Murine Studies Research Articles

Long-term in vitro expansion of a human fetal pancreas stem cell that generates all three pancreatic cell lineages

The mammalian pancreas consists of three epithelial compartments: the acini and ducts of the exocrine pancreas and the endocrine islets of Langerhans. Murine studies indicate that these three compartments derive from a transient, common pancreatic progenitor. Here, we report derivation of 18 human fetal pancreas organoid (hfPO) lines from gestational weeks 8-17 (8-17 GWs) fetal pancreas samples. Four of these lines, derived from 15 to 16 GWs samples, generate acinar-, ductal-, and endocrine-lineage cells while expanding exponentially for >2 years under optimized culture conditions. Single-cell RNA sequencing identifies rare LGR5+ cells in fetal pancreas and in hfPOs as the root of the developmental hierarchy. These LGR5+ cells share multiple markers with adult gastrointestinal tract stem cells. Organoids derived from single LGR5+ organoid-derived cells recapitulate this tripotency invitro. We describe a human fetal tripotent stem/progenitor cell capable of long-term expansion invitro and of generating all three pancreatic cell lineages.

Exploring the regenerative capacity of the spleen following irradiation

IntroductionHaematopoietic stem cell transplantation (HSCT) is commonly used to treat patients with haematological disorders. Myeloablative conditioning is an important preparation for patients receiving haematopoietic stem cells (HSC) or haematopoietic stem and progenitor cells (HSPC). While widely successful, HSCT is still associated with high rates of mortality. The recovery time between complete myeloablation and haematopoietic recovery is a large factor in the recovery rate. Successful engraftment of HSC is also directly correlated with the number of HSC niches available. This highlights the importance of the haematopoietic niche and its recovery from myeloablation as an important therapeutic target.MethodsThis murine model study specifically considers changes in spleen tissue architecture and cellular composition involving stromal and vascular cells that occur following lethal irradiation.ResultsSpleen recovered fully between 4- and 8-weeks after irradiation due to reconstitution by HSPC from bone marrow. Specific temporal changes in spleen architecture were identified, and these were linked to the cell types that constitute the white pulp, red pulp and marginal zones. Mesenchymal stromal cells returned before endothelial cells, and reticular cell types recovered more quickly in spleen following irradiation. Losses in gp38+ fibroblastic reticular cells and MAdCAM-1+ marginal reticular cells were associated with loss of the white pulp in the first 4 weeks following irradiation. White pulp was restored following recovery of supporting reticular cells.DiscussionThis study tests how spleen regeneration following a lethal dose of irradiation can be influenced by co-infusion of bone marrow HSPC together with either neonatal spleen stromal cells, or cells of the stromal STX3 line. Both the infusion of neonatal spleen stromal cells and STX3 stromal cells hastened recovery of both mesenchymal and vascular compartments. Following neonatal spleen stromal cell infusion, endothelial cells increased early, but a delay in structural reformation of distinct red and white pulp areas was found. Results from this study show that spleen regeneration can be influenced and even hastened through cellular therapy. Neonatal spleen stromal cells, co-infused together with HSPC following irradiation conditioning, represent a potential therapeutic opportunity for hastening spleen regeneration.

EXTH-76. REDIRECTING STROMAL CELLS TO IGNITE ANTI-TUMOR IMMUNITY AGAINST GLIOBLASTOMA

Abstract BACKGROUND The dismal prognosis of high-grade gliomas demands novel treatment approaches. Through the development of a novel mRNA lipid particle aggregate (RNA-LPA) vaccine, our lab has shown anti-tumor efficacy in preclinical murine studies and promising immunological responses in canine glioma models and human studies (Cell. 2024 May 9;187(10):2521-2535.e21). The translational potential necessitates further investigating the immune activation mechanism to allow greater applicability of this therapy in the future. OBJECTIVE We sought to unravel mechanistic cross-talk for immune initiation following intravenous administration of RNA-LPAs. METHODS We used Ai14 reporter mice to determine RNA-LPA transfected cellular subsets and dual reporter mice to assess cellular infiltration into murine glioma models. Murine gliomas were generated via implantation of K2 or KR158b-luc cell lines. RESULTS Following intravenous administration, we found fibroblastic reticular cells in secondary lymphoid organs take up RNA-LPAs. We show that FRCs have direct contact with antigen presenting cells and mediate release of full-length proteins following RNA-LPA transfection. Similar to the periphery, there is transfection of stromal cells in the brain tumor microenvironment (TME) and recruitment of infiltrating leukocytes. In response to RNA-LPAs, transfected stromal cells release type I interferon (IFN-I), which is requisite for anti-tumor efficacy. IFN-I mediates upregulation of CD69 on lymphocytes leading to sequestration following co-localization with RNA-LPAs. IFN-I is necessary for LFA-I expression on T cells, which is important for T cell trafficking across the blood-brain barrier. In preclinical models to track antigen specific immunity, we find systemic RNA administration elicits an increase in tumor reactive T cells with decreased S1P1 expression suggestive of a ‘locked-in’ response within the TME. CONCLUSION While stromal cells are regulatory niches in glioblastoma ecosystems, we show they can be leveraged for immunologic cross-talk that facilitates T cell recruitment, trafficking and sequestration in the TME critical for immunologic reprogramming and long-lasting immunotherapy.

Screening and validating the optimal panel of housekeeping genes for 4T1 breast carcinoma and metastasis studies in mice

The 4T1 model is extensively employed in murine studies to elucidate the mechanisms underlying the carcinogenesis of triple-negative breast cancer. Molecular biology serves as a cornerstone in these investigations. However, accurate gene expression analyses necessitate data normalization employing housekeeping genes (HKGs) to avert spurious results. Here, we initially delve into the characteristics of the tumor evolution induced by 4T1 in mice, underscoring the imperative for additional tools for tumor monitoring and assessment methods for tracking the animals, thereby facilitating prospective studies employing this methodology. Subsequently, leveraging various software platforms, we assessed ten distinct HKGs (GAPDH, 18 S, ACTB, HPRT1, B2M, GUSB, PGK1, CCSER2, SYMPK, ANKRD17) not hitherto evaluated in the 4T1 breast cancer model, across tumors and diverse tissues afflicted by metastasis. Our principal findings underscore GAPDH as the optimal HKG for gene expression analyses in tumors, while HPRT1 emerged as the most stable in the liver and CCSER2 in the lung. These genes demonstrated consistent expression and minimal variation among experimental groups. Furthermore, employing these HKGs for normalization, we assessed TNF-α and VEGF expression in tissues and discerned significant disparities among groups. We posit that this constitutes the inaugural delineation of an ideal HKG for experiments utilizing the 4T1 model, particularly in vivo settings.

A novel solvent-extruded tacrolimus-eluting suture for attenuated inflammation and scarring in skin repair

This study aimed to design an absorbable suture that locally delivers the immunosuppressive agent, tacrolimus (FK506), to attenuate scar tissue formation by modulating inflammation. As the suture degrades, it releases FK506 directly into the repair site. The poly (l-lactide-co-caprolactone) (PLLA-PCL) polymer and 0.2 % wt FK506 were co-extruded using a solvent extrusion technique to generate the drug-eluting sutures. The properties of the generated sutures were assessed regarding FK506 release in vitro, wound closure in an in vivo murine model, murine skin histology for structural and inflammatory markers, and mechanical strength of both sutures and healed murine skin. The generated suture consistently released FK506 at ≤10 ng/day rates. Following annealing, the sutures retained 60 % of their anticipated tensile strength, exhibiting enhanced ductility. The histological examination and in vivo murine study indicated that wounds treated with FK506-eluting PLLA-PCL sutures produced a thinner epidermal layer, enhanced integrin β4 expression, increased keratinocyte presence, attenuated inflammation, and reduced scarring, demonstrating healed tissue improvements compared with those treated with the placebo, poly (glycolic-co-caprolactone) (PGCL), sutures. Mechanical evaluations of healed tissue indicated that drug-PLLA-PCL sutures did not reduce the mechanical strength of the skin compared to placebo PGCL sutures. The FK506-eluting PLLA-PCL sutures show promising results in advancing wound healing by offering targeted drug delivery. This innovative method can improve wound healing and provide a streamlined approach to scar management. Continued research, optimization, and broad-scale studies are needed to refine the manufacturing process and ensure that the properties of the suture meet clinical requirements.

Development of a murine laser interstitial thermotherapy system.

The objective of this study was to develop a murine system for the delivery of laser interstitial thermotherapy (LITT) with probe-based thermometry as a model for human glioblastoma treatment to investigate thermal diffusion in heterogeneous brain tissue. First, the tissue heating properties were characterized using a diode-pumped solid-state near-infrared laser in a homogeneous tissue model. The laser was adapted for use with a repurposed stereotactic surgery frame utilizing a micro laser probe and Hamilton syringe. The authors designed and manufactured a stereotactic frame attachment to work as a temperature probe stabilizer. Application of this novel design was used as a precise method for real-time thermometry at known distances from the thermal ablative center mass during murine LITT studies. Temperature measurements were achieved during LITT that verified the direct thermometry capability of the system without the need for MR-based thermal monitoring. Application of multiple stereotactic design iterations led to an accurately reproducible surgical laser ablation procedure. Histological staining confirmed precise thermal ablation and controllable lesion size based on time and temperature control. Treatment of a syngeneic intracranial glioma model highly resistant to conventional therapy resulted in a modest survival benefit. The authors have successfully developed a murine model system of LITT with direct in situ thermometry for investigation into the effects of thermal ablation and combinatorial treatments in murine brain tumor models.

Antibiotic-associated neutropenia is marked by the depletion of intestinal Lachnospiraceae and associated metabolites in pediatric patients.

Prolonged antibiotic exposure causes dangerous hematologic side effects, including neutropenia, in up to 34% of patients. Murine studies established a link between the intestinal microbiota and hematopoiesis. To identify factors that predispose to neutropenia in pediatric patients, we evaluated changes in microbiota-derived metabolites and intestinal microbiota composition after prolonged courses of antibiotics. In this multi-center study, patients with infections requiring anticipated antibiotic treatment of two or more weeks were enrolled. Stool samples were obtained at the start and completion of antibiotics or at neutropenia onset (prospective arm). Some patients were enrolled in a retrospective arm in which a stool sample was collected at the time of neutropenia during antibiotic therapy and 2-4 weeks after completion of antibiotics with recovery of blood counts. We identified 10 patients who developed neutropenia on antibiotics and 29 controls matched for age, sex, race, and ethnicity. Clinical data demonstrated no association between neutropenia and the type of infection or antibiotic used; however, patients with neutropenia were admitted to the intensive care unit more often and received longer courses of antibiotics. Reduced intestinal microbiome richness and, specifically, decreased abundance of Lachnospiraceae family members correlated with neutropenia. Untargeted stool metabolomic profiling revealed several metabolites that were depleted exclusively in patients with neutropenia, including members of the urea cycle pathway, pyrimidine metabolism, and fatty acid metabolism that are known to be produced by Lachnospiraceae. Our study shows a relationship between intestinal microbiota disruption and abnormal hematopoiesis and identifies taxa and metabolites likely to contribute to microbiota-sustained hematopoiesis.

Efficacy of Myricetin Supplementation on Glucose and Lipid Metabolism: A Systematic Review and Meta-Analysis of In Vivo Mice Studies.

Type 2 diabetes mellitus (T2DM) is a disorder characterized by insulin resistance, hyperglycemia, and dyslipidemia. Myricetin, a flavonoid found in various plants, has shown potential anti-diabetic effects in murine studies. This meta-analysis aimed to evaluate the impact of myricetin supplementation on glucose metabolism and lipid profiles in mouse models of metabolic diseases. A systematic review and meta-analysis were conducted in accordance with PRISMA guidelines (PROSPERO: CRD42024591569). Studies involving mice with metabolic disease models and exclusively using myricetin supplementation were checked across four databases (Embase, Scopus, PubMed, and WoS) until 23rd September 2024. The primary outcomes assessed were blood glucose (BG), insulin levels, triacylglycerol (TAG), total cholesterol (TC), HDL, and LDL. A random-effects model was applied to estimate standardized mean differences (SMD), and SYRCLE's risk-of-bias tool for animal studies was used. Twenty-one studies with 514 mice met the inclusion criteria. Myricetin supplementation significantly reduced BG (SMD = -1.45, CI: -1.91 to -0.99, p < 0.00001, I2 = 74%), insulin (SMD = -1.78, CI: -2.89 to -0.68, p = 0.002, I2 = 86%), TAG (SMD = -2.60, CI: -3.24 to -1.96, p < 0.00001, I2 = 81%), TC (SMD = -1.86, CI: -2.29 to -1.44, p < 0.00001, I2 = 62%), and LDL (SMD = -2.95, CI: -3.75 to -2.14, p < 0.00001, I2 = 74%). However, the effect on HDL was not statistically significant (SMD = 0.71, CI: -0.01 to 1.43, p = 0.05, I2 = 83%). Myricetin supplementation improved glucose metabolism and lipid profiles in mouse models, suggesting its potential as a therapeutic agent for managing T2DM. However, further research is needed to confirm these findings in human studies.

Endometriosis development in relation to hypoxia: a murine model study.

Endometriosis, due to its ambiguous symptoms, still remains one of the most difficult female diseases to treat, with an average diagnosis time of 7-9years. The changing level of hypoxia plays an important role in a healthy endometrium during menstruation and an elevated expression of the hypoxia-inducible factor 1-alpha (HIF-1α) has been demonstrated in ectopic endometria. HIF-1α mediates the induction of proangiogenic factors and the development of angiogenesis is a critical step in the establishment and pathogenesis of endometriosis. Although the inhibition of angiogenesis has been proposed as one of the actionable therapeutic modalities, vascular normalization and re-oxygenation may become a possible new approach for therapeutic intervention. Our goal was to investigate whether a selected murine model of endometriosis would be suitable for future studies on new methods for treating endometriosis. Non-invasive, high-resolution ultrasound-monitored observation was selected as the preclinical approach to obtain imaging of the presence and volume of the endometriotic-like lesions. The EF5 (2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide) compound that selectively binds to reduced proteins in hypoxic cells was used for hypoxia detection. The expression of Pten and other crucial genes linking endometriosis and hypoxia were also assessed. Using EF5, a pentafluorinated derivative of the 2-nitroimidazole that is metabolically reduced by oxygen-inhibitable nitroreductase, we confirmed that hypoxia did develop in the selected model and was detected in uterine and ectopic endometriotic lesions. Moreover, the changes in oxygen tension also influenced the expression level of significant genes related to endometriosis, like Pten, Trp53, Hif1a, Epas1, and Vegfa. Their strong modulation evidenced here is indicative of model reliability. Using high-resolution ultrasound-based imaging, we present a non-invasive method of visualization that enables the detection and observation of lesion evolution throughout the duration of the experiment, which is fundamental for further preclinical studies and treatment evaluation. The selected model and method of visualization appear to be suitable for the study of new treatment strategies based on hypoxia alleviation and blood flow restoration.

Profiling the L-arginine/arginase 1 metabolism in early and advanced atherosclerosis

Abstract Background Atherosclerosis is a chronic inflammatory disease and the primary underlying cause of cardiovascular disease (CVD). Recently, intracellular metabolic pathways have been identified as master switches of immune cell function and thus seem promising targets for therapeutic interventions aimed at lowering inflammation and thereby atherogenesis. Decades of research have demonstrated the importance of the amino acid (AA) L-arginine (Arg) in CVD. Although most research has focused on endothelium-dependent effects of Arg by the nitric oxide synthase (NOS), Arg metabolism - mainly through its metabolizing enzyme arginase 1 (Arg1) - has also been shown to regulate immune cell responses independent of its endothelial functions. Despite these findings, the cell-specific contribution of Arg/Arg1 metabolism on atherogenesis is still largely unknown. Purpose Profiling the Arg metabolism in endothelial and immune cells to identify cell-specific changes during athero-progression. Methods To investigate Arg metabolism in atherogenesis, we fed apolipoprotein E deficient (Apoe-­/­-) mice a Western diet (WD) for 6 and 12 weeks. Chow diet-fed aged-matched Apoe-­/-­ mice were used as littermate controls and C57BL/6 mice as steady-state controls. Systemic and local Arg concentrations were measured by mass-spectrometry. Arg metabolism in organs was studied by qPCR. Arg1 expression in atherosclerotic lesions was examined by immunohistochemistry (IHC). To assess cell-specific expression of Arg-related enzymes, aortic leukocytes from WD-fed Apoe-­/-­ mice were subjected to CITE-seq analysis. The role of T cell Arg1 was determined by nor-NOHA inhibition in Jurkat cells. Results AA measurements showed that systemic Arg was reduced in early and advanced atherogenesis (p&amp;lt;0.02), and splenic Arg was reduced in advanced atherogenesis (p=0.006) compared with steady state. In advanced atherosclerosis, Arg1 was decreased in the liver but increased in the spleen and aorta, whereas Nos2 was increased in the liver and aorta compared with steady state. To identify the cell types driving these local changes, we performed IHC on atherosclerotic lesions. Arg1+ lesional macrophages accumulated in advanced compared to early lesions (p=0.03), whereas Arg1+ endothelial cells tended to decrease (p=0.2). Surprisingly, we also found an accumulation of Arg1+ CD4+ T cells in lesions and a further upward trend with athero-progression. CITE-seq confirmed a constitutive expression of Arg1 in lymphoid clusters. In vitro experiments with Jurkat cells showed that arginase inhibition suppressed T cell proliferation and promoted apoptosis and migration (all p&amp;lt;0.0001). Conclusion Our data indicate that Arg metabolism is strongly regulated during athero-progression and suggest a so far unknown role of T cell-intrinsic Arg/Arg1 metabolism in the disease. Murine atherosclerosis studies are now being performed to decipher the cell-specific contribution of Arg/Arg1 metabolism in atherosclerosis.

Major contributors to FLASH sparing efficacy emerge from murine skin studies: dose rate, total dose per fraction, anesthesia and oxygenation.

Normal tissue sparing from radiation damage upon ultra-high dose rate irradiation, known as the FLASH effect with an equivalent tumor response, has been widely reported in murine skin models, and translation of this type of radiotherapy to humans has already begun, with skin sparing being a primary outcome expected. This study reviews the status of the field, focusing on the proposed mechanisms and skin response assays, outlining what has become known in terms of input parameters that might control the magnitude of the FLASH effect. Murine studies have largely focused on acute damage responses, developing over 3-8 weeks, to single doses of FLASH versus conventional dose rate (CDR), suggesting that at dose rates above tens of Gray per second, with a total dose of more than 20 Gy, the FLASH effect is induced. Fractionated delivery appears to be possible, although fraction sizes >17 Gy appear to be needed for sparing efficacy. The interplay between the dose rate and total dose per fraction remains to be fully elucidated. Oxygen is a modulator of efficacy, with both hypoxia and hyperoxia diminishing the effect of FLASH. Measurement of transient changes in oxygen levels is possible and may be a marker of treatment efficacy. Taken together, murine skin data provide important information for translational studies, despite the associated limitations. Studies of later-term sparing effects, as well as studies on pig skin, are needed to take the next step in assessing translational FLASH efficacy. The control of biological factors, such as tissue oxygenation, may be required to understand and control the response.

Why hematopoietic stem cells fail in Fanconi anemia: Mechanisms and models.

Fanconi anemia (FA) is generally classified as a DNA repair disorder, conferring a genetic predisposition to cancer and prominent bone marrow failure (BMF) in early childhood. Corroborative human and murine studies point to a fetal origin of hematopoietic stem cell (HSC) attrition under replicative stress. Along with intriguing recent insights into non-canonical roles and domain-specific functions of FA proteins, these studies have raised the possibility of a DNA repair-independent BMF etiology. However, deeper mechanistic insight is critical as current curative options of allogeneic stem cell transplantation and emerging gene therapyhave limited eligibility, carry significant side effects, and involve complex procedures restricted to resource-rich environments. To develop rational and broadly accessible therapies for FA patients, the field will need more faithful disease models that overcome the scarcity of patient samples, leverage technological advances, and adopt investigational clinical trial designs tailored for rare diseases.

Evaluate the safety of a novel photohydrolysis technology used to clean and disinfect indoor air: A murine study.

There is a pressing need to develop new technologies that continuously eliminates harmful pollutants and pathogens in occupied indoor spaces without compromising safety. This study was undertaken to test the safety of a novel air cleaning and disinfection technology called Advanced Photohydrolysis. Advanced Photohydrolysis generates a complex mixture of ions and molecules that are released into the air and has been shown to reduce airborne and surface pathogens. Mice (6-8-week-old) were exposed to therapeutic levels of Advanced Photohydrolysis for 90-days. During the study, the Advanced-Photohydrolysis-exposed and control mice were monitored for food consumption, body weight gain, and any overt adverse effects. In addition, at the conclusion of the study, the blood chemistry and hematology values of both groups were determined. Finally, the tissues of the conduction and respiratory portions of the airways of mice from both groups were examined for any pathological changes. The mice of both groups were found to be normal and healthy throughout the 90-day study; there were no differences in the behavior, food consumption and weight gain. Analysis of clinical chemistry values found no differences in hepatocellular function or other markers of cellular and organ function, and clinical hematology values were also unremarkable. Finally, and importantly, histopathology of the upper and lower airway tissues showed no deleterious effects. These results are the first to demonstrate directly the safety of Advanced Photohydrolysis on live mammals and encourage additional studies.

Lymphovascular Tumoral Emboli in Inflammatory Breast Cancer Result from Haptotaxis-Mediated Encircling Lymphangiogenesis

Inflammatory breast cancer (IBC) is characterized by numerous tumor emboli within lymphatics. In a recent study, we observed tumor embolic budding both in vitro and in vivo within lymphovascular spaces and proposed this to account for the plethora of tumor emboli seen in IBC. These observations did not address, however, how lymphovascular invasion is initiated or the mechanisms involved. In the present study, using the well-characterized patient-derived xenograft (PDX), Mary-X, which exhibited florid lymphovascular invasion (LVI) in athymic mice (LVI) as defined by E-cadherin-positive tumor emboli within lymphatic channels distinguished by podoplanin and LYVE1 membrane and Prox1 nuclear immunoreactivities and spontaneous spheroidgenesis in vitro and human cases of IBC which showed similar LVI, we compared laser-captured microdissected emboli from Mary-X and from the cases of human IBC to non-embolic areas. Mary-X and IBC emboli expressed high levels of E-cadherin and no evidence of epithelial–mesenchymal transition (EMT). Mary-X spheroids expressed high levels of VEGF, especially VEGF-C, and stimulated both vascular and lymphatic endothelial haptotaxis. We then transplanted Mary-X serially into green, cyano, red, and nestin-green fluorescing protein (GFP-, CFP-, RFP-, and nestin-GFP) transgenic reporter mice in various combinations. Multicolor murine imaging studies indicated that reporter-labeled stroma initially encircled clumps of tumor cells and then served as a scaffold that supported nestin-GFP-labeled endothelial haptotaxis resulting in encircling lymphangiogenesis, confirmed by dual LYVE1 immunofluorescence. The present studies demonstrate a possible mechanism of a critical step of the tumor emboli formation of IBC.

Spectinamide MBX-4888A exhibits favorable lesion and tissue distribution and promotes treatment shortening in advanced murine models of tuberculosis.

The spectinamides are novel, narrow-spectrum semisynthetic analogs of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis. Spectinamides, including lead MBX-4888A (Lee-1810), exhibit promising therapeutic profiles in mice, as single drugs and as partner agents with other anti-tuberculosis antibiotics including rifampin and/or pyrazinamide. Here, we show that MBX-4888A, given by injection with the front-line standard of care regimen, is treatment shortening in multiple murine tuberculosis infection models. The positive treatment responses to MBX-4888A combination therapy in multiple mouse models, including mice exhibiting advanced pulmonary disease, can be attributed to favorable distribution in tissues and lesions, retention in caseum, along with favorable effects with rifampin and pyrazinamide under conditions achieved in necrotic lesions. This study also provides an additional data point regarding the safety and tolerability of spectinamide MBX-4888A in long-term murine efficacy studies.

Breast cancer cell derived exosomes reduces glycolysis of activated CD8 + T cells in a AKT-mTOR dependent manner.

Cytotoxic CD8+ T cells plays a pivotal role in the adaptive immune system to protect the organism against infections and cancer. During activation and response, T cells undergo a metabolic reprogramming that involves various metabolic pathways, with a predominant reliance on glycolysis to meet their increased energy demands and enhanced effector response. Recently, extracellular vesicles (EVs) known as exosomes have been recognized as crucial signaling mediators in regulating the tumor microenvironment (TME). Recent reports indicates that exosomes may transfer biologically functional molecules to the recipient cells, thereby facilitate cancer progression, angiogenesis, metastasis, drug resistance, and immunosuppression by reprogramming the metabolism of cancer cells. This study sought to enlighten possible involvement of cancer-derived exosomes in CD8 + T cell glucose metabolism and discover a regulated signalome as a mechanism of action. We observed reduction in glucose metabolism due to downregulation of AKT/mTOR signalome in activated CD8 + T cells after cancer derived exosome exposure. In-vivo murine breast tumor studies showed better tumor control and antitumor CD8 + T cell glycolysis and effector response after abrogation of exosome release from breast cancer cells. Summarizing, the present study establishes an immune evasion mechanism of breast cancer cell secreted exosomes that will act as a foundation for future precision cancer therapeutics.

Pro-efferocytic nanotherapies reduce vascular inflammation without inducing anemia in a large animal model of atherosclerosis

Atherosclerosis is an inflammatory disorder responsible for cardiovascular disease. Reactivation of efferocytosis, the phagocytic removal of cells by macrophages, has emerged as a translational target for atherosclerosis. Systemic blockade of the key ‘don’t-eat-me’ molecule, CD47, triggers the engulfment of apoptotic vascular tissue and potently reduces plaque burden. However, it also induces red blood cell clearance, leading to anemia. To overcome this, we previously developed a macrophage-specific nanotherapy loaded with a chemical inhibitor that promotes efferocytosis. Because it was found to be safe and effective in murine studies, we aimed to advance our nanoparticle into a porcine model of atherosclerosis. Here, we demonstrate that production can be scaled without impairing nanoparticle function. At an early stage of disease, we find our nanotherapy reduces apoptotic cell accumulation and inflammation in the atherosclerotic lesion. Notably, this therapy does not induce anemia, highlighting the translational potential of targeted macrophage checkpoint inhibitors.

The bacterial quorum sensing signal 2'-aminoacetophenone rewires immune cell bioenergetics through the Ppargc1a/Esrra axis to mediate tolerance to infection.

How bacterial pathogens exploit host metabolism to promote immune tolerance and persist in infected hosts remains elusive. To achieve this, we show that Pseudomonas aeruginosa (PA), a recalcitrant pathogen, utilizes the quorum sensing (QS) signal 2'-aminoacetophenone (2-AA). Here, we unveil how 2-AA-driven immune tolerization causes distinct metabolic perturbations in murine macrophages' mitochondrial respiration and bioenergetics. We present evidence indicating that these effects stem from decreased pyruvate transport into mitochondria. This reduction is attributed to decreased expression of the mitochondrial pyruvate carrier (Mpc1), which is mediated by diminished expression and nuclear presence of its transcriptional regulator, estrogen-related nuclear receptor alpha (Esrra). Consequently, Esrra exhibits weakened binding to the Mpc1 promoter. This outcome arises from the impaired interaction between Esrra and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a). Ultimately, this cascade results in diminished pyruvate influx into mitochondria and, consequently reduced ATP production in tolerized murine and human macrophages. Exogenously added ATP in infected macrophages restores the transcript levels of Mpc1 and Esrra and enhances cytokine production and intracellular bacterial clearance. Consistent with the in vitro findings, murine infection studies corroborate the 2-AA-mediated long-lasting decrease in ATP and acetyl-CoA and its association with PA persistence, further supporting this QS signaling molecule as the culprit of the host bioenergetic alterations and PA persistence. These findings unveil 2-AA as a modulator of cellular immunometabolism and reveal an unprecedented mechanism of host tolerance to infection involving the Ppargc1a/Esrra axis in its influence on Mpc1/OXPHOS-dependent energy production and PA clearance. These paradigmatic findings pave the way for developing treatments to bolster host resilience to pathogen-induced damage. Given that QS is a common characteristic of prokaryotes, it is likely that 2-AA-like molecules with similar functions may be present in other pathogens.

Links between fecal microplastics and parameters related to metabolic dysfunction-associated steatotic liver disease (MASLD) in humans: An exploratory study

Microplastics (MPs) can persist in the environment and human body. Murine studies showed that exposure to MPs could cause metabolic dysregulation, contributing metabolic dysfunction-associated steatotic liver disease (MASLD) or steatohepatitis (MASH). However, research on the role of MPs in humans is limited. Thus, we aimed to assess links between human fecal MPs and liver histology, gene expression, immune cells and intestinal microbiota (IM). We included 6 lean healthy liver donors and 6 normal liver (obese) and 11 MASH patients. Overall, pre-BSx, we observed no significant differences in fecal MPs between groups. However, fecal MP fibers and total MPs positively correlated with portal and total macrophages and total killer T cells while total fecal MPs were positively correlated with natural killer cells. Additionally, 19 genes related to immune system and apoptosis correlated with fecal MPs at baseline. Fecal MP fibers correlated positively with fecal Bifidobacterium and negatively with Lachnospiraceae. Patients with MASH (n = 11) were re-assessed 12-months post-bariatric surgery (BSx) and we found that those with persistent disease (n = 4) had higher fecal MP fragments than those with normalized liver histology (n = 7). At 12-month post-BSx, MP fragments positively correlated with helper T cells and total MPs positively correlated with natural killer T cells and B cells. Our study is the first to look at 1) the role of MPs in MASH and its association with IM, immune cells and hepatic gene expression and 2) look at the role of MPs longitudinally in MASH persistence following BSx. Future research should further explore this relationship.

Childhood anaesthesia and autism risk: population and murine study.

Early childhood exposure to general anaesthesia has been linked to potential changes in infant brain morphology and behaviour in preclinical studies, contributing to long-term behaviours associated with autism spectrum disorder. This study investigates the association between early childhood exposure to general anaesthesia and the risk of autism, using a population-based cohort study with matching for baseline characteristics and evaluates the effect of sevoflurane exposure on autism-like behaviour in mice, using the Taiwan Maternal and Child Health Database. Children aged 0-3 who received at least one exposure to general anaesthesia between 2004 and 2014 were matched 1:1 with children who were not exposed. Risk ratios and confidence intervals were used to assess the relationship between general anaesthesia and the occurrence of autism. Additionally, mice were exposed to sevoflurane for 2 h on postnatal days 5-7, and changes in behaviour related to autism were evaluated. Propensity score matching resulted in 7530 children in each group. The incidence rates (IRs) of autism were 11.26 and 6.05 per 100 000 person-years in the exposed and unexposed groups, respectively. The incidence ratio for autism following exposure to general anaesthesia was 1.86 (95% confidence interval, 1.34-2.59). In mice, sevoflurane exposure induced autism-like behaviours and led to the downregulation of high-risk autism genes, including ARID1B, GABRA5, GABRB3, GRIN2B, SHANK3 and SUV420H1. Early childhood exposure to general anaesthesia is associated with an increased risk of autism. Repeated exposure to sevoflurane in mice induces autism-like behaviours, suggesting a potential link between anaesthesia and the development of autism.