Cellular activation patterns of CD10+ fibro-adipogenic progenitors across acquired disease states in human skeletal muscle biopsies.

Background: Fibro-adipogenic progenitors (FAP) are muscle resident mesenchymal stem cells pivotal for regulation of myofiber repair. Experimental results show in addition involvement in a range of other pathological conditions and potential for pharmacological intervention. FAP histopathology in human muscle biopsies is largely unknown, but has potential to inform translational research. Methods: CD10+ FAPs in 32 archival muscle biopsies from 8 groups (normal, dermatomyositis, inclusion body myositis (IBM), anti-synthetase syndrome, immune-mediated necrotizing myopathy (IMNM), denervation, type 2 atrophy, rhabdomyolysis) were visualized by CD10 immunohistochemistry and their histology compared. Groups are compared by semi-quantitative scoring. Results: Histological activation of endomysial CD10+ FAPs includes prominent expansion of a network of cell processes surrounding muscle fibers, as well as endomysial cell clusters evidencing proliferation. Prominence of periarteriolar processes is a notable feature in some pathologies. FAP activation is often associated with fiber degeneration/regeneration, foci of inflammation, and denervation in keeping with experimental results. Type 2 atrophy shows no evidence of FAP activation. Dermatomyositis and anti-synthetase syndrome associated myositis demonstrate diffuse activation. Conclusion: Assessment of CD10+ FAP activation is routinely possible using CD10 immunohistochemistry and demonstrates several patterns in keeping with preclinical results. Prominent expansion of FAP processes surrounding myofibers suggests enhanced interaction between myofiber/basement membranes and FAPs during activation. The presence of diffuse FAP activation in dermatomyositis biopsies unrelated to fiber repair raises the possibility of FAP activation as part of the autoimmune process. Future diagnostic applications, clinical significance and therapeutic potential remain to be elucidated.

Defining the Parameters for Endovenous Microwave Ablation to Achieve Equivalence With Endovenous Laser Ablation, Using the Porcine Liver Model.

AIMS: Endovenous microwave ablation (EMWA) is an endovenous thermoablation (EVTA) system to ablate incompetent truncal veins. Early results suggest that EMWA uses more power than endovenous laser ablation (EVLA) to get the same results. We aimed to define the parameters for EMWA, which give the same tissue ablation as EVLA, using the validated porcine liver model. METHODS: EVLA (1470 nm 600 micron radial fibre) treatments were performed at 6 W, 8 W and 10 W, at pullback speeds of 6, 7, 8 and 9 s/cm, giving Linear Endovenous Energy Densities (LEEDs) between 36 - 90 J/cm. Each combination of power and pullback was repeated 5 times. We then used EMWA in the same model. Powers of 35-75 W and pullback speeds of 4-9 s/cm were used (LEEDs 140-675 J/cm). Ablation tracts from both devices were analysed by 2 blinded observers, noting thermal spread and carbonisation. RESULTS: For each commonly used parameter for EVLA, we identified a range of parameters for EMWA that produced similar tissue ablation in the porcine liver model. To keep the pullback speeds within the usual range, powers of 35-75 W were needed with EMWA, with mean EMWA LEEDs 3.9 - 5.8 times higher than EVLA LEEDs. We found the quicker the pullback speed, the higher the multiple of EMWA LEED we needed to get the same effect. CONCLUSION: We have identified parameters for EMWA that gave equivalent tissue ablation in the porcine liver model to commonly used parameters used for EVLA. These need to be validated clinically, but as the model used has already been validated against clinical outcome in endovenous thermal ablation, there is little reason to suspect that these results would not be valid. As the power during EMWA is higher than EVLA, EVMA LEEDs are approximately 4-6 times higher than EVLA LEEDs to achieve the same thermal effect on the tissues.

Uroplakin-IIIb as a novel immunohistochemical marker for mesothelioma.

Distinguishing mesothelioma from non-small cell lung carcinoma often requires a battery of immunohistochemical stains, as many traditional markers used in mesothelioma lack sufficient specificity to allow them to be used alone. A recent large-scale TMA screen identified uroplakin-IIIb (UpIIIb; clone MSVA-736M) as a potentially specific marker for mesothelioma. We examined the performance of this antibody using tissue microarrays containing a panel of 48 epithelioid mesotheliomas, 26 sarcomatoid mesotheliomas, and 144 non-small cell lung carcinomas (NSCLCs). Here we show that UpIIIb has good sensitivity (37/47 evaluable cases positive, 79%) and excellent specificity for distinguishing epithelioid mesothelioma from NSCLC (0/140 evaluable cases positive). UPIIIb sensitivity for epithelioid mesotheliomas was only slightly inferior to the established highly specific mesothelioma marker HEG1 (41/46 evaluable cases positive on the same TMA, 89%). However, UpIIIb did not stain any sarcomatoid mesotheliomas (0/24 evaluable cases positive). We also found that UpIIIb stained a proportion of high-grade serous ovarian carcinomas, a perennial diagnostic confounder in the context of mesotheliomas. Taken together, our data suggest that UpIIIb can be used as a highly specific and sensitive mesothelial marker when the diagnostic question is epithelioid mesothelioma versus NSCLC; in particular, UpIIIb staining will pick up some number of epithelioid mesotheliomas that are HEG1 negative. Since UpIIIb is known to stain some proportion of urothelial carcinomas as well as gynecologic and a few pancreatic tumors, it should be used with caution in the peritoneal cavity or when the differential diagnosis includes carcinomas from these locations.

C-terminal TMEM106B fragments in human brain correlate with disease-associated TMEM106B haplotypes.

Transmembrane protein 106B (TMEM106B) is a tightly regulated glycoprotein predominantly localized to endosomes and lysosomes. Genetic studies have implicated TMEM106B haplotypes in the development of multiple neurodegenerative diseases with the strongest effect in frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP), especially in progranulin (GRN) mutation carriers. Recently, cryo-electron microscopy studies showed that a C-terminal fragment (CTF) of TMEM106B (amino acid residues 120-254) forms amyloid fibrils in the brain of patients with FTLD-TDP, but also in brains with other neurodegenerative conditions and normal ageing brain. The functional implication of these fibrils and their relationship to the disease-associated TMEM106B haplotype remain unknown. We performed immunoblotting using a newly developed antibody to detect TMEM106B CTFs in the sarkosyl-insoluble fraction of post-mortem human brain tissue from patients with different proteinopathies (n = 64) as well as neuropathologically normal individuals (n = 10) and correlated the results with age and TMEM106B haplotype. We further compared the immunoblot results with immunohistochemical analyses performed in the same study population. Immunoblot analysis showed the expected ∼30 kDa band in the sarkosyl-insoluble fraction of frontal cortex tissue in at least some individuals with each of the conditions evaluated. Most patients with GRN mutations showed an intense band representing TMEM106B CTF, whereas in most neurologically normal individuals it was absent or much weaker. In the overall cohort, the presence of TMEM106B CTFs correlated strongly with both age (rs = 0.539, P < 0.001) and the presence of the TMEM106B risk haplotype (rs = 0.469, P < 0.001). Although there was a strong overall correlation between the results of immunoblot and immunohistochemistry (rs = 0.662, P < 0.001), 27 cases (37%) were found to have higher amounts of TMEM106B CTFs detected by immunohistochemistry, including most of the older individuals who were neuropathologically normal and individuals who carried two protective TMEM106B haplotypes. Our findings suggest that the formation of sarkosyl-insoluble TMEM106B CTFs is an age-related feature which is modified by TMEM106B haplotype, potentially underlying its disease-modifying effect. The discrepancies between immunoblot and immunohistochemistry in detecting TMEM106B pathology suggests the existence of multiple species of TMEM106B CTFs with possible biological relevance and disease implications.

Accumulation of TMEM106B C-terminal fragments in neurodegenerative disease and aging.

Several studies using cryogenic electron microscopy (cryo-EM) techniques recently reported the isolation and characterization of novel protein filaments, composed of a C-terminal fragment (CTF) of the endolysosomal transmembrane protein 106B (TMEM106B), from human post-mortem brain tissue with various neurodegenerative conditions and normal aging. Genetic variation in TMEM106B is known to influence the risk and presentation of several neurodegenerative diseases, especially frontotemporal dementia (FTD) caused by mutations in the progranulin gene (GRN). To further elucidate the significance of TMEM106B CTF, we performed immunohistochemistry with antibodies directed against epitopes within the filament-forming C-terminal region of TMEM106B. Accumulation of TMEM106B C-terminal immunoreactive (TMEM-ir) material was a common finding in all the conditions evaluated, including frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP), Alzheimer's disease, tauopathies, synucleinopathies and neurologically normal aging. TMEM-ir material was present in a wide range of brain cell types and in a broad neuroanatomical distribution; however, there was no co-localization of TMEM-ir material with other neurodegenerative proteins in cellular inclusions. In most conditions, the presence and abundance of TMEM-ir aggregates correlated strongly with patient age and showed only a weak correlation with the TMEM106B haplotype or the primary pathological diagnosis. However, all patients with FTD caused by GRN mutations were found to have high levels of TMEM-ir material, including several who were relatively young (< 60 years). These findings suggest that the accumulation of TMEM106B CTF is a common age-related phenomenon, which may reflect lysosomal dysfunction. Although its significance in most neurodegenerative conditions remains uncertain, the consistent finding of extensive TMEM-ir material in cases of FTLD-TDP with GRN mutations further supports a pathomechanistic role of TMEM106B and lysosomal dysfunction in this specific disease population.

UHRF1 Immunohistochemical Staining Separates Benign Reactive Spindle Cell Mesothelial Proliferations From Sarcomatoid Mesotheliomas.

The separation of benign from malignant mesothelial proliferations is often a difficult pathologic problem. UHRF1 (ubiquitin-like with plant homeodomain and ring finger domains-1) is a regulator of DNA methylation and an epigenetic driver of various human cancers. It has recently been reported that UHRF1 is overexpressed in mesotheliomas. We asked whether UHRF1 immunohistochemistry could be used to separate benign from malignant mesothelial proliferations. Initial studies showed that UHRF1 stained mesothelial cells but also endothelial and other non-neoplastic cells, so that accurate counting of positive mesothelial cells was difficult. Therefore, we ran dual UHRF1-AE1/AE3 stains on 2 tissue microarrays containing 40 reactive mesothelial proliferations and 61 mesotheliomas and only counted UHRF1 staining in keratin-positive cells. On average 10.3±8.6% (mean±SD; range: 0% to 36, median: 6.8%) of epithelioid mesothelioma cells stained compared with 5.3±4.8% (range: 0% to 15%, median: 4.1%) of reactive epithelial mesothelial cells. This difference was statistically significant but there was too much overlap to use diagnostically. In contrast, 37±26% (range: 2.5% to 95%, median: 31%) of cells in sarcomatoid mesotheliomas compared with 1.2±1.2% (range: 0% to 3.0%, median: 1.0%) of cells in reactive spindle cell mesothelial proliferations stained. To confirm this difference we stained whole sections of 21 sarcomatoid mesotheliomas and 19 cases of organizing pleuritis. Staining of mesothelial cells was seen in 2.1±2.4% (range: 0% to 6.8%, median: 1.0%) of organizing pleuritis cases and 44±22% (range: 14% to 90%, median: 41%) of sarcomatoid mesotheliomas. We conclude that dual UHRF1-AE1/AE3 immunohistochemistry is very useful for separating benign spindle cell mesothelial proliferations from sarcomatoid mesotheliomas.

Perceived usefulness of open educational resources: Impact of switching to online learning for face-to-face and distance learners.

This paper reports a study on the perceived usefulness of university students on open educational resources (OER) in relation to the switch of learning mode to online learning during the COVID-19 pandemic. The participants involved two groups of students, one studying in a face-to-face mode and the other in a distance learning mode. They took part in a survey which was conducted in 2019 before the pandemic (with a total of 912 responses) and 2021 during the pandemic (with a total of 1,018 responses). The results show that both groups of students generally perceived OER to be more useful during the pandemic. The specific types of OER which were perceived as relatively more useful include open online courses and open access textbooks. Face-to-face students showed a higher level of perceived usefulness of OER for preparing tests and examinations, while distance learning students perceived OER as more useful for supplementing course materials. They both concerned about the limitations of OER, especially on accuracy and comprehensiveness. The findings suggest the importance of recognizing the diverse needs of the two groups of students and offering appropriate OER support for them.

Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway.

Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.

Modelling hereditary diffuse gastric cancer initiation using transgenic mouse-derived gastric organoids and single-cell sequencing.

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell-cell adhesion molecule E-cadherin. Loss of E-cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single-cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage-specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation-dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Utility of SOX6 and DAB2 for the Diagnosis of Malignant Mesothelioma.

The separation of malignant mesothelioma from non-small cell lung carcinomas can be a difficult problem. Sex-determining region Y box 6 (SOX6) and disabled homolog 2 (DAB2) have recently been proposed as sensitive/specific markers of mesothelial lineage, but have not yet been independently tested for utility in mesothelioma diagnosis. Using tissue microarrays containing mesotheliomas (epithelioid: n=40, sarcomatoid: n=23) and non-small cell lung carcinomas (adenocarcinoma: n=52, squamous cell carcinoma: n=57, large cell carcinoma: n=12) we evaluated the performance of SOX6 and DAB2 by themselves, in conjunction with other established mesothelioma markers (calretinin, WT1, D2-40, CK5/6, HEG1) and combined with 3 broad-spectrum established carcinoma markers: claudin-4, MOC31, and BerEP4. For epithelioid mesothelioma, SOX6 and DAB2 had sensitivities of 85% and 98%, respectively. For sarcomatoid mesothelioma, SOX6 had a sensitivity of 13% and DAB2 could not be assessed due to background stromal staining. For SOX6 alone, specificity for mesothelioma versus adenocarcinoma, squamous cell carcinoma, and large cell carcinoma was 94%, 79%, and 92%, respectively, while for DAB2 specificity was 77%, 86%, and 67%. Combinations of SOX6 and established mesothelioma markers produced sensitivities of 95% or greater. A combination of SOX6 positive/claudin-4 negative staining was 95% to 100% specific for mesothelioma versus carcinoma with a sensitivity of 85%. SOX6 is a promising marker for the diagnosis of mesothelioma and potentially could be combined with other mesothelial markers or a broad-spectrum carcinoma marker to reach an accurate diagnosis with relatively few immunostains, The relatively low specificity and difficulty of interpreting DAB2 staining limits its utility for mesothelioma diagnosis.

Efficacy, safety and response predictors of adjuvant astragalus for diabetic kidney disease (READY): study protocol of an add-on, assessor-blind, parallel, pragmatic randomised controlled trial.

INTRODUCTION: Diabetic kidney disease (DKD) is a prevalent and costly complication of diabetes with limited therapeutic options, being the leading cause of end-stage kidney disease in most developed regions. Recent big data studies showed that add-on Chinese medicine (CM) led to a reduced risk of end-stage kidney disease and mortality among patients with chronic kidney disease (CKD) and diabetes. Astragalus, commonly known as huang-qi, is the most prescribed CM or used dietary herb in China for diabetes and DKD. In vivo and in vitro studies showed that astragalus ameliorated podocyte apoptosis, foot process effacement, mesangial expansion, glomerulosclerosis and interstitial fibrosis. Nevertheless, the clinical effect of astragalus remains uncharacterised. This pragmatic clinical trial aims to evaluate the effectiveness of add-on astragalus in patients with type 2 diabetes, stage 2-3 CKD and macroalbuminuria, and to identify related response predictors. METHODS AND ANALYSIS: This is an add-on, assessor-blind, parallel, pragmatic randomised controlled clinical trial. 118 patients diagnosed with DKD will be recruited and randomised 1:1 to receive 48 weeks of add-on astragalus or standard medical care. Primary endpoints are the changes in estimated glomerular filtration rate and urine albumin-to-creatinine ratio between baseline and treatment endpoint. Secondary endpoints include adverse events, fasting blood glucose, glycated haemoglobin, lipids and other biomarkers. Adverse events are monitored through self-complete questionnaire and clinical visits. Outcomes will be analysed by regression models. Subgroup and sensitivity analyses will be conducted for different epidemiological subgroups and statistical analyses. Enrolment started in July 2018. ETHICS AND DISSEMINATION: This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West/East/Kowloon Central clusters (UW 16-553/HKEC-2019-026/REC (KC/KE)-19-0049/ER-4). We will report the findings in medical journals and conferences. The dataset will be available on reasonable request. TRIAL REGISTRATION NUMBER: NCT03535935.

Long Term Performance of an Uncemented, Proximally Hydroxyapatite Coated, Double Tapered, Titanium-Alloy Femoral Stem: Results From 1465 Hips at 10 years Minimum Follow-Up.

BACKGROUND: We assessed the survivorship of a proximally hydroxyapatite coated, double tapered, titanium-alloy femoral stem in a single center, at an average follow up of 12.5 years (10.1-15.8). The majority of stems were inserted as part of a Metal on Metal (MoM) Total Hip Replacement (THR). METHODS: Data was collected prospectively in a local database. A retrospective review was performed of all patients undergoing a primary THR with the prosthesis between 2003 and 2010. Primary outcome was revision of the stem for any cause. Analysis was also performed for stem revision for aseptic loosening, stem revision in the MoM setting and a worst case scenario whereby lost to follow up were presumed to have failed. True stem failure was considered if revision occurred for a stem related complication. RESULTS: 1465 stems were included (1310 patients, 155 bilateral). The bearing surface was cobalt chrome on cobalt chrome in 1351 cases (92%). Seven hips were lost to follow up. Thirty-two stems (31 part of a MoM THR) underwent revision for any cause. Kaplan Meier survival analysis demonstrates an overall 97.4% survivorship. Subset analysis demonstrates 100% survivorship for aseptic loosening, 97.3% in the MoM setting and 96.7% for the worst case senario. Of the 32 cases of stem revision, only 13 were classified as 'true' stem failure. CONCLUSION: This study represents the largest cohort of this uncemented femoral component with a minimum follow-up longer than 10 years. Our results demonstrate excellent long-term survivorship even in the presence of a challenging MoM environment.