Extracellular Levels Research Articles

Purinergic P2X7R expressed on regulatory T cells potentially links molecular mimicry to autoimmune responses

<abstract> <p>The immune system is meant to protect against invading microbes. Although this system is effective against many microbes, some can use molecular mimicry to turn the immune system against the host and activate autoimmune responses. The resulting autoimmunity has significant implications for public health and healthcare costs. It is well known that regulatory T cells (Tregs) are crucial for self-tolerance and that their function is impaired after exposure to self-antigens or antigens with molecular mimicry, leading to the activation of autoimmune responses. How molecular mimicry disrupts Tregs in this manner remains under debate. This review contributes to the field of the pathogenesis of autoimmunity by proposing that purinergic signaling in the lymph nodes, with extracellular ATP, ADP, and adenosine as ligands, plays a pivotal role in this process. Repeated or high-dose microbial infection causes the release of large amounts of extracellular ATP sufficient to reach the threshold of extracellular ATP levels for activating P2X7 purinergic receptors (P2X7Rs) on dendritic cells and Tregs. This hampers the ability of Tregs to suppress autoimmune responses. Crucially, P2X7Rs are activated at very high extracellular ATP levels, thus only after repeated or high-dose infection with microbes. Arguably, in contrast to the rapid elimination of microbes with foreign antigens, the clearance of invading microbes that employ molecular mimicry requires the activation of P2X7Rs at the expense of self-tolerance. Because all processes required to activate autoimmune responses occur in secondary lymphoid organs, this article hypothesizes that, contrary to current convention, microbes do not need to enter organs to initiate autoimmunity. However, some types of microbes can prevent P2X7R-induced Treg disruption by converting extracellular ATP to adenosine, mitigating autoimmune responses resulting in chronic diseases with less severe inflammation. The proposed hypothesized mechanism has potential implications for the understanding and treatment of autoimmune disorders.</p> </abstract>

Transplantation and Noninvasive Longitudinal In Vivo Imaging of Parathyroid Cells: A Proof-of-Concept Study.

The parathyroid cell is a vital regulator of extracellular calcium levels, operating through the secretion of parathyroid hormone (PTH). Despite its importance, the regulation of PTH secretion remains complex and not fully understood, representing a unique interplay between extracellular and intracellular calcium, and hormone secretion. One significant challenge in parathyroid research has been the difficulty in maintaining cells ex vivo for in-depth cellular investigations. To address this issue, we introduce a novel platform for parathyroid cell transplantation and noninvasive in vivo imaging using the anterior chamber of the eye as a transplantation site. We found that parathyroid adenoma tissue transplanted into the mouse eye engrafted onto the iris, became vascularized, and retained cellular composition. Transplanted animals exhibited elevated PTH levels, indicating a functional graft. With in vivo confocal microscopy, we were able to repetitively monitor parathyroid graft morphology and vascularization. In summary, there is a pressing need for new methods to study complex cellular processes in parathyroid cells. Our study provides a novel approach for noninvasive in vivo investigations that can be applied to understand parathyroid physiology and pathology under physiological and pathological conditions. This innovative strategy can deepen our knowledge on parathyroid function and disease.

Increased Parenchymal Free Water May Be Decreased by Revascularization Surgery in Patients with Moyamoya Disease.

Moyamoya disease (MMD) is a cerebrovascular disease associated with steno-occlusive changes in the arteries of the circle of Willis and with hemodynamic impairment. Previous studies have reported that parenchymal extracellular free water levels may be increased and the number of neurites may be decreased in patients with MMD. The aim of the present study was to investigate the postoperative changes in parenchymal free water and neurites and their relationship with cognitive improvement. Multi-shell diffusion MRI (neurite orientation dispersion and density imaging and free water imaging using a bi-tensor model) was performed in 15 hemispheres of 13 adult patients with MMD (11 female, mean age 37.9 years) who had undergone revascularization surgery as well as age- and sex-matched normal controls. Parameter maps of free water and free-water-eliminated neurites were created, and the regional parameter values were compared among controls, patients before surgery, and patients after surgery. The anterior and middle cerebral artery territories of patients showed higher preoperative free water levels (P ≤ 0.007) and lower postoperative free water levels (P ≤ 0.001) than those of normal controls. The change in the dispersion of the white matter in the anterior cerebral artery territory correlated with cognitive improvement (r = -0.75; P = 0.004). Our study suggests that increased parenchymal free water levels decreased after surgery and that postoperative changes in neurite parameters are related to postoperative cognitive improvement in adult patients with MMD. Diffusion analytical methods separately calculating free water and neurites may be useful for unraveling the pathophysiology of chronic ischemia and the postoperative changes that occur after revascularization surgery in this disease population.

Analysis of extracellular vesicle microRNA profiles reveals distinct blood and lymphatic endothelial cell origins

AbstractExtracellular vesicles (EVs) are crucial mediators of cell‐to‐cell communication in physiological and pathological conditions. Specifically, EVs released from the vasculature into blood were found to be quantitatively and qualitatively different in diseases compared to healthy states. However, our understanding of EVs derived from the lymphatic system is still scarce. In this study, we compared the mRNA and microRNA (miRNA) expression in blood vascular (BEC) and lymphatic (LEC) endothelial cells. After characterization of the EVs by fluorescence‐triggered flow cytometry, nanoparticle tracking analysis and cryo‐transmission electron microscopy (cryo‐TEM) we utilized small RNA‐sequencing to characterize miRNA signatures in the EVs and identify cell‐type specific miRNAs in BEC and LEC. We found miRNAs specifically enriched in BEC and LEC on the cellular as well as the extracellular vesicle level. Our data provide a solid basis for further functional in vitro and in vivo studies addressing the role of EVs in the blood and lymphatic vasculature.

Glycosylation signature of plasma IgA of critically ill COVID-19 patients.

Thromboembolic complications are common in severe COVID-19 and are thought to result from excessive neutrophil-extracellular-trap (NET)-driven immunothrombosis. Glycosylation plays a vital role in the efficiency of immunoglobulin A (IgA) effector functions, with significant implications for NET formation in infectious diseases. This study represents the first comprehensive analysis of plasma IgA glycosylation during severe SARS-CoV-2 or Influenza A infection, revealing lower sialylation and higher galactosylation of IgA1 O-glycans in acute respiratory distress syndrome (ARDS), regardless of the underlying cause of the disease. Importantly, N-glycans displayed an infection-specific pattern, with N47 of IgA2 showing diminished sialylation and bisection, and N340/N327 of IgA1/2 demonstrating lower fucosylation and antennarity along with higher non-complex glycans in COVID-19 compared to Influenza. Notably, COVID-19 IgA possessed strong ability to induce NET formation and its glycosylation patterns correlated with extracellular DNA levels in plasma of critically ill COVID-19 patients. Our data underscores the necessity of further research on the role of IgA glycosylation in the modulation of pathogen-specific immune responses in COVID-19 and other infectious diseases.

A Method to Collect Cerebrospinal Fluid from Mouse Cisterna Magna to Determine Extracellular Tau Levels.

Despite being a cytoplasmic protein abundant in neurons, tau is detectable in various extracellular fluids. In addition to being passively released from dying/degenerating neurons, tau is also actively released from living neurons in a neuronal activity-dependent mechanism. In vivo, tau released from neurons first appears in brain interstitial fluid (ISF) and subsequently drains into cerebrospinal fluid (CSF) by glymphatic system. Changes in CSF tau levels alter during the course of AD pathogenesis and are considered to predict the disease-progression of AD. A method to collect CSF from various mouse models of AD will serve as a valuable tool to investigate the dynamics of physiological/pathological tau released from neurons. In this chapter, we describe and characterize a method that reliably collects a relatively large volume of CSF from anesthetized mice.

The GLP-1 receptor agonist exendin-4 reduces taurine and glycine in nucleus accumbens of male rats, an effect tentatively involving the nucleus tractus solitarius.

The physiological effects of glucagon-like peptide-1 (GLP-1) are mainly centered on its ability to decrease blood glucose levels and facilitate satiety. Additional physiological functions have been identified by means of GLP-1 agonists such as exenatide (exendin-4; Ex4). In particular, Ex4 reduces the intake of natural and artificial rewards, effects that to some extent involve activation of GLP-1 receptors in the nucleus tractus solitarius (NTS). Although Ex4 acts in the brain, the neurochemical mechanisms underlying this activation are not fully elucidated. Investigating Ex4-induced neurochemical alterations in the nucleus accumbens (NAc) would be valuable for understanding its impact on reward-related behaviors. The aim of the present exploratory in vivo microdialysis study was therefore to study how Ex4, administered either systemically or locally into the NTS, influences classical neurotransmitters like dopamine, serotonin, noradrenaline, glutamate and GABA as well as additional players such as glycine, taurine and serine in NAc of male rats. We showed that Ex4 reduced extracellular levels of serine, taurine and glycine, where the latter two declines appear to involve activation of GLP-1R in the NTS. Besides, after systemic Ex4 injection the metabolites DOPAC, HVA, and 5HIAA are elevated. Where the increase in metabolites related to dopamine, but not serotonin, involves GLP-1 receptors in other areas than the NTS. Although the descriptive nature of the present data does not provide causality, it may however serve as an indication of mechanisms underlying how Ex4 may modulate reward-related behaviors.

Quantifying ABCA1/apoA-1 Signaling Pathways with AFM Imaging and Lipidomic Analysis.

The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).

In Vivo Brain Microdialysis.

Many biological molecules in the brain interstitial fluid are involved in neuronal functions. Therefore, measuring the levels of these molecules in the extracellular fluid would provide deep insights into the physiological/pathological mechanisms underlying brain functions/disorders. In vivo microdialysis is a powerful technique used to examine the extracellular levels of various molecules in the brains of living animals. In neuroscience research, this technique has been widely used to investigate relatively small molecules including neurotransmitters and amino acids. However, recent advances in technology have made it possible to assess large molecules in the brain interstitial fluid, such as signaling peptides and proteins, using microdialysis probes with high-molecular-weight cutoff membranes. This chapter describes an in vivo microdialysis method to collect and measure the levels of large biological molecules in the extracellular fluid of the brains of freely moving mice.

Rab26 alleviates sepsis-induced immunosuppression as a master regulator of macrophage ferroptosis and polarization shift

Macrophage dysfunction is a significant contributor to more than 70 % of sepsis-related deaths, specifically secondary bacterial infections, during the immunosuppression stage of sepsis. Nevertheless, the role of Rab26 in this context remains unclear. In this study, we observed a substantial decrease in Rab26 expression in macrophages during the immunosuppressive phase of sepsis, which was also found to be suppressed by high extracellular levels of HMGB1. During the progression of sepsis, Rab26 deficiency promotes a polarization shift from the M1 to the M2-like phenotype in macrophages, rendering them susceptible to ferroptosis. Subsequent experimentation has revealed that Rab26 deficiency facilitates the degradation of GPX4, thereby aggravating macrophage ferroptosis through the upregulation of levels of lipid ROS, MDA, and ferrous iron induced by RSL3, a ferroptosis inducer. Additionally, Rab26-deficient mice in the immunosuppressed phase of sepsis exhibit heightened susceptibility to secondary infections, leading to exacerbated lung tissue damage and increased mortality rate. Overall, these findings indicate that Rab26 plays a crucial role in sepsis-induced macrophage immunosuppression by regulating macrophage ferroptosis and polarization. Hence, it represents a potential novel target for sepsis therapy.

Serum exosomes from hepatitis B virus–infected patients inhibit glycolysis in Sertoli cells via miR-122-5p/ALDOA axis

Hepatitis B virus (HBV) infection is associated with male infertility. The mechanism includes an increase in chromosomal instability in sperm, which has an adverse effect on sperm viability and function. Sertoli cells (SCs) are vital in spermatogenesis because they use glycolysis to provide energy to germ cells and themselves. HBV infection impairs sperm function. However, whether HBV infection disrupts energy metabolism in SCs remains unclear. This study aimed to determine the role of serum exosomes of HBV-infected patients in SC viability and glycolysis. Serum exosomes were obtained from 30 patients with (HBV+_exo) or without (HBV–_exo) HBV infection using high-speed centrifugation and identified by transmission electron microscopy and western blot analysis. Cell viability is determined by CCK-8 assay. Glycolysis is determined by detecting extracellular acidification rate and ATP levels. miR-122–5p expression levels are detected by quantitative RT-PCR, and a dual-luciferase gene reporter assay confirms the downstream target gene of miR-122–5p. Protein expression is determined by western blot analysis. The results show that HBV+ _exo inhibited cell viability, extracellular acidification rate, and ATP production of SCs. miR-122–5p is highly expressed in HBV+ _exo compared with that in HBV–_exo. Furthermore, HBV+ _exo is efficiently taken up by SCs, whereas miR-122–5p is efficiently transported to SCs. miR-122–5p overexpression downregulates ALDOA expression and inhibits SC viability and glycolysis. However, ALDOA overexpression reverses the effects of miR-122–5p and HBV+ _exo on SC viability and glycolysis. HBV+ _exo may deliver miR-122–5p to target ALDOA and inhibit SC viability and glycolysis, thus providing new therapeutic ideas for treating HBV-associated male infertility.

A New Role for Lactate in Breast Cancer

Lactate dehydrogenase (LDH) is an enzyme that plays a key role in cellular metabolism by converting pyruvate to lactate during anaerobic glycolysis. LDH has been implicated in various cancer types, including breast cancer, where increased LDH activity is associated with tumor growth, invasion, and resistance to therapy. LDH inhibitors are being investigated as potential therapeutic agents in cancer treatment, including breast cancer. Khajah and colleagues investigated the role of LDH inhibitors in both estrogen receptor (ER)+ and ER‑ breast cancer cell lines and in normal breast epithelial cells. A significant reduction in extracellular lactate level, cell proliferation, motility, and invasion was observed after treatment with LDH inhibitors. Subsequently, changes in the levels of vimentin, E‑cadherin, p38 MAPK, ERK1/2, and AKT were also observed. Cancer cell proliferation was preferentially blocked compared with normal epithelial cell inhibition by LDH inhibitors such as quercetin and lonidamine. These results further highlight the role of lactate in breast cancer progression and indicate the practical use of various commercially available LDH inhibitors as promising therapeutic agents to oppose the processes leading to cancer progression. While LDH inhibitors show promise, challenges include achieving selectivity for cancer cells and minimizing toxicity to normal tissues. The development of specific LDH inhibitors with favorable pharmacokinetic properties is an ongoing area of research. Mol Med Rep. 2024 Jan;29(1):12. doi: 10.3892/mmr.2023.1313.

Post-translational secretion stress regulation in Bacillus subtilis is controlled by intra- and extracellular proteases

The Gram-positive bacterium Bacillus subtilis is a prolific producer of industrial enzymes that are effectively harvested from the fermentation broth. However, the high capacity of B. subtilis for protein secretion has so far not been exploited to the full due to particular bottlenecks, including product degradation by extracellular proteases and counterproductive secretion stress responses. To unlock the Bacillus secretion pathway for difficult-to-produce proteins, various cellular interventions have been explored, including genome engineering. Our previous research has shown a superior performance of genome-reduced B. subtilis strains in the production of staphylococcal antigens compared to the parental strain 168. This was attributed, at least in part, to redirected secretion stress responses, including the presentation of elevated levels of the quality control proteases HtrA and HtrB that also catalyse protein folding. Here we show that this relates to the elimination of two homologous serine proteases, namely the cytosolic protease AprX and the extracellular protease AprE. This unprecedented posttranslational regulation of secretion stress effectors, like HtrA and HtrB, by the concerted action of cytosolic and extracellular proteases has important implications for the biotechnological application of microbial cell factories. In B. subtilis, this conclusion is underscored by extracellular degradation of the staphylococcal antigen IsaA by both AprX and AprE. Extracellular activity of the cytosolic protease AprX is remarkable since it shows that not only extracellular, but also intracellular proteases impact extracellular product levels. We therefore conclude that intracellular proteases represent new targets for improved recombinant protein production in microbial cell factories like B. subtilis.

Molecular and Medical Aspects of Psychedelics.

Psychedelics belong to the oldest psychoactive drugs. They arouse recent interest due to their therapeutic applications in the treatment of major depressive disorder, substance use disorder, end-of-life anxiety,= and anxiety symptoms, and obsessive-compulsive disorder. In this review, the current state of preclinical research on the mechanism of action, neurotoxicity, and behavioral impact of psychedelics is summarized. The effect of selective 5-HT2A receptor agonists, 25I- and 25B-NBOMe, after acute and repeated administration is characterized and compared with the effects of a less selective drug, psilocybin. The data show a significant effect of NBOMes on glutamatergic, dopaminergic, serotonergic, and cholinergic neurotransmission in the frontal cortex, striatum, and nucleus accumbens. The increases in extracellular levels of neurotransmitters were not dose-dependent, which most likely resulted from the stimulation of the 5-HT2A receptor and subsequent activation of the 5-HT2C receptors. This effect was also observed in the wet dog shake test and locomotor activity. Chronic administration of NBOMes elicited rapid development of tolerance, genotoxicity, and activation of microglia. Acute treatment with psilocybin affected monoaminergic and aminoacidic neurotransmitters in the frontal cortex, nucleus accumbens, and hippocampus but not in the amygdala. Psilocybin exhibited anxiolytic properties resulting from intensification of GABAergic neurotransmission. The data indicate that NBOMes as selective 5-HT2A agonists exert a significant effect on neurotransmission and behavior of rats while also inducing oxidative DNA damage. In contrast to NBOMes, the effects induced by psilocybin suggest a broader therapeutic index of this drug.

A Fiber Sensor for Long-Term Monitoring of Extracellular Potassium Ion Fluctuations in Chronic Neuropsychiatric Diseases.

The extracellular potassium ion concentration in the brain exerts a significant influence on cellular excitability and intercellular communication. Perturbations in the extracellular potassium ion level are closely correlated with various chronic neuropsychiatric disorders including depression. However, a critical gap persists in performing real-time and long-term monitoring of extracellular potassium ions, which is necessary for comprehensive profiling of chronic neuropsychiatric diseases. Here, a fiber potassium ion sensor (FKS) that consists of a soft conductive fiber with a rough surface and a hydrophobic-treated transduction layer interfaced with a potassium ion-selective membrane is found to solve this problem. The FKS demonstrates stable interfaces between its distinct functional layers in an aqueous environment, conferring an exceptional stability of 6 months in vivo, in stark contrast to previous reports with working durations from hours to days. Upon implantation into the mouse brain, the FKS enables effective monitoring of extracellular potassium ion dynamics under diverse physiological states including anesthesia, forced swimming, and tail suspension. Using this FKS, tracking of extracellular potassium ion fluctuations that align with behaviors associated with the progression of depression over months is achieved, demonstrating its usability in studying chronic neuropsychiatric disorders from a new biochemical perspective.

Low-dose imiquimod induces melanogenesis in melanoma cells through an ROS-mediated pathway

BackgroundMelanogenesis is the process of melanin maturation which not only protects skin from UV radiation but also plays an important role in antigenicity of melanomas. Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) agonist that exhibits antiviral and anticancer activity. ObjectiveTo explore whether IMQ could induce melanogenesis in melanoma cells. MethodsThe mouse melanoma cell line B16F10, the mouse immortalized melanocyte Melan-A, and human melanoma cell lines MNT-1, C32 and A375 were utilized in this study. The pigmented level was observed by the centrifuged cell pellet. The intracellular and extracellular melanin levels were examined in the absorbance in NaOH-extracted cell lysate and cell-cultured medium, respectively. The expression of melanogenesis related proteins was examined by immunoblotting. The intracellular cyclic AMP amount was evaluated by the cAMP Glo assay kit. The activity of phosphodiesterase 4B (PDE4B) was investigated by CREB reporter assay with overexpressed PDE4B or not. ResultsWe demonstrated that a low dose of IMQ could trigger melanogenesis in B16F10 cells. IMQ induced microphthalmia-associated transcription factor (MITF) nuclear translocation, upregulated the expression of melanogenesis-related proteins, increased tyrosinase (TYR) activity, and led to pigmentation in B16F10 cells. Next, we found that IMQ-induced melanogenesis was activated by excessive intracellular cAMP accumulation, which was regulated through IMQ-mediated PDE4B inhibition. Finally, IMQ-induced ROS production was found to be involved in melanogenesis by its control of PDE4B activity. ConclusionsLow dose of IMQ could activate melanogenesis through the ROS/PDE4B/PKA pathway in melanoma cells.

Metabolic signatures of tear extracellular vesicles caused by herpes simplex keratitis

PurposeHerpes simplex keratitis (HSK), caused by type 1 herpes simplex virus (HSV) reactivation, is a severe infectious disease that leads to vision loss. HSV can trigger metabolic reprogramming in the host cell and change the extracellular vesicles (EV) cargos; however, little is known about the EV metabolic signatures during ocular HSV infection. Here, we aimed to depict the EV-associated metabolic landscape in HSK patients’ tears. MethodsWe collected 82 samples from 41 participants with unilateral HSK (contralateral unaffected tears were set as negative control), including subtype cohorts of 13 epithelial, 20 stromal, and 8 endothelial HSK. We isolated tear EVs via our previously established platform and conducted metabolic analysis using LC-MS/MS. The metabolic signatures for recognizing HSK and subtypes were assessed through differential analysis and machine learning algorithms. ResultsHypopsia and increased extracellular CD63 levels were observed in affected eyes. We identified 339 metabolites based on sEVs isolated from tears. Differential analysis revealed alterations in energy and amino acid metabolism, as well as the infectious microenvironment. Furthermore, we observed dysregulated metabolite such as methyldopa, which is associated with inappropriate neovascularization and corneal sensation loss, contributing to the HSK severity particularly in the stromal subtype. Moreover, machine learning classification also suggested a set of EV metabolic signatures that have potential for pan-keratitis detection. ConclusionsOur findings demonstrate that tear EV metabolites can serve as valuable indicators for comprehending the underlying pathological mechanisms. This knowledge is expected to facilitate the development of liquid biopsy means and therapeutic target discovery.

Extracellular DNA level as an indicator of the activity of inflammatory reactions in patients with rheumatoid arthritis and asthma

BACKGROUND: We performed a clinical and laboratory examination of patients with rheumatoid arthritis and bronchial asthma. Nosologies were selected to examine extracellular DNA changes in the blood during immunopathological processes based on generally accepted views on the role of helper balance in disease pathogenesis. The degree of disease activity in rheumatoid arthritis primarily depends on the severity of inflammatory changes in the joints; therefore, it is closely associated with a shift in the balance of helpers toward the Th1. According to today’s concepts, the pathogenesis of asthma is determined by the intensive production of specific antibodies, which are controlled by activated Th2 lymphocytes. AIM: To study extracellular DNA changes in the blood of patients during immunopathological processes depending on the Th1/Th2 balance ratio and compare these data with the parameters of standard laboratory tests and indicators of neutrophil activities and their ability to netosis. METHODS: Extracellular DNA concentration was determined using the Quant-ITTM PicoGreen fluorescent reagent for double-stranded DNA. Neutrophils isolated in a double-density gradient of ficoll–urografin were cultured with or without the addition of phorbol myristate acetate followed by Sytox Green. RESULTS: The studied immunopathological conditions had significantly decreased extracellular DNA levels in the plasma of patients with asthma, which sharply contrasted with its increase in patients with arthritis. Laboratory parameters confirmed the presence of local inflammation in patients with asthma without obvious symptoms of a systemic inflammatory response, while in patients with arthritis, inflammatory changes in the joints were accompanied by an increased C-reactive protein level, indicating a systemic reaction of the body in response to inflammatory stimuli. CONCLUSIONS: Thus, the state of the Th1/Th2 balance is assumed as one of the significant regulators that determined the extracellular DNA concentration in the blood. According to the proposed hypothesis, the shift in this ratio toward the predominance of Th1 cells should promote the development of inflammatory processes in the body and increase the amount of extracellular DNA, while a shift in the balance of helpers toward the dominance of Th2 lymphocytes should suppress these processes and consequently decrease the extracellular DNA in the blood.

Highly Sensitive Diagnosis of Extracellular Calcium Ions Associated Brain Diseases using Ca2+‐Dependent T2‐T1 Switchable Magnetic Nanosensors

AbstractCalcium ions (Ca2+) play crucial roles as second messengers in living cells and are associated with the onset and development of chronic brain diseases, including epilepsy. Dynamic and real‐time imaging of extracellular calcium levels ([Ca2+]ex) in the brain holds great potential for identifying disease foci. Magnetic resonance imaging (MRI) is a powerful non‐invasive tool for Ca2+ imaging with unlimited depth penetration. However, current available Ca2+ MR sensors lack the sensitivity to monitor small [Ca2+]ex alterations associated with chronic brain diseases in vivo. In this study, a highly sensitive Ca2+‐dependent T2‐T1 switchable magnetic nanosensor is reported, consisting of Ca2+‐specific ligands engineered near‐paramagnetic iron oxide nanoparticles. Surface ligand modifications can be adjusted to regulate the interaction energy between sensors and Ca2+, allowing for customizable response range and sensitivity to fulfill the in vivo imaging requirements. The optimized sensors enable non‐invasive visualization of early‐stage seizure focus in mice undergoing epilepsy.

The protective role of CD73 in periodontitis: preventing hyper-inflammatory fibroblasts and driving osteoclast energy metabolism.

Periodontitis is an immune-mediated inflammatory disease affecting almost half of the adult population and is the leading cause of tooth loss in the United States. The role of extracellular nucleotide signaling including nucleotide metabolizing enzyme CD73 adds an important layer of interaction of purine mediators capable of orchestrating inflammatory outcomes. CD73 is able to catabolize 5'-adenosine monophosphate into adenosine at the extracellular level, playing a critical role in regulating many processes under physiological and pathological conditions. Here, we explored the role of CD73 in ligature-induced periodontitis in vivo comparing wild-type C57Bl/6J and CD73-deficient mice. We assessed gingival levels of inflammatory cytokines in vivo and in murine gingival fibroblasts in vitro, as well as bone loss, and RANKL-induced osteoclastogenesis. We have also analyzed CD73 mRNA in samples derived from patients diagnosed with severe periodontitis. Our results in mice show that lack of CD73 resulted in increased inflammatory cytokines and chemokines such as IL-1β, IL-17, Cxcl1 and Cxcl2 in diseased gingiva relative to the healthy-controls and in comparison with the wild type. CD73-deficient gingival fibroblasts also manifested a defective healing response with higher MMP-13 levels. CD73-deficient animals also showed increased osteoclastogenesis in vitro with increased mitochondrial metabolism typified by excessive activation of oxidative phosphorylation, increased mitochondrial membrane potential and accumulation of hydrogen peroxide. Micro-CT analysis revealed that lack of CD73 resulted in decreased bone mineral density, decreased trabecular bone volume and thickness as well as decreased bone volume in long bones. CD73 deficiency also resulted in increased alveolar bone loss in experimental periodontitis. Correlative studies of gingival samples from severe (Grade C) periodontitis showed decreased levels of CD73 compared to healthy controls, further supporting the relevance of our murine results. In conclusion, CD73 appears to play a protective role in the gingival periodontal tissue and bone homeostasis, regulating hyper-inflammatory state of stromal fibroblasts and osteoclast energy metabolism and being an important candidate for future target therapies to prevent or control immune-mediated inflammatory and osteolytic diseases.