Epithelial Alterations Research Articles

Consequences of Exposure to Hypobaric Hypoxia Associated with High Altitude on Spermatogenesis and Seminal Parameters: A Literature Review.

Preclinical research has provided compelling evidence indicating that exposure to hypobaric hypoxia (HH) results in a deterioration of spermatogenesis. This adverse effect extends to the underlying molecular mechanisms, progressively leading to impairments in the seminiferous epithelium and germ cells and alterations in semen parameters. Indeed, several studies have demonstrated that animals exposed to HH, whether in natural high-altitude environments or under simulated hypoxic conditions, exhibit damage to the self-renewal and differentiation of spermatogenesis, an increase in germline cell apoptosis, and structural alterations in the seminiferous tubules. One of the primary mechanisms associated with the inhibition of differentiation and an increase in apoptosis among germ cells is an elevated level of oxidative stress, which has been closely associated with HH exposure. Human studies have shown that individuals exposed to HH, such as mountaineers and alpinists, exhibit decreased sperm count, reduced motility, diminished viability, and increased sperm with abnormal morphology in their semen. This evidence strongly suggests that exposure to HH may be considered a significant risk factor that could elevate the prevalence of male infertility. This literature review aims to provide a comprehensive description and propose potential mechanisms that could elucidate the infertility processes induced by HH. By doing so, it contributes to expanding our understanding of the challenges posed by extreme environments on human physiology, opening new avenues for research in this field.

Abstract 2691: Mapping the landscape of innate immune pattern recognition receptors in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all pancreatic cancer cases, and is among the most aggressive and lethal malignancies worldwide. PDAC is driven by genetic alterations in the pancreatic epithelium (e.g. KRAS, TRP53) coupled with a dysregulated innate immune response, the latter leading to an inflammatory tumor microenvironment enriched in innate immune cells (e.g. macrophages). However, immune-based treatment regimens for PDAC patients have primarily focused on immunotherapy with adaptive immune checkpoint inhibitors (e.g. PD-1) which disappointingly have yielded minimal clinical benefit. Thus, there is an unmet clinical need to identity disease-associated innate immune regulators as therapeutic targets in PDAC. Innate immune responses depend on a series of cell surface, endosomal and cytosolic pattern recognition receptors (PRRs) that are expressed in immune and non-immune cells. PRRs are classified into several structurally and functionally conserved subfamilies, including Toll-like receptors (TLRs), Absent in melanoma 2 (AIM2)-like receptors (ALRs), and Nod-like receptors (NLRs). A subset of NLRs and AIM2 are also well documented for their formation of multiprotein complexes called ‘inflammasomes’ - comprising the adaptor ASC and Caspase-1 - which direct the maturation and release of bioactive pro-inflammatory cytokines, IL-18 and IL-1β. However, the role of these critical innate immune regulators in PDAC has been underexplored, and is virtually unknown. By coupling endoscopic ultrasound-guided fine needle aspiration (EUS FNA) to capture tumor biopsies from all stages of PDAC, with whole transcriptome profiling of PDAC patient primary tumors, we reveal specific enrichment of the innate immune TLR2 and ASC inflammasome molecular pathways. Augmented TLR2 and ASC expression was prognostic for low survival, and a “TLR2 activation” signature was also predictive for chemoresistance. Antibody-mediated anti-TLR2 therapy suppressed tumor growth in human PDAC tumor xenografts (patient and cell line) in both immunocompromised and ‘humanized’ mice. With respect to ASC, using the genetically engineered KPC PDAC mouse model, we show that Caspase-1 activation, a read-out for inflammasome activation, is upregulated in pancreatic tumors. Moreover, genetic ablation of ASC in KPC mice markedly suppressed the development of pancreatic tumors, which was associated with significant reductions in levels of both Caspase-1 activation and inflammation. Transcriptome profiling further identified unique downstream molecular networks associated with a range of oncogenic cellular processes. Taken together, our results support innate immune TLR2- and ASC inflammasome-based therapeutic targeting in PDAC, with further clinical utility that ASC inflammasome and TLR2 activation is prognostic, with TLR2 also predictive for chemoresponsiveness. Citation Format: Bassam Kashgari, Louise McLeod, Linden J. Gearing, Daniel Croagh, Brendan J. Jenkins. Mapping the landscape of innate immune pattern recognition receptors in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2691.

Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy.

Chronic rhinosinusitis with nasal polyp (CRSwNP) is a typical type 2 inflammation involving interleukin (IL)-4 and IL-13. Dupilumab is a fully human monoclonal antibody targeting IL-4 receptor α subunit, thereby blocking signaling by both cytokines. Our hypothesis was that IL-4 and IL-13, by inducing a severe epithelial dysregulation, are involved in CRSwNP pathogenesis. This study aimed to evaluate the in vitro direct effect of IL-4, IL-13, and dupilumab on nasal epithelial functions. Nasal polyps and control mucosa from 28 patients, as well as human nasal epithelial cells (HNEC) from 35 patients with CRSwNP were used. Three major epithelial functions were investigated: the epithelial barrier function (characterized by transepithelial electrical resistance measurements and tight junction protein expression), the ciliary motion (characterized by the ciliary beating efficiency index), and wound healing (characterized by the wound repair rate) under various stimulations (IL-4, IL-13, and dupilumab). The main outcome was a significant change in epithelial functions following exposure to IL-4, IL-13, and dupilumab for 48h in the basal media. IL-4 (1, 10, and 100ng/mL) but not IL-13 induced a significant decrease in occludin and zonula-occludens protein expression, ciliary beating efficiency, and wound repair rate in HNEC. Dupilumab (0.04mg/mL) had no effect on HNEC and specifically restored all epithelial functions altered when cells were exposed to a 48-h IL-4 stimulation. Dupilumab, in vitro, restored epithelial integrity by counteracting the effect of IL-4 on the epithelial barrier (increased epithelial permeability, decreased ciliary beating efficiency, and decreased wound repair rate).

Epithelial MAPK signaling directs endothelial NRF2 signaling and IL-8 secretion in a tri-culture model of the alveolar-microvascular interface following diesel exhaust particulate (DEP) exposure

BackgroundParticulate matter 2.5 (PM2.5) deposition in the lung’s alveolar capillary region (ACR) is significantly associated with respiratory disease development, yet the molecular mechanisms are not completely understood. Adverse responses that promote respiratory disease development involve orchestrated, intercellular signaling between multiple cell types within the ACR. We investigated the molecular mechanisms elicited in response to PM2.5 deposition in the ACR, in an in vitro model that enables intercellular communication between multiple resident cell types of the ACR.MethodsAn in vitro, tri-culture model of the ACR, incorporating alveolar-like epithelial cells (NCI-H441), pulmonary fibroblasts (IMR90), and pulmonary microvascular endothelial cells (HULEC) was developed to investigate cell type-specific molecular responses to a PM2.5 exposure in an in-vivo-like model. This tri-culture in vitro model was termed the alveolar capillary region exposure (ACRE) model. Alveolar epithelial cells in the ACRE model were exposed to a suspension of diesel exhaust particulates (DEP) (20 µg/cm2) with an average diameter of 2.5 µm. Alveolar epithelial barrier formation, and transcriptional and protein expression alterations in the directly exposed alveolar epithelial and the underlying endothelial cells were investigated over a 24 h DEP exposure.ResultsAlveolar epithelial barrier formation was not perturbed by the 24 h DEP exposure. Despite no alteration in barrier formation, we demonstrate that alveolar epithelial DEP exposure induces transcriptional and protein changes in both the alveolar epithelial cells and the underlying microvascular endothelial cells. Specifically, we show that the underlying microvascular endothelial cells develop redox dysfunction and increase proinflammatory cytokine secretion. Furthermore, we demonstrate that alveolar epithelial MAPK signaling modulates the activation of NRF2 and IL-8 secretion in the underlying microvascular endothelial cells.ConclusionsEndothelial redox dysfunction and increased proinflammatory cytokine secretion are two common events in respiratory disease development. These findings highlight new, cell-type specific roles of the alveolar epithelium and microvascular endothelium in the ACR in respiratory disease development following PM2.5 exposure. Ultimately, these data expand our current understanding of respiratory disease development following particle exposures and illustrate the utility of multicellular in vitro systems for investigating respiratory tract health.

Disruption of gonocyte development following neonatal exposure to di (2-ethylhexyl) phthalate

Pre- and/or post-natal administrations of di(2-ethylhexyl) phthalate (DEHP) in experimental animals cause alterations in the spermatogenesis. However, the mechanism by which DEHP affects fertility is unknown and could be through alterations in the survival and differentiation of the gonocytes. The aim of the present study was to evaluate the effect of a single administration of DEHP in newborn mice on gonocytic proliferation, differentiation and survival and its long-term effects on seminiferous epithelium and sperm quality. BALB/c mice distributed into Control and DEHP groups were used. Each animal in the DEHP group was given a single dose of 500 mg/Kg at birth. The animals were analyzed at 1, 2, 4, 6, 8, 10 and 70 days postpartum (dpp). Testicular tissues were processed for morphological analysis to determine the different types of gonocytes, differentiation index, seminiferous epithelial alterations, and immunoreactivity to Stra8, Pcna and Vimentin proteins. Long-term evaluation of the seminiferous epithelium and sperm quality were carried out at 70 dpp. The DEHP animal group presented gonocytic degeneration with delayed differentiation, causing a reduction in the population of spermatogonia (Stra8 +) in the cellular proliferation (Pcna+) and disorganization of Vimentin filaments. These events had long-term repercussions on the quality of the seminiferous epithelium and semen. Our study demonstrates that at birth, there is a period that the testes are extremely sensitive to DEHP exposure, which leads to gonocytic degeneration and delay in their differentiation. This situation can have long-term repercussions or permanent effects on the quality of the seminiferous epithelium and sperm parameters.

Juglans regia L. fruit pellicle extract-based bioreduction of silver nanoparticles: Structural features and in vivo therapeutic effects against ethanol-induced peptic ulcers

Context: Peptic ulcers are a challenging problem. Silver nanoparticles (AgNPs) play a substantial role in bioactivity and have distinct characteristics. There are several biomedical uses for bioreduction nanotechnology. Historically, traditional healthcare has employed Juglans regia L. Aims: To evaluate how well fruit pellicle extract-derived bio-reduced silver nanoparticles (AgNP/FP) can treat peptic ulcers. Methods: Ultraviolet spectra, dynamic light scattering, transmittance electron microscopy, and Fourier transform infrared were used to characterize AgNPs and AgNP/FP. AgNPs and extracts were compared to omeprazole in ethanol-induced stomach ulcers in mice. Six groups of 36 mice were created at random. AgNPs were given orally to the experimental groups, using 300 mg/kg as a dose. The reference proton pump inhibitor, omeprazole, was employed as a control. Results: As evidenced by a falling ulcer index and an increase in the percentage of ulcer inhibition, bio-reduced AgNPs decreased the adverse effect of ethanol-induced stomach injury. Reduced mucosal damage and hemorrhagic lesions, necrobiotic alterations in the stomach epithelium, submucosal edema, and blood vessel congestion were all indicators of substantially reduced ethanol-induced gastric lesions and greater anti-inflammatory results. Conclusions: Fruit pellicle extract-derived bio-reduced silver nanoparticles have an efficacy similar to proton pump inhibitors like omeprazole and a lower incidence of possible side effects, making them a safe herbal therapy for ulcers.

Downregulation of praja2 restrains endocytosis and boosts tyrosine kinase receptors in kidney cancer

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC will provide clues to treat this aggressive malignant tumor. Here, we report that the ubiquitin ligase praja2 forms a complex with-and ubiquitylates the AP2 adapter complex, contributing to receptor endocytosis and clearance. In human RCC tissues and cells, downregulation of praja2 by oncogenic miRNAs (oncomiRs) and the proteasome markedly impairs endocytosis and clearance of the epidermal growth factor receptor (EGFR), and amplifies downstream mitogenic and proliferative signaling. Restoring praja2 levels in RCC cells downregulates EGFR, rewires cancer cell metabolism and ultimately inhibits tumor cell growth and metastasis. Accordingly, genetic ablation of praja2 in mice upregulates RTKs (i.e. EGFR and VEGFR) and induces epithelial and vascular alterations in the kidney tissue.In summary, our findings identify a regulatory loop between oncomiRs and the ubiquitin proteasome system that finely controls RTKs endocytosis and clearance, positively impacting mitogenic signaling and kidney cancer growth.

Oral infection with Porphyromonas gingivalis augmented gingival epithelial barrier molecules alteration with aging

ObjectiveDisruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. MethodsIn vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. ResultsLPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. ConclusionIn conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.

FABP6 serves as a new therapeutic target in esophageal tumor.

Esophageal cancer is one of the most common malignant tumors with high incidence and mortality rates. Despite the continuous development of treatment options, the prognosis for esophageal cancer patients remains poor. Therefore, there is an urgent need for new diagnostic and therapeutic targets in clinical practice to improve the survival of patients with esophageal cancer. In this study, we conducted a comprehensive scRNA-seq analysis of the tumor microenvironment in primary esophageal tumors to elucidate cell composition and heterogeneity. Using Seurat, we identified eight clusters, encompassing non-immune cells (fibroblasts, myofibroblasts, endothelial cells, and epithelial cells) and immunocytes (myeloid-derived cells, T cells, B cells, and plasma cells). Compared to normal tissues, tumors exhibited an increased proportion of epithelial cells and alterations in immune cell infiltration. Analysis of epithelial cells revealed a cluster (cluster 0) with a high differentiation score and early distribution, suggesting its importance as a precursor cell. Cluster 0 was characterized by high expression of FABP6, indicating a potential role in fatty acid metabolism and tumor growth. T cell analysis revealed shifts in the balance between Treg and CD8+ effector T cells in tumor tissues. Cellular communication analysis identified increased interactions between FABP6+ tumor cells and T cells, with the involvement of the MIF-related pathway and the CD74-CD44 interaction. This study provides insights into the cellular landscape and immune interactions within esophageal tumors, contributing to a better understanding of tumor heterogeneity and potential therapeutic targets.

Abstract A019: Neoplastic co-option of epithelial-immune interactions in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is initiated by a complex interplay between genetic and environmental insults that induce a marked remodeling of the pancreatic epithelium and its immune cell landscape. These tissue changes are highly reminiscent of wound healing responses, yet they critically contribute to disease pathogenesis. How regenerative tissue plasticity is co-opted to drive PDAC pathogenesis remains poorly understood. Our recent work identified cancer-associated chromatin alterations in the pre-malignant pancreatic epithelium that are induced by cooperative effects of mutant KRAS and tissue damage (inflammation). These synergistic processes aberrantly activate several cell communication factors, such as IL-33, that establish an expansive communication network among selective KRAS-mutant subpopulations and their immune environment that contributes to tumor development (Alonso-Curbelo et al., Nature 2021; Burdziak*, Alonso-Curbelo* et al., Science 2023). These epigenetically-activated communication networks are not shared with physiological regenerative processes, and represent a molecular groundwork to understand (onco)gene-environment cooperation and identify early, tumor-specific cancer mechanisms. They also provide new opportunities at the intersection of cancer biology, epigenetics and immuno-oncology for exploiting cell-cell communication traits of early KRAS-mutant cells and their niches to block the emergence and progression of neoplastic lineages. To address these questions, we are integrating single-cell genomics profiling, functional perturbations, and multiplexed tissue analyses to identify early molecular programs responsible for co-opting immune responses in PDAC, define how these shape influence tumor evolution, and identify crosstalk networks sustaining disease maintenance. Ultimately, a deeper mechanistic understanding of how early epigenetic dysregulation directs regenerative immune responses towards cancer will expose new therapeutic concepts to selectively target pro-tumor inflammation without impairing homeostatic tissue responses. Citation Format: Direna Alonso-Curbelo. Neoplastic co-option of epithelial-immune interactions in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A019.

A toxicokinetic–toxicodynamic model with a transgenerational damage to explain toxicity changes over generations (in Daphnia magna exposed to depleted uranium).

We used a toxicokinetic–toxicodynamic (TKTD) model to analyze chronic effects, in Daphnia magna exposed to waterborne depleted uranium (DU) for two or three successive generations (F0, F1 and F2). Our aim was to understand how DU toxicity for growth and reproduction increased across generations. For the first time in a TKTD model, we introduced a novel transgenerational damage compartment, whose level was transmitted from parents to progeny upon egg deposition. Various exposure regimes took account of differences in exposure among generations. We used a simplified dynamic energy budget applied to toxicology (DEBtox) including two independent physiological modes of action (pMoA). The first pMoA, a reduction in assimilation linked to internal concentration, was previously confirmed by complementary analyses (direct measurements of carbon assimilation, histological observations of gut epithelium alterations). The second pMoA, an increase in costs for growth and maturation linked to transgenerational damage, was the most likely among three possible pMoA affecting both growth and reproduction. Modelling results showed that internal concentration and transgenerational damage followed strongly different kinetics across generations, suggesting that the two pMoA played very contrasting roles in long-term DU toxicity for D. magna. Internal concentration only increased between generations F0 and F1, showing no further difference between generations F1 and F2. Our model was able to correctly describe and predict DU toxicity data, in all tested generations and concentrations, and provided a mechanistic explanation for the increase in DU toxicity across generations.

Short Term Exposure of Sheep Tracheal Epithelium to Cigarette Smoke Extract Reduces ENaC Current: A Pilot Study.

Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle. Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (Isc) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10-5M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations. The amiloride effect on normal epithelium led to a significant decrease in Isc [ΔI=33±5.92 μA/cm2; p<0.001 versus control experiments (ΔI=1.44±0.71 μA/cm2)]. After incubation with CSE, ENaC Isc was significantly reduced (ΔI=14.80±1.96 μA/cm2; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells. Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium.

Detecting Keratoconus in Adolescents with Anterior Segment Optical Coherence Tomography.

Assessing the applicability of an algorithm developed for keratoconus detection in adolescents. This algorithm relies on optical coherence tomography (OCT) and incorporates features related to corneal pachymetric and epithelial thickness alterations. We retrospectively reviewed charts of patients under the age of 18 and divided them into four groups according to the Belin-Ambrosio display (Pentacam): normal, manifest, and subclinical keratoconus, as well as very asymmetric eye with normal topography and tomography (VAE-NTT). Corneal and epithelial thickness maps (Cirrus 5000 HD-OCT, Carl Zeiss Meditec, Germany) were evaluated by a human grader. In the first step, if at least one of four parameters (pachymetry minimum (pachy min), pachy minimum-median (min-med), pachy superonasal-inferotemporal (SN-IT), or epithelial (epi SN-IT)) exceeded its cut-off value, the eye was considered as suspect. In the second step, the combined presence of coincident thinning of total cornea and epithelium as well as concentric epithelial thinning lead to the diagnosis of keratoconus. Receiver operating characteristic (ROC) curves were generated to determine area under the curve (AUC), sensitivity, and specificity for the parameters. The study involved 19 pediatric patients diagnosed with keratoconus, comprising 29 manifest keratoconic eyes, 3 eyes with subclinical keratoconus, and 5 VAE-NTT eyes. In addition, 22 eyes from 11 normal adolescents were included in the analysis. The AUC values of parameters in step 1 were 0.889 for pachy min, 0.997 for pachy min-med, 0.893 for pachy SN-IT, and 0.998 for epi SN-IT. When both steps were performed, this algorithm captured all manifest and subclinical pediatric keratoconic eyes. When all eyes of the keratoconus patients were combined, step 1 had 97.3% sensitivity and step 2 had 100% specificity. Using this OCT-based approach in adolescents yielded a high level of agreement with the current gold standard, tomography. Using them together, potentially also with other examinations may improve the diagnostic accuracy of KC in the pediatric population. Integration of this approach into the software of the device to facilitate automated evaluations is desired.

Colonic mucosa barrier defects in collagenous and ischemic colitis.

The subepithelial myofibroblasts (SEMFs) and the subepithelial band of macrophages (SEBM) are major components of the colonic mucosa barrier. Although their role in homeostasis is widely recognized, their contribution to disease states is largely unknown. Our aim was to explore histological characteristics of SEMFs and SEBM in collagenous and ischemic colitis in order to identify specific changes in distinct mucosa backgrounds lacking significant inflammation. SEMFs, SEBM and lamina propria (LP) macrophages were identified immunohistochemically by alpha smooth muscle Actin and Cluster of Differentiation 68 respectively in 38 colonic biopsies [14 collagenous colitis (CC), 14 ischemic colitis (IC), 10 normal mucosa]. In CC, SEMFs were rarely detectable in the collagenous band while aSMA-negative pericryptal fibroblast-like cells appeared. In lower LP interconnecting SEMFs processes were formed. SEBM was preserved in areas with a collagenous layer up to 20 μm. In thicker layers, it was fragmented and gradually disappeared in parallel with engulfment of enlarged macrophages. LP macrophages were usually increased. In IC, slight SEMFs changes preceded discernible epithelial alterations. Rounding, disintegration and extinction of SEMFs constituted successive alterations coinciding with crypt shrinkage and denudation. SEBM displayed total or almost total abolishment in areas with crypt damage but also in sites with minimal changes and in adjacent normal mucosa. Our findings provide evidence of impairment of both mucosa barrier constituents in CC and IC. In CC, histological alterations are closely related to the collagenous layer which seems to affect SEMFs differentiation and migration as well as SEBM integrity. The early extinction of SEBM in IC is indicative of its high sensitivity to hypoxia and hypoperfusion.

Stromal and Epithelial Architectural Alterations Mimicking Invasion (Pseudoinvasion) in Noninvasive Papillary Urothelial Carcinoma.

Retraction artifact, paradoxic maturation/differentiation, desmoplasia, and complex irregular growth are morphologic criteria of invasion in urothelial carcinoma. To describe changes mimicking invasion in noninvasive papillary urothelial carcinoma (NPUC). We reviewed 159 consecutive in-house patients with NPUC for either the presence of pseudoinvasion (irregular carcinoma nests within dense hyalinized stroma in the absence of other criteria of invasion) or precursor findings (stromal hyalinization not yet associated with epithelial architectural alteration). We assessed the correlation of these findings with age, sex, evidence of peripheral vascular disease, tumor grade, tumor infarction, and tumor size. We then followed up the patients clinically for tumor recurrence or progression. We identified 233 separate NPUCs (136 high grade and 97 low grade) in 125 men and 34 women. Of the 233 tumors, 26 (11.2%) had pseudoinvasion and 24 of 233 tumors (10.3%) had precursor findings. Except for complex irregular growth, no other criteria for invasion were seen. Pseudoinvasion and precursor findings were more common in men (47 of 183 [26%] versus 3 of 50 [6%]; P = .003), larger tumors (mean size, 2.6 versus 1.2 cm; P < .001), and tumors with infarction (33 of 50 [66%] versus 29 of 183 [15.8%]; P < .001). In multivariable analysis, tumor size (odds ratio, 1.49; P =.006), male sex (odds ratio, 6.48; P = .007), and the presence of infarction (odds ratio, 6.59; P < .001) were significant variables. Recurrence rates did not differ between patients with and those without pseudoinvasion (31% [5 of 16] versus 42% [45 of 107], respectively; P = .41). None of the tumors with pseudoinvasion progressed to invasive carcinoma. Given the correlation with size and presence of infarcted papillae, we suggest the possibility of tumor ischemia/infarction as a plausible etiology of pseudoinvasion. Awareness of this phenomenon is important for the accurate diagnosis of invasion in papillary urothelial carcinoma.

Effectiveness of a New Active Tear Substitute Containing 0.2% Hyaluronic Acid and 0.001% Hydrocortisone on Signs and Symptoms of Dry Eye Disease by Means of Low- and High-Tech Assessments.

An innovative eye drops formulation containing 0.2% hyaluronic acid and a low concentration of hydrocortisone (0.001%; hereafter HALH) has been recently placed on the market (Idroflog®, Alfa Intes, Italy) to manage the dysregulated parainflammation in patients with dry eye disease (DED). In the present paper, the effectiveness of HALH on the signs and symptoms of DED was retrospectively evaluated and compared with that one obtained using standard tear substitutes (STS) by means of low- and high-tech (Keratograph®) assessments. This was a multicenter retrospective study carried out between February and April 2023, involving adult patients with DED diagnosis owing to post-cataract surgery, meibomian gland dysfunction, allergy, or glaucoma medications. The primary aim was to compare the changes induced by different therapies on Keratograph® parameters (noninvasive Keratograph tear breakup time [NIKBUT], tear meniscus height [TMH], eyelid meibography, conjunctival hyperemia, and conjunctivochalasis) or collected by traditional low-tech measures (tear breakup time [TBUT], Schirmer test, Efron score, and epithelial alterations) and the Ocular Surface Disease Index score. Data from 155 patients were analyzed. The effectiveness of HALH and STS was reported by both high- and low-tech measures. NIKBUT-first showed a significant improvement in the HALH group versus the STS one at 15days (6.4 ± 3.6 vs 5.4 ± 3.7s, p = 0.02), whereas this difference was latent with low-tech TBUT until 45days (6.8 ± 2.6 vs 5.6 ± 2.3s, p = 0.03). Patients with DED occurring after cataract surgery reported an enhanced activity of HALH versus STS, particularly for NIKBUT-first, TMH, Schirmer test, and hyperemia stage. These findings highlighted the effectiveness of HALH in all DED subtypes, especially in patients with post-cataract surgery, as well as its superiority versus STS in terms of tear film stability improvement. We recommend longer observation (i.e., 3-6months) to fully ascertain whether the early improvement detected by high-tech measures will be confirmed in subsequent time points even using low-tech tests.

A deep learning model for generating fundus autofluorescence images from color fundus photography

BackgroundFundus Autofluorescence (FAF) is a valuable imaging technique used to assess metabolic alterations in the retinal pigment epithelium (RPE) associated with various age-related and disease-related changes. The practical uses of FAF are ever-growing. This study aimed to evaluate the effectiveness of a generative deep learning (DL) model in translating color fundus (CF) images into synthetic FAF images and explore its potential for enhancing screening of age-related macular degeneration (AMD). MethodsA generative adversarial network (GAN) model was trained on pairs of CF and FAF images to generate synthetic FAF images. The quality of synthesized FAF images was assessed objectively by common generation metrics. Additionally, the clinical effectiveness of the generated FAF images in AMD classification was evaluated by measuring the area under the curve (AUC), using the LabelMe dataset. ResultsA total of 8410 FAF images from 2586 patients were analyzed. The synthesized FAF images exhibited an impressive objectively assessed quality, achieving a multi-scale structural similarity index (MS-SSIM) of 0.67. When evaluated on the LabelMe dataset, the combination of generated FAF images and CF images resulted in a noteworthy improvement in AMD classification accuracy, with the AUC increasing from 0.931 to 0.968. ConclusionsThis study presents the first attempt to use a generative deep learning model to create authentic and high-quality FAF images from CF images. The incorporation of the translated FAF images on top of CF images improved the accuracy of AMD classification. Overall, this study presents a promising approach to enhance large-scale AMD screening.

PSV-8 Early Life Adversity Impacts Intestinal Epithelial Development and Stem Cells Activities in Pigs

Abstract Early life adversity significantly impacts organ development and predisposes animals towards greater risk of diseases later in life. In the previous research in pigs, long lasting intestinal barrier dysfunctions have been found to be associated with early weaning stress. However, the underlying mechanism is not fully elucidated. Intestinal epithelial functions are associated with the number and maturation of multicellular components, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells (EEC). Intestinal epithelial stem cells (IESC) give rise to the whole epithelium within 5 to 7 days. Such rapid turnover rate allows intestinal adaptation to stress conditions. We hypothesized that the early life stress impacted stem cells activities which would contribute to the long-lasting post-stress functional changes in intestinal epithelium. The objective of the present study is to evaluate the epithelium component cells and IESC proliferation and differentiation activities in an early weaning stress model in pigs. Piglets (n = 18) were obtained from commercial litters and were pair-weaned at the age of d 15 (early weaning, EW) and d 26 (late weaning, LW). Piglets were co-housed and raised under the same conditions. At the age of d 35, ileal tissues were isolated and fixed for morphological measurements. Intestinal crypts were isolated and cultured in the Matrigel matrix to form enteroids. The proliferation activities of IESCs were measured by enteroids morphology; differentiation capacity was measured by quantification of epithelial cell markers with and without chemicals-induced enteroids differentiation. Gene expressions and (or) staining of specific markers for IESCs (Olfm4, BMI1), enterocytes (ALP1), goblet cells (Muc2), Paneth cells (LYZ), and EECs (ChgA) in ileal tissue and ileal enteroids were also quantified. Data were analyzed by the student’s t-test. Our results showed that EW significantly (P &amp;lt; 0.05) reduced ileal villus width and crypt fission rate, while no changes were found on villus height and crypt depth compared with LW control. The alterations of ileal epithelium components in EW pigs were indicated by significantly fewer number of EECs, greater number of the goblet cells, and reduced ALP1 marker expression. The enteroids formation and budding rates were both significantly suppressed in EW pigs, which indicated a reduced IESC proliferating activity. In the fully developed enteroids, gene expressions of LYZ, ALP1, BMI1, Olfm4, and Hes1 were least in EW, but Muc2 expression was greater compared with control. In the differentiation culture media, the enteroids from EW pigs exhibited significantly enhanced potential for goblet cell differentiation but reduced capacity for enterocyte differentiation compared with LW pigs. Overall, the results indicate that early weaning stress affects the programming and activity of IESCs, which leads to an altered epithelium cell population and intestinal functions. Further understanding of mechanisms regulating IESCs activity will shed light on novel strategies mitigating stress-associated intestinal dysfunctions.

Characterizing the T Cell Repertoire in the Proximal Airway in Health and Disease.

Recent translational scientific efforts in subglottic stenosis (SGS) support a disease model where epithelial alterations facilitate microbiome displacement, dysregulated immune activation, and localized fibrosis. Given the observed immune cell infiltrate in SGS, we sought to test the hypothesis that SGS cases possessed a low diversity (highly clonal) adaptive immune response when compared with healthy controls. Single cell RNA sequencing (scRNA-seq) of subglottic mucosal scar in iSGS (n = 24), iLTS (n = 8), and healthy controls (n = 7) was performed. T cell receptor (TCR) sequences were extracted, analyzed, and used to construct repertoire structure, compare diversity, interrogate overlap, and define antigenic targets using the Immunarch bioinformatics pipeline. The proximal airway mucosa in health and disease are equally diverse via Hill framework quantitation (iSGS vs. iLTS vs. Control, p > 0.05). Repertoires do not significantly overlap between individuals (Morisita <0.02). Among iSGS patients, clonality of the TCR repertoire is driven by CD8+ T cells, and iSGS patients possess numerous TCRs targeting viral and intercellular pathogens. High frequency clonotypes do not map to known targets in public datasets. SGS cases do not possess a lower diversity adaptive immune infiltrate when compared with healthy controls. Interestingly, the TCR repertoire in both health and disease contains a restricted number of high frequency clonotypes that do not significantly overlap between individuals. The target of the high frequency clonotypes in health and disease remain unresolved. NA Laryngoscope, 134:1757-1764, 2024.

Salivary microRNAs as innovative biomarkers for early diagnosis of oral diseases: a comparison of conventional cigarette smokers and tobacco heating system 2.2 users.

MicroRNAs (miRNAs) are considered valid prognostic and diagnostic biomarkers. The different miRNA expression profiles in cancer cells compared to normal cells make them potential biomarkers used for the early diagnosis of oral diseases. Following exposure to cigarette smoking, miRNA altered profile expression is associated with resistance mechanisms against anticancer therapies. Cellular models showed a reduced human gingival epithelium alteration after exposure to THS2.2 and a lower pathogenicity than 3R4F CS. The aim of the study was to compare the expression of saliva miRNA profile of THS2.2 and 3R4F CS users compared to patients not exposed to the risk factor and to identify and study the modulation of miRNAs associated with the development of oral diseases. In particular, we will focus on the analysis of a group of miRNAs know to be involved in the development of smoking-related diseases. The study will be performed in 18 months and dentists and biochemists will be involved in the different phases. To perform the study, healthy volunteers, including smokers of THS2.2 or 3R4F CS, will be enrolled. The samples will be collected from 3 experimental groups, each consisting of 30 subjects: group 1 (no smoking subjects), group 2 (subjects exposed to THS2.2), group 3 (subjects exposed to 3R4F CS). The collection of the saliva sample will be conducted in a standardized way. Following the collection, saliva will be processed. Previous studies have suggested that miRNAs are prognostic biomarkers for various smoking-related diseases. Based on the post-transcriptional regulation of some mRNAs connected to different oral pathologies, we expect a specific miRNA-mRNA interaction, which could be a starting point for the development of new possible diagnostic, therapeutic and prognostic approaches.