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Paracrine signaling is challenging to study in vitro, as conventional culture tools dilute soluble factors and offer little to no spatiotemporal control over signaling. Microfluidic chips offer potential to address both of these issues. However, few solutions offer both control over onset and duration of cell-cell communication, and high throughput. We have developed a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. The chip features 16 fluidic inputs and 128 individually-addressable chambers arranged in 32 sets of 4 chambers. Media can be continuously perfused or delivered by diffusion, which we model under different culture conditions to ensure normal cell viability. Immunocytochemistry assays can be performed in the chip, which we modeled and fine-tuned to reduce total assay time to 1 h. Finally, we validate the use of the chip for co-culture studies by showing that HEK293Ta cells respond to signals secreted by RAW 264.7 immune cells in adjacent chambers, only when the valve between the chambers is opened.
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Técnicas Analíticas Microfluídicas , Microfluídica , Técnicas de Cocultivo , Técnicas de Cultivo de Célula , BioensayoRESUMEN
Polydimethyl siloxane (PDMS) has been used extensively for microfluidic devices due to its chemical properties allowing for rapid molding and versatile biological application. Soft lithography based PDMS fabrication primarily comprises casting from patterned photoresist on a silicon wafer. The patterned photoresist is often replaced with the cast PDMS as a more durable template mold for final PDMS fabrication that is less fragile and expensive. PDMS-PDMS double casting prolongs the longevity of soft lithography molds and reduces overall costs to microfuidic applications. A common end to the lifetime of PDMS negative masters is the risk of bonding between the replicate and mold and distorted topographrical features. This review examines common chemical and physical debonding approaches between PDMS-PDMS castings to exend the lifetime of PDMS masters.
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We present a fully-integrated solution for controlling pneumatically-driven microfluidic chips, featuring a pump, one or more pressure regulators and up to 32 solenoid valves, controlled by a microcontroller. The microfluidics control system requires only a power source and a computer or mobile device for its operation. A touchscreen interface communicates with the microcontroller over USB or Bluetooth and allows users to control the system with ease either manually or autonomously, allowing experiments to run with no user intervention. The pressure regulators were purpose-built, enabling integrated pressure sources on-board, rather than relying on external equipment. These regulators can also be used as stand-alone devices in any other application.
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Microfluidic diagnostic devices have the potential to transform the practice of medicine. We engineered a multiplexed digital-analog microfluidic platform for the rapid and highly sensitive detection of 3-4 biomarkers in quadruplicate in 16 independent and isolated microfluidic unit cells requiring only a single 5 µL sample. We comprehensively characterized the platform by performing single enzyme and digital immunoassays, achieving single molecule detection and measured as low as â¼10 fM (330 fg/mL) GFP in buffer and â¼12 fM GFP in human serum. We applied our integrated digital detection mechanism to multiplexed detection of 1pM anti-Ebola IgG in human serum and were able to differentiate three common Ebola strains. To ascertain that the device can be applied in environments beyond clinical point-of-care settings, we developed a low-cost, portable hardware system to control and read out the microfluidic device and detected anti-Ebola IgG in ultralow volume whole blood samples to levels of 100 pM in a multiplexed assay format.
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BACKGROUND: This study aimed to have international experts converge on a harmonized definition of whole hippocampus boundaries and segmentation procedures, to define standard operating procedures for magnetic resonance (MR)-based manual hippocampal segmentation. METHODS: The panel received a questionnaire regarding whole hippocampus boundaries and segmentation procedures. Quantitative information was supplied to allow evidence-based answers. A recursive and anonymous Delphi procedure was used to achieve convergence. Significance of agreement among panelists was assessed by exact probability on Fisher's and binomial tests. RESULTS: Agreement was significant on the inclusion of alveus/fimbria (P = .021), whole hippocampal tail (P = .013), medial border of the body according to visible morphology (P = .0006), and on this combined set of features (P = .001). This definition captures 100% of hippocampal tissue, 100% of Alzheimer's disease-related atrophy, and demonstrated good reliability on preliminary intrarater (0.98) and inter-rater (0.94) estimates. DISCUSSION: Consensus was achieved among international experts with respect to hippocampal segmentation using MR resulting in a harmonized segmentation protocol.
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Hipocampo/patología , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Neuroimagen/métodos , Enfermedad de Alzheimer/patología , Atrofia , Consenso , Técnica Delphi , Hipocampo/anatomía & histología , Humanos , Imagenología Tridimensional/métodos , InternacionalidadRESUMEN
BACKGROUND: An international Delphi panel has defined a harmonized protocol (HarP) for the manual segmentation of the hippocampus on MR. The aim of this study is to study the concurrent validity of the HarP toward local protocols, and its major sources of variance. METHODS: Fourteen tracers segmented 10 Alzheimer's Disease Neuroimaging Initiative (ADNI) cases scanned at 1.5 T and 3T following local protocols, qualified for segmentation based on the HarP through a standard web-platform and resegmented following the HarP. The five most accurate tracers followed the HarP to segment 15 ADNI cases acquired at three time points on both 1.5 T and 3T. RESULTS: The agreement among tracers was relatively low with the local protocols (absolute left/right ICC 0.44/0.43) and much higher with the HarP (absolute left/right ICC 0.88/0.89). On the larger set of 15 cases, the HarP agreement within (left/right ICC range: 0.94/0.95 to 0.99/0.99) and among tracers (left/right ICC range: 0.89/0.90) was very high. The volume variance due to different tracers was 0.9% of the total, comparing favorably to variance due to scanner manufacturer (1.2), atrophy rates (3.5), hemispheric asymmetry (3.7), field strength (4.4), and significantly smaller than the variance due to atrophy (33.5%, P < .001), and physiological variability (49.2%, P < .001). CONCLUSIONS: The HarP has high measurement stability compared with local segmentation protocols, and good reproducibility within and among human tracers. Hippocampi segmented with the HarP can be used as a reference for the qualification of human tracers and automated segmentation algorithms.
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Hipocampo/patología , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Neuroimagen/métodos , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Atrofia , Femenino , Lateralidad Funcional , Humanos , Imagenología Tridimensional/métodos , Internet , Imagen por Resonancia Magnética/instrumentación , Masculino , Persona de Mediana Edad , Tamaño de los Órganos , Reproducibilidad de los ResultadosRESUMEN
Pufferfish species of the Tetraodontidae family carry the smallest genomes among vertebrates. Their compressed genomes are thought to be enriched for functional DNA compared to larger vertebrate genomes, and they are important models for comparative genomics. The significance of pufferfish as model organisms in comparative genomics is due to the availability of two sequenced genomes, that of spotted green pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes). However, there is only a very limited utilization of pufferfish as an experimental model organism, due to the lack of established husbandry and developmental genetics protocols. In this study, we provide the first description of the normal embryonic development of Tetraodon nigroviridis. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored by brightfield microscopy at constant temperature. Tetraodon development was divided into distinct stages based on diagnostic morphological features, which were adopted from published literature on normal development of other fish species like medaka (Oryzias latipes), zebrafish (Danio rerio), and fugu. Tetraodon embryos show more similar morphologies to medaka than to zebrafish, reflecting its phylogenetic position. The early developmental stage series described in this study forms the foundation for the utilization of tetraodon as an experimental model organism for comparative developmental studies.
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Biología Evolutiva/métodos , Genoma/genética , Estadios del Ciclo de Vida/fisiología , Modelos Animales , Tetraodontiformes/embriología , Tetraodontiformes/genética , Animales , Técnicas de Cultivo de Embriones , Femenino , Fertilización In Vitro , Masculino , Especificidad de la EspecieRESUMEN
Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates.
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Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Purinas/metabolismo , Sitio de Iniciación de la Transcripción , Pez Cebra/embriología , Pez Cebra/genética , Animales , Evolución Molecular , Perfilación de la Expresión Génica , Genes , Genoma , Filogenia , Regiones Promotoras Genéticas , ARN/genética , ARN/metabolismo , Caperuzas de ARN/genética , Empalme del ARN , Transcriptoma , Vertebrados/genéticaAsunto(s)
Epilepsia del Lóbulo Temporal/complicaciones , Convulsiones/etiología , Síncope/etiología , Adolescente , Anticonvulsivantes/uso terapéutico , Carbamazepina/análogos & derivados , Carbamazepina/uso terapéutico , Electrocardiografía , Electroencefalografía , Femenino , Humanos , Malformaciones Arteriovenosas Intracraneales/patología , Malformaciones Arteriovenosas Intracraneales/cirugía , Imagen por Resonancia Magnética , Examen Neurológico , Oxcarbazepina , Marcapaso ArtificialRESUMEN
Manual segmentation from magnetic resonance imaging (MR) is the gold standard for evaluating hippocampal atrophy in Alzheimer's disease (AD). Nonetheless, different segmentation protocols provide up to 2.5-fold volume differences. Here we surveyed the most frequently used segmentation protocols in the AD literature as a preliminary step for international harmonization. The anatomical landmarks (anteriormost and posteriormost slices, superior, inferior, medial, and lateral borders) were identified from 12 published protocols for hippocampal manual segmentation ([Abbreviation] first author, publication year: [B] Bartzokis, 1998; [C] Convit, 1997; [dTM] deToledo-Morrell, 2004; [H] Haller, 1997; [J] Jack, 1994; [K] Killiany, 1993; [L] Lehericy, 1994; [M] Malykhin, 2007; [Pa] Pantel, 2000; [Pr] Pruessner, 2000; [S] Soininen, 1994; [W] Watson, 1992). The hippocampi of one healthy control and one AD patient taken from the 1.5T MR ADNI database were segmented by a single rater according to each protocol. The accuracy of the protocols' interpretation and translation into practice was checked with lead authors of protocols through individual interactive web conferences. Semantically harmonized landmarks and differences were then extracted, regarding: (a) the posteriormost slice, protocol [B] being the most restrictive, and [H, M, Pa, Pr, S] the most inclusive; (b) inclusion [C, dTM, J, L, M, Pr, W] or exclusion [B, H, K, Pa, S] of alveus/fimbria; (c) separation from the parahippocampal gyrus, [C] being the most restrictive, [B, dTM, H, J, Pa, S] the most inclusive. There were no substantial differences in the definition of the anteriormost slice. This survey will allow us to operationalize differences among protocols into tracing units, measure their impact on the repeatability and diagnostic accuracy of manual hippocampal segmentation, and finally develop a harmonized protocol.
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Enfermedad de Alzheimer/patología , Mapeo Encefálico/normas , Hipocampo/patología , Imagen por Resonancia Magnética/normas , Comités Consultivos , Algoritmos , Atrofia , Encuestas Epidemiológicas , Humanos , Interpretación de Imagen Asistida por Computador , Imagen por Resonancia Magnética/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The effect of 0.2% emamectin benzoate (SLICE; Intervet/ Schering-Plough Animal Health, Roseland, New Jersey) administered in top-dressed, pelleted commercial fish feed was evaluated for control of freshwater Argulus sp. in goldfish Carassius auratus and koi carp, a variant of common carp Cyprinus carpio, in freshwater aquaria at 24-25 degrees C. Sixteen individually housed goldfish were each exposed to 37 Argulus. The number of fish lice attached to each fish at the start of the experiment was not determined; however, the total number of motile fish lice in each aquarium (on fish and in the water) was determined at the start and end of each experiment. Eight goldfish were fed the control diet (0 microg x kg fish biomass(-1) x d(-1)) and eight were fed the medicated diet (50 microg x kg fish biomass(-1) x d(-1)) for seven consecutive days. After treatment, fish louse infestation in controls was 20.5 +/- 1.5 (mean +/- SE) lice per fish. No Argulus were found on fish in the treated group. In a separate experiment, 10 individually housed koi were each exposed to 128 Argulus. Five koi were fed the control diet and five were fed a low-dose medicated diet (5 microg x kg fish biomass(-1) x d(-1)) for 7 d. After treatment, fish louse infestation among the controls was 14.6 +/- 3.8 lice per koi. No Argulus were found on koi in the treated group. Hence, a 7-d regimen of oral emamectin benzoate controlled experimental infestation of Argulus when administered to goldfish at 50 microg x kg fish biomass(-1) x d(-1) and to koi at 5 microg x kg fish biomass(-1) x d(-1).
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Arguloida/efectos de los fármacos , Carpas , Enfermedades de los Peces/tratamiento farmacológico , Ivermectina/análogos & derivados , Enfermedades Parasitarias en Animales/tratamiento farmacológico , Animales , Antiparasitarios/uso terapéutico , Enfermedades de los Peces/parasitología , Ivermectina/uso terapéutico , Enfermedades Parasitarias en Animales/parasitologíaRESUMEN
PURPOSE: Some patients with pharmacoresistant epilepsy undergoing the Wada test experience transient shivering. The purpose of this study was to investigate various clinical and radiographic characteristics of these individuals to delineate underlying mechanisms of this phenomenon. METHODS: A systematic review of prospectively collected information on patients undergoing the Wada test was performed. All demographic, clinical, and radiographic information was obtained and reviewed by the appropriate expert in the field; statistical analysis was performed to determine the predictors of transient shivering. RESULTS: A total of 120 consecutive carotid artery injections in 59 patients were included in the study. Shivering was observed in 46% of the patients, and it was not significantly affected by gender, age, location of epileptogenic zone, brain lesion on magnetic resonance imaging (MRI), side of the first injection, duration of the hemiparesis, or excess slow wave activity on electroencephalography (EEG). However, shivering was more likely to follow sodium amobarbital injection if there was no filling of the posterior circulation on cerebral angiogram. DISCUSSION: Transient shivering during the Wada test is common. A transient but selective functional lesion of the anterior hypothalamus produced by the effects of sodium amobarbital may result in disinhibition of the posterior hypothalamus and other brainstem thermoregulatory centers, thereby inducing transient shivering.
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Amobarbital , Regulación de la Temperatura Corporal/fisiología , Encéfalo/fisiología , Dominancia Cerebral/fisiología , Epilepsia/diagnóstico , Lenguaje , Tiritona/fisiología , Adolescente , Adulto , Anciano , Amobarbital/administración & dosificación , Amobarbital/farmacología , Regulación de la Temperatura Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Arteria Carótida Interna , Niño , Dominancia Cerebral/efectos de los fármacos , Electroencefalografía/efectos de los fármacos , Epilepsia/fisiopatología , Epilepsia/cirugía , Femenino , Lateralidad Funcional/efectos de los fármacos , Lateralidad Funcional/fisiología , Humanos , Inyecciones Intraarteriales , Masculino , Memoria/efectos de los fármacos , Memoria/fisiología , Persona de Mediana Edad , Cuidados Preoperatorios/métodos , Estudios ProspectivosRESUMEN
During shipping, ornamental fish can be stressed due to handling, high stocking densities, and deteriorating water quality. Adding sedatives, such as metomidate hydrochloride, to shipping water may improve fish survival rates and the percentage of fish in saleable condition. Although the effects of metomidate hydrochloride on the stress response in fish have been studied, its application as a shipping additive has not been well investigated, particularly for tropical ornamental fishes shipped under industry conditions. Convict cichlids Cichlasoma nigrofasciatum and black mollies Poecilia sphenops were evaluated for 7 d after a 24-h period of exposure (including ground and air transport) to one of four metomidate hydrochloride concentrations: 0.0, 0.2, 0.5, and 1.0 mg/L. Immediate posttransport and cumulative mortality data, as well as 12-h and 7-d posttransport appearance and behavior scores, were generated. In convict cichlids, the highest dose of metomidate hydrochloride (1.0 mg/L) reduced mortality (0% compared with cumulative means of 5.5-9.2% in other groups) and increased the percentage of saleable fish (91.7% were immediately saleable compared with 12.5-50% in other groups). No effect was detected in black mollies at any concentration tested. Metomidate hydrochloride showed promise as a shipping additive for convict cichlids, but further studies are warranted to evaluate species-specific responses in other ornamental species.
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Conducta Animal/efectos de los fármacos , Cíclidos/fisiología , Hipnóticos y Sedantes/farmacología , Ácidos Isonipecóticos/farmacología , Poecilia/fisiología , Animales , Estrés Fisiológico/efectos de los fármacos , TransportesRESUMEN
The spotted green pufferfish (Tetraodon nigroviridis) is an important genetics model animal due to its small, well-mapped genome. However, only wild-caught juveniles and adults are available to researchers. A lack of gametes, fertilized eggs, developing embryos, and other early life stages hampers development of the full potential of T. nigroviridis as a model research species. We report on successful spawning trials using a novel induced spawning technique, ovarian lavage. Chorulon(®) (human chorionic gonadotropin, hCG) was injected into a catheter inserted into the oviduct at a rate of 3 µl/g body weight. In one trial, a female paired with a male spawned in an aquarium at about 72 h post-treatment. In other trials, females were hand-stripped of eggs at 36 h post-treatment. There were 3680 eggs/g of eggs and females produced up to 24% of their body weight in eggs. Hatch resulted from all trials on the 4th day post-fertilization. Ovarian lavage is a simple method for administering spawning hormones, uses a catheter technique similar to that frequently performed to determine egg maturity in broodstock, and eliminates the need for injection.
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Gliomas represent approximately one-third of all intracranial tumors in adults and commonly present clinically with seizures. We report two seizure patients with paradoxical imaging findings on preoperative grading of their cerebral gliomas. A 53-year-old man with a history of temporal lobe epilepsy originating from a mass in the right medial temporal region (patient 1) and a 44-year-old man with a history of predominantly left sided sensory seizures with a mass in the right posterior parietal region (patient 2) underwent presurgical evaluation including MRI and glucose PET, followed by surgery to remove cerebral tumors associated with seizure onset. Preoperatively, patient 1 had a homogenous non-enhancing lesion on MRI and hypometabolism on PET imaging, suggesting a low-grade tumor. Postoperative histopathology was consistent with a glioblastoma multiforme (grade IV). Patient 2 had a heterogeneous lesion with cyst formation, edema, and contrast enhancement on preoperative MRI imaging, and interictal hypermetabolism on PET scan, thus suggesting a high-grade tumor. Postoperative histopathology was consistent with an oligodendroglioma (grade II) without anaplastic features. We conclude preoperative grading of cerebral gliomas may be inaccurate occasionally even in cases with concordant structural and functional imaging findings. This should be considered when counseling patients.
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Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/diagnóstico , Glioma/complicaciones , Glioma/diagnóstico , Adulto , Neoplasias Encefálicas/cirugía , Epilepsia del Lóbulo Temporal/etiología , Epilepsia del Lóbulo Temporal/patología , Fluorodesoxiglucosa F18 , Glioma/cirugía , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Tomografía de Emisión de PositronesRESUMEN
PURPOSE: We sought to determine whether hypersalivation helps lateralize seizure onset during complex partial seizures of temporal lobe origin. Several clinical signs, which help lateralize seizure onset, have been reported in temporal lobe epilepsy (TLE). Increased salivation only occasionally has been reported as a manifestation of partial epilepsy. METHODS: Of 590 consecutive patients admitted for video-EEG monitoring, either as a part of a presurgical evaluation of medically intractable epilepsy or for diagnosis and clarification of their paroxysmal symptoms, we identified 10 patients with ictal hypersalivation as a prominent manifestation of complex partial seizures. We reviewed the clinical features, scalp-sphenoidal video-EEG monitoring, intracarotid amytal (Wada) testing, hippocampal volumetric magnetic resonance imaging (MRI), and fluorodeoxyglucose-positron emission tomography (FDG-PET) scans of these patients. RESULTS: Of the 10 patients with ictal hypersalivation, seven patients had nondominant/right TLE, and three patients had dominant/left TLE. All patients had hippocampal atrophy on volumetric MRI. Eight of the 10 patients underwent standard temporal lobectomy with amygdalohippocampectomy (six right, two left). All of the operated-on patients had a seizure-free (Engel class I) outcome, and their increased salivation resolved. Two patients, who did not undergo surgical treatment, continue to have complex partial seizures with increased salivation. CONCLUSIONS: We conclude that increased salivation as a prominent ictal finding in complex partial seizures of temporal lobe origin is more likely to be of nondominant temporal lobe origin. Further studies with larger numbers of patients are needed to replicate this finding.
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Epilepsia del Lóbulo Temporal/diagnóstico , Sialorrea/diagnóstico , Adulto , Edad de Inicio , Amobarbital/administración & dosificación , Amígdala del Cerebelo/cirugía , Lobectomía Temporal Anterior , Arteria Carótida Interna , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Comorbilidad , Supervivencia sin Enfermedad , Electroencefalografía/estadística & datos numéricos , Epilepsia del Lóbulo Temporal/epidemiología , Epilepsia del Lóbulo Temporal/cirugía , Lateralidad Funcional/efectos de los fármacos , Lateralidad Funcional/fisiología , Hipocampo/patología , Hipocampo/cirugía , Humanos , Inyecciones Intraarteriales , Lenguaje , Imagen por Resonancia Magnética , Persona de Mediana Edad , Monitoreo Fisiológico , Cuidados Preoperatorios , Sialorrea/epidemiología , Resultado del Tratamiento , Grabación de Cinta de VideoRESUMEN
Abnormal metabolism of tryptophan has been implicated in modulation of tumor cell proliferation and immunoresistance. alpha-[(11)C]Methyl-L-tryptophan (AMT) is a PET tracer to measure cerebral tryptophan metabolism in vivo. In the present study, we have measured tumor tryptophan uptake in 40 patients with primary brain tumors using AMT PET and standard uptake values (SUV). Tryptophan metabolism was further quantified in 23 patients using blood input data. Estimates of the volume of distribution (VD') and the metabolic rate constant (k(3)') were calculated and related to magnetic resonance imaging (MRI) and histology findings. All grade II to IV gliomas and glioneuronal tumors showed increased AMT SUV, including all recurrent/residual tumors. Gadolinium enhancement on MRI was associated with high VD' values, suggesting impaired blood-brain barrier, while k(3)' values were not related to contrast enhancement. Low-grade astrocytic gliomas showed increased tryptophan metabolism, as measured by k(3)'. In contrast, oligodendrogliomas showed high VD' values but lower k(3)' as compared with normal cortex. In astrocytic tumors, low grade was associated with high k(3)' and lower VD', while high-grade tumors showed the reverse pattern. The findings show high AMT uptake in primary and residual/recurrent gliomas and glioneuronal tumors. Increased AMT uptake can be due to increased metabolism of tryptophan and/or high volume of distribution, depending on tumor type and grade. High tryptophan metabolic rates in low-grade tumors may indicate activation of the kynurenine pathway, a mechanism regulating tumor cell growth. AMT PET might be a useful molecular imaging method to guide therapeutic approaches aimed at controlling tumor cell proliferation by acting on tryptophan metabolism.