CD8+ T cells induce cachexia during chronic viral infection.

Cachexia represents a leading cause of morbidity and mortality in various cancers, chronic inflammation and infections. Understanding of the mechanisms that drive cachexia has remained limited, especially for infection-associated cachexia (IAC). In the present paper we describe a model of reversible cachexia in mice with chronic viral infection and identify an essential role for CD8+ T cells in IAC. Cytokines linked to cancer-associated cachexia did not contribute to IAC. Instead, virus-specific CD8+ T cells caused morphologic and molecular changes in the adipose tissue, which led to depletion of lipid stores. These changes occurred at a time point that preceded the peak of the CD8+ T cell response and required T cell-intrinsic type I interferon signaling and antigen-specific priming. Our results link systemic antiviral immune responses to adipose-tissue remodeling and reveal an underappreciated role of CD8+ T cells in IAC.

Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function.

CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.

Evaluation of Hepatic Glucose and Palmitic Acid Metabolism in Rodents on High-Fat Diet Using Deuterium Metabolic Imaging.

BACKGROUND: One of the main features of several metabolic disorders is dysregulation of hepatic glucose and lipid metabolism. Deuterium metabolic imaging (DMI) allows for assessing the uptake and breakdown of 2H-labeled substrates, giving specific insight into nutrient processing in healthy and diseased organs. Thus, DMI could be a useful approach for analyzing the differences in liver metabolism of healthy and diseased subjects to gain a deeper understanding of the alterations related to metabolic disorders. PURPOSE: Evaluating the feasibility of DMI as a tool for the assessment of metabolic differences in rodents with healthy and fatty livers (FLs). STUDY TYPE: Animal Model. POPULATION: 18 male Sprague Dawley rats on standard (SD, n = 9, healthy) and high-fat diet (HFD, n = 9, FL disease). FIELD STRENGTH/SEQUENCE: Phase-encoded 1D pulse-acquire sequence and anatomy co-registered phase-encoded 3D pulse-acquire chemical shift imaging for 2H at 9.4T. ASSESSMENT: Localized and nonlocalized liver spectroscopy was applied at eight time points over 104 minutes post injection. The obtained spectra were preprocessed and quantified using jMRUI (v7.0) and the resulting amplitudes translated to absolute concentration (mM) according to the 2H natural abundance water peak. STATISTICAL TESTS: Two-way repeated measures ANOVA were employed to assess between-group differences, with statistical significance at P < 0.05. RESULTS: DMI measurements demonstrated no significant difference (P = 0.98) in the uptake of [6,6'-2H2]glucose between healthy and impaired animals (AUCSD = 1966.0 ± 151.5 mM - minutes vs. AUCHFD = 2027.0 ± 167.6 mM·minutes). In the diseased group, the intrahepatic uptake of palmitic acid d-31 was higher (AUCHFD = 57.4 ± 17.0 mM·minutes, AUCSD = 33.3 ± 10.5 mM·minutes), but without statistical significance owing to substantial in-group variation (P = 0.73). DATA CONCLUSION: DMI revealed higher concentrations of palmitic acid in rats with FL disease and no difference in hepatic glucose concentration between healthy and impaired animals. Thus, DMI appears to be a useful tool for evaluating metabolism in rodents with FL disease. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 3.

Iron deficiency-induced thrombocytosis increases thrombotic tendency in rats.

Iron deficiency (ID) is globally prevalent, and apart from anemia is associated with thrombocytosis. While considered benign, studies linking thrombotic events with prior ID anemia suggest otherwise. Herein we used animal models to assess the influence of ID on thrombotic tendency. Sprague-Dawley rats were fed control or iron deficient diets and ferric carboxymaltose was used to reverse ID. Thrombosis was induced via stenosis of the inferior vena cava or damage to the right carotid artery using ferric chloride. Thrombi were evaluated histologically and via high frequency ultrasound in the venous model. ID consistently induced thrombocytosis alongside anemia. Venous thrombus growth and final dimensions in both arterial and venous thrombi were largest in ID. In both models, platelet numbers correlated with the final thrombus size, with ID thrombi having the largest platelet areas. Platelet function was also evaluated in surgically naive rats. Coagulability on thromboelastography and hemostasis on tail transection were augmented in ID. Platelet and plasma P-selectin expression were both higher in ID. Platelet adhesion and aggregation in ID was impaired under shear flow but was intact on static assays. Iron replacement therapy reversed all ID-related changes in hematological parameters, thrombus dimensions, and platelet assays. In summary, ID alone increases thrombotic tendency. Iron replacement therapy reverses these changes, making it a viable strategy for prevention of ID-related thrombotic disease. This may be of importance in patients with chronic illnesses which may already be at increased risk for thrombosis such as inflammatory bowel disease, chronic kidney disease, or cancer.

Superfluorinated, Highly Water-Soluble Polyphosphazenes as Potential 19F Magnetic Resonance Imaging (MRI) Contrast Agents.

"Hot spot" 19F magnetic resonance imaging (MRI) has garnered significant attention recently for its ability to image various disease markers quantitatively. Unlike conventional gadolinium-based MRI contrast agents, which rely on proton signal modulation, 19F-MRI's direct detection has a unique advantage in vivo, as the human body exhibits a negligible background 19F-signal. However, existing perfluorocarbon (PFC) or PFC-based contrast materials suffer from several limitations, including low longitudinal relaxation rates and relatively low imaging efficiency. Hence, we designed a macromolecular contrast agent featuring a high number of magnetically equivalent 19F-nuclei in a single macromolecule, adequate fluorine nucleus mobility, and excellent water solubility. This design utilizes superfluorinated polyphosphazene (PPz) polymers as the 19F-source; these are modified with sodium mercaptoethanesulfonate (MESNa) to achieve water solubility exceeding 360 mg/mL, which is a similar solubility to that of sodium chloride. We observed substantial signal enhancement in MRI with these novel macromolecular carriers compared to non-enhanced surroundings and aqueous trifluoroacetic acid (TFA) used as a positive control. In conclusion, these novel water-soluble macromolecular carriers represent a promising platform for future MRI contrast agents.

Non-Contrast-Enhanced Multiparametric MRI of the Hypoxic Tumor Microenvironment Allows Molecular Subtyping of Breast Cancer: A Pilot Study.

Tumor neoangiogenesis is an important hallmark of cancer progression, triggered by alternating selective pressures from the hypoxic tumor microenvironment. Non-invasive, non-contrast-enhanced multiparametric MRI combining blood-oxygen-level-dependent (BOLD) MRI, which depicts blood oxygen saturation, and intravoxel-incoherent-motion (IVIM) MRI, which captures intravascular and extravascular diffusion, can provide insights into tumor oxygenation and neovascularization simultaneously. Our objective was to identify imaging markers that can predict hypoxia-induced angiogenesis and to validate our findings using multiplexed immunohistochemical analyses. We present an in vivo study involving 36 female athymic nude mice inoculated with luminal A, Her2+, and triple-negative breast cancer cells. We used a high-field 9.4-tesla MRI system for imaging and subsequently analyzed the tumors using multiplex immunohistochemistry for CD-31, PDGFR-ß, and Hif1-α. We found that the hyperoxic-BOLD-MRI-derived parameter ΔR2* discriminated luminal A from Her2+ and triple-negative breast cancers, while the IVIM-derived parameter fIVIM discriminated luminal A and Her2+ from triple-negative breast cancers. A comprehensive analysis using principal-component analysis of both multiparametric MRI- and mpIHC-derived data highlighted the differences between triple-negative and luminal A breast cancers. We conclude that multiparametric MRI combining hyperoxic BOLD MRI and IVIM MRI, without the need for contrast agents, offers promising non-invasive markers for evaluating hypoxia-induced angiogenesis.

Hyperoxic BOLD-MRI-Based Characterization of Breast Cancer Molecular Subtypes Is Independent of the Supplied Amount of Oxygen: A Preclinical Study.

Hyperoxic BOLD-MRI targeting tumor hypoxia may provide imaging biomarkers that represent breast cancer molecular subtypes without the use of injected contrast agents. However, the diagnostic performance of hyperoxic BOLD-MRI using different levels of oxygen remains unclear. We hypothesized that molecular subtype characterization with hyperoxic BOLD-MRI is feasible independently of the amount of oxygen. Twenty-three nude mice that were inoculated into the flank with luminal A (n = 9), Her2+ (n = 5), and triple-negative (n = 9) human breast cancer cells were imaged using a 9.4 T Bruker BioSpin system. During BOLD-MRI, anesthesia was supplemented with four different levels of oxygen (normoxic: 21%; hyperoxic: 41%, 71%, 100%). The change in the spin-spin relaxation rate in relation to the normoxic state, ΔR2*, dependent on the amount of erythrocyte-bound oxygen, was calculated using in-house MATLAB code. ΔR2* was significantly different between luminal A and Her2+ as well as between luminal A and triple-negative breast cancer, reflective of the less aggressive luminal A breast cancer's ability to better deliver oxygen-rich hemoglobin to its tissue. Differences in ΔR2* between subtypes were independent of the amount of oxygen, with robust distinction already achieved with 41% oxygen. In conclusion, hyperoxic BOLD-MRI may be used as a biomarker for luminal A breast cancer identification without the use of exogenous contrast agents.

CAM-Xenograft Model Provides Preclinical Evidence for the Applicability of [68Ga]Ga-Pentixafor in CRC Imaging.

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. Increased expression of CXCR4 has been associated with liver metastasis, disease progression, and shortened survival. Using in vitro cell binding studies and the in ovo model, we aimed to investigate the potential of [68Ga]Ga-Pentixafor, a radiotracer specifically targeting human CXCR4, for CRC imaging. Specific membrane binding and internalisation of [68Ga]Ga-Pentixafor was shown for HT29 cells, but not for HCT116 cells. Accordingly, [68Ga]Ga-Pentixafor accumulated specifically in CAM-xenografts derived from HT29 cells, but not in HCT116 xenografts, as determined by µPET/MRI. The CAM-grown xenografts were histologically characterised, demonstrating vascularisation of the graft, preserved expression of human CXCR4, and viability of the tumour cells within the grafts. In vivo viability was further confirmed by µPET/MRI measurements using 2-[18F]FDG as a surrogate for glucose metabolism. [68Ga]Ga-Pentixafor µPET/MRI scans showed distinct radiotracer accumulation in the chick embryonal heart, liver, and kidneys, whereas 2-[18F]FDG uptake was predominantly found in the kidneys and joints of the chick embryos. Our findings suggest that [68Ga]Ga-Pentixafor is an interesting novel radiotracer for CRC imaging that is worth further investigation. Moreover, this study further supports the suitability of the CAM-xenograft model for the initial preclinical evaluation of targeted radiopharmaceuticals.

Novel Salinomycin-Based Paramagnetic Complexes-First Evaluation of Their Potential Theranostic Properties.

Combining therapeutic with diagnostic agents (theranostics) can revolutionize the course of malignant diseases. Chemotherapy, hyperthermia, or radiation are used together with diagnostic methods such as magnetic resonance imaging (MRI). In contrast to conventional contrast agents (CAs), which only enable non-specific visualization of tissues and organs, the theranostic probe offers targeted diagnostic imaging and therapy simultaneously. METHODS: Novel salinomycin (Sal)-based theranostic probes comprising two different paramagnetic metal ions, gadolinium(III) (Gd(III)) or manganese(II) (Mn(II)), as signal emitting motifs for MRI were synthesized and characterized by elemental analysis, infrared spectral analysis (IR), electroparamagnetic resonance (EPR), thermogravimetry (TG) differential scanning calorimetry (DSC) and electrospray ionization mass spectrometry (ESI-MS). To overcome the water insolubility of the two Sal-complexes, they were loaded into empty bacterial ghosts (BGs) cells as transport devices. The potential of the free and BGs-loaded metal complexes as theranostics was evaluated by in vitro relaxivity measurements in a high-field MR scanner and in cell culture studies. RESULTS: Both the free Sal-complexes (Gd(III) salinomycinate (Sal-Gd(III) and Mn(II) salinomycinate (Sal-Mn(II)) and loaded into BGs demonstrated enhanced cytotoxic efficacy against three human tumor cell lines (A549, SW480, CH1/PA-1) relative to the free salinomycinic acid (Sal-H) and its sodium complex (Sal-Na) applied as controls with IC50 in a submicromolar concentration range. Moreover, Sal-H, Sal-Gd(III), and Sal-Mn(II) were able to induce perturbations in the cell cycle of treated colorectal and breast human cancer cell lines (SW480 and MCF-7, respectively). The relaxivity (r1) values of both complexes as well as of the loaded BGs, were higher or comparable to the relaxivity values of the clinically applied contrast agents gadopentetate dimeglumine and gadoteridol. CONCLUSION: This research is the first assessment that demonstrates the potential of Gd(III) and Mn(II) complexes of Sal as theranostic agents for MRI. Due to the remarkable selectivity and mode of action of Sal as part of the compounds, they could revolutionize cancer therapy and allow for early diagnosis and monitoring of therapeutic follow-up.

Modified amino-dextrans as carriers of Gd-chelates for retrograde transport and visualization of peripheral nerves by magnetic resonance imaging (MRI).

Amino-dextrans (AD) conjugated with gadolinium (Gd3+) were developed as neuro-specific contrast agents (CA) for the visualization of the sciatic nerve in rats by magnetic resonance imaging (MRI). AD with 3, 10, and 70 kDa molecular weights were assessed as carrier molecules known to be transported with various speed by axonal microtubules. Detailed spectroscopic characterizations, analyses by Fast Protein Liquid Chromatography (FPLC), Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and inductively coupled plasma-mass spectrometry (ICP-MS), were carried out. For MRI, the paramagnetic Gd3+ ion was coupled as a T1 signal enhancer. The well-established linear chelator, diethylenetriaminepentaacetic acid (DTPA), was used and subsequently replaced by the more stable cyclic chelator 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In addition, a fluorescently labeled AD-DTPA-Gd was prepared to demonstrate an active transport to the spinal cord by histochemistry. After successful synthesis and characterization, molecular migration of the AD-DTPA-Gd in the sciatic nerve of healthy Sprague Dawley rats was monitored by MRI for up to seven days. Enhancement of nerve structures was evaluated by MRI and correlated with ICP-MS analyses. To investigate the distribution of CA along the neuraxis, all animals were sacrificed after the final MRI monitoring. Nerves, spinal ganglions, and corresponding spinal cord sections were harvested, to determine the localization and concentration of the paramagnetic element. This is the first report that demonstrates the active uptake and transport of AD-Gd conjugates within the sciatic nerve. This new concept may serve as a potential diagnostic tool for the direct visualization and monitoring of the continuity of injured nerves.

MR Imaging of Peripheral Nerves Using Targeted Application of Contrast Agents: An Experimental Proof-of-Concept Study.

Introduction: Current imaging modalities for peripheral nerves display the nerve's structure but not its function. Based on a nerve's capacity for axonal transport, it may be visualized by targeted application of a contrast agent and assessing the distribution through radiological imaging, thus revealing a nerve's continuity. This concept has not been explored, however, may potentially guide the treatment of peripheral nerve injuries. In this experimental proof-of-concept study, we tested imaging through MRI after administering gadolinium-based contrast agents which were then retrogradely transported. Methods: We synthesized MRI contrast agents consisting of paramagnetic agents and various axonal transport facilitators (HSA-DTPA-Gd, chitosan-DTPA-Gd or PLA/HSA-DTPA-Gd). First, we measured their relaxivity values in vitro to assess their radiological suitability. Subsequently, the sciatic nerve of 24 rats was cut and labeled with one of the contrast agents to achieve retrograde distribution along the nerve. One week after surgery, the spinal cords and sciatic nerves were harvested to visualize the distribution of the respective contrast agent using 7T MRI. In vivo MRI measurements were performed using 9.4 T MRI on the 1st, 3rd, and the 7th day after surgery. Following radiological imaging, the concentration of gadolinium in the harvested samples was analyzed using inductively coupled mass spectrometry (ICP-MS). Results: All contrast agents demonstrated high relaxivity values, varying between 12.1 and 116.0 mM-1s-1. HSA-DTPA-Gd and PLA/HSA-DTPA-Gd application resulted in signal enhancement in the vertebral canal and in the sciatic nerve in ex vivo MRI. In vivo measurements revealed significant signal enhancement in the sciatic nerve on the 3rd and 7th day after HSA-DTPA-Gd and chitosan-DTPA-Gd (p < 0.05) application. Chemical evaluation showed high gadolinium concentration in the sciatic nerve for HSA-DTPA-Gd (5.218 ± 0.860 ng/mg) and chitosan-DTPA-Gd (4.291 ± 1.290 ng/mg). Discussion: In this study a novel imaging approach for the evaluation of a peripheral nerve's integrity was implemented. The findings provide radiological and chemical evidence of successful contrast agent uptake along the sciatic nerve and its distribution within the spinal canal in rats. This novel concept may assist in the diagnostic process of peripheral nerve injuries in the future.