Detection of Sensitization Profiles with Cellular In Vitro Tests in Wheat Allergy Dependent on Augmentation Factors (WALDA).

Wheat allergy dependent on augmentation factors (WALDA) is the most common gluten allergy in adults. IgE-mediated sensitizations are directed towards ω5-gliadin but also to other wheat allergens. The value of the different in vitro cellular tests, namely the basophil activation test (BAT) and the active (aBHRA) and passive basophil histamine-release assays (pBHRA), in the detection of sensitization profiles beyond ω5-gliadin has not been compared. Therefore, 13 patients with challenge-confirmed, ω5-gliadin-positive WALDA and 11 healthy controls were enrolled. Specific IgE (sIgE), skin prick tests, BATs, aBHRA, and pBHRA were performed with allergen test solutions derived from wheat and other cereals, and results were analyzed and compared. This study reveals a distinct and highly individual reactivity of ω5-gliadin-positive WALDA patients to a range of wheat allergens beyond ω5-gliadin in cellular in vitro tests and SPT. In the BAT, for all tested allergens (gluten, high-molecular-weight glutenin subunits, α-amylase/trypsin inhibitors (ATIs), alcohol-free wheat beer, hydrolyzed wheat proteins (HWPs), rye gluten and secalins), basophil activation in patients was significantly higher than in controls (p = 0.004-p < 0.001). Similarly, significant histamine release was detected in the aBHRA for all test substances, exceeding the cut-off of 10 ng/mL in all tested allergens in 50% of patients. The dependency of tests on sIgE levels against ω5-gliadin differed; in the pBHRA, histamine release to any test substances could only be detected in patients with sIgE against ω5-gliadin ≥ 7.7 kU/L, whereas aBHRA also showed high reactivity in less sensitized patients. In most patients, reactivity to HWPs, ATIs, and rye allergens was observed. Additionally, alcohol-free wheat beer was first described as a promising test substance in ω5-gliadin-positive WALDA. Thus, BAT and aBHRA are valuable tools for the identification of sensitization profiles in WALDA.

Oral Drug Product Administration via Enteral Feeding Tubes: In Vitro Testing.

Medication administration via enteral feeding tubes (EFT) is a necessary practice for patients unable to swallow oral dosage forms due to a medical condition or treatment that affects the ability to swallow or the function of the gastrointestinal tract. Off-label administration of oral drug products via EFT raises concerns for pharmaceutical sponsors, regulators, and healthcare practitioners (HCPs) because of the potential risks this practice introduces to both the patient and the caregiver. These risks can be mitigated by generating data-supported instructions that patients and HCPs can use to ensure safe and accurate administration of oral drug products via EFT. This commentary presents an industry perspective on the testing that should be conducted to enable development of product-specific instructions in the labeling to support or advise against administration of oral drug products via enteral feeding tube. The proposal outlined in this commentary takes a risk-based approach, addressing recommendations from both regulatory agencies as well as considerations for expanding this testing to address needs specific to neonatal and pediatric populations.

Combination effects of phytonutrient pellet and lemongrass (Cymbopogon citratus) powder on rumen fermentation efficiency and nutrient degradability using in vitro technique.

Phytonutrients (PTN) namely saponins (SP) and condensed tannins (CT) have been demonstrated to assess the effect of rumen fermentation and methane mitigation. Phytonutrient pellet containing mangosteen, rambutan, and banana flower (MARABAC) and lemongrass including PTN, hence these plant-phytonutrients supplementation could be an alternative plant with a positive effect on rumen fermentation. The aim of this experiment was to evaluate the effect of supplementation of MARABAC and lemongrass (Cymbopogon citratus) powder on in vitro fermentation modulation and the ability to mitigate methane production. The treatments were arranged according to a 3 × 3 Factorial arrangement in a completely randomized design. The two experimental factors consisted of MARABAC pellet levels (0%, 1%, and 2% of the total substrate) and lemongrass supplementation levels (0%, 1%, and 2% of the total substrate). The results of this study revealed that supplementation with MARABAC pellet and lemongrass powder significantly improved gas production kinetics (P < 0.01) and rumen fermentation end-products especially the propionate production (P < 0.01). While rumen methane production was subsequently reduced by both factors. Additionally, the in vitro dry matter degradability (IVDMD) and organic matter degradability (IVOMD) were greatly improved (P < 0.05) by the respective treatments. MARABAC pellet and lemongrass powder combination showed effective methane mitigation by enhancing rumen fermentation end-products especially the propionate concentration and both the IVDMD and IVOMD, while mitigated methane production. The combined level of both sources at 2% MARABAC pellet and 2% lemongrass powder of total substrates offered the best results. Therefore, MARABAC pellet and lemongrass powder supplementation could be used as an alternative source of phytonutrient in dietary ruminant.

Regulation of myocardial contraction as revealed by intracellular Ca2+ measurements using aequorin.

Of the ions involved in myocardial function, Ca2+ is the most important. Ca2+ is crucial to the process that allows myocardium to repeatedly contract and relax in a well-organized fashion; it is the process called excitation-contraction coupling. In order, therefore, for accurate comprehension of the physiology of the heart, it is fundamentally important to understand the detailed mechanism by which the intracellular Ca2+ concentration is regulated to elicit excitation-contraction coupling. Aequorin was discovered by Shimomura, Johnson and Saiga in 1962. By taking advantage of the fact that aequorin emits blue light when it binds to Ca2+ within the physiologically relevant concentration range, in the 1970s and 1980s, physiologists microinjected it into myocardial preparations. By doing so, they proved that Ca2+ transients occur upon membrane depolarization, and tension development (i.e., actomyosin interaction) subsequently follows, dramatically advancing the research on cardiac excitation-contraction coupling.

Comparison of forages' digestion levels for different in vitro digestion techniques in horses.

BACKGROUND: Forages are widely used in horse diets. Different in vitro techniques are being tried to determine the fermentation levels of forages in the horse digestive tract. OBJECTIVES: This study aimed to evaluate the digestion levels of four dry forages commonly used in horse nutrition: alfalfa herbage, meadow hay, wheat straw, and Italian ryegrass. In vitro total digestion (TDT), in vitro Sunvold-large intestine digestion (SDT) and in vitro Menke-large intestine digestion (MDT) techniques were compared. METHODS: The study determined in vitro true dry matter digestion (T-DMD), in vitro true organic matter digestion (T-OMD) and in vitro true neutral detergent fibre digestion (T-NDFD). Additionally, concentrations of straight short-chain fatty acids (SCFAs; acetic acid - AA, propionic acid , butyric acid, and valeric acid ) and branched short-chain fatty acids (BSCFA) were assessed. RESULTS: The highest in vitro T-DMD, T-OMD and T-NDFD values were determined by the in vitro TDT for the four forages (p < 0.05). In vitro T-DMD and T-OMD values of alfalfa herbage were higher than those of Italian ryegrass, meadow hay and wheat straw in the in vitro TDT (p < 0.001). In addition, in vitro T-DMD and T-OMD values of alfalfa herbage in the in vitro SDT were higher than those of meadow hay and wheat straw (p < 0.001). In the in vitro TDT, the molarity of AA, total SCFA and BSCFA in the digestion fluid of alfalfa herbage was higher than those of other forages (p < 0.05). CONCLUSION: The in vitro total enzymatic + fermentative digestion technique for horse forages revealed higher values than the in vitro fermentative digestion techniques. In general, the higher the non-structural carbohydrate and crude protein contents in the forage, the higher the results of the in vitro TDT compared to the other techniques.

In vitro Evaluation Method of UV Protecting Ability of Sunscreens: Clarifying and Overcoming Problems to Develop New Method.

Factors influencing on in vitro evaluation of UV protecting ability of sunscreens were analyzed. It was found that any factors making the sunscreen layer spatially inhomogeneous, such as directional viscous fingering during the sunscreen application, dewetting of applied sunscreen layer, and the surface roughness of the standard PMMA plate, alter the UV transmittance. New application procedure and new type of flat hydrophilic plate were developed for inhibiting the generation of spatial inhomogeneity in applied sunscreen layer. The method created by the combination of these newly developed technologies succeeded in providing reliable and reproducible in vitro evaluation of UV protecting ability.

In vitro test methods for evaluating high molecular weight polyethylene oxide polymer induced hemolytic and thrombotic potential.

To combat opioid abuse, the U.S. Food and Drug Administration (FDA) released a comprehensive action plan to address opioid addiction, abuse, and overdose that included increasing the prevalence of abuse-deterrent formulations (ADFs) in opioid tablets. Polyethylene oxide (PEO) has been widely used as an excipient to deter abuse via nasal insufflation. However, changes in abuse patterns have led to unexpected shifts in abuse from the nasal route to intravenous injection. Case reports identify adverse effects similar to thrombotic thrombocytopenic purpura (TTP) syndrome following the intravenous (IV) abuse of opioids containing PEO excipient. Increased risk of IV opioid ADF abuse compared to clinical benefit of the drug led to the removal of one opioid product from the market in 2017. Because many generic drugs containing PEO are still in development, there is interest in assessing safety consistent with generic drug regulation and unintended uses. Currently, there are no guidelines or in vitro assessment tools to characterize the safety of PEO excipients taken via intravenous injection. To create a more robust excipient safety evaluation tool and to study the mechanistic basis of HMW PEO-induced TMA, a dynamic in vitro test system involving blood flow through a needle model has been developed.

An integrated in vitro carcinogenicity test that distinguishes between genotoxic carcinogens, non-genotoxic carcinogens, and non-carcinogens.

Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not always accurately predict the chemicals that will be in vivo carcinogens. If chemicals test positive in vitro for genotoxicity but negative in vivo, this can contribute to unnecessary testing in animals used to confirm erroneous in vitro positive results. Current in vitro tests typically evaluate only genotoxicity endpoints, which limits their potential to detect non-genotoxic carcinogens. The frequency of misleading in vitro positive results can be high, leading to a requirement for more informative in vitro tests. It is now recognized that multiple-endpoint genotoxicity testing may aid more accurate detection of carcinogens and non-carcinogens. The objective of this review was to evaluate the utility of our novel, multiple-endpoint in vitro test, which uses multiple cancer-relevant endpoints to predict carcinogenic potential. The tool assessed micronucleus frequency, p53 expression, p21 expression, mitochondrial respiration, cell cycle abnormalities and, uniquely, cell morphology changes in human lymphoblastoid cell lines, TK6 and MCL-5. The endpoints were used to observe cellular responses to 18 chemicals within the following categories: genotoxic carcinogens, non-genotoxic carcinogens, toxic non-carcinogens, and misleading in vitro positive and negative agents. The number of endpoints significantly altered for each chemical was considered, alongside the holistic Integrated Signature of Carcinogenicity score, derived from the sum of fold changes for all endpoints. Following the calculation of an overall score from these measures, carcinogens exhibited greater potency than non-carcinogens. Genotoxic carcinogens were generally more potent than non-genotoxic carcinogens. This novel approach therefore demonstrated potential for correctly predicting whether chemicals with unknown mechanism may be considered carcinogens. Overall, while further validation is recommended, the test demonstrates potential for the identification of carcinogenic compounds. Adoption of the approach could enable reduced animal use in carcinogenicity testing.

In vitro activity of cefiderocol and other newly approved antimicrobials against multi-drug resistant Gram-negative pathogens recovered in intensive care units in Spain and Portugal / Actividad in vitro de cefiderocol y otros antimicrobianos recientemente aprobados frente a gramnegativos multirresistentes aislados en unidades de cuidados intensivos en España y Portugal

Introduction. The antimicrobial resistance is a significant public health threat, particularly for healthcare-associated infections caused by carbapenem-resistant Gram-negative pathogens which are increasingly reported worldwide. The aim of this study was to provide data on the in vitro antimicrobial activity of cefiderocol and that of commercially available comparator antibiotics against a defined collection of recent clinical multi-drug resistant (MDR) microorganisms, including carbapenem resistant Gram-negative bacteria collected from different regions in Spain and Portugal. Material and methods. A total of 477 clinical isolates of Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia were prospectively (n=265) and retrospectively (n=212) included (2016-2019). Susceptibility testing was performed using standard broad microdilution and results were interpreted using CLSI-2021 and EUCAST-2021 criteria. Results. Overall, cefiderocol showed a good activity against Enterobacterales isolates, being 99.5% susceptible by CLSI and 94.5% by EUCAST criteria. It also demonstrated excellent activity against P. aeruginosa and S. maltophilia isolates, all being susceptible to this compound considering CLSI breakpoints. Regarding A. baumannii (n=64), only one isolate was resistant to cefiderocol. Conclusions. Our results are in agreement with other studies performed outside Spain and Portugal highlighting its excellent activity against MDR gram-negative bacteria. Cefiderocol is a therapeutic alternative to those available for the treatment of infections caused by these MDR bacteria. (AU)

Introducción. La resistencia a los antimicrobianos constituye una importante amenaza para la salud pública, especialmente en el caso de las infecciones relacionadas con la asistencia sanitaria causadas por patógenos gramnegativos resistentes a los carbapenémicos, las cuales están aumentando en todo el mundo. El objetivo de este estudio fue proporcionar datos sobre la actividad antimicrobiana in vitro de cefiderocol y la de antibióticos comparadores disponibles en el arsenal terapéutico frente a una colección definida de microorganismos multirresistentes (MDR) obtenidos de muestras clínicas, incluidas bacterias gramnegativas resistentes a carbapenemas procedentes de diferentes regiones de España y Portugal. Material y métodos. Se recogieron un total de 477 aislados clínicos de Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii y Stenotrophomonas maltophilia de forma prospectiva (n=265) y retrospectiva (n=212) (2016-2019). El estudio de sensibilidad se realizó por microdilución standard y los resultados se analizaron empleando criterios del CLSI de 2021 y de EUCAST de 2021. Resultados. En general, cefiderocol demostró una buena actividad frente a aislados de Enterobacterales, siendo 99,5% sensible según criterios del CLSI y 94,5% según los de EUCAST. Cefiderocol demostró una excelente actividad frente a aislados de P. aeruginosa y S. maltophilia, siendo todos ellos sensibles a este compuesto considerando los puntos de corte del CLSI. En relación a A. baumannii (n=64), sólo un aislado fue resistente a cefiderocol. Conclusiones. Nuestros resultados concuerdan con los de otros estudios realizados fuera de España y Portugal en los que se destaca la excelente actividad de cefiderocol frente a bacterias gramnegativas MDR. Cefiderocol constituye una alternativa terapéutica a las disponibles en el tratamiento de las infecciones causadas por estos microorganismos. (AU)

Small Molecules Accelerate Skin Wound Healing: Shikonin Efficacy and Mechanism of Action in Mice / Las Moléculas Pequeñas Aceleran la Curación de Heridas en la Piel: Eficacia de Shikonin y Mecanismo de Acción en Ratones

SUMMARY: The objective of this study was to investigate the therapeutic wound healing potential and molecular mechanisms of shikonin as small molecules in vitro. A mouse burn model was used to explore the potential therapeutic effect of shikonin; we traced proliferating cells in vivo to locate the active area of skin cell proliferation. Through the results of conventional pathological staining, we found that shikonin has a good effect on the treatment of burned skin and promoted the normal distribution of skin keratin at the damaged site. At the same time, shikonin also promoted the proliferation of skin cells at the damaged site; importantly, we found a significant increase in the number of fibroblasts at the damaged site treated with shikonin. Most importantly, shikonin promotes fibroblasts to repair skin wounds by regulating the PI3K/AKT signaling pathway. This study shows that shikonin can effectively promote the proliferation of skin cell, and local injection of fibroblasts in burned skin can play a certain therapeutic role.

El objetivo de este trabajo fue investigar el potencial terapéutico de cicatrización de heridas y los mecanismos moleculares de la shikonina como moléculas pequeñas in vitro. Se utilizó un modelo de quemaduras en ratones para explorar el posible efecto terapéutico de la shikonina; Rastreamos las células en proliferación in vivo para localizar el área activa de proliferación de células de la piel. A través de los resultados de la tinción para patología convencional, encontramos que la shikonina tiene un buen efecto en el tratamiento de la piel quemada y promueve la distribución normal de la queratina de la piel en el sitio dañado. Al mismo tiempo, la shikonina también promovió la proliferación de células de la piel en el sitio dañado. Es importante destacar que encontramos un aumento significativo en la cantidad de fibroblastos en el sitio dañado tratado con shikonina. Lo más importante es que la shikonina promueve la función reparadora de fibroblastos en las heridas de la piel regulando la vía de señalización PI3K/ AKT. Este estudio muestra que la shikonina puede promover eficazmente la proliferación de células de la piel y que la inyección local de fibroblastos en la piel quemada puede desempeñar un cierto papel terapéutico.

Evaluation of the efficacy of hydroxyl radical (OH) release for disinfection of the air and surfaces in the dental clinic: an in vitro study / Evaluación de la eficacia de la liberación de radicales hidroxilo (OH) para la desinfección del aire y superficies en la clínica dental: un estudio in vitro

Background: Concerning about the quality of room air has increased exponentially. Specially in dental clinicswhere diary practice is characterized by the important generation of aerosols.Material and Methods: An in vitro model was used in which samples were collected from the surfaces and roomair of a dental clinic before and after the use of an OH˙ radical generator.Results: A total of 1260 samples were collected for bacteriological analysis and 14 samples for the detection ofSARS-CoV-2. Following OH˙ treatment, the tested surface samples showed a decrease in the number of colonyforming units (CFUs) of 76.9% in TSA culture medium. The circulating room air samples in turn showed adecrease in CFUs of 66.7% in Sabouraud medium and 71.4% in Mannitol agar medium. No presence of SARS-CoV-2 was observed on the surface of the face shield.Conclusions: The disinfectant technology based on the use of hydroxyl radicals (OH˙) is effective in reducing thepresence of moulds and yeasts and Staphylococcus in the air, and in reducing total aerobic bacteria on the testedsurfaces.(AU)

Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.

Review on In-vitro Techniques and In-vivo Animals Models for Screening Diabetes and Diabetic Complications.

Diabetes mellitus is a type of metabolic disorders. Various pharmaceutical interventions and animal models have been used to investigate the genetic, environmental, and etiological aspects of diabetes and its effects. In recent years for the development of ant-diabetic remedies, numerous novel genetically modified animals, pharmaceutical substances, medical techniques, viruses, and hormones have been developed to screen diabetic complications. A unique disease-treating drug with new properties is still being sought after. The current review tried to include all published models and cutting-edge techniques. Experimental induction of diabetes mellitus in animal models and in vitro methods are essential for advancing our knowledge, a thorough grasp of pathophysiology, and the creation of novel therapeutics. Animal models and in vitro techniques are necessary to develop innovative diabetic medications. New approaches and additional animal models are required for diabetes research to advance. This is particularly true for models produced via dietary modifications, which have various macronutrient compositions. In this article, we review the rodent models of diet-induced diabetic peripheral neuropathy, diabetic retinopathy, and diabetic nephropathy and critically compare the key characteristics of these micro-vascular complications in humans and the diagnostic criteria with the parameters used in preclinical research using rodent models, taking into consideration the potential need for factors that can accelerate or aggravate these conditions.

Skin barrier function for regulatory skin absorption tests and effects on testosterone and sucrose absorption.

In vitro absorption through human skin is a critical component in the safety assessment of chemicals, crop protection products, consumer healthcare products and cosmetics. A barrier integrity assay is used to identify skin samples which are potentially damaged. A retrospective analysis of 9978 electrical resistance (ER) measurements generated in a single laboratory (DTL) over a 15-year period was performed. Skin absorption experiments were performed using two model penetrants, testosterone and sucrose, utilising no ER acceptance criteria, and the results assessed. Using a barrier integrity test, to remove potentially damaged samples, was offset against one that can be used to remove intact skin samples with a poorer barrier function (i.e. false positives). The previously identified barrier integrity limit (10 kΩ for a 2.54 cm2 diffusion cell; Davies et al., 2004) was demonstrated to identify half of all samples tested, many of which would be false positive samples. This retrospective analysis identified 5.0 kΩ (17.5th percentile) as an acceptance criterion for a 2.54 cm2 diffusion cell, whilst not considerably changing results generated in skin absorption studies. This was confirmed from the cumulative absorption of the model penetrants tested. Using this limit would, therefore, provide suitable skin samples for regulatory skin absorption studies.

A comprehensively validated IVRT method reliably discriminates sameness and differences between several topical clotrimazole creams.

In vitro release testing (IVRT) has gained increasing acceptance for use as a biowaiver for topical products intended for local action. Whereas the United States Food and Drug Administration (US FDA) has issued product specific guidances (PSGs) recommending IVRT for several products, the PSG for clotrimazole cream does not include an IVRT option. However, an important requirement to include supplemental selectivity in the validation process as described in the recent FDA draft guidance on IVRT studies for topical drug products has generally been conspicuously absent in the published literature describing the application of IVRT as a biowaiver. Supplemental selectivity involves the comparison of a reference product and altered formulations containing the same strength of the active pharmaceutical ingredient (API). In order to demonstrate supplemental selectivity, cream formulation containing the same API (clotrimazole), at the same strength (1 %) and in the same dosage form (cream) but manufactured using different excipients were used. This will help assess the impact that excipients may have on the release rate of clotrimazole and whether the method is capable of identifying differences in the microstructure and arrangement of matter (Q3) as an important performance parameter. In addition, products containing <30 % or >40 % clotrimazole to serve as negative controls were also included for the discriminatory power assessment. Hence, the primary objective was to develop and validate a simple, reliable, reproducible, and cost-effective in vitro technique in accordance with the recent draft FDA guidance to assess the "sameness" of topical creams containing 1 % clotrimazole. An in vitro release testing (IVRT) system was used and an IVRT method was developed and accordingly validated. The validated IVRT method showed the potential to accurately measure the release from 1 % clotrimazole creams and demonstrated supplemental selectivity and appropriate discriminatory power to identify "sameness" and/ or differences.

Key Challenges for In Vitro Testing of Tobacco Products for Regulatory Applications: Recommendations for the In Vitro Mouse Lymphoma Assay.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across traditional tobacco and various tobacco and nicotine next-generation products (NGPs), including Heated Tobacco Products (HTPs) and Electronic Nicotine Delivery Systems (ENDS). This report was developed by a working group composed of attendees of the seventh IIVS workshop, 'Approaches and recommendations for conducting the mouse lymphoma gene mutation assay (MLA) and introduction to in vitro disease models', which was held virtually on 21-23 June 2022. This publication provides a background overview of the MLA, and includes the description of assay conduct and data interpretation, key challenges and recommended best practices for evaluating tobacco and nicotine products, with a focus on the evaluation of NGPs, and a summary of how the assay has been used to evaluate and compare tobacco and nicotine products.

Skin tropism during Usutu virus and West Nile virus infection: an amplifying and immunological role.

Usutu virus (USUV) and West Nile virus (WNV) are closely related emerging arboviruses belonging to the Flavivirus genus and posing global public health concerns. Although human infection by these viruses is mainly asymptomatic, both have been associated with neurological disorders such as encephalitis and meningoencephalitis. Since USUV and WNV are transmitted through the bite of an infected mosquito, the skin represents the initial site of virus inoculation and provides the first line of host defense. Although some data on the early stages of WNV skin infection are available, very little is known about USUV. Herein, USUV-skin resident cell interactions were characterized. Using primary human keratinocytes and fibroblasts, an early replication of USUV during the first 24 hours was shown in both skin cells. In human skin explants, a high viral tropism for keratinocytes was observed. USUV infection of these models induced type I and III interferon responses associated with upregulated expression of various interferon-stimulated genes as well as pro-inflammatory cytokine and chemokine genes. Among the four USUV lineages studied, the Europe 2 strain replicated more efficiently in skin cells and induced a higher innate immune response. In vivo, USUV and WNV disseminated quickly from the inoculation site to distal cutaneous tissues. In addition, viral replication and persistence in skin cells were associated with an antiviral response. Taken together, these results provide a better understanding of the pathophysiology of the early steps of USUV infection and suggest that the skin constitutes a major amplifying organ for USUV and WNV infection.IMPORTANCEUsutu virus (USUV) and West Nile virus (WNV) are closely related emerging Flaviviruses transmitted through the bite of an infected mosquito. Since they are directly inoculated within the upper skin layers, the interactions between the virus and skin cells are critical in the pathophysiology of USUV and WNV infection. Here, during the early steps of infection, we showed that USUV can efficiently infect two human resident skin cell types at the inoculation site: the epidermal keratinocytes and the dermal fibroblasts, leading to the induction of an antiviral innate immune response. Moreover, following cutaneous inoculation, we demonstrated that both viruses can rapidly spread, replicate, and persist in all distal cutaneous tissues in mice, a phenomenon associated with a generalized skin inflammatory response. These results highlight the key amplifying and immunological role of the skin during USUV and WNV infection.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.