IL7 increases targeted lipid nanoparticle-mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation.

The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4+ and CD8+ T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo.

Inhibition of Adult Neurogenesis in Male Mice after Repeated Exposure to Paracetamol Overdose.

Paracetamol, or acetaminophen (N-acetyl-para-aminophenol, APAP), is an analgesic and antipyretic drug that is commonly used worldwide, implicated in numerous intoxications due to overdose, and causes serious liver damage. APAP can cross the blood-brain barrier and affects brain function in numerous ways, including pain signals, temperature regulation, neuroimmune response, and emotional behavior; however, its effect on adult neurogenesis has not been thoroughly investigated. We analyze, in a mouse model of hepatotoxicity, the effect of APAP overdose (750 mg/kg/day) for 3 and 4 consecutive days and after the cessation of APAP administration for 6 and 15 days on cell proliferation and survival in two relevant neurogenic zones: the subgranular zone of the dentate gyrus and the hypothalamus. The involvement of liver damage (plasma transaminases), neuronal activity (c-Fos), and astroglia (glial fibrillar acidic protein, GFAP) were also evaluated. Our results indicated that repeated APAP overdoses are associated with the inhibition of adult neurogenesis in the context of elevated liver transaminase levels, neuronal hyperactivity, and astrogliosis. These effects were partially reversed after the cessation of APAP administration for 6 and 15 days. In conclusion, these results suggest that APAP overdose impairs adult neurogenesis in the hippocampus and hypothalamus, a fact that may contribute to the effects of APAP on brain function.

Sex-specific variations in spatial reference memory acquisition: Insights from a comprehensive behavioral test battery in C57BL/6JRj mice.

Sex differences in declarative memory are described in humans, revealing a female or a male advantage depending on the task. Specifically, spatial memory (i.e., spatial navigation) is typically most efficient in men. This sexual dimorphism has been replicated in male rats but not clearly in mice. In this study, sex differences in spatial memory were assessed in thirty-six C57BL/6 J mice (Janvier Labs; i.e., C57BL/6JRj mice), a widely used mouse substrain. Both male and female mice (12 weeks-old) were subjected to standard behavioral paradigms: the elevated plus maze, the open field test, the novel object and place tests, the forced swimming test, and the water maze test for spatial navigation. Across assessment, no sex differences were found in measures of locomotor activity, emotional and behavioral responses, and object and place recognition memories. In the water maze, male mice were faster in learning the platform location in the reference memory training and used more spatial strategies during the first training days. However, both sexes reached a similar asymptotic performance and performed similarly in the probe trial for long-term memory consolidation. No sex differences were found in the cued training, platform inversion sessions, or spatial working memory sessions. Hippocampal expression of the brain-derived neurotrophic factor was similar in both sexes, either in basal conditions or after performing the behavioral training battery. Importantly, female mice were not more variable than males in any measure analyzed. This outcome encourages the investigation of sex differences in animal models and the usefulness of including female mice in behavioral research.

Plasma Lysophosphatidic Acid Concentrations in Sex Differences and Psychiatric Comorbidity in Patients with Cocaine Use Disorder.

We have recently reported sex differences in the plasma concentrations of lysophosphatidic acid (LPA) and alterations in LPA species in patients with alcohol and cocaine use disorders. Preclinical evidence suggests a main role of lysophosphatidic acid (LPA) signaling in anxiogenic responses and drug addiction. To further explore the potential role of the LPA signaling system in sex differences and psychiatric comorbidity in cocaine use disorder (CUD), we conducted a cross-sectional study with 88 patients diagnosed with CUD in outpatient treatment and 60 healthy controls. Plasma concentrations of total LPA and LPA species (16:0, 18:0, 18:1, 18:2 and 20:4) were quantified and correlated with cortisol and tryptophan metabolites [tryptophan (TRP), serotonin (5-HT), kynurenine (KYN), quinolinic acid (QUIN) and kynurenic acid (KYNA)]. We found sexual dimorphism for the total LPA and most LPA species in the control and CUD groups. The total LPA and LPA species were not altered in CUD patients compared to the controls. There was a significant correlation between 18:2 LPA and age at CUD diagnosis (years) in the total sample, but total LPA, 16:0 LPA and 18:2 LPA correlated with age at onset of CUD in male patients. Women with CUD had more comorbid anxiety and eating disorders, whereas men had more cannabis use disorders. Total LPA, 18:0 LPA and 20:4 LPA were significantly decreased in CUD patients with anxiety disorders. Both 20:4 LPA and total LPA were significantly higher in women without anxiety disorders compared to men with and without anxiety disorders. Total LPA and 16:0 LPA were significantly decreased in CUD patients with childhood ADHD. Both 18:1 LPA and 20:4 LPA were significantly augmented in CUD patients with personality disorders. KYNA significantly correlated with total LPA, 16:0 LPA and 18:2 LPA species, while TRP correlated with the 18:1 LPA species. Our results demonstrate that LPA signaling is affected by sex and psychiatric comorbidity in CUD patients, playing an essential role in mediating their anxiety symptoms.

A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

Moving From Transactional to Relational: How Funders Can Work in Partnership With Black, Indigenous and People of Color Communities.

BACKGROUND: The Racial Equity Coalition (REC) formed to address persistent educational disparities. The coalition was composed of 14 Black, Indigenous and People of Color (BIPOC) organizations that provide culturally integrative youth services. OBJECTIVES: REC, with support from United Way of King County, engaged in participatory research to identify commonalities and shared struggles to inform collective action. Participatory research aligns with REC's commitment to equitable participatory processes. This article focuses on REC's experiences with funders. The objective was to understand what creates positive and challenging experiences with funders, and to identify recommendations for funders to become more culturally responsive. METHODS: A research committee was formed including representatives of nine REC organizations and United Way of King County staff. The committee conducted interviews with each of the 14 REC organizations and conducted thematic analysis of interview transcripts. Through participatory analysis, the committee drafted narratives that were further refined through a series of research retreats attended by all REC organizations. RESULTS: Recommendations were to incentivize collaboration, listen to communities to create culturally responsive definitions of success and measurement strategies, arrive at mutually agreed upon approaches with organizations, honor the connections BIPOC organizations have with their communities, and provide unrestricted funding to allow BIPOC organizations greater agency. CONCLUSIONS: A major challenge for BIPOC organizations is navigating White dominant culture that too often shows up in funding requirements. Having to fit dominant culture standards stifles BIPOC organizations' abilities to meet community needs and the responsiveness of their approaches. REC identified recommendations for funders to be more culturally responsive and community centered.

A Functional Pipeline of Genome-Wide Association Data Leads to Midostaurin as a Repurposed Drug for Alzheimer's Disease.

Genome-wide association studies (GWAS) constitute a powerful tool to identify the different biochemical pathways associated with disease. This knowledge can be used to prioritize drugs targeting these routes, paving the road to clinical application. Here, we describe DAGGER (Drug Repositioning by Analysis of GWAS and Gene Expression in R), a straightforward pipeline to find currently approved drugs with repurposing potential. As a proof of concept, we analyzed a meta-GWAS of 1.6 × 107 single-nucleotide polymorphisms performed on Alzheimer's disease (AD). Our pipeline uses the Genotype-Tissue Expression (GTEx) and Drug Gene Interaction (DGI) databases for a rational prioritization of 22 druggable targets. Next, we performed a two-stage in vivo functional assay. We used a C. elegans humanized model over-expressing the Aß1-42 peptide. We assayed the five top-scoring candidate drugs, finding midostaurin, a multitarget protein kinase inhibitor, to be a protective drug. Next, 3xTg AD transgenic mice were used for a final evaluation of midostaurin's effect. Behavioral testing after three weeks of 20 mg/kg intraperitoneal treatment revealed a significant improvement in behavior, including locomotion, anxiety-like behavior, and new-place recognition. Altogether, we consider that our pipeline might be a useful tool for drug repurposing in complex diseases.

Desmoplastic stroma restricts T cell extravasation and mediates immune exclusion and immunosuppression in solid tumors.

The desmoplastic stroma in solid tumors presents a formidable challenge to immunotherapies that rely on endogenous or adoptively transferred T cells, however, the mechanisms are poorly understood. To define mechanisms involved, here we treat established desmoplastic pancreatic tumors with CAR T cells directed to fibroblast activation protein (FAP), an enzyme highly overexpressed on a subset of cancer-associated fibroblasts (CAFs). Depletion of FAP+ CAFs results in loss of the structural integrity of desmoplastic matrix. This renders these highly treatment-resistant cancers susceptible to subsequent treatment with a tumor antigen (mesothelin)-targeted CAR T cells and to anti-PD-1 antibody therapy. Mechanisms include overcoming stroma-dependent restriction of T cell extravasation and/or perivascular invasion, reversing immune exclusion, relieving T cell suppression, and altering the immune landscape by reducing myeloid cell accumulation and increasing endogenous CD8+ T cell and NK cell infiltration. These data provide strong rationale for combining tumor stroma- and malignant cell-targeted therapies to be tested in clinical trials.

How Healthy Is It to Fortify Cocoa-Based Products with Cocoa Flavanols? A Comprehensive Review.

(1) Background: Cocoa's healthy benefits may be attributed to the potent antioxidant activity of cocoa polyphenols, mainly flavanols, which have been characterised as existing in a high concentration in cocoa. However, the phenolic composition of cocoa and cocoa-derived products is highly variable, and manufacturing processes might significantly reduce their phenolic content. For that reason, the full characterisation of cocoa and cocoa-derived products before evaluating their bioactivity is crucial. The aim of this review is to analyse the available evidence on the effect of flavanol-fortified cocoa-derived products on human health. (2) Methods: Forty-eight clinical trials focused on the health effect of consuming flavanol-fortified drinks, bars and chocolate have been reviewed, with a total of 1523 subjects. (3) Results: Although studies differ widely in methodology, dosage, duration, and target population, beneficial effects of flavanol-rich cocoa consumption have been observed at doses ranging from 45.3 mg/d to 1078 mg/d, especially on cardiovascular health and cognitive function. (4) Conclusions: Considering the high consumption and acceptability of cocoa and cocoa-derived products, the fortification of cocoa products as well as other highly consumed foods with cocoa flavanols could be an effective strategy for health promotion.

miRNA-132/212 Deficiency Disrupts Selective Corticosterone Modulation of Dorsal vs. Ventral Hippocampal Metaplasticity.

Cortisol is a potent human steroid hormone that plays key roles in the central nervous system, influencing processes such as brain neuronal synaptic plasticity and regulating the expression of emotional and behavioral responses. The relevance of cortisol stands out in the disease, as its dysregulation is associated with debilitating conditions such as Alzheimer's Disease, chronic stress, anxiety and depression. Among other brain regions, cortisol importantly influences the function of the hippocampus, a structure central for memory and emotional information processing. The mechanisms fine-tuning the different synaptic responses of the hippocampus to steroid hormone signaling remain, however, poorly understood. Using ex vivo electrophysiology and wild type (WT) and miR-132/miR-212 microRNAs knockout (miRNA-132/212-/-) mice, we examined the effects of corticosterone (the rodent's equivalent to cortisol in humans) on the synaptic properties of the dorsal and ventral hippocampus. In WT mice, corticosterone predominantly inhibited metaplasticity in the dorsal WT hippocampi, whereas it significantly dysregulated both synaptic transmission and metaplasticity at dorsal and ventral regions of miR-132/212-/- hippocampi. Western blotting further revealed significantly augmented levels of endogenous CREB and a significant CREB reduction in response to corticosterone only in miR-132/212-/- hippocampi. Sirt1 levels were also endogenously enhanced in the miR-132/212-/- hippocampi but unaltered by corticosterone, whereas the levels of phospo-MSK1 were only reduced by corticosterone in WT, not in miR-132/212-/- hippocampi. In behavioral studies using the elevated plus maze, miRNA-132/212-/- mice further showed reduced anxiety-like behavior. These observations propose miRNA-132/212 as potential region-selective regulators of the effects of steroid hormones on hippocampal functions, thus likely fine-tuning hippocampus-dependent memory and emotional processing.

Plasma concentrations of lysophosphatidic acid and the expression of its receptors in peripheral blood mononuclear cells are altered in patients with cocaine use disorders.

We have recently reported alterations in the plasma concentrations of lysophosphatidic acid (LPA) in patients with substance use disorders. In order to further explore the potential role of the LPA signaling system as biomarker in cocaine use disorders (CUD) we conducted a cross-sectional study with 105 patients diagnosed with CUD and 92 healthy controls. Participants were clinically evaluated and blood samples were collected to determine plasma concentrations of total LPA and LPA species (16:0-, 18:0-, 18:1-, 18:2-, and 20:4-LPA), and the gene expression of LPA1 and LPA2 receptors in peripheral blood mononuclear cells. We found that patients with CUD had significantly lower plasma concentration of the majority of LPA species, while the mRNA expression of LPA1 receptor was found to be higher than controls. Moreover, we found a positive association between plasma concentration of 20:4-LPA and relevant CUD-related variables: age of onset cocaine use and length of cocaine abstinence. The statistical analysis revealed sex differences in concentrations of total LPA and LPA species, and women showed higher LPA concentrations than men. Furthermore, studies in rats of both sexes showed that plasma concentrations of total LPA were also altered after acute and chronic cocaine administration, revealing a sexual dimorphism in these effects. This study found alterations on the LPA signaling system in both, patients with CUD and rats treated with cocaine. Our results demonstrate that LPA signaling is impacted by CUD and sex, which must be taken into consideration in future studies evaluating LPA as a reliable biomarker for CUD.

Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors.

Chimeric antigen receptor (CAR) T cells demonstrate remarkable success in treating hematological malignancies, but their effectiveness in non-hematopoietic cancers remains limited. This study proposes enhancing CAR T cell function and localization in solid tumors by modifying the epigenome governing tissue-residency adaptation and early memory differentiation. We identify that a key factor in human tissue-resident memory CAR T cell (CAR-TRM) formation is activation in the presence of the pleotropic cytokine, transforming growth factor ß (TGF-ß), which enforces a core program of both "stemness" and sustained tissue residency by mediating chromatin remodeling and concurrent transcriptional changes. This approach leads to a practical and clinically actionable in vitro production method for engineering peripheral blood T cells into a large number of "stem-like" CAR-TRM cells resistant to tumor-associated dysfunction, possessing an enhanced ability to accumulate in situ and rapidly eliminate cancer cells for more effective immunotherapy.

Desmoplastic stroma restricts T cell extravasation and mediates immune exclusion and immunosuppression in solid tumors.

The desmoplastic stroma in solid tumors presents a formidable challenge to immunotherapies that rely on endogenous or adoptively transferred T cells, however, the mechanisms are poorly understood. To define mechanisms involved, we treat established desmoplastic pancreatic tumors with CAR T cells directed to fibroblast activation protein (FAP), an enzyme highly overexpressed on a subset of cancer-associated fibroblasts (CAFs). Depletion of FAP+CAFs results in loss of the structural integrity of desmoplastic matrix. This renders these highly treatment-resistant cancers susceptible to subsequent treatment with a tumor antigen (mesothelin)-targeted CAR and to anti-PD1 antibody therapy. Mechanisms include overcoming stroma-dependent restriction of T cell extravasation and/or perivascular invasion, reversing immune exclusion, relieving T cell suppression, and altering the immune landscape by reducing myeloid cell accumulation and increasing endogenous CD8+ T cell and NK cell infiltration. These data provide strong rationale for combining tumor stroma- and malignant cell-targeted therapies to be tested in clinical trials.

Exploring the Nutritional Composition and Bioactive Compounds in Different Cocoa Powders.

Cocoa, the main derivative of the seeds of Theobroma cacao L., has been recognized to have several effects on human health including antioxidant and neuro- and cardio-protective effects, among others. These effects have been attributed mainly to its bioactive compounds. In this context, the aim of this work is to evaluate the nutritional composition, bioactive compounds (i.e., phenolic compounds, procyanidins and methylxanthines) and the antioxidant activity of seven different cocoas (alkalized and non-alkalized) from different origins (Peru, Venezuela, Ivory Coast, Dominican Republic, and West Africa). It represents the first stage of a larger project aiming to find high polyphenol cocoa-based nutritional strategies and related biomarkers that may potentiate brain plasticity and cognitive function. Cocoa powders were extracted by ultrasound-assisted technology, and the total phenolic content (TPC) was measured by Folin-Ciocalteu. Methylxanthines (caffeine and theobromine) and procyanidin contents were determined by HPLC-FLD-DAD, and the antioxidant activity was assessed through DPPH, ABTS and FRAP assays. Non-alkalized cocoas showed higher phenolic and procyanidin contents and higher antioxidant activity compared to the alkalized ones. A strongly significant (p < 0.05) positive correlation between the antioxidant activity and the TPC, especially with the total procyanidin content, but not with methylxanthines was found. In conclusion, the non-alkalized cocoas, especially the one from Peru, were the best candidates in terms of bioactive compounds. The cocoa from Peru had a TPC of 57.4 ± 14.4 mg of gallic acid equivalent/g d.w., 28,575.06 ± 62.37 µg of catechin equivalents/g d.w., and 39.15 ± 2.12 mg/g of methylxanthines. Further studies should be undertaken to evaluate its effect on brain plasticity and cognitive function.

Nutrition and adult neurogenesis in the hippocampus: Does what you eat help you remember?

Neurogenesis is a complex process by which neural progenitor cells (NPCs)/neural stem cells (NSCs) proliferate and differentiate into new neurons and other brain cells. In adulthood, the hippocampus is one of the areas with more neurogenesis activity, which is involved in the modulation of both emotional and cognitive hippocampal functions. This complex process is affected by many intrinsic and extrinsic factors, including nutrition. In this regard, preclinical studies performed in rats and mice demonstrate that high fats and/or sugars diets have a negative effect on adult hippocampal neurogenesis (AHN). In contrast, diets enriched with bioactive compounds, such as polyunsaturated fatty acids and polyphenols, as well as intermittent fasting or caloric restriction, can induce AHN. Interestingly, there is also growing evidence demonstrating that offspring AHN can be affected by maternal nutrition in the perinatal period. Therefore, nutritional interventions from early stages and throughout life are a promising perspective to alleviate neurodegenerative diseases by stimulating neurogenesis. The underlying mechanisms by which nutrients and dietary factors affect AHN are still being studied. Interestingly, recent evidence suggests that additional peripheral mediators may be involved. In this sense, the microbiota-gut-brain axis mediates bidirectional communication between the gut and the brain and could act as a link between nutritional factors and AHN. The aim of this mini-review is to summarize, the most recent findings related to the influence of nutrition and diet in the modulation of AHN. The importance of maternal nutrition in the AHN of the offspring and the role of the microbiota-gut-brain axis in the nutrition-neurogenesis relationship have also been included.

Temozolomide treatment inhibits spontaneous motivation for exploring a complex object in mice: A potential role of adult hippocampal neurogenesis in "curiosity".

Intrinsic exploratory biases are an innate motivation for exploring certain types of stimuli or environments over others, and they may be associated with cognitive, emotional, and even personality-like traits. However, their neurobiological basis has been scarcely investigated. Considering the involvement of the hippocampus in novelty recognition and in spatial and pattern separation tasks, this work researched the role of adult hippocampal neurogenesis (AHN) in intrinsic exploratory bias for a perceptually complex object in mice. Spontaneous object preference tasks revealed that both male and female C57BL/6J mice showed a consistent unconditioned preference for exploring "complex"-irregular-objects over simpler ones. Furthermore, increasing objects' complexity resulted in an augmented time of object exploration. In a different experiment, male mice received either vehicle or the DNA alkylating agent temozolomide (TMZ) for 4 weeks, a pharmacological treatment that reduced AHN as evidenced by immunohistochemistry. After assessment in a behavioral test battery, the TMZ-treated mice did not show any alterations in general exploratory and anxiety-like responses. However, when tested in the spontaneous object preference task, the TMZ-treated mice did not display enhanced exploration of the complex object, as evidenced both by a reduced exploration time-specifically for the complex object-and a lack of preference for the complex object over the simple one. This study supports a novel role of AHN in intrinsic exploratory bias for perceptual complexity. Moreover, the spontaneous complex object preference task as a rodent model of "curiosity" is discussed.

Environmental enrichment alleviates cognitive and psychomotor alterations and increases adult hippocampal neurogenesis in cocaine withdrawn mice.

Cocaine is a widely used psychostimulant drug whose repeated exposure induces persistent cognitive/emotional dysregulation, which could be a predictor of relapse in users. However, there is scarce evidence on effective treatments to alleviate these symptoms. Environmental enrichment (EE) has been shown to be associated with improved synaptic function and cellular plasticity changes related to adult hippocampal neurogenesis (AHN), resulting in cognitive enhancement. Therefore, EE could mitigate the negative impact of chronic administration of cocaine in mice and reduce the emotional and cognitive symptoms present during cocaine abstinence. In this study, mice were chronically administered with cocaine for 14 days, and control mice received saline. After the last cocaine or saline dose, mice were submitted to control or EE housing conditions, and they stayed undisturbed for 28 days. Subsequently, mice were evaluated with a battery of behavioural tests for exploratory activity, emotional behaviour, and cognitive performance. EE attenuated hyperlocomotion, induced anxiolytic-like behaviour and alleviated cognitive impairment in spatial memory in the cocaine-abstinent mice. The EE protocol notably upregulated AHN in both control and cocaine-treated mice, though cocaine slightly reduced the number of immature neurons. Altogether, these results demonstrate that EE could enhance hippocampal neuroplasticity ameliorating the behavioural and cognitive consequences of repeated administration of cocaine. Therefore, environmental stimulation may be a useful strategy in the treatment cocaine addiction.