Synergistic induction of blood-brain barrier properties.

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/ß-catenin signaling and inhibition of the TGF-ß pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/ß-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

Unravelling the complexities of resistance mechanism in pancreatic cancer: insights from in vitro and ex-vivo model systems.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis and rising global deaths. Late diagnosis, due to absent early symptoms and biomarkers, limits treatment mainly to chemotherapy, which soon encounters resistance. PDAC treatment innovation is hampered by its complex and heterogeneous resistant nature, including mutations in key genes and a stromal-rich, immunosuppressive tumour microenvironment. Recent studies on PDAC resistance stress the need for suitable in vitro and ex vivo models to replicate its complex molecular and microenvironmental landscape. This review summarises advances in these models, which can aid in combating chemoresistance and serve as platforms for discovering new therapeutics. Immortalised cell lines offer homogeneity, unlimited proliferation, and reproducibility, but while many gemcitabine-resistant PDAC cell lines exist, fewer models are available for resistance to other drugs. Organoids from PDAC patients show promise in mimicking tumour heterogeneity and chemosensitivity. Bioreactors, co-culture systems and organotypic slices, incorporating stromal and immune cells, are being developed to understand tumour-stroma interactions and the tumour microenvironment's role in drug resistance. Lastly, another innovative approach is three-dimensional bioprinting, which creates tissue-like structures resembling PDAC architecture, allowing for drug screening. These advanced models can guide researchers in selecting optimal in vitro tests, potentially improving therapeutic strategies and patient outcomes.

Experimental Models of SARS-CoV-2 Infection: Possible Platforms to Study COVID-19 Pathogenesis and Potential Treatments.

In December 2019, a novel coronavirus crossed species barriers to infect humans and was effectively transmitted from person to person, leading to a worldwide pandemic. Development of effective clinical interventions, including vaccines and antiviral drugs that could prevent or limit theburden or transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global health priority. It is thus of utmost importance to assess possible therapeutic strategies against SARS-CoV-2 using experimental models that recapitulate aspects of the human disease. Here, we review available models currently being developed and used to study SARS-CoV-2 infection and highlight their application to screen potential therapeutic approaches, including repurposed antiviral drugs and vaccines. Each identified model provides a valuable insight into SARS-CoV-2 cellular tropism, replication kinetics, and cell damage that could ultimately enhance understanding of SARS-CoV-2 pathogenesis and protective immunity.

Approaches to investigating metabolism in human neurodevelopment using organoids: insights from intestinal and cancer studies.

Interrogating the impact of metabolism during development is important for understanding cellular and tissue formation, organ and systemic homeostasis, and dysregulation in disease states. To evaluate the vital functions metabolism coordinates during human brain development and disease, pluripotent stem cell-derived models, such as organoids, provide tractable access to neurodevelopmental processes. Despite many strengths of neural organoid models, the extent of their replication of endogenous metabolic programs is currently unclear and requires direct investigation. Studies in intestinal and cancer organoids that functionally evaluate dynamic bioenergetic changes provide a framework that can be adapted for the study of neural metabolism. Validation of in vitro models remains a significant challenge; investigation using in vivo models and primary tissue samples is required to improve our in vitro model systems and, concomitantly, improve our understanding of human development.

Microfluidic systems for modeling human development.

The proper development and patterning of organs rely on concerted signaling events emanating from intracellular and extracellular molecular and biophysical cues. The ability to model and understand how these microenvironmental factors contribute to cell fate decisions and physiological processes is crucial for uncovering the biology and mechanisms of life. Recent advances in microfluidic systems have provided novel tools and strategies for studying aspects of human tissue and organ development in ways that have previously been challenging to explore ex vivo. Here, we discuss how microfluidic systems and organs-on-chips provide new ways to understand how extracellular signals affect cell differentiation, how cells interact with each other, and how different tissues and organs are formed for specialized functions. We also highlight key advancements in the field that are contributing to a broad understanding of human embryogenesis, organogenesis and physiology. We conclude by summarizing the key advantages of using dynamic microfluidic or microphysiological platforms to study intricate developmental processes that cannot be accurately modeled by using traditional tissue culture vessels. We also suggest some exciting prospects and potential future applications of these emerging technologies.

Kidney Disease Modeling with Organoids and Organs-on-Chips.

Kidney disease is a global health crisis affecting more than 850 million people worldwide. In the United States, annual Medicare expenditures for kidney disease and organ failure exceed $81 billion. Efforts to develop targeted therapeutics are limited by a poor understanding of the molecular mechanisms underlying human kidney disease onset and progression. Additionally, 90% of drug candidates fail in human clinical trials, often due to toxicity and efficacy not accurately predicted in animal models. The advent of ex vivo kidney models, such as those engineered from induced pluripotent stem (iPS) cells and organ-on-a-chip (organ-chip) systems, has garnered considerable interest owing to their ability to more accurately model tissue development and patient-specific responses and drug toxicity. This review describes recent advances in developing kidney organoids and organ-chips by harnessing iPS cell biology to model human-specific kidney functions and disease states. We also discuss challenges that must be overcome to realize the potential of organoids and organ-chips as dynamic and functional conduits of the human kidney. Achieving these technological advances could revolutionize personalized medicine applications and therapeutic discovery for kidney disease.

Opposing roles for ADAMTS2 and ADAMTS14 in myofibroblast differentiation and function.

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor ß availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

In vitro Models for Predicting Bioadhesion Fracture Strength to Ex Vivo Animal Buccal Tissue.

Commitment to the 3Rs principle (Replacement, Reduction, and Refinement) led to the development of a cell-based system to measure buccal bioadhesion in vitro and replace the use of porcine buccal and esophageal tissues (PBT and PET, respectively). Additionally, the aim is to bridge the gap in knowledge regarding the bioadhesion properties of PBT and PET. The in vitro models are based on the human buccal epithelial cell line-TR146 without ("Model I") or with ("Model II") 5% (w/v) mucous layer. The in vitro setup also provides a method to evaluate the bioadhesion between two soft materials. Standard bioadhesive hydrogels (alginate, chitosan, and gelatin) are used to test and compare the results from the in vitro models to the ex vivo tissues. The ex vivo and in vitro models show increased bioadhesion as the applied force and contact time increases. Furthermore, Model I exhibits bioadhesion values-of alginate, chitosan, and gelatin-comparable to those obtained with PBT. It is also found that contact time and applied force similarly affect PBT and PET bioadhesion, while PET exhibits greater values. In conclusion, Model I can replace PBT for measuring bioadhesion and be incorporated into the experimental design of bioadhesive DDS, thus minimizing animal tissue usage.

Long-term culture of human Sertoli cells from adult Klinefelter patients as a first step to develop new tools for unravelling the testicular physiopathology.

STUDY QUESTION: Are Sertoli cells (SCs) from adult Klinefelter men (47,XXY) capable of proliferating in vitro and maintaining their main phenotypical and functional characteristics as do SCs from adult 46,XY patients? SUMMARY ANSWER: Isolated SCs from patients with Klinefelter syndrome (KS) can be expanded in vitro while maintaining their characteristics and a stable karyotype, similar to SCs from 46,XY patients. WHAT IS KNOWN ALREADY: The mechanism leading to testicular tissue degeneration in KS is still unknown. A few recent studies highlight the main role played by SCs in the physiopathology of the disease, but new study models based on co-culture or testicular organoids are needed to further understand the SC's involvement in the mechanism of testicular degeneration and fibrosis, and to find therapeutical targets. KS SC expansion could be the first step towards developing such in vitro study models. SCs have been isolated from 46,XY men and expanded in vitro while maintaining the expression of phenotypical and functional markers, but propagation of SCs from KS men has not been achieved yet. STUDY DESIGN, SIZE, DURATION: Testicular tissue was obtained during a testicular sperm extraction procedure for infertility treatment between 2019 and 2021 from three azoospermic adult KS (47,XXY) men (33±3.6 years old) and from three control patients (46,XY) (36±2 years old) presenting with obstructive azoospermia. SCs isolated from frozen-thawed tissue of KS and 46,XY patients were cultured for 60 days and compared. All patients signed an informed consent according to the ethical board approval of the study protocol. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular biopsies obtained from KS (n = 3) and 46,XY (n = 3) adult patients were slow-frozen. After tissue thawing SCs were isolated using a double-step enzymatic digestion and differential plating, and cultured for 60 days in DMEM medium containing FBS. Analyses were performed at different culture times (passages 5 (P5) and 10 (P10)). Quantification of cells using immunofluorescence (IF) for cell type-specific markers (Sox9, GATA4, ACTA2, INSL3, MAGEA4), SCs characterization using both IF and quantitative real-time PCR for GDNF, BMP4, AR and CLDN11 and cells karyotyping were performed. MAIN RESULTS AND THE ROLE OF CHANCE: We demonstrate for the first time that a small population of human SCs isolated from frozen-thawed testis of adult KS patients can be expanded in vitro while retaining expression of characteristic markers of SCs and the 47,XXY karyotype, and exhibiting cell-specific functional proteins and gene expression (GDNF, BMP4, AR, and CLDN11) after 60 days in culture. At P10, 83.39 ± 4.2% of cultured cells from KS men and 85.34 ± 4.1% from 46,XY men expressed Sox9, and 88.8 ± 3.9% of KS cells versus 82.9 ± 3.2% of the control cells were positive for GATA4 without any differences between two groups; both Sox9 and GATA4 are typical SC markers. No differences were found between KS and 46,XY SCs in vitro in terms of cells expansion (exponential growth between P1 and P10 with an average cell count of 2.8±1.5×107 versus 3.8±1.2×107 respectively for the KS and control groups at P10). There was no significant statistical difference for functional proteins and genes expressions (GDNF, BMP4, AR, and CLDN11) neither between KS SCs and control SCs nor between P5 and P10. LIMITATIONS, REASONS FOR CAUTION: The small number of donor samples is a limitation but it is due to limited availability of tissue for research in KS populations. Although no differences were observed in SCs function in the culture of isolated SCs after 60 days, the possibility of a SCs dysfunction needs to be investigated in more complex 3-dimensional models allowing the establishment of a proper cell organization and further analyses of cell functions and interactions during longer culture periods. WIDER IMPLICATIONS OF THE FINDINGS: The demonstration of the possibility to propagate KS SCs in vitro could be useful to build new in vitro models for deciphering testicular cell interactions, determining deficient signalling pathways involved in impaired spermatogenesis, and identifying targets for infertility treatment in KS. As the cell numbers achieved in this study are higher than cell numbers used to develop testicular organoids, we may expect to be able to understand the behaviour and physiopathology of SCs in the disease during the long-term culture of these organoids. Such models could be further applied to understand other causes of deficiencies in seminiferous tubules. STUDY FUNDING/COMPETING INTEREST(S): M.G.G is funded by a grant from the Cliniques Universitaires Saint-Luc (FRC) for the research project on Klinefelter Syndrome Physiopathology. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: NCT05997706.

Single-cell RNA sequencing reveals 2D cytokine assay can model atopic dermatitis more accurately than immune-competent 3D setup.

Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL-4 and IL-13 induce AD-like features in keratinocytes in vitro. However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure is superior to model AD compared to simple 2D cell culture with cytokines. For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single-cell RNA sequencing data (scRNA-seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL-4 and IL-13 induced the closest in vivo-like AD phenotype which was observed in the scRNA-seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM-associated genes such as POSTN. While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo. Taken together, our comprehensive study shows that the simple model using IL-4 or IL-13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro, which may aid the discovery of novel treatment options.

Multidimensional fragmentomic profiling of cell-free DNA released from patient-derived organoids.

BACKGROUND: Fragmentomics, the investigation of fragmentation patterns of cell-free DNA (cfDNA), has emerged as a promising strategy for the early detection of multiple cancers in the field of liquid biopsy. However, the clinical application of this approach has been hindered by a limited understanding of cfDNA biology. Furthermore, the prevalence of hematopoietic cell-derived cfDNA in plasma complicates the in vivo investigation of tissue-specific cfDNA other than that of hematopoietic origin. While conventional two-dimensional cell lines have contributed to research on cfDNA biology, their limited representation of in vivo tissue contexts underscores the need for more robust models. In this study, we propose three-dimensional organoids as a novel in vitro model for studying cfDNA biology, focusing on multifaceted fragmentomic analyses. RESULTS: We established nine patient-derived organoid lines from normal lung airway, normal gastric, and gastric cancer tissues. We then extracted cfDNA from the culture medium of these organoids in both proliferative and apoptotic states. Using whole-genome sequencing data from cfDNA, we analyzed various fragmentomic features, including fragment size, footprints, end motifs, and repeat types at the end. The distribution of cfDNA fragment sizes in organoids, especially in apoptosis samples, was similar to that found in plasma, implying occupancy by mononucleosomes. The footprints determined by sequencing depth exhibited distinct patterns depending on fragment sizes, reflecting occupancy by a variety of DNA-binding proteins. Notably, we discovered that short fragments (< 118 bp) were exclusively enriched in the proliferative state and exhibited distinct fragmentomic profiles, characterized by 3 bp palindromic end motifs and specific repeats. CONCLUSIONS: In conclusion, our results highlight the utility of in vitro organoid models as a valuable tool for studying cfDNA biology and its associated fragmentation patterns. This, in turn, will pave the way for further enhancements in noninvasive cancer detection methodologies based on fragmentomics.

More than a number: Incorporating the aged phenotype to improve in vitro and in vivo modeling of neurodegenerative disease.

Age is the number one risk factor for developing a neurodegenerative disease (ND), such as Alzheimer's disease (AD) or Parkinson's disease (PD). With our rapidly ageing world population, there will be an increased burden of ND and need for disease-modifying treatments. Currently, however, translation of research from bench to bedside in NDs is poor. This may be due, at least in part, to the failure to account for the potential effect of ageing in preclinical modelling of NDs. While ageing can impact upon physiological response in multiple ways, only a limited number of preclinical studies of ND have incorporated ageing as a factor of interest. Here, we evaluate the aged phenotype and highlight the critical, but unmet, need to incorporate aspects of this phenotype into both the in vitro and in vivo models used in ND research. Given technological advances in the field over the past several years, we discuss how these could be harnessed to create novel models of ND that more readily incorporate aspects of the aged phenotype. This includes a recently described in vitro panel of ageing markers, which could help lead to more standardised models and improve reproducibility across studies. Importantly, we cannot assume that young cells or animals yield the same responses as seen in the context of ageing; thus, an improved understanding of the biology of ageing, and how to appropriately incorporate this into the modelling of ND, will ensure the best chance for successful translation of new therapies to the aged patient.

Tumor-microenvironment-on-a-chip: the construction and application.

Currently, despite the vast amounts of time and money invested in cancer treatment, cancer remains one of the primary threats to human life. The primary factor contributing to the low treatment efficacy is cancer heterogeneity. The unclear molecular mechanisms underlying tumorigenesis, coupled with the complexity of human physiology, and the inability of animal models to accurately replicate the human tumor microenvironment, pose significant hurdles in the development of novel cancer therapies. Tumor-microenvironment-on-chip (TMOC) represents a research platform that integrates three-dimensional cell culture with microfluidic systems, simulating the essential components and physiological traits of the in vivo tumor microenvironment. It offers a dynamic setting within the chip system to study tumor progression, potentially heralding a breakthrough in cancer research. In this review, we will summarize the current advancements in this platform, encompassing various types of TMOCs and their applications in different types of cancer. From our perspective, the TMOC platform necessitates enhanced integration with tissue engineering techniques and microphysiological environments before it can evolve into a more refined preclinical model for cancer research.

Engineering a 3D Biomimetic Peptides Functionalized-Polyethylene Glycol Hydrogel Model Cocultured with Endothelial Cells and Astrocytes: Enhancing In Vitro Blood-Brain Barrier Biomimicry.

The blood-brain barrier (BBB) poses a significant challenge for drug delivery and is linked to various neurovascular disorders. In vitro BBB models provide a tool to investigate drug permeation across the BBB and the barrier's response to external injury events. Yet, existing models lack fidelity in replicating the BBB's complexity, hindering a comprehensive understanding of its functions. This study introduces a three-dimensional (3D) model using polyethylene glycol (PEG) hydrogels modified with biomimetic peptides that represent recognition sequences of key proteins in the brain. Hydrogels were functionalized with recognition sequences for laminin (IKVAV) and fibronectin peptides (RGD) and chemically cross-linked with matrix metalloprotease-sensitive peptides (MMPs) to mimic the extracellular matrix of the BBB. Astrocytes and endothelial cells were seeded within and on the surface of the hydrogels, respectively. The barrier integrity was assessed through different tests including transendothelial electrical resistance (TEER), the permeability of sodium fluorescence (Na-F), the permeability of Evan's blue bound to albumin (EBA), and the expression of zonula occluden-1 (ZO-1) in seeded endothelial cells. Hydrogels with a combination of RGD and IKVAV peptides displayed superior performance, exhibiting significantly higher TEER values (55.33 ± 1.47 Ω·cm2) at day 5 compared to other 2D controls including HAECs-monoculture and HAECs-cocultured with NHAs seeded on well inserts and 3D controls including RGD hydrogel and RGD-IKVAV monoculture with HAECs and RGD hydrogel cocultured with HAECs and NHAs. The designed 3D system resulted in the lowest Evan's blue permeability at 120 min (0.215 ± 0.055 µg/mL) compared to controls. ZO-1 expression was significantly higher and formed a relatively larger network in the functionalized hydrogel cocultured with astrocytes and endothelial cells compared to the controls. Thus, the designed 3D model effectively recapitulates the main BBB structure and function in vitro and is expected to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

In vitro models for neuropathic pain phenotypic screening in brain therapeutics.

The discovery of brain therapeutics faces a significant challenge due to the low translatability of preclinical results into clinical success. To address this gap, several efforts have been made to obtain more translatable neuronal models for phenotypic screening. These models allow the selection of active compounds without predetermined knowledge of drug targets. In this review, we present an overview of various existing models within the field, examining their strengths and limitations, particularly in the context of neuropathic pain research. We illustrate the usefulness of these models through a comparative review in three crucial areas: i) the development of novel phenotypic screening strategies specifically for neuropathic pain, ii) the validation of the models for both primary and secondary screening assays, and iii) the use of the models in target deconvolution processes.

Blood-tumor barrier in focus - investigation of glioblastoma-induced effects on the blood-brain barrier.

PURPOSE: Glioblastoma (GBM) is the most prevalent, malignant, primary brain tumor in adults, characterized by limited treatment options, frequent relapse, and short survival after diagnosis. Until now, none of the existing therapy and treatment approaches have proven to be an effective cure. The availability of predictive human blood-tumor barrier (BTB) test systems that can mimic in-vivo pathophysiology of GBM would be of great interest in preclinical research. Here, we present the establishment of a new BTB in-vitro test system combining GBM spheroids and BBB models derived from human induced pluripotent stem cells (hiPSCs). METHODS: We co-cultured hiPSC-derived brain capillary endothelial-like cells (iBCECs) with GBM spheroids derived from U87-MG and U373-MG cell lines in a cell culture insert-based format. Spheroids were monitored over 168 hours (h) of culture, characterized for GBM-specific marker expression and treated with standard chemotherapeutics to distinguish inhibitory effects between 2D mono-culture and 3D spheroids. GBM-induced changes on iBCECs barrier integrity were verified via measurement of transendothelial electrical resistance (TEER), immunocytochemical staining of tight junction (TJ) proteins claudin-5 and occludin as well as the glucose transporter-1 (Glut-1). GBM-induced secretion of vascular endothelial growth factor (VEGF) was additionally quantified. RESULTS: Our hypothesis was validated by reduced expression of TJ proteins, occludin and claudin-5 together with significant barrier breakdown in iBCECs after only 24 h of co-culture, demonstrated by reduction in TEER from 1313 ± 265 Ω*cm2 to 712 ± 299 Ω*cm2 (iBCECs + U87-MG) and 762 ± 316 Ω*cm2 (iBCECs + U373-MG). Furthermore, 3D spheroids show more resistance to standard GBM chemotherapeutics in-vitro compared to 2D cultures. CONCLUSIONS: We demonstrate the establishment of a simplified, robust in-vitro BTB test system, with potential application in preclinical therapeutic screening and in studying GBM-induced pathological changes at the BBB.

Concentration-resistance relationship and PK/PD evaluation of danofloxacin against emergence of resistant Pasteurella multocida in an in vitro dynamic model.

AIMS: This study aimed to assess the pharmacokinetic/pharmacodynamic (PK/PD) targets of danofloxacin to minimize the risk of selecting resistant Pasteurella multocida mutants and to identify the mechanisms underlying their resistance in an in vitro dynamic model, attaining the optimum dosing regimen of danofloxacin to improve its clinical efficacy based on the mutant selection window (MSW) hypothesis. METHODS AND RESULTS: Danofloxacin at seven dosing regimens and 5 days of treatment were simulated to quantify the bactericidal kinetics and enrichment of resistant mutants upon continuous antibiotic exposure. The magnitudes of PK/PD targets associated with different efficacies were determined in the model. The 24 h area under the concentration-time curve (AUC) to minimum inhibitory concentration (MIC) ratios (AUC24h/MIC) of danofloxacin associated with bacteriostatic, bactericidal and eradication effects against P. multocida were 34, 52, and 64 h. This translates to average danofloxacin concentrations (Cav) over 24 h being 1.42, 2.17, and 2.67 times the MIC, respectively. An AUC/MIC-dependent antibacterial efficacy and AUC/mutant prevention concentration (MPC)-dependent enrichment of P. multocida mutants in which maximum losses in danofloxacin susceptibility occurred at a simulated AUC24h/MIC ratio of 72 h (i.e. Cav of three times the MIC). The overexpression of efflux pumps (acrAB-tolC) and their regulatory genes (marA, soxS, and ramA) was associated with reduced susceptibility in danofloxacin-exposed P. multocida. The AUC24h/MPC ratio of 19 h (i.e. Cav of 0.8 times the MPC) was determined to be the minimum mutant prevention target value for the selection of resistant P. multocida mutants. CONCLUSIONS: The emergence of P. multocida resistance to danofloxacin exhibited a concentration-dependent pattern and was consistent with the MSW hypothesis. The current clinical dosing regimen of danofloxacin (2.5 mg kg-1) may have a risk of treatment failure due to inducible fluoroquinolone resistance.

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches.

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

Co-existing ambient fine particulate matter exacerbated electronic cigarette toxicity on human respiratory cells.

Respiratory co-exposure to ambient PM2.5 and electronic cigarettes (e-cigarettes) frequently occurs in public. However, the combined effects on human respiratory health have not been well documented. To discuss potential co-effects and possible biological mechanisms, A549/THP-1 co-cultures and BEAS-2B cells were exposed to unvapedtobacco or mint-flavored e-liquids (0-7.2% v/v), e-cigarette aerosol extract (ECE, 0-50% v/v), PM2.5 (60 µg/mL), or PM2.5 + ECE for 24 h. Cell viability assessments on e-liquids, ECE, PM2.5 + ECE showed that the mint flavor exhibited higher cytotoxicity compared to the tobacco flavor in both A549/THP-1 and BEAS-2B. However, the influence of flavors on ROS levels and mRNA expression of inflammatory markers (IL-6, TNF-α, IL-8, IL-1ß) after ECE exposure demonstrated inconsistency in the two cell models. PM2.5 + ECE treatment notably elevated ROS production and inflammation responses compared to ECE alone exposure. Only co-exposure induced a significant increase in nuclear transcription factor-κB p65 (NF-κB p65) and NOD-like receptor 3 (NLRP3) protein expression regardless of flavors. Our results indicate that PM2.5-treated cells exacerbate the adverse effects induced by ECE in both A549/THP-1 and BEAS-2B cells. Flavors in unvaped e-liquids affect cytotoxicity, oxidative stress and inflammation response, but these effects vary depending on the vaping process and the specific cell line.

The effect of the mechanodynamic lung environment on fibroblast phenotype via the Flexcell.

The lung is a highly mechanical organ as it is exposed to approximately 109 strain cycles, (where strain is the length change of tissue structure per unit initial length), with an approximately 4% amplitude change during quiet tidal breathing or 107 strain cycles at a 25% amplitude during heavy exercises, sighs, and deep inspirations. These mechanical indices have been reported to become aberrant in lung diseases such as acute respiratory distress syndrome (ARDS), pulmonary hypertension, bronchopulmonary dysplasia (BPD), idiopathic pulmonary fibrosis (IPF), and asthma. Through recent innovations, various in vitro systems/bioreactors used to mimic the lung's mechanical strain have been developed. Among these, the Flexcell tension system which is composed of bioreactors that utilize a variety of programs in vitro to apply static and cyclic strain on different cell-types established as 2D monolayer cultures or cell-embedded 3D hydrogel models, has enabled the assessment of the response of different cells such as fibroblasts to the lung's mechanical strain in health and disease. Fibroblasts are the main effector cells responsible for the production of extracellular matrix (ECM) proteins to repair and maintain tissue homeostasis and are implicated in the excessive deposition of matrix proteins that leads to lung fibrosis. In this review, we summarise, studies that have used the Flexcell tension bioreactor to assess effects of the mechanical lung on the structure, function, and phenotype of lung fibroblasts in homeostatic conditions and abnormal environments associated with lung injury and disease. We show that these studies have revealed that different strain conditions regulate fibroblast proliferation, ECM protein production, and inflammation in normal repair and the diseased lung.