Ex Vivo Evaluation of the Function of Hematopoietic Stem Cells in Toxicology of Metals.

A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Identify the impact of a metal on the competence of HSCs Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure.

Sodium MRI of the Lumbar Intervertebral Discs of the Human Spine: An Ex Vivo Study.

BACKGROUND: Lower back pain affects 75%-85% of people at some point in their lives. The detection of biochemical changes with sodium (23Na) MRI has potential to enable an earlier and more accurate diagnosis. PURPOSE: To measure 23Na relaxation times and apparent tissue sodium concentration (aTSC) in ex-vivo intervertebral discs (IVDs), and to investigate the relationship between aTSC and histological Thompson grade. STUDY TYPE: Ex-vivo. SPECIMEN: Thirty IVDs from the lumbar spines of 11 human body donors (4 female, 7 male, mean age 86 ± 8 years). FIELD STRENGTH/SEQUENCE: 3 T; density-adapted 3D radial sequence (DA-3D-RAD). ASSESSMENT: IVD 23Na longitudinal (T1), short and long transverse (T2s* and T2l*) relaxation times and the proportion of the short transverse relaxation (ps) were calculated for one IVD per spine sample (11 IVDs). Furthermore, aTSCs were calculated for all IVDs. The degradation of the IVDs was assessed via histological Thompson grading. STATISTICAL TESTS: A Kendall Tau correlation (τ) test was performed between the aTSCs and the Thompson grades. The significance level was set to P < 0.05. RESULTS: Mean 23Na relaxation parameters of a subset of 11 IVDs were T1 = 9.8 ± 1.3 msec, T2s* = 0.7 ± 0.1 msec, T2l* = 7.3 ± 1.1 msec, and ps = 32.7 ± 4.0%. A total of 30 IVDs were examined, of which 3 had Thompson grade 1, 4 had grade 2, 5 had grade 3, 5 had grade 4, and 13 had grade 5. The aTSC decreased with increasing degradation, being 274.6 ± 18.9 mM for Thompson grade 1 and 190.5 ± 29.5 mM for Thompson grade 5. The correlation between whole IVD aTSC and Thompson grade was significant and strongly negative (τ = -0.56). DATA CONCLUSION: This study showed a significant correlation between aTSC and degenerative IVD changes. Consequently, aTSC has potential to be useful as an indicator of degenerative spinal changes. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 1.

Recovery kinetics of dual AAV-mediated human otoferlin expression.

Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.

Light-activated conjugated polymer nanoparticles to defeat pathogens associated with bovine mastitis.

Bovine mastitis (BM) represents a significant challenge in the dairy industry. Limitations of conventional treatments have prompted the exploration of alternative approaches, such as photodynamic inactivation (PDI). In this study, we developed a PDI protocol to eliminate BM-associated pathogens using porphyrin-doped conjugated polymer nanoparticles (CPN). The PDI-CPN protocol was evaluated in four mastitis isolates of Staphylococcus and in a hyper-biofilm-forming reference strain. The results in planktonic cultures demonstrated that PDI-CPN exhibited a bactericidal profile upon relatively low light doses (∼9.6 J/cm2). Furthermore, following a seven-hour incubation period, no evidence of cellular reactivation was observed, indicating a highly efficient post-photodynamic inactivation effect. The successful elimination of bacterial suspensions encouraged us to test the PDI-CPN protocol on mature biofilms. Treatment using moderate light dose (∼64.8 J/cm2) reduced biofilm biomass and metabolic activity by up to 74% and 88%, respectively. The impact of PDI-CPN therapy on biofilms was investigated using scanning electron microscopy (SEM), which revealed nearly complete removal of the extracellular matrix and cocci. Moreover, ex vivo studies conducted on bovine udder skin demonstrated the efficacy of the therapy in eliminating bacteria from these scaffolds and its potential as a prophylactic method. Notably, the histological analysis of skin revealed no signs of cellular degeneration, suggesting that the protocol is safe and effective for BM treatment. Overall, this study demonstrates the potential of PDI-CPN in treating and preventing BM pathogens. It also provides insights into the effects of PDI-CPN on bacterial growth, metabolism, and survival over extended periods, aiding the development of effective control strategies and the optimization of future treatments.

Pharmacokinetics and Histotoxic Profile of a Novel Azithromycin-Loaded Lipid-Based Nanoformulation.

Azithromycin traditional formulations possesses poor oral bioavailability which necessitates development of new formulation with enhanced bioavailability of the drug. The objective of current research was to explore the kinetics and safety profile of the newly developed azithromycin lipid-based nanoformulation (AZM-NF). In the in-vitro study of kinetics profiling, azithromycin (AZM) release was assessed using dialysis membrane enclosing equal quantity of either AZM-NF, oral suspension of azithromycin commercial product (AZM-CP), or azithromycin pure drug (AZM-PD) in simulated intestinal fluid. The ex-vivo study was performed using rabbit intestinal segments in physiological salts solution in a tissue bath. The in-vivo study was investigated by oral administration of AZM to rabbits while taking blood samples at predetermined time-intervals, followed by HPLC analysis. The toxicity study was conducted in rats to observe histopathological changes in rat's internal organs. In the in-vitro study, maximum release was 95.38 ± 4.58% for AZM-NF, 72.79 ± 8.85% for AZM-CP, and 46.13 ± 8.19% for AZM-PD (p < 0.0001). The ex-vivo investigation revealed maximum permeation of 85.68 ± 5.87 for AZM-NF and 64.88 ± 5.87% for AZM-CP (p < 0.001). The in-vivo kinetics showed Cmax 0.738 ± 0.038, and 0.599 ± 0.082 µg/ml with Tmax of 4 and 2 h for AZM-NF and AZM-CP respectively (p < 0.01). Histopathological examination revealed compromised myocardial fibers integrity by AZM-CP only, liver and kidney showed mild aberrations by both formulations, with no remarkable changes in the rest of studied organs. The results showed that AZM-NF exhibited significantly enhanced bioavailability with comparative safer profile to AZM-CP investigated.

Engineering a 3-dimensional tissue construct with adipose-derived stem cells for healing bone defect: An ex vivo study with femur head.

The compatibility of bone graft substitutes (BGS) with mesenchymal stem cells (MSCs) is an important parameter to consider for their use in repairing bone defects as it eventually affects the clinical outcome. In the present study, a few commercially available BGS - ß-tricalcium phosphate (ß-TCP), calcium sulfate, gelatin sponge, and different forms of hydroxyapatite (HAP) were screened for their interactions with MSCs from adipose tissue (ADSCs). It was demonstrated that HAP block favorably supported ADSC viability, morphology, migration, and differentiation compared to other scaffolds. The results strongly suggest the importance of preclinical evaluation of bone scaffolds for their cellular compatibility. Furthermore, the bone regenerative potential of HAP block with ADSCs was evaluated in an ex vivo bone defect model developed using patient derived trabecular bone explants. The explants were cultured for 45 days in vitro and bone formation was assessed by expression of osteogenic genes, ALP secretion, and high resolution computed tomography. Our findings confirmed active bone repair process in ex vivo settings. Addition of ADSCs significantly accelerated the repair process and improved bone microarchitecture. This ex vivo bone defect model can emerge as a viable alternative to animal experimentation and also as a potent tool to evaluate patient specific bone therapeutics under controlled conditions.

Short-term cultured tumor fragments to study immunotherapy combinations based on CD137 (4-1BB) agonism.

Biomarkers for cancer immunotherapy are an unmet medical need. The group of Daniela Thommen at the NKI recently reported on novel methodologies based on short-term cultures of patient-derived tumor fragments whose cytokine concentrations in the supernatants and activation markers on infiltrating T cells were associated with clinical response to PD-1 blockade. We set up a similar culture technology with tumor-derived fragments using mouse tumors transplanted into syngeneic immunocompetent mice to test an agonist anti-CD137 mAb and its combinations with anti-PD-1 and/or anti-TGF-ß. Increases in IFNγ concentrations in the tissue culture supernatants were detected upon in-culture activation with the anti-CD137 and anti-PD-1 mAb combinations or concanavalin A as a positive control. No other cytokine from a wide array was informative of stimulation with these mAbs. Interestingly, increases in Ki67 and other activation markers were substantiated in lymphocytes from cell suspensions gathered at the end of 72 h cultures. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137 + anti-PD-1 treatment to perform the fragment culture evaluation, no association was found between IFNγ production from the fragments and the in vivo therapeutic outcome in the non-resected contralateral tumors. The experimental system permitted freezing and thawing of the fragments with similar functional outcomes. Using a series of patient-derived tumor fragments from excised solid malignancies, we showed IFNγ production in a fraction of the studied cases, that was conserved in frozen/thawed fragments. The small tumor fragment culture technique seems suitable to preclinically explore immunotherapy combinations.

How to apply ex-vivo split liver transplantation safely and feasibly: A three-step approach.

BACKGROUND: Given the current organ shortage crisis, split liver transplantation (SLT) has emerged as a promising alternative for select end-stage liver disease patients. AIM: To introduce an ex-vivo liver graft splitting approach and evaluate its safety and feasibility in SLT. METHODS: A retrospective analysis was conducted on the liver transplantation data from cases performed at our center between April 1, 2022, and May 31, 2023. The study included 25 SLT cases and 81 whole liver transplantation (WLT) cases. Total ex-vivo liver splitting was employed for SLT graft procurement in three steps. Patient outcomes were determined, including liver function parameters, postoperative complications, and perioperative mortality. Group comparisons for categorical variables were performed using the χ²-test. RESULTS: In the study, postoperative complications in the 25 SLT cases included hepatic artery thrombosis (n = 1) and pulmonary infections (n = 3), with no perioperative mortality. In contrast, among the 81 patients who underwent WLT, complications included perioperative mortality (n = 1), postoperative pulmonary infections (n = 8), abdominal infection (n = 1), hepatic artery thromboses (n = 3), portal vein thrombosis (n = 1), and intra-abdominal bleeding (n = 5). Comparative analysis demonstrated significant differences in alanine aminotransferase (176.0 vs 73.5, P = 0.000) and aspartate aminotransferase (AST) (42.0 vs 29.0, P = 0.004) at 1 wk postoperatively, and in total bilirubin (11.8 vs 20.8, P = 0.003) and AST (41.5 vs 26.0, P = 0.014) at 2 wk postoperatively. However, the overall incidence of complications was comparable between the two groups (P > 0.05). CONCLUSION: Our findings suggest that the total ex-vivo liver graft splitting technique is a safe and feasible approach, especially under the expertise of an experienced transplant center. The approach developed by our center can serve as a valuable reference for other transplantation centers.

Porcine lungs perfused with three different flows using the 8-h open-atrium cellular ex vivo lung perfusion technique.

The number of lung transplantations is limited due to the shortage of donor lungs fulfilling the standard criteria. The ex vivo lung perfusion (EVLP) technique provides the ability of re-evaluating and potentially improving and treating marginal donor lungs. Accordingly, the technique has emerged as an essential tool to increase the much-needed donor lung pool. One of the major EVLP protocols, the Lund protocol, characterized by high pulmonary artery flow (100% of cardiac output [CO]), an open atrium, and a cellular perfusate, has demonstrated encouraging short-EVLP duration results. However, the potential of the longer EVLP duration of the protocol is yet to be investigated, a duration which is considered necessary to rescue more marginal donor lungs in future. This study aimed to achieve stable 8-h EVLP using an open-atrium cellular model with three different pulmonary artery flows in addition to determining the most optimal flow in terms of best lung performance, including lung electrolytes and least lung edema formation, perfusate and tissue inflammation, and histopathological changes, using the porcine model. EVLP was performed using a flow of either 40% (n = 6), 80% (n = 6), or 100% (n = 6) of CO. No flow rate demonstrated stable 8-h EVLP. Stable 2-h EVLP was observed in all three groups. Insignificant deterioration was observed in dynamic compliance, peak airway pressure, and oxygenation between the groups. Pulmonary vascular resistance increased significantly in the 40% group (p < .05). Electrolytes demonstrated an insignificant worsening trend with longer EVLP. Interleukin-8 (IL-8) in perfusate and tissue, wet-to-dry weight ratio, and histopathological changes after EVLP were insignificantly time dependent between the groups. This study demonstrated that stable 8-h EVLP was not feasible in an open-atrium cellular model regardless of the flow of 40%, 80%, or 100% of CO. No flow was superior in terms of lung performance, lung electrolytes changes, least lung edema formation, minimal IL-8 expression in perfusate and tissue, and histopathological changes.

Encapsulation of Alpinia leaf essential oil in nanophytosome-embedded gel as novel strategy to treat periodontal infections: evaluation of antimicrobial effectiveness, pharmacokinetic, in vitro-ex vivo correlation and in silico studies.

AIM: The work reports a novel nanophytosomal gel encapsulating Alpinia galanga (L.) Willd leaf essential oil to treat periodontal infections. METHODS: Alpinia oil-loaded nanophytosomes (ANPs) were formulated by lipid layer hydration technique and were evaluated by FESEM, cryo-TEM, loading efficiency, zeta potential, particle size, release profile etc. Selected ANPs-loaded gel (ANPsG) was evaluated by both in vitro and in vivo methods. RESULTS: Selected ANPs were spherical, unilamellar, 49.32 ± 2.1 nm size, 0.45 PDI, -46.7 ± 0.8 mV zeta potential, 9.8 ± 0.5% (w/w) loading, 86.4 ± 3.02% (w/w) loading efficiency with sustained release profile. ANPsG showed good spreadability (6.8 ± 0.3 gm.cm/sec), extrudability (79.33 ± 1.5%), viscosity (36522 ± 0.82 cps), mucoadhesive strength (44.56 ± 3.5 gf) with sustained ex vivo release tendency. Satisfied ZOI and MIC was observed for ANPsG against periodontal bacteria vs. standard/control. ANPsG efficiently treated infection in ligature induced periodontitis model. Key pharmacokinetic parameters like AUC, MRT, Vd were enhanced for ANPsG. CONCLUSION: ANPsG may be investigated for futuristic clinical studies.

Challenges in Psoriasis Research: A Systematic Review of Preclinical Models.

INTRODUCTION: Psoriasis is a chronic inflammatory skin disease with variable clinical presentation, multifactorial etiology and an immunogenetic basis. Several studies demonstrate that it results from a dysregulated interaction between skin keratinocytes, immune cells, and the environment that leads to a persistent inflammatory process modulated by cytokines and T cells. The development of new treatment options requires increased understanding of pathogenesis. However, the successful implementation of effective drugs requires well-characterized and highly available preclinical models that allow researchers to quickly and reproducibly determine their safety and efficacy. METHODS: A systematic search on PubMed and Scopus databases was performed and assessed to find appropriate articles about psoriasis models applying the key words previously defined. The PRISMA guidelines were employed. RESULTS: A total of 45 original articles were selected that met the selection criteria. Among these, there are articles on in vivo, in vitro, and ex vivo models, with the in vitro model being the majority due to its ease of use. Within animal models, the most widely used in recent years are chemically induced models using a compound known as imiquimod. However, the rest of the animal models used throughout the disease's research were also discussed. On the other hand, in vitro models were divided into two and three dimensions. The latter were the most used due to their similarity to human skin. Lastly, the ex vivo models were discussed, although they were the least used due to their difficulty in obtaining them. CONCLUSIONS: Therefore, this review summarizes the current preclinical models (in vivo, in vitro, and ex vivo), discussing how to develop them, their advantages, limitations, and applications. There are many challenges to improve the development of the different models. However, research in these in vitro model studies could reduce the use of animals. This is favored with the use of future technologies such as 3D bioprinting or organ-on-a-chip technologies.

Mechanism of Ocular Penetration of Lipophilic Drugs from Lipophilic Vehicles.

Since eyedrops have conventionally been formulated in aqueous vehicles, ocular pharmacokinetic studies are generally performed using aqueous buffers to identify physicochemical properties of the drug and the vehicles that influence drug absorption. In recent years, biocompatible lipophilic vehicles are increasingly finding application in ocular drug delivery; however, the mechanism of drug penetration from these non-aqueous vehicles is poorly understood. This study aims to compare ocular penetration of the model lipophilic drug curcumin when incorporated into lipophilic vehicles. To elucidate whether intrinsic solubility in the lipophilic vehicle influences ocular penetration, a curcumin solution and suspension were prepared in medium chain triglycerides (MCT) and squalane, respectively. Ocular penetration and distribution of curcumin from both vehicles was compared and evaluated qualitatively and quantitatively ex vivo. Significantly greater and faster penetration was observed from the squalane suspension than from the MCT solution in all ocular tissues. Our results suggest that the ability of lipophilic drugs to partition out of lipophilic vehicles and into cell membranes, rather than their intrinsic solubility in the lipophilic vehicle, determines the rate and extent of their ocular penetration.

Ex-Vivo 13C NMR Spectroscopy of Rodent Brain: TNF Restricts Neuronal Utilization of Astrocyte-Derived Metabolites.

Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly understood. Here, we microinjected TNF into the brain to study its impact on glial and neuronal metabolism (glycolysis, pentose phosphate pathway, citric acid cycle, pyruvate dehydrogenase, and pyruvate carboxylase pathways) using 13C NMR spectroscopy on brain extracts following intravenous [1,2-13C]-glucose (to probe glia and neuron metabolism), [2-13C]-acetate (probing astrocyte-specific metabolites), or [3-13C]-lactate. An increase in [4,5-13C]-glutamine and [2,3-13C]-lactate coupled with a decrease in [4,5-13C]-glutamate was observed in the [1,2-13C]-glucose-infused animals treated with TNF. As glutamine is produced from glutamate by astrocyte-specific glutamine synthetase the increase in [4,5-13C]-glutamine reflects increased production of glutamine by astrocytes. This was confirmed by infusion with astrocyte substrate [2-13C]-acetate. As lactate is metabolized in the brain to produce glutamate, the simultaneous increase in [2,3-13C]-lactate and decrease in [4,5-13C]-glutamate suggests decreased lactate utilization, which was confirmed using [3-13C]-lactate as a metabolic precursor. These results suggest that TNF rearranges the metabolic network, disrupting the energy supply chain perturbing the glutamine-glutamate shuttle between astrocytes and the neurons. These insights pave the way for developing astrocyte-targeted therapeutic strategies aimed at modulating effects of TNF to restore metabolic homeostasis in neuroinflammatory disorders.

Biodegradable Silk Fibroin Matrices for Wound Closure in a Human 3D Ex Vivo Approach.

In this study, the potential of silk fibroin biomaterials for enhancing wound healing is explored, focusing on their integration into a human 3D ex vivo wound model derived from abdominoplasties. For this purpose, cast silk fibroin membranes and electrospun nonwoven matrices from Bombyx mori silk cocoons were compared to untreated controls over 20 days. Keratinocyte behavior and wound healing were analyzed qualitatively and quantitatively by histomorphometric and immune histochemical methods (HE, Ki67, TUNEL). Findings reveal rapid keratinocyte proliferation on both silk fibroin membrane and nonwoven matrices, along with enhanced infiltration in the matrix, suggesting improved early wound closure. Silk fibroin membranes exhibited a significantly improved early regeneration, followed by nonwoven matrices (p < 0.05) compared to untreated wounds, resulting in the formation of multi-layered epidermal structures with complete regeneration. Overall, the materials demonstrated excellent biocompatibility, supporting cell activity with no signs of increased apoptosis or early degradation. These results underscore silk fibroin's potential in clinical wound care, particularly in tissue integration and re-epithelialization, offering valuable insights for advanced and-as a result of the electrospinning technique-individual wound care development. Furthermore, the use of an ex vivo wound model appears to be a viable option for pre-clinical testing.

Development of an Ex Vivo Porcine Eye Model for Exploring the Pathogenicity of Acanthamoeba.

Acanthamoeba, a widely distributed free-living amoeba found in various environments, is an opportunistic pathogen responsible for causing Acanthamoeba keratitis, a condition that may lead to blindness. However, identifying the pathogenicity of Acanthamoeba is challenging due to its complex life cycle, ability to adapt to different environments, variable virulence factors, and intricate interactions with the host immune system. Additionally, the development of an effective model for studying Acanthamoeba pathogenicity is limited, hindering a comprehensive understanding of the mechanisms underlying its virulence and host interactions. The aim of this study was to develop an ex vivo model for Acanthamoeba infection using porcine eyeballs and to evaluate the pathogenicity of the Acanthamoeba isolates. Based on slit lamp and biopsy analysis, the developed ex vivo model is capable of successfully infecting Acanthamoeba within 3 days. Histopathological staining revealed that clinical isolates of Acanthamoeba exhibited greater corneal stroma destruction and invasion in this model than environmental isolates. Our results highlight the importance of an ex vivo porcine eye model in elucidating the pathogenesis of Acanthamoeba infection and its potential implications for understanding and managing Acanthamoeba-related ocular diseases.

Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations.

Background: Mesenchymal stem cells (MSCs) from gestational tissues represent promising strategies for in utero treatment of congenital malformations, but plasticity and required high-risk surgical procedures limit their use. Here we propose natural exosomes (EXOs) isolated from amniotic fluid-MSCs (AF-MSCs), and their mimetic counterparts (MIMs), as valid, stable, and minimally invasive therapeutic alternatives. Methods: MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. Physiochemical and molecular characterization was performed to compare them to EXOs released from the same number of cells. The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake (using two different cell types, fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams. Results: Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a 3-fold greater yield. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein (GFP) in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo. Conclusions: The present data confirms the potential application of EXOs for the prenatal repair of neural tube defects and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.

Fetus Exposure to Drugs and Chemicals: A Holistic Overview on the Assessment of Their Transport and Metabolism across the Human Placental Barrier.

BACKGROUND: The placenta exerts a crucial role in fetus growth and development during gestation, protecting the fetus from maternal drugs and chemical exposure. However, diverse drugs and chemicals (xenobiotics) can penetrate the maternal placental barrier, leading to deleterious, adverse effects concerning fetus health. Moreover, placental enzymes can metabolize drugs and chemicals into more toxic compounds for the fetus. Thus, evaluating the molecular mechanisms through which drugs and chemicals transfer and undergo metabolism across the placental barrier is of vital importance. In this aspect, this comprehensive literature review aims to provide a holistic approach by critically summarizing and scrutinizing the potential molecular processes and mechanisms governing drugs and chemical transfer and metabolism across the placental barrier, which may lead to fetotoxicity effects, as well as analyzing the currently available experimental methodologies used to assess xenobiotics placental transfer and metabolism. METHODS: A comprehensive and in-depth literature review was conducted in the most accurate scientific databases such as PubMed, Scopus, and Web of Science by using relevant and effective keywords related to xenobiotic placental transfer and metabolism, retrieving 8830 published articles until 5 February 2024. After applying several strict exclusion and inclusion criteria, a final number of 148 relevant published articles were included. RESULTS: During pregnancy, several drugs and chemicals can be transferred from the mother to the fetus across the placental barrier by either passive diffusion or through placental transporters, resulting in fetus exposure and potential fetotoxicity effects. Some drugs and chemicals also appear to be metabolized across the placental barrier, leading to more toxic products for both the mother and the fetus. At present, there is increasing research development of diverse experimental methodologies to determine the potential molecular processes and mechanisms of drug and chemical placental transfer and metabolism. All the currently available methodologies have specific strengths and limitations, highlighting the strong demand to utilize an efficient combination of them to obtain reliable evidence concerning drug and chemical transfer and metabolism across the placental barrier. To derive the most consistent and safe evidence, in vitro studies, ex vivo perfusion methods, and in vivo animal and human studies can be applied together with the final aim to minimize potential fetotoxicity effects. CONCLUSIONS: Research is being increasingly carried out to obtain an accurate and safe evaluation of drug and chemical transport and metabolism across the placental barrier, applying a combination of advanced techniques to avoid potential fetotoxic effects. The improvement of the currently available techniques and the development of novel experimental protocols and methodologies are of major importance to protect both the mother and the fetus from xenobiotic exposure, as well as to minimize potential fetotoxicity effects.

Utilizing a Disposable Sensor with Polyaniline-Doped Multi-Walled Carbon Nanotubes to Enable Dopamine Detection in Ex Vivo Mouse Brain Tissue Homogenates.

Disposable sensors are inexpensive, user-friendly sensing tools designed for rapid single-point measurements of a target. Disposable sensors have become more and more essential as diagnostic tools due to the growing demand for quick, easy-to-access, and reliable information related to the target. Dopamine (DA), a prevalent catecholamine neurotransmitter in the human brain, is associated with central nervous system activities and directly promotes neuronal communication. For the sensitive and selective estimation of DA, an enzyme-free amperometric sensor based on polyaniline-doped multi-walled carbon nanotubes (PANI-MWCNTs) drop-coated disposable screen-printed carbon electrodes (SPCEs) was fabricated. This PANI-MWCNTs-2/SPCE sensor boasts exceptional accuracy and sensitivity when working directly with ex vivo mouse brain homogenates. The sensor exhibited a detection limit of 0.05 µM (S/N = 3), and a wide linear range from 1.0 to 200 µM. The sensor's high selectivity to DA amidst other endogenous interferents was recognized. Since the constructed sensor is enzyme-free yet biocompatible, it exhibited high stability in DA detection using ex vivo mouse brain homogenates extracted from both Parkinson's disease and control mice models. This research thus presents new insights into understanding DA release dynamics at the tissue level in both of these models.

Human Skin as an Ex Vivo Model for Maintaining Mycobacterium leprae and Leprosy Studies.

The in vitro cultivation of M. leprae has not been possible since it was described as causing leprosy, and the limitation of animal models for clinical aspects makes studies on leprosy and bacteria-human host interaction a challenge. Our aim was to standardize the ex vivo skin model (hOSEC) to maintenance and study of M. leprae as an alternative animal model. Bacillary suspensions were inoculated into human skin explants and sustained in DMEM medium for 60 days. Explants were evaluated by RT-PCR-16SrRNA and cytokine gene expression. The viability and infectivity of bacilli recovered from explants (D28 and D60) were evaluated using the Shepard's model. All explants were RT-PCR-16SrRNA positive. The viability and infectivity of recovered bacilli from explants, analyzed after 5 months of inoculation in mice, showed an average positivity of 31%, with the highest positivity in the D28 groups (80%). Furthermore, our work showed different patterns in cytokine gene expression (TGF-ß, IL-10, IL-8, and TNF-α) in the presence of alive or dead bacilli. Although changes can be made to improve future experiments, our results have demonstrated that it is possible to use the hOSEC to maintain M. leprae for 60 days, interacting with the host system, an important step in the development of experimental models for studies on the biology of the bacillus, its interactions, and drug susceptibility.

Preclinical techniques for drug discovery in psoriasis.

Psoriasis is a chronic, inflammatory, papulosquamous, noncontagious disease characterized by scaly, demarcated erythematous plaque, affecting skin, nails, and scalp. The IL-23/Th17 axis is the main operator in the development of psoriasis. Psoriasis is affecting worldwide, and new treatment options are urgently needed. Various local and systemic treatments are available for psoriasis but they only provide symptomatic relief because of numerous unknown mechanisms. Clinical trials demand overwhelming resources; therefore, drug development predominantly depends on the in-vivo, in-vitro, and ex-vivo techniques. Immediate attention is required to develop experimental techniques that completely imitate human psoriasis to assist drug development. This review portrays the various in-vivo, in-vitro, and ex-vivo techniques used in psoriasis research. It describes these techniques' characteristics, pathological presentations, and mechanisms. The experimental techniques of psoriasis provide significant information on disease progression mechanisms and possible therapeutic targets. However, until now, it has been challenging to invent a timely, affordable model that precisely imitates a human disease. Only the xenotransplantation model is reckoned as the closer, that mimics the complete genetic, and immunopathogenic event. Imiquimod-induced psoriasis and HaCat cell lines are popular among researchers because of their convenience, ease of use, and cost-effectiveness. There need to further improve the experimental techniques to best serve the disease imitation and meet the research goal.