Staging of neuroendocrine tumours: comparison of [68Ga]DOTATOC multiphase PET/CT and whole-body MRI.

PURPOSE: In patients with a neuroendocrine tumour (NET), the extent of disease strongly influences the outcome and multidisciplinary therapeutic management. Thus, systematic analysis of the diagnostic performance of the existing staging modalities is necessary. The aim of this study was to compare the diagnostic performance of 2 whole-body imaging modalities, [(68)Ga]DOTATOC positron emission tomography (PET)/computed tomography (CT) and magnetic resonance imaging (MRI) in patients with NET with regard to possible impact on treatment decisions. MATERIALS AND METHODS: [(68)Ga]DOTATOC-PET/CT and whole-body magnetic resonance imaging (wbMRI) were performed on 51 patients (25 females, 26 males, mean age 57 years) with histologically proven NET and suspicion of metastatic spread within a mean interval of 2.4 days (range 0-28 days). PET/CT was performed after intravenous administration of 150 MBq [(68)Ga]DOTATOC. The CT protocol comprised multiphase contrast-enhanced imaging. The MRI protocol consisted of standard sequences before and after intravenous contrast administration at 1.5 T. Each modality (PET, CT, PET/CT, wbMRI) was evaluated independently by 2 experienced readers. Consensus decision based on correlation of all imaging data, histologic and surgical findings and clinical follow-up was established as the standard of reference. Lesion-based and patient-based analysis was performed. Detection rates and accuracy were compared using the McNemar test. P values <0.05 were considered significant. The impact of whole-body imaging on the treatment decision was evaluated by the interdisciplinary tumour board of our institution. RESULTS: 593 metastatic lesions were detected in 41 of 51 (80%) patients with NET (lung 54, liver 266, bone 131, lymph node 99, other 43). One hundred and twenty PET-negative lesions were detected by CT or MRI. Of all 593 lesions detected, PET identified 381 (64%) true-positive lesions, CT 482 (81%), PET/CT 545 (92%) and wbMRI 540 (91%). Comparison of lesion-based detection rates between PET/CT and wbMRI revealed significantly higher sensitivity of PET/CT for metastatic lymph nodes (100% vs 73%; P < 0.0001) and pulmonary lesions (100% vs 87%; P = 0.0233), whereas wbMRI had significantly higher detection rates for liver (99% vs 92%; P < 0.0001) and bone lesions (96% vs 82%; P < 0.0001). Of all 593 lesions, 22 were found only in PET, 11 only in CT and 47 only in wbMRI. The patient-based overall assessment of the metastatic status of the patient showed comparable sensitivity of PET/CT and MRI with slightly higher accuracy of PET/CT. Patient-based analysis of metastatic organ involvement revealed significantly higher accuracy of PET/CT for bone and lymph node metastases (100% vs 88%; P = 0.0412 and 98% vs 78%; P = 0.0044) and for the overall comparison (99% vs 89%; P < 0.0001). The imaging results influenced the treatment decision in 30 patients (59%) with comparable information from PET/CT and wbMRI in 30 patients, additional relevant information from PET/CT in 16 patients and from wbMRI in 7 patients. CONCLUSION: PET/CT and wbMRI showed comparable overall lesion-based detection rates for metastatic involvement in NET but significantly differed in organ-based detection rates with superiority of PET/CT for lymph node and pulmonary lesions and of wbMRI for liver and bone metastases. Patient-based analysis revealed superiority of PET/CT for NET staging. Individual treatment strategies benefit from complementary information from PET/CT and MRI.

The neuroprotective adenosine-activated signal transduction pathway involves activation of phospholipase C.

We have demonstrated before that exposure of neuronal cultures to poisoning by iodoacetic acid (IAA) followed by "reperfusion" (IAA-R insult), results in severe cytotoxicity, which could be markedly attenuated by prior activation of the adenosine A1 receptors. We also have demonstrated that adenosine activates a signal transduction pathway (STP), which involves activation of PKC epsilon and opening of KATP channels. Here, we provide proof for the involvement also of phospholipase C (PLC) in the neuronal protective adenosine-activated STP. R-PIA, a specific A1 adenosine receptor agonist, was found to enhance neuronal PLC activity and protect against the IAA-R insult. The PLC inhibitor U73122, abrogated both R-PIA-induced effects. These results demonstrate that activation of PLC is a vital step in the neuronal protective adenosine-induced STP.

Clinical and biochemical manifestations and molecular characterization of the mutation HPRT Jerusalem.

A novel point mutation (I137T) was identified in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5-phosphoribosyl-1-pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch-Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).

Decelerated rate of dendrite outgrowth from dopaminergic neurons in primary cultures from brains of hypoxanthine phosphoribosyltransferase-deficient knockout mice.

Lesch-Nyhan syndrome (LNS), caused by the complete deficiency of hypoxanthine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is still unclear. Evidence has accumulated indicating that it reflects dopamine deficiency associated with defective arborization of dopaminergic dendrites. We monitored the differentiation in vitro of dopaminergic neurons, cultured from HPRT-deficient knockout mice. The HPRT-deficient dopaminergic neurons exhibited a decelerated rate of outgrowth of dendrites in comparison to that of control neurons resulting, after 8 days in culture, in 32% smaller average total length of dendrites per neuron (P<0.025). The results suggest that the abnormal dendrite outgrowth in LNS reflects a defective developmental process.

The adenosine-induced mechanism for the acquisition of ischemic tolerance in primary rat neuronal cultures.

Neurons can be preconditioned by various procedures to resist ischemic insult. The preconditioning mechanism induced by adenosine ("the adenosine mechanism") was characterized in primary rat neuronal cultures, employing a model of chemical ischemia. The protective mechanism, initiated by activation of adenosine receptors, consists of a signal transduction pathway, involving activation of protein kinase C (PKC) and opening of ATP-sensitive potassium (K(ATP)) channels. Direct activation (and inhibition) of PKC, as well as opening of K(ATP) channels, also confers protection. The opening of the K(ATP) channels mediates the signal activated by the adenosine receptors, and probably also that activated by PKC. The acquired ischemic resistance lasts up to 5 days, depending on the activating substance. The adenosine-activated cascade of events leading to ischemic tolerance in neurons is similar to that operating in cardiomyocytes.

Ischemic tolerance conferred to cultured rat neurons by heat shock is not mediated by opening of adenosine triphosphate-sensitive potassium channels.

The effect of sublethal heat shock on the capacity of neurons to resist subsequent ischemia-reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to chemical ischemia. Exposure of the cultures to sublethal heat shock (42 degrees C; 20 min) resulted in elevation in cellular content of heat shock protein (HSP)-70, at 4 h following the shock, and in acquisition of a 15 h 'time window of protection' against ischemia-reperfusion insult, with maximum protection at 4 h. Presence in the culture medium of glibenclamide (2 microM), a blocker of ATP sensitive potassium (K(ATP)) channels, did not abolish the acquisition of protection throughout the entire duration of the acquired 'time window of protection'. The results demonstrate that heat shock induces in neurons a protective mechanism against ischemia-reperfusion insult, probably associated with enhanced expression of HSPs, which does not depend on opening of K(ATP) channels. In this respect, the neuronal 'heat-shock mechanism' for the acquisition of ischemic tolerance differs from the neuronal 'adenosine mechanism' and probably also from the heart 'heat shock mechanism' for the acquisition of protection.

Elevated UTP and CTP content in cultured neurons from HPRT-deficient transgenic mice.

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8.; HPRT) catalyzes the salvage synthesis of inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP) from the purine bases hypoxanthine and guanine, respectively. Complete deficiency of HPRT activity is associated with the Lesch-Nyhan syndrome (LNS), characterized by excessive purine production and severe neurological manifestations. The etiology of the metabolic consequences of HPRT deficiency is clarified, but that of the neurological manifestations is not yet understood. HPRT-deficient mice represent an experimental animal model of LNS. In search for a possible metabolic abnormality in LNS brains, connecting the neurological deficit to HPRT deficiency, the purine and pyrimidine nucleotide content of cultured neurons, prepared from HPRT-deficient transgenic mice, was now determined. The HPRT-deficient neuronal cultures exhibited a significantly elevated content of the pyrimidine nucleotides UTP (1.33-fold the normal level, p = 0.0002) and CTP (1.28-fold the normal level, p = 0.02), but normal content of the purine nucleotides ATP and GTP. This abnormality in neuronal pyrimidine nucleotide content may be associated with the pathophysiology of the neurological deficit in LNS.

XDH gene mutation is the underlying cause of classical xanthinuria: a second report.

BACKGROUND: Classical xanthinuria is a rare autosomal recessive disorder characterized by excessive excretion of xanthine in urine. Type I disease results from the isolated deficiency of xanthine dehydrogenase (XDH), and type II results from dual deficiency of XDH and aldehyde oxidase. The XDH gene has been cloned and localized to chromosome 2p22-23. The aim of this study was to characterize the molecular basis of classical xanthinuria in an Iranian-Jewish family. METHODS: The apparently unrelated parents originated from a community in which consanguineous marriages are common. Subtyping xanthinuria was attempted by homozygosity mapping using microsatellite markers D2S352, D2S367, and D2S2374 in the vicinity of the XDH gene. Mutation detection was accomplished by PCR-SSCP screening of all 36 exons and exon-intron junctions of the XDH gene, followed by direct sequencing and confirmation of sequence alteration by restriction analysis. RESULTS: The index case was homozygous for all three microsatellite markers analyzed. The expected frequency of this genotype in a control population was 0. 0002. These results suggested that xanthinuria in the patient is linked to the XDH gene. Consequently, a 1658insC mutation in exon 16 of the XDH gene was identified. The 1658insC mutation was not detected in 65 control DNA samples. CONCLUSION: A molecular approach to the diagnosis of classical xanthinuria type I in a female patient with profound hypouricemia is described. Linkage of xanthinuria to the XDH locus was demonstrated by homozygosity mapping, and a 1658insC mutation, predicting a truncated inactive XDH protein, was identified. These results reinforce the notion that mutations in the XDH gene are the underlying cause of classical xanthinuria type I.

Opening of K(ATP) channels is mandatory for acquisition of ischemic tolerance by adenosine.

Binding of adenosine to neuronal adenosine receptors activates a signal transduction pathway (the adenosine mechanism), leading to a temporary ischemic tolerance. We have demonstrated before that induction of this mechanism in primary rat neuronal cultures, by activation of adenosine receptors, or by activation of protein kinase C (PKC), confers a wide time window of ischemic tolerance, lasting up to 72h, the early (immediate) part of which depends on opening of K(ATP) channels (glibenclamide sensitive). Here we demonstrate that the entire duration of the ischemic tolerance conferred by activation of the adenosine mechanism depends on opening of the K(ATP) channels. Thus, opening of the K(ATP) channels appears to be a mandatory step in the adenosine mechanism, leading to the creation of the wide time window of ischemic tolerance.

Kelley-Seegmiller syndrome due to a unique variant of hypoxanthine-guanine phosphoribosyltransferase: reduced affinity for 5-phosphoribosyl-1-pyrophosphate manifested only at low, physiological substrate concentrations.

A male child, who presented at the age of 3.5 years with acute renal failure, was diagnosed as having partial deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8). The underlying HPRT mutation was unique in that the specific activity of HPRT in erythrocyte and in fibroblast lysates was normal, but the rate of uptake of hypoxanthine into nucleotides of intact cultured fibroblasts was markedly reduced (23% of normal). The low functioning of HPRT in the intact fibroblasts was associated with decreased utilization of endogenously generated hypoxanthine and with decreased utilization of the cosubstrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The non-utilized hypoxanthine was excreted into the incubation medium. The accumulation of PRPP was indicated by the 2.3-fold increase in the rate of uptake of adenine into intact cell nucleotides and by the 7. 5-fold enhancement of the rate of de novo purine synthesis. Kinetic studies of HPRT activity in fibroblast lysates revealed reduced affinity of the enzyme for PRPP (apparent K(m) 500 microM in comparison to 25 microM in control lysates), manifested in low activity at low (physiological), but not at high PRPP concentrations. The apparent K(m) for hypoxanthine was normal (23 microM in comparison to 14.2 microM in control lysates). With allopurinol treatment, our patient has had no problems since presentation, and is developing normally at 5 years of age.

Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice.

Lesch-Nyhan syndrome is a pediatric metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT-deficient transgenic mice. The HGPRT-deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT-deficient cells. The following abnormalities were found: absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides; 27.8% increase in the availability of 5-phosphoribosyl-1-pyrophosphate; 9.4-fold acceleration of the rate of de novo nucleotide synthesis; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine); enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2-fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT-deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch-Nyhan syndrome.

Opening of ATP-sensitive potassium channels by cromakalim confers tolerance against chemical ischemia in rat neuronal cultures.

The effect of opening and of blocking of ATP-sensitive potassium (K(ATP)) channels on the short-term capacity of neurons to resist ischemia-reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to metabolic poisoning by iodoacetic acid (150 microM, 150 min), followed by reperfusion (1 h). The metabolic poisoning resulted in a marked decrease in cellular ATP content (from 65.3 +/- 13.4 to 21.6 +/- 11.7 nmole/mg protein), simulating an ischemia, or hypoxia-induced condition of energy crisis. The degree of neuronal damage was assessed by the trypan blue exclusion test. Exposure of the neurons to the channel-opener cromakalim (10 microM; 15 min), prior to the insult, induced resistance, which could be abolished by the specific channel blocker glibenclamide (2 microM). Glibenclamide also abolished the protection acquired by preconditioning of the neurons with iodoacetate (IA; 100 microM), the adenosine A1 agonist N6-(R)-phenylisopropyladenosine (R-PIA; 100 microM), or with the protein kinase C (PKC) activator 1,2 dioctanoyl-rac-glycerol (DOG; 1 microM). The results indicate that in the neurons, opening of the K(ATP) channels confers protection against an ATP-depleting crisis, and suggest that the protective effects induced by adenosine and by activation of PKC, are mediated by the opening of these channels.

Cyclic AMP decreases the availability of 5-phosphoribosyl-1-pyrophosphate and decelerates de novo purine synthesis in rat hepatocytes.

Cyclic adenosine monophosphate (cAMP) was found to decrease the availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) and to decelerate the rate of de novo purine synthesis in suspensions of adult rat hepatocytes. Glucagon did not affect these parameters. The glucagon antagonist des-His1[Glu9]glucagon amide (DHGA), and the protein kinase C activator 1,2-dioctanoyl-sn-glycerol (DOG) were also found to lower PRPP availability. Incubation of the hepatocytes with dbcAMP or with DHGA, did not alter the activity of PRPP synthetase in the hepatocyte lysates, indicating that the above effects are not mediated through the activity of this enzyme. The possibility that the decrease in PRPP availability reflects increased consumption associated with accelerated pyrimidine synthesis is discussed. The decelerated rate of de novo purine synthesis is probably secondary to the decreased PRPP availability.

Activation and inhibition of protein kinase C protect rat neuronal cultures against ischemia-reperfusion insult.

The effect of activation and inhibition of protein kinase C (PKC) on the capacity of neurons to resist subsequent ischemic and ischemia-reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to chemical ischemia. Activation of PKC by 1,2 dioctanoyl-rac-glycerol (DOG; 1 microM), or phorbol 12-myristate 13-acetate (PMA; 1 microM), as well as inhibition of the enzyme by chelerythrine (10 microM), or by calphostin C (0.2 microM), 10 min before the ischemic insult, resulted in acquisition of resistance against the two insults. The length of the 'time window of protection' induced by exposure to DOG and to chelerythrine was studied and found to last for several days. The results demonstrate an apparently 'paradoxical' phenomenon, in which both activation and inhibition of PKC in the same tissue induce protection. This may be explained by differential activation of various PKC isoforms.

Preconditioning of primary rat neuronal cultures against ischemic injury: characterization of the "time window of protection'.

Primary rat neuronal cultures can be preconditioned against ischemic damage by several mechanisms. In the present study we established a new model system in order to characterize the "time window of protection' obtained by preconditioning of neurons with adenosine. Ischemia was simulated by exposure of the cultures to iodoacetate (100 microM) for 150 min, with a post-ischemic reperfusion period of 60 min. Ischemic injury was assessed by the release of lactic dehydrogenase (LDH) to the medium during the ischemic period and ischemia-reperfusion damage by the Trypan blue exclusion test. Exposure of the neuronal cultures to the ischemic or ischemia-reperfusion insult resulted in severe damage to the neurons, manifested for the former insult in a 5.4-fold increase in the release of LDH and for the latter insult in an 8.5-fold increase in the proportion of stained cells by the Trypan blue exclusion test. Preconditioning by short exposure (5 min) of the cultures to iodoacetic acid (simulating sublethal ischemia), or to adenosine (1 mM) and the A1 adenosine receptor agonist N6-(R)-phenylisopropyladenosine (R-PIA; 1 and 100 microM), prior to the insult, partially protected the neurons against the damage. The time-course of the development and waning of the resistance against the two insults following preconditioning exhibited different patterns. The resistance obtained against the ischemic insult developed rapidly, being maximal for all substances at 10 min (the shortest time window studied), and lasted up to 1 h for iodoacetate, 3 h for R-PIA and 24 h for adenosine. In contrast, the protection induced by adenosine and R-PIA against ischemia-reperfusion injury developed relatively slowly, being maximal at 3 h, but lasted longer, up to 48 h. At this time the time-response curve exhibited a second peak of protection. The waning of protection against the two insults was found to continue into a period of increased sensitivity to the insults. This phenomenon was more intense for preconditioning with iodoacetate, and especially against the ischemic injury. The results suggest that in the neurons, different mechanisms may mediate the adenosine-induced preconditioning against the ischemic or ischemia-reperfusion injury. In addition, the results support the possibility that the relatively long "time window of protection', induced by adenosine and R-PIA against ischemia-reperfusion insult, reflects a combination of two different preconditioning mechanisms.

Adenine nucleotide metabolism in primary rat neuronal cultures.

The metabolism of adenine nucleotides (AdRN) has been studied previously in whole brains, brain slices and brain extracts, containing mixed populations of neurons and glia. The availability of primary neuronal cultures enables us to study these pathways in almost pure neuronal preparations. The aim of the present study was to characterize the relative importance of the pathways of AdRN metabolism in the neurons. The metabolic fate of (8-14C) adenine and of AdRN prelabeled with (8-14C)adenine were studied in immature and mature primary rat neuronal cultures. Specific inhibitors were used to clarify the various metabolic fluxes, which were evaluated based on the time-related changes in the distribution of label (the cellular nucleotide content did not change during incubation). The turnover rate of AdRN was found to reflect mainly conversion of label to acid insoluble derivatives (AID) and partly degradation to hypoxanthine. The turnover was faster in the immature neurons. The combined addition of 2'-deoxycoformycin (2'-dCF) and of 5'-amino-5'-deoxyadenosine, inhibiting adenosine metabolism, resulted in both cultures in enhanced loss of label from AdRN, mainly to adenosine and adenine. This finding indicates the activity of the futile cycle AMP-->adenosine-->AMP. In both cultures, in the presence of these inhibitors, the ratio (hypoxanthine + inosine)/(adenine + adenosine) was 1.1, indicating that the fluxes through AMP deamination and AMP dephosphorylation are about equal. Addition of L-alanosine, inhibiting the conversion of IMP to AMP, resulted in both cultures, but especially in the mature neurons, in enhanced loss of label from AdRN to hypoxanthine and inosine. This finding indicates the functioning of the adenine nucleotide cycle (AMP-->IMP-->adenylosuccinic acid-->AMP). Under conditions of enhanced degradation of ATP (induced by iodoacetate and antimycin A), addition of 2'-dCF resulted in the immature cultures in lowering the ratio (hypoxanthine + inosine + IMP)/(adenine + adenosine) to 0.62, indicating a shift in favor of AMP dephosphorylation.