Exploring the potential of laser-textured metal alloys: Fine-tuning vascular cells responses through in vitro and ex vivo analysis.

Medical stents are vital for treating vascular complications and restoring blood flow in millions of patients. Despite its widespread effectiveness, restenosis, driven by the complex interplay of cellular responses, remains a concern. This study investigated the reactions of vascular cells to nano/microscale wrinkle (nano-W and micro-W) patterns created on laser-textured nitinol (NiTi) surfaces by adjusting laser processing parameters, such as spot overlap ratio and line overlap ratio. Evaluation of topographical effects on endothelial and smooth muscle cells (SMCs) revealed diverse morphologies, proliferation rates, and gene expressions. Notably, microscale wrinkle patterns exhibited reduced monocyte adhesion and inflammation-related gene expression, demonstrating their potential applications in mitigating vascular complications after stent insertion. Additionally, an ex vivo metatarsal assay was utilized to bridge the gap between in vitro and in vivo studies, demonstrating enhanced angiogenesis on laser-textured NiTi surfaces. Laser-textured NiTi exhibits a guided formation process, emphasizing their potential to promote swift endothelialization. These findings underscore the efficacy of laser texturing for tailored cellular interactions on metallic surfaces and offer valuable insights into optimizing biocompatibility and controlling cellular responses, which may pave the way for innovative advances in vascular care and contribute to the ongoing improvement of stent insertion.

Calcium Imaging in Drosophila.

Ex vivo calcium imaging in Drosophila opens an expansive amount of research avenues for the study of live signal propagation through complex tissue. Here, we describe how to isolate Drosophila organs of interest, like the developing wing imaginal disc and larval brain, culture them for extended periods, up to 10 h, and how to image the calcium dynamics occurring within them using genetically encoded biosensors like GCaMP. This protocol enables the study of complex calcium signaling dynamics, which is conserved throughout biology in such processes as cell differentiation and proliferation, immune reactions, wound healing, and cell-to-cell and organ-to-organ communication, among others. These methods also allow pharmacological compounds to be tested to observe effects on calcium dynamics with the applications of target identification and therapeutic development.

A Tensioned Human Skin Explant Model Used for Preliminary Assessment of Chemexfoliant-Stimulated Bioeffects.

A tensioned ex vivo full-thickness human skin explant platform was used to assess the bioeffects arising from application of several commercial chemexfoliation agents. Although such treatments are well-established, and improved understanding of the underlying mechanistic processes continues to emerge, research into the optimum treatments for specific skin types/conditions is still needed for enhanced efficacy while minimizing recovery time. The 3 commercial chemexfoliation agents employed all contained trichloroacetic acid at well-defined concentrations (6, 10, and 20%) and were applied to the explants' stratum corneum. Subsequently, measurements of dermal remodeling factors (COL1A1, ELN, HAS2, HAS3, and procollagen type I) and inflammatory marker (IL-1b) were undertaken using qPCR and immunofluorescent analyses. Statistical analysis of these data facilitated the establishment of benchmarking biological responses to these trichloroacetic acid-containing agents against untreated controls. The performance of an innovative trichloroacetic acid-free chemexfoliation agent was then measured and, upon comparison with the previous benchmarking data, indicated that dermal remodeling factors could be upregulated in fashion comparable with that of the trichloroacetic acid-containing agents but with significant suppression of inflammatory response. Our measurements thus underscore the promise of the tensioned explant over prolonged study periods and also that potentially valuable insights to guide preclinical strategies may be forthcoming from the protocol developed.

Retinal Explant Culture from Mouse, Human, and Nonhuman Primates and Its Applications in Vision Research.

The retinal explant culture system is a valuable tool for studying the pharmacological, toxicological, and developmental aspects of the retina. It is also used for translational studies such as gene therapy. While no photoreceptor-like cell lines are available for in vitro studies of photoreceptor cell biology, the retinal explant culture maintains the laminated retinal structure ex vivo for as long as a month. Human and nonhuman primate (NHP) postmortem retinal explants cut into small pieces offer the possibility of testing multiple conditions for safety and adeno-associated viral (AAV) vector optimization. In addition, the cone-enriched foveal area can be studied using the retinal explants. Here, we present a detailed working protocol for retinal explant isolation and culture from mouse, human, and NHP for testing drug efficacy and AAV transduction. Future applications of this protocol include combining live imaging and multiwell retinal explant culture for high-throughput drug screening systems in rodent and human retinal explants to identify new drugs against retinal degeneration.

Automated manufacturing and characterization of clinical grade autologous CD20 CAR T cells for the treatment of patients with stage III/IV melanoma.

Introduction: Point-of-care (POC) manufacturing of chimeric antigen receptor (CAR) modified T cell has expanded rapidly over the last decade. In addition to the use of CD19 CAR T cells for hematological diseases, there is a growing interest in targeting a variety of tumor-associated epitopes. Methods: Here, we report the manufacturing and characterization of autologous anti-CD20 CAR T cells from melanoma patients within phase I clinical trial (NCT03893019). Using a second-generation lentiviral vector for the production of the CD20 CAR T cells on the CliniMACS Prodigy®. Results: We demonstrated consistency in cell composition and functionality of the products manufactured at two different production sites. The T cell purity was >98.5%, a CD4/CD8 ratio between 2.5 and 5.5 and transduction rate between 34% and 61% on day 12 (harvest). Median expansion rate was 53-fold (range, 42-65-fold) with 1.7-3.8×109 CAR T cells at harvest, a sufficient number for the planned dose escalation steps (1×105/kg, 1×106/kg, 1×107/kg BW). Complementary research of some of the products pointed out that the CAR+ cells expressed mainly central memory T-cell phenotype. All tested CAR T cell products were capable to translate into T cell activation upon engagement of CAR target cells, indicated by the increase in pro-inflammatory cytokine release and by the increase in CAR T cell amplification. Notably, there were some interindividual, cell-intrinsic differences at the level of cytokine release and amplification. CAR-mediated T cell activation depended on the level of CAR cognate antigen. Discussion: In conclusion, the CliniMACS Prodigy® platform is well suited for decentralized POC manufacturing of anti-CD20 CAR T cells and may be likewise applicable for the rapid and automated manufacturing of CAR T cells directed against other targets. Clinical trial registration: https://clinicaltrials.gov/study/NCT03893019?cond=Melanoma&term=NCT03893019&rank=1, identifier NCT03893019.

Outcomes of Donation After Circulatory Death (DCD) and Ex-Vivo Lung Perfusion (EVLP) Lung Transplantation.

BACKGROUND: Donation after circulatory death (DCD) and ex-vivo lung perfusion (EVLP) have been adopted to expand the donor pool in lung transplantation, but outcomes data have been conflicting. This study explores mid-term outcomes of DCD lung transplantation in the modern era, with a focus on EVLP and risk factors for graft failure. METHODS: The United Network for Organ Sharing (UNOS) database was queried for adult lung transplants from 1/1/2015 to 3/1/2023. Loss to follow-up, multiorgan and prior lung transplants were excluded. DCD vs DBD (donation after brain death) lung transplants were compared, with subgroup analysis +/- EVLP. Outcomes were survival and postoperative complications. Overall survival was analyzed separately for an early era (2015-2018) and modern era (2019-2023). RESULTS: The study included 1103 DCD (221 with EVLP, and 882 without) and 17973 donation after brain death (DBD) lung transplants (524 with EVLP, and 17449 without). Median follow-up was 3 years. DCD donors were less likely to be CDC high risk (19.3% vs 24.1%, p<0.001), have purulence on bronchoscopy (13.3% vs 18.3%, p<0.001) or infiltrates on chest x-ray (66.7% vs 67.8%, p=0.013). EVLP was more likely to be used for DCD transplants (20.0% vs 2.9%, p<0.001). After transplant, DCD recipients were more likely to be reintubated (24.3% vs 18.5%, p<0.001) and require ECMO within 72 hours (14.9% vs 7.8%, p<0.001), and DCD donation was an independent risk factor for these complications on multivariable logistic regression. Overall survival did not differ significantly between DCD and DBD transplants on adjusted survival analysis in the early or modern era (p=0.774 and p=0.468 respectively). On multivariable Cox regression, DCD and EVLP were not independent risk factors for mortality. On subgroup analysis, the DCD+EVLP cohort had significantly worse survival in the modern era, which remained significant after adjusting for donor and recipient factors (p=0.005). EVLP was an independent risk factor for graft failure in the DCD cohort (HR 1.33, 95% CI 1.00-1.77, p=0.047), but did not significantly affect DBD graft survival (p=0.870). Risk factors for graft failure and mortality in the DCD+EVLP cohort included pulmonary hypertension (HR 77.5, 95% CI 6.15-979, p<0.001), transfusion prior to transplant (HR 2.60, 95% CI 1.07-6.31, p=0.035), elevated creatinine (HR 2.82, 95% CI 1.34-5.90, p=0.006), and higher allocation score (HR 1.02, 95% CI 1.00-1.04, p=0.017) CONCLUSION: Study findings suggest increased risks of mortality and perioperative complications following transplantation with DCD lungs that have undergone EVLP. DCD lung transplantation without EVLP confers equivalent survival but with some increase in perioperative complications. Further investigation and careful recipient selection is warranted to optimize the use of these extended criteria donors in the modern era.

Tumor slice culture system for ex vivo immunotherapy studies.

Personalized immunotherapy is emerging as a promising approach for cancer treatment, aiming to harness the patient's own immune system to target and eliminate tumor cells. One key aspect of developing effective personalized immunotherapies is the utilization of tumor slices derived from individual patient tumors. Tumor slice models retain the complexity and heterogeneity of the original tumor microenvironment, including interactions with immune cells, stromal elements, and vasculature. These ex vivo models serve as valuable tools for studying tumor-immune interactions and for testing the efficacy of immunotherapeutic agents tailored to the specific characteristics of each patient's tumor. In this chapter, we set up a protocol for immunotherapy strategies in mouse models highlighting their translational potential to guide treatment decisions and improve therapeutic outcomes in cancer patients.

Development of an ex vivo model to study Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network.

Staphylococcus aureus has multiple mechanisms to evade the host's immune system and antibiotic treatment. One such mechanism is the invasion of the osteocyte lacuno-canalicular network (OLCN), which may be particularly important in recurrence of infection after debridement and antibiotic therapy. The aim of this study was to develop an ex vivo model to facilitate further study of S. aureus invasion of the OLCN and early-stage testing of antibacterial strategies against bacteria in this niche. The diameter of the canaliculi of non-infected human, sheep, and mouse bones was measured microscopically on Schmorl's picrothionin stained sections, showing a large overlap in canalicular diameter. S. aureus successfully invaded the OLCN in all species in vitro as revealed by presence in osteocyte lacunae in Brown and Brenn-stained sections and by scanning electron microscopy. Murine bones were then selected for further experiments, and titanium pins with either a wild-type or ΔPBP4 mutant S. aureus USA300 were placed trans-cortically and incubated for 2 weeks in tryptic soy broth. Wild-type S. aureus readily invaded the osteocyte lacunae in mouse bones while the ΔPBP4 showed a significantly lower invasion of the OLCN (p = 0.0005). Bone specimens were then treated with gentamicin, sitafloxacin, R14 bacteriophages, or left untreated. Gentamicin (p = 0.0027) and sitafloxacin (p = 0.0280) significantly reduced the proportion of S. aureus-occupied lacunae, whilst bacteriophage treatment had no effect. This study shows that S. aureus is able to invade the OLCN in an ex vivo model. This ex vivo model can be used for future early-stage studies before proceeding to in vivo studies.

Trivalent arsenicals induce skin toxicity through thiol depletion.

Arsenic, a widespread environmental contaminant, is highly toxic to human health. Arsenic exposure is associated with the occurrence of skin lesions and diseases. This study investigated the dermal toxicity of trivalent arsenicals (AsIII and MMAIII) and its underlying mechanism using human keratinocyte cell line and ex vivo porcine skin. AsIII and MMAIII induced concentration-dependent cell apoptosis and necrosis in HaCaT cells, which was confirmed in ex vivo porcine skin. AsIII and MMAIII increased reactive oxygen species generation and GSH depletion. Interestingly, radical scavenger antioxidants such as Vitamin C failed to mitigate arsenic-induced cytotoxicity, while thiol-containing compounds effectively alleviated it, suggesting a key role of thiol depletion in the trivalent arsenical-induced dermal toxicity. DMSA showed the strongest protective effects against AsIII and MMAIII-induced cytotoxicity in HaCaT cells. Of note, DMSA restored arsenical-induced tissue damage, and reduced the apoptosis in ex vivo porcine skin, highlighting its potential use to alleviate arsenic-induced skin lesions and diseases.

Myelin water imaging of in vivo and ex vivo human brains using multi-echo gradient echo at 3 T and 7 T.

PURPOSE: To compare the myelin water fraction (MWF) measurements between 3 T and 7 T and between in vivo and ex vivo human brains, and to investigate the relationship between multi-echo gradient-echo (mGRE)-based 3D MWF and myelin content using histological staining, which has not been validated in the human brain. METHODS: In this study, we performed 3D mGRE-based MWF measurements on five ex vivo human brain hemispheres and five healthy volunteers at 3 T and 7 T with 1 mm isotropic resolution. The data were fitted with the T 2 * $$ {\mathrm{T}}_2^{\ast } $$ based on a three compartment complex-valued model to estimate MWF. We obtained myelin basic protein (MBP) staining from two tissue blocks and co-registered the MWF map and histology image for voxel-wise correlation between the two. RESULTS: The MWF values measured from 7 T were overall higher than 7 T, but data between the two field strength demonstrated high correlations both in vivo (r = 0.88) and ex vivo (r = 0.83) across 19 white matter regions. Moreover, the MWF measurements showed a good agreement between in vivo and ex vivo assessments at 3 T (r = 0.61) and 7 T (r = 0.54). Based on MBP staining, the MWF values exhibited strong positive correlations with myelin content on both 3 T (r = 0.68 and r = 0.78 for the two tissue blocks) and 7 T (r = 0.64 and r = 0.82 for the two tissue blocks). CONCLUSION: The findings demonstrated that the mGRE-based MWF mapping can be used to quantify myelin content in the human brain, despite the field-strength dependency of the measurements.

Tumoral Pharmacokinetic-Pharmacodynamic Simulation of Doxorubicin-Encapsulated Polymeric Micelles Using a Tissue-Isolated Tumor Perfusion System.

Pharmacokinetic (PK) elucidation of polymeric micelles delivering anticancer drugs is crucial for accurate antitumor PK-pharmacodynamic (PK-PD) simulations. Particularly, establishing a methodology to quantify the tumor inflow and outflow of anticancer drugs encapsulated in polymeric micelles is an essential challenge. General tumor biodistribution experiments are disadvantageous in that inflow quantification is easy, but outflow quantification is challenging. We addressed this issue by proposing a quantification method that combines a tissue-isolated tumor perfusion system with microdialysis. This method aims to determine tumoral drug inflow and outflow by quantifying the drugs released from the polymeric micelles via a tumor-embedded microdialysis probe and perfusate, respectively. Furthermore, we evaluated the feasibility of this method by perfusing pH-sensitive polyethylene glycol-poly(aspartate-hydrazone-doxorubicin/phenylalanine)n (PPDF-Hyd-DOX) in a tissue-isolated tumor perfusion system, and we quantified tumor inflow and outflow released DOX. Based on the quantitative results, we performed compartmental analyses by incorporating the gamma-distributed delay function and calculated the PK rate constants. These parameters were input into a tumor-bearing rat compartment model for ex vivo-in vivo extrapolation (EVIVE) of the rat plasma PPDF-Hyd-DOX concentrations and simulated intratumorally released DOX concentrations. The simulation profiles demonstrated a good fit with the Walker 256 intratumoral released DOX concentration profiles previously reported. This EVIVE-PK model was coupled with the threshold natural-growth tumor PD model, and PK-PD analysis was performed. This model exhibited a better fit to the tumor weight profile of Walker 256-bearing rats treated with PPDF-Hyd-DOX than that of our previously reported PK-PD model. Thus, EVIVE, based on a tissue-isolated tumor perfusion system with microdialysis, is a promising approach for the PK-PD simulation of polymeric micelle anticancer therapy.

Evaluating the suitability of placental bovine explants for ex vivo modelling of host-pathogen interactions in Neospora caninum infections.

Bovine abortions, often caused by infectious agents like Neospora caninum, inflict substantial economic losses. Studying host-pathogen interactions in pregnant cows is challenging, and existing cell cultures lack the intricate complexity of real tissues. To bridge the gap between in vitro and in vivo models, we explored the use of cryopreserved bovine placental explants. Building upon our successful development of protocols for obtaining, culturing, and cryopreserving sheep placental explants, we applied these methods to bovine tissues. Here, we compared fresh and cryopreserved bovine explants, evaluating their integrity and functionality over culture time. Additionally, we investigated their susceptibility to N. caninum infection. Our findings revealed that bovine explants deteriorate faster in culture compared to sheep explants, exhibiting diminished viability and function. Cryopreservation further exacerbated this deterioration. While fresh explants were successfully infected with N. caninum, parasite replication was limited. Notably, cryopreservation reduced infection efficiency. This pioneering work paves the way for developing ex vivo models to study reproductive pathogens in cattle. However, further optimization of the model is essential. These improved models will have the potential to significantly reduce the reliance on animals in research.

A human ex vivo skin model breaking boundaries.

Ex vivo human skin models are valuable tools in skin research due to their physiological relevance. Traditionally, standard cultivation is performed in a cell culture incubator with a defined temperature of 37 °C and a specific atmosphere enriched with CO2 to ensure media stability. Maintaining the model under these specific conditions limits its flexibility in assessing exposures to which the skin is exposed to in daily life, for example changes in atmospheric compositions. In this study we demonstrated that the foreskin-derived skin model can be successfully cultured at room temperature outside a CO2 incubator using a CO2-independent, serum-free media. Over a cultivation period of three days, the integrity of the tissue and the preservation of immune cells is well maintained, indicating the model's stability and resilience under the given conditions. Exposing our Medical University of Graz - human Organotypic Skin Explant Culture (MUG-hOSEC) model to cytotoxic and inflammatory stimuli results in responses analyzable within the supernatant. Besides the common analysis of released proteins upon treatment, such as cytokines and enzymes, we have included extracellular vesicle to obtain a more comprehensive picture of cell communication.

AURKA inhibition shows promise as a therapeutic strategy for ARID1A-mutant colorectal cancer.

PURPOSE: Mutations in ARID1A frequently occur in colorectal cancer (CRC) cells. However, there are currently no clinical treatment options specifically addressing this aberration. The preliminary in vitro experiments revealed a synthetic lethal interaction between ARID1A and Aurora kinase A (AURKA) in colorectal cancer (CRC) cells. METHODS: We collected samples from 80 CRC patients and evaluated the efficacy of AURKA inhibitor (AURKAi) using the ATP-tumor chemosensitivity assay (ATP-TCA) on untreated ARID1A-proficient (ARID1A +) and ARID1A-deficient (ARID1A-) CRC patient samples. In addition, we validated this result by a clonogenic assay. Additionally, we examined the effects of AURKA inhibitors on cell cycle progression and apoptosis in ARID1A + and ARID1A- CRC patient samples using flow cytometry. RESULTS: The results showed that AURKAi selectively inhibited the growth of ARID1A- CRC cells. Furthermore, AURKA inhibitors significantly increased G2/M arrest and induced apoptosis in ARID1A- cells. CONCLUSION: We believe that AURKAi hold promise as potential therapeutics for ARID1A mutation colorectal cancer patients.

Optimization, Formulation, and Ex vivo Evaluation of Solid Lipid Nanoparticles for Transdermal Delivery of Diltiazem Hydrochloride.

BACKGROUND: Cardiac arrhythmia, is a medical condition that reduces the heart's efficiency in pumping blood, and can be fatal, requires long-term management with conventional drugs, despite their limited efficacy. Diltiazem hydrochloride, chosen as a model drug, has a short biological half-life and extensive metabolism. Administering drug through skin is challenging, particularly due to the penetration via stratum corneum. However, solid lipid nanoparticles as a particulate carrier system can enhance its permeation and bioavailability. OBJECTIVE: The study aimed to develop a matrix type transdermal patch with diltiazem hydrochloride encapsulated in solid lipid nanoparticles Methods: The study used the solvent diffusion technique to prepare SLNs by mixing the drug and solid lipid in an organic phase at 80°C, then slowly adding it to an aqueous phase with continuous stirring for 45 minutes. The resulting nanodispersion was freeze-dried and analyzed for morphological studies, encapsulation efficiency & drug content. A patch was formulated using solvent evaporation technique, incorporating HPMC E50 (2% w/v), propylene glycol, and ethanolic oleic acid (1.5% v/v). SLNs loaded with diltiazem hydrochloride taken equivalent to diltiazem hydrochloride dose in the transdermal patch. The patch was then evaluated for In vitro and skin permeation studies. RESULTS: The result showed a positive correlation between lipid concentration and particle size. Probe sonication and homogenization increased particle size, while stirring speed reduced it. SEM and TEM images confirmed spherical particles with a size of 488.1±4.01nm and an entrapment efficiency of 55.03±1.99%. Drug release studies demonstrated 70.7% drug release from lipid matrix over 24 hrs. The formulated patch with uniform SLN distribution, had a drug content 89.37 ± 0.04% with a surface pH of 6.1 ± 0.53, close to skin pH. The uniformity of content in 3x3 patch estimated to be 14.587 ± 1.404 mg, close to the theoretical content 16.318 ± 1.08 mg, confirmed homogenous distribution of diltiazem hydrochloride SLNs throughout the patch diameter. Cumulative amount released from patch formulation at pH 5.6 and pH 7.4 was 518.1414µg/cm2 and 404.4466 µg/cm2. Synergistic flux enhancement was observed with oleic acid propylene glycol blend. Ex vivo study of the patch showed steady-state flux of 6.9 µg/cm2/hr, permeability coefficient 0.00362 cm/hr, diffusion coefficient 0.000103 cm/hr, cumulative drug permeation (Dmax) 814.885 µg after 24 hrs, and followed a Higuchi-matrix release model. CONCLUSION: The developed patch possessed improved bioavailability with reduced dosing and enhanced patient compliance.

Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue.

Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic and adult skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of skin, including nucleated/enucleated epithelium, mesenchyme, and hair follicle, as well as the role of collagens in maintaining its integrity. Furthermore, quantitative analysis comparing stiffness distributions in live tissue samples with those in preserved tissues, including formalin-fixed and cryopreserved tissue samples, unveiled the distinct impacts of preservation processes on tissue stiffness patterns. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples to accurately capture the true spatiotemporal variations in mechanical properties. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology. STATEMENT OF SIGNIFICANCE: Stiffness, a simple mechanical parameter, has drawn attention in understanding the mechanobiological principles underlying the homeostasis and pathology of living tissues. To explore tissue-scale mechanobiology, we propose a technique integrating an upright structured illumination microscope and an atomic force microscope. This technique enables live measurements of stiffness distribution and fluorescent observation of thick living tissue slices. Experiments revealed the highly heterogeneous mechanical nature of mouse embryonic and adult skin in three dimensions and the previously unnoticed influences of preservation techniques on the mechanical properties of tissue at microscopic resolution. This study provides a new technical platform for live stiffness measurement and biomolecular observation of tissue-scale samples with micron-scale resolution, thus contributing to future studies of tissue- and organ-scale mechanobiology.

A New Frontier in Cystic Fibrosis Pathophysiology: How and When Clock Genes Can Affect the Inflammatory/Immune Response in a Genetic Disease Model.

Cystic fibrosis (CF) is a monogenic syndrome caused by variants in the CF Transmembrane Conductance Regulator (CFTR) gene, affecting various organ and systems, in particular the lung, pancreas, sweat glands, liver, gastrointestinal tract, vas deferens, and vascular system. While for some organs, e.g., the pancreas, a strict genotype-phenotype occurs, others, such as the lung, display a different pathophysiologic outcome in the presence of the same mutational asset, arguing for genetic and environmental modifiers influencing severity and clinical trajectory. CFTR variants trigger a pathophysiological cascade of events responsible for chronic inflammatory responses, many aspects of which, especially related to immunity, are not ascertained yet. Although clock genes expression and function are known modulators of the innate and adaptive immunity, their involvement in CF has been only observed in relation to sleep abnormalities. The aim of this review is to present current evidence on the clock genes role in immune-inflammatory responses at the lung level. While information on this topic is known in other chronic airway diseases (chronic obstructive pulmonary disease and asthma), CF lung disease (CFLD) is lacking in this knowledge. We will present the bidirectional effect between clock genes and inflammatory factors that could possibly be implicated in the CFLD. It must be stressed that besides sleep disturbance and its mechanisms, there are not studies directly addressing the exact nature of clock genes' involvement in inflammation and immunity in CF, pointing out the directions of new and deepened studies in this monogenic affection. Importantly, clock genes have been found to be druggable by means of genetic tools or pharmacological agents, and this could have therapeutic implications in CFLD.

Development of Glycerosomal pH Triggered In Situ Gelling System to Ameliorate the Nasal Delivery of Sulpiride for Pediatric Psychosis.

Sulpiride (Sul) is a medication that blocks dopamine D2 receptors. It is used to treat gastrointestinal disturbances and has antipsychotic effects depending on the dose given. Sulpiride is subject to P-glycoprotein efflux, resulting in limited bioavailability and erratic absorption. Hence, the aim of this study was to generate a glycerosomal in situ gel of sulpiride for intranasal administration, specifically targeting children with schizophrenia who may have difficulty swallowing traditional solid medications, for enhancing its bioavailability. This study aimed to demonstrate the efficacy of intranasal administration of glycerin-encapsulated lipid-nanovesicles (glycerosomes) mixed with in situ gels for prolonged release of anti-psychotic medication. A Box-Behnken design was utilized to create sulpiride-loaded glycerosomes (Sul-GMs), with the lipid amount (A), glycerin concentration (B), and sonication time (C) acting as independent variables. Their impact on the entrapment efficiency, EE% (Y1), and in vitro drug release (Y2) were evaluated. The sulpiride EE% showed an increase when the glycerin concentration was raised to 25% v/v. Nevertheless, when the glycerin concentration was raised to 40% v/v, there was a notable decrease in the EE%. The optimized glycerosome was added to pH triggered carbopol 974P in situ gel formulations including HPMC K15M with different concentrations. The in situ gel formulation (G3) comprising 0.6% carbopol 974P and 0.6% hydroxypropyl methyl cellulose-K15M (HPMC K15M) demonstrated suitable pH, viscosity, desired gel strength, spreadability, and mucoadhesive strength. Consequently, it was selected for in vitro study, ex vivo permeation investigation, and in vivo evaluations. The glycerosomal in situ gel exhibited favorable ex vivo permeability of SU when applied to the nasal mucosa. The pharmacokinetic investigation revealed that the optimized Sul-loaded glycerosomal in situ gel exhibited a significant fourfold and twofold enhancement in systemic bioavailability compared to both the control gel and the commercially available formulation. Finally, the intranasal administration of Sul-loaded glycerosomal in situ gel is a promising alternative to oral treatment for pediatric patients with psychosis.

Staurosporine as a Potential Treatment for Acanthamoeba Keratitis Using Mouse Cornea as an Ex Vivo Model.

Acanthamoeba is a ubiquitous genus of amoebae that can trigger a severe and progressive ocular disease known as Acanthamoeba Keratitis (AK). Furthermore, current treatment protocols are based on the combination of different compounds that are not fully effective. Therefore, an urgent need to find new compounds to treat Acanthamoeba infections is clear. In the present study, we evaluated staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and a comparative proteomic analysis was conducted to elucidate a mechanism of action. The obtained results indicate that staurosporine altered the conformation of actin and tubulin in treated trophozoites of A. castellanii. In addition, proteomic analysis of treated trophozoites revealed that this molecule induced overexpression and a downregulation of proteins related to key functions for Acanthamoeba infection pathways. Additionally, the ex vivo assay used validated this model for the study of the pathogenesis and therapies of AK. Finally, staurosporine eliminated the entire amoebic population and prevented the adhesion and infection of amoebae to the epithelium of treated mouse corneas.

Ex Vivo Simultaneous H215O Positron Emission Tomography and Magnetic Resonance Imaging of Porcine Kidneys-A Feasibility Study.

The aim was to establish combined H215O PET/MRI during ex vivo normothermic machine perfusion (NMP) of isolated porcine kidneys. We examined whether changes in renal arterial blood flow (RABF) are accompanied by changes of a similar magnitude in renal blood perfusion (RBP) as well as the relation between RBP and renal parenchymal oxygenation (RPO). METHODS: Pig kidneys (n = 7) were connected to a NMP circuit. PET/MRI was performed at two different pump flow levels: a blood-oxygenation-level-dependent (BOLD) MRI sequence performed simultaneously with a H215O PET sequence for determination of RBP. RESULTS: RBP was measured using H215O PET in all kidneys (flow 1: 0.42-0.76 mL/min/g, flow 2: 0.7-1.6 mL/min/g). We found a linear correlation between changes in delivered blood flow from the perfusion pump and changes in the measured RBP using PET imaging (r2 = 0.87). CONCLUSION: Our study demonstrated the feasibility of combined H215O PET/MRI during NMP of isolated porcine kidneys with tissue oxygenation being stable over time. The introduction of H215O PET/MRI in nephrological research could be highly relevant for future pre-transplant kidney evaluation and as a tool for studying renal physiology in healthy and diseased kidneys.