Imaging of Adrenal Masses with Emphasis on Adrenocortical Tumors

Because of the more widespread and frequent use of cross-sectional techniques, mainly computed tomography (CT), an increasing number of adrenal tumors are detected as incidental findings (“incidentalomas”). These incidentaloma patients are much more frequent than those undergoing imaging because of symptoms related to adrenal disease. CT and magnetic resonance imaging (MRI) are in most patients sufficient for characterization and follow-up of the incidentaloma. In a minor portion of patients, biochemical screening reveals a functional tumor and further diagnostic work-up and therapy need to be performed according to the type of hormonal overproduction. In oncological patients, especially when the morphological imaging criteria indicate an adrenal metastasis, biopsy of the lesion should be considered after pheochromocytoma is ruled out biochemically. In the minority of patients in whom CT and MRI fail to characterize the tumor and when time is of essence, functional imaging mainly by positron emission tomography (PET) is available using various tracers. The most used PET tracer, [18F]fluoro-deoxy-glucose (18FDG), is able to differentiate benign from malignant adrenal tumors in many patients. 11C-metomidate (11C-MTO) is a more specialized PET tracer that binds to the 11-beta-hydroxylase enzyme in the adrenal cortex and thus makes it possible to differ adrenal tumors (benign adrenocortical adenoma and adrenocortical cancer) from those of non-adrenocortical origin

Environmental benefits of circular food systems: The case of upcycled protein recovered using genome edited potato

Although essential in the human diet, large quantities of available protein are currently lost or under-utilized within the food system, including protein rich side streams from conventional potato starch production. By using the genome editing technique CRISPR-Cas9, conventional starch potato cultivars can be upgraded to facilitate high-value recovery of potato protein fit for human consumption. In turn, this could support the nessecary transition towards more circular food systems. The aim of this study was to assess what environmental benefits could be gained by shifting from conventional protein recovery practice to a novel approach using genome edited potato. Our results, using consequential life cycle assessment, showed that the novel protein recovery scenario provided substantial environmental savings for every ton potato starch produced, with a reduction in global warming impact, terrestrial acidification, land use and ecosystem damage of −720 kgCO2eq, −13 kgSO2eq, −760 m2a crop eq, and −1.1 × 10−5 species.yr respectively. The potential environmental benefits of using genome edited potato were maintained even when simulating reduced tuber yield, increased production inputs, and substitution of various protein sources. Although currently limited by EU legislation and technical maturity, high-value protein recovery from food side streams holds a promising potential to support sustainable production and circularity within the food system

Decreased defluorination using the novel beta-cell imaging agent [18F]FE-DTBZ-d4 in pigs examined by PET

The aim of the thesis was twofold. The first aim was to radiolabel small molecules by using carbon-11 and fluorine-18 for visualising beta cell mass (BCM) in the pancreas by PET. Diabetes Mellitus (DM) is a chronic metabolic disorder that results from an absolute or relative lack of BCM of endocrine pancreas. The lack of an adequate non-invasive imaging PET probe prevents detailed examination of beta cell loss during onset and progression of DM as well as development of novel treatments and islets transplantation progress. The second aim of the thesis was to radiolabel peptide molecules with fluorine-18 to visualise beta amyloid in Alzheimer’s disease (AD) brain. AD is a chronic, progressive neurodegenerative disorder. Brain penetration study of a labelled peptide, specific for beta amyloid that can cross blood-brain-barrier (BBB), is important to gain knowledge about the fate of the molecule as a diagnostic probe. A series of three novel radioligands for BCM imaging has been developed in this thesis. In paper I, a vesicular monoamine transporter type 2 (VMAT2) specific radioligand [18F]FE-DTBZ-d4 was synthesised in two steps. First step is the nucleophilic [18F]fluorination to produce deuterated-[18F]fluoroethylbromide followed by the O- alkylation of desmethyl-DTBZ precursor to produce [18F]FE-DTBZ-d4. The in vivo pharmacokinetics (PK) studies in pigs by PET/CT demonstrated reduced in vivo defluorination; therefore, it may be an improved potential candidate for imaging VMAT2 dense tissue i.e. islets transplantation in proximity to cortical bone structure. In Paper II, a glucokinase (GK) specific radioligand, [11C]AZ12504948, was synthesised in one step via alkylation of O-desmethyl precursor using [11C]methyl iodide. Both in vitro and in vivo (pig and monkey) studies with [11C]AZ12504948 for imaging GK in the pancreas and liver indicated low specificity. Increased target specificity is required for further progress in GK imaging using PET radioligands. In Paper III, a radioligand for G-protein coupled receptor 44 (GPR44), [11C/3H]AZ Compound X, was synthesised via S-methylation of sodium sulfinate salt in one step using [11C/3H]methyl iodide. In vitro binding of the radioligand, evaluated by autoradiography (ARG) on human and rat pancreatic tissues, confirmed higher specific binding in islets of human pancreatic tissue and no measurable binding in rat pancreas, which is devoid of GPR44. These studies indicate that the radioligand has suitable properties for beta cell imaging with high potential for further preclinical and clinical evaluation. Three novel D-peptides were radiolabelled with fluorine-18 ([18F]ACI-87, [18F]ACI- 88, [18F]ACI-89) by using prosthetic group N-succinimidyl-4-[18F]fluorobenzoate, [18F]SFB, with epsilon (ε)-amino groups of lysine residues of peptide precursors in two steps. First step is the synthesis of [18F]SFB followed by the addition of [18F]SFB via acylation to the peptide molecule. Trimethylammonium salt [N(CH3)3+] precursor for synthesising [18F]SFB as well as the reference standard SFB were synthesised with good yields. Three 19F-peptide reference standards were also synthesised by using SFB. Preliminary ARG measurements were performed in AD and control human brains. ARG demonstrated higher radioligand uptake in the AD brain compared to age-matched control brain, which makes them potential for further use in in vivo testing by PET. However, preliminary PET (in vivo) studies in cynomolgus monkey brain, using these 18F-D-peptides, confirmed too low BBB penetration, making them unsuitable for further use as in vivo PET probes

Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis

Background: The thermostable beta-glucosidase (TnBgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e. g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of TnBgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-beta-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3. Results: Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on beta-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as T-m by differential scanning calorimetry (101.9 degrees C for wt), was kept in the mutated variants and significant decrease (Delta T of 5 -10 degrees C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in K-M and turnover. The K-M-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 x increase for pNPGlc, while the K-M decreased a corresponding extent for Q3. Turnover was only significantly changed using pNPGlc, and was decreased 2-3 x in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 x compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in K-M for this substrate. Conclusion: These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both K-M and turnover. An affinity change, leading to a decreased K-M, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site

Haemophilus influenzae surface fibril (Hsf) is a unique twisted hairpin-like trimeric autotransporter.

The Haemophilus surface fibril (Hsf) is an extraordinary large (2413 amino acids) trimeric autotransporter, present in all encapsulated Haemophilus influenzae. It contributes to virulence by directly functioning as an adhesin. Furthermore, Hsf recruits the host factor vitronectin thereby inhibiting the host innate immune response resulting in enhanced survival in serum. Here we observed by electron microscopy that Hsf appears as an 100nm long fibril at the bacterial surface albeit the length is approximately 200nm according to a bioinformatics based model. To unveil this discrepancy, we denaturated Hsf at the surface of Hib by using guanidine hydrochloride (GuHCl). Partial denaturation induced in the presence of GuHCl unfolded the Hsf molecules, and resulted in an increased length of fibres in comparison to the native trimeric form. Importantly, our findings were also verified by E. coli expressing Hsf at its surface. In addition, a set of Hsf-specific peptide antibodies also indicated that the N-terminal of Hsf is located near the C-terminal at the base of the fibril. Taken together, our results demonstrated that Hsf is not a straight molecule but is folded and doubled over. This is the first report that provides the unique structural features of the trimeric autotransporter Hsf

Lessons on Tumour Response: Imaging during Therapy with 177Lu-DOTA-octreotate. A Case Report on a Patient with a Large Volume of Poorly Differentiated Neuroendocrine Carcinoma

Favourable outcomes of peptide receptor radiotherapy (PRRT) of neuroendocrine tumours have been reported during the last years. Still, there are uncertainties on the radionuclides to be used, the treatment planning, and the indication in patients with a high proliferation rate

Para-chloro-2-[18F]fluoroethyl-etomidate: A promising new PET radiotracer for adrenocortical imaging.

Introduction: [11C]Metomidate ([11C]MTO), the methyl ester analogue of etomidate, was developed as a positron emission tomography (PET) radiotracer for adrenocortical tumours and has also been suggested for imaging in primary aldosteronism (PA). A disadvantage of [11C]MTO is the rather high non-specific binding in the liver, which impacts both visualization and quantification of the uptake in the right adrenal gland. Furthermore, the short 20-minute half-life of carbon-11 is a logistic challenge in the clinical setting. Objectives: The aim of this study was to further evaluate the previously published fluorine-18 (T1/2=109.5 min) etomidate analogue, para-chloro-2-[18F]fluoroethyl etomidate; [18F]CETO, as an adrenal PET tracer. Methods: In vitro experiments included autoradiography on human and cynomolgus monkey (non-human primate, NHP) tissues and binding studies on adrenal tissue from NHPs. In vivo studies with [18F]CETO in mice, rats and NHP, using PET and CT/MRI, assessed biodistribution and binding specificity in comparison to [11C]MTO. Results: The binding of [18F]CETO in the normal adrenal cortex, as well as in human adrenocortical adenomas and adrenocortical carcinomas, was shown to be specific, both in vitro (in humans) and in vivo (in rats and NHP) with an in vitro Kd of 0.66 nM. Non-specific uptake of [18F]CETO in NHP liver was found to be low compared to that of [11C]MTO. Conclusions: High specificity of [18F]CETO to the adrenal cortex was demonstrated, with in vivo binding properties qualitatively surpassing those of [11C]MTO. Non-specific binding to the liver was significantly lower than that of [11C]MTO. [18F]CETO is a promising new PET tracer for imaging of adrenocortical disease and should be evaluated further in humans

Geographic Differentials in Mortality of Children in Mozambique: Their Implications for Achievement of Millennium Development Goal 4

In the light of Mozambique's progress towards the achievement of Millennium Development Goal 4 of reducing mortality of children aged less than five years (under-five mortality) by two-thirds within 2015, this study investigated the relationship between the province of mother's residence and under-five mortality in Mozambique, using data from the 2003 Mozambican Demographic and Health Survey. The analyses included 10,326 children born within 10 years before the survey. Results of univariate and multivariate analyses showed a significant association between under-five mortality and province (region) of mother's residence. Children of mothers living in the North provinces (Niassa, Cabo Delgado, and Nampula) and the Central provinces (Zambezia, Sofala, Manica, and Tete) had higher risks of mortality than children whose mothers lived in the South provinces, especially Maputo province and Maputo city. However, controlling for the demographic, socioeconomic and environmental variables, the significance found between the place of mother's residence and under-five mortality reduced slightly. This suggests that other variables (income distribution and trade, density of population, distribution of the basic infrastructure, including healthcare services, climatic and ecologic factors), which were not included in the study, may have confounding effects. This study supports the thought that interventions aimed at reducing under-five mortality should be tailored to take into account the subnational/regional variation in economic development. However, research is warranted to further investigate the potential determinants behind the observed differences in under-five mortality

imaging and biomarkers in gastroenteropancreatic neuroendocrine tumor disease management

The complexity of the clinical management of neuroendocrine neoplasia (NEN) is exacerbated by limitations in imaging modalities and a paucity of clinically useful biomarkers. Limitations in currently available imaging modalities reflect difficulties in measuring an intrinsically indolent disease, resolution inadequacies and inter-/intra-facility device variability and that RECIST (Response Evaluation Criteria in Solid Tumors) criteria are not optimal for NEN. Limitations of currently used biomarkers are that they are secretory biomarkers (chromogranin A, serotonin, neuron-specific enolase and pancreastatin); monoanalyte measurements; and lack sensitivity, specificity and predictive capacity. None of them meet the NIH metrics for clinical usage. A multinational, multidisciplinary Delphi consensus meeting of NEN experts (n = 33) assessed current imaging strategies and biomarkers in NEN management. Consensus (>75%) was achieved for 78% of the 142 questions. The panel concluded that morphological imaging has a diagnostic value. However, both imaging and current single-analyte biomarkers exhibit substantial limitations in measuring the disease status and predicting the therapeutic efficacy. RECIST remains suboptimal as a metric. A critical unmet need is the development of a clinico-biological tool to provide enhanced information regarding precise disease status and treatment response. The group considered that circulating RNA was better than current general NEN biomarkers and preliminary clinical data were considered promising. It was resolved that circulating multianalyte mRNA (NETest) had clinical utility in both diagnosis and monitoring disease status and therapeutic efficacy. Overall, it was concluded that a combination of tumor spatial and functional imaging with circulating transcripts (mRNA) would represent the future strategy for real- time monitoring of disease progress and therapeutic efficacy