Diacylglycerol lipases (DAGLα and DAGLβ) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that ...can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.
The cytokines interleukin 13 and 4 share a common heterodimeric receptor and are important modulators of peripheral allergic reactions. Produced primarily by T-helper type 2 lymphocytes, they are ...typically considered as anti-inflammatory cytokines because they can downregulate the synthesis of T-helper type 1 pro-inflammatory cytokines. Their presence and role in the brain is only beginning to be investigated and the data collected so far shows that these molecules can be produced by microglial cells and possibly by neurons. Attention has so far been given to the possible role of these molecules in neurodegeneration. Both neuroprotective or neurotoxic effects have been proposed based on evidence that interleukin 13 and 4 can reduce inflammation by promoting the M2 microglia phenotype and contributing to the death of microglia M1 phenotype, or by potentiating the effects of oxidative stress on neurons during neuro-inflammation. Remarkably, the heterodimeric subunit IL-13Rα1 of their common receptor was recently demonstrated in dopaminergic neurons of the ventral tegmental area and the substantia nigra pars compacta, suggesting the possibility that both cytokines may affect the activity of these neurons regulating reward, mood, and motor coordination. In mice and man, the gene encoding for IL-13Rα1 is expressed on the X chromosome within the PARK12 region of susceptibility to Parkinson's disease (PD). This, together with finding that IL-13Rα1 contributes to loss of dopaminergic neurons during inflammation, indicates the possibility that these cytokines may contribute to the etiology or the progression of PD.
Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse ...enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and -beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function.
Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by serving as negative regulators of adaptive immune system effector cell responses. Reduced production or function of ...Tregs has been implicated in several human autoimmune diseases. The cytokine interleukin 2 plays a central role in promoting Treg differentiation, survival, and function in vivo and may therefore have therapeutic benefits for autoimmune diseases. mRNA‐6231 is an investigational, lipid nanoparticle‐encapsulated, mRNA‐based therapy that encodes a modified human interleukin 2 mutein fused to human serum albumin (HSA‐IL2m). Herein, we report the development of a semi‐mechanistic kinetic‐pharmacodynamic model to quantify the relationship between subcutaneous dose(s) of mRNA‐6231, HSA‐IL2m protein expression, and Treg expansion in nonhuman primates. The nonclinical kinetic‐pharmacodynamic model was extrapolated to humans using allometric scaling principles and the physiological basis of pharmacological mechanisms to predict the clinical response to therapy a priori. Model‐based simulations were used to inform the dose selection and design of the first‐in‐human clinical study (NCT04916431). The modeling approach used to predict human responses was validated when data became available from the phase I clinical study. This validation indicates that the approach is valuable in informing clinical decision‐making.
Remote inflammation monitoring with digital health technologies (DHTs) would provide valuable information for both clinical research and care. Controlled perturbations of the immune system may reveal ...physiological signatures which could be used to develop a digital biomarker of inflammatory state. In this study, molecular and physiological profiling was performed following an in vivo lipopolysaccharide (LPS) challenge to develop a digital biomarker of inflammation. Ten healthy volunteers received an intravenous LPS challenge and were monitored for 24 h using the VitalConnect VitalPatch (VitalPatch). VitalPatch measurements included heart rate (HR), heart rate variability (HRV), respiratory rate (RR), and skin temperature (TEMP). Conventional episodic inpatient vital signs and serum proteins were measured pre‐ and post‐LPS challenge. The VitalPatch provided vital signs that were comparable to conventional methods for assessing HR, RR, and TEMP. A pronounced increase was observed in HR, RR, and TEMP as well as a decrease in HRV 1–4 h post‐LPS challenge. The ordering of participants by magnitude of inflammatory cytokine response 2 h post‐LPS challenge was consistent with ordering of participants by change from baseline in vital signs when measured by VitalPatch (r = 0.73) but not when measured by conventional methods (r = −0.04). A machine learning model trained on VitalPatch data predicted change from baseline in inflammatory protein response (R2 = 0.67). DHTs, such as VitalPatch, can improve upon existing episodic measurements of vital signs by enabling continuous sensing and have the potential for future use as tools to remotely monitor inflammation.
Hypovitaminosis D and COVID-19 Vassallo, Gabriele A.; Fiorino, Sirio; Mori, Simone ...
Italian journal of medicine,
12/2021, Letnik:
14, Številka:
4
Journal Article
Recenzirano
Odprti dostop
As the main title ‘COVID-19 revolution: a new challenge for the internist’ states, the global coronavirus infection disease 2019 (COVID-19) pandemic represented a new challenge for the internists. ...This paper is part of a series of articles written during the difficult period of the ongoing global pandemic and published all together in this fourth issue of the Italian Journal of Medicine, with the aim of sharing the direct experiences of those who were the first to face this severe emergency, expressing each point of view in the management of COVID-19 in relation to other diseases. Each article is therefore the result of many efforts and a joint collaboration between many colleagues from the Departments of Internal Medicine or Emergency Medicine of several Italian hospitals, engaged in the front line during the pandemic. These preliminary studies therefore cover diagnostic tools available to health care personnel, epidemiological reflections, possible new therapeutic approaches, discharge and reintegration procedures to daily life, the involvement of the disease not only in the lung, aspects related to various comorbidities, such as: coagulopathies, vasculitis, vitamin D deficiency, gender differences, etc.. The goal is to offer a perspective, as broad as possible, of everything that has been done to initially face the pandemic in its first phase and provide the tools for an increasingly better approach, in the hope of not arriving unprepared to a possible second wave. This paper in particular deals with hypovitaminosis D and COVID-19.
The majority of Parkinson's disease (PD) cases are sporadic and idiopathic suggesting that this neurodegenerative disorder is the result of both environmental and genetic factors. Stress and ...neuroinflammation are among the factors being investigated for their possible contributions to PD. Experiments in rodents showed that severe chronic stress can reduce the number of dopaminergic neurons in the substantia nigra pars compacta (SNc); the same cells that are lost in PD. These actions are at least in part mediated by increased oxidative stress. Here, we tested the hypothesis that the interleukin-13 receptor alpha 1 (IL-13Rα1), a cytokine receptor whose activation increases the vulnerability of dopaminergic neurons to oxidative damage, participates in the stress-dependent damage of these neurons.
Mice were subject to daily sessions of 8 h (acute) stress for 16 weeks (5 days a week), a procedure previously showed to induce loss of dopaminergic neurons in the SNc. The source and the kinetics of interleukin-13 (IL-13), the endogenous ligand of IL-13Rα1, were evaluated 0, 1, 3, 6, and 8 h and at 16 weeks of stress. Identification of IL-13 producing cell-type was performed by immunofluorescent and by in situ hybridization experiments. Markers of oxidative stress, microglia activation, and the number of dopaminergic neurons in IL-13Rα1 knock-out animals (Il13ra1
) and their wild-type littermates (Il13ra1
) were evaluated at 16 weeks of stress and at 20 weeks, following a 4 week non-stressed period and compared to non-stressed mice.
IL-13 was expressed in microglial cells within the SN and in a fraction of the tyrosine hydroxylase-positive neurons in the SNc. IL-13 levels were elevated during daily stress and peaked at 6 h. 16 weeks of chronic restraint stress significantly reduced the number of SNc dopaminergic neurons in Il13ra1
mice. Neuronal loss at 16 weeks was significantly lower in Il13ra1
mice. However, the loss of dopaminergic neurons measured at 20 weeks, after 4 weeks of non-stress following the 16 weeks of stress, was similar in Il13ra1
and Il13ra1
mice.
IL-13, a cytokine previously demonstrated to increase the susceptibility of SNc dopaminergic neurons to oxidative stress, is elevated in the SN by restraint stress. Lack of IL-13Rα1 did not prevent nor halted but delayed neuronal loss in the mouse model of chronic restraint stress. IL-13/IL-13Rα1 may represent a target to reduce the rate of DA neuronal loss that can occur during severe chronic restraint stress.
Cyclooxygenase inhibitors such as ibuprofen have been used for decades to control fever through reducing the levels of the pyrogenic lipid transmitter prostaglandin E2 (PGE2). Historically, ...phospholipases have been considered to be the primary generator of the arachidonic acid (AA) precursor pool for generating PGE2 and other eicosanoids. However, recent studies have demonstrated that monoacyglycerol lipase (MAGL), through hydrolysis of the endocannabinoid 2-arachidonoylglycerol, provides a major source of AA for PGE2 synthesis in the mammalian brain under basal and neuroinflammatory states. We show here that either genetic or pharmacological ablation of MAGL leads to significantly reduced fever responses in both centrally or peripherally-administered lipopolysaccharide or interleukin-1β-induced fever models in mice. We also show that a cannabinoid CB1 receptor antagonist does not attenuate these anti-pyrogenic effects of MAGL inhibitors. Thus, much like traditional nonsteroidal anti-inflammatory drugs, MAGL inhibitors can control fever, but appear to do so through restricted control over prostaglandin production in the nervous system.
BackgroundMacrophages, monocytes, and dendritic cells are sentinel cells of the innate immune system that play a central role in phagocytosis, inflammation, immune cell recruitment, and antigen ...presentation. Genetically redirecting myeloid cells against tumor-associated antigens represents a novel strategy for cancer immunotherapy. Ex vivo chimeric antigen receptor (CAR) macrophage and monocyte cell therapies have demonstrated robust anti-tumor immunity via targeted phagocytosis, cytokine/chemokine release, activation of the tumor microenvironment (TME), T cell recruitment, and epitope spreading in pre-clinical models. Here, we describe a novel strategy to deliver modified messenger RNA (mRNA) encapsulated in lipid nanoparticles (LNPs) to generate CAR-M (macrophages and monocytes) directly in vivo.MethodsHuman and murine primary monocytes and macrophages were utilized to characterize mRNA/LNP driven CAR expression kinetics and anti-tumor functionality in vitro using flow cytometry, live cell imaging, single cell RNA sequencing (scRNAseq), and cytokine release assays. The efficacy, safety, and myeloid cell tropism of CAR-encoding mRNA/LNP were evaluated in vivo in syngeneic and humanized mouse models of solid tumors. Tissues were collected and CAR expression along with dynamic changes in immune cell frequency and phenotype were assessed using flow cytometry and scRNAseq. All animal studies were conducted in accordance with the Animal Welfare Act and Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the Wistar Institute.ResultsHuman macrophages and monocytes engineered with CAR-encoding mRNA/LNP in vitro demonstrated high CAR expression and viability. CAR expression conferred antigen specificity leading to target-specific proinflammatory cytokine secretion and tumor cell killing, with serial killing demonstrated upon tumor rechallenge. The myeloid tropism of the LNP was demonstrated both in vitro and in vivo, with significant CAR expression observed in macrophages, monocytes, and dendritic cells compared to immune cells of non-myeloid origin in mice. In vivo, regional and systemic administration of CAR-encoding mRNA/LNP led to significant tumor regression in subcutaneous and systemically disseminated metastatic solid tumor models, respectively. Treated animals tolerated repeat mRNA/LNP administration with no signs of toxicity.ConclusionsThese data demonstrate that CAR-M can be directly produced in vivo and directed against tumor associated antigens using mRNA/LNP technology. This novel cancer immunotherapy platform offers an off-the-shelf solution that has the potential to increase access to CAR-based therapies and can be applied to numerous target antigens and indications.Ethics ApprovalAll animal studies were conducted in accordance with the Animal Welfare Act and Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the Wistar Institute.
Alcohol produces complex effects on the immune system. Moderate alcohol use (1–2 drinks per day) has been shown to produce anti-inflammatory responses in human blood monocytes, whereas, the post ...mortem brains of severe alcoholics show increased immune gene expression and activated microglial markers. The present study was conducted to evaluate the time course of alcohol effects during exposure and after withdrawal, and to determine the relationship between microglial and cytokine responses in brain and blood. Forty-eight adult, male Wistar rats were exposed to chronic ethanol vapors, or air control, for 5 weeks. Following ethanol/air exposure blood and brains were collected at three time points: 1) while intoxicated, following 35 days of air/vapor exposure; 2) following 24 h of withdrawal from exposure, and 3) 28 days after withdrawal. One hemisphere of the brain was flash-frozen for cytokine analysis, and the other was fixed for immunohistochemical analysis. The ionized calcium-binding adapter molecule 1 (Iba-1) was used to evaluate microglia activation at the three time points, and rat cytokine/chemokine Magnetic Bead Panels (Millipore) were used to analyze frontal cortex tissue lysate and serum. Ethanol induced a significant increase in Iba-1 that peaked at day 35, remained significant after 1 day of withdrawal, and was elevated at day 28 in frontal cortex, amygdala, and substantia nigra. Ethanol exposure was associated with a transient reduction of the serum level of the major pro- and anti-inflammatory cytokines and chemokines and a transient increase of effectors of sterile inflammation. Little or no changes in these molecules were seen in the frontal cortex except for HMG1 and fractalkine that were reduced and elevated, respectively, at day 28 following withdrawal. These data show that ethanol exposure produces robust microglial activation; however, measures of inflammation in the blood differ from those in the brain over a protracted time course.
•Chronic ethanol exposure induced microglia activation, which remained elevated at day 28 of withdrawal in select brain areas.•Ethanol exposure induced a transient reduction in serum cytokine/chemokines and an increase in sterile inflammation effectors.•Chronic ethanol exposure induced little or no changes in major cytokine/chemokines in the frontal cortex.•Measures of inflammation in the blood differed from those in the brain over a protracted time course.
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