Release Of Tumor Necrosis Factor Research Articles

Regulation of astrocyte metabolism by mitochondrial translocator protein 18 kDa.

The mitochondrial translocator protein 18 kDa (TSPO) has been linked to functions from steroidogenesis to regulation of cellular metabolism and is an attractive therapeutic target for chronic CNS inflammation. Studies in Leydig cells and microglia indicate that TSPO function may vary between cells depending on their specialized roles. Astrocytes are critical for providing trophic and metabolic support in the brain. Recent work has highlighted that TSPO expression increases in astrocytes under inflamed conditions and may drive astrocyte reactivity. Relatively little is known about the role TSPO plays in regulating astrocyte metabolism and whether this protein is involved in immunometabolic processes in these cells. Using TSPO-deficient (TSPO-/- ) mouse primary astrocytes invitro (MPAs) and a human astrocytoma cell line (U373 cells), we performed extracellular metabolic flux analyses. We found that TSPO deficiency reduced basal cellular respiration and attenuated the bioenergetic response to glucopenia. Fatty acid oxidation was increased, and lactate production was reduced in TSPO-/- MPAs and U373 cells. Co-immunoprecipitation studies revealed that TSPO forms a complex with carnitine palmitoyltransferase 1a in U373 and MPAs, presenting a mechanism wherein TSPO may regulate FAO in these cells. Compared to TSPO+/+ cells, in TSPO-/- MPAs we observed attenuated tumor necrosis factor release following 3 h lipopolysaccharide (LPS) stimulation, which was enhanced at 24 h post-LPS stimulation. Together these data suggest that while TSPO acts as a regulator of metabolic flexibility, TSPO deficiency does not appear to modulate the metabolic response of MPAs to inflammation, at least in response to the model used in this study.

Anti-Inflammatory Effects and Immunomodulatory Efficacy of Unitein (Fermented Glycine max, Panax ginseng, and Chenpi Mixture) in RAW 264.7 Macrophages and Mice Splenocytes along with Component Analysis

Inflammation and the immune system are intricately linked, with the immune system serving as a vital defense mechanism in the human body. Consequently, there is great emphasis placed on the regulation of both the body’s inflammatory response and the immune system. This study investigated the anti-inflammatory properties and immunomodulatory effects of Unitein (fermented Glycine max, Panax ginseng, and chenpi mixture) in both RAW 264.7 macrophages and mice splenocytes while also comprehensively analyzing its components. To this end, the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was employed to evaluate the cytotoxicity of the samples to RAW 264.7 cells. The Griess assay was utilized to determine nitrite concentrations, while the enzyme-linked immunosorbent assay (ELISA) was employed to assess the levels of inflammation-related cytokines in RAW 264.7 cells and isolated mouse splenocytes. Additionally, quantitative polymerase chain reaction was employed to quantify the mRNA expression of inflammation-related genes in the cells, while the lactate dehydrogenase assay was used to analyze natural killer (NK) cell activity. Our results revealed that Unitein exhibited no toxicity to RAW 264.7 cells at concentrations below 1.5 mg/mL. However, Unitein effectively suppressed the release of proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and interferon (IFN)-γ, as well as enzymes COX-2 and iNOS from RAW 264.7 cells, while concurrently promoting the release of anti-inflammatory cytokines IκB-α, IL-10, and IL-4. Furthermore, Unitein inhibited the increase in TNF-α, IL-1β, IFN-γ, IL-12, nuclear factor (NF)-κB p50, NF-κB p65, and iNOS induced by lipopolysaccharides (LPSs) in isolated mouse splenocytes. Notably, Unitein also exhibited the potential to stimulate NK cell activity. Metabolite analysis indicated that Unitein contained more active compounds with anti-inflammatory and immunity-enhancing properties than did unfermented Unitein. This study highlights the potential therapeutic role of Unitein in mitigating inflammation and enhancing immune responses, thereby deepening our understanding of its biological activities and underlying mechanisms. These findings underscore the significance of Unitein as a potential candidate for the development of novel anti-inflammatory and immunomodulatory interventions.

Potency assay to predict the anti-inflammatory capacity of a cell therapy product for macrophage-driven diseases: overcoming the challenges of assay development and validation

BackgroundGiven the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment. MethodsAn in vitro inflammation model was established by coculturing skin-derived ABCB5+ MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay. ResultsIL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors. ConclusionsWe describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications.

Administration of Gracilariopsis lemaneiformis polysaccharide attenuates cisplatin-induced inflammation and intestinal mucosal damage in colon-26 carcinoma tumor-bearing mice.

Our preliminary research revealed that the polysaccharide GP90 from Gracilariopsis lemaneiformis enhanced the antitumor effect of cisplatin, indicating that GP90 may increase the chemotherapeutic sensitivity. However, it is still necessary to fully understand whether GP90 can also improve the intestinal barrier dysfunction and systemic inflammation induced by cisplatin. GP90 has been demonstrated to inhibit the excessive release of nitirc oxide, interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α induced by lipopolysaccharide in RAW264.7 cells. In vivo, GP90 effectively ameliorated the decrease in the serum CD4+ /CD8+ T-cell ratio induced by cisplatin and significantly reduced the increase in the inflammatory cytokines, CD4+ Foxp3+ , CD4+ granzyme B+ and CD4+ TNF-α induced by cisplatin. Furthermore, when combined with cisplatin, GP90 increases the protein expression levels of mucin-2 and zonula occludens-1 in the mouse small intestine. Additionally, GP90 combined with cisplatin has a modulatory effect on the intestinal microbiota by elevating the Firmicutes-to-Bacteroidetes ratio and the relative abundance of beneficial microorganisms (Lachnospiraceae bacterium), at the same time as reducing the abundance of cisplatin specific Bacteroides acidifaciens and elevating the content of butyric acid and isobutyric acid. Collectively, these findings indicate that GP90 potentially mitigates inflammation and protects the intestinal barrier in tumor-bearing organisms undergoing chemotherapy. © 2024 Society of Chemical Industry.

Blocking of microglia-astrocyte proinflammatory signaling is beneficial following stroke.

Microglia and astrocytes play an important role in the neuroinflammatory response and contribute to both the destruction of neighboring tissue as well as the resolution of inflammation following stroke. These reactive glial cells are highly heterogeneous at both the transcriptomic and functional level. Depending upon the stimulus, microglia and astrocytes mount a complex, and specific response composed of distinct microglial and astrocyte substates. These substates ultimately drive the landscape of the initiation and recovery from the adverse stimulus. In one state, inflammation- and damage-induced microglia release tumor necrosis factor (TNF), interleukin 1α (IL1α), and complement component 1q (C1q), together "TIC." This cocktail of cytokines drives astrocytes into a neurotoxic reactive astrocyte (nRA) substate. This nRA substate is associated with loss of many physiological astrocyte functions (e.g., synapse formation and maturation, phagocytosis, among others), as well as a gain-of-function release of neurotoxic long-chain fatty acids which kill neighboring cells. Here we report that transgenic removal of TIC led to reduction of gliosis, infarct expansion, and worsened functional deficits in the acute and delayed stages following stroke. Our results suggest that TIC cytokines, and likely nRAs play an important role that may maintain neuroinflammation and inhibit functional motor recovery after ischemic stroke. This is the first report that this paradigm is relevant in stroke and that therapies against nRAs may be a novel means to treat patients. Since nRAs are evolutionarily conserved from rodents to humans and present in multiple neurodegenerative diseases and injuries, further identification of mechanistic role of nRAs will lead to a better understanding of the neuroinflammatory response and the development of new therapies.

Assessment of Nailfold Capillaroscopy Findings in Pregnant Women having Preeclampsia: A Research Protocol

Introduction: Pregnancy causes significant vascular and cutaneous changes in a woman’s body. Preeclampsia is one of the life-threatening conditions associated with high blood pressure and proteinuria after the 20th week of pregnancy. It can cause placental hypoperfusion and placental insufficiency, leading to foetal defects or, in severe cases, foetal loss, which can be prevented by early diagnosis and treatment. There are many ways to observe and study the microcirculation non invasively the safest and most accessible method is nailfold capillaroscopy. Nail-fold Video Capillaroscopy (NVC) is a handheld digital microscope used to observe the microcirculation at the nailfold of the hands. Thus, comparing the capillary status with healthy subjects will help establish an early diagnosis and manage cases to prevent further complications in the mother and newborn. Need of the Study: The pathophysiology states that preeclampsia is related to systemic endothelial dysfunction, causing the release of Tumour Necrosis Factor (TNF) alpha, a proinflammatory mediator, leading to hypoxia of the placenta and the loss of endothelial cell-lined capillaries. Thus, there is a need to find if there is an established correlation in association of decreased Capillary Density (CD) in individuals with preeclampsia. Although evidence supporting a link between decreased CD and preeclampsia is limited, timely assessment and prevention are crucial. Nailfold capillaroscopy, an accessible and non invasive imaging method, holds promising calibre for understanding microvascular changes in preeclampsia. This could facilitate post-conception monitoring and early interventions to identify and address circulatory issues in affected women with preeclampsia. Aim: To study and compare the nailfold capillary changes in women diagnosed with preeclampsia and women having normal and uneventful pregnancies. Materials and Methods: A comparative cross-sectional study will be done in the Outpatient Department (OPD) and Inpatient Department (IPD) of Obstetrics and Gynaecology and the Outpatient Department of Dermatology at AVBRH Sawangi Meghe, Wardha, Maharashtra, India, from March 2023 to March 2025. A total of 150 pregnant women will be considered in the study, with 75 women being normal pregnant women (controls) and 75 pregnant women diagnosed with preeclampsia (cases). In order to confirm the gestational age of the patients, fundal height and abdominal circumference measurements will be taken. Demographic data, Basal Metabolic Index (BMI), weight gain during pregnancy, laboratory reports including Complete Blood Count (CBC), coagulation profile (Prothrombin Time, International Normalised Ratio, Activated Partial Thromboplastin Time), liver function tests, kidney function tests, urine examinations, and serial ultrasonography reports available for the patients will be noted. Pre-examination blood pressure will be recorded and compared with the baseline to confirm the status of preeclampsia after obtaining written consent from the patients. Nailfold capillaroscopy will be performed using a (Dinolite -AF4115ZT) dermoscope equipped with a 200X magnification, high-resolution lens in a non polarised light setting with oil immersion as a fluid coupling. The observations will be used to interpret the results of the study. Results will be calculated for finding differences between the two comparative groups of cases and controls at a p-value of ≤0.05.

Mesenchymal stromal cells suppress microglial activation and tumor necrosis factor production

Background aimsWhite matter diseases are commonly associated with microglial activation and neuroinflammation. Mesenchymal stromal cells (MSCs) have immunomodulatory properties and thus have the potential to be developed as cell therapy for white matter disease. MSCs interact with resident macrophages to alter the trajectory of inflammation; however, the impact MSCs have on central nervous system macrophages and the effect this has on the progression of white matter disease are unclear. MethodsIn this study, we utilized numerous assays of varying complexity to model different aspects of white matter disease. These assays ranged from an in vivo spinal cord acute demyelination model to a simple microglial cell line activation assay. Our goal was to investigate the influence of human umbilical cord tissue MSCs on the activation of microglia. ResultsMSCs reduced the production of tumor necrosis factor (TNF) by microglia and decreased demyelinated lesions in the spinal cord after acute focal injury. To determine if MSCs could directly suppress the activation of microglia and to develop an efficient potency assay, we utilized isolated primary microglia from mouse brains and the Immortalized MicroGlial Cell Line (IMG). MSCs suppressed the activation of microglia and the release of TNF after stimulation with lipopolysaccharide, a toll-like receptor agonist. ConclusionsIn this study, we demonstrated that MSCs altered the immune response after acute injury in the spinal cord. In numerous assays, MSCs suppressed activation of microglia and release of the pro-inflammatory cytokine TNF. Of these assays, IMG could be standardized and used as an effective potency assay to determine the efficacy of MSCs for treating white matter disease or other neuroinflammatory conditions associated with microglial activation.

Understanding the Macrophage's Role in Regulating Cancer-Associated Thrombosis

Background: With the risk of thrombosis elevated in cancer patients, there is a need to find treatments that can regulate clotting abnormalities during this disease. The immune system has become a target for treating cancer-associated thrombosis (CAT). Notably, the macrophage, a key immune cell, has been shown to play a role in mediating cancer progression and thrombosis. A regulator of the coagulation process, urokinase plasminogen activator (uPA) also mediates cancer progression. uPA regulates blood clot breakdown through fibrinolysis. Monocytes have receptors for uPA. In addition, upon stimulation, they can release tumor necrosis factor alpha (TNF-α), a prothrombotic cytokine. Furthermore, macrophages can release higher quantities of tissue factor upon polarization through interleukins (IL-4 and IL-13). However, no known studies show the macrophage phenotypes' (pro-inflammatory or anti-inflammatory) effects on clot formation, structure, and fibrinolysis. Given that uPA has a role in cancer progression, monocytes contain receptors for uPA, and macrophage phenotype influences tissue factor expression, there is a need to determine how macrophage phenotype and components of the urokinase system can mediate clot formation, structure, and resolution. Aim: We aim to elucidate the biochemical and structural mechanism(s) by which both pro-inflammatory (M1) and anti-inflammatory (M2) macrophages can induce CAT in the presence of the urokinase system. Methods: Human monocytes, THP-1 cells, were differentiated into M0, then attenuated into M1 or M2 macrophages using LPS or LPS+PGE2, respectively. Human plasma was coincubated with the respective macrophage phenotypes, and clot formation was tracked over time by collecting turbidity measurements using a microplate reader (n=2-3). This same coincubation experiment was also conducted with the addition of uPA to track fibrinolysis kinetics (n=3). Confocal microscopy was used to characterize clot structure; the fibrin network was labeled with a 488 nm fluorophore. Images (n=6-7) were measured for area percentage in FIJI to assess clot density. Results: Clotting kinetics showed that all macrophage phenotypes resulted in a >1.4-fold faster onset of clotting compared to controls without cells (p<0.002). The faster onset of clotting corresponded to an increased fibrin area density relative to no cells, where proinflammatory M1 macrophages had the most pronounced effect (p<0.001) (Figure 1A). Fibrinolysis triggered by uPA indicated that M1 and M2 attenuated macrophages delayed the time to 50% lysis relative to M0 macrophages and no cells (Figure 1B). Conclusion: Attenuation to the M1 and M2 macrophage phenotype promoted resistance to uPA-based lysis compared to M0 and no cell cohorts, suggesting that the change of phenotype can slow uPA-based lysis. Overall, all macrophage phenotypes accelerated clotting relative to controls without cells, which suggests macrophages can induce a hypercoagulable state leading to a denser fibrin network. We propose that the denser fibrin network is more resistant to fibrinolysis, thereby shedding light on a potential mechanism for the role of macrophages in immunothrombosis and CAT.

Role of perfusion medium in rewarming machine perfusion from hypo- to normothermia.

Gradual warming up of cold stored organ grafts using a controlled machine perfusion protocol facilitates restitution of cellular homeostasis and mitigates rewarming injury by adapted increase of temperature and metabolism. The aim of the present study was to compare intra- and extracellular type perfusion media for the use in machine perfusion-assisted rewarming from hypo- to normothermia. Rat livers were retrieved 20 min after cardiac arrest. After 18 h of cold storage (CS) with or without additional 2 h of rewarming machine perfusion from 8°C up to 35°C with either diluted Steen solution or with Belzer MPS, liver functional parameters were evaluated by an established exvivo reperfusion system. Rewarming machine perfusion with either solution significantly improved graft performance upon reperfusion in terms of increased bile production, less enzyme release, and reduced lipid peroxidation compared to CS alone. Cellular apoptosis (release of caspase-cleaved keratin 18) and release of tumor necrosis factor were only reduced significantly after machine perfusion with Belzer MPS. Histological evaluation did not disclose any major morphological damage in any of the groups. Within the limitation of our model, the use of Belzer MPS seems to be an at least adequate alternative to a normothermic medium like Steen solution for rewarming machine perfusion of cold liver grafts.

The role of melatonin in amyloid beta-induced inflammation mediated by inflammasome signaling in neuronal cell lines

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. In addition to amyloid beta (Aβ) and tau, neuroinflammation is a crucial element in the etiology of this disease. However, the relevance of inflammasome-induced pyroptosis to AD is unknown. We aimed to clarify whether the anti-inflammatory effects of melatonin could prevent Aβ-mediated activation of the inflammasome. We demonstrated that Aβ upregulated NOD-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD, and cysteinyl aspartate-specific proteinase caspase (caspase 1) expression in SH-SY5Y neuroblastoma cells, resulting in the release of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-18 (IL-18) and tumor necrosis factor (TNF-α). Melatonin prevented inflammasome signaling and excessive cytokine release caused by Aβ. We found that ethyl 2[(2-chlorophenyl)(hydroxy) methyl]acrylate (INF-4E, NLRP3 and caspase 1 inhibitor) significantly abolished Aβ-induced proinflammatory cytokine expression. The increase in cleaved-caspase 1, pro-IL18, and cleaved-IL18 caused by Aβ suggested the occurrence of pyroptosis, which was further confirmed by the increased expression of N-terminal gasdermin D (N-GSDMD). Melatonin plays a protective role against Aβ-induced inflammation via an inflammasome-associated mechanism that is essential in inducing the active forms of cytokines and pyroptosis. The ability of melatonin to inhibit inflammasome may represent a turning point in the treatment of AD progression.

Abstract 29 Umbilical Cord-derived Mesenchymal Stromal Cells Suppress Microglia Activation Induced by Demyelination

Abstract Introduction White matter diseases are often accompanied by microglial activation and neuroinflammation. Mesenchymal stromal cells (MSCs) manufactured from umbilical cord tissue possess immunomodulatory properties, making them promising candidates for cell therapy in white matter disease. MSCs can interact with resident macrophages and modify the course of inflammation. However, the influence MSCs have on microglia, the resident macrophages of the central nervous system (CNS), and its implications for white matter disease remain uncertain. Objectives From the blood, MSCs traffic to lung and interact with resident macrophages to alter the trajectory of inflammation. We hypothesized that MSCs injected into the cerebrospinal fluid (CSF) would similarly suppress the activation of microglia. Our goals in this study were to test the efficacy MSCs have on microglia activation and develop a relevant potency assay. Methods In this study, we employed a range of assays, including an in vivo acute spinal cord demyelination model, organotypic brain slice cultures, and microglia activation assays with primary microglia and an immortalized cell line. Using these assays, we assessed the impact cord tissue derived MSCs have on microglial activation. Results In spinal cords and brain slices, lysophosphatidylcholine produced robust demyelination and microglia activation that was suppressed by MSCs. To determine if MSCs could directly impact microglia, we isolated primary microglia and activated them in culture with lipopolysachride (LPS), a toll-like receptor agonist. We screen 6 cytokines induced by LPS and determined that MSCs suppress the release of tumor necrosis factor (TNF) from LPS-activated microglia. We confirmed these results in vivo as MSCs blocked TNF production by spinal cord microglia after acute demyelination in the spinal cord. Additionally, we confirmed the ability of MSCs to suppress TNF in a simple assay with a microglia cell line (IMG). Discussion In this study, we demonstrated that cord tissue MSCs altered the immune response, suppressed microglia activation, and the release of TNF, a cytokine known to contribute to white matter damage. Among these assays, IMG could be standardized as an effective potency assay to determine the efficacy of MSC for the use in treatment of white matter disease.

Receptor-interacting protein kinase 1 (RIPK1) regulates cervical cancer cells via NF-κB–TNF-α pathway: An in vitro study

IntroductionCervical cancer (CC) is associated with high morbidity and mortality rates in women. Members of the receptor-interacting protein kinase (RIPK) family are important regulators of inflammation and cell death. However, the characteristics, molecular functions, and expression mechanisms of RIPK1 in CC remain unclear. Material and methodsTo determine whether RIPK1 can be used for targeted therapy of CC, we assessed the clinical importance, biological function, and potential impact of RIPK1 in CC in 50 patients with CC. We utilized immunohistochemical staining, transfection, western blotting, cell counting kit-8 assay, colony formation assay, and wound healing assays among others, to elucidate the role of RIPK1 in CC. ResultsRIPK1 expression was higher in tumor tissues than in paracancerous tissues. Poor prognosis of CC was linked to RIPK1 upregulation. Furthermore, silencing RIPK1 significantly inhibited the proliferation, migration, and invasion of CC cells in vitro. We also established that RIPK1 increased cell migration, invasion, and multiplication by regulating nuclear factor kappa-B (NF-κB) and tumor necrosis factor (TNF). DiscussionRIPK1 activates NF-κB and regulates TNF release to enhance the proliferation and spread of CC cells while suppressing their apoptosis. Therefore, RIPK1 plays a key role in the formation and progression of CC and is a potential target for CC treatment.

Microglial Priming in Bilirubin-Induced Neurotoxicity.

Neuroinflammation is a major contributor to bilirubin-induced neurotoxicity, which results in severe neurological deficits. Microglia are the primary immune cells in the brain, with M1 microglia promoting inflammatory injury and M2 microglia inhibiting neuroinflammation. Controlling microglial inflammation could be a promising therapeutic strategy for reducing bilirubin-induced neurotoxicity. Primary microglial cultures were prepared from 1-3-day-old rats. In the early stages of bilirubin treatment, pro-/anti-inflammatory (M1/M2) microglia mixed polarization was observed. In the late stages, bilirubin persistence induced dominant proinflammatory microglia, forming an inflammatory microenvironment and inducing iNOS expression as well as the release of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Simultaneously, nuclear factor-kappa B (NF-κB) was activated and translocated into the nucleus, upregulating inflammatory target genes. As well known, neuroinflammation can have an effect on N-methyl-D-aspartate receptor (NMDAR) expression or function, which is linked to cognition. Treatment with bilirubin-treated microglia-conditioned medium did affect the expression of IL-1β, NMDA receptor subunit 2A (NR2A), and NMDA receptor subunit 2B (NR2B) in neurons. However, VX-765 effectively reduces the levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β, as well as the expressions of CD86, and increases the expressions of anti-inflammatory related Arg-1. A timely reduction in proinflammatory microglia could protect against bilirubin-induced neurotoxicity.

Protective Effect of Limosilactobacillus fermentum ME-3 against the Increase in Paracellular Permeability Induced by Chemotherapy or Inflammatory Conditions in Caco-2 Cell Models.

Chemotherapy- or inflammation-induced increase in intestinal permeability represents a severe element in disease evolution in patients suffering from colorectal cancer and gut inflammatory conditions. Emerging data strongly support the gut microbiota's role in preserving intestinal barrier integrity, whilst both chemotherapy and gut inflammation alter microbiota composition. Some probiotics might have a strong re-balancing effect on the gut microbiota, also positively affecting intestinal barrier integrity. In this study, we asked whether Limosilactobacillus fermentum ME-3 can prevent the intestinal paracellular permeability increase caused by the chemotherapeutic drug Irinotecan or by inflammatory stimuli, such as lipopolysaccharide (LPS). As an intestinal barrier model, we used a confluent and polarized Caco-2 cell monolayer and assessed the ME-3-induced effect on paracellular permeability by transepithelial electrical resistance (TEER) and fluorescent-dextran flux assays. The integrity of tight and adherens junctions was examined by confocal microscopy analysis. Transwell co-cultures of Caco-2 cells and U937-derived macrophages were used as models of LPS-induced intestinal inflammation to test the effect of ME-3 on release of the pro-inflammatory cytokines Tumor Necrosis Factor α, Interleukin-6, and Interleukin-8, was measured by ELISA. The results demonstrate that ME-3 prevents the IRI-induced increment in paracellular permeability, possibly by modulating the expression and localization of cell junction components. In addition, ME-3 inhibited both the increase in paracellular permeability and the release of pro-inflammatory cytokines in the co-culture model of LPS-induced inflammation. Our findings sustain the validity of L. fermentum ME-3 as a valuable therapeutic tool for preventing leaky gut syndrome, still currently without an available specific treatment.

Asprosin Enhances Cytokine Production by a Co-Culture of Fully Differentiated Mature Adipocytes and Macrophages Leading to the Exacerbation of the Condition Typical of Obesity-Related Inflammation.

Asprosin, a fasting-induced, glucogenic, and orexigenic adipokine, has gained popularity in recent years as a potential target in the fight against obesity and its complications. However, the contribution of asprosin to the development of moderate obesity-related inflammation remains still unknown. The present study aimed to evaluate the effect of asprosin on the inflammatory activation of adipocyte-macrophage co-cultures at various stages of differentiation. The study was performed on co-cultures of the murine 3T3L1 adipocyte and the RAW264.7 macrophage cell lines treated with asprosin before, during, and after 3T3L1 cell differentiation, with or without lipopolysaccharide (LPS) stimulation. Cell viability, overall cell activity, and the expression and release of key inflammatory cytokines were analyzed. In the concentration range of 50-100 nM, asprosin increased the pro-inflammatory activity in the mature co-culture and enhanced the expression and release of tumor necrosis factor α (TNF-α), high-mobility group box protein 1 (HMGB1), and interleukin 6 (IL-6). Macrophage migration was also increased, which could be related to the upregulated expression and release of monocyte chemoattractant protein-1 (MCP-1) by the adipocytes. In summary, asprosin exerted a pro-inflammatory effect on the mature adipocyte-macrophage co-culture and may contribute to the spread of moderate obesity-associated inflammation. Nevertheless, further research is needed to fully elucidate this process.

GARCINIA COWA ROXB. ETHANOL EXTRACT INHIBITS INFLAMMATION IN LPS-INDUCED RAW 264.7 MACROPHAGES

Objective: The purpose of this study was to examine the effect of Garcinia cowa Roxb. Ethanol (EGC) extract in LPS-induced Raw 264.7 macrophages by observing the release of Tumor Necrosis Factor (TNF) and Interleukin-6 (IL-6).
 Methods: Using the MTT method, a cell viability assay was performed to observe the cytotoxic effect on Raw 264.7 macrophages. For 24 h, Raw 264.7 macrophages were incubated with various EGC concentrations (100, 50, 10, 1 and 0.1 µg/ml). The medium was taken out after 48 h of incubation, and 100 µl of MTT 0.5 mg/ml was then added. 100 µl DMSO was used to dissolve the crystals and absorbance was measured using a microplate reader. To investigate the activity of EGC to LPS-induced Raw 264.7 macrophages, the ELISA method was used. Supernatant was obtained after treating Raw 264.7 macrophages with complete medium, EGC samples, and LPS (10 g/ml) for 24 h. IL-6 and TNF-α levels were assessed using supernatants with ELISA kit.
 Results: Cytotoxic effect of EGC to Raw 264.7 macrophages occurred at a concentration of 100 µg/ml with the cell viability value of 59.5%. At a concentration of 50 µg/ml, no cytotoxic effect occurred and the cell viability value was 105.5%. So, the higher concentration of EGC used for further investigation is 50 µg/ml. It was shown that the production of IL 6 was suppressed by EGC at a concentration of 12.5 µg/ml. The inhibition of TNF-α production was only seen at the concentration of 12.5, 25 and 50 µg/ml; there was an increase of TNF-α production.
 Conclusion: It can be concluded that EGC can be developed as a natural immunomodulator that can inhibit inflammation by suppressing IL-6 production to prevent immune system disorders.

Effects of Bacterial Biofilm on Regulation of Neurovascular Unit Functions and Neuroinflammation of Patients with Ischemic Cerebral Stroke by Immunocyte.

In this experiment, the effects of biofilm on neurovascular unit functions and neuroinflammation of patients with ischemic cerebral stroke were investigated. For this purpose, 20 adult male rats were purchased from Taconic (8 to 10 weeks old, weighing between 20 and 24g) and selected as the research objects. Then, they were randomly divided into an experimental group (10 rats) and a control group (10 rats). Ischemic cerebral stroke rat models were established. Besides, pseudomonas aeruginosa (PAO1) was prepared manually and implanted into the bodies of rats in the experimental group. mNSS scores, cerebral infarction area, and the release of inflammatory cytokines of rats in the two groups were compared. Results showed that mNSS scores for rats in the experimental group at all periods were remarkably higher than those for rats in the control group (P<0.05), which demonstrated that the rats in the experimental group suffered much severer neurological impairment than those in the control group. In addition, the release of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, inducible nitric oxide synthase (iNOS), and IL-10 were all higher than those of the control group (P<0.05). The cerebral infarction area of the experimental group at all periods was remarkably larger than that of the control group (P<0.05). In conclusion, the formation of biofilm led to the aggravation of neurological impairment and inflammatory reactions among patients with ischemic cerebral stroke.

Development and Validation of UV-Visible spectroscopic method for estimation of Nimesulide in bulk and its pharmaceutical formulation

The aim of the present work was to develop and validate a simple UV Spectroscopic method for the determination of Nimesulide, which is the inhibitor of prostaglandin synthesis from arachidonic acid and platelet aggregation. It has the ability to exert analgesic and anti-inflammatory actions through various mechanisms (ie, COX independent pathways) and also it inhibits the release of tumour necrosis factor (TNFα) and interleukins, and stops the release of histamine from mast cells and reduce the synthesis of platelet activating factor (PAF) from the basophil cells. The UV visible Spectrophotometric analysis was performed using systronics UV-Spectrophotometer 2704 X visible double beam by using solvent KOH. Detection was performed at a wavelength of 395nm. Method validation was carried out according to ICH Q2R1 guidelines by taking the parameters such as accuracy, linearity, precision, ruggedness, and robustness, LOD and LOQ. The UV spectrophotometric was found linear in the range 5- 25µg/ml. The method was rugged and robust with % relative standard deviation less than 2. The extraction recoveries was found to be higher than 99.19% in all experimental conditions. Based upon the performance characteristics, the proposed method was found accurate, precise, rapid and suitable for the determination of nimesulide for routine analysis. The aim of the present work was to develop and validate a simple UV Spectroscopic method for the determination of Nimesulide, which is the inhibitor of prostaglandin synthesis from arachidonic acid and platelet aggregation. It has the ability to exert analgesic and anti-inflammatory actions through various mechanisms (ie, COX independent pathways) and also it inhibits the release of tumour necrosis factor (TNFα) and interleukins, and stops the release of histamine from mast cells and reduce the synthesis of platelet activating factor (PAF) from the basophil cells. The UV visible Spectrophotometric analysis was performed using systronics UV-Spectrophotometer 2704 X visible double beam by using solvent KOH. Detection was performed at a wavelength of 395nm. Method validation was carried out according to ICH Q2R1 guidelines by taking the parameters such as accuracy, linearity, precision, ruggedness, and robustness, LOD and LOQ. The UV spectrophotometric was found linear in the range 5- 25µg/ml. The method was rugged and robust with % relative standard deviation less than 2. The extraction recoveries was found to be higher than 99.19% in all experimental conditions. Based upon the performance characteristics, the proposed method was found accurate, precise, rapid and suitable for the determination of nimesulide for routine analysis.

Prediction of COPD acute exacerbation in response to air pollution using exosomal circRNA profile and Machine learning.

Ambient fine particulate matter (PM2.5) is linked to an increased risk of chronic obstructive pulmonary disease (COPD) exacerbations, which significantly increase the risk of mortality in COPD patients. Identifying the subtype of COPD patients who are sensitive to environmental aggressions is necessary. Using in vitro and in vivo PM2.5 exposure models, we demonstrate that exosomal hsa_circ_0005045 is upregulated by PM2.5 and binds to the protein cargo peroxiredoxin2, which functionally aggravates hallmarks of COPD by recruiting neutrophil elastase and triggering in situ release of tumor necrosis factor (TNF)-α by inflammatory cells. The biological function of hsa_circ_0005045 associated with aggravation of COPD is validated using exosome-transplantation and conditional circRNA-knockdown murine models. By sorting the major components of PM2.5, we find that PM2.5-bound heavy metals, which are distinguishable from the components of cigarette smoke, trigger the elevation of exosomal hsa_circ_0005045. Finally, using machine learning models in a cohort with 327 COPD patients, the PM2.5 exposure-sensitive COPD patients are characterized by relatively high hsa_circ_0005045 expression, non-smoking, and group C (mMRC 0-1 (or CAT<10) and≥2 exacerbations (or≥1 exacerbation leading to hospital admission) in the past year). Thus, our results suggest that environmental reduction in PM2.5 emission provides a targeted approach to protecting non-smoking COPD patients against air pollution-related disease exacerbation.

Concentration‐dependent effects of proinflammatory cytokines on barrier function and tight junction protein expression in brain microvascular endothelial cells and the hypothermic and hyperthermic effects on tight junction protein expression

AbstractObjectiveThe mechanisms underlying therapeutic hypothermia, which protects neurons following severe brain damage, are only partially understood. We previously demonstrated that hypothermia reduced, whereas hyperthermia augmented, the release of tumor necrosis factor (TNF)‐α and interleukin (IL)‐17. Cerebral ischemia causes the loss of the blood–brain barrier (BBB) integrity, thereby increasing cerebral vascular permeability, which directly contributes to vasogenic edema, hemorrhagic transformation, and increased mortality. Brain microvascular endothelial cells (BMVECs) are a major component of BBB and tight junction proteins (TJPs) in these cells maintain the BBB integrity. In this study we determined the mechanisms underlying this treatment by measuring the effects of TNF‐α and IL‐17 on BMVEC barrier function and TJP expression in BMVECs, and by evaluating the effects of hypothermia and hyperthermia on TJP expression.MethodsThe barrier function of BMVECs was evaluated by measuring transepithelial electrical resistance (TEER). The expression of several TJPs, such as claudin‐5 and junctional adhesion molecule (JAM)‐B, was measured at the mRNA and protein levels using real‐time polymerase chain reaction and immunocytochemistry, respectively.ResultsTNF‐α and IL‐17 decreased TEER values, and TNF‐α decreased claudin‐5 and JAM‐B mRNA and protein levels, whereas IL‐17 decreased JAM‐B mRNA and protein levels, and all of these effects were concentration‐dependent. Compared with normothermia, claudin‐5 and JAM‐B proteins were not affected by hypothermia, whereas JAM‐B protein was reduced by hyperthermia.ConclusionThe hypothermic suppression of TNF‐α and IL‐17 release may contribute to the maintenance of BBB function by ameliorating the decrease of TJP(s). In contrast, hyperthermia may decrease barrier function through a decrease in JAM‐B expression. However, the contribution of changes in the JAM‐B expression to the barrier function of BMVECs remains to be clarified.