In vivo mapping of vascular inflammation using the translocator protein tracer 18F-FEDAA1106.

Noninvasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro-2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5-methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18F-FEDAA1106 and 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG, a marker of glucose metabolism) for positron emission tomographic (PET) imaging of vascular inflammation. This was tested using a murine model in which focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18F-FEDAA1106 or 18F-FDG was registered to anatomic data generated by computed tomographic (CT)/CT angiography. Standardized uptake values were significantly increased at cuffed compared to contralateral arteries using either 18F-FEDAA1106 (p < .01) or FDG (p < .05). However, the 18F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the noninflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18F-FEDAA1106 corresponded with vascular inflammation more specifically than FDG uptake.

Improving IVF Utilization with Patient-Centric Artificial Intelligence-Machine Learning (AI/ML): A Retrospective Multicenter Experience.

Objectives: In vitro fertilization (IVF) has the potential to give babies to millions more people globally, yet it continues to be underutilized. We established a globally applicable and locally adaptable IVF prognostics report and framework to support patient-provider counseling and enable validated, data-driven treatment decisions. This study investigates the IVF utilization rates associated with the usage of machine learning, center-specific (MLCS) prognostic reports (the Univfy® report) in provider-patient pre-treatment and IVF counseling. Methods: We used a retrospective cohort comprising 24,238 patients with new patient visits (NPV) from 2016 to 2022 across seven fertility centers in 17 locations in seven US states and Ontario, Canada. We tested the association of Univfy report usage and first intra-uterine insemination (IUI) and/or first IVF usage (a.k.a. conversion) within 180 days, 360 days, and "Ever" of NPV as primary outcomes. Results: Univfy report usage was associated with higher direct IVF conversion (without prior IUI), with odds ratios (OR) 3.13 (95% CI 2.83, 3.46), 2.89 (95% CI 2.63, 3.17), and 2.04 (95% CI 1.90, 2.20) and total IVF conversion (with or without prior IUI), OR 3.41 (95% CI 3.09, 3.75), 3.81 (95% CI 3.49, 4.16), and 2.78 (95% CI 2.59, 2.98) in 180-day, 360-day, and Ever analyses, respectively; p < 0.05. Among patients with Univfy report usage, after accounting for center as a factor, older age was a small yet independent predictor of IVF conversion. Conclusions: Usage of a patient-centric, MLCS-based prognostics report was associated with increased IVF conversion among new fertility patients. Further research to study factors influencing treatment decision making and real-world optimization of patient-centric workflows utilizing the MLCS reports is warranted.

Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease.

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.

Comparison of Next-Generation Assays for Fecal Calprotectin vs the PhiCal Assay.

OBJECTIVES: To compare the PhiCal assay (CALPRO), the first US Food and Drug Administration-approved assay for fecal calprotectin, to 4 next-generation assays. METHODS: Stool samples from 50 patients were selected, and relevant clinical information was collected. Comparisons were performed using the PhiCal, fCAL turbo (BÜHLMANN), LIAISON Calprotectin (DiaSorin), QUANTA Lite Calprotectin ELISA (Inova Diagnostics), and Calprotectin Chemiluminescence ELISA (ALPCO) assays. RESULTS: All 4 assays had acceptable agreement with PhiCal when qualitatively categorizing results. Within the PhiCal reportable range of 16 to 1,250 µg/g, the DiaSorin, Inova Diagnostics, and ALPCO assays had Spearman correlation coefficients of 0.98, 0.97, and 0.95 and positive biases of 17%, 20%, and 15%, respectively. The BÜHLMANN assay ran approximately 2-fold higher than the PhiCal assay but had a correlation coefficient of 0.98, with similar result categorization. CONCLUSIONS: Our results demonstrate good comparison between PhiCal and 4 next-generation assays. Laboratories performing fecal calprotectin assays may have compelling reasons to adopt next-generation fecal calprotectin testing, such as greater automation, a decreased number of replicates needed per test, and the use of stool-extraction devices. These benefits could decrease turnaround times and lower costs. Although the results of the assays correlated, they are not standardized. Laboratories adopting the newer assays will need to further investigate their performance through validation studies.

Clinical and Analytical Characterization of the DiaSorin and ScheBo Fecal Pancreatic Elastase 1 Assays.

OBJECTIVES: Fecal pancreatic elastase (PE) assays are screening tests for exocrine pancreatic insufficiency (EPI). We analytically evaluated a new PE assay and retrospectively analyzed data from an academic hospital and reference laboratory to understand the clinical utility. METHODS: Forty stool samples with different PE concentrations were tested on the ScheBo enzyme-linked immunosorbent assay (ELISA) versus DiaSorin LIAISON immunoassay; a simple-to-use extraction device was assessed. The cross-reactivity of porcine enzymes was investigated in the immunoassay. Charts of 207 patients with PE results less than 250 µg/g at an academic hospital were reviewed, and data were analyzed for 5136 patients with repeat PE results from a reference laboratory. RESULTS: The LIAISON immunoassay gave comparable results to the ScheBo ELISA, with 87.5% agreement of PE results in classifying as sufficient, mild/moderate insufficiency, or severe insufficiency. The extraction device worked well compared with manual weighing, and no cross reactivity with porcine enzymes was observed. In agreement with prior studies, our clinical data suggested that PE assays were most useful in detecting severe EPI. CONCLUSIONS: The new DiaSorin LIAISON immunoassay preforms similarly to the well-known ScheBo ELISA. Pancreatic elastase assays can help identify patients with severe EPI but are not as useful in classifying mild/moderate EPI.

Clinical and Analytical Verification of an Automated Fecal Calprotectin Immunoassay with Extraction Device.

BACKGROUND: Fecal calprotectin (FC) is a screening test for intestinal inflammation, and often used by clinicians to help identify and monitor patients with inflammatory bowel disease (IBD). Improvements in FC assays include moving to more automated immunoassays compared to ELISAs and simple-to-use extraction devices compared to manual weighing for the extraction process. METHODS: A method comparison was performed between the PhiCal ELISA and LIAISON immunoassay for 53 stool samples, and the screening results were compared to the gold standard endoscopy with biopsy results. Clinical accuracy was assessed by comparing the FC results from each assay to the presence or absence of inflammation determined from the biopsy report. The performance of the extraction device was compared to manually weighing. Additional studies were completed to verify the manufacturer's claims. RESULTS: The FC results were compared to the biopsy results for detecting inflammation. PhiCal ELISA had a sensitivity of 86% and specificity of 100%, while the LIAISON immunoassay had a sensitivity of 97% with specificity of 94%. Therefore, the LIAISON immunoassay performed better than the PhiCal ELISA. The extraction device performed well compared to manual weighing if stool samples were <800 µg/g, within Bristol stool types 2-6, and did not contain a significant amount of undigested material, fibrous material, or mucus. CONCLUSION: The LIAISON immunoassay with extraction device has acceptable performance for clinical use in measuring fecal calprotectin.

Study on the association of wall shear stress and vessel structural stress with atherosclerosis: An experimental animal study.

BACKGROUND AND AIMS: Artery is subject to wall shear stress (WSS) and vessel structural stress (VSS) simultaneously. This study is designed to explore the role of VSS in development of atherosclerosis. METHODS: Silastic collars were deployed on the carotid to create two constrictions on 13 rabbits for a distinct mechanical environment at the constriction. MRI was performed to visualize arteries' configuration. Animals with high fat (n = 9; Model-group) and normal diet (n = 4; Control-group) were sacrificed after 16 weeks. 3D fluid-structure interaction analysis was performed to quantify WSS and VSS simultaneously. RESULTS: Twenty plaques were found in Model-group and 3 in Control-group. In Model-group, 8 plaques located proximally to the first constriction (Region-1, close to the heart) and 7 distally to the second (Region-2, close to the head) and 5 plaques were found on the contralateral side of 3 rabbits. Plaques at Region-1 tended to be bigger than those at Region-2 and the macrophage density at these locations was comparable. Minimum time-averaged WSS (TAWSS) in Region-1 was significantly higher than that in Region-2, and both maximum oscillatory shear index (OSI) and particle relative residence time (RRT) were significantly lower. Peak and mean VSS in Region-1 were significantly higher than those in Region-2. Correlation analyses indicated that low TAWSS, high OSI and RRT were only associated with plaque in Region-2, while lesions in Region-1 were only associated with high VSS. Moreover, only VSS was associated with wall thickness of plaque-free regions in both regions. CONCLUSIONS: VSS might contribute to the initialization and development of atherosclerosis solely or in combination with WSS.

Strategy for improved [(11)C]DAA1106 radiosynthesis and in vivo peripheral benzodiazepine receptor imaging using microPET, evaluation of [(11)C]DAA1106.

INTRODUCTION: The peripheral benzodiazepine receptor (PBR) has shown considerable potential as a clinical marker of neuroinflammation and tumour progression. [(11)C]DAA1106 ([(11)C]N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)-acetamide) is a promising positron emission tomography (PET) radioligand for imaging PBRs. METHODS: A four-step synthetic route was devised to prepare DAA1123, the precursor for [(11)C]DAA1106. Two robust, high yielding methods for radiosynthesis based on [(11)C]-O-methylation of DAA1123 were developed and implemented on a nuclear interface methylation module, producing [(11)C]DAA1106 with up to 25% radiochemical yields at end-of-synthesis based on [(11)C]CH(3)I trapped. Evaluation of [(11)C]DAA1106 for in vivo imaging was performed in a rabbit model with microPET, and the presence of PBR receptor in the target organ was further corroborated by immunohistochemistry. RESULTS: The standard solution method produced 2.6-5.2 GBq (n=19) of [(11)C]DAA1106, whilst the captive solvent method produced 1.6-6.3 GBq (n=10) of [(11)C]DAA1106. Radiochemical purities obtained were 99% and specific radioactivity at end-of-synthesis was up to 200 GBq/micromol for both methods. Based on radiochemical product, shorter preparation times and simplicity of synthesis, the captive solvent method was chosen for routine productions of [(11)C]DAA1106. In vivo microPET [(11)C]DAA1106 scans of rabbit kidney demonstrated high levels of binding in the cortex. The subsequent introduction of nonradioactive DAA1106 (0.2 micromol) produced considerable displacement of the radioactive signal in this region. The presence of PBR in kidney cortex was further corroborated by immunohistochemistry. CONCLUSIONS: A robust, high yielding captive solvent method of [(11)C]DAA1106 production was developed which enabled efficacious in vivo imaging of PBR expressing tissues in an animal model.

Bridging the gap: ageing, pharmacokinetics and pharmacodynamics.

Changes in pharmacokinetics and pharmacodynamics in elderly patients generally result in an increase in the incidence of drug toxicity and adverse drug reactions. Molecular alterations associated with ageing could bring about biological changes, a consequence of which is an altered response to pharmacological agents. Unfortunately, research in this area has yet to progress beyond the cataloguing of the pharmacokinetic and pharmacodynamic changes observed in the elderly. Therefore, real progress in our understanding of pharmacogerontology could be achieved if it were possible to merge pharmacokinetic and pharmacodynamic studies with recent advances in our understanding of the causal processes bringing about ageing changes at the cellular level. Therefore, this review will focus on the mechanisms of ageing in the hope that the information will be of value to those planning independent studies.

Identifying active vascular microcalcification by (18)F-sodium fluoride positron emission tomography.

Vascular calcification is a complex biological process that is a hallmark of atherosclerosis. While macrocalcification confers plaque stability, microcalcification is a key feature of high-risk atheroma and is associated with increased morbidity and mortality. Positron emission tomography and X-ray computed tomography (PET/CT) imaging of atherosclerosis using (18)F-sodium fluoride ((18)F-NaF) has the potential to identify pathologically high-risk nascent microcalcification. However, the precise molecular mechanism of (18)F-NaF vascular uptake is still unknown. Here we use electron microscopy, autoradiography, histology and preclinical and clinical PET/CT to analyse (18)F-NaF binding. We show that (18)F-NaF adsorbs to calcified deposits within plaque with high affinity and is selective and specific. (18)F-NaF PET/CT imaging can distinguish between areas of macro- and microcalcification. This is the only currently available clinical imaging platform that can non-invasively detect microcalcification in active unstable atherosclerosis. The use of (18)F-NaF may foster new approaches to developing treatments for vascular calcification.

Can we say that senescent cells cause ageing?

Replicative senescence, the irreversible loss of proliferative capacity, is a common feature of somatic cells derived from many different species. The molecular mechanisms controlling senescence in mammals, and especially in humans, have now been substantively elucidated. However, to date, attempts to link the senescence of cells with the ageing of the organisms they comprise has not met with any similar degree of success, largely due to a lack of systematic investigation and the absence of the necessary biochemical tools. This review will summarise current data linking replicative senescence and organismal ageing. It will also suggest some essential tests of the cell senescence hypothesis and some necessary ground work which must be carried out before such tests can be fruitfully performed. It will not discuss the detailed molecular 'clockwork' controlling the decision to exit the cell cycle irreversibly because this is covered by other authors in this special issue.

Quantitative phosphor imaging autoradiography of radioligands for positron emission tomography.

Imaging using phosphor screens have increasingly been employed for the analysis of radioactive samples in molecular biology, pharmacology, and receptor autoradiography. The major advantages of phosphor screens compared to radiation sensitive film are their greatly increased sensitivity, reducing exposure times with at least one order of magnitude, and their increased linear dynamic range. These features make phosphor screens ideal for imaging short-lived radionuclides, where exposure times are limited, such as (11)C and (18)F widely used to label radioligands for positron emission tomography (PET). Phosphor imaging can also considerably reduce exposure times for weak ß-particle emitters such as (3)H. In this chapter, we present methods for the characterization and evaluation of novel PET radioligands using quantitative phosphor imaging autoradiography.

Dynamic in vivo imaging of receptors in small animals using positron emission tomography.

Positron emission tomography (PET) is a functional imaging technique with the potential to image and quantify receptors in vivo with high sensitivity. PET has been used extensively to study major neurotransmitters such as dopamine, serotonin, and benzodiazepine in humans as well as proving to be a very powerful tool to accelerate development and assessment of existing and novel drugs. With the recent development of dedicated PET scanners for small animals, such as the microPET, it is now possible to perform functional imaging in small animals such as rodents at high resolution. This will allow the study of animal models of disease and longitudinal studies in these models to monitor disease progression or effect of treatment in the same animal. Furthermore, the complete pharmacokinetics of a drug as well as pharmacodynamic information can be obtained in a single animal. Thus, small animal imaging will significantly reduce the number of animals needed for this type of experiment as well as reducing the effect of inter-animal variation. Experimental protocols in small animal imaging potentially can be very labor intensive. In this chapter, we discuss methods and practical aspects related to this type of experiment using the microPET system.

FDG-PET can distinguish inflamed from non-inflamed plaque in an animal model of atherosclerosis.

The presence of activated macrophages is an important predictor of atherosclerotic plaque rupture. In this study, our aim was to determine the accuracy of (18)F- fluorodeoxyglucose (FDG) microPET imaging for quantifying aortic wall macrophage content in a rabbit model of atherosclerosis. Rabbits were divided into a control group and two groups post aortic balloon injury: 6 months high-cholesterol diet (HC); and 3 months HC followed by 3 months low-cholesterol diet plus statin (LCS). In vivo and ex vivo microPET, ex vivo well counting and histological quantification of the atherosclerotic aortas were performed for all groups. Macrophage density was greater in the HC group than the LCS group (5.1 +/- 1.4% vs. 0.6 +/- 0.7%, P < 0.001) with a trend towards greater macrophage density in LCS compared to controls (P = 0.08). There was a strong correlation across all groups between macrophage density and standardized uptake value (SUV) derived from ex vivo microPET (r = 0.95, P < 0.001) and well counting (r = 0.96, P < 0.001). Ex vivo FDG SUV was significantly different between the three groups (P < 0.001). However, the correlation between in vivo microPET FDG SUV and macrophage density was insignificant (r = 0.16, P = 0.57) with no statistical differences in FDG SUV seen between the three groups. This study confirms that in an animal model of inflamed and non-inflamed atherosclerosis, significant differences in FDG SUV allow differentiation of highly inflamed atherosclerotic aortas from those stabilized by statin therapy and low cholesterol diet and controls.

Notochordal cell produce and assemble extracellular matrix in a distinct manner, which may be responsible for the maintenance of healthy nucleus pulposus.

STUDY DESIGN: Analysis of proteoglycan synthesis, distribution and assembly of notochordal cells and small nucleus pulposus cells embedded in alginate beads and cultured in presence of [S]-Na2SO4. OBJECTIVE: To determine whether the degeneration of the nucleus pulposus of the intervertebral disc is associated with a change in the cell phenotype. SUMMARY OF BACKGROUND DATA: The loss of the notochordal cell from the nucleus pulposus is associated with ageing and disc degeneration. The reduction in their numbers after birth in humans and in the chondrodystrophoid dog has been suggested to result from cell death and replacement or differentiation by chondrocytes. The almost total disappearance of the notochordal cells in the nucleus pulposus correlates with early degenerative changes in the disc and a concomitant reduction in proteoglycan content, increased collagen, and loss of water content. The basic mechanism of this accelerated degeneration with ageing is poorly understood. METHODS: Nucleus pulposus and anulus fibrosus cells were isolated from the lumbar intervertebral discs of chondrodystrophoid and nonchondrodystrophoid dogs. The cells from the nucleus pulposus were further separated by size into notochordal cells and small nucleus pulposus cells. Cells were embedded in alginate beads and cultured in the presence of [S]-Na2SO4 to measure proteoglycan size, rate of synthesis, and distribution into the pericellular and intercellular compartments. RESULTS: Large notochordal cells in the nucleus pulposus of chondrodystrophoid dogs formed 13% of the cell population in young dogs and fell to 0.4% in adults, whereas they were the predominant cell type in the nonchondrodystrophoid dogs at all ages. These cells were capable of 1.5-fold greater rate of synthesis of proteoglycans than the small nucleus pulpous cells. Proteoglycans secreted by the large cells were evenly distributed between the pericellular and intercellular compartments,whereas the small cells distributed 3-fold more proteoglycan into the intercellular phase. By size exclusion chromatography, the proteoglycans synthesized by the small cells of the chondrodystrophoid dogs formed large-size aggregates (Kav = 0.1) within the pericellular region, which then moved to the intercellular region over 5 to 10 days. In contrast, proteoglycans secreted by the notochordal cells were capable of rapid migration to the intercellular phase before assembly into large-sized aggregates. The ability to form aggregates was independent of age of the animal. CONCLUSIONS: Our model shows that a change in intervertebral disc cell phenotype correlates with the grade of disc degeneration and that the notochordal cells synthesize proteoglycans, which exhibit delayed aggregation than those synthesized by the small nucleus pulposus cells. This implies that the cell type composition of the nucleus pulposus of the chondrodystrophoid and nonchondrodystrophoid dogs produces an extracellular matrix that is assembled in a distinct manner, which may affect tissue integrity.

Age-related changes in the composition, the molecular stoichiometry and the stability of proteoglycan aggregates extracted from human articular cartilage.

The heterogeneity of the components of proteoglycan aggregates, their stoichiometry within the aggregate and the aggregates' stability was investigated in normal human articular cartilage specimens (age-range newborn to 63 years). Proteoglycans were extracted from tissue by sequentially extracting them with PBS alone, PBS containing oligosaccharides of hyaluronan, and PBS containing solutions of increasing guanidinium chloride concentration (1 M, 2 M, 3 M and 4 M). A high proportion of each of the components of the proteoglycan aggregate, i.e. uronic acid, sulphated glycosaminoglycan, hyaluronan binding domain of aggrecan (G1-domain), link protein (LP) and hyaluronan, was extracted from immature cartilage by PBS alone and PBS containing oligosaccharides of hyaluronan. This was in marked contrast to adult cartilage, which required high concentrations of guanidinium chloride for the efficient extraction of these components. The molar ratios of total G1-domain:LP and the G1-domain associated with aggrecan:LP also differed markedly between immature and mature cartilage and between each of the sequential extracts. The concentration of LP was less than that of the G1-domain in all extracts of cartilage from individuals over 13 years, but this was particularly noticeable in the 1 M guanidinium chloride extracts, and it was surmised that a deficiency in LP produces unstable aggregates in situ. The fragmentation of LP, which is known to occur with advancing age, did not influence the extractability of LP, and fragments were present in each of the sequential extracts. Therefore the generally accepted model of proteoglycan aggregation presented in the literature, which is mostly derived from analysis of immature animal cartilage, cannot be used to describe the structure and organization of aggregates in adult human articular cartilage, where a heterogeneous population of complexes exist that have varying degrees of stability.