Mediated Gene Expression Research Articles

Reprograming of Gene Expression of Key Inflammatory Signaling Pathways in Human Peripheral Blood Mononuclear Cells by Soybean Lectin and Resveratrol.

Inflammation is linked to several human diseases like microbial infections, cancer, heart disease, asthma, diabetes, and neurological disorders. We have shown that the prototype inflammatory agonist LPS modulates the activity of Ubiquitin-Proteasome System (UPS) and regulates transcription factors such as NF-κB, leading to inflammation, tolerance, hypoxia, autophagy, and apoptosis of cells. We hypothesized that proteasome modulators resveratrol and soybean lectin would alter the gene expression of mediators involved in inflammation-induced signaling pathways, when administered ex vivo to human peripheral blood mononuclear blood cells (PBMCs) obtained from normal healthy controls. To test this hypothesis, analysis of RNA derived from LPS-treated human PBMCs, with or without resveratrol and soybean lectin, was carried out using Next Generation Sequencing (NGS). Collectively, the findings described herein suggest that proteasome modulators, resveratrol (proteasome inhibitor) and lectins (proteasome activator), have a profound capacity to modulate cytokine expression in response to proteasome modulators, as well as expression of mediators in multiple signaling pathways in PBMCs of control subjects. We show for the first-time that resveratrol downregulates expression of mediators involved in several key signaling pathways IFN-γ, IL-4, PSMB8 (LMP7), and a subset of LPS-induced genes, while lectins induced IFN-γ, IL-4, PSMB8, and many of the same genes as LPS that are important for innate and adaptive immunity. These findings suggest that inflammation may be influenced by common dietary components and this knowledge may be used to prevent or reverse inflammation-based diseases.

A Survey of Transcription Factors in Cell Fate Control.

Transcription factors (TFs) play a cardinal role in the development and maintenance of human physiology by acting as mediators of gene expression and cell state control. Recent advancements have broadened our knowledge on the potency of TFs in governing cell physiology and have deepened our understanding of the mechanisms through which they exert this control. The ability of TFs to program cell fates has gathered significant interest in recent decades, and high-throughput technologies now allow for the systematic discovery of forward programming factors to convert pluripotent stem cells into numerous differentiated cell types. The next generation of these technologies has the potential to improve our understanding and control of cell fates and states and provide advanced therapeutic modalities to address many medical conditions.

HIV infection drives pro-inflammatory immunothrombotic pathway activation and organ dysfunction among adults with sepsis in Uganda.

The global burden of sepsis is concentrated in high HIV-burden settings in sub-Saharan Africa (SSA). Despite this, little is known about the immunopathology of sepsis in persons with HIV (PWH) in the region. We sought to determine the influence of HIV on host immune responses and organ dysfunction among adults hospitalized with suspected sepsis in Uganda. Prospective cohort study. We compared organ dysfunction and 30-day outcome profiles of PWH and those without HIV. We quantified 14 soluble immune mediators, reflective of key domains of sepsis immunopathology, and performed whole-blood RNA-sequencing on samples from a subset of patients. We used propensity score methods to match PWH and those without HIV by demographics, illness duration, and clinical severity, and compared immune mediator concentrations and gene expression profiles across propensity score-matched groups. Among 299 patients, 157 (52.5%) were PWH (clinical stage 3 or 4 in 80.3%, 67.7% with known HIV on antiretroviral therapy). PWH presented with more severe physiologic derangement and shock, and had higher 30-day mortality (34.5% vs. 10.2%; P < 0.001). Across propensity score-matched groups, PWH exhibited greater pro-inflammatory immune activation, including upregulation of interleukin (IL)-6, IL-8, IL-15, IL-17 and HMGB1 signaling, with concomitant T-cell exhaustion, prothrombotic pathway activation, and angiopoeitin-2-related endothelial dysfunction. Sepsis-related organ dysfunction and mortality in Uganda disproportionately affect PWH, who demonstrate exaggerated activation of multiple immunothrombotic and metabolic pathways implicated in sepsis pathogenesis. Further investigations are needed to refine understanding of sepsis immunopathology in PWH, particularly mechanisms amenable to therapeutic manipulation.

The CaT stretcher: An open-source system for delivering uniaxial strain to cells and tissues (CaT).

Integration of mechanical cues in conventional 2D or 3D cell culture platforms is an important consideration for in vivo and ex vivo models of lung health and disease. Available commercial and published custom-made devices are frequently limited in breadth of applications, scalability, and customization. Herein we present a technical report on an open-source, cell and tissue (CaT) stretcher, with modularity for different in vitro and ex vivo systems, that includes the following features: 1) Programmability for modeling different breathing patterns, 2) scalability to support low to high-throughput experimentation, and 3) modularity for submerged cell culture, organ-on-chips, hydrogels, and live tissues. The strategy for connecting the experimental cell or tissue samples to the stretching device were designed to ensure that traditional biomedical outcome measurements including, but not limited to microscopy, soluble mediator measurement, and gene and protein expression remained possible. Lastly, to increase the uptake of the device within the community, the system was built with economically feasible and available components. To accommodate diverse in vitro and ex vivo model systems we developed a variety of chips made of compliant polydimethylsiloxane (PDMS) and optimized coating strategies to increase cell adherence and viability during stretch. The CaT stretcher was validated for studying mechanotransduction pathways in lung cells and tissues, with an increase in alpha smooth muscle actin protein following stretch for 24h observed in independent submerged monolayer, 3D hydrogel, and live lung tissue experiments. We anticipate that the open-source CaT stretcher design will increase accessibility to studies of the dynamic lung microenvironment through direct implementation by other research groups or custom iterations on our designs.

A2A adenosine receptor agonist reduced MMP8 expression in healthy M2-like macrophages but not in macrophages from ankylosing spondylitis patients

BackgroundAnkylosing spondylitis (AS) is an inflammatory autoimmune disease that mostly affects different joints of the body. Macrophages are the predominant cells that mediate disease progression by secreting several pro-inflammatory mediators. Different receptors are involved in macrophages’ function including the adenosine receptors (AR). Our main objective in this study was to assess the effect of applying A2A adenosine receptor agonist (CGS-21,680) on the gene expression of inflammatory mediators including bone morphogenetic proteins (BMP)-2, 4 and matrix metalloproteinases (MMP)-3, 8, 9, and 13 on the macrophages from AS patients compared to healthy macrophages.MethodsMonocytes were isolated from the whole blood of 28 individuals (AS patients and healthy controls in a 1:1 ratio). Macrophages were differentiated using macrophage colony-stimulating factor (M-CSF), and flow cytometry was performed to confirm surface markers. CGS-21,680 was used to treat cells that had been differentiated. Using SYBR green real-time PCR, relative gene expression was determined.ResultsActivating A2AAR diminished MMP8 expression in healthy macrophages while it cannot reduce MMP8 expression in patients’ macrophages. The effect of A2AAR activation on the expression of BMP2 and MMP9 reached statistical significance neither in healthy macrophages nor in the patients’ group. We also discovered a significant positive connection between MMP8 expression and patient scores on the Bath ankylosing spondylitis functional index (BASFI).ConclusionDue to the disability of A2AAR activation in the reduction of MMP8 expression in patients’ macrophages and the correlation of MMP8 expression with BASFI index in patients, these results represent defects and dysregulations in the related signaling pathway in patients’ macrophages.

Replication of Human Norovirus in Mice after Antibiotic-Mediated Intestinal Bacteria Depletion.

Human noroviruses (HuNoVs) are the main cause of acute gastroenteritis causing more than 50,000 deaths per year. Recent evidence shows that the gut microbiota plays a key role in enteric virus infectivity. In this context, we tested whether microbiota depletion or microbiota replacement with that of human individuals susceptible to HuNoVs infection could favor viral replication in mice. Four groups of mice (n = 5) were used, including a control group and three groups that were treated with antibiotics to eliminate the autochthonous intestinal microbiota. Two of the antibiotic-treated groups received fecal microbiota transplantation from a pool of feces from infants (age 1–3 months) or an auto-transplantation with mouse feces that obtained prior antibiotic treatment. The inoculation of the different mouse groups with a HuNoVs strain (GII.4 Sydney [P16] genotype) showed that the virus replicated more efficiently in animals only treated with antibiotics but not subject to microbiota transplantation. Viral replication in animals receiving fecal microbiota from newborn infants was intermediate, whereas virus excretion in feces from auto-transplanted mice was as low as in the control mice. The analysis of the fecal microbiota by 16S rDNA NGS showed deep variations in the composition in the different mice groups. Furthermore, differences were observed in the gene expression of relevant immunological mediators, such as IL4, CXCL15, IL13, TNFα and TLR2, at the small intestine. Our results suggest that microbiota depletion eliminates bacteria that restrict HuNoVs infectivity and that the mechanism(s) could involve immune mediators.

Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials.

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA (“biomaterial risk management”) an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 “benchmark” nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses.

Regulation, functions and transmission of bivalent chromatin during mammalian development.

Cells differentiate and progress through development guided by a dynamic chromatin landscape that mediates gene expression programmes. During development, mammalian cells display a paradoxical chromatin state: histone modifications associated with gene activation (trimethylated histone H3 Lys4 (H3K4me3)) and with gene repression (trimethylated H3 Lys27 (H3K27me3)) co-occur at promoters of developmental genes. This bivalent chromatin modification state is thought to poise important regulatory genes for expression or repression during cell-lineage specification. In this Review, we discuss recent work that has expanded our understanding of the molecular basis of bivalent chromatin and its contributions to mammalian development. We describe the factors that establish bivalency, especially histone-lysine N-methyltransferase 2B (KMT2B) and Polycomb repressive complex 2 (PRC2), and consider evidence indicating that PRC1 shapes bivalency and may contribute to its transmission between generations. We posit that bivalency is a key feature of germline and embryonic stem cells, as well as other types of stem and progenitor cells. Finally, we discuss the relevance of bivalent chromtin to human development and cancer, and outline avenues of future research.

The Gut Microbial Metabolite Pyrogallol Is a More Potent Inducer of Nrf2-Associated Gene Expression Than Its Parent Compound Green Tea (-)-Epigallocatechin Gallate.

(-)-Epigallocatechin gallate (EGCG) has been associated with multiple beneficial effects. However, EGCG is known to be degraded by the gut microbiota. The present study investigated the hypothesis that microbial metabolism would create major catechol-moiety-containing microbial metabolites with different ability from EGCG to induce nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated gene expression. A reporter gene bioassay, label-free quantitative proteomics and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were combined to investigate the regulation of Nrf2-related gene expression after exposure of U2OS reporter gene or Hepa1c1c7 cells in vitro to EGCG or to its major microbial catechol-moiety-containing metabolites: (-)-epigallocatechin (EGC), gallic acid (GA) and pyrogallol (PG). Results show that PG was a more potent inducer of Nrf2-mediated gene expression than EGCG, with a 5% benchmark dose (BMD5) of 0.35 µM as compared to 2.45 µM for EGCG in the reporter gene assay. EGC and GA were unable to induce Nrf2-mediated gene expression up to the highest concentration tested (75 µM). Bioinformatical analysis of the proteomics data indicated that Nrf2 induction by PG relates to glutathione metabolism, drug and/or xenobiotics metabolism and the pentose phosphate pathway. Taken together, our findings demonstrate that the microbial metabolite PG is a more potent inducer of Nrf2-associated gene expression than its parent compound EGCG.

Concentration-dependent transcriptional switching through a collective action of cis-elements.

Gene expression specificity of homeobox transcription factors has remained paradoxical. WUSCHEL activates and represses CLAVATA3 transcription at lower and higher concentrations, respectively. We use computational modeling and experimental analysis to investigate the properties of the cis-regulatory module. We find that intrinsically each cis-element can only activate CLAVATA3 at a higher WUSCHEL concentration. However, together, they repress CLAVATA3 at higher WUSCHEL and activate only at lower WUSCHEL, showing that the concentration-dependent interactions among cis-elements regulate both activation and repression. Biochemical experiments show that two adjacent functional cis-elements bind WUSCHEL with higher affinity and dimerize at relatively lower levels. Moreover, increasing the distance between cis-elements prolongs WUSCHEL monomer binding window, resulting in higher CLAVATA3 activation. Our work showing a constellation of optimally spaced cis-elements of defined affinities determining activation and repression thresholds in regulating CLAVATA3 transcription provides a previously unknown mechanism of cofactor-independent regulation of transcription factor binding in mediating gene expression specificity.

Gambogenic acid alleviates kidney fibrosis via epigenetic inhibition of EZH2 to regulate Smad7-dependent mechanism

BackgroundEpigenetics regulating gene expression plays important role in kidney fibrosis. Natural products originating from diverse sources including plants and microorganisms are capable to influence epigenetic modifications. Gambogenic acid (GNA) is a caged xanthone extracted from gamboge resin, exudation of Garcinia hanburyi Hook.f., and the effect of GNA on kidney fibrosis with its underlying mechanism on epigenetics remains unknown. PurposeThis study aimed to explore the role of GNA against kidney fibrogenesis by histone methylation mediating gene expression. MethodsTwo experimental mice of unilateral ureteral obstruction (UUO) and folic acid (FA) were given two dosages of GNA (3 and 6 mg/kg/d). TGF-β1 was used to stimulate mouse tubular epithelial (TCMK-1) cells and siRNAs were transfected to verify the underlying mechanisms of GNA. Histological changes were evaluated by HE, MASSON stainings, immunohistochemistry and immunofluorescence. Western blot and qPCR were used to measure protein/gene transcription levels. ResultsGNA dose-dependently alleviated UUO-induced kidney fibrosis and FA-induced kidney early fibrosis, indicated by the pathology and fibrotic factor changes (α-SMA, collagen I, collagen VI, and fibronectin). Mechanically, GNA reduced enhancer of zeste homolog 2 (EZH2) and H3K27me3, promoted Smad7 transcription, and inhibited TGF-β/Smad3 fibrotic signaling in injured kidneys. Moreover, with TGF-β1-induced EZH2 increasing, GNA suppressed α-SMA, fibronectin and collagen levels in tubular epithelial TCMK-1 cells. Although partially decreasing EZH2, GNA did not influence fibrotic signaling in Smad7 siRNA-transfected TCMK-1 cells. ConclusionEpigenetic inhibition of EZH2 by GNA ameliorated kidney fibrogenesis via regulating Smad7-meidated TGF-β/Smad3 signaling.

DbEmbryo multi-omics database for analyses of synergistic regulation in early mammalian embryo development.

During early mammalian embryo development, different epigenetic marks undergo reprogramming and play crucial roles in the mediation of gene expression. Currently, several databases provide multi-omics information on early embryos. However, how interconnected epigenetic markers function together to coordinate the expression of the genetic code in a spatiotemporal manner remains difficult to analyze, markedly limiting scientific and clinical research. Here, we present dbEmbryo, an integrated and interactive multi-omics database for human and mouse early embryos. dbEmbryo integrates data on gene expression, DNA methylation, histone modifications, chromatin accessibility, and higher-order chromatin structure profiles for human and mouse early embryos. It incorporates customized analysis tools, such as “multi-omics visualization,” “Gene&Peak annotation,” “ZGA gene cluster,” “cis-regulation,” “synergistic regulation,” “promoter signal enrichment,” and “3D genome.” Users can retrieve gene expression and epigenetic profile patterns to analyze synergistic changes across different early embryo developmental stages. We showed the uniqueness of dbEmbryo among extant databases containing data on early embryo development and provided an overview. Using dbEmbryo, we obtained a phase-separated model of transcriptional control during early embryo development. dbEmbryo offers web-based analytical tools and a comprehensive resource for biologists and clinicians to decipher molecular regulatory mechanisms of human and mouse early embryo development.

Evidence for Monocyte Reprogramming in a Long-Term Postsepsis Study.

OBJECTIVES:This study sought to identify monocyte alterations from septic patients after hospital discharge by evaluating gene expression of inflammatory mediators and monocyte polarization markers. It was hypothesized that sepsis reprograms the inflammatory state of monocytes, causing effects that persist after hospital discharge and influencing patient outcomes.DESIGN:The gene expression patterns of inflammatory receptors, M1 and M2 macrophage polarization markers, NLRP3 inflammasome components, and pro- and anti-inflammatory cytokines in monocytes were assessed.PATIENTS:Thirty-four patients from the University of São Paulo Hospital, during the acute sepsis phase (phase A), immediately after ICU discharge (phase B), and 3 months (phase C), 6 months (phase D), 1 year (phase E), and 3 years (phase F) after discharge, were included. Patients that died during phases A and B were grouped separately, and the remaining patients were collectively termed the survivor group.MEASUREMENTS AND MAIN RESULTS:The gene expression of toll-like receptor (TLR)2 and TLR4 (inflammatory receptors), NLRP3, NFκB1, adaptor molecule apoptosis-associated speck-like protein containing a CARD, caspase 1, caspase 11, and caspase 12 (NLRP3 inflammasome components), interleukin-1α, interleukin-1β, interleukin-18, and high-mobility group box 1 protein (proinflammatory cytokines), interleukin-10 (anti-inflammatory cytokine), C-X-C motif chemokine ligand 10, C-X-C motif chemokine ligand 11, and interleukin-12p35 (M1 inflammatory polarization markers), and C-C motif chemokine ligand 14, C-C motif chemokine ligand 22, transforming growth factor-beta (TGF-β), SR-B1, and peroxisome proliferator-activated receptor γ (M2 anti-inflammatory polarization and tissue repair markers) was upregulated in monocytes from phase A until phase E compared with the control group.CONCLUSIONS:Sepsis reprograms the inflammatory state of monocytes, probably contributing to postsepsis syndrome development and mortality.

Effects of dietary marine sulphated polysaccharides (Algimun®) on growth performance, immune responses and disease resistance of juvenile gilthead seabream (Sparus aurata) to Photobacterium damselae subsp. piscicida

The present study evaluated the effects of a dietary mix of marine sulphated polysaccharides, named Algimun® (AL), supplementation to gilthead seabream (Sparus aurata) juveniles in terms of growth performance, immune responses, and resistance against Photobacterium damselae subsp. piscicida. A total of 240 fish (initial mean weight of 6.00 ± 0.03 g) was randomly separated into 12 tanks (400 L, 20 fish per tank) distributed in four replicates. Fish were fed three experimental diets: a basal diet (Control), and a basal diet with two inclusion rates of Algimun® as 3 g/kg (AL0.3) and 5 g/kg (AL0.5) for 30 days before bacterial infection with P. damselae subsp. piscicida. After a 30-day feeding-period, growth performance was significantly improved in AL0.3 and AL0.5 groups compared to the control group (P < 0.05). AL0.3 and AL0.5 groups showed significantly higher lysozyme activity and myeloperoxidase activity when compared to the control group (P < 0.05). The gene expression of immune mediators (IL-1β, IL-6, IL-10, IL-18, TNF-α and COX-2) was significantly upregulated in the intestine, spleen and head kidney in AL0.3 and AL0.5 groups when compared to the control group (P < 0.05). Eight days post-challenge, the survival rate against P. damselae subsp. piscicida was numerically higher in fish within AL0.3 and AL0.5 groups compared to control (+20%). The study findings suggest that marine sulphated polysaccharides (Algimun®) could be used as an immunomodulator in gilthead seabream to support animal's health and boost resistance in case of disease outbreak.

Surface layer protein A from hypervirulent Clostridioides difficile ribotype 001 can induce autophagy process in human intestinal epithelial cells

Clostridioides difficile is the leading cause of nosocomial diarrhea with high morbidity and mortality worldwide. C. difficile strains produce a crystalline surface layer protein A (SlpA), which is an absolute necessity for its pathogenesis. However, its pathogenic mechanisms and its pro-inflammatory behavior are not yet fully elucidated. Herein, we report for the first time that SlpA extracted from C. difficile can induce autophagy process in Caco-2 cells. SlpA protein was purified from two C. difficile strains (RT001 and ATCC 700075). The cell viability of Caco-2 cells after exposure with different concentrations (15, 20, 25 μg/mL) of SlpA at various time points (3, 6, 12, 24 h) was measured by MTT assay. Acridine orange staining was used to visualize the hypothetical acidic vesicular organelles. The gene expression of autophagy mediators including LC3B, Atg5, Atg16L, and Beclin-1 was determined by quantitative real-time PCR assay. Western blotting assay was used to detect the expression of LC3B protein. MTT assay showed that different concentrations of SlpA did not induce significant changes in the viability of Caco-2 cells. SlpA at concentration of 20 μg/mL enhanced the formation of acidic vesicular organelles in Caco-2 cells after 12 h of exposure. Moreover, SlpA treatment significantly increased the expression of autophagy-associated genes, and increased the expression of LC3B protein in Caco-2 cells. In conclusion, our study demonstrated that SlpA is capable to induce autophagy in intestinal epithelial cells. These findings reveal a novel mechanism for the pathogenesis of C. difficile mediated by its SLPs.

Quercetin Decreases Corneal Haze In Vivo and Influences Gene Expression of TGF-Beta Mediators In Vitro.

We have previously reported the flavonoid, quercetin, as a metabolic regulator and inhibitor of myofibroblast differentiation in vitro. Our current study evaluated the effects of topical application of quercetin on corneal scar development using two different animal models followed by RNA analysis in vitro. Wild-type C57BL/6J mice were anesthetized and the corneal epithelium and stroma were manually debrided, followed by quercetin (0.5, 1, 5, or 50 mM) or vehicle application. Corneal scarring was assessed for 3 weeks by slit lamp imaging and clinically scored. In a separate animal study, six New Zealand White rabbits underwent lamellar keratectomy surgery, followed by treatment with 5 mM quercetin or vehicle twice daily for three days. Stromal backscattering was assessed at week 3 by in vivo confocal microscopy. In mice, a single dose of 5 mM quercetin reduced corneal scar formation. In rabbits, stromal backscattering was substantially lower in two out of three animals in the quercetin-treated group. In vitro studies of human corneal fibroblasts showed that quercetin modulated select factors of the transforming growth factor-β (TGF-β) signaling pathway. These results provide evidence that quercetin may inhibit corneal scarring. Further studies in a larger cohort are required to validate the efficacy and safety of quercetin for clinical applications.

Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts.

Interleukin (IL-) 6 is a key factor in the inflammatory processes of rheumatoid arthritis. Several biologic agents target the IL-6 signaling pathway, including sarilumab, a monoclonal antibody that blocks the IL-6 receptor and inhibits IL-6-mediated cis- and trans-signaling. A careful analysis of the IL-6 signaling blockade should consider not only inflammatory processes but also the regenerative functions of IL-6. The purpose of this study was to investigate whether inhibition of the IL-6 receptors affects differentiation of human primary osteoblasts (hOB). The effects of sarilumab on viability and the differentiation capacity in unstimulated osteoblasts as well as after stimulation with various IL-6 and sIL6-R concentrations were determined. Sarilumab treatment alone did not affect the differentiation or induction of inflammatory processes in hOB. However, the significant induction of alkaline phosphatase activity which was observed after exogenous IL-6/sIL-6R costimulation at the highest concentrations was reduced back to baseline levels by the addition of sarilumab. The IL-6 receptor blockade also decreased gene expression of mediators required for osteogenesis and bone matrix maintenance. Our results demonstrate that concomitant administration of the IL-6 receptor blocker sarilumab can inhibit IL-6/sIL-6R-induced osteogenic differentiation.

Zinc-loaded whey protein nanoparticles alleviate the oxidative damage and enhance the gene expression of inflammatory mediators in rats

BackgroundZinc (Zn) is an essential trace element required for the function of the immune system. However, Zn fortification of food has faced some challenges, although excess Zn may be induced obesity and other related. This study aimed to use Zn-loaded whey protein nanoparticles (Zn-WPNPs) to enhance the immunomodulatory activity of Zn in rats treated with CCl4. MethodsZn was loaded to WPNPs at a level of 14 mg/g. Four experimental groups of male albino Wistar rats were treated for 30 days including the control group, CCl4-treated group (0.5 ml/100 g b.w), Zn plus CCl4-treated group (50 mg/kg b.w), and CCl4 plus Zn-WPNPs-treated group (50 mg/kg b.w). Blood and tissue samples were collected for different assays and histological examinations. ResultsThe results revealed that CCl4 disturbs the serum biochemical, hematological, and immune indicators in different organs besides the liver as a target organ. Animals that received CCl4 showed a significant increase in oxidative stress markers, cytokines, and the mRNA expression of inflammatory mediators in the lung and spleen accompanied by a significant decrease in the hepatic and renal antioxidant enzymes along with histological changes in the liver, kidney, spleen, and lung. Zn or Zn-WPNPs could improve these parameters and the histological picture of the tested organs and Zn-WPNPs were more effective than Zn alone. ConclusionWPNPs induced synergistic immune-modulating effects which may control Zn release and may be a suitable candidate to enhance the immune system during any pandemic or the exposure to any chemicals that affect the immune system.

Quercetin-Rich Ethanolic Extract of Polygonum odoratum var Pakphai Leaves Decreased Gene Expression and Secretion of Pro-Inflammatory Mediators in Lipopolysaccharide-Induced Murine RAW264.7 Macrophages.

Polygonum odoratum var. Pakphai has been used in traditional Thai medicine for the treatment of flatulence and constipation and to relieve the inflammation caused by insect bites. Quercetin (Q), which is abundant in plant-based foods, has been found to exert anti-inflammatory properties. This study evaluated the anti-inflammatory activity of P. odoratum ethanolic extract in RAW264.7 macrophage cells. Leaves were extracted with 50% ethanol, phenolics and flavonoids were then analyzed using UHPLC-QTOF-MS and HPLC-DAD. RAW264.7 cells were induced with lipopolysaccharides (LPSs). They were then treated with the extract and prostaglandin E2 (PGE2), and interleukin-6 (IL-6) and tumor necrotic factor-alpha (TNF-α) concentrations were determined. Levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IL-6 and TNF-α mRNAs were analyzed using qRT-PCR. Chemical analysis demonstrated that the extract was abundant with Q while also containing catechin, gallic acid, epicatechin gallate and coumarin. The extract increased the viability of RAW264.7 cells and dose-dependently decreased nitric oxide production, PGE2, IL-6 and TNF-α levels in the medium from the LPS-induced RAW264.7 cell culture. Consistently, COX-2, iNOS, IL-6 and TNF-α mRNA levels were decreased in a concentration-dependent manner (p < 0.05). Thus, the quercetin-rich ethanolic extract derived from P. odoratum var Pakphai leaves can exert anti-inflammatory activity in LPS-induced RAW264.7 cells through a reduction of the pro-inflammatory mediator response.

ALDH1A1 overexpression in melanoma cells promotes tumor angiogenesis by activating the IL‑8/Notch signaling cascade.

ALDH1A1 is a cytosolic enzyme upregulated in tumor cells, involved in detoxifying cells from reactive aldehydes and in acquiring resistance to chemotherapeutic drugs. Its expression correlates with poor clinical outcomes in a number of cancers, including melanoma. The present study hypothesized that the increased ALDH1A1 expression and activity upregulated the release of proangiogenic factors from melanoma cells, which regulate angiogenic features in endothelial cells (ECs) through a rearrangement of the Notch pathway. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing melanoma cells displayed a higher microvessel density. In a 3D multicellular system, obtained co-culturing melanoma cancer cells with stromal cells, including ECs, melanoma ALDH1A1 overexpression induced the recruitment of ECs into the core of the tumorspheres. By using a genes array, overexpression of ALDH1A1 in tumor cells also promoted modulation of Notch cascade gene expression in ECs, suggesting an interaction between tumor cells and ECs mediated by enrichment of angiogenic factors in the tumor microenvironment. To confirm this hypothesis, inactivation of ALDH1A1 by the pharmacological inhibitor CM037 significantly affected the release of angiogenic factors, including IL-8, from melanoma cells. High levels of ALDH1A1, through the retinoic acid pathway, regulated the activation of NF-κB-p65 and IL-8. Further, in a 2D co-culture system, the addition of an IL-8 neutralizing antibody to ECs co-cultured with melanoma cells forced to express ALDH1A1 dampened endothelial angiogenic features, both at the molecular (in terms of gene and protein expression of mediators of the Notch pathway) and at the functional level (proliferation, scratch assay, tube formation and permeability). In conclusion, these findings demonstrated the existence of a link between melanoma ALDH1A1 expression and EC Notch signaling modification that results in a pro-angiogenic phenotype. Based on the crucial role of ALDH1A1 in melanoma control of the tumor microenvironment, the enzyme seems a promising target for the development of novel drugs able to interrupt the cross-talk between cancer (stem) cells and endothelial cells.