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Despite recent progress in cancer treatment, liver metastases persist as an unmet clinical need. Here, we show that arming liver and tumor-associated macrophages in vivo to co-express tumor antigens (TAs), IFNα, and IL-12 unleashes robust anti-tumor immune responses, leading to the regression of liver metastases. Mechanistically, in vivo armed macrophages expand tumor reactive CD8 + T cells, which acquire features of progenitor exhausted T cells and kill cancer cells independently of CD4 + T cell help. IFNα and IL-12 produced by armed macrophages reprogram antigen presenting cells and rewire cellular interactions, rescuing tumor reactive T cell functions. In vivo armed macrophages trigger anti-tumor immunity in distinct liver metastasis mouse models of colorectal cancer and melanoma, expressing either surrogate tumor antigens, naturally occurring neoantigens or tumor-associated antigens. Altogether, our findings support the translational potential of in vivo armed liver macrophages to expand and rejuvenate tumor reactive T cells for the treatment of liver metastases. Liver metastases are a clinical problem, with low responses to immunotherapy. Here, authors coordinate expression of tumor antigens IFNα and IL-12 in liver and tumor associated macrophages to rejuvenate tumor reactive T cells and eliminate liver metastases.

Obesity is a chronic, complex pathology associated with a risk of developing secondary pathologies, including cardiovascular diseases, cancer, type 2 diabetes (T2DM) and musculoskeletal disorders. Since skeletal muscle accounts for more than 70% of total glucose disposal, metabolic alterations are strictly associated with the onset of insulin resistance and T2DM. The present study relies on the proteomic analysis of gastrocnemius muscle from 15 male and 15 female C56BL/J mice fed for 14 weeks with standard, 45% or 60% high-fat diets (HFD) adopting a label-free LC–MS/MS approach followed by bioinformatic pathway analysis. Results indicate changes in males due to HFD, with increased muscular stiffness (Col1a1, Col1a2, Actb), fiber-type switch from slow/oxidative to fast/glycolytic (decreased Myh7, Myl2, Myl3 and increased Myh2, Mylpf, Mybpc2, Myl1), increased oxidative stress and mitochondrial dysfunction (decreased respiratory chain complex I and V and increased complex III subunits). At variance, females show few alterations and activation of compensatory mechanisms to counteract the increase of fatty acids. Bioinformatics analysis allows identifying upstream molecules involved in regulating pathways identified at variance in our analysis (Ppargc1a, Pparg, Cpt1b, Clpp, Tp53, Kdm5a, Hif1a). These findings underline the presence of a gender-specific response to be considered when approaching obesity and related comorbidities.

Pulmonary infections caused by Mycobacterium abscessus (MA) have increased over recent decades, affecting individuals with underlying pathologies such as chronic obstructive pulmonary disease, bronchiectasis and, especially, cystic fibrosis. The lack of a representative and standardized model of chronic infection in mice has limited steps forward in the field of MA pulmonary infection. To overcome this challenge, we refined the method of agar beads to establish MA chronic infection in immunocompetent mice. We evaluated bacterial count, lung pathology and markers of inflammation and we performed longitudinal studies with magnetic resonance imaging (MRI) up to three months after MA infection. In this model, MA was able to establish a persistent lung infection for up to two months and with minimal systemic spread. Lung histopathological analysis revealed granulomatous inflammation around bronchi characterized by the presence of lymphocytes, aggregates of vacuolated histiocytes and a few neutrophils, mimicking the damage observed in humans. Furthermore, MA lung lesions were successfully monitored for the first time by MRI. The availability of this murine model and the introduction of the successfully longitudinal monitoring of the murine lung lesions with MRI pave the way for further investigations on the impact of MA pathogenesis and the efficacy of novel treatments.

Ceruloplasmin (Cp) is a ferroxidase that plays a role in cellular iron homeostasis and is mainly expressed in the liver and secreted into the blood. Cp is also produced by adipose tissue, which releases it as an adipokine. Although a dysfunctional interaction of iron with the metabolism of lipids has been associated with several metabolic diseases, the role of Cp in adipose tissue metabolism and in the interplay between hepatocytes and adipocytes has been poorly investigated. We previously found that Cp‐deficient (CpKO) mice become overweight and demonstrate adipose tissue accumulation together with liver steatosis during aging, suggestive of lipid dysmetabolism. In the present study, we investigated the lipid alterations which occur during aging in adipose tissue and liver of CpKO and wild‐type mice both in vivo and ex vivo . During aging of CpKO mice, we observed adipose tissue accumulation and liver lipid deposition, both of which are associated with macrophage infiltration. Liver lipid deposition was characterized by accumulation of triglycerides, fatty acids and ω‐3 fatty acids, as well as by a switch from unsaturated to saturated fatty acids, which is characteristic of lipid storage. Liver steatosis was preceded by iron deposition and macrophage infiltration, and this was observed to be already occurring in younger CpKO mice. The accumulation of ω‐3 fatty acids, which can only be acquired through diet, was associated with body weight increase in CpKO mice despite food intake being equal to that of wild‐type mice, thus underlining the alterations in lipid metabolism/catabolism in Cp‐deficient animals.

Habitual Physical Activity Is Associated With Intrahepatic Fat Content in Humans Gianluca Perseghin , MD 1 2 3 , Guido Lattuada , PHD 1 , Francesco De Cobelli , MD 2 4 , Francesca Ragogna , PHD 1 , Georgia Ntali , MD 1 , Antonio Esposito , MD 4 , Elena Belloni , MD 4 , Tamara Canu 4 , Ileana Terruzzi , PHD 1 , Paola Scifo , PHD 5 , Alessandro Del Maschio , MD 2 4 6 and Livio Luzi , MD 1 2 3 1 Internal Medicine, Section of Nutrition/Metabolism, San Raffaele Scientific Institute, Milan, Italy 2 Unit of Clinical Spectroscopy, San Raffaele Scientific Institute, Milan, Italy 3 Center “Physical Exercise for Health and Wellness,” Faculty of Exercise Sciences, Università degli Studi di Milano, Milan, Italy 4 Diagnostic Radiology, San Raffaele Scientific Institute, Milan, Italy 5 Nuclear Medicine, San Raffaele Scientific Institute, Milan, Italy 6 Università Vita e Salute San Raffaele, Milan, Italy Address correspondence and reprint requests to Gianluca Perseghin, MD, Faculty of Exercise Sciences, Università degli Studi di Milano and San Raffaele Scientific Institute, Internal Medicine, via Olgettina 60, 20132, Milan, Italy. E-mail: perseghin.gianluca{at}hsr.it Abstract OBJECTIVE —Fatty liver may be involved in the pathogenesis of type 2 diabetes. Physical exercise is a tool to improve insulin sensitivity, but little is known about its effect on intrahepatic fat (IHF) content. The purpose of this study was to examine the association of habitual physical activity, insulin resistance, and adiponectin with IHF content. RESEARCH DESIGN AND METHODS —Participants were 191 (77 female and 114 male) apparently healthy, nonalcoholic individuals (aged 19–62 years; BMI 17.0–35.5 kg/m 2 ). IHF content was assessed in a quantitative fashion and noninvasively as a continuous variable by means of 1 H magnetic resonance spectroscopy (MRS), and habitual physical activity was assessed by means of a questionnaire. Fatty liver was defined as IHF content of >5% wet weight, and insulin sensitivity was estimated using the computer homeostasis model assessment (HOMA)-2 indexes. RESULTS —A reduced prevalence of fatty liver in the quartile of the most physically active individuals (25, 11, 25, and 2% in quartile 1, 2, 3, and 4, respectively; χ 2 = 15.63; P = 0.001) was found along with an inverse correlation between the physical activity index and the IHF content when plotted as continuous variables (Pearson’s r = −0.27; P < 0.000). This association was not attenuated when adjusted for age, sex, BMI, HOMA-2, and adiponectin (partial correlation r = −0.25; P < 0.001). CONCLUSIONS —This study demonstrated that a higher level of habitual physical activity is associated with a lower IHF content and suggested that this relationship may be due to the effect of exercise per se. FFA, free fatty acid HOMA, homeostasis model assessment HOMA2-%B, HOMA2-derived index of β-cell insulin sensitivity HOMA2-%S, HOMA2-derived index of insulin sensitivity IHF, intrahepatic fat MRS, magnetic resonance spectroscopy NAFLD, nonalcoholic fatty liver disease TSH, thyroid-stimulating hormone Footnotes A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C Section 1734 solely to indicate this fact. Accepted November 30, 2006. Received October 2, 2006. DIABETES CARE

Magnetic resonance imaging (MRI) provides non-invasive, repetitive measures in the same individual, allowing the study of a physio-pathological event over time. In this study, we tested the performance of 7 Tesla multi-parametric MRI to monitor the dynamic changes of mouse skeletal muscle injury and regeneration upon acute ischemia induced by femoral artery dissection. T2-mapping (T2 relaxation time), diffusion-tensor imaging (Fractional Anisotropy) and perfusion by Dynamic Contrast-Enhanced MRI (K-trans) were measured and imaging results were correlated with histological morphometric analysis in both Gastrocnemius and Tibialis anterior muscles. We found that tissue damage positively correlated with T2-relaxation time, while myofiber regeneration and capillary density positively correlated with Fractional Anisotropy. Interestingly, K-trans positively correlated with capillary density. Accordingly, repeated MRI measurements between day 1 and day 28 after surgery in ischemic muscles showed that: 1) T2-relaxation time rapidly increased upon ischemia and then gradually declined, returning almost to basal level in the last phases of the regeneration process; 2) Fractional Anisotropy dropped upon ischemic damage induction and then recovered along with muscle regeneration and neoangiogenesis; 3) K-trans reached a minimum upon ischemia, then progressively recovered. Overall, Gastrocnemius and Tibialis anterior muscles displayed similar patterns of MRI parameters dynamic, with more marked responses and less variability in Tibialis anterior. We conclude that MRI provides quantitative information about both tissue damage after ischemia and the subsequent vascular and muscle regeneration, accounting for the differences between subjects and, within the same individual, between different muscles.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T‐cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene‐modified postnatal murine TECs into three‐dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline‐induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4‐expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107–1122 Transient Oct4 expression promoted thymic epithelial cells expansion without drastically changing the cell lineage identity of adult thymic epithelial cells. iOCT4 thymic epithelial cells were able to grow into three‐dimensional collagen type I scaffolds both in vitro and in vivo demonstrating that the collagen structure reproduced a three‐dimensional environment similar to the thymic extracellular matrix, perfectly recognized by thymic epithelial cells. in vivo results showed that thymic organoids transplanted subcutaneously in athymis nude mice were vascularized but failed to support thymopoiesis because of the limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of three‐dimensional biomimetic scaffolds, represents a novel approach allowing the use of postnatal thymic epithelial cells for thymic regeneration.

Skeletal muscle remodeling in response to various noxae physiologically includes structural changes and inflammatory events. The possibility to study those phenomena in-vivo has been hampered by the lack of validated imaging tools. In our study, we have relied on multiparametric magnetic resonance imaging for quantitative monitoring of muscle changes in mice experiencing age-related sarcopenia or active regeneration after sterile acute injury of tibialis anterior muscle induced by cardiotoxin (CTX) injection. The extent of myofibrils' necrosis, leukocyte infiltration, and regeneration have been evaluated and compared with parameters from magnetic resonance imaging: T2-mapping (T2 relaxation time; T2-rt), diffusion-tensor imaging (fractional anisotropy, F.A.) and diffusion weighted imaging (apparent diffusion coefficient, ADC). Inflammatory leukocytes within the perimysium and heterogeneous size of fibers characterized aged muscles. They displayed significantly increased T2-rt (P<0.05) and F.A. (P<0.05) compared with young muscles. After acute damage T2-rt increased in otherwise healthy young muscles with a peak at day 3, followed by a progressive decrease to basal values. F.A. dropped 24 hours after injury and afterward increased above the basal level in the regenerated muscle (from day 7 to day 15) returning to the basal value at the end of the follow up period. The ADC displayed opposite kinetics. T2-rt positively correlated with the number of infiltrating leucocytes retrieved by immunomagnetic bead sorting from the tissue (r = 0.92) and with the damage/infiltration score (r = 0.88) while F.A. correlated with the extent of tissue regeneration evaluated at various time points after injury (r = 0.88). Our results indicate that multiparametric MRI is a sensitive and informative tool for monitoring inflammatory and structural muscle changes in living experimental animals; particularly, it allows identifying the increase of T2-rt and F.A. as common events reflecting inflammatory infiltration and muscle regeneration in the transient response of the tissue to acute injury and in the persistent adaptation to aging.

BackgroundImmune effector cell-associated neurotoxicity syndrome (ICANS) is an adverse event commonly observed in cancer patients receiving CAR-T cell therapies. Despite the clinical features of ICANS being recognizable, its pathophysiology remains poorly understood, current hypothesis are mainly based on findings in autopsies performed on individuals who suffered fatal neurotoxicity after CAR-T cell therapy and preclinical models are limited. Recently, a syngeneic mouse model of CAR-related neurotoxicity has been described; unfortunately, it does not allow to study ICANS that develops upon interaction of human CAR-T cells with human immune and tumor cells.MethodsHere we proposed a humanized mouse model, previously characterized for its ability to recapitulate CAR-related cytokine release syndrome (CRS) toxicity, in which we observed the development of multifocal brain haemorrhages, reminiscent of neurotoxic manifestations observed in patients with high-grade ICANS.ResultsInterestingly, we found that microhaemorrhages did not occur in mice treated with untransduced T cells, while they were observed in about the 45% of mice treated with human CD19 CAR-T cells. Moreover, we observed that this event is associated with CRS severity, as it occurs in patients, and by assessing the number and dimensions of microhaemorrhages by brain tissue area we found them to be greater in terms of extension and incidence in the cerebellum.By magnetic resonance imaging we observed the onset of hypointense lesions in deep brain structures and noteworthily an extravasation event of the injected contrast agent occurred, suggesting changes in blood vessels permeability. Moreover, according to the most affected mentioned brain areas, we find changes in locomotor abilities likely providing a clinical relevance to the phenomena occurring in the brain.ConclusionsThese findings suggest that our humanized mouse model is promisingly able to recapitulate ICANS-related complications, thus providing us with a preclinical tool to deepen ICANS pathophysiology studying.

OBJECTIVE:--Perturbations in cardiac energy metabolism might represent early alterations in diabetes preceding functional and pathological changes. We evaluated left ventricular (LV) structure/geometry and function in relation to energy metabolism and cardiovascular risk factors in overweight/obese men using magnetic resonance techniques. RESEARCH DESIGN AND METHODS--We studied 81 healthy men (aged 22-55 years, with BMI between 19 and 35 kg/m²) by means of cardiac magnetic resonance imaging and ³¹P-magnetic resonance spectroscopy in the resting and fasted conditions and stratified them in quartiles of BMI (cut offs: 23.2, 25.5 and 29.0 kg/m²). RESULTS:--LV mass increased across quartiles of BMI; meanwhile, the volumes did not differ. Parameters of LV systolic and diastolic function were not different among quartiles. The phosphocreatine-to-ATP ratio was reduced across increasing quartiles of mean ± SD BMI (2.25 ± 0.52, 1.89 ± 0.26, 1.99 ± 0.38, and 1.79 ± 0.29; P < 0.006) in association with insulin sensitivity (computer homeostasis model assessment 2 model); this relation was independent of age, BMI, blood pressure, wall mass, HDL cholesterol, triglycerides, smoking habits, and metabolic syndrome. CONCLUSIONS:--Abnormal LV energy metabolism was detectable in obese men in the presence of normal function, supporting the hypothesis that metabolic remodeling in insulin resistant states precedes functional and structural/geometrical remodeling of the heart regardless of the onset of overt hyperglycemia.