Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis.

Metastasis is the leading cause of cancer-related deaths, and greater knowledge of the metastatic microenvironment is necessary to effectively target this process. Microenvironmental changes occur at distant sites prior to clinically detectable metastatic disease; however, the key niche regulatory signals during metastatic progression remain poorly characterized. Here, we identify a core immune suppression gene signature in pre-metastatic niche formation that is expressed predominantly by myeloid cells. We target this immune suppression program by utilizing genetically engineered myeloid cells (GEMys) to deliver IL-12 to modulate the metastatic microenvironment. Our data demonstrate that IL12-GEMy treatment reverses immune suppression in the pre-metastatic niche by activating antigen presentation and T cell activation, resulting in reduced metastatic and primary tumor burden and improved survival of tumor-bearing mice. We demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.

Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

MicroRNA-409-3p/BTG2 signaling axis improves impaired angiogenesis and wound healing in obese mice.

Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.

Aversion-associated drug and alcohol seeking in females.

Compulsive drug intake is characterized by the continuation of use regardless of negative consequences. This is modeled preclinically using procedures where a negative stimulus is delivered contingently with consumption of the reinforcer. In humans, women and men exhibit different drug taking behavior as it pertains to overall use, withdrawal symptoms, and rate of dependence. In substance use research, females have often been excluded from many studies due to concerns that circulating sex hormones may affect drug seeking behavior. However, the more recent inclusion of females in preclinical studies has identified interesting sex differences in aversion-resistant intake of drugs and alcohol. This review will serve to summarize key findings in aversion-related intake of alcohol, psychostimulants, and opioids in females by examining studies that have included female subjects. Further discussion will examine the effect of intake model, neuroanatomical pathways, and sex hormones in the expression of aversion-resistant drug and alcohol consumption.

Sex differences in neuronal activation in the cortex and midbrain during quinine-adulterated alcohol intake.

AIMS: Continued alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This is modeled in mice by pairing negative stimuli with alcohol, such as adulterating alcohol solution with quinine. Mice consuming alcohol under these conditions are considered to be engaging in aversion-resistant intake. Previously, we have observed sex differences in this behavior, with females more readily expressing aversion-resistant consumption. We also identified three brain regions that exhibited sex differences in neuronal activation during quinine-alcohol drinking: ventromedial prefrontal cortex (vmPFC), posterior insular cortex (PIC), and ventral tegmental area (VTA). Specifically, male mice showed increased activation in vmPFC and PIC, while females exhibited increased activation in VTA. In this study, we aimed to identify what specific type of neurons are activated in these regions during quinine-alcohol drinking. METHOD: We assessed quinine-adulterated alcohol intake using the two-bottle choice procedure. We also utilized RNAscope in situ hybridization in the three brain regions that previously exhibited a sex difference to examine colocalization of Fos, glutamate, GABA, and dopamine. RESULT: Females showed increased aversion-resistant alcohol consumption compared to males. We also found that males had higher colocalization of glutamate and Fos in vmPFC and PIC, while females had greater dopamine and Fos colocalization in the VTA. CONCLUSIONS: Collectively, these experiments suggest that glutamatergic output from the vmPFC and PIC may have a role in suppressing, and dopaminergic activity in the VTA may promote, aversion-resistant alcohol consumption. Future experiments will examine neuronal circuits that contribute to sex differences in aversion resistant consumption.

Vicarious defeat stress induces increased alcohol consumption in female mice: Role of neurokinin-1 receptor and interleukin-6.

There is a high frequency of comorbidity of alcohol use disorder (AUD) and depression in human populations. We have studied this relationship in our lab using the social defeat stress (SDS) model, which results in both depression-like behaviours and increased alcohol consumption in male mice. However, standard SDS procedures are difficult to use in female mice due to a lack of territorial aggression. In the experiments presented here, we used vicarious defeat stress (VDS) to assess social withdrawal and alcohol consumption in female C57BL6/J mice. We also assessed the expression of interleukin-6 (IL6), which is a proinflammatory cytokine that is associated with depression in humans and sensitivity to SDS in mice. In these experiments, C57BL/6 female mice underwent 10 days of VDS where they witnessed the physical defeat of a male conspecific by an aggressive CD1 mouse. After the end of VDS, mice were either given access to alcohol or sacrificed for the measurement of IL6 expression. We found that VDS increased alcohol consumption and IL6 expression in the frontal cortex and hippocampus. Given that the neurokinin-1 receptor (NK1R) can mediate both stress-induced alcohol consumption and IL6 expression, we tested the ability of NK1R antagonism to reduce VDS-induced alcohol consumption and found that this treatment reduced alcohol intake in both VDS-exposed mice and in unstressed controls. The observed increase in alcohol consumption suggests that VDS is a model that can be utilized to study stress-induced alcohol consumption in female mice, and that this is sensitive to NK1R antagonism.

Pathology of hereditary renal cell carcinoma syndromes: Tuberous sclerosis complex (TSC).

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease characterized by hamartomatous tumors involving multiple organs such as the brain, skin, heart, lung and kidney. TSC is caused by inactivating mutations in TSC1/TSC2, which encodes hamartin and tuberin, respectively, and forms a complex that regulates mechanistic target of rapamycin complex 1 (mTORC1), resulting in cell overgrowth and oncogenesis. Since a leading cause of morbidity and mortality in TSC relates to chronic kidney disease and the ability to preserve renal function, this review describes the important pathologic findings in TSC-associated renal neoplasms and their correlating sporadic counterparts. The most common renal tumor in TSC patients are AMLs, followed by a heterogeneous spectrum of renal epithelial tumors, which may provide clues to establishing a diagnosis of TSC.

Can rapid maxillary expansion affect speech sound production in growing patients? A systematic review.

Rapid maxillary expansion (RME) may change speech sound parameters due to the enlargement of oral and nasal cavities. This study aimed to systematically review the current evidence on speech changes as a side effect of RME. An electronic search was conducted in nine databases, and two of them accessed the 'grey literature'. The eligibility criteria included clinical studies assessing orthodontic patients with maxillary transverse deficiency and the relationship with speech alterations without restricting publication year or language. Only interventional studies were included. The JBI Critical Appraisal Tool assessed the risk of bias. The initial search provided 4853 studies. Seven articles (n = 200 patients) met the inclusion criteria and were analysed. The primary source of bias was the absence of a control group in four studies. RME altered speech production by changing vowel fundamental frequency and fricative phoneme formant frequency. Shimmer and jitter rates changed in one and two studies, respectively. Two studies presented deterioration during orthodontic treatment, but speech improved after appliance removal. Despite the limited evidence, RME affects speech during and after treatment.

Middle East respiratory syndrome coronavirus vaccine based on a propagation-defective RNA replicon elicited sterilizing immunity in mice.

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.

Spatial proteomics of hippocampal subfield-specific pathology in Alzheimer's disease and primary age-related tauopathy.

INTRODUCTION: Alzheimer's disease (AD) and primary age-related tauopathy (PART) both harbor 3R/4R hyperphosphorylated-tau (p-tau)-positive neurofibrillary tangles (NFTs) but differ in the spatial p-tau development in the hippocampus. METHODS: Using Nanostring GeoMx Digital Spatial Profiling, we compared protein expression within hippocampal subregions in NFT-bearing and non-NFT-bearing neurons in AD (n = 7) and PART (n = 7) subjects. RESULTS: Proteomic measures of synaptic health were inversely correlated with the subregional p-tau burden in AD and PART, and there were numerous differences in proteins involved in proteostasis, amyloid beta (Aß) processing, inflammation, microglia, oxidative stress, and neuronal/synaptic health between AD and PART and between definite PART and possible PART. DISCUSSION: These results suggest subfield-specific proteome differences that may explain some of the differences in Aß and p-tau distribution and apparent pathogenicity. In addition, hippocampal neurons in possible PART may have more in common with AD than with definite PART, highlighting the importance of Aß in the pathologic process. HIGHLIGHTS: Synaptic health is inversely correlated with local p-tau burden. The proteome of NFT- and non-NFT-bearing neurons is influenced by the presence of Aß in the hippocampus. Neurons in possible PART cases share more proteomic similarities with neurons in ADNC than they do with neurons in definite PART cases.

Exploring microtubule dynamics in Alzheimer's disease: Longitudinal assessment using [11C]MPC-6827 PET imaging in rodent models of Alzheimer's-related pathology.

INTRODUCTION: Microtubule (MT) stability is crucial for proper neuronal function. Understanding MT dysregulation is critical for connecting amyloid beta (Aß) and tau-based degenerative events and early changes in presymptomatic Alzheimer's disease (AD). Herein we present positron emission tomography (PET) imaging properties of our MT-PET radiotracer, [11C]MPC-6827, in multiple established AD mouse models. METHODS: Longitudinal PET, biodistribution, autoradiography, immunohistochemistry, and behavioral studies were conducted at multiple time points in APPswe/PSEN1dE9 (APP/PS1), P301S-PS19 (P301S), 5xFAD, and age-matched control mice. RESULTS: Longitudinal [11C]MPC-6827 brain imaging showed significant increases in APP/PS1, P301S, and 5xFAD mice compared to controls. Longitudinal MT-PET correlated positively with biodistribution, autoradiography, and immunohistochemistry results and negatively with behavior data. DISCUSSION: Our study demonstrated significant longitudinal [11C]MPC-6827 PET increases in multiple AD mouse models for the first time. Strong correlations between PET and biomarker data underscored the interplay of MT destabilization, amyloid, and tau pathology in AD. These results suggest [11C]MPC-6827 PET as a promising tool for monitoring MT dysregulation early in AD progression. HIGHLIGHTS: Longitudinal positron emission tomography (PET) imaging studies using [11C]MPC-6827 in multiple established Alzheimer's disease (AD) mouse models revealed an early onset of microtubule dysregulation, with significant changes in brain radiotracer uptake evident from 2 to 4 months of age. Intra-group analysis showed a progressive increase in microtubule dysregulation with increasing AD burden, supported by significant correlations between PET imaging data and biodistribution, autoradiography, and molecular pathological markers. [11C]MPC-6827 PET imaging demonstrated its efficacy in detecting early microtubule alterations preceding observable behavioral changes in AD mouse models, suggesting its potential for early AD imaging. The inclusion of the 5xFAD mouse model further elucidated the impact of amyloid beta (Aß) toxicity on inducing tau hyperphosphorylation-mediated microtubule dysregulation, highlighting the versatility of [11C]MPC-6827 in delineating various aspects of AD pathology. Our study provides immediate clarity on high uptake of the microtubule-based radiotracer in AD brains in a longitudinal setting, which directly informs clinical utility in Aß/tau-based studies.

Viral Chemotaxis of Paramecium Bursaria Altered by Algal Endosymbionts.

Chemotaxis is widespread across many taxa and often aids resource acquisition or predator avoidance. Species interactions can modify the degree of movement facilitated by chemotaxis. In this study, we investigated the influence of symbionts on Paramecium bursaria's chemotactic behavior toward chloroviruses. To achieve this, we performed choice experiments using chlorovirus and control candidate attractors (virus stabilization buffer and pond water). We quantified the movement of Paramecia grown with or without algal and viral symbionts toward each attractor. All Paramecia showed some chemotaxis toward viruses, but cells without algae and viruses showed the most movement toward viruses. Thus, the endosymbiotic algae (zoochlorellae) appeared to alter the movement of Paramecia toward chloroviruses, but it was not clear that ectosymbiotic viruses (chlorovirus) also had this effect. The change in behavior was consistent with a change in swimming speed, but a change in attraction remains possible. The potential costs and benefits of chemotactic movement toward chloroviruses for either the Paramecia hosts or its symbionts remain unclear.

Polymerogenic neuroserpin causes mitochondrial alterations and activates NFκB but not the UPR in a neuronal model of neurodegeneration FENIB.

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients.

Multinational comparison study of aircraft pilot healthcare avoidance behaviour.

BACKGROUND: US and Canadian pilots are required to meet medical standards to secure their active flying status, but a subgroup exhibit healthcare avoidance behaviour due to fear of loss of that status. This phenomenon has the potential to impact pilot health, aeromedical screening and aviation safety. No international comparison study of pilot healthcare avoidance currently exists between US and Canadian pilots. AIMS: To compare the rate and subtypes of healthcare avoidance behaviour secondary to fear for loss of flying status between US and Canadian pilots. METHODS: A comparison analysis of data collected during two independent, non-probabilistic, cross-sectional internet surveys including any individual certified to perform flying duties in the USA (US survey) or Canada (Canadian survey). RESULTS: There were 4320 US pilots and 1415 Canadian pilots who completed informed consent and 3765 US pilots and 1405 Canadian pilots were included in the results. There were 56% of US pilots who reported a history of healthcare avoidance behaviour compared to 55% of Canadian pilots (P = 0.578). A multivariable logistic regression that included age, pilot type and gender showed that US pilots were slightly more likely than Canadian pilots to report this behaviour (odds ratio 1.22, 95% confidence interval 1.06-1.4). CONCLUSIONS: Healthcare avoidance behaviour due to fear of loss of flying status has a relatively high prevalence in both US and Canadian pilot populations.

Preliminary psychometric properties of an inhibition task in young children with Down syndrome.

Background: Executive function (EF) skills are important treatment targets for people with Down syndrome (DS); however, few EF measures have been evaluated for use with young children in this population. Methods: The present study evaluated preliminary psychometric properties of a measure of the EF component of inhibition. Participants were 73 children with DS between 2.5 and 8.67 years old who completed an adapted ability to delay task using a desirable toy. Results: Across two separate trials, latencies to touch the toys were significantly correlated. Latencies increased overall with chronological and mental age, with caveats for the youngest and oldest participants. Conclusion: Findings suggest that an adapted prohibition task is an appropriate method of measuring inhibition for children with DS between 4 and 7 years old, though many children in this chronological age range are at early stages of acquiring this skill set.

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure.

BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Smooth muscle cell FTO regulates contractile function.

The fat mass and obesity gene (FTO) is a N6-methyladenosine RNA demethylase that was initially linked by Genome-wide association studies to increased rates of obesity. Subsequent studies have revealed multiple mass-independent effects of the gene, including cardiac myocyte contractility. We created a mouse with a conditional and inducible smooth muscle cell deletion of Fto (Myh11 Cre+ Ftofl/fl) and did not observe any changes in mouse body mass or mitochondrial metabolism. However, the mice had significantly decreased blood pressure (hypotensive), despite increased heart rate and sodium, and significantly increased plasma renin. Remarkably, the third-order mesenteric arteries from these mice had almost no myogenic tone or capacity to constrict to smooth muscle depolarization or phenylephrine. Microarray analysis from Fto-/--isolated smooth muscle cells demonstrated a significant decrease in serum response factor (Srf) and the downstream effectors Acta2, Myocd, and Tagln; this was confirmed in cultured human coronary arteries with FTO siRNA. We conclude Fto is an important component to the contractility of smooth muscle cells.NEW & NOTEWORTHY We show a key role for the fat mass obesity (FTO) gene in regulating smooth muscle contractility, possibly by methylation of serum response factor (Srf).

Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study.

BACKGROUND: We evaluated a new chemoimmunotherapy combination based on the anti-PD1 monoclonal antibody pembrolizumab and the pyrimidine antimetabolite gemcitabine in HER2- advanced breast cancer (ABC) patients previously treated in the advanced setting, in order to explore a potential synergism that could eventually obtain long term benefit in these patients. METHODS: HER2-negative ABC patients received 21-day cycles of pembrolizumab 200 mg (day 1) and gemcitabine (days 1 and 8). A run-in-phase (6 + 6 design) was planned with two dose levels (DL) of gemcitabine (1,250 mg/m2 [DL0]; 1,000 mg/m2 [DL1]) to determine the recommended phase II dose (RP2D). The primary objective was objective response rate (ORR). Tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumors and myeloid-derived suppressor cells (MDSCs) levels in peripheral blood were analyzed. RESULTS: Fourteen patients were treated with DL0, resulting in RP2D. Thirty-six patients were evaluated during the first stage of Simon's design. Recruitment was stopped as statistical assumptions were not met. The median age was 52; 21 (58%) patients had triple-negative disease, 28 (78%) visceral involvement, and 27 (75%) ≥ 2 metastatic locations. Progression disease was observed in 29 patients. ORR was 15% (95% CI, 5-32). Eight patients were treated ≥ 6 months before progression. Fourteen patients reported grade ≥ 3 treatment-related adverse events. Due to the small sample size, we did not find any clear association between immune tumor biomarkers and treatment efficacy that could identify a subgroup with higher probability of response or better survival. However, patients that experienced a clinical benefit showed decreased MDSCs levels in peripheral blood along the treatment. CONCLUSION: Pembrolizumab 200 mg and gemcitabine 1,250 mg/m2 were considered as RP2D. The objective of ORR was not met; however, 22% patients were on treatment for ≥ 6 months. ABC patients that could benefit of chemoimmunotherapy strategies must be carefully selected by robust and validated biomarkers. In our heavily pretreated population, TILs, PD-L1 expression and MDSCs levels could not identify a subgroup of patients for whom the combination of gemcitabine and pembrolizumab would induce long term benefit. TRIAL REGISTRATION: ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001,779-54, respectively). Registration dates: 20/01/2017 and 18/11/2016, respectively.

Loss of Endothelial FTO Antagonizes Obesity-Induced Metabolic and Vascular Dysfunction.

RATIONALE: Increasing prevalence of obesity and its associated risk with cardiovascular diseases demands a better understanding of the contribution of different cell types within this complex disease for developing new treatment options. Previous studies could prove a fundamental role of FTO (fat mass and obesity-associated protein) within obesity; however, its functional role within different cell types is less understood. OBJECTIVES: We identify endothelial FTO as a previously unknown central regulator of both obesity-induced metabolic and vascular alterations. METHODS AND RESULTS: We generated endothelial Fto-deficient mice and analyzed the impact of obesity on those mice. While the loss of endothelial FTO did not influence the development of obesity and dyslipidemia, it protected mice from high-fat diet-induced glucose intolerance and insulin resistance by increasing AKT (protein kinase B) phosphorylation in endothelial cells and skeletal muscle. Furthermore, loss of endothelial FTO prevented the development of obesity-induced hypertension by preserving myogenic tone in resistance arteries. In Fto-deficient arteries, microarray analysis identified upregulation of L-Pgds with significant increases in prostaglandin D2 levels. Blockade of prostaglandin D2 synthesis inhibited the myogenic tone protection in resistance arteries of endothelial Fto-deficient mice on high-fat diet; conversely, direct addition of prostaglandin D2 rescued myogenic tone in high-fat diet-fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D2 application. CONCLUSIONS: These data identify endothelial FTO as a previously unknown regulator in the development of obesity-induced metabolic and vascular changes, which is independent of its known function in regulation of obesity.

Transactivation of receptor tyrosine kinases by purinergic P2Y and adenosine receptors.

Transactivation of receptor tyrosine kinases (RTK) is a crosstalk mechanism exhibited by G-protein-coupled receptors (GPCR) to activate signaling pathways classically associated with growth factors. The discovery of RTK transactivation was a breakthrough in signal transduction that contributed to developing current concepts in intracellular signaling. RTK transactivation links GPCR signaling to important cellular processes, such as cell proliferation and differentiation, and explains the functional diversity of these receptors. Purinergic (P2Y and adenosine) receptors belong to class A of GPCR; in the present work, we systematically review the experimental evidence showing that purinergic receptors have the ability to transactivate RTK in multiple tissues and physiopathological conditions resulting in the modulation of cellular physiology. Of particular relevance, the crosstalk between purinergic receptors and epidermal growth factor receptor is a redundant pathway that participates in multiple pathophysiological processes. Specific and detailed knowledge of purinergic receptor-regulated pathways advances our understanding of the complexity of GPCR signal transduction and opens the way for pharmacologic intervention in the pathological context.