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.

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.

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.

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.

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.

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.

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.

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.

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.

Endocytosis as a critical regulator of hematopoietic stem cell fate -implications for hematopoietic stem cell and gene therapy.

Hematopoietic stem cells (HSCs) have emerged as one of the most therapeutically significant adult stem cells, paving way for a range of novel curative regimens over decades. HSCs are transplanted, either directly or post restorative genetic engineering in order to repopulate a healthy hematopoietic homeostasis in patients with disorders affecting the blood and immune cells. Despite being an extensively studied system, the maintenance and expansion of functional HSCs ex vivo remains a major bottleneck. The challenge primarily stems from difficulties in reproducing HSC self-renewal divisions and gradual depletion of stemness characters, in vitro. Refining the in vitro culture can be particularly beneficial in the case of cord blood HSCs (CB-HSCs), as inadequate numbers in a single umbilical cord limits its therapeutic potential. In recent years, molecular dissection of HSC stemness has significantly improved in vitro hematopoietic stem and progenitor cells (HSPCs) culture. Despite such significant progress, lacunae exist in fully understanding all the underlying mechanisms and their interplay active in bona fide HSCs, and how it transforms when cells proliferate in culture. A new groundbreaking study titled "MYCT1 controls environmental sensing in human haematopoietic stem cells", published in Nature in June 2024, sheds light on this complex field. Through a series of experiments, including knock-down, overexpression, single-cell RNA sequencing, and transplantation, the study identifies a previously unknown role of the MYC target 1 (MYCT1) protein in HSC maintenance. This protein acts as a crucial regulator of human HSCs, with high expression in primitive HSCs and subsequently downregulated during ex vivo culture. The study reveals that MYCT1 plays a vital role in moderating endocytosis and environmental sensing in HSCs, processes thereby essential for maintaining HSC stemness and function. This commentary will discuss the implications of the new findings for cord blood expansion in cell therapies and HSPC culture for gene therapy applications, providing valuable insights for the field of hematopoietic regenerative medicine.

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.

The Fibrotic Phenotype of Human Precision-Cut Lung Slices Is Maintained after Cryopreservation.

Human precision-cut lung slices (hPCLS) prepared from fibrotic lungs recapitulate the pathophysiological hallmarks of fibrosis. These hallmark features can also be induced by treating non-fibrotic hPCLS with a fibrotic cocktail (FC). As a result, the fibrotic and fibrosis-induced hPCLS are rapidly emerging as preferred models for disease modeling and drug discovery. However, current hPCLS models are limited by tissue viability in culture, as they are usually only viable for one week after harvesting. Here, we demonstrate that the fibrotic hPCLS can be cryopreserved, stored for months, and then thawed on demand without loss of hPCLS viability or protein content for 14 days post-thawing. Cryopreservation also preserves the pro-fibrotic potential of non-fibrotic hPCLS. Specifically, when we treated the thawed non-fibrotic hPCLS with an FC, we observed significant pro-fibrotic cytokine secretion and elevated tissue stiffness. These pro-fibrotic changes were inhibited by the small-molecule tyrosine kinase inhibitor, Nintedanib. Taken together, our work indicates that a feasible solution to prolong the pre-clinical utility of fibrotic and fibrosis-induced hPCLS is cryopreservation. We anticipate that cryopreserved hPCLS will serve as an advantageous predictive model for the evaluation of pro-fibrotic pathways during acute and chronic toxicity testing.

Low-Volume Ex Situ Lung Perfusion System for Single Lung Application in a Small Animal Model Enables Optimal Compliance With "Reduction" in 3R Principles of Animal Research.

Ex situ lung perfusion (ESLP) is used for organ reconditioning, repair, and re-evaluation prior to transplantation. Since valid preclinical animal models are required for translationally relevant studies, we developed a 17 mL low-volume ESLP for double- and single-lung application that enables cost-effective optimal compliance "reduction" of the 3R principles of animal research. In single-lung mode, ten Fischer344 and Lewis rat lungs were subjected to ESLP and static cold storage using STEEN or PerfadexPlus. Key perfusion parameters, thermal lung imaging, blood gas analysis (BGA), colloid oncotic pressure (COP), lung weight gain, histological work-up, and cytokine analysis were performed. Significant differences between perfusion solutions but not between the rat strains were detected. Most relevant perfusion parameters confirmed valid ESLP with homogeneous lung perfusion, evidenced by uniform lung surface temperature. BGA showed temperature-dependent metabolic activities with differences depending on perfusion solution composition. COP is not decisive for pulmonary oedema and associated weight gain, but possibly rather observed chemokine profile and dextran sensitivity of rats. Histological examination confirmed intact lung architecture without infarcts or hemorrhages due to optimal organ procurement and single-lung application protocol using our in-house-designed ESLP system.

Pushing the Survival Bar Higher: Two Decades of Innovation in Lung Transplantation.

Survival after lung transplantation has significantly improved during the last two decades. The refinement of the already existing extracorporeal life support (ECLS) systems, such as extracorporeal membrane oxygenation (ECMO), and the introduction of new techniques for donor lung optimization, such as ex vivo lung perfusion (EVLP), have allowed the extension of transplant indication to patients with end-stage lung failure after acute respiratory distress syndrome (ARDS) and the expansion of the donor organ pool, due to the better evaluation and optimization of extended-criteria donor (ECD) lungs and of donors after circulatory death (DCD). The close monitoring of anti-HLA donor-specific antibodies (DSAs) has allowed the early recognition of pulmonary antibody-mediated rejection (AMR), which requires a completely different treatment and has a worse prognosis than acute cellular rejection (ACR). As such, the standardization of patient selection and post-transplant management has significantly contributed to this positive trend, especially at high-volume centers. This review focuses on lung transplantation after ARDS, on the role of EVLP in lung donor expansion, on ECMO as a principal cardiopulmonary support system in lung transplantation, and on the diagnosis and therapy of pulmonary AMR.

In Vitro Study of Cyano-Phycocyanin Release from Hydrogels and Ex Vivo Study of Skin Penetration.

BACKGROUND: This study explored the most suitable materials for incorporating cyano-phycocyanin (C-PC) into hydrogels, focusing on maintaining the C-PC's long-term structural integrity and stabilityNext, the release of C-PC from the hydrogels and its skin penetration were investigated. METHODS: A series of 1% (w/w) C-PC hydrogels was prepared using various gelling agents and preservatives. Spectrophotometric measurements compared the amount of C-PC in the hydrogels to the initially added amount. After selecting the most suitable gelling agent and preservative, two C-PC hydrogels, with and without propylene glycol (PG) (Sigma-Aldrich, St. Louis, MO, USA), were produced for further testing. In vitro release studies utilized modified Franz-type diffusion cells, while ex vivo skin-permeation studies employed Bronaugh-type cells and human skin. Confocal laser scanning microscopy analyzed C-PC accumulation in the skin. RESULTS: The findings demonstrated that sodium alginate (Sigma-Aldrich, St. Louis, MO, USA), hydroxyethyl cellulose (HEC) (Sigma-Aldrich, St. Louis, MO, USA), and SoligelTM (Givaudan, Vernier, Switzerland) are effective biopolymers for formulating hydrogels while maintaining C-PC stability. After 6 h, C-PC release from the hydrogel containing PG was approximately 10% or 728.07 (±19.35) µg/cm2, significantly higher than the nearly 7% or 531.44 (±26.81) µg/cm2 release from the hydrogel without PG (p < 0.05). The ex vivo qualitative skin-permeation study indicated that PG enhances C-PC penetration into the outermost skin layer. CONCLUSION: PG's ability to enhance the release of C-PC from the hydrogel, coupled with its capacity to modify the skin barrier ex vivo, facilitates the penetration of C-PC into the stratum corneum.

Ex Vivo Analysis of the Association of GFP-Expressing L. aethiopica and L. mexicana with Human Peripheral Blood-Derived (PBD) Leukocytes over 24 Hours.

Leishmania parasites are transmitted to mammalian hosts through the bite of sandflies. These parasites can infect phagocytic cells (macrophages, dendritic cells, and neutrophils) and non-phagocytic cells (B cells and fibroblasts). In mice models, the disease development or resolution is linked to T cell responses involving inflammatory cytokines and the activation of macrophages with the M1/M2 phenotype. However, this mechanism does not apply to human infection where a more complex immunological response occurs. The understanding of interactions between immune cells during Leishmania infection in humans is still limited, as current infection models focus on individual cell types or late infection using controlled human infection models (CHIMs). This study investigated the early parasite infection in freshly isolated peripheral blood-derived (PBD) leukocytes over 24 h. Flow cytometer analysis is used in immunophenotyping to identify different subpopulations. The study found that among the L. aethiopicaGFP-associated leukocytes, most cells were neutrophils (55.87% ± 0.09 at 4 h) and monocytes (23.50% ± 0.05% at 24 h). B cells were 12.43% ± 0.10% at 24 h. Additionally, 10-20% of GFP+ leukocytes did not belong to the aforementioned cell types, and further investigation revealed their identity as CD4+ T cells. Data not only confirm previous findings of Leishmania infection with PBD leukocytes and association with B cells but also suggest that CD4+ T cells might influence the early-stage of infection.

Luteolin-loaded Invasomes Gel for Transdermal Delivery: Development, Optimization, in-vitro, and Preclinical Evaluation.

Luteolin (LN), is an herbal bioactive flavone and exhibits many pharmacological activities. However, the bioavailability of LN is limited due to its inadequate solubility and significant first-pass metabolism. The present study developed transdermal LN-loaded invasomes (IVM) gel to improve the therapeutic efficacy. The LN-IVM was prepared and optimized by 2 3 factorial designs. LN-IVM was characterized for physicochemical parameters. The optimized LN-IVM (LN-IVMopt) was incorporated into HPMC-K4M gel and evaluated for viscosity, spreadability, and irritation. Further LN-IVM gel was evaluated for drug release, ex-vivo permeation, pharmacokinetic and pharmacodynamics study. LN-IVMopt showed 300.8±2.67 nm of VS, 0.258 of PDI, 89.92±1.29% of EE, and a zeta potential of -18.2 mV. LN-IVM exhibited spherical morphology. FTIR and XRD results demonstrated that LN was encapsulated into IVM matrix. The optimized IVM gel (LN-IVMoptG2) exhibited excellent viscosity, spreadability, and sustained release of LN (91.32±2.95% in 24 h). LN-IVMoptG2 exhibited statistically significant (p < 0.05) higher flux (5.79 µg/h/cm2 ) than LN-gel (2.09 µg/h/cm2 ). The apparent permeability coefficient of plain LN gel and LN- IVMoptG was 1.15×10-5 cm/min and 3.22×10-5 cm/min respectively. LN-IVMoptG2 showed no irritation (score 0.0) throughout the study (60 min). The relative bioavailability of LN from LN-IVMopt-G2 (transdermal) was 2.38±0.19 fold as compared to LN-Sus (oral) and 1.81±0.15-fold than plain LN-gel (transdermal). The LN-IVMoptG2 showed a substantial lessening in the paw volume up to 12 h (17.48±1.94% swelling) than plain LN-gel (44.77±2.82% swelling). The finding concluded that the IVM gel is a novel, effective, and safe approach for the delivery of LN transdermally to improve its therapeutic efficacy.

A Comparative Ex Vivo Study on Apex Locator Precision in Mature Teeth and Immature Teeth With Divergent Canals.

AIM: This study investigates the accuracy of an apex locator in mature and immature teeth with divergent root canals ex vivo. MATERIALS AND METHODS: Fifty extracted premolar teeth were utilized for the study, with 25 mature teeth (Nolla stage 9 or 10) and 25 immature teeth (Nolla stages ≤ 8). The discrepancies between the actual length (AL) and the electronic length (EL), measured using a Bingo 1020 apex locator, were compared and analyzed. Statistical analysis included Pearson's correlation analysis, a T-test for independent samples, and multiple linear regression. Statistical significance was set at p < 0.05. RESULTS: The difference between AL and EL in immature teeth was significantly higher than in mature premolar teeth. Nonetheless, patient gender or jaw type (upper/lower) did not affect the accuracy of electronic apex locator (EAL) measurements. CONCLUSION: EAL readings are less accurate in immature teeth than in mature teeth. Supplementary measures, such as radiographic length determination and wet-dry paper point tests, are required to confirm the working length for improved treatment outcomes and patient compliance.