Utility of pathologist panels for achieving consensus in NASH histologic scoring in clinical trials: Data from a phase 3 study

Copyright \ua9 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.Background: Liver histopathologic assessment is the accepted surrogate endpoint in NASH trials; however, the scoring of NASH Clinical Research Network (CRN) histologic parameters is limited by intraobserver and interobserver variability. We designed a consensus panel approach to minimize variability when using this scoring system. We assessed agreement between readers, estimated linear weighted kappas between 2 panels, compared them with published pairwise kappa estimates, and addressed how agreement or disagreement might impact the precision and validity of the surrogate efficacy endpoint in NASH trials. Methods: Two panels, each comprising 3 liver fellowship-trained pathologists who underwent NASH histology training, independently evaluated scanned whole slide images, scoring fibrosis, inflammation, hepatocyte ballooning, and steatosis from baseline and month 18 biopsies for 100 patients from the precirrhotic NASH study REGENERATE. The consensus score for each parameter was defined as agreement by ≥2 pathologists. If consensus was not reached, all 3 pathologists read the slide jointly to achieve a consensus score. Results: Between the 2 panels, the consensus was 97%-99% for steatosis, 91%-93% for fibrosis, 88%-92% for hepatocyte ballooning, and 84%-91% for inflammation. Linear weighted kappa scores between panels were similar to published NASH CRN values. Conclusions: A panel of 3 trained pathologists independently scoring 4 NASH CRN histology parameters produced high consensus rates. Interpanel kappa values were comparable to NASH CRN metrics, supporting the accuracy and reproducibility of this method. The high concordance for fibrosis scoring was reassuring, as fibrosis is predictive of liver-specific outcomes and all-cause mortality

Microvascular engineering in perfusion culture: immunohistochemistry and CLSM findings

BACKGROUND: One of the most challenging problems in tissue engineering is the establishment of vascular supply. A possible approach might be the engineering of microvasculature in vitro and the supply by engineered feeder vessels. METHODS: An in vitro model for a small-diameter vessel was developed and made from adipose tissue stromal cells and human umbilical vein endothelial cells in a tube-like gelatine scaffold. The number of "branches" emerging from the central lumen and the morphology of the central lumen of the vessel equivalent were assessed after 16 days of either pulsatile perfusion culture or culture in rotating containers by evaluation of immunohistochemically stained sections (n = 6 pairs of cultures). Intramural capillary network formation was demonstrated in five experiments with confocal laser scanning microscopy. RESULTS: Perfused specimens showed a round or oval lumen lined by a single layer of endothelial cells, whereas following rotation culture the lumen tended to collapse. Confocal laser scanning microscopy showed more extended network formation in perfused specimens as compared to specimens after rotation culture. Partially highly interconected capillary-like networks were imaged which showed orientation around the central lumen. Perfused specimens exhibited significantly more branches emerging from the central lumen. There were, however, hardly any capillary branches crossing the whole vessel wall. CONCLUSION: Pulsatile perfusion supports the development of vascular networks with physiological appearance. Advances in reactor development, acquisition of functional data and imaging procedures are however necessary in order to attain the ultimate goal of a fully functional engineered supplying vessel

The claudin gene family: expression in normal and neoplastic tissues

BACKGROUND: The claudin (CLDN) genes encode a family of proteins important in tight junction formation and function. Recently, it has become apparent that CLDN gene expression is frequently altered in several human cancers. However, the exact patterns of CLDN expression in various cancers is unknown, as only a limited number of CLDN genes have been investigated in a few tumors. METHODS: We identified all the human CLDN genes from Genbank and we used the large public SAGE database to ascertain the gene expression of all 21 CLDN in 266 normal and neoplastic tissues. Using real-time RT-PCR, we also surveyed a subset of 13 CLDN genes in 24 normal and 24 neoplastic tissues. RESULTS: We show that claudins represent a family of highly related proteins, with claudin-16, and -23 being the most different from the others. From in silico analysis and RT-PCR data, we find that most claudin genes appear decreased in cancer, while CLDN3, CLDN4, and CLDN7 are elevated in several malignancies such as those originating from the pancreas, bladder, thyroid, fallopian tubes, ovary, stomach, colon, breast, uterus, and the prostate. Interestingly, CLDN5 is highly expressed in vascular endothelial cells, providing a possible target for antiangiogenic therapy. CLDN18 might represent a biomarker for gastric cancer. CONCLUSION: Our study confirms previously known CLDN gene expression patterns and identifies new ones, which may have applications in the detection, prognosis and therapy of several human cancers. In particular we identify several malignancies that express CLDN3 and CLDN4. These cancers may represent ideal candidates for a novel therapy being developed based on CPE, a toxin that specifically binds claudin-3 and claudin-4

Congenital beta cell defects are not associated with markers of islet autoimmunity, even in the context of high genetic risk for type 1 diabetes

This is the final version. Available on open access from Springer via the DOI in this recordData availability: Access to data is open only through collaboration. Requests for collaboration will be considered following an application to the Genetic Beta Cell Research Bank (https://www.diabetesgenes.org/current-research/genetic-beta-cell-research-bank/). Contact by email should be directed to the Lead Nurse, Bridget Knight ([email protected]).Aims/hypothesis A key unanswered question in type 1 diabetes is whether beta cells initiate their own destruction or are victims of an aberrant immune response (beta cell suicide or homicide?). To investigate this, we assessed islet autoantibodies in individuals with congenital beta cell defects causing neonatal diabetes mellitus (NDM). Methods We measured autoantibodies to GAD (GADA), islet antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) in 242 individuals with NDM (median age diagnosed 1.8 months [IQR 0.39–2.9 months]; median age collected 4.6 months [IQR 1.8–27.6 months]; median diabetes duration 2 months [IQR 0.6–23 months]), including 75 whose NDM resulted from severe beta cell endoplasmic reticulum (ER) stress. As a control cohort we also tested samples from 69 diabetes-free individuals (median age collected 9.9 months [IQR 9.0–48.6 months]) for autoantibodies. Results We found low prevalence of islet autoantibodies in individuals with monogenic NDM; 13/242 (5.4% [95% CI 2.9, 9.0%]) had detectable GADA, IA-2A and/or ZnT8A. This was similar to the proportion in the control participants who did not have diabetes (1/69 positive [1.4%, 95% CI 0.03, 7.8%], p=0.3). Importantly, monogenic individuals with beta cell ER stress had a similar rate of GADA/IA-2A/ZnT8A positivity to non-ER stress aetiologies (2.7% [95% CI 0.3, 9.3%] vs 6.6% [95% CI 3.3, 11.5%] p=0.4). We observed no association between islet autoimmunity and genetic risk, age at testing (including 30 individuals >10 years at testing) or diabetes duration (p>0.4 for all). Conclusions/interpretation Our data support the hypothesis that beta cell stress/dysfunction alone does not lead to the production of islet autoantibodies, even in the context of high-risk HLA types. This suggests that additional factors are required to trigger an autoimmune response towards beta cells.Wellcome TrustNational Institute for Health Research (NIHR)JDRFDutch Diabetes Research FoundationStichting DONEuropean CommissionWanek Family Project for Type 1 DiabetesDiabetes UKRoyal SocietyResearch EnglandHelmsley FoundationDiabetes Research and Wellness FoundationUniversity of Exete

Replication of an empirical approach to delineate the heterogeneity of chronic unexplained fatigue

<p>Abstract</p> <p>Background</p> <p>Chronic fatigue syndrome (CFS) is defined by self-reported symptoms. There are no diagnostic signs or laboratory markers, and the pathophysiology remains inchoate. In part, difficulties identifying and replicating biomarkers and elucidating the pathophysiology reflect the heterogeneous nature of the syndromic illness CFS. We conducted this analysis of people from defined metropolitan, urban, and rural populations to replicate our earlier empirical delineation of medically unexplained chronic fatigue and CFS into discrete endophenotypes. Both the earlier and current analyses utilized quantitative measures of functional impairment and symptoms as well as laboratory data. This study and the earlier one enrolled participants from defined populations and measured the internal milieu, which differentiates them from studies of clinic referrals that examine only clinical phenotypes.</p> <p>Methods</p> <p>This analysis evaluated 386 women identified in a population-based survey of chronic fatigue and unwellness in metropolitan, urban, and rural populations of the state of Georgia, USA. We used variables previously demonstrated to effectively delineate endophenotypes in an attempt to replicate identification of these endophenotypes. Latent class analyses were used to derive the classes, and these were compared and contrasted to those described in the previous study based in Wichita, Kansas.</p> <p>Results</p> <p>We identified five classes in the best fit analysis. Participants in Class 1 (25%) were polysymptomatic, with sleep problems and depressed mood. Class 2 (24%) was also polysymptomatic, with insomnia and depression, but participants were also obese with associated metabolic strain. Class 3 (20%) had more selective symptoms but was equally obese with metabolic strain. Class 4 (20%) and Class 5 (11%) consisted of nonfatigued, less symptomatic individuals, Class 4 being older and Class 5 younger. The classes were generally validated by independent variables. People with CFS fell equally into Classes 1 and 2. Similarities to the Wichita findings included the same four main defining variables of obesity, sleep problems, depression, and the multiplicity of symptoms. Four out of five classes were similar across both studies.</p> <p>Conclusion</p> <p>These data support the hypothesis that chronic medically unexplained fatigue is heterogeneous and can be delineated into discrete endophenotypes that can be replicated. The data do not support the current perception that CFS represents a unique homogeneous disease and suggests broader criteria may be more explanatory. This replication suggests that delineation of endophenotypes of CFS and associated ill health may be necessary in order to better understand etiology and provide more patient-focused treatments.</p

RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent

Cranial Pathologies in a Specimen of Pachycephalosaurus

. The specimen features two large oval depressions on the dorsal surface, accompanied by numerous circular pits on the margin and inner surface of the larger depressions.In order to identify the origin of these structures, computed tomography (CT) data and morphological characteristics of the specimen are analyzed and compared with similar osteological structures in fossil and extant archosaurs caused by taphonomic processes, non-pathologic bone resorption, and traumatic infection/inflammatory origins. The results of these analyses suggest that the structures are pathologic lesions likely resulting from a traumatic injury and followed by secondary infection at the site.The presence of lesions on a frontoparietal dome, and the exclusivity of their distribution along the dorsal dome surface, offers further insight into frontoparietal dome function and supports previously hypothesized agonistic behavior in pachycephalosaurids

Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation

Brain iron homeostasis, the choroid plexus, and localization of iron transport proteins

Maintenance of appropriate iron homeostasis in the brain is important, but the mechanisms involved in brain iron uptake are incompletely understood. Here, we have analyzed where messenger RNAs that encode iron transport proteins are expressed in the brain, using the Allen Brain atlas, and we conclude that several important iron transporters are highly expressed in the choroid plexus. Based on recent estimates of the surface area of the choroid plexus and on MRI imaging studies of manganese uptake in the brain, we propose that the choroid plexus may have a much greater role than has been previously appreciated in brain iron transport