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1.
J Hosp Infect ; 140: 90-95, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37562590

ABSTRACT

OBJECTIVES: To compare intensivist-diagnosed ventilator-associated pneumonia (iVAP) with four established definitions, assessing their agreement in detecting new episodes. METHODS: A multi-centric prospective study on pulmonary microbiota was carried out in patients requiring mechanical ventilation (MV). Data collected were used to compare hypothetical VAP onset according to iVAP with the study consensus criteria, the European Centre for Disease Control and Prevention definition, and two versions of the latter adjusted for leukocyte count and fever. RESULTS: In our cohort of 186 adult patients, iVAPs were 36.6% (68/186, 95% confidence interval 30.0-44.0%), with an incidence rate of 4.64/100 patient-MV-days, and median MV-day at diagnosis of 6. Forty-seven percent of patients (87/186) were identified as VAP by at least one criterion, with a median MV-day at diagnosis of 5. Agreement between intensivist judgement (iVAP/no-iVAP) and the criteria was highest for the study consensus criteria (50/87, 57.4%), but still one-third of iVAP were not identified and 9% of patients were identified as VAP contrary to intensivist diagnosis. VAP proportion differed between criteria (25.2-30.1%). CONCLUSIONS: Caution is needed when evaluating studies describing VAP incidence. Pre-agreed criteria and definitions that capture VAP's evolving nature provide greater consistency, but new clinically driven definitions are needed to align surveillance and diagnostic criteria with clinical practice.


Subject(s)
Pneumonia, Ventilator-Associated , Adult , Humans , Pneumonia, Ventilator-Associated/diagnosis , Pneumonia, Ventilator-Associated/epidemiology , Pneumonia, Ventilator-Associated/prevention & control , Respiration, Artificial/adverse effects , Prospective Studies , Preliminary Data , Incidence , Intensive Care Units
2.
Cytopathology ; 26(5): 318-24, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25040579

ABSTRACT

OBJECTIVE: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique proteomic technology that explores the spatial distribution of biomolecules directly in situ, thus integrating molecular and morphological information. The possibility of correlating distribution maps of multiple analyses with cytological features makes it an ideal research tool for discovering new diagnostic markers. A previous study showed that MALDI-IMS could help discrimination between different types of thyroid lesions, especially papillary thyroid carcinoma (PTC); the present feasibility study on ex vivo fine needle aspiration (FNA) smears describes its potential in detecting new proteomic targets of other thyroid lesions (follicular lesions, medullary carcinoma). METHODS: MALDI-IMS was conducted on ex vivo FNAs obtained from surgical specimens and corresponding in vivo samples. Differences between proteomic profiles of different thyroid lesions were compared. RESULTS: Comparing the protein profiles of hyperplastic nodules obtained from three different patients with each other, and with a new PTC, showed a high degree of concordance, indicating good reproducibility of the IMS technology on cytological samples, suggesting its potential as a tool for biomarker discovery. Furthermore, comparison of the average proteomic profiles of hyperplastic nodules with a Hürthle cell adenoma revealed significant differences, underlying the capability of MALDI-IMS to distinguish between different thyroid lesions. Finally, the proteomic profile of medullary thyroid carcinoma was also characterized. CONCLUSIONS: Our results confirmed the possible role of MALDI-IMS in the search for diagnostic targets of PTC and follicular lesions, which could be applied in larger trials aimed at the identification of proteins, convertible to cost-effective diagnostic tools such as immunohistochemistry. These tests could be used to analyse in vivo cytological smears, improving the preoperative diagnosis of indeterminate thyroid nodules.


Subject(s)
Thyroid Gland/metabolism , Thyroid Gland/pathology , Thyroid Neoplasms/diagnosis , Thyroid Neoplasms/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers/metabolism , Biopsy, Fine-Needle/methods , Carcinoma/metabolism , Carcinoma/pathology , Carcinoma, Medullary/metabolism , Carcinoma, Medullary/pathology , Carcinoma, Neuroendocrine/metabolism , Carcinoma, Neuroendocrine/pathology , Carcinoma, Papillary , Female , Humans , Male , Middle Aged , Proteomics/methods , Thyroid Cancer, Papillary , Thyroid Neoplasms/pathology
3.
Mol Biosyst ; 9(6): 1220-33, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23511837

ABSTRACT

Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties. Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE. Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery.


Subject(s)
Carcinoma, Renal Cell/metabolism , Exosomes/metabolism , Kidney Neoplasms/metabolism , Proteome/analysis , Adult , Aged , Aged, 80 and over , Biomarkers , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Female , Humans , Kidney/metabolism , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Male , Middle Aged , Protein Array Analysis , Proteins/analysis , Proteomics
4.
Mol Biosyst ; 9(6): 1139-46, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23344851

ABSTRACT

Urinary exosomes (UE) are nanovesicles released by every epithelial cell facing the urinary space and they are considered a promising source of molecular markers for renal dysfunction and structural injury. Exosomal proteomics has emerged as a powerful tool for understanding the molecular composition of exosomes and has potential to accelerate biomarker discovery. We employed this strategy in the study of diabetic nephropathy (DN) and the consequent end stage renal disease, which represent the dramatic evolution of diabetes, often leading the patients to dialysis or kidney transplantation. The identification of DN biomarkers is likely to help monitoring the disease onset and progression. A label free LC-MS/MS approach was applied to investigate the alteration of the proteome of urinary exosomes isolated from the Zucker diabetic fatty rats (ZDF), as a model of type 2 DN. We collected 24 hour urine samples from 7 ZDF and from 7 control rats at different ages (6, 12 and 20 weeks old) to monitor the development of DN. Exosomes were isolated by ultracentrifugation and their purity assessed by immunoblotting for known exosomal markers. Exosomal proteins from urine samples of 20 week old rats were pooled and analyzed by nLC-ESI-UHR-QToF-MS/MS after pre-filtration and tryptic digestion, leading to the identification and label free quantification of 286 proteins. Subcellular localization and molecular functions were assigned to each protein by UniprotKB, showing that the majority of identified proteins were membrane-associated or cytoplasmic and involved in transport, signalling and cellular adhesion, typical functions of exosomal proteins. We further validated label free mass spectrometry results by immunoblotting, as exemplified by: Xaa-Pro dipeptidase, Major Urinary Protein 1 and Neprilysin, which resulted increased, decreased and not different, respectively, in exosomes isolated from diabetic urine samples compared to controls, by both techniques. In conclusion we show the potential of exosome proteomics for DN biomarker discovery.


Subject(s)
Diabetes Mellitus, Type 2/metabolism , Diabetic Nephropathies/metabolism , Exosomes/chemistry , Proteins/analysis , Proteomics , Urinary Tract/metabolism , Animals , Biomarkers/metabolism , Biomarkers/urine , Diabetes Mellitus, Type 2/pathology , Diabetic Nephropathies/genetics , Diabetic Nephropathies/pathology , Exosomes/genetics , Male , Mass Spectrometry , Proteome/analysis , Proteome/metabolism , Rats , Rats, Zucker , Urinary Tract/pathology
5.
Mol Biosyst ; 8(4): 1007-16, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22159573

ABSTRACT

Renal cell carcinoma (RCC) is representing about 3% of all adult cancers. A promising strategy for cancer biomarker discovery is subcellular comparative proteomics, allowing enriching specific cell compartments and assessing differences in protein expression patterns. We investigated the proteomic profile of a peculiar RCC subcellular compartment, plasma membrane microdomains (MD), involved in cell signalling, transport, proliferation and in many human diseases, such as cancer. Subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after Triton X-100 treatment and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues were analyzed after SDS-PAGE separation by LC-ESI-MS/MS. We identified 93 proteins from MD isolated from RCC tissue, and 98 proteins from ANK MD. About 70% of the identified proteins are membrane-associated and about half of these are known as microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. We chose a panel of proteins to validate their differential expression by WB. In conclusion, our work shows that RCC microdomain proteome is reproducibly different from ANK, and suggests that mining into such differences may support new biomarker discovery.


Subject(s)
Carcinoma, Renal Cell/genetics , Kidney Neoplasms/genetics , Kidney/cytology , Neoplasm Proteins/genetics , Protein Array Analysis/methods , Adult , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Electrophoresis, Polyacrylamide Gel , Female , Gene Expression Profiling , Humans , Kidney/metabolism , Kidney/pathology , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Male , Membrane Microdomains/genetics , Membrane Microdomains/metabolism , Neoplasm Proteins/metabolism , Octoxynol , Proteome/genetics , Proteomics/methods , Tandem Mass Spectrometry
6.
J Proteomics ; 73(3): 593-601, 2010 Jan 03.
Article in English | MEDLINE | ID: mdl-19631771

ABSTRACT

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease affecting about 0.12% of the world's population. Diabetic nephropathy (DN) is a major long-term complication of both types of diabetes and retains a high human, social and economic cost. Thus, the identification of markers for the early detection of DN represents a relevant target of diabetic research. The present work is a pilot study focused on proteomic analysis of serum of controls (n=9), IDDM patients (n=10) and DN patients (n=4) by the ClinProt profiling technology based on mass spectrometry. This approach allowed to identify a pattern of peptides able to differentiate the studied populations with sensitivity and specificity close to 100%. Variance of the results allowed to estimate the sample size needed to keep the expected False Discovery Rate low. Moreover, three peptides differentially expressed in the serum of patients as compared to controls were identified by LC-ESI MS/MS as the whole fibrinopeptide A peptide and two of its fragments, respectively. The two fragments were under-expressed in diabetic patients, while Fibrinopeptide A was over-expressed, suggesting that anomalous turnover of Fibrinopeptide A could be involved in the pathogenesis of DN.


Subject(s)
Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/complications , Diabetic Nephropathies/blood , Fibrinopeptide A/analysis , Peptide Fragments/blood , Adult , Area Under Curve , Blood Pressure/physiology , Case-Control Studies , Chromatography, Liquid/methods , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 1/physiopathology , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/physiopathology , Female , Fibrinopeptide A/chemistry , Fibrinopeptide A/metabolism , Humans , Male , Metabolome , Middle Aged , Peptide Fragments/analysis , Peptide Fragments/metabolism , Pilot Projects , Proteome/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods
7.
Appl Radiat Isot ; 64(2): 182-6, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16169239

ABSTRACT

An improved synthesis of the precursor acetic acid-piperidine-4-yl ester by acetylation of 4-hydroxypiperidine hydrochloride in anhydrous chloroform was developed. A procedure for fast evaluation and characterization of products originated by acetylation of the 4-piperidinol using LC-APCI/MS with an acetonitrile-water gradient method on a Merck Purosphere RP-18 column was also developed. The highly purified precursor allowed the production of [11C]MP4A for PET studies of acetylcholine neurotransmission system. The tracer was produced with >98% radiochemical purity, with yields ranging 20-60% (decay-corrected) from EOB.


Subject(s)
Acetates/chemical synthesis , Hydrocarbons, Iodinated/chemistry , Piperidines/chemical synthesis , Radiopharmaceuticals/chemical synthesis , Acetates/chemistry , Acetylation , Acetylcholinesterase/analysis , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Humans , Isotope Labeling/methods , Magnetic Resonance Spectroscopy , Piperidines/chemistry , Positron-Emission Tomography/methods , Radiopharmaceuticals/chemistry
8.
Rapid Commun Mass Spectrom ; 16(15): 1485-93, 2002.
Article in English | MEDLINE | ID: mdl-12125026

ABSTRACT

Alzheimer's disease is characterised by brain neuritic plaques composed of a 39-44 amino acid peptide (Abeta). Lipid peroxidation is an early event induced by these amyloid beta-peptides, leading to the formation of 4-hydroxy-2-nonenal (HNE), which is one of the major end products of this process. HNE has been reported to form adducts via a stable covalent binding to proteins through a Michael addition to amino acid residues with a nucleophilic side chain. The present study reports an investigation of the conditions for formation of Abeta-HNE (Abeta 1-28 and Abeta 1-42) adducts, and their characterisation by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). The results suggest that one or more HNE moieties are localised in the 6-16 region of these adducts, while Asp-1, Lys-16 and Lys-28 are not modified under the described reaction conditions.


Subject(s)
Aldehydes/analysis , Amyloid beta-Peptides/analysis , Lipid Peroxidation , Amino Acid Sequence , Amino Acids/analysis , Chromatography, Liquid , Hydrolysis , Molecular Sequence Data , Spectrometry, Mass, Electrospray Ionization , Trypsin
9.
Theor Appl Genet ; 104(6-7): 1035-1041, 2002 May.
Article in English | MEDLINE | ID: mdl-12582609

ABSTRACT

The development of microsatellite markers can be a time-consuming process, especially in species such as conifers where many microsatellites have been shown to be associated with the repetitive fraction of the genome and to produce complex banding patterns following electrophoresis. Therefore, procedures to eliminate this fraction from further processing are sought. In this paper, we report on the development of 53 dinucleotide SSR markers in Norway spruce, 35 of which (66%) produce simple, polymorphic patterns. This high efficiency is obtained by introducing a dot-blot selection against high copy number sequences, performed on the microsatellite-containing clones. The resulting markers turned out to be polymorphic and useful for population genetic studies and for linkage mapping. Seven additional markers that were not subject to the dot-blot selection are also presented.

10.
Theor Appl Genet ; 106(1): 40-50, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12582869

ABSTRACT

Trinucleotide microsatellites have proven to be the markers of choice in human genetic analysis because they are easier to genotype than dinucleotides. Their development can be more time-consuming due to their lower abundance in the genome. We isolated trinucleotide microsatellites in Norway spruce ( Picea abies K.) using an enrichment procedure for the genomic-library construction. Here we report on the characterisation of 85 ATC microsatellite-containing clones, from which 39 markers were developed. Many of the clones showed the occurrence of tandem repeats of higher order than the trinucleotide ones, often resembling minisatellite repeats. The sequencing of a sample of the alleles at one of the loci revealed size homoplasy due to base substitutions within the microsatellite region. The presence of ATC motifs within repetitive sequence families was observed. We found a significant relationship between the level of polymorphism and the length of the microsatellite. The levels of variability for ATC trinucleotide markers were lower than those for dinucleotides, both when tested on all loci in a set of six individuals and on a subset of loci in four natural populations. This difference is most likely attributable to lower mutation rates for trinucleotide than for dinucleotide loci. The availability of markers with different mutation rates allows one to select the proper marker set to investigate population processes on different time scales.


Subject(s)
Genetic Markers , Picea/genetics , Trinucleotide Repeats , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Polymorphism, Genetic
11.
Anal Biochem ; 298(2): 181-8, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11700972

ABSTRACT

Tumor pretargeting with biotinylated antibody/avidin complexes improves the therapeutic index of systemically administered biotin-tumor necrosis factor (TNF) conjugates. Since the number of biotins in this conjugate is known to be critical for activity, we have characterized the structure of different biotin-TNF conjugates, prepared by reaction with d-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester and identified the biotinylation sites by trypsin digestion, reverse-phase chromatography, and electrospray mass spectrometry analyses. The results have shown that N-terminal valine is a preferential biotinylation site at pH 5.8, half of biotins being located on the alpha-amino group of this residue in a conjugate bearing one biotin/trimer (on average). Moreover, evidence has been obtained to suggest that the remaining part of biotins are linked to the epsilon-amino group of lysine 128, 112, and 65, while lysine 11, 90, and 98 were practically unmodified. No evidence of O-biotinylation of serine, threonine and tyrosine was obtained.


Subject(s)
Biotin/metabolism , Tumor Necrosis Factor-alpha/metabolism , Amino Acid Sequence , Animals , Binding Sites , Biotinylation , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Fibroblasts/physiology , Humans , Mass Spectrometry/methods , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Trypsin/metabolism
12.
J Mass Spectrom ; 36(6): 670-6, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11433541

ABSTRACT

Therapy with cyclosporin A (CsA) for immunosuppression after organ transplantation requires monitoring of its levels in blood owing to the narrow therapeutic index of the drug and to the high inter-individual variability of the drug absorption and metabolism. We describe the preparation of CsA labelled with stable isotopes ((13)C and (2)H) with an isotopic enrichment of about 99% using labelled glucose and its use as internal standard for quantification of CsA blood levels by isotope dilution/electrospray ionization mass spectrometry. The method was found to be linear in the tested range (1-1000 ng) with and without the matrix. The accuracy of the bracketting calibration curves prepared using 100 ng ml(-1) labelled CsA was within +/-1.7% (bias). The results confirmed the usefulness of the procedure as a reference method for the external quality assessment of the field methods for the evaluation of CsA blood concentration, the imprecision (relative standard deviation) and accuracy (bias) being <2%.


Subject(s)
Cyclosporine/blood , Immunosuppressive Agents/blood , Chromatography, High Pressure Liquid , Chromatography, Liquid , Humans , Solutions , Spectrometry, Mass, Electrospray Ionization
13.
Mol Ecol ; 10(5): 1255-63, 2001 May.
Article in English | MEDLINE | ID: mdl-11380882

ABSTRACT

Populations from 13 elevational transects of Norway spruce [Picea abies (L.) Karst] across the Alpine range were sampled to elucidate the geographical pattern of genetic variation in relation to postglacial re-colonization and to study elevational effects on haplotypic diversity. We assessed fragment length variation in a tandem repeat region of the mitochondrial (mt) nad1 intron 2. This maternally inherited genetic marker is suited to infer migration as it is dispersed by seed only. A total of 10 haplotypes was found, most of which were due to repeat copy number variation. An analysis of molecular variance (amova) showed that overall population differentiation was high (F(ST)=0.41), and it revealed a significant differentiation between monomorphic western and moderately to highly variable eastern Alpine populations. This phylogeographic pattern may be explained by a founder effect during postglacial re-colonization. An early arriving haplotype, assumed to originate from a western Carpathian refugium, could expand into suitable habitats, reducing the chances for establishment of subsequently arriving haplotypes. On the other hand, the high variation in populations within an Italian transect of the south-eastern Alps may be the consequence of merging migration pathways from and close distance to putative glacial refugia, most likely those assumed in the Carpathian mountains and on the Balkan peninsula or possibly in the central plains of Italy. An effect of elevation on haplotypic diversity was not evident, though a low, but significant, partition of total genetic variation was attributed to among-population variation in one Italian transect. Various factors, such as vertical seed dispersal and forest management, may account for blurring an otherwise established pattern of genetic variation on a small geographical scale.


Subject(s)
DNA, Mitochondrial , Genetics, Population , Haplotypes , Tandem Repeat Sequences , Trees/genetics , Analysis of Variance , Biological Evolution , Europe , Founder Effect , Trees/physiology
14.
Diabetes Care ; 24(5): 875-80, 2001 May.
Article in English | MEDLINE | ID: mdl-11347747

ABSTRACT

UNLABELLED: The aim of this study was to evaluate whether long-term administration of arginine acting through a normalization of NO/cyclic-guanosine-3' 5'-cyclic monophosphate (cGMP) pathway was able to ameliorate peripheral and hepatic insulin sensitivity in 12 lean type 2 diabetic patients. RESEARCH DESIGN AND METHODS: A double-blind study was performed for 3 months. In the first month, patients were treated with their usual diet. Then they were randomly allocated into to groups. In group 1, patients were treated with diet plus placebo (orally three times per day) for 2 months. In group 2 patients were treated for 1 month with diet plus placebo orally, three times per day) and then for 1 month with diet plus L-arginine (3 g three times per day). At the end of the first and the second month of therapy, patients underwent a euglycemic-hyperinsulinemic clamp combined with [6,6-2H2] glucose infusion. A total of 10 normal subjects underwent the same test as control subjects. RESULTS: In group 1, no changes in basal cGMP levels, systolic blood pressure, forearm blood flow, glucose disposal, and endogenous glucose production were observed throughout. In group 2, L-arginine normalized basal cGMP levels and significantly increased forearm blood flow by 36% and glucose disposal during the clamp by 34% whereas it decreased systolic blood pressure and endogenous glucose production by 14 and 29%, respectively. However, compared with normal subjects, L-arginine treatment was not able to completely overcome the defect in glucose disposal. CONCLUSIONS: L-Arginine treatment significantly improves but does not completely normalizc peripheral and hepatic insulin sensitivity in type 2 diabetic patients.


Subject(s)
Arginine/therapeutic use , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/physiopathology , Insulin/blood , Liver/physiopathology , Administration, Oral , Arginine/administration & dosage , Blood Glucose/metabolism , Blood Pressure , Body Weight , Cyclic GMP/blood , Diabetes Mellitus, Type 2/diet therapy , Diet, Diabetic , Double-Blind Method , Forearm/blood supply , Glucose Clamp Technique , Glycated Hemoglobin/analysis , Heart Rate , Humans , Insulin/metabolism , Insulin Secretion , Liver/drug effects , Middle Aged , Potassium/blood , Reference Values , Regional Blood Flow
15.
J Med Chem ; 44(4): 579-85, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11170647

ABSTRACT

The novel quinoline-2-carboxamide derivatives N-[methyl-11C]-3-methyl-4-phenyl-N-(phenylmethyl)quinoline-2-carboxamide ([11C]4), (+/-)-N-[methyl-11C]-3-methyl-N-(1-methylpropyl)-4-phenylquinoline-2-carboxamide ([11C]5), and (+/-)-N-[methyl-11C]-3-methyl-4-(2-fluorophenyl)-N-(1-methylpropyl)quinoline-2-carboxamide ([11C]6) were labeled with carbon-11 (t1/2 = 20.4 min, beta+ = 99.8%) as potential radioligands for the noninvasive assessment of peripheral benzodiazepine type receptors (PBR) in vivo with positron emission tomography (PET). The radiosynthesis consisted of N-methylation of the desmethyl precursors 3-methyl-4-phenyl-N-(phenylmethyl)quinoline-2-carboxamide (4a), (+/-)-3-methyl-N-(1-methylpropyl)-4-phenylquinoline-2-carboxamide (5a), and (+/-)-4-(2-fluorophenyl)-3-methyl-N-(1-methylpropyl)quinoline-2-carboxamide (6a) with either [11C]methyl iodide or [11C]methyl triflate in the presence of tetrabutylammonium hydroxide or potassium hydroxide in dimethylformamide. The radioligands [11C]4, [11C]5, and [11C]6 were synthesized with over 99% radiochemical purity in 30 min, 30 +/- 5% radiochemical yield, calculated at the end of synthesis (EOS) non-decay-corrected, and 2.5 +/- 1.2 Ci/micromol of specific radioactivity. Inhibition studies in rats following intravenous pre-administration of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195, 1) showed high specific binding to PBR of [11C]4, [11C]5, and [11C]6 in heart, lung, kidney, adrenal gland, spleen, and brain. The biological data suggest that [11C]5, [11C]6, and particularly [11C]4 are promising radioligands for PBR imaging in vivo with PET.


Subject(s)
Amides/chemical synthesis , Quinolines/chemical synthesis , Radiopharmaceuticals/chemical synthesis , Receptors, GABA-A/metabolism , Amides/chemistry , Amides/metabolism , Animals , Carbon Radioisotopes , Isotope Labeling , Ligands , Male , Methylation , Quinolines/chemistry , Quinolines/metabolism , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/metabolism , Rats , Tissue Distribution , Tomography, Emission-Computed
16.
Neurochem Int ; 38(2): 169-80, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11137886

ABSTRACT

(+/-)-1-[4-(2-Isopropoxyethoxymethyl)-phenoxy]-3-isopropylamino-2-propanol (bisoprolol) is a potent, clinically used beta(1)-adrenergic agent. (R)-(+) and (S)-(-) enantiomers of bisoprolol were labelled with carbon-11 (t(1/2)=20.4 min) as putative tracers for the non-invasive assessment of the beta(1)-adrenoceptor subtype in the human heart and brain with positron emission tomography (PET). The radiosynthesis consisted of reductive alkylation of des-iso-propyl precursor with [2-11C]acetone in the presence of sodium cyanoborohydride and acetic acid. The stereo-conservative synthesis of (R)-(+) and (S)-(-)-1-[4-(2-isopropoxyethoxymethyl)-phenoxy]-3-amino-2-propanol to be used as the precursors for the radiosynthesis of [11C]bisoprolol enantiomers was readily accomplished by the use of the corresponding chiral epoxide in three steps starting from the commercially available hydroxybenzyl alcohol. The final labelled product (either (+) or (-)-1-[4-(-isopropoxyethoxymethyl)-phenoxy]-3- [11C]isopropylamino-2-propanol) was obtained in 99% radiochemical purity in 30 min with 15+/-5% (EOS, non-decay corrected) radiochemical yield and 3.5+/-1 Ci/micromol specific radioactivity. Preliminary biological evaluation of the tracer in rats showed that about 30% of heart uptake of [11C](S)-bisoprolol is due to specific binding. The high non-specific uptake in lung might mask the heart uptake, thus precluding the use of [11C](S)-bisoprolol for heart and lung studies by PET. The remarkably high uptake of the tracer in rat brain areas rich of beta-adrenergic receptors such as pituitary (1.8+/-0.3% I.D. at 30 min) was blocked by pre-treatment with the beta-adrenergic antagonists propranolol (45%) and bisoprolol (51%, p<0.05). [11C](S)-bisoprolol deserves further evaluation in other animal models as a putative beta(1) selective radioligand for in vivo investigation of central adrenoceptors.


Subject(s)
Bisoprolol/metabolism , Receptors, Adrenergic, beta/metabolism , Animals , Bisoprolol/chemical synthesis , Bisoprolol/chemistry , Brain/metabolism , Ligands , Lung/metabolism , Male , Myocardium/metabolism , Rats , Rats, Inbred Strains , Stereoisomerism , Tissue Distribution
17.
Appl Radiat Isot ; 54(1): 93-5, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11144258

ABSTRACT

The procedure previously reported for the radiosynthesis of [123I]betaCIT was modified in order to improve both radiochemical yield and purity of betaCIT (2beta-carbomethoxy-3beta(4-iodophenyl) tropane) to be injected for SPECT (Single Photon Emission Computed Tomography) analysis imaging. The overall procedure, involving a HPLC purification step, results in quite good and reproducible yields of a highly purified tracer.


Subject(s)
Brain/diagnostic imaging , Brain/metabolism , Cocaine/chemical synthesis , Dopamine/metabolism , Iodine Radioisotopes , Radiopharmaceuticals/chemical synthesis , Serotonin/metabolism , Tomography, Emission-Computed, Single-Photon/methods , Chromatography, High Pressure Liquid , Cocaine/analogs & derivatives , Cocaine/pharmacokinetics , Humans , Ligands , Radiopharmaceuticals/pharmacokinetics
18.
Nat Biotechnol ; 18(11): 1185-90, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11062439

ABSTRACT

The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local treatments because of its dose-limiting systemic toxicity. We show here that murine TNF fused with CNGRC peptide (NGR-TNF), an aminopeptidase N (CD13) ligand that targets activated blood vessels in tumors, is 12-15 times more efficient than murine TNF in decreasing the tumor burden in lymphoma and melanoma animal models, whereas its toxicity is similar. Similarly, human NGR-TNF induced stronger antitumor effects than human TNF, even with 30 times lower doses. Coadministration of murine NGR-TNF with a CNGRC peptide or an anti-CD13 antibody markedly decreased its antitumor effects. Tumor regression, induced by doses of murine NGR-TNF lower than the LD50, was accompanied by protective immunity. In contrast, no cure was induced by TNF at any dose. These results suggest that targeted delivery of TNF to CD13 may enhance its immunotherapeutic properties. Moreover, these findings reveal the potential of tumor homing peptides to generate a new class of recombinant cytokines that compared to immunocytokines have a simpler structure, could be easier to produce and are potentially less immunogenic.


Subject(s)
CD13 Antigens/metabolism , Neoplasms/therapy , Tumor Necrosis Factor-alpha/therapeutic use , Animals , CD13 Antigens/genetics , CD13 Antigens/therapeutic use , Cell Line , Cell Separation , Chromatography, Gel , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , HL-60 Cells , Humans , Ligands , Lymphoma/drug therapy , Mass Spectrometry , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Neoplasms/drug therapy , Neoplasms, Experimental/drug therapy , Neovascularization, Pathologic , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time Factors , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , U937 Cells
19.
Nucl Med Biol ; 27(6): 565-9, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11056370

ABSTRACT

This paper describes the radiosynthesis of [(11)C]CGP62349, a potential ligand to assess GABA(B) receptors in vivo. (11)C was introduced by O-methylation of the corresponding des-methyl precursor, namely CGP67780. The final product was obtained with a reliable method in good yield. The radioligand was tested in monkey, revealing negligible blood-brain barrier penetration and brain uptake, thus prompting us to search for a new target structure with a better lipophilicity.


Subject(s)
Benzoates/chemical synthesis , Benzoates/pharmacokinetics , Brain/metabolism , GABA-B Receptor Antagonists , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/pharmacokinetics , Animals , Benzoates/blood , Benzoates/chemistry , Blood-Brain Barrier/drug effects , Brain/blood supply , Brain/diagnostic imaging , Carbon Radioisotopes/chemistry , Chromatography, High Pressure Liquid , Female , Hydrocarbons, Iodinated/chemistry , Ligands , Macaca nemestrina , Mass Spectrometry , Organophosphorus Compounds/blood , Phosphinic Acids/chemistry , Radioactive Tracers , Radioligand Assay , Receptors, GABA-B/metabolism , Tomography, Emission-Computed
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