Stromal Layer Research Articles

Drug Response Profiling Informs Personalized Bridging to Cell Therapy for Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia

Introduction Treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL) remains challenging in spite of recent progress with immunotherapy and targeted small-molecule combinations. Growing awareness of the molecular complexity of the disease with subtype-specific cancer dependencies positions functional drug testing as a promising precision tool to improve the prediction of clinical drug efficacy and adapt treatment. We leverage automated, image-based Drug Response Profiling (DRP) and machine learning to deliver individualized therapeutic options in real time for ALL patients with urgent medical needs. Here we report on the DRP experience of 307 primary samples from 278 patients with BCP-ALL, T-ALL/T-LBL and mixed phenotypic acute leukemia (MPAL) registered from 2016 to 2022 in a non-interventional setting. Methods We have generated a reference set of 141 diagnostic samples from patients with high-risk (75%) or standard/medium-risk (25%) disease as defined by the clinical trial AIEOP-BFM ALL 2017, alongside 122 samples with first and 44 with more advanced relapse. We assayed cell viability at single-cell level from viably frozen bone marrow (BM) samples, co-cultured with hTERT-immortalized mesenchymal stromal cells (MSC) and following a 72h drug incubation with a library of up to 110 compounds. We quantified cell counts at five concentrations in duplicates by segmenting and classifying MSC, live and dead leukemia cells using a modular image processing pipeline deployed on cloud infrastructure. We longitudinally confirmed assay consistency with PDX models and monitored integrity of the stromal layer on a per-sample basis. In a subset of cases (15-20%), relevant stromal detachment or low leukemia survival led to samples not meeting all quality control standards. Drugs were ranked by the area under the dose response curve (AUC) and referenced to all previous samples. This patient-specific ex vivo drug fingerprint was reported to the treating physician with a typical turn-around time of less than two weeks. Results Dexamethasone sensitivities measured ex vivo in BM samples from pediatric front-line patients, who were treated on a one-week cytoreductive steroid pre-phase followed by a four-drug induction regimen, correlated significantly with clinical responses at d8 (PGR vs. PPR, p<0.001), d15 (flow MRD below 10%, p<0.001) and d33 (PCR MRD below E-2, p<0.001). By integrating dose-response profiles from all patients, we observed differential drug activities across cytogenetic groups and identified exceptional responders to Venetoclax in very high-risk subtypes including TCF3::HLF (5 patients, AUC percent rank: 0.03 - 0.19) as well as refractory T-ALL. In 77 r/r T-ALL patients from 12 different countries, DRP supported selection of a personalized bridging regimen prior to consolidation with cellular therapy. Top-scoring drug sensitivities (10th percentile of AUC) were found for a variety of agents including Imatinib, Dasatinib, Venetoclax, Bortezomib, Selinexor and different nucleoside analogs. Eleven T-ALL and 5 BCP-ALL patients received a DRP-guided bridging element involving compounds with high ex vivo drug efficacy and complemented by chemotherapy (Table 1). The other 66 r/r patients were treated according to physician's choice incorporating information on drug resistance detected by DRP in real time. Bridging strategies implementing DRP-guided therapy blocks stabilized or reduced disease burden in 15/16 patients allowing HSCT or infusion of CAR-T cells in 12 patients with 8 patients still being in remission at last follow-up. Conclusion We describe an array of pharmacological sensitivity and resistance patterns across a heterogeneous, high-risk cohort of BCP- and T-ALL patients. We identified individual susceptibilities to targeted agents of different classes including BH3-mimetics, proteasome, tyrosine kinase and exportin inhibitors as well as standard-of-care chemotherapeutics. Prospective recording of drug response profiles offers opportunities to systematically correlate ex vivo and clinical responses and inform personalized therapies. These data strongly advocate inclusion of DRP into randomized, early and late phase clinical trials for high-risk pediatric ALL, comprehensively integrating molecular and functional precision information to improve patient stratification and long-term outcome after HSCT and CAR-T.

The Biological Characteristics of Eutopic and Ectopic Endometrial Progenitor Cells in Endometriosis.

The aim of this study was to identify the biological characteristics and potential roles of endometrial progenitor cells in the pathogenesis of endometriosis. It is generally believed that progenitor cells in human endometrium are responsible for rapid endometrial regeneration. However, the biological characteristics and potential roles of the paired eutopic and ectopic endometrial progenitor cells in endometriosis remain unclear. This study intends to isolate the epithelial progenitor (EP) cells and endometrial mesenchymal stem cells (eMSCs) from the eutopic and ectopic endometria from endometriosis patients, further to reveal their features and functions respectively. The distributions of EP cells and eMSCs and the expression of steroid hormone receptors in the endometrium and endometriotic tissues were assessed by immunohistochemistry. EP cells and eMSCs were sorted from paired eutopic and ectopic endometria with epithelial cell adhesion molecule (EpCAM) magnetic beads. The clonogenicity, cell viability after being treated with estradiol and progesterone, and cell markers expression were evaluated with colony forming on Matrigel, CCK-8 and immunofluorescence staining, respectively. The differentially expressed genes (DEGs) were further identified with RNA sequencing. SSEA-1- and PDGFRβ-positive cells were distributed in the epithelial and stromal layers. The ERβ staining was much more intense in endometriotic tissues, but PR expression was almost absent. The ectopic EP cells exhibit strong clonogenicity and ERβ expression but weak PR expression, leading to progesterone resistance. There are 12604 and 13242 DEGs revealed by RNA sequencing between eutopic and ectopic EP cells or eMSCs. GO and KEGG analyses revealed that the functions and pathways of DEGs enriched in cellular energy metabolism and regulation of the immune response, respectively. Additionally, ERβ targets were mainly enriched in ectopic EP cells. Both EP cells and eMSCs may engage in ectopic lesion formation in endometriosis by modifying the metabolic mode and immune tolerance. These data not only help to understand the molecular mechanism of endometriosis but also could potentially contribute to the discovery of therapeutic targets for endometriosis.

Stromal cell inhibition of anti-CD20 antibody mediated killing of B-cell malignancies.

Introduction: The glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab has been licensed for treatment in follicular non-Hodgkin lymphoma and B-CLL following clinical trials demonstrating superior outcomes to standard of care treatment. However, ultimately many patients still relapse, highlighting the need to understand the mechanisms behind treatment failure to improve patient care. Resistance to chemotherapy is often caused by the ability of malignant B-cells to migrate to the bone marrow and home into the stromal layer. Therefore, this study aimed to investigate whether stromal cells were also able to inhibit type II anti-CD20 antibody mechanisms of action, contributing to resistance to therapy. Methods: A stromal-tumor co-culture was established in vitro between Raji or Daudi B-cell tumor cells and M210B4 stromal cells in 24 well plates. Results: Contact with stromal cells was able to protect tumor cells from obinutuzumab mediated programmed cell death (PCD), antibody dependent cellular phagocytosis and antibody dependent cellular cytotoxicity. Furthermore, such protection required direct contact between stroma and tumor cells. Stromal cells appeared to interfere with obinutuzumab mediated B-cell homotypic adhesion through inhibiting and reversing actin remodelling, potentially as a result of stromal-tumor cell contact leading to downregulation of CD20 on the surface of tumor cells. Further evidence for the potential role of CD20 downregulation comes through the reduction in surface CD20 expression and inhibition of obinutuzumab mediated PCD when tumor cells are treated with Ibrutinib in the presence of stromal cells. The proteomic analysis of tumor cells after contact with stromal cells led to the identification of a number of altered pathways including those involved in cell adhesion and the actin cytoskeleton and remodeling. Discussion: This work demonstrates that contact between tumor cells and stromal cells leads to inhibition of Obinutuzumab effector functions and has important implications for future therapies to improve outcomes to anti-CD20 antibodies. A deeper understanding of how anti-CD20 antibodies interact with stromal cells could prove a useful tool to define better strategies to target the micro-environment and ultimately improve patient outcomes in B-cell malignancies.

Nectin-3 and shed forms of CSPG4 can serve as epithelial cell receptors for Clostridioides difficile TcdB.

Toxin B (TcdB) is a major virulence factor of Clostridioides difficile, a Gram-positive pathogen that is a leading cause of hospital-acquired diarrhea. While previous studies have established that TcdB can engage multiple cell surface receptors in vitro, little is known about how these interactions promote disease and where these receptors localize on colonic tissue. Here, we used immunofluorescence microscopy to visualize Nectin-3 and CSPG4 on tissue, revealing unexpected localization of both receptors on colonic epithelial cells. We show that Nectin-3, which was previously characterized as an adherens junction protein, is also localized to the brush border of colonocytes. Staining for CSPG4 revealed that it is present along epithelial cell junctions, suggesting that it is shed by fibroblasts along the crypt-surface axis. Collectively, our study provides new insights into how TcdB can gain access to the receptors Nectin-3 and CSPG4 to intoxicate colonic epithelial cells.

Macular Hole Hydrodissection Technique with Human Amniotic Membrane for Repair of Large Macular Holes.

To describe a combined surgical technique utilizing the Macular Hole Hydrodissection (MHH) with human amniotic membrane (hAM) for repair of large macular holes. A step-by-step procedure and a surgical video using the combined MHH and hAM technique are presented. As the first step, the MHH separates the adhesions of the macular hole to the underlying retinal pigment epithelium (RPE) with a soft tipped cannula through proportional reflux followed by gentle passive aspiration. The hAM graft is marked to identify the non-sticky epithelial side and ensure that the stromal layer (sticky and non-shinny) is facing downward towards the RPE. The graft is then tucked into the space created with MHH between the macular hole edges and the RPE with closed forceps to decrease the likelihood of the graft from dislocating post-operatively. The MHH in combination with the hAM is a practical and effective technique for addressing challenging large macular holes.

CLINICAL AND IN VIVO CONFOCAL MICROSCOPY FINDINGS IN MGUS - A CASE REPORT

Purpose The purpose of this study was to describe a case of paraproteinemic keratopathy (PPK) in a patient with monoclonal gammopathy of undetermined significance (MGUS) Methods A 56 years old man was assessed using a slit lamp and in vivo confocal microscopy (IVCM) with Heidelberg HRT Rostock II. Results The patient showed bilateral diffuse, symmetric, non-polychromatic, punctiform crystals in all layers of the cornea. The remaining part of the ophthalmic clinical assessment was normal. Esthesiometry was normal in all quadrants. Anterior segment OCT confirmed diffused hyperreflectivity of the epithelium and corneal stroma. IVCM was then executed, showing polymorphic (granular, needle-like, stellate) hyperreflective crystals in all layers of corneal epithelium and anterior stroma, subbasal plexus nerves beading, cytoplasmatic hyperreflectivity of the keratocytes of the posterior stroma without any endothelial involvement After considering the age, health status, and ocular signs the primary entities to be investigated were adult benign cystinosis and paraproteinemic disorder. All the diagnostic tests were negative except for a monoclonal component IgG/k of 1.37 d/dl. A hematological examination was then performed with the resulting diagnosis of MGUS. In this paper, we analyzed 11 articles about IVCM in PPK in MGUS and other paraproteinemias highlighting IVCM features compatible with our findings allowing us to generalize a first diagnostic IVCM pattern for PPK. Conclusions In this case report we suggest the IVCM diagnostic patterns that may be used to identify PPK without corneal biopsy.

FRI390 The Effects Of Chronic Asprosin Administration On Ovarian Tissue

Abstract Disclosure: H. Kelestimur: None. Z. Oz: None. M. Ozdede: None. I. Serhatlioglu: None. N. Kaya Tektemur: None. E. Kacar: None. Purpose: Asprosin is a novel glucogenic and orexigenic hormone produced by the fibrillin 1 (FBN1) gene that is secreted by white adipose tissue during fasting. Fibrillins (FBNs) are structural and signalling proteins found mainly in the ovarian stroma, tunica albuginea and theca layers. The aim of this study was to determine the effects of asprosin on ovarian tissue in the female rats. Materials and methods: Twenty-four female Sprague-Dawley rats were randomly divided into 2 groups (n=12) as control and asprosin. Asprosin and control groups were given intraperitoneal saline at doses of 500 ng/kg and 1 ml/kg, respectively, at 14.00 every day for eight weeks. After the application, decapitation was performed, and the rapidly removed ovarian tissues were placed in a 10% formaldehyde solution for fixation. Formalin-fixed ovaries were dehydrated in a graded alcohol series and graded ethanol, respectively After that, all tissues were embedded in paraffin wax. The ovaries were cut into 5-µm sections and stained with hematoxylin and eosin (H&E) and also Masson trichrome staining. Ovarian follicular reserve and fibrosis were evaluated by light microscopy. The follicles were histologically classified as primordial, primary, secondary, and Graaf and all follicular structures were counted in each section. The fibrosis was evaluated with Masson’s trichrome staining and scored from 0 to 3 semi-quantitatively (0 = no fibrosis, 1 = low fibrosis, 2 = intermediate fibrosis, 3 = severe fibrosis). Results: In the asprosin group, unlike the control group, occasional congestion was observed in the vessels located in the ovarian medulla. Apart from this, no histopathological changes were found. An increase in fibrosis was detected in the ovarian sections of the asprosin group when compared to the control group (p = 0,035). In the light microscopic evaluation of the ovarian sections of the rats in the asprosin group, the primary and secondary follicle numbers were significantly higher than those in the control group (p = 0,008 and p = 0,047 respectively). However, the difference between the control and asprosin groups in terms of the primordial follicle, Graafian follicle and corpus luteum numbers were not statistically significant. Conclusion: Our results have shown that chronic asprosin administration causes an increase in the number of primary and secondary follicles, suggesting that changes in ovarian steroidogenesis and folliculogenesis depending on the application may lead to positive effects on female fertility. Keywords: Asprosin, adipokine, ovarian follicle. Acknowledgement: This study was supported by TUBITAK (Project: 220S744). Presentation: Friday, June 16, 2023

Integrated analysis of microRNA expression in tears of Kazakh patients with climatic droplet keratopathy in Xinjiang, China

Climatic droplet keratopathy (CDK) is a corneal diseases, which is characterized by increased oil-like deposits on the anterior elastic lamina and anterior stromal layer. Severe CDK can even cause blindness, with no specific available treatment. Besides. CDK is poorly understood in terms of its pathogenic mechanisms. Thus, to determine potential biomarkers for CDK, we analyzed the microRNA expression profile in tear samples from CDK patients and investigated their putative roles in the pathogenesis of CDK. Herein, miRNA sequencing and following bioinformatics analysis was performed to explore the roles of their target genes in CDK. A total of 67 differentially expressed miRNAs were identified, of which 25 were upregulated and 42 were downregulated. qPCR verification showed that among the up- and down-regulated miRNAs, expression of five and six, respectively, was most significantly different.The target genes of the differentially expressed miRNAs are involved in the FoxO signaling pathway, tumor necrosis factor (TNF) signaling pathway, and steroid hormone biosynthesis. Protein–protein interaction network analyses identified 20 hub genes, including PTEN, GSK3B, and SMAD3. In conclusion, the panel of differentially expressed miRNAs identified may have potential utility as early diagnostic biomarkers for CDK. Moreover, the TNF signaling pathway is a new potential target in CDK for the development of treatments.

Application of intraoperative optical coherence tomography in deep lamellar keratoplasty

Objective: To evaluate the clinical application value of intraoperative optical coherence tomography (iOCT) in deep anterior lamellar keratoplasty (DALK) using the big-bubble technique to bare Descemet's membrane. Methods: Retrospective case series. Clinical data of 92 patients (92 eyes) with monocular stromal corneal diseases who underwent big-bubble DALK in the Eye Hospital of Shandong First Medical University from January 2020 to August 2021 were collected. There were 53 males and 39 females. The average age was (53.2±16.0) years old. All patients underwent iOCT scanning to determine the location and depth of the injection needle after initial removal of the corneal lesion, to observe the integrity of the recipient bed, Descemet's membrane, after complete lesion removal, and to observe the adhesion between the corneal graft and the recipient bed and check folds on the recipient bed after suturing of the corneal graft. The intraoperative perforation of Descemet's membrane, postoperative thickness of the cornea and the recipient bed, visual acuity, and corneal astigmatism were recorded. Results: By iOCT, the thickness of the recipient bed was found to be about 1/2 of the corneal thickness and relatively uniform in all directions in 62 eyes (67.4%), so the sterile air was injected from the center of the recipient bed to separate it from the stromal layer. In 30 eyes (32.6%) with an uneven thickness of the recipient bed, the sterile air was injected from the paracentral area of the recipient bed. Under the guidance of iOCT scanning, 89 eyes (96.7%) did not experience any perforation of Descemet's membrane during surgery. The Descemet's membrane folds in the central 5-mm area of the recipient bed was observed and flattened in 20 eyes with the assistance of iOCT scanning. The postoperative corneal thickness was (578.95±108.26) μm, and the recipient bed thickness was (36.06±23.11) μm. The best corrected visual acuity of all patients at 6 months after surgery was 0.57±0.25 logMAR, which was significantly better than that before surgery (1.61±1.27 logMAR; P<0.001). The average corneal astigmatism at 6 months after surgery was (2.72±2.44) diopters. Conclusions: The application of iOCT scanning in DALK surgery assisted by the big-bubble method can provide safe guidance for surgeons to adopt correct surgical procedures, decrease the risk of Descemet's membrane perforation, reduce the recipient bed folds, and facilitate corneal interlayer adhesion, thereby improving the visual prognosis.

Amniotic Membrane and Its Derivatives: Novel Therapeutic Modalities in Liver Disorders.

The liver is a vital organ responsible for metabolic and digestive functions, protein synthesis, detoxification, and numerous other necessary functions. Various acute, chronic, and neoplastic disorders affect the liver and hamper its biological functions. Most of the untreated liver diseases lead to inflammation and fibrosis which develop into cirrhosis. The human amniotic membrane (hAM), the innermost layer of the fetal placenta, is composed of multiple layers that include growth-factor rich basement membrane, epithelial and mesenchymal stromal cell layers. hAM possesses distinct beneficial anti-fibrotic, anti-inflammatory and pro-regenerative properties via the secretion of multiple potent trophic factors and/or direct differentiation into hepatic cells which place hAM-based therapies as potential therapeutic strategies for the treatment of chronic liver diseases. Decellularized hAM is also an ideal scaffold for liver tissue engineering as this biocompatible niche provides an excellent milieu for cell proliferation and hepatocytic differentiation. Therefore, the current review discusses the therapeutic potential of hAM and its derivatives in providing therapeutic solutions for liver pathologies including acute liver failure, metabolic disorders, liver fibrosis as well as its application in liver tissue engineering.

The blood-prostate barrier: an obstacle to drug delivery into the prostate.

The blood-prostate barrier (BPB), a non-static physical barrier, stands as an obstacle between the prostate stroma and the lumen of the prostate gland tube. The barrier has the ability to dynamically regulate and strictly control the mass exchange between the blood and the prostate, thereby limiting drug penetration into the prostate. The basement membrane, fibrous stromal layer, capillary endothelial cell, prostatic ductal epithelial cell, lipid layer, etc., have been confirmed to be involved in the composition of the barrier structure and altered membrane permeability mainly by regulating the size of paracellular ion pores. Various studies have been conducted to improve the efficiency of drug therapy for prostate diseases by changing the administration approaches, improving barrier permeability and increasing drug penetration. To gain a full understanding of BPB, the composition of BPB, the methodology for evaluating the permeability of BPB and alterations in barrier function under pathological conditions are summarized in this review. To find a shortcut for drug delivery across BPB, the biodistribution of drugs in the prostate and different methods of improving drug penetration across BPB are outlined. This review offers an applied perspective on recent advances in drug delivery across BPB.

The role of anterior segment optical coherence tomography in the evaluation of the pterygium

BackgroundTo evaluate the ability of anterior segment optical coherence tomography (AS-OCT) to visualize the anatomic features of the pterygium and its invasion of the corneal layers. MethodsSeventy-five eyes of 54 patients diagnosed with pterygium were included. All subjects underwent complete ophthalmologic examinations, including AS-OCT. The limbus–apex distance, vertical height at the limbus, invasion of the Bowman's and stromal layers, and other morphologic structures of the pterygium tissue were analyzed. ResultsThe mean age of the patients was 49.67 ± 16.49 (20–85) years. The mean apex–limbus distance was 2548.37 ± 1026.32 (933–4597) μm, and the mean vertical height at the limbus was 4843.89 ± 1374.10 (1740–7784) μm. A space was observed beneath the pterygium tissue in 44 (58.67%) eyes. The mean width and height of this space were 1756.33 ± 560.22 (1009–3095) μm and 231.70 ± 85.88 (109–465) μm, respectively. Invasion of the Bowman's layer was apparent in 74 (98.67%) eyes, and invasion of the stromal layer was detected in 33 (44%) eyes. A hyperreflective layer was observed beneath the epithelial layer at the edge of the pterygium apex in 31 (41.33%) eyes. In 24 (92.31%) of the 26 advanced pterygium cases and 20 (40.82%) of the 49 early pterygium cases, a subpterygium space was found beneath the lesion (p = 0.0001). ConclusionAS-OCT enables measurement of the actual size and thickness of pterygia, assessment of invasion of the Bowman's and stromal layers of the cornea, and evaluation of the pterygium structure. Over half of the eyes exhibited space beneath the pterygium.

Removal of grass awn from the stromal layer of cornea in a dog: A case report

Removal of grass awn from the stromal layer of cornea in a dog: A case report

P-407 Artificial ovary construction: short-term in vitro culture of human primordial and primary follicles in a 3D environment on a stromal cell feeder layer

Abstract Study question Do ovarian stromal cells (OSCs) influence viability and growth of human pre-antral follicles in vitro? Summary answer Feeder layer of OSCs advanced growth and transition of primordial follicles to primary/secondary stage while keeping a high proportion of viable follicles. What is known already In the ovary, follicles require the support of ovarian cells through the secretion of essential factors for their survival and development. This was also demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of ovarian stromal cells. This co-culture significantly increased follicle survival and growth. Study design, size, duration Pre-antral follicles were isolated from human frozen-thawed ovarian tissue (OT) biopsies and encapsulated in 1% alginate scaffolds. Embedded pre-antral follicles were placed directly on the OSCs feeder layer or bottom of a culture dish for a 7-day in vitro culture (control). Follicle viability and growth and hormone production were compared between groups. Participants/materials, setting, methods Primordial and primary follicles were isolated from frozen-thawed OT of cancer patients (n = 6). OSCs were isolated from OT of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 under an inverted microscope. Viability was assessed by staining a proportion of follicles (n = 87) with calcein AM and ethidium homodimer-I. They were classified (V1/V2: healthy/minimally damaged; V3/V4: damaged/dead follicles) using confocal fluorescence microscopy. Estradiol levels were measured by ELISA. Main results and the role of chance A total of 379 human pre-antral follicles (primordial=360; primary=19) were isolated and embedded in 1% alginate hydrogels and classified according to their viability. Most follicles (96%) were viable after isolation with a diameter of 40.8±9.9 µm (mean±SD). At Day 7, pre-antral follicles have grown significantly in size in both culture conditions: on the OSCs feeder layer and without it (p &amp;lt; 0.0001 for D0 vs. D7). However, no significant difference (p = 0.07) between culture conditions was observed. The mean diameter of follicles cultured on the OSCs layer was 80.6±11.0 μm and without it, 67.3±7.2 μm. Nonetheless, the distribution pattern of different quality grade follicles was significantly different on OSCs (V1: 62%, V2: 25%, V3: 2%, V4: 11%) and follicles without OSCs (V1: 35%, V2: 38%, V3: 0%, V4: 23%; p = 0.03). Additionally, more follicles have been activated and reached a higher developmental stage on OSCs (D0 primordial: 184, primary: 7 vs. D7 primordial: 53, primary/secondary: 93) than without stromal cells (D0 primordial: 186, primary: 4 vs. D7 primordial: 84, primary/secondary: 64; p &amp;lt; 0.001) with 66 and 43 follicles reaching a secondary stage (75&amp;lt;x&amp;lt;200 μm), respectively. Estradiol level was significantly (p = 0.006) higher in media of follicles cultured on the OSCs, 54.1±14.2 pg/ml vs. 29.9±4.0 pg/ml. Limitations, reasons for caution This study was performed only in a short-term culture and no primordial/primary follicles have reached the antral stage. Further in vitro studies on follicular developmental capacity, physiology and steroidogenesis in alginate scaffold with human ovarian stromal cells are needed. Wider implications of the findings Activation and growth of human primordial follicles during in vitro short-term culture to a secondary stage has been a long-lasting challenge. Co-culture with human ovarian stromal cells can help to overcome this limitation. Trial registration number not applicable

O-093 Endometrium-blastocyst cross talk: how far are we from understanding?

Abstract The early events characterising implantation of the human embryo represent a critical step in which proper activation of orchestrated molecular pathways regulate the success of pregnancy. Understanding this complex network is considered by many the last frontier of human reproduction. Although outstanding research advancements have provided clinicians tools to select the best quality embryo, identification of the optimal timing in which the endometrium is ready to accommodate it remains a challenge. Over the last decade several studies have identified many key players involved in the preparation of the endometrium to implantation and several diffusible signals guiding the embryo in its journey toward the maternal endometrial tissue have been uncovered (Massimiani et al. 2020). However, pieces of the puzzle are still missing. For convenience, the process of implantation is subdivided into several sequential processes, the completion of one being propaedeutic to the following. Once the blastocyst has arrived in close proximity to the uterine epithelium, the first event occurring is its escape from the zona pellucida. The molecular basis of blastocyst hatching remains poorly understood, and species-specific differences have been reported. Using the mouse model, we recently demonstrated that thyroid hormone up-regulates the expression of specific lytic enzymes (Isp1 and Isp2) known mediators of mouse embryo zona lysis. Interestingly, we observed that TH-mediated upregulation of Isp1 and Isp2 is strictly dependent on the presence of endometrial stromal cell feeder layer, clearly indicating that blastocyst–endometrium interaction is indispensable for TH-mediated increased expression of these proteases. So far, no molecular signals regulating hatching in humans have been identified, and only mechanical forces have been proposed to induce zona opening; however very recently the possibility that proteases may be involved also in humans has been suggested (Almagor et al., 2020), although further studies are needed to support this hypothesis. Once free from the zona, the embryo contacts the endometrial epithelium and starts its journey to further proceed in the consolidation of pregnancy. Hormones, pro- and anti-inflammatory mediators, growth factors and their receptors, adhesion molecules, and proteases and protease inhibitors have been reported in the regulation of the process of embryo apposition, adhesion, and invasion (Massimiani et al., 2020). Among the many molecular signals, the Notch pathway has been recognized key in embryo-endometrium cross talk (Cuman et al., 2014). Transient activation of the Notch1 receptor has been demonstrated in the process of decidualization (Afshar et al., 2012), a limiting step to implantation, and blastocyst-conditioned medium has been shown to induce the expression of Notch family members in decidual cells (Hess et al., 2007). Expression of notch receptors and ligands has been reported in the blastocyst trophectoderm as well (Cuman et al., 2014). We previously demonstrated that the secreted factor epidermal growth factor-like domain 7 (EGFL7) is a novel modulator of the Notch pathway, it is expressed by trophoblast cells of the mouse blastocyst (Fitch et al., 2004; Lacko et al., 2014) and it regulates human trophoblast migration and invasion ability by activating the Notch pathway (Massimiani et al., 2015). Data will be presented demonstrating that EGFL7 is expressed in the human endometrium and its expression is mainly localized in the glandular epithelium throughout the menstrual cycle. EGFL7 is significantly upregulated in whole endometrial tissue during the secretory phase, particularly in the stromal compartment; this observation is further supported by in vitro studies showing its upregulation in decidualized endometrial stromal cells, suggesting a role for EGFL7 in implantation. Further support is provided by the evidence that its levels are strongly downregulated in the endometrium of women with impaired fertility. We propose EGFL7 as a novel player involved in the embryo-endometrium cross talk.

Diabetes Exacerbates Pseudomonas aeruginosa Keratitis in Streptozotocin-Induced and db/db Mice via Altering Programmed Cell Death Pathways.

Patients with diabetes have a higher incidence of infections, which are often more severe. This study aimed to investigate the impact of hyperglycemia on bacterial keratitis caused by Pseudomonas aeruginosa (Pa) in two mouse models of diabetes, streptozotocin-induced type 1 diabetes mellitus (T1DM) and db/db type 2 diabetes mellitus. The susceptibility of corneas to Pa was assessed by determining the inocula required to cause infectious keratitis. Dead or dying cells were identified using TUNEL staining or immunohistochemistry. Specific inhibitors were used to evaluate the role of cell death modulators in Pa keratitis. Cytokines and Treml4 expressions were analyzed using quantitative PCR, and the role of Treml4 in keratitis was determined using small interfering RNA technology. DM corneas required significantly fewer inocula to develop Pa keratitis, with T1DM corneas requiring 750 inocula and type 2 diabetes mellitus corneas requiring 2000 inocula, compared with 10,000 inocula required for normal (NL) mice. T1DM corneas had more TUNEL-positive and fewer F4/80-positive cells than NL corneas. Phospho-caspase 8 (apoptosis) and -RIPK3 (necroptosis) staining was more intense in the epithelial and stromal layers of NL and T1DM corneas, respectively. Pa keratitis was augmented by targeting caspase-8 and prevented by RIPK3 inhibition in both NL and T1DM mice. Hyperglycemia suppressed IL-17A/F and augmented IL-17C, IL-1β, IL-1Ra, and TREML4, the downregulation of which protected T1DM corneas from Pa infection by suppressing necroptosis. RIPK3 inhibition blocked Pa infection in db/+ mice and significantly decreased the severity of keratitis in db/db mice. Hyperglycemia exacerbates bacterial keratitis in B6 mice by skewing apoptosis toward necroptosis. Preventing or reversing this transition may serve as an adjunct therapy for treating microbial keratitis in patients with diabetes.

MORPHOLOGICAL CHANGES OF THE RABBIT CORNEA IN THE LATE TERMS AFTER THE SIMULATION OF A MECHANICAL NON-PENETRATING CORNEA INJURY AND ITS CORRECTION USING DECELLULARIZED XENOSTROMA

Abstract. The global incidence of vision loss is estimated at 36 million per year, and one of the fi ve leading causes is injury or disease aff ecting the cornea. In some cases, damage to the cornea requires surgical intervention using xenografts.This study aims to investigate the peculiarities of the morphological changes of the rabbit cornea in the dynamics of experimental mechanical non-penetrating corneal injury and its correction with decellularized xenocorneal stroma.Material and methods. Experiments were performed on Chinchilla rabbits in compliance with the rules of bioethics. The animals were divided into three groups: I – animals without simulated pathology, II – animals with simulated mechanical nonenetrating corneal trauma, III – animals with corneal trauma that underwent surgical correction using pig corneal stroma obtained by the decellularization method. Animals were removed from the experiment 14 and 28 days after the beginning of pathology simulation. Rabbits of the second and third groups were subjected to a concentric epithelial incision in the upper half of the cornea of both eyes under local anaesthesia, within which the anterior epithelium was removed together with the upper layer of the corneal stroma with a disposable ophthalmic scalpel. The rabbits of the third group underwent corneal injury correction by closing the defect with decellularized pig xenocorneal stroma. For histological examination, pieces of the cornea were processed according to generally accepted methods. Para ffi n sections of the cornea were stained with hematoxylin and eosin. The thickness of the anterior corneal epithelium in the central and peripheral areas of the injury, the stroma’s thickness, and the cornea’s general thickness were determined morphometrically. The normal distribution determined the reliability of the diff erence between the data according to the student’s t-test. Diff erences were considered signifi cant at p&lt;0,05.Results. Morphometrically established, the dynamic increase in the thickness of the anterior epithelium in the marginal zones up to 103.49±5.17 μm on the 14th day of the experiment, which is 1.44 times greater (p˂0,001) compared to the norm, and the thickness of the cornea, with the maximum value in the same period, to 759.42±27.12 μm, which exceeded the intact indicator by 2.39 times (p˂0.001). On the 28th day of the experiment, under the conditions of simulation of mechanical injury at the microscopic level, the closure of the defect with an epithelium of average thickness 60.27±2.01 μm with an uneven relief of the basal surface and areas of detachment from the substantia propria was revealed. In the upper layers of the stroma, signs of maturation of young connective tissue with the presence of fi broblastic and leukocyte cells, capillaries and disordered orientation of collagen fi bers are microscopically revealed. Morphometrically, on the 28th day, the substantia propria thickness of the second group animals is signifi cantly (p˂0.001) bigger than the norm but 1.56 times less compared to the 14th day of the experiment. Microscopically and morphometrically, it was established that under the condition of using a decellularized pig cornea for the correction of mechanical corneal trauma, hyperplasia of the epithelium of the marginal areas was most pronounced on the 14th day 126.37±5.06 μm, which resulted in the closure of the defect with a well-structured epithelial regenerate 35.20±1.07 μm without areas of exfoliation. These morphometric parameters signifi cantly (p˂0.001) exceeded those of similar groups of animals in the cornea without correction. In the same term, the dominance of cells of the fi broblastic series and the absence of morphological manifestations of infl ammation were recorded in the granulation tissue. It was histologically investigated that using a decellularized pig cornea 28 days after correction ensures the restoration of the typical structure of the cornea with the approximation of morphometric indicators to the group of intact animals. In contrast to the second experimental group, where no correction was performed, the renewed anterior epithelium of the central corneal zone of the animals of the third group had a clear diff erentiation into layers, an even relief of the basal surface and a contoured Bowman’s membrane. Its thickness 54.19±2.09 μm was unreliably diff erent from the norm. During this period of the experiment, when correction was applied, mature connective tissue and single capillaries in a state of reduction were histologically detected in the stroma, while the phenomena of maturation of connective tissue components were often observed in the cornea of the animals of the second experimental group. Microscopically, in the substantia propria, bundles of collagen fi brils were oriented parallel, which is one of the factors that ensure the cornea’s transparency.Conclusions. Microscopically and morphometrically, it was established that the use of decellularized pig cornea for the correction of mechanical corneal trauma improves the dynamics of regeneration, accelerates the maturation of granulation tissue, the healing of the defect, contributes to the structuring and restoration of the corneal morphology as close to normal as possible.

Investigating the effectiveness of Difluprednate-Loaded core-shell lipid-polymeric hybrid nanoparticles for ocular delivery

Uveitis is a sight-threatening disease that causes inflammation in the uvea; difluprednate (DFB) is the first approved drug molecule for postoperative pain, inflammation, and endogenous uveitis. Complex ocular physiology and structure make it difficult to deliver drugs to the eye. Increased permeation and retention in the layer of the eye are required to improve the bioavailability of ocular drugs. In the current research investigation, DFB-loaded lipid polymer hybrid nanoparticles (LPHNPs) were designed and fabricated to enhance the corneal permeation and sustained release of DFB. A well-established two-step approach was used to fabricate the DFB-LPHNPs, comprising of Poly-Lactic-co-Glycolic Acid (PLGA) core that entrapped the DFB and DFB loaded PLGA NPs covered by lipid shell. The manufacturing parameters were optimized for the preparation of DFB-LPHNPs; the optimal DFB-LPHNPs showed a mean particle size of 117.3 ± 2.9 nm, suitable for ocular administration and high entrapment efficiency of 92.45 ± 2.17 % with neutral pH (7.18 ± 0.02) and isotonic Osmolality (301 ± 3 mOsm/kg). Microscopic examination confirms the core–shell morphological structure of DFB-LPHNPs. The prepared DFB-LPHNPs were extensively characterized using spectroscopic techniques and physicochemical characterization, which confirms the entrapment of the drug and the formation of the DFB-LPHNPs. The confocal laser scanning microscopy studies revealed that Rhodamine B-loaded LPHNPs were penetrated into stromal layers of the cornea in ex-vivo conditions. The DFB-LPHNPs showed a sustained release pattern in simulated tear fluid and 4- folds enhanced permeation of DFB as compared to pure DFB solution. The ex-vivo histopathological studies revealed that DFB-LPHNPs didn't cause any damage or no alteration in the cellular structure of the cornea. Additionally, the results of the HET-CAM assay confirmed that the DFB-LPHNPs were not toxic for ophthalmic administration.

Corneal layer thickness in keratoconus using optical coherence tomography

ABSTRACT Clinical relevance Accurate thickness measurement of corneal layers using anterior segment OCT can be used to improve visual outcomes. Understanding its applications is essential for optometric practices to enhance eye care procedures. Background To evaluate the thicknesses of different corneal layers for identifying keratoconus (KCN) and subclinical keratoconus (SKCN) using spectral-domain optical coherence tomography (SD-OCT). Methods This prospective study analyzed 60 eyes with KCN, 48 eyes with SKCN, and 53 normal eyes. The central corneal thickness (CCT) and thicknesses of the epithelium, Bowman, stroma, and Descemet-endothelium layers were measured using SD-OCT. One way analysis of variance and the area under the curve (AUC) were used to evaluate the parameters. The Delong method was used to compare AUCs. Results In KCN, CCT and thicknesses of epithelium, Bowman, stroma, and Descemet-endothelium layers were 495.5 ± 41.7, 52.6 ± 6.4,11.5 ± 1.4, 415.5 ± 38.9, and 12.3 ± 1.7 µm, respectively. These thickness values were respectively 524.5 ± 33.3, 56.8 ± 6.8, 11.5 ± 1.6, 439.8 ± 30.6, and 12.4 ± 1.7 µm in SKCN and 563.8 ± 37.9, 57.7 ± 6.9, 12.2 ± 1.6, 469.5 ± 33.7, and 12.8 ± 2.1µm in normal group. Total cornea and stroma in KCN and SKCN, and epithelium in KCN were significantly thinner compared to the normal group (P < 0.001). The highest AUC values were observed for CCT in KCN (AUC 0.90) and SKCN (AUC 0.782). The diagnostic accuracy was significantly higher for stromal thickness in KCN (sensitivity 81.7%, specificity 73.6%, AUC 0.871) and SKCN (sensitivity 80.0%, specificity 56.6%, AUC 0.751) than other individual corneal layers (Delong, P < 0.001) . Conclusion CCT can accurately distinguish keratoconus from normal eyes. However, central corneal stromal thinning was the most sensitive diagnostic index for early detection of SKCN. Developing standardized stromal maps may be helpful for detecting SKCN.

The Corneal Ectasia Model of Rabbit: A Validity and Stability Study.

Keratoconus is a bilateral progressive degenerative corneal disease characterized by localized corneal thinning and dilatation. The pathogenesis of keratoconus is not fully elucidated. To gain a better understanding of the pathophysiology of this disease and to explore potential treatments, animal models are essential for basic research. Several attempts have been made to establish animal models of corneal ectasia by using collagenase. However, continuous changes of the cornea have not been well-tracked for the model. In this study, corneal morphology and biomechanical behavior in vivo were determined before and after collagenase Ⅱ treatment at 2, 4, and 8 weeks. The elastic modulus and histology of cornea tissues ex vivo were measured at 8 weeks postoperatively. The results showed that the posterior corneal curvature (Km B) increased and central corneal thickness (CCT) decreased after collagenase treatment. The mechanical properties of ectatic corneas weakened significantly and the collagen fiber interval in the stromal layer was increased and disorganized. This study provides insights into the changes of corneal morphology and biomechanical properties in a rabbit model of corneal ectasia. Changes observed at 8 weeks indicated that the cornea was still undergoing remodeling.