cognitive disturbances. One of the most well-studied pharmacological models of HD is produced by local injection in the rat brain striatum of the excitotoxin quinolinic acid (QA), which produces many of the distinctive features of this human neurodegenerative disorder. Here, we report a detailed analysis, obtained both in vivo and in vitro of this pharmacological model of HD. MATERIALS AND METHODS: By combining emission tomography (PET) with autoradiographic and immunocytochemical confocal laser techniques, we quantified in the QA-injected striatum the temporal behavior (from 1 to 60 days from the excitotoxic insult) of neuronal cell density and receptor availability (adenosine A(2A) and dopamine D(2) receptors) together with the degree of microglia activation. RESULTS: Both approaches showed a loss of adenosine A(2A) and dopamine D(2) receptors paralleled by an increase of microglial activation. CONCLUSION: This combined longitudinal analysis of the disease progression, which suggested an impairment of neurotransmission, neuronal integrity and a reversible activation of brain inflammatory processes, might represent a more quantitative approach to compare the differential effects of treatments in slowing down or reversing HD in rodent models with potential applications to human patients.

In vivo imaging of adenosine A2A receptors in rat and primate brain using [11C]SCH442416.

PURPOSE: The aim of this study was to evaluate the suitability of [11C]SCH442416 for the in vivo imaging of adenosine A2A receptors. METHODS: In rats and Macaca nemestrina, we evaluated the time course of the cerebral distribution of [11C]SCH442416. Furthermore, in rats we investigated the rate of metabolic degradation, the inhibitory effects of different drugs acting on adenosine or dopamine receptors and the modification induced by the intrastriatal administration of quinolinic acid (QA). RESULTS: The rate of metabolic degradation of [11C]SCH442416 in rats was slow; 60 min after tracer injection, more than 40% of total plasma activity was due to unmetabolised [11C]SCH442416. At the time of maximum uptake, radioactive metabolites represented only 6% of total extractable activity in the cerebellum and less than 1% in the striatum. In the striatum, the region with the highest expression of A2A receptors, the in vivo uptake of [11C]SCH442416 was significantly reduced only by drugs acting on A2A receptors or by QA, a neurotoxin that selectively reduces the number of intrastriatal GABAergic neurons. Position emission tomography (PET) studies in monkeys indicated that the tracer rapidly accumulates in brain, reaching maximum uptake between 5 and 10 min. Twenty minutes after the injection, radioactivity concentration in the striatum was two times that in the cerebellum. CONCLUSION: The specificity of binding, the rank order of regional distribution in the brain of rats and M. nemestrina, the good signal to noise ratios and the low amount of radioactive metabolites in brain and periphery indicate that [11C]SCH442416 is a promising tracer for the in vivo imaging of A2A adenosine receptors using PET.

Evaluation of three quinoline-carboxamide derivatives as potential radioligands for the in vivo pet imaging of neurodegeneration.

The peripheral-type benzodiazepine receptors (PBRs) are only minimally expressed in normal brain parenchyma, where they are primarily localized in glial cells. Their basal expression rises in different neurodegenerative disorders, due to the presence of infiltrating inflammatory cells and activated microglia. [11C]PK11195, a selective PBR antagonist, has been used for the in vivo PET monitoring of neurodegeneration in clinical observations. We recently developed and labeled with carbon-11 three new carboxamide derivatives: [11C]VC193M, [11C]VC195 and [11C]VC198M. Aim of this study was to evaluate these ligands for the in vivo measuring of PBRs expression in neurodegenerations and compare their kinetic behavior with that of the reference tracer [11C]PK11195. Radioligands were evaluated in a preclinical model of Huntington's disease consisting in the monolateral striatal injection of quinolinic acid (QA). Activated microglia and astrocytic gliosis was present only within the affected striatum. A concomitant increase in radioactivity accumulation was observed for all the tracers examined (P<0.01). Among the new compounds, [11C]VC195 showed higher levels of lesioned/unlesioned striatum ratios (3.28+/-0.44), in comparison with [11C]VC193M and [11C]VC198M (2.69+/-0.53 and 1.52+/-0.36, respectively), but slightly inferior to that observed for [11C]PK11195 (3.76+/-1.41).In conclusion, the results of the study indicate that [11C]VC195 is a promising candidate for in vivo PET monitoring of neurodegenerative processes but its in vivo behavior overlap that of [11C]PK11195.

Isomer-specific activity of dichlorodyphenyltrichloroethane with estrogen receptor in adult and suckling estrogen reporter mice.

We investigated the tissue-specific effects of dichlorodyphenyltrichloroethane (DDT) isomers in adult and suckling newborn mice, using a novel mouse line engineered to express a reporter of estrogen receptor transcriptional activity (ERE-tkLUC mouse). The DDT isomers p,p'-DDT [1,1,1-trichloro2,2-bis(p-chlorophenyl) ethane] and o,p'-DDT [1,1,1-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl) ethane] were specifically selected as a weak and a strong estrogen, respectively. In adult male mice, p,p'-DDT induced luciferase activity in liver, brain, thymus, and prostate but not in heart and lung. The effect of p,p'-DDT was dose-dependent, maximal at 16 h after sc treatment, and completely blocked by the estrogen receptor antagonist ICI-182,780. In all the organs analyzed, except the liver, administration of o,p'-DDT showed a pattern of luciferase induction superimposable to that of its isomer p,p'-DDT. In liver, o,p'-DDT significantly decreased basal luciferase activity and blocked the reporter induction by 17beta-estradiol. These data lead us to hypothesize that a modulation of ER activity may be involved in the toxic effects of DDT demonstrated by epidemiological and experimental studies. Luciferase activity was also studied in 4-d-old mice lactating from a mother injected with either p,p'-DDT or o,p'-DDT. Both isomers induced a 2-fold increase in the newborn brain. An opposite effect was observed in liver, where p,p'-DDT increased and o,p'-DDT decreased luciferase, thus indicating that these compounds modulate ER activity in adult and newborn tissues by use of a similar mechanism. The ERE-tkLUC mouse proves to be a suitable tool to functionally assess the tissue specificity of estrogenic/antiestrogenic compounds in adult (as well as in suckling) mice.

Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: identification of the new mutation C14G.

BACKGROUND: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. METHODS: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5' untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. RESULTS: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 microg/L. The new mutation G14C was identified. CONCLUSIONS: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

A simple method for the quantitative description of sleep microstructure.

A simple method for the quantitative description of sleep microstructure is proposed. This method is based on the computation of descriptors which provide a normalized measure of how the amplitude of the activity in a frequency band differs, at a given instant, from its background. The use of these descriptors makes it possible to identify epochs of transient increase in band activity, to measure their length, and to measure the time distance between two successive epochs.

Evaluation of cytokeratin 19 serum fragments (CYFRA 21-1) in patients with lung cancer: results of a multicenter trial.

Recently, a new immunometric assay (Cyfra 21-1) was developed to measure serum concentrations of a soluble fragment of cytokeratin subunit 19. With this method, supplied by Boehringer Mannheim (EIA Test Cyfra 21-1), an Italian multicenter trial was performed in patients with lung cancer. Cyfra 21-1 serum levels were determined in 568 normal subjects (blood donors), 607 patients with non-malignant diseases (491 respiratory diseases) and 730 patients with malignancies. In the latter group 584 had lung cancer. All these 584 patients had pathologically confirmed disease; 314 were epidermoid tumors, 166 adenocarcinomas, 88 small cell cancers and 16 large cell cancers. In the 568 healthy blood donors the mean Cyfra 21-1 value was 0.91 ng/ml (SD 0.47 ng/ml; range 0.05-2.90 ng/ml). A threshold of 1.9 ng/ml was chosen as the upper limit of normality. High levels of Cyfra 21-1 were observed in patients with chronic hepatitis (positivity rate: 17/51-33.3%) and with pancreatitis (positivity rate 5/16-31.3%). In 114 out of 491 (23.2%) patients with respiratory diseases Cyfra 21-1 showed values greater than 1.9 ng/ml. The overall sensitivity (all stages) of Cyfra 21-1 in lung cancer was 65.6% (383/584). When the histology was considered the highest positivity rates were found in patients with squamous cell tumors (226/314; 72%) followed by adenocarcinomas (105/166; 63%). In patients with SCLC the global sensitivity was 52.3% (46/88). Higher sensitivity of Cyfra 21-1 was observed from stage I to stage IV (53.9% vs 85.7%; Chi square: p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

A new approach to tumour marker assessment by perioperative determination in breast and colorectal cancer.

Preoperative serum tumour markers are currently classified as positive or negative according to a predetermined cut-off point. In the present study we examined the dynamic variation of marker levels after radical surgery of breast and colorectal cancer. CEA and CA15.3 were measured in 93 patients with breast cancer, CEA and CA19.9 in 97 patients with colorectal carcinoma before and 30 days after radical surgery. Any variation higher than 3-fold the analytical coefficient of variation of the assay was considered significant. In patients with negative preoperative marker levels a significant decrease was noted after surgery in 15.6% of cases for CEA and 27.8% for CA15.3 in breast cancer and in 46.8% for CEA and 25.7% for CA19.9 in colorectal cancer. Using both cut-off-based and dynamic criteria, we found an overall positivity rate of 19.6% for CEA and 33.3% for CA15.3 in breast cancer; 60.0% for CEA and 37.1% for CA19.9 in colorectal cancer. From the present findings we conclude that the dynamic study of perioperative variations of tumour markers is a sensitive method additional to cut-off-based criteria for the assessment of the phenotypic expression of the marker by the tumour.

Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.

Protective effect of a prostacyclin-mimetic on the ischaemic-reperfused rabbit myocardium.

To assess whether the administration of the stable prostacyclin-mimetic ZK 36374 (iloprost) protects the myocardium in a dose-dependent manner against ischaemia and reperfusion, isolated rabbit hearts were infused with three different concentrations of iloprost: 2.7, 27 and 270 nM. Diastolic and developed pressures were monitored; coronary effluent was collected and assayed for creatine phosphokinase (CPK) activity and for noradrenaline concentration; mitochondria were harvested and assayed for respiratory activity; ATP production and calcium content and tissue concentration of adenosine triphosphate (ATP) and creatine phosphate (CP) were determined. Treatment with iloprost altered neither developed pressure under normoxic conditions nor the rate and extent of depletion of ATP and CP during ischaemia. The ischaemic-induced deterioration of mitochondrial function, however, was attenuated. On reperfusion, hearts treated with iloprost recovered better than the untreated hearts with respect to left ventricular performance, replenishment of ATP and CP stores and mitochondrial function. The reperfusion-induced mitochondrial calcium overload and release of CPK and of noradrenaline were also significantly reduced. The effect of iloprost was dose-dependent. The lower concentration (2.7 nM) failed to modify ischaemic and reperfusion damage. The best protective effect was found at 27 nM. An increase of the dose to 270 nM did not result in further protection. It is concluded that iloprost infusion provides a dose-dependent protection of the heart against some of the deleterious effects of ischaemia and reperfusion and, in particular, prevents mitochondrial calcium overload and maintains mitochondrial function. Because this protection occurred in the absence of negative inotropic effect during normoxia or of a coronary dilatory effect during ischaemia, it cannot be attributed to an energy sparing effect or to improvement of oxygen delivery. Therefore, alternative mechanisms of action are to be considered.

Metabolic changes during post-ischaemic reperfusion.

We attempted to identify the nature and time-course of metabolic changes occurring during ischaemia followed by reperfusion either in coronary artery disease patients undergoing intracoronary thrombolysis or in isolated and perfused rabbit hearts. Arterial and coronary sinus differences for oxygen, lactate, glucose, free fatty acid and creatine kinase were measured in patients undergoing successful intracoronary thrombolysis of left anterior descending occlusion. Early reperfusion (after 160 mins of ischaemia) restored aerobic metabolism and myocardial contractility. In contrast, reperfusion after more prolonged ischaemia (335 mins) did not restore mitochondrial function or contractile activity of the myocytes. Results obtained using isolated and perfused rabbit hearts also confirm that the likelihood of recovery during reperfusion depends on the rapidity of recanalization. Furthermore the data reported indicate that on reperfusion after prolonged ischaemia (90 mins) cell damage occurs, leading to a breakdown of the permeability barrier to ions and to larger molecules such as creatine phosphokinase. As a consequence, reperfusion produces a large increase of intracellular calcium, whilst the intracellular magnesium content is severely reduced. Under these conditions, with the observed loss of magnesium from the cell, mitochondrial calcium transport is highly stimulated and the equilibrium between ATP synthesis and calcium influx is shifted towards calcium influx. This sequence of events leads to mitochondrial calcium overload with subsequent damage of mitochondrial structure and loss of the ability to synthesize ATP. Reperfusion of the isolated rabbit hearts with solutions containing high magnesium and low calcium for 10 mins reduced mitochondrial calcium overload. This, in turn, resulted in maintenance of ATP synthesis and, on return to normal perfusate, in partial recovery of developed pressure and myocardial ATP content. These findings may be of importance in the restoration of blood flow to ischaemic heart muscle during thrombolysis.

Use of a reference standard to improve the accuracy and precision of seven kits for determination of ferritin in serum.

We assessed the accuracy and precision of seven commercial kits for serum ferritin. The concentration of ferritin as determined by these assays for a liver ferritin reference standard, a human heart ferritin standard, and normal sera correlated highly, but the absolute values varied widely. Use of an international reference standard for ferritin to prepare the standard curve greatly diminished the variability, but did not eliminate it. Although the seven kits differed in specificity for various human isoferritins, this did not appear substantially to affect accuracy. Six of the seven kits appeared sufficiently precise for clinical use over a wide range of ferritin concentrations.