ROCKETS - a novel one-for-all toolbox for light sheet microscopy in drug discovery.

Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified gustatory papillae of the tongue, choroid plexi in the brain and duodenal papillae as previously unanticipated locations of high EpCAM expression. Subsequently, we confirmed high EpCAM expression also in human tongue and duodenal specimens. Choroid plexi and duodenal papillae may be considered as particularly sensitive sites due to their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. In conclusion, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.

New Advances in Metabolic Syndrome, from Prevention to Treatment: The Role of Diet and Food.

The definition of metabolic syndrome (MetS) has undergone several changes over the years due to the difficulty in establishing universal criteria for it. Underlying the disorders related to MetS is almost invariably a pro-inflammatory state related to altered glucose metabolism, which could lead to elevated cardiovascular risk. Indeed, the complications closely related to MetS are cardiovascular diseases (CVDs) and type 2 diabetes (T2D). It has been observed that the predisposition to metabolic syndrome is modulated by complex interactions between human microbiota, genetic factors, and diet. This review provides a summary of the last decade of literature related to three principal aspects of MetS: (i) the syndrome's definition and classification, pathophysiology, and treatment approaches; (ii) prediction and diagnosis underlying the biomarkers identified by means of advanced methodologies (NMR, LC/GC-MS, and LC, LC-MS); and (iii) the role of foods and food components in prevention and/or treatment of MetS, demonstrating a possible role of specific foods intake in the development of MetS.

Industrial Hemp (Cannabis sativa L.) Inflorescences as Novel Food: The Effect of Different Agronomical Practices on Chemical Profile.

In this study, the effect of several agronomical practices on the chemical composition of hemp inflorescences, a potential novel food that needs to be further studied, was observed. Here, the case study of inflorescences from Ferimon cultivars is discussed and submitted to different agronomical practices (irrigation and fertilizers) in different years, and the inflorescences harvested in different periods were analyzed by a multimethodological approach. Targeted and untargeted methodologies allowed cannabinoids, total phenolic content, metabolite profile and antioxidant activity to be determined. The biomass and inflorescence yields were also reported. The whole data set was submitted to ANOVA-simultaneous component analysis. The statistic results allowed us to observe that irrigation was responsible for the (-)-Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) increment. THC, cannabichromene (CBC), cannabigerol (CBG), succinate, and fructose resulted as higher in full female flowering than in the period of seed maturity. On the other hand, nitrogen supplementation led to an increase of iso-leucine, valine, and threonine. The obtained results underlined both the potential food application of hemp inflorescences, due to the rich chemical profile, and the strong effect of agronomical practices, mainly irrigation and harvesting, on the qualitative and quantitative characteristics of its metabolite profile.

The influence of front-of-pack nutritional labels on eating and purchasing behaviors: a narrative review of the literature.

BACKGROUND: Front-of-Pack Nutritional Labels are considered a useful tool to help consumers orient themselves in their food choices and direct their behavior toward a healthier diet. FOPNL development and use are part of a framework that includes cognitive, biological, hedonic and cultural aspects, able to affect consumers' eating and purchasing behavior. AIM: Given the complexity of the matter, the aim of this narrative review is to analyze the combination of different factors that drive food choices and eating behaviors and to highlight some aspects that are not fully studied. METHODS: The authors conducted the research using a top-down approach at first, followed by a bottom-up approach; starting with general considerations about the purchasing process, gradually narrowing the discussion to a specific sub-population, and finally extending the discussion back to more general reasonings about the direction to adopt in future, or at least to evaluate, for effective communication. RESULTS: Biases and attitudes toward food products were found to regularly interfere with buying behavior patterns, making it impossible to standardize an average consumer. This reflects in current research, increasing the complexity of the topic. All determinants influencing food choices are often assessed individually rather than in a synergistic and multidimensional context, while the purchasing scenario is characterized by multiple stimuli to which the consumer is subjected. FOPNLs' impact on perceived healthiness has been studied in different conditions, but some population subgroups have not been sufficiently represented. In particular, the effect of FOPNLs on consumers suffering from eating disorders is understudied and needs further attention. Furthermore, some approaches can be compared to "negative nutrition" or "loss-framed communication", putting nutrients out of context, emphasizing losses more than gains and risking promoting negative feelings in consumers. CONCLUSION: Due to the heterogeneity of studies, evidence on what works best in driving people to adopt lasting lifestyle changes is still mixed. Science communicators and policymakers should consider the possibility that a multi-component approach incorporating nutrition information and education may be a key strategy to promote consumers' self-consciousness and to support them in their cognitive efforts toward a healthy and sustainable diet. LEVEL OF EVIDENCE: Level V, narrative review.

Efficacy of front-of-pack nutrition labels in improving health status.

Nutrition labels advise consumers about the nutritional value of packaged foods and their contribution to the overall composition of the diet. They have been proposed as an instrument for the promotion of healthy diets and as a fundamental tool in the prevention of obesity and diet-related non-communicable diseases (NCDs). The aim of this review is to discuss the effectiveness of front-of-pack nutrition labels (FOPNLs) in improving health status, the concern about focusing on single nutrients/foods rather than on eating patterns to prevent obesity and NCDs, and the strength of positive rather than negative messages to promote a healthy and sustainable diet. Although nutrition science investigates individual foods or nutrients, when communicating to the public most of the significant evidence of the favorable health effect largely depends on dietary patterns and not on a single food component or individual nutrient. Therefore, we suggest that a new tool based on positive communication should be developed and implemented to highlight the importance of the diet as a complex matrix.

Integrated approaches to testing and assessment for grouping nanomaterials following dermal exposure.

Exposure to different nanoforms (NFs) via the dermal route is expected in occupational and consumer settings and thus it is important to assess their dermal toxicity and the contribution of dermal exposure to systemic bioavailability. We have formulated four grouping hypotheses for dermal toxicity endpoints which allow NFs to be grouped to streamline and facilitate risk assessment. The grouping hypotheses are developed based on insight into how physicochemical properties of NFs (i.e. composition, dissolution kinetics, size, and flexibility) influence their fate and hazard following dermal exposure. Each hypothesis is accompanied by a tailored Integrated Approach to Testing and Assessment (IATA) that is structured as a decision tree and tiered testing strategies (TTS) for each relevant question (at decision nodes) that indicate what information is needed to guide the user to accept or reject the grouping hypothesis. To develop these hypotheses and IATAs, we gathered and analyzed existing information on skin irritation, skin sensitization, and dermal penetration of NFs from the published literature and performed experimental work to generate data on NF dissolution in sweat simulant fluids. We investigated the dissolution of zinc oxide and silicon dioxide NFs in different artificial sweat fluids, demonstrating the importance of using physiologically relevant conditions for dermal exposure. All existing and generated data informed the formulation of the grouping hypotheses, the IATAs, and the design of the TTS. It is expected that the presented IATAs will accelerate the NF risk assessment for dermal toxicity via the application of read-across.

Determining nanoform similarity via assessment of surface reactivity by abiotic and in vitro assays.

Grouping of substances is a method used to streamline hazard and risk assessment. Assessment of similarity provides the scientific evidence needed for formation of groups. This work reports on justification of grouping of nanoforms (NFs) via similarity of their surface reactivity. Four reactivity assays were used for concentration dependent detection of reactive oxygen species (ROS) generated by NFs: abiotic assays FRAS, EPR and DCFH2-DA, as well as the in vitro assay of NRF2/ARE responsive luciferase reporter activation in the HEK293 cell line. Representative materials (CuO, Mn2O3, BaSO4, CeO2 and ZnO) and three case studies of each several NFs of iron oxides, Diketopyrrolopyrroles (DPP)-based organic pigments and silicas were assessed. A novel similarity assessment algorithm was applied to quantify similarities between pairs of NFs, in a four-step workflow on concentration-response curves, individual concentration and response ranges, and finally the representative materials. We found this algorithm to be applicable to all abiotic and in vitro assays that were tested. Justification of grouping must include the increased potency of smaller particles via the scaling of effects with specific surface, and hence quantitative similarity analysis was performed on concentration-response in mass-metrics. CuO and BaSO4 were the most and least reactive representative materials respectively, and all assays found BaSO4/CuO not similar, as confirmed by their different NOAECs of in vivo studies. However, similarity outcomes from different reactivity assays were not always in agreement, highlighting the need to generate data by one assay for the representative materials and the candidate group of NFs. Despite low similarity scores in vitro some pairs of case study NFs can be accepted as sufficiently similar because the in vivo NOAECs are similar, highlighting the conservative assessment by the abiotic assays.

Modulatory Properties of Food and Nutraceutical Components Targeting NLRP3 Inflammasome Activation.

Inflammasomes are key intracellular multimeric proteins able to initiate the cellular inflammatory signaling pathway. NLRP3 inflammasome represents one of the main protein complexes involved in the development of inflammatory events, and its activity has been largely demonstrated to be connected with inflammatory or autoinflammatory disorders, including diabetes, gouty arthritis, liver fibrosis, Alzheimer's disease, respiratory syndromes, atherosclerosis, and cancer initiation. In recent years, it has been demonstrated how dietary intake and nutritional status represent important environmental elements that can modulate metabolic inflammation, since food matrices are an important source of several bioactive compounds. In this review, an updated status of knowledge regarding food bioactive compounds as NLRP3 inflammasome modulators is discussed. Several chemical classes, namely polyphenols, organosulfurs, terpenes, fatty acids, proteins, amino acids, saponins, sterols, polysaccharides, carotenoids, vitamins, and probiotics, have been shown to possess NLRP3 inflammasome-modulating activity through in vitro and in vivo assays, mainly demonstrating an anti-NLRP3 inflammasome activity. Plant foods are particularly rich in important bioactive compounds, each of them can have different effects on the pathway of inflammatory response, confirming the importance of the nutritional pattern (food model) as a whole rather than any single nutrient or functional compound.

Dissecting the mechanism of cytokine release induced by T-cell engagers highlights the contribution of neutrophils.

T cell engagers represent a novel promising class of cancer-immunotherapies redirecting T cells to tumor cells and have some promising outcomes in the clinic. These molecules can be associated with a mode-of-action related risk of cytokine release syndrome (CRS) in patients. CRS is characterized by the rapid release of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-6 and IL-1ß and immune cell activation eliciting clinical symptoms of fever, hypoxia and hypotension. In this work, we investigated the biological mechanisms triggering and amplifying cytokine release after treatment with T cell bispecific antibodies (TCBs) employing an in vitro co-culture assay of human PBMCs or total leukocytes (PBMCs + neutrophils) and corresponding target antigen-expressing cells with four different TCBs. We identified T cells as the triggers of the TCB-mediated cytokine cascade and monocytes and neutrophils as downstream amplifier cells. Furthermore, we assessed the chronology of events by neutralization of T-cell derived cytokines. For the first time, we demonstrate the contribution of neutrophils to TCB-mediated cytokine release and confirm these findings by single-cell RNA sequencing of human whole blood incubated with a B-cell depleting TCB. This work could contribute to the construction of mechanistic models of cytokine release and definition of more specific molecular and cellular biomarkers of CRS in the context of treatment with T-cell engagers. In addition, it provides insight for the elaboration of prophylactic mitigation strategies that can reduce the occurrence of CRS and increase the therapeutic index of TCBs.

Characterization of the Phytochemical Composition and Bioactivities of Anacyclus maroccanus Ball. and Anacyclus radiatus Loisel Aerial Parts: Preliminary Evidence for the Possible Development of Moroccan Plants.

In the present study, the phytochemical composition and bioactivities of A. maroccanus (AM) and A. radiatus (AR), two ecotypes collected in the Demnate road and Essaouira regions, respectively, were studied to highlight a pharmacological interest and to enable possible pharmaceutical development. To this end, methanolic and ethyl acetate extracts were prepared for each ecotype by fractionation; next, their phytochemical composition was evaluated by spectrophotometric and chromatographic analysis. Moreover, in line with the available evidence for Anacyclus spp. and their traditional use, a screening of bioactivities, including antioxidant, hypoglycemic, antiglycative, chelating, and antibacterial activities, was performed. The extracts were characterized by high amounts of polyphenols, tannins, and flavonoids, especially in the methanolic extracts; these samples were also enriched in carotenoids despite a lower chlorophyll content. Chlorogenic acid and rutin were the major identified compounds. The extracts also showed interesting hypoglycemic, antiglycative, and antibacterial properties, although with differences in efficacy and potency. Present results provide more scientific basis to the ethnopharmacological uses of Anacyclus spp. and suggest a further interest in AM and AR ecotypes as natural sources of bioactive compounds and/or phytocomplexes for possible pharmaceutical and nutraceutical developments.

Human immunocompetent choroid-on-chip: a novel tool for studying ocular effects of biological drugs.

Disorders of the eye leading to visual impairment are a major issue that affects millions of people. On the other side ocular toxicities were described for e.g. molecularly targeted therapies in oncology and may hamper their development. Current ocular model systems feature a number of limitations affecting human-relevance and availability. To find new options for pharmacological treatment and assess mechanisms of toxicity, hence, novel complex model systems that are human-relevant and readily available are urgently required. Here, we report the development of a human immunocompetent Choroid-on-Chip (CoC), a human cell-based in vitro model of the choroid layer of the eye integrating melanocytes and microvascular endothelial cells, covered by a layer of retinal pigmented epithelial cells. Immunocompetence is achieved by perfusion of peripheral immune cells. We demonstrate controlled immune cell recruitment into the stromal compartments through a vascular monolayer and in vivo-like cytokine release profiles. To investigate applicability for both efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies, we exposed the CoCs to cyclosporine and tested CD3 bispecific antibodies.

JAK and mTOR inhibitors prevent cytokine release while retaining T cell bispecific antibody in vivo efficacy.

BACKGROUND: T cell engaging therapies, like chimeric antigen receptor T cells and T cell bispecific antibodies (TCBs), efficiently redirect T cells towards tumor cells, facilitating the formation of a cytotoxic synapse and resulting in subsequent tumor cell killing, a process that is accompanied by the release of cytokines. Despite their promising efficacy in the clinic, treatment with TCBs is associated with a risk of cytokine release syndrome (CRS). The aim of this study was to identify small molecules able to mitigate cytokine release while retaining T cell-mediated tumor killing. METHODS: By screening a library of 52 Food and Drug Administration approved kinase inhibitors for their impact on T cell proliferation and cytokine release after CD3 stimulation, we identified mTOR, JAK and Src kinases inhibitors as potential candidates to modulate TCB-mediated cytokine release at pharmacologically active doses. Using an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the effects of mTOR, JAK and Src kinase inhibitors combined with 2+1 T cell bispecific antibodies (TCBs) including CEA-TCB and CD19-TCB on T cell activation, proliferation and target cell killing measured by flow cytometry and cytokine release measured by Luminex. The combination of mTOR, JAK and Src kinase inhibitors together with CD19-TCB was evaluated in vivo in non-tumor bearing stem cell humanized NSG mice in terms of B cell depletion and in a lymphoma patient-derived xenograft (PDX) model in humanized NSG mice in terms of antitumor efficacy. RESULTS: The effect of Src inhibitors differed from those of mTOR and JAK inhibitors with the suppression of CD19-TCB-induced tumor cell lysis in vitro, whereas mTOR and JAK inhibitors primarily affected TCB-mediated cytokine release. Importantly, we confirmed in vivo that Src, JAK and mTOR inhibitors strongly reduced CD19-TCB-induced cytokine release. In humanized NSG mice, continuous treatment with a Src inhibitor prevented CD19-TCB-mediated B cell depletion in contrast to mTOR and JAK inhibitors, which retained CD19-TCB efficacy. Ultimately, transient treatment with Src, mTOR and JAK inhibitors minimally interfered with antitumor efficacy in a lymphoma PDX model. CONCLUSIONS: Taken together, these data support further evaluation of the use of Src, JAK and mTOR inhibitors as prophylactic treatment to prevent occurrence of CRS.

Development of a standard operating procedure for the DCFH2-DA acellular assessment of reactive oxygen species produced by nanomaterials.

Improved strategies are required for testing nanomaterials (NMs) to make hazard and risk assessment more efficient and sustainable. Including reduced reliance on animal models, without decreasing the level of human health protection. Acellular detection of reactive oxygen species (ROS) may be useful as a screening assay to prioritize NMs of high concern. To improve reliability and reproducibility, and minimize uncertainty, a standard operating procedure (SOP) has been developed for the detection of ROS using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) assay. The SOP has undergone an inter- and intra-laboratory comparison, to evaluate robustness, reliability, and reproducibility, using representative materials (ZnO, CuO, Mn2O3, and BaSO4 NMs), and a number of calibration tools to normalize data. The SOP includes an NM positive control (nanoparticle carbon black (NPCB)), a chemical positive control (SIN-1), and a standard curve of fluorescein fluorescence. The interlaboratory comparison demonstrated that arbitrary fluorescence units show high levels of partner variability; however, data normalization improved variability. With statistical analysis, it was shown that the SIN-1 positive control provided an extremely high level of reliability and reproducibility as a positive control and as a normalization tool. The NPCB positive control can be used with a relatively high level of reproducibility, and in terms of the representative materials, the reproducibility CuO induced-effects was better than for Mn2O3. Using this DCFH2-DA acellular assay SOP resulted in a robust intra-laboratory reproduction of ROS measurements from all NMs tested, while effective reproduction across different laboratories was also demonstrated; the effectiveness of attaining reproducibility within the interlaboratory assessment was particle-type-specific.

Commercial Bio-Packaging to Preserve the Quality and Extend the Shelf-Life of Vegetables: The Case-Study of Pumpkin Samples Studied by a Multimethodological Approach.

A multidisciplinary protocol is proposed to monitor the preservation of fresh pumpkin samples (FP) using three commercial polymeric films: A made of biodegradable cellophane from regenerated cellulose pulp; B from corn starch, cassava and eucalyptus, C made of polylactic acid from corn starch, and a polyethylene film used as reference (REF). Chemical, mechanical and microbiological analyses were applied on packaging and fresh and packaged samples at different times. After an 11-day period, NMR spectroscopy results showed a sucrose increase and a malic acid decrease in all the biofilms with respect to FP; fructose, glucose, galactose levels remained quite constant in biofilms B and C; the most abundant amino acids remained quite constant in biofilm A and decreased significantly in biofilm B. From microbiological analyses total microbial count was below the threshold value up to 7 days for samples in all the films, and 11 days for biofilm C. The lactic acid bacteria, and yeasts and molds counts were below the acceptability limit during the 11 days for all packages. In the case of biofilm C, the most promising packaging for microbiological point of view, aroma analysis was also carried out. In this paper, you can find all the analysis performed and all the values found.

Metabolomic Profiling of Fresh Goji (Lycium barbarum L.) Berries from Two Cultivars Grown in Central Italy: A Multi-Methodological Approach.

The metabolite profile of fresh Goji berries from two cultivars, namely Big Lifeberry (BL) and Sweet Lifeberry (SL), grown in the Lazio region (Central Italy) and harvested at two different periods, August and October, corresponding at the beginning and the end of the maturation, was characterized by means of nuclear magnetic resonance (NMR) and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR MS) methodologies. Several classes of compounds such as sugars, amino acids, organic acids, fatty acids, polyphenols, and terpenes were identified and quantified in hydroalcoholic and organic Bligh-Dyer extracts. Sweet Lifeberry extracts were characterized by a higher content of sucrose with respect to the Big Lifeberry ones and high levels of amino acids (glycine, betaine, proline) were observed in SL berries harvested in October. Spectrophotometric analysis of chlorophylls and total carotenoids was also carried out, showing a decrease of carotenoids during the time. These results can be useful not only to valorize local products but also to suggest the best harvesting period to obtain a product with a chemical composition suitable for specific industrial use. Finally, preliminary studies regarding both the chemical characterization of Goji leaves generally considered a waste product, and the biological activity of Big Lifeberry berries extracts was also investigated. Goji leaves showed a chemical profile rich in healthy compounds (polyphenols, flavonoids, etc.) confirming their promising use in the supplements/nutraceutical/cosmetic field. MG63 cells treated with Big Lifeberry berries extracts showed a decrease of iNOS, COX-2, IL-6, and IL-8 expression indicating their significant biological activity.

Human immunocompetent Organ-on-Chip platforms allow safety profiling of tumor-targeted T-cell bispecific antibodies.

Traditional drug safety assessment often fails to predict complications in humans, especially when the drug targets the immune system. Here, we show the unprecedented capability of two human Organs-on-Chips to evaluate the safety profile of T-cell bispecific antibodies (TCBs) targeting tumor antigens. Although promising for cancer immunotherapy, TCBs are associated with an on-target, off-tumor risk due to low levels of expression of tumor antigens in healthy tissues. We leveraged in vivo target expression and toxicity data of TCBs targeting folate receptor 1 (FOLR1) or carcinoembryonic antigen (CEA) to design and validate human immunocompetent Organs-on-Chips safety platforms. We discovered that the Lung-Chip and Intestine-Chip could reproduce and predict target-dependent TCB safety liabilities, based on sensitivity to key determinants thereof, such as target expression and antibody affinity. These novel tools broaden the research options available for mechanistic understandings of engineered therapeutic antibodies and assessing safety in tissues susceptible to adverse events.

Src/lck inhibitor dasatinib reversibly switches off cytokine release and T cell cytotoxicity following stimulation with T cell bispecific antibodies.

BACKGROUND: T cell engagers are bispecific antibodies recognizing, with one moiety, the CD3ε chain of the T cell receptor and, with the other moiety, specific tumor surface antigens. Crosslinking of CD3 upon simultaneous binding to tumor antigens triggers T cell activation, proliferation and cytokine release, leading to tumor cell killing. Treatment with T cell engagers can be associated with safety liabilities due to on-target on-tumor, on-target off-tumor cytotoxic activity and cytokine release syndrome (CRS). Tyrosine kinases such as SRC, LCK or ZAP70 are involved in downstream signaling pathways after engagement of the T cell receptor and blocking these kinases might serve to abrogate T cell activation when required (online supplemental material 1). Dasatinib was previously identified as a potent kinase inhibitor that switches off CAR T cell functionality. METHODS: Using an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the effects of dasatinib combined with 2+1 T cell bispecific antibodies (TCBs) including CEA-TCB, CD19-TCB or HLA-A2 WT1-TCB on T cell activation, proliferation and target cell killing measured by flow cytometry and cytokine release measured by Luminex. To determine the effective dose of dasatinib, the Incucyte system was used to monitor the kinetics of TCB-mediated target cell killing in the presence of escalating concentrations of dasatinib. Last, the effects of dasatinib were evaluated in vivo in humanized NSG mice co-treated with CD19-TCB. The count of CD20+ blood B cells was used as a readout of efficacy of TCB-mediated killing and cytokine levels were measured in the serum. RESULTS: Dasatinib concentrations above 50 nM prevented cytokine release and switched off-target cell killing, which were subsequently restored on removal of dasatinib. In addition, dasatinib prevented CD19-TCB-mediated B cell depletion in humanized NSG mice. These data confirm that dasatinib can act as a rapid and reversible on/off switch for activated T cells at pharmacologically relevant doses as they are applied in patients according to the label. CONCLUSION: Taken together, we provide evidence for the use of dasatinib as a pharmacological on/off switch to mitigate off-tumor toxicities or CRS by T cell bispecific antibodies.

A Multimethodological Characterization of Cannabis sativa L. Inflorescences from Seven Dioecious Cultivars Grown in Italy: The Effect of Different Harvesting Stages.

The chemical profile of the female inflorescence extracts from seven Cannabis sativa L. dioecious cultivars (Carmagnola, Fibranova, Eletta Campana, Antal, Tiborszallasi, Kompolti, and Tisza) was monitored at three harvesting stages (4, 14, and 30 September), reaching from the beginning of flowering to end of flowering/beginning of seed formation, using untargeted nuclear magnetic resonance (NMR) and targeted (ultra-high-performance liquid chromatography (UHPLC) and spectrophotometry) analyses. The tetrahydrocannabinol content was always below the legal limits (<0.6%) in all the analyzed samples. The NMR metabolite profile (sugars, organic acids, amino acids, and minor compounds) subjected to principal components analysis (PCA) showed a strong variability according to the harvesting stages: samples harvested in stage I were characterized by a high content of sucrose and myo-inositol, whereas the ones harvested in stage II showed high levels of succinic acid, alanine, valine, isoleucine, phenylalanine, and threonine. Samples harvested in stage III were characterized by high levels of glucose, fructose, choline, trigonelline, malic acid, formic acid, and some amino acids. The ratio between chlorophylls and carotenoids content indicated that all plants grew up exposed to the sun, the Eletta Campana cultivar having the highest pigment amount. Tiborszallasi cultivar showed the highest polyphenol content. The highest antioxidant activity was generally observed in stage II. All these results suggested that the Cannabis sativa L. inflorescences of each analyzed dioecious hemp cultivar presented a peculiar chemical profile affected by the harvesting stage. This information could be useful for producers and industries to harvest inflorescences in the appropriate stage to obtain samples with a peculiar chemical profile suitable for proper applications.

Towards FAIR nanosafety data.

Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology.

Fibroblast Activation Protein α-Targeted CD40 Agonism Abrogates Systemic Toxicity and Enables Administration of High Doses to Induce Effective Antitumor Immunity.

PURPOSE: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. EXPERIMENTAL DESIGN: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. RESULTS: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. CONCLUSIONS: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.