Strong Labeling Research Articles

Seasonal Variation in TLR4 Expression in The Testis and Epididymis of Anatolian Ground Squirrels (Spermophilus xanthoprymnus): Insights from Non-Breeding Period of Pre-Hibernation and Hibernation

This study investigates the modulation of Toll-like receptor 4 (TLR4) expression within the testis and epididymis of Anatolian ground squirrels (Spermophilus xanthoprymnus) during the non-breeding period of pre-hibernation and hibernation. Immunohistochemical investigation showed that TLR4 was not detected in germ cells, Leydig cells, or Sertoli cells in the testicular tissue during the pre-hibernation period. Nevertheless, there was a presence of TLR4 in the vessels walls and certain interstitial cells within the intertubular regions. Epithelial cells in the caput, corpus, and cauda regions of the epididymis showed no TLR4 expression. However, it was observed in the vessel walls, smooth muscle layers, and some interstitial cells. TLR4 expression was seen in spermatogonia and primary spermatocytes during hibernation, with strong labeling observed in the vessel walls of the intertubular area. In contrast, TLR4 was detected in the epididymal epithelium, as well as in the smooth muscle layers and vessel walls throughout all segments. A notable upregulation in the expression of TLR4 in the testis was identified through quantitative image analysis during hibernation as compared to pre-hibernation. During pre-hibernation, the cauda segment of the epididymis exhibited the highest expression of TLR4, whereas during hibernation, the corpus segment demonstrated the highest expression. These findings suggest a dynamic modulation of TLR4 in response to hibernation, highlighting its potential role in reproductive function and immune adaptation.

Aerial and terrestrial root habits influence the composition of the cell walls of Vanilla phaeantha (Orchidaceae).

In response to the restrictions imposed by their epiphytic habit, orchids have developed structural traits that allow greater efficiency in water uptake and use, such as a complex adventitious root system with velamen. The composition of cell wall of this specialized epidermis can be altered according to the substrate to which it is fixed, influencing wall permeability, absorption, and storage of water in roots. The current study aimed to evaluate the cell wall composition of adventitious roots of Vanilla phaeantha (Orchidaceae) that grow attached to the phorophyte, fixed in the soil, or hung free. Immunocytochemical analyses were used to determine the protein, hemicellulose, and pectin composition of the cell walls of aerial and terrestrial roots. We observed that pectins are present in the different tissues of the aerial roots, while in the terrestrial roots, they are concentrated in the cortical parenchyma. The deposition of xyloglucans, extensins, and arabinogalactans was greater in the epidermis of the free side of the roots attached to the phorophyte. The strong labeling of pectins in aerial roots may be related to the influx of water and nutrients, which are generally scarce in this environment. The arrangement of hemicelluloses and proteins with the pectins may be associated with increased cell rigidity and sustainability, a feature of interest for the aerial roots. In summary, the habit of roots can interfere with the non-cellulosic composition of the cell walls of V. phaeantha, possibly related to changes in cell functionality.

Determining the Optimal Window Duration to Enhance Emotion Recognition Based on Galvanic Skin Response and Photoplethysmography Signals

Automatic emotion recognition using portable sensors is gaining attention due to its potential use in real-life scenarios. Existing studies have not explored Galvanic Skin Response and Photoplethysmography sensors exclusively for emotion recognition using nonlinear features with machine learning (ML) classifiers such as Random Forest, Support Vector Machine, Gradient Boosting Machine, K-Nearest Neighbor, and Decision Tree. In this study, we proposed a genuine window sensitivity analysis on a continuous annotation dataset to determine the window duration and percentage of overlap that optimize the classification performance using ML algorithms and nonlinear features, namely, Lyapunov Exponent, Approximate Entropy, and Poincaré indices. We found an optimum window duration of 3 s with 50% overlap and achieved accuracies of 0.75 and 0.74 for both arousal and valence, respectively. In addition, we proposed a Strong Labeling Scheme that kept only the extreme values of the labels, which raised the accuracy score to 0.94 for arousal. Under certain conditions mentioned, traditional ML models offer a good compromise between performance and low computational cost. Our results suggest that well-known ML algorithms can still contribute to the field of emotion recognition, provided that window duration, overlap percentage, and nonlinear features are carefully selected.

Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration.

AbstractThis study characterizes a fluorescentSlc17a6-tdTomato neuronal reporter mouse line offering strong labeling in axons throughout the optic nerve, dendrites and soma in 99% of retinal ganglion cells (RGCs). The model facilitates neuronal assessmentex vivowith wholemounts quantified to show neurodegeneration following optic nerve crush or elevated IOP as related to glaucoma,in vitrowith robust Ca2+responses to P2X7 receptor stimulation in neuronal cultures, andin vivousing a confocal scanning laser ophthalmoscope (cSLO). While the tdTomato signal showed strong overlap with RGC markers, BRN3A and RBPMS, there was no cross-labeling of displaced amacrine cells in the ganglion cell layer. Controls indicated no impact ofSlc17a6-tdTomato expression on light-dependent neuronal function, as determined with a microelectrode array (MEA), or on structure, as measured with optical coherence tomography (OCT). In summary, this novel neuronal reporter mouse model offers an effective means to increase the efficiency for real-time, specific visualization of retinal ganglion cells. It holds substantial promise for enhancing our understanding of RGC pathology in glaucoma and other diseases of the optic nerve, and could facilitate the screening of targeted therapeutic interventions for neurodegeneration.Abstract FigureGraphical abstract.Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration. Puttipong Sripinun, Wennan Lu, Sergei Nikonov, Suhani Patel, Sarah Hennessy, Claire H. Mitchell*. This study delves into a new mouse model, featuring a fluorescentSlc17a6-tdTomato neuronal reporter. This model effectively labels axons in the optic nerve, as well as dendrites and soma in 99% of retinal ganglion cells (RGCs). This allows for bothin vitroandin vivoassessment of neurodegeneration, offering a practical tool for real-time, precise visualization of RGCs, with potential applications in various fields of neuroscience and neurology. Created byBiorender.com.

Anti-HLA-B7/HLA-B44 strong cross immunoreactivity observed in flow cytometry HLA-B27 immunotyping.

Cross reactivities are known for human leukocyte antigen inside HLA-B7 related Cross-Reactive Group (B7CREG). Some CE-IVD flow-cytometry kits use double monoclonal antibodies (mAb) to distinguish HLA-B27 and HLA-B7 but practice reveals more complexes results. This study explores the performances of this test. Analysis of 466 consecutive cases using HLA-B27 IOTest™ kit on a Navios™ cytometer from Beckman-Coulter, partially compared to their genotypes. Expected haplotypes HLA-B27-/HLA-B7- (undoubtedly HLA-B27 negative) and HLA-B27+/HLA-B7- (undoubtedly HLA-B27+) were clearly identified according to the manufacturer's instructions. On the opposite, patients strongly labeled with anti-HLA-B7 showed three different phenotypes regarding anti-HLA-B27 labeling: (1) most of the cases were poorly labeled in accordance with cross reactivity inside B7CREG (HLA-B27-/HLA-B7+ haplotype); (2) rare cases had strong B7 and B27 labeling corresponding to HLA-B27+/HLA-B7+ haplotype; (3) even less cases had strong labeling by anti-HLA-B7 but non for anti-HLA-B27, all expressing HLA-B44 and no B7CREG molecules. Surprisingly, more cases were not labeled with anti-HLA-B7 antibody but partially labeled with anti-HLA-B27 suggesting another cross reactivity out of B7CREG. mAb HLA typing suggests new, cross reactivities of anti-HLA-B27 antibody to more molecules out of B7CREG and of anti-HLA-B7 antibody but not anti-HLA-B27 to HLA-B44 molecule also out of B7CREG. HLA-B27 could surely be excluded in most samples labeled with HLA-B27, below a "grey zone" on intermediate intensity. More comparison is needed in future studies.

Strong versus Weak Data Labeling for Artificial Intelligence Algorithms in the Measurement of Geographic Atrophy

PurposeTo gain an understanding of data labeling requirements to train deep learning models for measurement of geographic atrophy with autofluorescence (FAF) images. DesignEvaluation of AI algorithms SubjectsThe Age-Related Eye Disease Study 2 images were used for training and cross validation and GA clinical trial images were used for testing. MethodsTraining data consisted of two sets of FAF images; one with area measurements only and no indication of GA location (Weak-labeled) and the second with GA segmentation masks (Strong-labeled). Main outcome measuresBland Altman plots and scatter plots were used to compare GA area measurement between ground truth and AI measurements. Dice Coefficient was used to compare accuracy of segmentation of Strong model. ResultsIn the cross validation AREDS2 dataset (n = 601), mean area (SD) of GA measured by human grader, Weak-labeled and Strong-labeled AI models was 6.65 mm2 (6.3), 6.83 mm2 (6.29) and 6.58 mm2 (6.24). The mean difference between ground truth and AI was 0.18 mm2 (95% CI -7.57,7.92) for the Weak-labeled model and -0.07 mm2 (-1.61,1.47) for the Strong-labeled model. With GSK testing data (n = 156), the GA area (SD) was 9.79 mm2 (SD 5.6), 8.82 mm2 (4.61) and 9.55 mm2 (5.66) respectively. The mean difference between ground truth and AI for the 2 models was -0.97 mm2 (- 4.36,2.41) and -0.24 mm2 (- 4.98,4.49) respectively. The Dice Coefficient was 0.99 for intergrader agreement, 0.88 for the cross-validation data and 0.92 for the testing data. ConclusionsDeep learning models can achieve reasonable accuracy even with Weakly-labeled data. Training methods that integrate large volumes of Weak-labeled images with small number of Strong-labeled images offer a promising solution to overcome the burden of cost and time for data labeling. Words 3290

Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Immunohistochemistry Can Be a Useful Ancillary Tool to Identify Perivascular Epithelioid Cell Tumor

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal tumors that express smooth muscle and melanocytic makers. Diagnosis of PEComas can be challenging due to focal or lost expression of traditional immunohistochemical markers, limited availability of molecular testing, and morphological overlap with much more common smooth muscle tumors. This study evaluates the use of glycoprotein nonmetastatic melanoma protein B (GPNMB) immunohistochemical staining as a surrogate marker for TSC1/2/MTOR alteration or TFE3 rearrangement to differentiate PEComas from other mesenchymal tumors. Cathepsin K was also assessed for comparison. A total of 399 tumors, including PEComas, alveolar soft part sarcomas, and other histologic PEComa mimics, were analyzed using GPNMB and cathepsin K immunohistochemistry. GPNMB expression was seen in all PEComas and alveolar soft part sarcomas with the majority showing diffuse and moderate-to-strong labeling, whereas other sarcomas were negative or showed focal labeling. When a cutoff of diffuse and at least moderate staining was used, GPNMB demonstrated 95% sensitivity and 97% specificity in distinguishing PEComas from leiomyosarcoma, well-differentiated/dedifferentiated liposarcomas, and undifferentiated pleomorphic sarcomas. Cathepsin K with a cutoff of any labeling had lower sensitivity (78%) and similar specificity (94%) to GPNMB. This study highlights GPNMB as a highly sensitive marker for PEComas and suggests its potential use as an ancillary tool within a panel of markers for accurate classification of these tumors.

Transition to Chronic Fibrosis in an Animal Model of Retinal Detachment With Features of Proliferative Vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the most common cause of failure of surgically repaired rhegmatogenous retinal detachment (RRD). Chemically induced and cell injection PVR models do not fully simulate the clinical characteristics of PVR in the post-RRD context. There is an unmet need for translational models in which to study mechanisms and treatments specific to RRD-PVR. RRD was induced in adult Dutch Belted rabbits. Posterior segments were fixed or processed for RNA sequencing at 6 hours and 2, 7, 14, and 35 days after induction. Histochemical staining and immunolabeling for glial fibrillary acidic protein, alpha smooth muscle actin, vascular endothelial growth factor receptor 2, CD68, and RPE 65 kDa protein were performed, and labeling intensity was scored. Single cell RNA sequencing was performed. Acute histopathological changes included intravitreal and intraretinal hemorrhage, leukocytic vitritis, chorioretinitis, and retinal rarefaction. Chronic lesions showed retinal atrophy, gliosis, fibrotic subretinal membranes, and epiretinal fibrovascular proliferation. Fibrillar collagen was present in the fibrocellular and fibrovascular membranes in chronic lesions. Moderate to strong labeling of glia and vasculature was detected in chronic lesions. At day 14, most cells profiled by single cell sequencing were identified as Mϋller glia and microglia, consistent with immunolabeling. Expression of several fibrillar collagen genes was upregulated in chronic lesions. Histological and transcriptional features of this rabbit model simulate important features of human RRD-PVR, including the transition to chronic intraretinal and periretinal fibrosis. This animal model of RRD with features of PVR will enable further research on targeted treatment interventions.

Androgen Receptor Immunohistochemistry is Superior to PRAME for the Differentiation of Sebaceous Carcinoma From Primary Cutaneous Basaloid Mimics.

Cutaneous sebaceous neoplasia comprises a spectrum of disease ranging from benign adenomas to malignant carcinomas. The hallmark of these lesions is sebaceous differentiation. However, poorly-differentiated sebaceous carcinoma (SC), which lacks significant overt sebaceous differentiation, can show morphologic overlap with a variety of other basaloid cutaneous neoplasms. The accurate classification of SC is essential not only for diagnosis, but also because of the potential association with Muir-Torre syndrome. Androgen receptor (AR) is a sensitive, but not entirely specific immunohistochemical marker that has been used for the diagnosis of SC. PReferentially expressed Antigen in MElanoma (PRAME) demonstrates strong cytoplasmic labeling of mature sebocytes and has been reported to be expressed in a variety of sebaceous neoplasms, including in the basaloid cell component. Therefore, we sought to compare the diagnostic use of cytoplasmic PRAME expression with that of AR for the distinction of SC from a cohort of basaloid cutaneous mimics; namely basal cell carcinoma, basaloid squamous cell carcinoma, pilomatricoma, cutaneous lymphadenoma, and extra-mammary Paget disease. We report that cytoplasmic PRAME expression is uncommon in poorly differentiated SC, and although specific, it shows very low sensitivity (22%). In contrast, AR was moderately sensitive (66%) and highly specific (92%) for the distinction of SC from basaloid mimics. These attributes, in addition to the nuclear expression of AR in the sebocytic and basaloid components of SC, suggest that AR is superior to PRAME for the diagnosis of SC.

Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow

BackgroundDuring clotting, thrombin generates fibrin monomers and activates plasma-derived transglutaminase factor (F) XIIIa; collagen and thrombin-activated platelets offer thrombin-independent cellular FXIIIa (cFXIIIa) for clotting. Detecting fibrin on collagen and tissue factor surfaces in whole blood clotting typically uses complex reagents like fluorescent fibrinogen or antifibrin antibody. ObjectivesWe want to test whether the peptide using the α2- antiplasmin crosslinking mechanism by FXIIIa is a useful tool in both monitoring FXIIIa activity, and visualize and monitor fibrin formation, deposition, and extent of crosslinking within fibrin structures in whole blood clots formed under flow. MethodsWe tested a fluorescent peptide derived from α2-antiplasmin sequence (Ac-GNQEQVSPLTLLKWC-fluorescein) to monitor the location of transglutaminase activity and fibrin during whole blood clotting under microfluidic flow (wall shear rate, 100 s−1). ResultsThe peptide rapidly colocated with accumulating fibrin due to transglutaminase activity, confirmed by Phe-Pro-Arg-chloromethylketone inhibiting fibrin and peptide labeling. The FXIIIa inhibitor T101 had no effect on fibrin generation but ablated the labeling of fibrin by the peptide. Similarly, Gly-Pro-Arg-Pro abated fibrin formation and thus strongly attenuated the peptide signal. At arterial wall shear rate (1000 s−1), less fibrin was formed, and consequently, less peptide labeling of fibrin was detected compared with venous conditions. The addition of tissue plasminogen activator caused a reduction of both fibrin and peptide signals. Also, the peptide strongly colocalized with fibrin (but not platelets) in clots from laser-injured mouse cremaster arterioles. For clotting under flow, FXIIIa activity was most likely plasma-derived since a RhoA inhibitor did not block α2-antiplasmin fragment cross-linking to fibrin. ConclusionUnder flow, the majority of FXIIIa-dependent fibrin labeling with peptide during clotting was distal of thrombin activity. The synthetic peptide provided a strong and sustained labeling of fibrin as it formed under flow.

Static recording apparatus for soundscape analysis in Macau

The concept of Soundscape was initially proposed to study the relationship between humans and their sonic environment. It has gathered momentum from academia to environmentalists and policymakers throughout the years. The study and characterisation of Soundscapes can be complex as it tries to take a holistic and qualitative approach rather than simply quantifying sound pressure levels. This paper introduces a comprehensive Soundscape study process in an ongoing research project in Macao (China), a small territory (32.9 km2) and one of the most densely populated areas in the world. The paper seeks to show a first version of a technical solution to systematically capture the local soundscape, analyse it, classify it, and ultimately deliver a dataset library and the intangible qualities of the environmental sound. This implementation, including technical documentation, code, and sound library with strong labelling, is presented under an open-source license to encourage future collaborative research. Finally, the paper offers suggestions on further developing the apparatus to reach a systematic and near real-time soundscape analysis with the development of a machine learning system.

Ultrastructural and immunofluorescence features of the epidermal cells and its secretory granules in the amphioxus Branchiostoma lanceolatum L.

Background and purpose: In the vertebrate family, the epidermis of terrestrial animals is keratinized while in aquatic forms and amphibians can produce mucus. Amphioxus, a cephalochordate, is an important animal model in the study of chordate phylogeny. Major cytoskeletal and extracellular matrix proteins in epidermal cells of amphioxus (Branchiostoma lanceolatum L.) were investigated using transmission electron microscopy and immunofluorescence. Materials and methods: Amphioxus specimens were fresh caught in the Adriatic Sea. Tissue samples were fixed and prepared for transmission electron microscopy and immunofluorescence techniques. Results: The epidermis consisted of one-layered columnar epithelium. Epithelial cells contained voluminous nucleus, a well-developed supranuclear Golgi apparatus, abundant vesicles and apical secretory vesicles surrounded by cytokeratin filaments. A single row of smaller vesicles also appeared close to the basal cell membrane. Strong positive immunolocalization of hyaluronic acid and collagen type I was observed in the apical domain of the plasma membrane of amphioxus, respectively. The positive signal of hyaluronan and collagen proteins referred to the secretory granules in the apical domain of the epidermal cell. Strong positive labelling of aggrecan was seen around the cell nucleus, deep below the apical domain, suggesting that aggrecan is not located in the secretory granules. A strong signal of the cytokeratin 10 protein was seen throughout the cytoplasm of the epidermal cells. Conclusion: Due to its morphology and histochemical composition, epidermal cells of amphioxus may contribute to course surface protection and the mucous layer in amphioxus may serve as a physical barrier between the body and its environment.

Immunolocalization of SIFamide-like neuropeptides in the adult and developing central nervous system of the amphipod Parhyale hawaiensis (Malacostraca, Peracarida, Amphipoda)

Immunohistochemical analyses on the distribution of neuropeptides in the pancrustacean brain in the past have focussed mostly on representatives of the decapod (“ten-legged”) pancrustaceans whereas other taxa are understudied in this respect. The current report examines the post-embryogenic and adult brain and ventral nerve cord of the amphipod pancrustacean Parhyale hawaiensis (Dana. 1853; Peracarida, Amphipoda, Hyalide), a subtropical species with a body size of 1.5 cm and a direct post-embryonic development using immunohistochemistry to label the neuropeptide SIFamide and synaptic proteins (synapsins). We found strong SIFamide-like labelling in proto-, deuto- and tritocerebrum, especially in the lamina, the lateral protocerebrum, lateral assessory lobe, the central body, olfactory lobe, medial antenna 1 neuropil and antenna 2 neuropil. Out of a total of 28 ± 5 (N = 12) SIFamide-positive neurons in the central brain of adult P. hawaiensis, we found three individually identifiable somata which were consistently present within the brain of adult and subadult animals. Additionally, the subesophageal and two adjacent thoracic ganglia were analysed in only adult animals and also showed a strong SIFamide-like immunoreactivity. We compare our findings to other pancrustaceans including hexapods and discuss them in an evolutionary context.

Strong desmin immunoreactivity in the myocardial sleeves around pulmonary veins, superior caval vein and coronary sinus supports the presumed arrhythmogenicity of these regions.

Myocardial sleeve around human pulmonary veins plays a critical role in the pathomechanism of atrial fibrillation. Besides the well-known arrhythmogenicity of these veins, there is evidence that myocardial extensions into caval veins and coronary sinus may exhibit similar features. However, studies investigating histologic properties of these structures are limited. We aimed to investigate the immunoreactivity of myocardial sleeves for intermediate filament desmin, which was reported to be more abundant in Purkinje fibers than in ventricular working cardiomyocytes. Sections of 16 human (15 adult and 1 fetal) hearts were investigated. Specimens of atrial and ventricular myocardium, sinoatrial and atrioventricular nodes, pulmonary veins, superior caval vein and coronary sinus were stained with anti-desmin monoclonal antibody. Intensity of desmin immunoreactivity in different areas was quantified by the ImageJ program. Strong desmin labeling was detected at the pacemaker and conduction system as well as in the myocardial sleeves around pulmonary veins, superior caval vein, and coronary sinus of adult hearts irrespective of sex, age, and medical history. In the fetal heart, prominent desmin labeling was observed at the sinoatrial nodal region and in the myocardial extensions around the superior caval vein. Contrarily, atrial and ventricular working myocardium exhibited low desmin immunoreactivity in both adults and fetuses. These differences were confirmed by immunohistochemical quantitative analysis. In conclusion, this study indicates that desmin is abundant in the conduction system and venous myocardial sleeves of human hearts.

The proteome of extracellular vesicles of the lung fluke Paragonimus kellicotti produced in vitro and in the lung cyst

Paragonimiasis is a zoonotic, food-borne trematode infection that affects 21 million people globally. Trematodes interact with their hosts via extracellular vesicles (EV) that carry protein and RNA cargo. We analyzed EV in excretory-secretory products (ESP) released by Paragonimus kellicotti adult worms cultured in vitro (EV ESP) and EV isolated from lung cyst fluid (EV CFP) recovered from infected gerbils. The majority of EV were approximately 30–50 nm in diameter. We identified 548 P. kellicotti-derived proteins in EV ESP by mass spectrometry and 8 proteins in EV CFP of which 7 were also present in EV ESP. No parasite-derived proteins were reliably detected in EV isolated from plasma samples. A cysteine protease (MK050848, CP-6) was the most abundant protein found in EV CFP in all technical and biological replicates. Immunolocalization of CP-6 showed strong labeling in the tegument of P. kellicotti and in the adjacent cyst and lung tissue that contained worm eggs. It is likely that CP-6 present in EV is involved in parasite-host interactions. These results provide new insights into interactions between Paragonimus and their mammalian hosts, and they provide potential clues for development of novel diagnostic tools and treatments.

Appl1, Sortilin and Syndecan-1 immunohistochemistry on intraductal carcinoma of the prostate provides evidence of retrograde spread

The presence of intraductal carcinoma of the prostate (IDCP) correlates with late-stage disease and poor outcomes for patients with prostatic adenocarcinoma, but the accurate and reliable staging of disease severity remains challenging. Immunohistochemistry (IHC) has been utilised to overcome problems in assessing IDCP morphology, but the current markers have only demonstrated limited utility in characterising the complex biology of this lesion. In a retrospective study of a cohort of patients who had been diagnosed with IDCP, we utilised IHC on radical prostatectomy sections with a biomarker panel of Appl1, Sortilin and Syndecan-1, to interpret different architectural patterns and to explore the theory that IDCP occurs from retrograde spread of high-grade invasive prostatic adenocarcinoma. Cribriform IDCP displayed strong Appl1, Sortilin and Syndecan-1 labelling patterns, while solid IDCP architecture had high intensity Appl1 and Syndecan-1 labelling, but minimal Sortilin labelling. Notably, the expression pattern of the biomarker panel in regions of IDCP was similar to that of adjacent invasive prostatic adenocarcinoma, and also comparable to prostate cancer showing perineural and vascular invasion. The Appl1, Sortilin, and Syndecan-1 biomarker panel in IDCP provides evidence for the model of retrograde spread of invasive prostatic carcinoma into ducts/acini, and supports the inclusion of IDCP into the five-tier Gleason grading system.

German Shorthaired Pointer dogs with exfoliative cutaneous lupus erythematosus develop immune-complex membranous glomerulonephropathy.

German Shorthaired Pointer (GSHP) dogs with a UNC93B1 gene mutation develop exfoliative cutaneous lupus erythematosus (ECLE) and kidney disease resembling lupus nephritis in humans. The objective of this study was to characterize the kidney disease by light microscopy, immunofluorescence, and electron microscopy in a population of GSHP dogs with ECLE. Medical records were reviewed, and light microscopy of kidneys from 7 GSHP dogs with a previous histologic diagnosis of ECLE was performed. Immunofluorescence of fresh-frozen kidney from 1 dog and transmission electron microscopy of kidney from that dog and 2 additional dogs were performed. Five of 7 dogs had proteinuria diagnosed by urinalysis or urine protein-to-creatinine ratio. Two of 7 dogs were intermittently hypoalbuminemic, and none were azotemic. Histologic findings included early (2 dogs) to late (5 dogs) membranous glomerulonephropathy characterized by mild-to-severe glomerular capillary loop thickening and tubular proteinosis. In all 7 cases, trichrome staining revealed red granular immune deposits on the subepithelial surface of the glomerular basement membrane. Immunofluorescence revealed strong granular labeling for immunoglobulins and complement protein C3. Electron microscopy demonstrated subepithelial electron-dense immune deposits encircled by the remodeled glomerular basement membrane. These findings are diagnostic of immune-complex membranous glomerulonephropathy and are similar to class V lupus in humans. This cohort of GSHP dogs with ECLE developed immune-complex membranous glomerulonephropathy, which we hypothesize is a manifestation of systemic lupus erythematosus. GSHP dogs with ECLE should undergo clinical evaluation of renal function for early identification and treatment.

Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer's disease.

Ergothioneine (Ergo) is a naturally occurring dietary antioxidant. Ergo uptake is dependent on the transporter, organic cation transporter novel-type 1 (OCTN1) distribution. OCTN1 is highly expressed in blood cells (myeloid lineage cells), brain and ocular tissues that are likely predisposed to oxidative stress. Ergo may protect the brain and eye against oxidative damage and inflammation, however, the underlying mechanism remains unclear. Amyloid beta (Aβ) clearance is a complex process mediated by various systems and cell types including vascular transport across the blood-brain barrier, glymphatic drainage, and engulfment and degradation by resident microglia and infiltrating innate immune cells. Impaired Aβ clearance is a major cause for Alzheimer's disease (AD). Here we investigated neuroretinas to explore the neuroprotective effect of Ergo in a transgenic AD mouse model. Age-matched groups of Ergo-treated 5XFAD, non-treated 5XFAD, and C57BL/6J wildtype (WT controls) were used to assess Ergo transporter OCTN1 expression and Aβ load along with microglia/macrophage (IBA1) and astrocyte (GFAP) markers in wholemount neuroretinas (n = 26) and eye cross-sections (n = 18). Immunoreactivity was quantified by fluorescence or by semi-quantitative assessments. OCTN1 immunoreactivity was significantly low in the eye cross-sections of Ergo-treated and non-treated 5XFAD vs. WT controls. Strong Aβ labeling, detected in the superficial layers in the wholemounts of Ergo-treated 5XFAD vs. non-treated 5XFAD reflects the existence of an effective Aβ clearance system. This was supported by imaging of cross-sections where Aβ immunoreactivity was significantly low in the neuroretina of Ergo-treated 5XFAD vs. non-treated 5XFAD. Moreover, semi-quantitative analysis in wholemounts identified a significantly reduced number of large Aβ deposits or plaques, and a significantly increased number of IBA1(+)ve blood-derived phagocytic macrophages in Ergo-treated 5XFAD vs. non-treated 5XFAD. In sum, enhanced Aβ clearance in Ergo-treated 5XFAD suggests that Ergo uptake may promote Aβ clearance possibly by blood-derived phagocytic macrophages and via perivascular drainage.

Abstract P2-11-17: Prognostic value of Trop2 expression levels in non-metastatic triple-negative breast cancer and correlation with other biomarkers

Abstract Background: Since the development of anti Trop2 antibody-drug conjugates (ADC), Trop2 has been validated as a major therapeutic target for antibody-drug conjugates in metastatic triple-negative breast cancer (TNBC). However, very few data have been reported regarding the prognostic implication, or clinicopathological variables, associated with Trop2 expression levels in early TNBC. Our aim was to evaluate Trop2 expression and its prognostic value in a retrospective series of patients with non-metastatic TNBC and a long follow-up, characterized for Basal-like (BL) or molecular apocrine-like (MA) IHC profiles as well as TILs infiltrate, PDL1 expression and PIK3CA/PTEN mutations. Patients and methods: The analysis was performed in a series of 228 patients with non-metastatic TNBC treated in our center between 2002 and 2012 arrayed on 6 TMA. BL and MA profiles were defined as IHC CK5/6 and/or EGFR expression, and Androgen-receptor and FOXA1 IHC expression, respectively. Trop2 expression levels were tested for their association with baseline clinicopathological variables, and for Relapse-Free Survival (RFS) and Overall survival (OS). Trop2 IHC level of expression was evaluated using the ENZ-ABS380 mouse monoclonal antibody and the methodology described by Bardia et al. (Annals of Oncology, 2021) to quantify the membrane signal. First, we established a score grid, according to staining intensity (no labeling: 0; weak labeling: 1; moderate labeling: 2; strong labeling: 3). Then, for each sampled core, the percentage of labeled invasive tumor cells in each intensity was reported. The overall membrane expression was then calculated using the H-Score method (3 x % of cells with labeling intensity 3 + 2 x % of cells with intensity 2 + 1 x % of cells with intensity 1). The scores obtained ranged from 0 to 300. Results: Median age was 58.2 years (range 28.5-89.1). 43.9% of tumors were classified pT1 and 63.5% pN0. 83.8% of patients had ductal carcinomas. Histological grade 1-2 represented 22.7% of all tumors. A BL phenotype was observed in 68.1% of cases, and an MA profile in 39.4% of the cases. Adjuvant chemotherapy (ACT) was delivered in 74% of patients. We observed low Trop2 expression (H-Score < 100) in 12.3% of the cases (28/228 samples), moderate Trop2 expression (H-Score 100-200) in 28.9% of the cases (66/228 samples), and strong Trop2 expression (H-Score >200) in 58.8% of the cases (134/228 samples). We only identified 3 tumors without any Trop2 expression. Regarding baseline clinicopathological correlations, Trop2 levels were only found significantly associated with pT stage, pT1 tumors displaying more frequently high Trop2 scores compared to tumors >= pT2 (p=0.002). No significant correlation was found between Trop2 expression levels and HER2 levels (0 vs. 1+/2+), BL or MA status. With a median follow-up of 9.7 years, Trop2 levels were not associated with RFS nor OS in univariate analysis. In multivariate analysis, poor RFS was associated with classical variables in the early TNBC setting: tumors >=T2 stage (Hazard Ratio (HR) 2.13, p=0.02), N+ status (HR 3.51, p< 0.001), while high (>5%) TILs infiltration levels (HR 0.54, p=0.03) and adjuvant chemotherapy use (HR 0.49, p=0.007) were associated with improved RFS. Conclusions: Trop2 is expressed in nearly all early TNBC cases, and Trop2 levels of expression, while associated with T stage, did not impact survival in this population. These results are consistent with the ones reported in the metastatic setting in the ASCENT trial. Trop2 level of expression appearing homogeneous distributed in all the clinicopathological subtypes of TNBC, advocating for the evaluation of combined treatments with anti-Trop2 ADCs and dedicated targeted therapies in specific TNBC subgroups. Citation Format: William Jacot, Marie-Christine Chateau, Simon Thezenas, Séverine Guiu, Nelly Firmin, Florence Boissière-Michot. Prognostic value of Trop2 expression levels in non-metastatic triple-negative breast cancer and correlation with other biomarkers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-17.

Strong Labeling of Sound Events Using Crowdsourced Weak Labels and Annotator Competence Estimation

Crowdsourcing is a popular tool for collecting large amounts of annotated data, but the specific format of the strong labels necessary for sound event detection is not easily obtainable through crowdsourcing. In this work, we propose a novel annotation workflow that leverages the efficiency of crowdsourcing weak labels, and uses a high number of annotators to produce reliable and objective strong labels. The weak labels are collected in a highly redundant setup, to allow reconstruction of the temporal information. To obtain reliable labels, the annotators' competence is estimated using MACE (Multi-Annotator Competence Estimation) and incorporated into the strong labels estimation through weighing of individual opinions. We show that the proposed method produces consistently reliable strong annotations not only for synthetic audio mixtures, but also for audio recordings of real everyday environments. While only a maximum 80% coincidence with the complete and correct reference annotations was obtained for synthetic data, these results are explained by an extended study of how polyphony and SNR levels affect the identification rate of the sound events by the annotators. On real data, even though the estimated annotators' competence is significantly lower and the coincidence with reference labels is under 69%, the proposed majority opinion approach produces reliable aggregated strong labels in comparison with the more difficult task of crowdsourcing directly strong labels.