Generation of Induced-Primary Retinal Pigment Epithelium from Human Retinal Organoids.

Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.

Accuracy study of Angiotensin 1-7 composite index test to predict pulmonary fibrosis and guide treatment.

BACKGROUND: Pulmonary fibrosis can develop after acute respiratory distress syndrome (ARDS). The hypothesis is we are able to measure phenotypes that lie at the origin of ARDS severity and fibrosis development. The aim is an accuracy study of prognostic circulating biomarkers. METHODS: A longitudinal study followed COVID-related ARDS patients with medical imaging, pulmonary function tests and biomarker analysis, generating 444 laboratory data. Comparison to controls used non-parametrical statistics; p < 0·05 was considered significant. Cut-offs were obtained through receiver operating curve. Contingency tables revealed predictive values. Odds ratio was calculated through logistic regression. RESULTS: Angiotensin 1-7 beneath 138 pg/mL defined Angiotensin imbalance phenotype. Hyper-inflammatory phenotype showed a composite index test above 34, based on high Angiotensin 1-7, C-Reactive Protein, Ferritin and Transforming Growth Factor-ß. Analytical study showed conformity to predefined goals. Clinical performance gave a positive predictive value of 95 % (95 % confidence interval, 82 %-99 %), and a negative predictive value of 100 % (95 % confidence interval, 65 %-100 %). Those severe ARDS phenotypes represented 34 (Odds 95 % confidence interval, 3-355) times higher risk for pulmonary fibrosis development (p < 0·001). CONCLUSIONS: Angiotensin 1-7 composite index is an early and objective predictor of ARDS evolving to pulmonary fibrosis. It may guide therapeutic decisions in targeted phenotypes.

Development of an AI-based support system for controlled ovarian stimulation.

Purpose: Controlled ovarian stimulation (COS) is vital for IVF. We have developed an AI system to support the implementation of COS protocols in our clinical group. Methods: We developed two models as AI algorithms of the AI system. One was the oocyte retrieval decision model, to determine the timing of oocyte retrieval, and the other was the prescription inference model, to provide a prescription similar to that of an expert physician. Data was obtained from IVF treatment records from the In Vitro Fertilization (IVF) management system at the Asada Ladies Clinic, and these models were trained with this data. Results: The oocyte retrieval decision model achieved superior sensitivity and specificity with 0.964 area under the curve (AUC). The prescription inference model achieved an AUC value of 0.948. Four models, namely the hCG prediction model, the hMG prediction model, the Cetrorelix prediction model, and the Estradiol prediction model included in the prescription inference model, achieved AUC values of 0.914, 0.937, 0.966, and 0.976, respectively. Conclusion: The AI algorithm achieved high accuracy and was confirmed to be useful. The AI system has now been implemented as a COS tool in our clinical group for self-funded treatments.

In vitro digestive properties and the bioactive effect of walnut green husk on human gut microbiota.

Introduction: Walnut green husk (WGH) is a waste byproduct from walnut industry. However, it is not well-known about its bioactive effect on human gut health. Methods: This study conducted in vitro digestion and fermentation experiments to study the bioactive effect of WGH. Results: Microbial fermentation was the primary mechanism to efficiently release phenolics and flavonoids, resulting in more excellent antioxidant capacities (DPPH, ABTS, and FRAP assays), which reached a highest value with 14.82 ± 0.01 mg VcE/g DW, 3.47 ± 0.01 mmol TE/g DW, and 0.96 ± 0.07 mmol FeSO4·7H2O/g DW, respectively. The surface microstructure of WGH became loose and fragmented after microbial fermentation. The analytical results of gut microbiota demonstrated that WGH could significantly increase the relative abundance of Proteobacteria in phylum level and Phascolarctobacterium in genus level while certain pro-inflammatory bacteria (such as Clostridium_sensu_stricto_1, Dorea, Alistipes, and Bilophila) was inhibited. Additionally, 1,373 differential metabolites were identified and enriched in 283 KEGG pathways. Of which some metabolites were significantly upregulated including ferulic acid, chlorogenic acid, umbelliferone, scopolin, muricholic acid, and so forth. Discussion: These results indicated that WGH could have antioxidant and anti-inflammatory activities in the human gut, which could improve the economical value of WGH in the food industry.

Effects of Metabolic Syndrome on Pregnancy Outcomes in Women Without Polycystic Ovary Syndrome.

Context: Metabolic syndrome (MetS) is a cluster of metabolic risk factors that predict cardiovascular disease. Previous studies suggested that MetS impaired clinical outcomes in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF). Objective: To evaluate the effects of MetS on IVF/intracytoplasmic sperm injection (ICSI) outcomes in women without PCOS. Methods: This retrospective study collected 8539 eligible women without PCOS who came for their first cycle of IVF/ICSI to the Institute of Women, Children and Reproductive Health, Shandong University, from 2017 to 2020, including 1147 subjects in the MetS group and 7392 in the control group. The primary outcome was live birth. Secondary outcomes included other pregnancy outcomes and the risk of maternal and neonatal complications. Results: Women in the MetS group had a lower live birth rate (50.6% vs 54.9%, adjusted odds ratio [aOR] 0.87, 95% CI 0.75-1.00, P = .045) and higher risks of late miscarriage (5.8% vs 3.3%, aOR 1.52, 95% CI 1.02-2.27, P = .041), gestational diabetes mellitus (13.7% vs 7.0%, aOR 1.84, 95% CI 1.30-2.60, P = .001), hypertensive disorder of pregnancy (7.8% vs 3.5%, aOR 1.79, 95% CI 1.14-2.83, P = .012), and preterm birth (9.0% vs 4.4%, aOR 2.03, 95% CI 1.33-3.08, P = .001). Singleton newborns in the MetS group were at higher risk of large for gestational age (33.3% vs 20.5%, aOR 1.66, 95% CI (1.31-2.13), P < .001) but at lower risk of small for gestational age (2.7% vs 6.2%, aOR 0.48, 95% CI 0.25-0.90, P = .023). Conclusion: MetS was associated with adverse IVF/ICSI outcomes in women without PCOS.

Antioxidant effects and compatibility of zinc oxide nanoparticles during in vitro maturation of bovine oocytes and subsequent embryo development.

Zinc oxide nanoparticles (ZnO-NPs) have garnered significant attention in biological applications due to their known antioxidant properties. However, their potential impact on assisted reproduction techniques remains largely unexplored, particularly in the context of oocyte quality maintenance within in vitro culture systems, where free radicals can exert detrimental effects. This study investigated the effects of incorporating ZnO-NPs to in vitro maturation (IVM) media on the developmental, cryosurvival, and metabolic profiles of bovine embryos. Three concentrations of ZnO-NPs (0, 1.0, and 1.5 µg/mL) were evaluated. We observed, for the first time, that the inclusion of ZnO-NPs at a concentration of 1.0 µg/mL led to a significant increase in the number of embryonic cells (p < 0.05) accompanied by a reduction in reactive oxygen species production (p < 0.05). Notably, ZnO-NPs did not alter embryonic development, cryosurvival rates, or mitochondrial viability. These findings suggested that ZnO-NPs has antioxidant properties and are compatible with bovine oocytes. Consequently, they may serve as promising supplements to the IVM media, potentially enhancing the efficiency of assisted reproduction techniques.

Electrochemotherapy and Calcium Electroporation on Hepatocellular Carcinoma Cells: An In-Vitro Investigation.

PURPOSE: Electrochemotherapy, clinically established for treating (sub)cutaneous tumors, has been standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy (ESOPE). Due to common side effects of chemotherapeutic drugs, recent advances focus on non-cytotoxic agents, like calcium, to induce cell death (calcium electroporation). Therefore, this study aims to determine the efficacy of electrochemotherapy with bleomycin or cisplatin, or calcium electroporation on human hepatocellular carcinoma cells (HepG2) in vitro using the ESOPE protocol. METHODS: HepG2 cell viability was measured with a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after electrochemotherapy with the chemotherapeutic drugs bleomycin or cisplatin (0-20 µM), or after calcium electroporation (0-20 mM), to determine its efficacy on HepG2 cells in vitro using the ESOPE protocol (8 rectangular pulses, 1000 V/cm, 100 µs) compared to non-electroporated drug treatment. RESULTS: Cell viability was significantly lower in electroporated samples, compared to their non-electroporated controls (27-75% difference). Electrochemotherapy with bleomycin and calcium electroporation, reached (almost) complete cell death (- 1 ± 3% and 2.5 ± 2%), in the lowest concentration of 2.5 µM and 2.5 mM, respectively. Electrochemotherapy with 2.5 µM cisplatin, significantly decreased cell viability to only 68% (± 7%). CONCLUSION: Electrochemotherapy with bleomycin or cisplatin, or calcium electroporation were more effective in reducing the HepG2 cell viability in vitro using the ESOPE protocol compared to the non-electroporated drug treatments alone. When comparing electrochemotherapy, HepG2 cells are more sensitive to bleomycin than cisplatin, in similar concentrations. Calcium electroporation has the same effectiveness as electrochemotherapy with bleomycin, but calcium potentially has a better safety profile and several treatment advantages.

True empty follicle syndrome is a subtype of oocyte maturation abnormalities.

Objective: To review the outcomes of in vitro maturation (IVM) and in vitro fertilization (IVF) in women with empty follicle syndrome (EFS). The study evaluated the genetic underpinnings of EFS by analyzing mutations. Materials and Methods: This retrospective case series involving 17 women with EFS over at least 2 IVF cycles was conducted. The study also employed whole-exome sequencing to analyze the genetic mutations. The treatment approaches included letrozole-primed IVM, follicle-stimulating hormone (FSH)-human chorionic gonadotrophin (hCG)-primed IVM, and conventional IVF. Results: The average female age was 31.5±4.6 years, and the duration of infertility was 7.3±3.5 years. Four patients underwent IVF. IVM oocyte collections yielded oocytes in 12 of 13 subjects. Of these, 75% (9/12) yielded MII oocytes after 48 h of IVM media incubation. Six subjects had fertilized embryos, resulting in a 40.9% intracytoplasmic sperm injection (ICSI) fertilization rate (9 embryos/22 MII oocytes). Genetic analysis revealed mutations in seven patients. This study demonstrated the partial efficacy of letrozole-primed IVM plus growth hormone and FSH-hCG primed IVM protocols. No pregnancies or live births were recorded after IVM. One ongoing pregnancy post-IVF and one spontaneous live birth were observed. Conclusion: Inter-cycle variabilities were observed in women with oocyte maturation abnormalities (OMAs). Almost all patients with EFS had oocytes collected during IVM following IVF. These oocytes have limited potential for maturation, fertilization, and live birth, as demonstrated by the low rates observed after IVM culture and ICSI. These conditions are observed in OMAs due to defects in the oocyte machinery. The proposed flowchart provides a comprehensive classification approach for various forms of EFS.

A 3D in-vitro biomimicking Caco-2 intestinal permeability model-based assessment of physically modified telmisartan towards an alkalizer-free formulation development.

Efficient telmisartan delivery for hypertension management requires the incorporation of meglumine and/or sodium hydroxide as an alkalizer in the formulation. Long-term use of powerful alkalis with formulation as part of chronic therapy can cause metabolic alkalosis, ulcers, diarrhea, and body pain. Here, we aimed to design a telmisartan formulation without alkalizers. Telmisartan properties were tailor-made by microfluidizer-based physical modification. After microfluidization, telmisartan nanosuspension was lyophilized to obtain telmisartan premix powder. The optimized telmisartan nanosuspension had an average particle size of 579.85 ±â€¯32.14 nm. The lyophilized premix was characterized by FT-IR, DSC, and PXRD analysis to ensure its physicochemical characteristics. The solubility analysis of premix showed 2.2 times, 2.3 times, and 6 times solubility improvement in 0.1 N HCl, phosphate buffer pH 7.5, and pH 6.8 compared to pure telmisartan. A 3D in-vitro Caco-2 model was developed to compare apparent permeability of API and powder premix. It showed that the powder premix was more permeable than pure API. The tablet formulation prepared from the telmisartan premix showed a dissolution profile comparable to that of the marketed formulation. The technique present herein can be used as a platform technology for solubility and permeability improvement of similar classes of molecules.

Formulation and Evaluation of Butenafine Hydrochloride-Incorporated Solid Lipid Nanoparticles as Novel Excipients for the Treatment of Superficial Fungal Infections.

Objectives: The objective of the present study was to develop natural excipient-based solid lipid nanoparticles (SLN) of butenafine hydrochloride (BUTE) using a modified solvent emulsification technique and to evaluate the competence of aloe vera nanolipidgel in enhancing the penetration of BUTE. Materials and Methods: BUTE-SLNs were prepared using a 23 factorial design to correlate the effect of formulation components on the BUTE-SLN. Particle size, polydispersity index (PDI), zeta potential, entrapment performance, and drug loading were assessed in the formed SLNs. The fabricated BUTE-SLN was evaluated for transmission electron microscopy, fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction study studies and revealed the encapsulation of BUTE in lipid in the amorphous state. BUTE-SLN-based aloe vera gel was formulated and evaluated compared with the marketed product with respect to primary skin irritation, hydration, skin permeation, and antifungal activity. Results: The BUTE-SLN aloe vera gel, optimized for its formulation, features excellent slip properties and controlled drug release. DSC and XRD studies confirm its amorphous nature with effective drug entrapment. The gel provides enhanced skin deposition, improved antifungal activity, and reduced irritation. This makes it a cost-effective and innovative alternative to traditional dosage forms. BUTE-SLN promisingly showed no irritation, higher hydrating potential, slow and sustained release, and enhanced antifungal activity. With an aim to target deeper skin strata, minimize the side effects of drugs and symptomatic impact of fungal infection, and shorten the duration of therapy, BUTE-SLN was successfully prepared. The mean particle size and PDI were 261.25 ± 2.38 nm and 0.268 ± 0.01, respectively. Conclusion: BUTE-SLN gel offers improved topical delivery of BUTE with significantly higher compatibility and antifungal activity than the marketed formulation.

Gradient-induced instability in tumour spheroids unveils the impact of microenvironmental nutrient changes.

Tumours often display invasive behaviours that induce fingering, branching and fragmentation processes. The phenomenon, known as diffusional instability, is driven by differential cell proliferation, migration, and death due to the presence of metabolite and catabolite concentration gradients. An understanding of the intricate dynamics of this spatially heterogeneous process plays a key role in the investigation of tumour growth and invasion. In this study, we developed an in vitro tumour invasion assay to investigate cell invasiveness in tumour spheroids under a chemotactic stimulus. Our method, employing tumour spheroids seeded in a 3D collagen gel within a microfluidic chemotaxis chamber, focuses on the role of diffusive gradients. Using Time-Lapse Microscopy, the dynamic evolution of tumour spheroids was monitored in real-time, providing a comprehensive view of the morphological changes and cell migration patterns under different chemotactic conditions. Specifically, we explored the impact of fetal bovine serum (FBS) gradients on the behaviour of CT26 mouse colon carcinoma cells and compared the effects of varying FBS concentrations to two isotropic control conditions. Furthermore, a finite element in silico model was developed to quantify the diffusive flow of nutrients in the chemotaxis chamber and obtain a detailed understanding of tumour dynamics. Our findings reveal that the presence of a chemotactic gradient significantly influences tumour invasiveness, with higher concentrations of nutrients associated with increased cancer growth and cell migration.

Comparative influence of inappropriate gestational weight gain on pregnancy outcomes in IVF-conceived and spontaneously conceived twin pregnancies.

OBJECTIVE: To investigate the influence of inappropriate gestational weight gain (GWG) on pregnancy outcomes in twin pregnant women with in vitro fertilization (IVF) treatment. METHODS: This retrospective cohort study included 2992 twin pregnant women and categorized the participants as follows: (i) they were classified into spontaneous conception (SC) or IVF groups based on whether they received IVF treatment, and (ii) they were categorized into inadequate, optimal, or excessive GWG groups according to the International Organization for Migration Twin Pregnancy Guidelines. Initially, the study investigated the separate effects of IVF treatment and different levels of GWG on the outcomes of twin pregnancies. Subsequently, after adjusting for confounding factors, multifactorial logistic regression analysis was performed to further investigate the impact of IVF treatment and high GWG on twin pregnancy outcomes. Based on this, the analysis was stratified by whether IVF was used to explore the effects of different GWG levels on each subgroup (those who underwent IVF and those who conceived spontaneously). Finally, potential multiplicative interactions between IVF and different GWG categories were examined to identify their combined effect on pregnancy outcomes. RESULTS: The results showed that women with twin gestations conceived via IVF exhibited significantly higher maternal age, pre-pregnancy body mass index, and a greater incidence of GWG beyond recommended guidelines compared to the SC group. Furthermore, both IVF treatment and inappropriate GWG increased the risk of adverse pregnancy outcomes, respectively. Following adjustments for confounding variables through multifactorial logistic regression, it was demonstrated that both IVF treatment and high GWG significantly elevated the risk of adverse outcomes in twin pregnancies, such as admission to the neonatal intensive care unit. It is noteworthy that inappropriate GWG, combined with IVF treatment, will stepwise increase the incidence of intrahepatic cholestasis of pregnancy, respiratory failure, respiratory distress, pre-eclampsia, maternal intensive care unit admission, and postpartum hemorrhage risk. However, these outcomes were less affected by inappropriate GWG in the SC group. Lastly, this study did not unveil a significant interaction between the IVF procedure and disparate levels of GWG in relation to the adverse outcomes. CONCLUSION: A high incidence of inappropriate GWG in twin pregnancies with IVF treatment and inappropriate GWG conferred more adverse twin pregnancy outcomes in the IVF group relative to the SC group. This study indicates that proper management of GWG may be a breakthrough in reducing adverse outcomes in twin pregnancies associated with IVF. Therefore, implementing proactive interventions such as supervised exercise programs, prescribed physical or dietary plans, enhanced weight management, or personalized counseling, holds promise for lowering the risks associated with inappropriate GWG in twin pregnancies resulting from IVF.

Manifesting the Dasatinib-gallic acid co-amorphous system to augment anticancer potential: Physicochemical characterization, in silico molecular simulation, ex vivo permeability, and in vitro efficacy.

Dasatinib (DAB) has been explored for repurposing in the treatment of breast cancer (BC) due to its known effectiveness in treating leukemia, in addition to its role as a tyrosine kinase inhibitor. Gallic acid (GA) was chosen as a co-former due to its anticancer potential in BC, as demonstrated in several previous studies. DAB is a low-solubility drug, which is a significant hurdle for its oral bioavailability. To address this limitation, a DAB and GA co-amorphous (DAB-GA-CA) system was developed using liquid-assisted grinding and ball mill technology to enhance solubility, bioavailability, and anti-tumor efficacy. Physical characterization investigation revealed that the emergence of the halo diffractogram in PXRD, single glass transition temperature (Tg) value at 111.7 °C in DSC thermogram, and irregularly shaped blocks with loose, porous surfaces in SEM analysis indicated the formation of the DAB-GA-CA system at 1:1 M ratio. Furthermore, FTIR, Raman spectroscopy, in-silico molecular docking, and molecular dynamic studies confirmed the intermolecular hydrogen connections between DAB and GA. Moreover, the outcomes of the ligands (DAB and GA) and receptors (BCL-2, mTOR, estrogen receptor, and HER-2) docking studies demonstrated that both DAB and GA could interact with those receptors, leading to preventive action on BC cells. Additionally, the solubility and dissolution rate significantly improved at pH 6.8, and the permeability study indicated that DAB-GA-CA showed 1.9 times higher apparent permeability compared to crystalline DAB. Furthermore, in vitro cytotoxicity assessments of the DAB-GA-CA system revealed 3.42 times lower IC50 than free DAB. The mitochondrial membrane depolarization, apoptotic index, and reactive oxygen species formation in MCF-7 cells were also notably higher in the DAB-GA-CA system than in free DAB. Hence, this research suggests that the DAB-GA-CA system could substantially enhance oral delivery, solubility, and therapeutic efficacy.

Luteinizing hormone supplementation with human menopausal gonadotropin versus low dose human chorionic gonadotropin during ovarian stimulation does not affect live birth rates after fresh and frozen embryo transfer.

AIM: Luteinizing hormone (LH) plays an important role in ovarian follicle maturation. Human menopausal gonadotropin (hMG) or low dose human chorionic gonadotropin (hCG) can provide LH supplementation during in vitro fertilization (IVF) ovarian stimulation, though studies directly comparing their impact on IVF outcomes are limited. The aim of the study was to determine whether LH supplementation with hMG versus low dose hCG during IVF stimulation affects live birth rate. METHODS: Fresh and frozen embryo transfers (ET) from 2017 to 2021 after standard long or antagonist protocols supplemented with hMG (75-250 IU) or low dose hCG (50-100 IU) during stimulation cycles in our academic center were included. Statistical analysis was performed with T-tests, Mann-Whitney U tests, Chi-square, and multiple linear and logistic regression. RESULTS: Four hundred and sixty eight unique stimulation cycles resulting in 213 fresh and 412 frozen embryo transfers were analyzed. There was a lower mature oocyte yield (10.9 vs. 11.8, p = 0.044) but similar high-quality blastocyst yield (3.6 vs. 3.9, p = 0.11) for hMG vs low dose hCG. Live birth rates per transfer were comparable for fresh (42% vs. 49%, p = 0.24) and frozen (46% vs. 53%, p = 0.45) embryo transfers. Multiple logistic regressions showed no association between supplemental gonadotropin and live birth for both fresh and frozen embryo transfers. CONCLUSION: Fresh and frozen IVF-ET pregnancy outcomes were comparable after hMG versus low dose hCG supplementation, suggesting flexibility in supplemental LH dosing regimens that may address patient or physician preference or cost concerns.

In vitro neurotoxicity testing: lessons from chemotherapy-induced peripheral neurotoxicity.

INTRODUCTION: Chemotherapy induced peripheral neurotoxicity (CIPN) is a long-lasting, or even permanent, late toxicity caused by largely used anticancer drugs. CIPN affects a growing population of cancer survivors and diminishes their quality of life since there is no curative/preventive treatment. Among several reasons for this unmet clinical need, there is an incomplete knowledge on mechanisms leading to CIPN. Therefore, bench side research is still greatly needed: in vitro studies are pivotal to both evaluate neurotoxicity mechanisms and potential neuroprotection strategies. AREAS COVERED: Advantages and disadvantages of in vitro approaches are addressed with respect to their applicability to the CIPN field. Different cell cultures and techniques to assess neurotoxicity/neuroprotection are described. PubMed search-string: (chemotherapy-induced) AND (((neuropathy) OR neurotoxicity) OR neuropathic pain) AND (in vitro) AND (((((model) OR SH-SY5Y) OR PC12) OR iPSC) OR DRG neurons); (chemotherapy-induced) AND (((neuropathy) OR neurotoxicity) OR neuropathic pain) AND (model) AND (((neurite elongation) OR cell viability) OR morphology). No articles published before 1990 were selected. EXPERT OPINION: CIPN is an ideal experimental setting to test axonal damage and, in general, peripheral nervous system mechanisms of disease and neuroprotection. Therefore, starting from robust preclinical data in this field, potentially, relevant biological rationale can be transferred to other human spontaneous diseases of the peripheral nervous system.

ThymoSpheres culture: A model to study human polyclonal unconventional T cells.

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αß unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms.

Characterization of the C17.2 cell line as testing system for endocrine disruption-induced developmental neurotoxicity.

Hormone signaling plays an essential role during fetal life and is vital for brain development. Endocrine-disrupting chemicals can interfere with the hormonal milieu during this critical time-period, disrupting key neurodevelopmental processes. Hence, there is a need for the development of assays that evaluate developmental neurotoxicity (DNT) induced by an endocrine mode of action. Herein, we evaluated the applicability of the neural progenitor C17. 2 cell-line, as an in vitro test system to aid in the detection of endocrine disruption (ED) induced DNT. For this, C17.2 cells were exposed during 10 days of differentiation to agonists and antagonists of the thyroid hormone (Thr), glucocorticoid (Gr), retinoic acid (Rar), retinoic x (Rxr), oxysterols (Lxr), estrogen (Er), androgen (Ar), and peroxisome proliferator activated delta (Pparß/δ) receptors, as well as to the agonist of the vitamin D (Vdr) receptor. Upon exposure and differentiation, neuronal morphology (neurite outgrowth and branching), and the percentage of neurons in culture were assessed by immunofluorescence. For this, the cells were incubated with Hoechst (nuclear staining) and stained for ßIII-tubulin (neuronal marker). The C17.2 cells were responsive to the Rar, Rxr and Pparß/δ agonists which decreased neurite outgrowth and branching. Additionally, exposure to the Gr agonist increased the number of cells differentiating into neurons, while exposure to the Rxr agonist had the opposite effect. With this approach, we have identified that the C17.2 cells are responsive to Gr, Rar, Rxr, and Pparß/δ agonists, hence contributing to the development of test systems for hazard assessment of ED-induced DNT.

Endocrine disrupting chemicals (EDCs) interfere with hormonal signaling. As hormones play a vital role for an organism's development, EDC exposure is of high concern. In European regulations, the use of a chemical can be restricted if its toxicity is mediated by hormonal interference. A number of EDCs affect brain development. However, in animal tests, it is impossible to prove that a chemical induces developmental neurotoxicity (DNT) via endocrine disruption (ED). Furthermore, the regulatory DNT tests require large amounts of animals. Thus, there is an urgent need for in vitro test systems to identify ED-induced DNT. Herein we present the development of such a method based on the murine neural progenitor cell-line C17.2 with which neuronal differentiation processes can be mimicked. We show that differentiation of C17.2 cells are sensitive to retinoid, glucocorticoid, and peroxisome proliferator activated receptor signaling disruption, thus providing an alternative method for identifying ED-induced DNT.

An Improved Test Method for Assaying the Inhibition of Bioflavonoids on Xanthine Oxidase Activity in†vitro.

The difference on inhibitory effects of bioflavonoids inhibiting XOD activity assayed by varying test methods cause of us to be further in consideration. The reported test method creating a micro-environment surrounding XOD in the absence of ⋅O2 -, which is seemly different from the assay in vivo. So, the vitro test method for assaying XOD activity is necessary to be improved for selection of potential inhibitors in the presence of ⋅O2 -. The inhibitory results demonstrated that bioflavonoids of MY, DMY, QUE and LUT are capable to be on effective IC50 values, but others are not. As well, their resulting inhibitions determined by the improved test method are much less than that reported in the literature, indicating that their chemical affinities with XOD become weaker. Moreover, DMY assayed on the inhibitions of XOD in the improved test method performs to be a better inhibitor, as compared to the assay of the reported test methods. Abasing on the transformation of DMY into MY in the presence of ⋅O2 -, the good inhibition of DMY on XOD activity can be explained by the synergistic effect of MY.

Integrative Analysis of Probiotic-Mediated Remodeling in Canine Gut Microbiota and Metabolites Using a Fermenter for an Intestinal Microbiota Model.

In contemporary society, the increasing number of pet-owning households has significantly heightened interest in companion animal health, expanding the probiotics market aimed at enhancing pet well-being. Consequently, research into the gut microbiota of companion animals has gained momentum, however, ethical and societal challenges associated with experiments on intelligent and pain-sensitive animals necessitate alternative research methodologies to reduce reliance on live animal testing. To address this need, the Fermenter for Intestinal Microbiota Model (FIMM) is being investigated as an in vitro tool designed to replicate gastrointestinal conditions of living animals, offering a means to study gut microbiota while minimizing animal experimentation. The FIMM system explored interactions between intestinal microbiota and probiotics within a simulated gut environment. Two strains of commercial probiotic bacteria, Enterococcus faecium IDCC 2102 and Bifidobacterium lactis IDCC 4301, along with a newly isolated strain from domestic dogs, Lactobacillus acidophilus SLAM AK001, were introduced into the FIMM system with gut microbiota from a beagle model. Findings highlight the system's capacity to mirror and modulate the gut environment, evidenced by an increase in beneficial bacteria like Lactobacillus and Faecalibacterium and a decrease in the pathogen Clostridium. The study also verified the system's ability to facilitate accurate interactions between probiotics and commensal bacteria, demonstrated by the production of short-chain fatty acids and bacterial metabolites, including amino acids and gamma-aminobutyric acid precursors. Thus, the results advocate for FIMM as an in vitro system that authentically simulates the intestinal environment, presenting a viable alternative for examining gut microbiota and metabolites in companion animals.

Reliability and relevance of the ES®-RHE model for in vitro skin irritation test application.

INTRODUCTION: In vitro methods have been widely used to assess adverse effects. Reconstructed Human Epidermis (RHE) poses as a fascinating test system employed to assess the dermal irritation hazard potential of chemicals. Although several RHE models are reported in the OECD Test Guideline No. 439, the OECD Document No. 220 encourages the scientific community to develop and validate new RHE test systems due to its relevance for socio-economic advancement. METHODS: Following the criteria documented in the OECD No. 220, a blind study for skin irritation (OECD 439) was conducted employing the Minimum List of Reference Chemicals for Determination of Reproducibility and Predictive Capacity using ES®-RHE. Structural and functional characteristics were assessed alongside the prediction model. RESULTS: The model has shown reproducibility of optical density and barrier function, similarly to internationally validated methods. Furthermore, it shows the cell layers' development and differentiation ability due to Cytokeratin14, Cytokeratin10, and filaggrin expression. The prediction model resulted in sensitivity, specificity and accuracy rates of 100, 70, and 77 %, respectively. CONCLUSIONS: The ES®-RHE demonstrated reliability and relevance, with similar structural and functional characteristics comparable to internationally validated models, in addition to the accepted predictive capacity according to OECD required minimum criteria, thus confirming the suitability of the national ES®-RHE in the hazard prediction of dermal irritation based on OECD Test Guideline No. 439.