Isolation of human intrahepatic leukocytes for phenotypic and functional characterization by flow cytometry

With the growing appreciation of tissue-resident immunity, studying tissue-specific immune cells contributing to both homeostasis and disease is imperative. Here, we provide a protocol for the isolation of human intrahepatic leukocytes (IHL) maximizing viability, purity, and yield. Our protocol is scalable by tissue weight, allowing for reproducible and efficient IHL liberation suitable for functional characterization, cell isolation, and profiling by flow (or mass) cytometry. Furthermore, we provide a "guide" to determine an expected IHL yield per gram of tissue processed. For complete details on the use and execution of this protocol, please refer to Stegmann et al. (2016), Pallett et al. (2017), Easom et al. (2018), Swadling et al. (2020), Pallett et al. (2020), and Zakeri et al. (2022)

Feasibility of Different Tumor Delineation Approaches for 18F-PSMA-1007 PET/CT Imaging in Prostate Cancer Patients

Background: Delineation of PSMA-positive tumor volume on PET using PSMA-ligands is of highest clinical interest as changes of PSMA-PET/CT-derived whole tumor volume (WTV) have shown to correlate with treatment response in metastatic prostate cancer patients. So far, WTV estimation was performed on PET using 68Ga-labeled ligands; nonetheless, 18F-labeled PET ligands are gaining increasing importance due to advantages over 68Ga-labeled compounds. However, standardized tumor delineation methods for 18F-labeled PET ligands have not been established so far. As correlation of PET-based information and morphological extent in osseous and visceral metastases is hampered by morphological delineation, low contrast in liver tissue and movement artefacts, we correlated CT-based volume of lymph node metastases (LNM) and different PET-based delineation approaches for thresholding on 18F-PSMA-1007 PET. Methods: Fifty patients with metastatic prostate cancer, 18F-PSMA-1007 PET/CT and non-bulky LNM (short-axis diameter ≥10mm) were included. Fifty LNM were volumetrically assessed on contrast-enhanced CT (volumetric reference standard). Different approaches for tumor volume delineation were applied and correlated with the reference standard: I) fixed SUV threshold, II) isocontour thresholding relative to SUVmax (SUV%), and thresholds relative to III) liver (SUVliver), IV) parotis (SUVparotis) and V) spleen (SUVspleen). Results: A fixed SUV of 4.0 (r=0.807, r2 = 0.651, p0.05 each). Recently reported cut-offs for intraprostatic tumor delineation (isocontour 44% SUVmax, 42% SUVmax and 20% SUVmax) revealed inferior association for LNM delineation. Conclusions: A threshold of SUV 4.0 for tumor delineation showed highest association with volumetric reference standard irrespective of potential changes in PSMA-avidity of background tissues (e. g. parotis). This approach is easily applicable in clinical routine without specific software requirements. Further studies applying this approach for total tumor volume delineation are initiated

An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314

An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314

Mitochondrial DNA Polymorphism A4917G Is Independently Associated with Age-Related Macular Degeneration

The objective of this study was to determine if MTND2*LHON4917G (4917G), a specific non-synonymous polymorphism in the mitochondrial genome previously associated with neurodegenerative phenotypes, is associated with increased risk for age-related macular degeneration (AMD). A preliminary study of 393 individuals (293 cases and 100 controls) ascertained at Vanderbilt revealed an increased occurrence of 4917G in cases compared to controls (15.4% vs.9.0%, p = 0.11). Since there was a significant age difference between cases and controls in this initial analysis, we extended the study by selecting Caucasian pairs matched at the exact age at examination. From the 1547 individuals in the Vanderbilt/Duke AMD population association study (including 157 in the preliminary study), we were able to match 560 (280 cases and 280 unaffected) on exact age at examination. This study population was genotyped for 4917G plus specific AMD-associated nuclear genome polymorphisms in CFH, LOC387715 and ApoE. Following adjustment for the listed nuclear genome polymorphisms, 4917G independently predicts the presence of AMD (OR = 2.16, 95%CI 1.20–3.91, p = 0.01). In conclusion, a specific mitochondrial polymorphism previously implicated in other neurodegenerative phenotypes (4917G) appears to convey risk for AMD independent of recently discovered nuclear DNA polymorphisms

The Eighth Data Release of the Sloan Digital Sky Survey: First Data from SDSS-III

The Sloan Digital Sky Survey (SDSS) started a new phase in August 2008, with new instrumentation and new surveys focused on Galactic structure and chemical evolution, measurements of the baryon oscillation feature in the clustering of galaxies and the quasar Ly alpha forest, and a radial velocity search for planets around ~8000 stars. This paper describes the first data release of SDSS-III (and the eighth counting from the beginning of the SDSS). The release includes five-band imaging of roughly 5200 deg^2 in the Southern Galactic Cap, bringing the total footprint of the SDSS imaging to 14,555 deg^2, or over a third of the Celestial Sphere. All the imaging data have been reprocessed with an improved sky-subtraction algorithm and a final, self-consistent photometric recalibration and flat-field determination. This release also includes all data from the second phase of the Sloan Extension for Galactic Understanding and Evolution (SEGUE-2), consisting of spectroscopy of approximately 118,000 stars at both high and low Galactic latitudes. All the more than half a million stellar spectra obtained with the SDSS spectrograph have been reprocessed through an improved stellar parameters pipeline, which has better determination of metallicity for high metallicity stars.Comment: Astrophysical Journal Supplements, in press (minor updates from submitted version

The Baryon Oscillation Spectroscopic Survey of SDSS-III

The Baryon Oscillation Spectroscopic Survey (BOSS) is designed to measure the scale of baryon acoustic oscillations (BAO) in the clustering of matter over a larger volume than the combined efforts of all previous spectroscopic surveys of large scale structure. BOSS uses 1.5 million luminous galaxies as faint as i=19.9 over 10,000 square degrees to measure BAO to redshifts z<0.7. Observations of neutral hydrogen in the Lyman alpha forest in more than 150,000 quasar spectra (g<22) will constrain BAO over the redshift range 2.15<z<3.5. Early results from BOSS include the first detection of the large-scale three-dimensional clustering of the Lyman alpha forest and a strong detection from the Data Release 9 data set of the BAO in the clustering of massive galaxies at an effective redshift z = 0.57. We project that BOSS will yield measurements of the angular diameter distance D_A to an accuracy of 1.0% at redshifts z=0.3 and z=0.57 and measurements of H(z) to 1.8% and 1.7% at the same redshifts. Forecasts for Lyman alpha forest constraints predict a measurement of an overall dilation factor that scales the highly degenerate D_A(z) and H^{-1}(z) parameters to an accuracy of 1.9% at z~2.5 when the survey is complete. Here, we provide an overview of the selection of spectroscopic targets, planning of observations, and analysis of data and data quality of BOSS.Comment: 49 pages, 16 figures, accepted by A

Towards an In Vitro Model of Plasmodium Hypnozoites Suitable for Drug Discovery

Contains fulltext : 96475.pdf (publisher's version ) (Open Access)BACKGROUND: Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites. METHODOLOGY: P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured. RESULTS: The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine. CONCLUSION: Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

The Ninth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the SDSS-III Baryon Oscillation Spectroscopic Survey

The Sloan Digital Sky Survey III (SDSS-III) presents the first spectroscopic data from the Baryon Oscillation Spectroscopic Survey (BOSS). This ninth data release (DR9) of the SDSS project includes 535,995 new galaxy spectra (median z=0.52), 102,100 new quasar spectra (median z=2.32), and 90,897 new stellar spectra, along with the data presented in previous data releases. These spectra were obtained with the new BOSS spectrograph and were taken between 2009 December and 2011 July. In addition, the stellar parameters pipeline, which determines radial velocities, surface temperatures, surface gravities, and metallicities of stars, has been updated and refined with improvements in temperature estimates for stars with T_eff<5000 K and in metallicity estimates for stars with [Fe/H]>-0.5. DR9 includes new stellar parameters for all stars presented in DR8, including stars from SDSS-I and II, as well as those observed as part of the SDSS-III Sloan Extension for Galactic Understanding and Exploration-2 (SEGUE-2). The astrometry error introduced in the DR8 imaging catalogs has been corrected in the DR9 data products. The next data release for SDSS-III will be in Summer 2013, which will present the first data from the Apache Point Observatory Galactic Evolution Experiment (APOGEE) along with another year of data from BOSS, followed by the final SDSS-III data release in December 2014.Comment: 9 figures; 2 tables. Submitted to ApJS. DR9 is available at http://www.sdss3.org/dr