Tumor-Targeted Fluorescence Imaging and Mechanisms of Tumor Cell-Derived Carbon Nanodots

An ideal cancer diagnostic probe should possess precise tumor-targeted accumulation with negligible sojourn in normal tissues. Herein, tumor cell-derived carbon nanodots (C-CNDU87 and C-CNDHepG2) about 3~7 nm were prepared by a solvothermal method with stable fluorescence and negligible cytotoxicity. More interestingly, due to the differences in gene expression of cancers, C-CND structurally mimicked the corresponding precursors during carbonization in which carbon nanodots were functionalized with α-amino and carboxyl groups with different densities on their edges. With inherent homology and homing effect, C-CND were highly enriched in precursor tumor tissues. Mechanistic studies showed that under the mediation of the original configuration of α-amino and carboxyl groups, C-CND specifically bound to the large neutral amino acid transporter 1 (LAT1, overexpressed in cancer cells), achieving specific tumor fluorescence imaging. This work provided a new vision about tumor cell architecture-mimicked carbon nanodots for tumor-targeted fluorescence imaging.

IMMU-04. UNVEILING THE TUMOR-METABOLOME-IMMUNITY AXIS OF GLIOMA

The glioma microenvironment orchestrates tumor evolution, progression, and resistance to therapy. In high-grade gliomas, microglia and monocyte-derived macrophages constitute up to 70% of the tumor mass. However, the dynamics and phenotypes of intratumoral myeloid cells during tumor progression are poorly understood. Here we define myeloid cellular states in gliomas by longitudinal single-cell profiling and demonstrate their strict control by the tumor genotype. We report the unexpected and clinically highly relevant finding that human as well as murine gliomas with Isocitrate Dehydrogenase (IDH)1-R132H, a key oncogenic driver mutation of glioma, subdue their innate immune microenvironment by prompting a multifaceted reprogramming of myeloid and T cell metabolism. We employed integrated single-cell transcriptomic, time-of-flight mass cytometry and proteomic analyses of human healthy cortex control and glioma samples to identify myeloid cell subsets with distinct fates in IDH-mutated glioma that diverge from canonical trajectories of antigen-presenting cells as a result of a monocyte-to-macrophage differentiation block. Moving beyond single time point assessments, we now longitudinally describe differential immune cell infiltration and phenotype dynamics during glioma progression that are orchestrated by a fluctuating network of resident microglial cells and educated recruited immune cells. IDH mutations in glioma induce a tolerogenic alignment of their immune microenvironment through increased tryptophan uptake via large neutral amino acid transporter (LAT1)-CD98 and subsequent activation of the aryl hydrocarbon receptor (AHR) in educated blood-borne macrophages. In experimental tumor models, this immunosuppressive phenotype was reverted by LAT1-CD98 and AHR inhibitors. Taken together with direct effects on T cell activation, our findings not only link this oncogenic metabolic pathway to distinct immunosuppressive pathways but also provide the rationale and novel molecular targets for the development of immunotherapeutic concepts addressing the disease-defining microenvironmental effects of IDH mutations.

Macrophage metabolism in the intestine is compartment-specific and regulated by the microbiota

Intestinal macrophages play a vital role in the maintenance of gut homeostasis through signals derived from the microbiota. We previously demonstrated that microbial-derived metabolites can shape the metabolic functions of macrophages. Here, we show that antibiotic-induced disruption of the intestinal microbiota dramatically alters both the local metabolite environment, and the metabolic functions of macrophages in the colon. Broad-spectrum antibiotic administration in mice increased expression of the large neutral amino acid transporter and accordingly, amino acid uptake. Subsequently, antibiotic administration enhanced the metabolic functions of colonic macrophages, increasing phosphorylation of components of mammalian/mechanistic target of rapamycin (mTOR) signalling pathways, increasing expression of genes involved in glycolysis and oxidative phosphorylation (OXPHOS), increasing mitochondrial function and increased levels of ECAR and OCR as a direct measure of glycolysis and OXPHOS. Small bowel macrophages were less metabolically active than in the colon, with macrophage metabolism being independent of the microbiota. Finally, we reveal tissue resident Tim4+ CD4+ macrophages exhibit enhanced fatty acid uptake alongside reduced fatty acid synthesis compared to their recruited counterparts. Thus the microbiota shapes gut macrophage metabolism in a compartment-specific manner, with important implications for functions when monocyte recruitment and macrophage differentiation.

Large Neutral Amino acid uptake and mTOR activation within CD4+ T cells coordinate Type 2 immunity and host resistance to Trichuris muris

Trichuris trichiura (whipworm) is a gastrointestinal nematode that infects approximately 465 million people worldwide. T. muris is used as a tractable model for the human whipworm. In wild type mice, infection with a high dose of T. muris eggs leads to worm expulsion, which is dependent on a CD4+Th2 response and interleukin (IL-)13 production. It is known that T cells up-regulate glycolysis and uptake of substrates upon activation. The amino acid transporter SLC7A5 has been shown necessary for activation of mTORC1, a nutrient/energy/redox sensor critical for T cell differentiation into effector cells. We found that at the peak of the immune response to T. muris, mice lacking SLC7A5 in CD4+T cells have delayed worm expulsion, lower levels of IL-13, reduced pmTOR and glycolytic rates. However, at later stages of infection IL-13 levels partially recovered alongside resistance. The critical role of CD4+T cell metabolism per se and down-stream mTOR in CD4+T cells in resistance was shown in mice lacking mTOR in CD4+T cells, that failed to expel a high dose of parasites and developed chronic infection. Our study shows that mTOR is essential for effective functioning of T cells during whipworm infection and that deletion of Slc7a5 significantly delays worm clearance.

84 Effects of incremental conditioning and supplemental dietary tryptophan on pre, mid, and post-exercise heart rate and respiratory rate in sled dogs

Repetitive bouts of resistance and aerobic exercise can have dramatic effects on whole body physiology. Dietary tryptophan supplementation supports protein turnover and serotonin production, which assist in responses to exercise. The objective of this study was to investigate the effects of a 12-week incremental exercise regimen and supplemental dietary tryptophan on pre-, mid-, and post-exercise heart rate and respiratory rate in sled dogs. Sixteen dogs (mean age of 4.8 ± 2.5 years, body weight 24.3 ± 4.3 kg) were blocked for sex, age, and body weight, and randomly allocated to a control diet or the control plus tryptophan diet (tryptophan to large-neutral-amino-acid ratio of 0.075:1). All dogs participated in a 12-week conditioning regimen with controlled exercise challenges at week -1 and subsequently every 3 weeks. Electrocardiogram electrodes and thoracic rib bands were worn to record heart rate and respiratory rate (EMKA Tech., Falls Church, VA, USA) prior to, during, and following each exercise challenge. A trans regression, mixed, and correlation model were used where appropriate to assess the fixed effects of treatment and week, and explore linear relationships between recovery time and week in SAS (v 9.4). No differences were found in heart rate between treatment groups for any training level (P > 0.10). Working, recovery, and time required for heart rate to recover post-exercise decreased from week -1 to week 11 (P < 0.05). Correlation analysis indicated that treatment dogs recovered respiratory rate faster post-exercise compared to control (r = -0.421, P < 0.05). Resting, recovery and time required for respiratory rate to recover post-exercise decreased from baseline to week 11 (P < 0.05). This data suggests improvement in cardiorespiratory fitness was observed over 12 weeks of training and that tryptophan may support respiratory function during exercise recovery.

Exercise but Not Supplemental Dietary Tryptophan Influences Heart Rate and Respiratory Rate in Sled Dogs

Tryptophan (Trp), an indispensable amino acid for dogs, is the precursor of serotonin, a neurotransmitter with a variety of effects throughout the body, including the ability to modulate cardiac and pulmonary activity. This study aimed to investigate the effects of a 12-week incremental exercise regimen and supplemental dietary Trp on heart rate (HR) and respiratory rate (RR) in client-owned sled dogs. Sixteen Siberian huskies were randomly allocated to either treatment or control diet groups. Both groups were fed a control diet (Trp to large neutral amino acid ratio of 0.047:1); however, treatment dogs received a Trp supplement to achieve a Trp to large neutral amino acid ratio of 0.075:1. Every three weeks, external telemetry equipment was used to non-invasively measure and record HR and RR at a resting, working, and post-exercise state in a controlled exercise challenge. A mixed model was used to test differences between diet, activity parameter, and week. Dietary Trp supplementation had no effect on HR or RR. Independent of diet, resting, working, post-exercise HR, and time to recover post-exercise HR decreased from week −1 to week 11 (p < 0.05). Resting HR had the greatest reduction from week −1 to week 11 (21%, p < 0.05). Working RR did not change with exercise (p > 0.10), but rRR and postRR decreased from week −1 to week 11 (p < 0.05). These data suggest that the exercise regimen the dogs were subjected to may have positively impacted the dogs’ capacity to sustain aerobic exercise, whereas Trp supplementation had no effect on HR or RR.

Randomized Double-Blind Controlled Trial on the Effect of Proteins with Different Tryptophan/Large Neutral Amino Acid Ratios on Sleep in Adolescents: The PROTMORPHEUS Study

Disturbed sleep is common in adolescents. Ingested nutrients help regulate the internal clock and influence sleep quality. The purpose of this clinical trial is to assess the effect of protein tryptophan (Trp)/large neutral amino acids (LNAAs) ratio on sleep and circadian rhythm. Ingested Trp is involved in the regulation of the sleep/wake cycle and improvement of sleep quality. Since Trp transport through the blood–brain barrier is competing with LNAAs, protein with higher Trp/LNAAs were expected to increase sleep efficiency. This randomized double-blind controlled trial will enroll two samples of male adolescents predisposed to sleep disturbances: elite rugby players (n = 24) and youths with obesity (n = 24). They will take part randomly in three sessions each held over a week. They will undergo a washout period, when dietary intake will be calibrated (three days), followed by an intervention period (three days), when their diet will be supplemented with three proteins with different Trp/LNAAs ratios. Physical, cognitive, dietary intake, appetite, and sleepiness evaluations will be made on the last day of each session. The primary outcome is sleep efficiency measured through in-home electroencephalogram recordings. Secondary outcomes include sleep staging, circadian phase, and sleep-, food intake-, metabolism-, and inflammation-related biochemical markers. A fuller understanding of the effect of protein Trp/LNAAs ratio on sleep could help in developing nutritional strategies addressing sleep disturbances.

Possibility of Amino Acid Treatment to Prevent the Psychiatric Disorders via Modulation of the Production of Tryptophan Metabolite Kynurenic Acid

Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan catabolism, acts as an antagonist for both the α7 nicotinic acetylcholine receptor and glycine coagonist sites of the N-methyl-d-aspartic acid receptor at endogenous brain concentrations. Elevation of brain kynurenic acid levels reduces the release of neurotransmitters such as dopamine and glutamate, and kynurenic acid is considered to be involved in psychiatric disorders such as schizophrenia and depression. Thus, the control of kynurenine pathway, especially kynurenic acid production, in the brain is an important target for the improvement of brain function or the effective treatment of brain disorders. Astrocytes uptake kynurenine, the immediate precursor of kynurenic acid, via large neutral amino acid transporters, and metabolize kynurenine to kynurenic acid by kynurenine aminotransferases. The former transport both branched-chain and aromatic amino acids, and the latter have substrate specificity for amino acids and their metabolites. Recent studies have suggested the possibility that amino acids may suppress kynurenic acid production via the blockade of kynurenine transport or via kynurenic acid synthesis reactions. This approach may be useful in the treatment and prevention of neurological and psychiatric diseases associated with elevated kynurenic acid levels.