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The co-mitogenic effects of the α 1 -adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10 -10 -10 -6  M) and SAC (10 -6  M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α 1 -adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α 1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

Cyclooxygenase-2 (COX-2) is expressed in a variety of human colorectal cancer cells and can contribute to carcinogenesis. This study aimed to investigate the effect of diclofenac (DCF), a selective COX-2 inhibitor, on cell adhesion molecules and apoptosis in human colon adenocarcinoma cells. Levels of homing cell adhesion molecule (H-CAM, CD44), intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule-1 (VCAM-1, CD106), and epithelial cell adhesion molecule (EpCAM, CD326) were evaluated in cancer cells overexpressing (HT29) or not expressing (HCT116) COX-2. Cell viability was determined by MTT assay, COX-2 protein levels and activity were assessed by immunofluorescence and fluorometric analysis, respectively. Endogenous levels of polyunsaturated fatty acids (PUFAs) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) while expression of cell adhesion molecules was analyzed by flow cytometry. Annexin V-FITC/propidium iodide-labelling and fluorometric caspase-3 activity measurements were carried out to determine apoptosis. Flow cytometry analysis revealed that the percentage of CD44 and ICAM-1 staining in HCT116 cells was significantly lower compared to HT29 cells. In HT29 cells, phorbol 12-myristate 13-acetate (PMA) induced COX-2 expression and increased CD44 and ICAM-1 levels were down-regulated by diclofenac. Stimulation of COX-2 activity in HT29 cells via PMA significantly decreased diclofenac associated increase in PUFA levels. Treatment with both diclofenac and PMA significantly increased the number of apoptotic cells and caspase-3 activity in colon adenocarcinoma cells compared to control groups. In conclusion, diclofenac's effect to retard colorectal tumor growth and metastasis occurs in COX-2 overexpressing colon cancer cells by increased apoptosis and decreased expression of CD44 and ICAM-1., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Here, we propose a cancer cell membrane (CM) coated Au-Fe 3 O 4 complex (AFTP@CM), loaded with tannic acid and phorbol 12-myristate 13-acetate for targeted drug delivery and enhanced chem/photo therapy.

Objectives: The objective of this work was to evaluate the anti-inflammatory activity of the aqueous extract, fractions and major compounds, which are isolated and identified from Passiflora edulis f. edulis (purple passion fruit) leaves extract., Methods: For the isolation of the major compounds, reversed-phase chromatography and normal phase countercurrent chromatography were used. The separation was followed by thin layer chromatography and HPLC-DAD-ELSD. One-dimensional and two-dimensional NMR and ESI-TOF-MS/MS were used for structural elucidation. The anti-inflammatory activity was evaluated on a TPA multiple dose model of skin chronic inflammation in mice. Additionally, myeloperoxidase (MPO) and nitric oxide synthase (NOS) activity assays were performed as possible mechanisms of action studies., Key Findings and Conclusions: The study of the butanolic fraction mainly showed the presence of saponins and flavonoids. Three minor flavonoids were detected; and three known saponins, cyclopassiflosides IX, XI and III were isolated and identified. This is the first unequivocal report of the presence of these compounds in P. edulis f. edulis leaves. The most favourable results of anti-inflammatory activity were obtained for the flavonoid-rich fraction. All the fractions and isolated compounds evaluated, presented high percentages of inhibition of nitric oxide synthase activity., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Metabolism of progesterone (P4) by the enzyme 20α hydroxysteroid dehydrogenase (20α-HSD) in myometrial cells is postulated to be a mechanism for P4 withdrawal, which occurs concomitant to uterine inflammation (physiologic or infection-induced) and associated activation of transcription factors: NF-кB and AP-1, common to term and preterm labour. We found that 20α-HSD protein is significantly increased in human myometrium during term labour, and in mouse uterus during term and preterm labour. Treatment of human myometrial cells with the pro-inflammatory mediators, lipopolysaccharide (LPS, mimicking infection) and 12-O-tetradecanoylphorbol-13-acetate (TPA, mimicking inflammation), induced 20α-HSD gene expression and increased 20α-HSD protein abundance. LPS treatment decreased P4 release into the culture medium and resulted in up-regulation of GJA1 in the hTERT-HM cells. The NF-кB /AP-1 transcription factors mediated effects of LPS and TPA on 20α-HSD gene transcription. Both pro-inflammatory stimuli induced 20α-HSD promoter activity in LPS/TPA-treated cells which was significantly attenuated by inhibition of NF-кB (JSH: 20 µM) or AP-1 signalling (T5224: 10 µM). Deletion of NF-кB consensus sites abrogated LPS-mediated promoter induction, while removal of AP-1 sites reversed the TPA-mediated induction of 20α-HSD promoter. We conclude that inflammatory stimuli (physiologic or pathologic) that activate NF-кB or AP-1 induce 20α-HSD transcription and subsequent local P4 withdrawal resulting in up-regulation of GJA1 and activation of myometrium that precedes labour., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Cellular responses to implanted biomaterials are key to understanding osseointegration. The aim of this investigation was to determine the in vitro priming and activation of the respiratory burst activity of monocytes in response to surface-modified titanium. Human peripheral blood monocytes of healthy blood donors were separated, then incubated with surface-modified grade 2 commercially pure titanium (CPT) disks with a range of known surface energies and surface roughness for 30- or 60-min. Secondary stimulation by phorbol 12-myrisate 13-acetate (PMA) following the priming phase, and luminol-enhanced-chemiluminescence (LCL) was used to monitor oxygen-dependent activity. Comparison among groups was made by incubation time using one-way ANOVA. One sample from each group for each phase of the experiment was viewed under scanning electron microscopy (SEM) and qualitative comparisons made. The results indicate that titanium is capable of priming peripheral blood monocytes following 60-min incubation. In contrast, 30 min incubation time lead to reduced LCL on secondary stimulation as compared to cells alone. At both time intervals, the disk with the lowest surface energy produced significantly less LCL compared to other samples. SEM examination revealed differences in surface morphology at different time points but not between differently surface-modified disks. These results are consistent with the hypothesis that the titanium surface characteristics influenced the monocyte activity, which may be important in regulating the healing response to these materials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 De Poi, Kowolik, Oshida and El Kholy.)

Non-Steroidal Anti-inflammatory Drugs (NSAIDs) are some of the most prescribed medications for pain but the incidence of adverse effects -especially during chronic treatment- points out the requirement of new analgesics. In this study, we showed an efficient two-steps synthesis of diphenylamine-containing dipeptides consisting of a multicomponent process followed by a Buchwald-Hartwig cross-coupling reaction. We prepared 16 diphenylamine derivatives and evaluated their in vivo anti-inflammatory activity through an ear edema model using 12-O-tetradecanoylpholbol-13-acetate. Furthermore, the toxicity of the more potent compounds in the Artemia salina model and their cell viability using murine RAW 264.7 cells is reported. The fluorinated compound 10k becomes a reliable candidate since it reduced the TPA-induced edema to 92%, lacked cytotoxicity against murine macrophages, and had minimal toxicity in Artemia salina., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Low-grade persistent inflammation is a feature of diabetes-driven vascular complications, in particular activation of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome to trigger the maturation and release of the inflammatory cytokine interleukin-1β (IL-1β). We investigated whether inhibiting the NLRP3 inflammasome, through the use of the specific small-molecule NLRP3 inhibitor MCC950, could reduce inflammation, improve vascular function, and protect against diabetes-associated atherosclerosis in the streptozotocin-induced diabetic apolipoprotein E-knockout mouse. Diabetes led to an approximately fourfold increase in atherosclerotic lesions throughout the aorta, which were significantly attenuated with MCC950 ( P < 0.001). This reduction in lesions was associated with decreased monocyte-macrophage content, reduced necrotic core, attenuated inflammatory gene expression (IL-1β, tumor necrosis factor-α, intracellular adhesion molecule 1, and MCP-1; P < 0.05), and reduced oxidative stress, while maintaining fibrous cap thickness. Additionally, vascular function was improved in diabetic vessels of mice treated with MCC950 ( P < 0.05). In a range of cell lines (murine bone marrow-derived macrophages, human monocytic THP-1 cells, phorbol 12-myristate 13-acetate-differentiated human macrophages, and aortic smooth muscle cells from humans with diabetes), MCC950 significantly reduced IL-1β and/or caspase-1 secretion and attenuated leukocyte-smooth muscle cell interactions under high glucose or lipopolysaccharide conditions. In summary, MCC950 reduces plaque development, promotes plaque stability, and improves vascular function, suggesting that targeting NLRP3-mediated inflammation is a novel therapeutic strategy to improve diabetes-associated vascular disease., (© 2020 by the American Diabetes Association.)

More than a million cases of cutaneous squamous cell carcinoma are diagnosed in the USA each year, and its incidence is increasing. Most of these malignancies arise from premalignant lesions, providing an opportunity for intervention before malignant progression. We previously documented how cytoplasmic mislocalization of CDC25A in premalignant and malignant skin cancers confers resistance to apoptotic cell death via a mechanism that depends on its interaction with 14-3-3ε. From these data, we hypothesized that 14-3-3ε overexpression drives skin tumor development and progression, such that targeting 14-3-3ε may be a useful strategy for skin cancer treatment. Like CDC25A, 14-3-3ε was overexpressed and mislocalized to the cytoplasm of both benign and malignant human skin cancer. Skin-targeted deletion of the 14-3-3ε gene reduced skin tumor development by 75% and blocked malignant progression. 14-3-3ε suppressed apoptosis through activation of Akt, leading to inhibition of BCL2 associated agonist of cell death and upregulation of Survivin. Using virtual tetrapeptide libraries, we developed a novel peptide that specifically blocked 14-3-3ε heterodimerization and thereby prevented its interaction with CDC25A. The peptide reduced prosurvival signaling, killed skin cancer cells and reduced skin tumor growth in xenograft. Normal skin keratinocytes were unaffected by inhibition or deletion of 14-3-3ε. Thus, targeting of 14-3-3ε dimerization is a promising strategy for the treatment of premalignant skin lesions., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

There is an increasing interest in natural products and their derivatives with therapeutic benefits and less side effects compared to steroid therapy. Benzofuran derivatives display biological effects including anti-inflammatory effects. The present study aims to investigate whether (3-(7-methoxy-2-p-tolyl benzofuran-5-yl) propan-1-ol) (DK-1108), new synthetic benzofuran compound exerts anti-asthmatic effects in vitro and in vivo. DK-1108 strongly reduced the production of inflammatory mediators, cytokines and chemokines in RAW264.7 and A549 cells. DK-1108 significantly regulated the levels of AKT/MAPKs/c-Jun activation, AP-1 luciferase activity and ICAM-1 expression. Furthermore, DK-1108 effectively suppressed the adhesion of A549 and EOL-1 cells. In OVA-induced asthmatic mice, DK-1108 decreased the levels of IL-5/IL-13/IgE production, eosinophils/macrophages influx, ICAM-1/MCP-1 expression, mucus secretion and airway hyperresponsiveness (AHR). These effects of DK-1108 were accompanied by downregulation of MAPKs activation. Therefore, we suggest that DK-1108 exerts protective effect against airway inflammation and mucus overproduction, and therefore could be valuable therapeutic agent for treatment in asthma., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Whereas effector CD4 + and CD8 + T cells promote immune activation and can drive clearance of infections and cancer, CD4 + regulatory T (T reg ) cells suppress their function, contributing to both immune homeostasis and cancer immunosuppression. The transcription factor BACH2 functions as a pervasive regulator of T cell differentiation, promoting development of CD4 + T reg cells and suppressing the effector functions of multiple effector T cell (T eff ) lineages. Here, we report the development of a stable cell-based bioluminescence assay of the transcription factor activity of BACH2. Tetracycline-inducible BACH2 expression resulted in suppression of phorbol 12-myristate 13-acetate (PMA)/ionomycin-driven activation of a luciferase reporter containing BACH2/AP-1 target sequences from the mouse Ifng + 18k enhancer. BACH2 expression repressed the luciferase signal in a dose-dependent manner but this activity was abolished at high levels of AP-1 signalling, suggesting contextual regulation of AP-1 driven gene expression by BACH2. Finally, using the reporter assay developed, we find that the histone deacetylase 3 (HDAC3)-selective inhibitor, RGFP966, inhibits BACH2-mediated repression of signal-driven luciferase expression. In addition to enabling mechanistic studies, this cell-based reporter may enable identification of small molecule agonists or antagonists of BACH2 function for drug development.

Background/aims: The study aimed to explore the effects of Epstein-Barr virus--encoded BARF1 in human gastric epithelial cells (GES-1)., Materials and Methods: A eukaryotic expression vector carrying BARF1 gene (pcDNA3.1-BARF1) was constructed. The pcDNA3.1-BARF1 was transfected into GES-1 cells, and they were selected by G418. The GES-1 cells lines that expressed BARF1 (GES-1-BARF1) were obtained. The cycle of GES-1-pcDNA3.1 cells (GES-1 cells transfected with empty vector), GES-1-BARF1 cells (GES-1 cells transfected with BARF1), and TPA-GES-1-BARF1(GES-1-BARF1 cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) were analyzed by flow cytometry. Colony formation in soft agar and tumorigenicity of the transfected cells in mice with severe combined immunodeficiency (SCID) were also observed., Results: The morphology of GES-1-BARF1 cells were changed from the original shuttle to round, the adhesion between the cells and bottle wall was weakened, and the cells showed overlapping growth. The proliferation rate of GES-1-BARF1 and TPA-GES-1-BARF1 cells were faster than GES-1 and GES-1-pcDNA3.1 cells; the S phase was significantly prolonged for GES-1-BARF1 and TPA-GES-1-BARF1. GES-1-BARF1 and TPA-GES-1-BARF1 cells formed colonies in soft agar, with a cloning rate of 24.2% (58/240) and 40.0% (96/240), respectively; GES-1 and GES-1-pcDNA3.1 cells did not form colonies in soft agar. Tumors were formed in mice with SCID after injecting TPA-GES-1-BARF1 cell groups. Tumor formation did not occur in mice with SCID after injecting GES-1 and GES-1-pcDNA3.1 cell groups, but nodules were formed in the mice with SCID after injecting GES-1-BARF1 cell groups., Conclusion: GES-1-BARF1 cells malignant transformation was induced by transfected BARF1 gene and TPA stimulation. This result indicated that tumor formation not only require oncogenes, but also the stimulation of cancer-promoting substance.

Background: Pulicaria crispa ( P. crispa ) is a plant from the Compositae family that exhibits antioxidant, anti-inflammatory, antibacterial, and cytotoxic activities., Objective: The current study aimed at investigating the immunomodulatory effects of P. crispa extract in lipopolysaccharide- (LPS-) stimulated human monocytic THP-1 cells., Methods: To induce macrophage differentiation, THP-1 cell lines were treated with phorbol-12-myristate 13-acetate, followed by exposure to LPS with or without 50 or 100  μ g/ml of P. crispa extract. The following tests were employed to test the immunomodulatory effects of the extract: MTT assay, ELISA, Western blotting analysis, cell migration and phagocytosis assays, and Annexin V staining method., Results: Exposure to 100  μ g/ml P. crispa extract significantly reduced THP-1 cell proliferation, migration, and phagocytosis (in LPS-stimulated cells, but not in unstimulated cells). Moreover, the extract alone significantly reduced the rate of THP-1 cell apoptosis, while it increased the rate of late apoptosis. Molecular investigations showed that treatment with P. crispa extract significantly upregulated the expression of ERK1, p-MAPK, P-P38, and Bcl2, while it significantly reduced the expression of ERK5, Bax, NF- κ B, P-NF- κ B, CCL1, CCL2, CCL5, CCL22, CXCL1, and CXCL10., Conclusion: Pulicaria crispa extract exhibited anti-inflammatory, antiproliferative, antimigratory, and antiphagocytic effects in LPS-stimulated THP-1 cells. Future studies should investigate these mechanisms in animal models with chronic inflammatory diseases., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Tarfa Albrahim et al.)

RB1 gene is often homozygously deleted or mutated in prostate adenocarcinomas following acquirement of castration resistance and/or metastatic ability. We found that SUCLA2 gene is frequently involved in the deletion of the RB1 gene region in advanced prostate cancer. SUCLA2 constitutes the β-subunit of succinate CoA ligase heterodimer that reversibly converts succinyl CoA into succinate. We sought the possibility that deletion of SUCLA2 gives rise to a metabolic vulnerability that could be targeted therapeutically. We found a significant metabolic shift in SUCLA2-deleted prostate cancer cells, including lower mitochondrial respiratory activity. By screening a number of libraries for compounds that induce cell death selectively in SUCLA2-deficient prostate cancer cells, we identified thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) and PMA (phorbol-12-myristate-13-acetate) from a natural compound library. These findings indicate that the metabolic vulnerability in SUCLA2-deficient prostate cancer cells is pharmacologically targetable.

Scytonemin is a yellow-green ultraviolet sunscreen pigment present in different genera of aquatic and terrestrial blue-green algae, including marine cyanobacteria. In the present study, the anti-inflammatory activities of scytonemin were evaluated in vitro and in vivo. Topical application of scytonemin inhibited 12- O -tetradecanoylphorbol-13-acetate (TPA)-induced ear swelling in BALB/c mice. The expression of tumor necrosis factor-a (TNF-a) and inducible nitric oxide synthase (iNOS) was also suppressed by scytonemin treatment in the TPA-treated ear of BALB/c mice. In addition, scytonemin inhibited lipopolysaccharide (LPS)-induced production of TNF-a and nitric oxide (NO) in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expressions of TNF-a and iNOS were also suppressed by scytonemin in LPS-stimulated RAW 264.7 cells. Further study demonstrated that LPS-induced NF-kB activity was significantly suppressed by scytonemin treatment in RAW 264.7 cells. Our results also showed that the degradation of IkBa and nuclear translocation of the p65 subunit were blocked by scytonemin in LPS-stimulated RAW 264.7 cells. Collectively, these results suggest that scytonemin inhibits skin inflammation by blocking the expression of inflammatory mediators, and the anti-inflammatory effect of scytonemin is mediated, at least in part, by down-regulation of NF-kB activity. Our results also suggest that scytonemin might be used as a multi-function skin care ingredient for UV protection and anti-inflammation., Competing Interests: The authors declare no conflict of interest.

Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well-characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross-talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO-1 and ZO-2 localize at podosomes in response to phorbol-12-myristate-13-acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO-1 and ZO-2 are relocated from a pre-existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases., (© 2019 Wiley Periodicals, Inc.)

Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB , CR- ACTB ) and ACTG1 (CRISPR clones with inactivated ACTG1 , CR- ACTG1 ) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either β actin coded by ACTB or γ actin coded by ACTG1 . First, we observed different distributions of β and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than β actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR- ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR- ACTB and CR- ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells' FA formation and motility.

Background: Chrysoeriol is a flavone found in diverse dietary and medicinal herbs such as Lonicerae Japonicae Flos (the dried flower bud or newly bloomed flower of Lonicera japonica Thunb.). These herbs are commonly used for treating inflammatory diseases. Herbal extracts containing chrysoeriol have been shown to have anti-inflammatory effects and inhibit nuclear factor-kappa B (NF-κB) signaling. Some of these extracts can inhibit signal transducers and activators of transcription 3 (STAT3) signaling in cancer cells., Purpose: This study aimed to determine whether chrysoeriol has anti-inflammatory effects and whether NF-κB and STAT3 pathways are involved in the effects., Study Design and Methods: A TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model and LPS-stimulated RAW264.7 cells were used to evaluate the effects of chrysoeriol. Griess reagent was used to measure the production of nitric oxide (NO). Western blot and enzyme-linked immunosorbent assays were employed to detect protein levels. RT-qPCR analyses were used to detect mRNA levels. Haematoxylin and eosin (H&E) staining was employed to examine the pathological conditions in animal tissues., Results: In the mouse model, chrysoeriol ameliorated acute skin inflammation, evidenced by reduced ear thickness, ear weight and number of inflammatory cells in inflamed ear tissues. The compound lowered protein levels of phospho-p65 (Ser536), phospho-STAT3 (Tyr705), inducible nitric oxide synthases (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), IL-1β and tumor necrosis factor α (TNF-α) in mouse swollen ears. In LPS-stimulated RAW264.7 cells, chrysoeriol also lowered levels of these proteins. In addition, chrysoeriol decreased the production of NO and prostaglandin E2; inhibited the phosphorylation of inhibitor of κB (Ser32), p65 (Ser536) and Janus kinase 2 (Tyr1007/1008); decreased nuclear localization of p50, p65 and STAT3; and down-regulated mRNA levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α that are transcriptionally regulated by NF-κB and STAT3 in the cell model., Conclusion: We for the first time demonstrated that chrysoeriol ameliorates TPA-induced ear edema in mice, and that inhibition of JAK2/STAT3 and IκB/p65 NF-κB pathways are involved in the anti-inflammatory effects of chrysoeriol. This study provides chemical and pharmacological justifications for the use of chrysoeriol-containing herbs in treating inflammatory diseases, and provides pharmacological groundwork for developing chrysoeriol as a novel anti-inflammatory agent., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflicts of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

A nanobiocomposite was prepared from multiwalled carbon nanotubes and zein nanoparticles. It was dispersed in water/ethanol and drop cast onto a glassy carbon electrode. The modified electrode can be used for electroreduction of H 2 O 2 (typically at a working potential of -0.71 V vs. Ag/AgCl). The electrochemical properties of the electrode were investigated by cyclic voltammetry, linear sweep voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Response to H 2 O 2 is linear in the 0.049 to 22 μM concentration range, and the detection limit is 35 nM at pH 7.0. The sensor was successfully utilized for the measurement of H 2 O 2 in a synthetic urine sample, and for monitoring the release of H 2 O 2 from human dermal fibroblasts and human hepatocellular carcinoma cells. Graphical abstractSchematic representation of a novel metal- and enzyme-free electrochemical nanosensor. A glassy carbon electrode was modified with a nanocomposite prepared from multiwalled carbon nanotubes and zein nanoparticles. It was applied to the identification of liver cancer cells via sensing of H2O2 and has a very low detection limit.

Juglone (5-hydroxy-1, 4-naphthoquinone), is a natural phenolic compound that has been shown to relax smooth muscle. Therefore the aim of this study was to determine the effect of juglone on vascular tone using porcine coronary artery (PCA). Segments of PCA, with or without endothelium, were mounted for isometric tension recording in isolated tissue baths and precontracted with the thromboxane A 2 analog U46619 or KCl. After pre-contraction, cumulative concentrations of juglone were added to the tissues, in the presence or absence of a variety of inhibitors on intracellular signaling pathways. Juglone (10 -9 to 10 -5  M) produced a concentration-dependent relaxation of the PCA which was reduced in endothelium-denuded vessels, as well as in vessels pre-treated with the nitric oxide synthase inhibitor L-NAME, indicating that at least part of the effect of juglone is mediated through an endothelium, NO-dependent mechanism. Juglone also inhibited contractions in response to influx of extracellular calcium and release of intracellular calcium, indicating that juglone may inhibit a common signaling pathway downstream of calcium. Contractions to the protein kinase C activator Phorbol 12-myristate 13-acetate were also reduced by juglone, suggesting that juglone might be acting through inhibition of protein kinase C. In summary, juglone produces a relaxation of the porcine coronary artery through activation of the nitric oxide pathway and inhibition of calcium-induced contractions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Innate lymphoid cells (ILCs) are emerging as important effectors of innate immunity and play a critical role in maintaining intestinal immune homeostasis. They are tissue-residing immune cells that can be subdivided based on master transcription factor and cytokine expression, bearing striking resemblance to their CD4 + T helper (Th) cell counterparts. ILCs are increasingly recognized as potential mediators of inflammatory bowel disease (IBD) providing a need to explore their functional and phenotypic differences in health vs. disease. In this chapter we outline protocols for the characterization of human ILCs and intracellular cytokine expression using flow cytometry. We include protocols for isolating human peripheral blood and colonic lamina propria mononuclear cells essential for evaluating human IBD specimens.

Expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells and its interaction with programmed cell death protein 1 (PD-1) on tumor-infiltrating lymphocytes suppress anti-tumor immunity. In breast tumors, PD-L1 expression levels are the highest in estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative (triple-negative) cancers. In this study, we show that a portion of PD-L1 exogenously expressed in several triple-negative breast cancer cell lines, as well as endogenous PD-L1, is proteolytically cleaved by cell surface metalloproteases. The cleavage generates an ~ 37-kDa N-terminal PD-L1 fragment that is released to the media and a C-terminal PD-L1 fragment that remains associated with cells but is efficiently eliminated by lysosomal degradation. We identify ADAM10 and ADAM17, two closely related members of the ADAM family of cell surface metalloproteases, as enzymes mediating PD-L1 cleavage. Notably, treatment of cells with ionomycin, a calcium ionophore and a known activator of ADAM10, or with phorbol 12-myristate 13-acetate, an activator of ADAM17, dramatically increases the release of soluble PD-L1 to the media. We postulate that ADAM10 and/or ADAM17 may contribute to the regulation of the PD-L1/PD-1 pathway and, ultimately, to anti-tumor immunity in triple-negative breast cancer.

Background: HIV rapid progressors (RPs) present with a rapid decline of CD4 + T cells within a few years of infection. Determining the underlying mechanisms throughout this decline is important to identify prognostic biomarkers and intervention strategies. Determining the numbers of CD4 + and CD8 + T cells is essential for monitoring the immune status of HIV infected patients. There are additional kinds of cell subtypes in T cells, but their relationship to the rapid progression of HIV disease is not well defined., Methods: Nineteen RPs and twenty-one chronic progressors (CPs) were enrolled in this study. Based on the intensity of CD4 and CD8 expression, different T cell subtypes were identified, including CD4 + CD8 + T cells, CD4 - CD8 - T cells, CD4 + CD8 low T cells and CD4 - CD8 low T cells. Alterations in these T cell subtypes in early HIV infection (within 120 days of infection) between RPs and CPs were measured, and the relationships between these subtypes and HIV disease progression were investigated. In addition, expression of IFN-γ in T cell subtypes after PMA stimulation was analyzed by flow cytometry., Results: We found that during early HIV infection, CD4 + CD8 low T cells both significantly decreased in numbers and percentages in RPs compared to CPs. Furthermore, baseline CD4 + CD8 low T cells positively correlated not only with baseline CD4 + T cells but also with CD4 + T cells 12 months after infection. Moreover, survival analysis indicated that low levels of baseline CD4 + CD8low T cells significantly accelerated the decline in CD4 + T cells as well as increased viral loads. CD4 + CD8 low T cells secreted significantly more IFN-γ after PMA stimulation compared to CD4 + CD8 - T cells and CD4 - CD8 + T cells, which may be beneficial for the prevention of disease progression., Conclusions: Our results identified that in early stage HIV-1 infection, a subtype of T cells, CD4 + CD8 low , are associated with subsequent disease progression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Methylxanthines are important pharmacological agents in the treatment of asthma and of chronic obstructive pulmonary diseases. The present study was designed to compare the ability of doxofylline and theophylline to modulate inflammatory pathways in human monocytes., Methods: Monocytes isolated from healthy anonymous human buffy coats were treated with doxofylline or theophylline in the presence of phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS), and their phenotype, the oxidative burst, cytokine expression and release, cAMP production, and protein kinase C (PKC) activity were evaluated., Results: Doxofylline and theophylline did not have overlapping effects on human monocytes. While sharing some common characteristics, they differed significantly in their selectivity. Theophylline affected LPS- above PMA-induced cellular responsivity, while doxofylline behaved in the opposite manner. Furthermore, when testing PKC activity, we found an inhibitory effect of doxofylline but not of theophylline, at equimolar doses., Conclusions: In conclusion, our data support the growing hypothesis that doxofylline does not have a superimposable mechanism of action compared to theophylline, and this may both explain some differences in the risk/benefit ratio and may direct studies to tailor therapy for patients., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

ADAM17, prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Several of these play central roles in oncogenesis and inflammation, yet despite its importance, the mechanism by which ADAM17 is activated is not fully understood. We recently presented evidence that surface exposure of phosphatidylserine (PS) is the penultimate event required for sheddase activation, which occurs upon binding of a membrane-proximal, cationic binding motif to the anionic phospholipid headgroup. Here, we show that mutagenesis of the 3 amino acids constituting the PS-binding motif leads to embryonic lethality in mice. Heterozygotes showed no abnormalities. Primary hepatocytes and fibroblasts were analysed and found to express the mutant protease on the cell surface. However, PMA-stimulated release of ADAM17 substrates was completely abolished. The results directly support the novel concept of transiently externalised PS as essential trigger of extracellular protease function in vivo., (© 2019 Veit et al.)

The interleukin 7 receptor alpha chain (IL-7Rα) is predominately expressed by lymphocytes, and activation by its ligand IL-7 supports the development and maintenance of T cells and boosts T-cell mediated immunity. We recently reported that lymphatic endothelial cells (LECs) in dermal lymphatics also express IL-7 and its receptor chains (IL-7Rα and CD132) and that IL-7 supports lymphatic drainage. This suggested that activation of IL-7Rα signaling in lymphatics could exert inflammation-resolving activity, by promoting the clearance of excess tissue fluid. Here we investigated how the potentially opposing effects of IL-7Rα signaling in immune cells and in the lymphatic vasculature would affect the development and progression of psoriasis-like skin inflammation. We found that during acute and chronic skin inflammation mice with an endothelial-specific deletion of IL-7Rα (IL-7Rα ΔEC mice) developed more edema compared to control mice, as a consequence of impaired lymphatic drainage. However, systemic treatment of wild-type mice with IL-7 exacerbated edema and immune cell infiltration in spite of increasing lymphatic drainage, whereas treatment with IL-7Rα blocking antibody ameliorated inflammatory symptoms. These data identify IL-7Rα signaling as a new pathway in psoriasis-like skin inflammation and show that its pro-inflammatory effects on the immune compartment override its anti-inflammatory, drainage-enhancing effects on the endothelium.

Methanolic extract of Clinacanthus nutans Lindau leaves (MECN) has been reported to exert antinociceptive activity. The present study aimed to elucidate the possible antinociceptive mechanisms of a lipid-soluble fraction of MECN, which was obtained after sequential extraction in petroleum ether. The petroleum ether fraction of C. nutans (PECN), administered orally to mice, was (i) subjected to capsaicin-, glutamate-, phorbol 12-myristate 13-acetate-, bradykinin-induced nociception model; (ii) prechallenged (intraperitoneal (i.p.)) with 0.15 mg/kg yohimbine, 1 mg/kg pindolol, 3 mg/kg caffeine, 0.2 mg/kg haloperidol, or 10 mg/kg atropine, which were the respective antagonist of α 2 -adrenergic, β -adrenergic, adenosinergic, dopaminergic, or muscarinic receptors; and (iii) prechallenged (i.p.) with 10 mg/kg glibenclamide, 0.04 mg/kg apamin, 0.02 mg/kg charybdotoxin, or 4 mg/kg tetraethylammonium chloride, which were the respective inhibitor of ATP sensitive-, small conductance Ca 2+ -activated-, large conductance Ca 2+ -activated-, or nonselective voltage-activated-K + channel. Results obtained demonstrated that PECN (100, 250, and 500 mg/kg) significantly (P<0.05) inhibited all models of nociception described earlier. The antinociceptive activity of 500 mg/kg PECN was significantly (P<0.05) attenuated when prechallenged with all antagonists or K + channel blockers. However, only pretreatment with apamin and charybdotoxin caused full inhibition of PECN-induced antinociception. The rest of the K + channel blockers and all antagonists caused only partial inhibition of PECN antinociception, respectively. Analyses on PECN's phytoconstituents revealed the presence of antinociceptive-bearing bioactive compounds of volatile (i.e., derivatives of γ -tocopherol, α -tocopherol, and lupeol) and nonvolatile (i.e., cinnamic acid) nature. In conclusion, PECN exerts a non-opioid-mediated antinociceptive activity involving mainly activation of adenosinergic and cholinergic receptors or small- and large-conductance Ca 2+ -activated-K + channels., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper.

Background: High expression of secreted matricellular protein cysteine-rich 61 (CYR61) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation has been shown to correlate with expression of key genes involved in cancer progression. However, such mechanisms in CYR61 transcription regulation remain unexplored., Methods: Expression of CYR61 was determined by immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and western blotting (WB) in CRC patients paraffin specimens and colon cell lines. ChIP-seq data of enhancer-characteristic histone modifications, in CRC tissues from the Gene Expression Omnibus (GEO) database, were reanalyzed to search for putative enhancers of CYR61. Dual-luciferase reporter assay was used to detected enhancer activity. Physical interactions between putative enhancers and CYR61 promoter were detected by chromosome conformation capture (3C) assay. Histone modification and transcription factors (TFs) enrichment were detected by ChIP-qPCR. Additionally, biological function of enhancers was investigated by transwell migration assays., Results: CRC tissues and cell lines expressed higher level of CYR61 than normal colon mucosa. Three putative enhancers located downstream of CYR61 were found in CRC tissues by ChIP-seq data reanalysis. Consistent with the ChIP-seq analysis results in the GEO database, the normal colon mucosal epithelial cell line NCM460 possessed no active CYR61 enhancers, whereas colon cancer cells exhibited different patterns of active CYR61 enhancers. HCT116 cells had an active Enhancer3, whereas RKO cells had both Enhancer1 and Enhancer3 active. Pioneer factor FOXA1 promoted CYR61 expression by recruiting CBP histone acetyltransferase binding and increasing promoter-enhancer looping frequencies and enhancer activity. CBP knockdown attenuated H3K27ac enrichment, promoter-enhancer looping frequencies, and enhancer activity. Small molecule compound 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment, which stimulated CYR61 expression, and verteporfin (VP) treatment, which inhibited CYR61 expression, confirmed that the enhancers regulated CYR61 expression. Knockdown and ectopic expression of CYR61 rescued cell migration changes induced by over-expressing and knockdown of FOXA1, respectively., Conclusions: CYR61 enhancer activation, mediated by FOXA1 and CBP, occurs during CRC progression to up-regulate CYR61 expression and promote cell migration in CRC, suggesting inhibition of recruitment of FOXA1 and/or CBP to CYR61 enhancers may have therapeutic implications.

Circulating leukocytes are indispensable components of the immune system, and rapid analysis of their native state or functionalities can help to unravel their pathophysiological roles and identify novel prognostic biomarkers in health and diseases. Herein we report a novel high throughput "sample-in-answer-out" integrated platform for continuous leukocyte sorting and single-cell electrical profiling in a label-free manner. The multi-staged platform enables isolation of neutrophils and monocytes from diluted or lysed blood samples directly within minutes based on Dean flow fractionation (DFF) (stage 1). Next DFF-purified leukocytes are inertially focused in serpentine channels into a single stream (stage 2) prior to impedance detection (stage 3). As a proof-of-concept for neutrophil functional characterization towards diabetes testing, we characterized the formation of neutrophil extracellular traps (NETosis) of healthy and glucose-treated neutrophils and observed significant changes in dielectric properties (size and opacity) between both groups. Interestingly, the NETosis profiles induced by calcium ionophore (CaI) and phorbol 12-myristate 13-acetate (PMA) were also electrically different, which could be attributed to the differential rates of cell enlargement and attenuated membrane permeability. Taken together, these results clearly demonstrated the potential of the developed platform for rapid (∼mins) and label-free leukocyte profiling and the use of impedance signatures as novel functional biomarkers for point-of-care testing in diabetes.

In recent years, skin reactions such as phytophotodermatitis, contact dermatitis, and other inflammatory responses after contact with chemicals from various plants, e.g., Heracleum mantegazzianum or Hippomane mancinella , are one of the hot topics in phytobiology. Occupational skin inflammation after contact with latices of plants from Euphorbiaceae are common among people who work with plants of this family. Activation of protein kinase C by G protein-coupled receptors such as protease-activated receptors is associated with skin inflammation. In this study, we focused on the inflammatory modulation potential of proteases combined with diterpenes on human skin. Because of its role as a proinflammatory cytokine, we concentrated on the release of IL-8 by fibroblasts and keratinocytes. Therefore, primary human dermal fibroblasts and the HaCaT keratinocytes cell line were used as a model. The results indicated that the combination of the protease mauritanicain from Euphorbia mauritanica and phorbol-12-myristate-13-acetate induced a significantly increased IL-8 release in HaCaT keratinocytes compared to single treatments. The obtained results also suggest that mauritanicain has an anti-inflammatory effect on primary human dermal fibroblasts., Competing Interests: The authors declare no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Viral infection is often accompanied with alteration of intracellular redox state, especially an imbalance between reactive oxygen species (ROS) production and antioxidant cellular defenses. The previous studies showed that an antioxidant cellular defense system, the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), played an important role against spring viraemia of carp virus (SVCV) infection in fish. To further reveal the mediated mechanism that Nrf2 active state was affected by protein kinase C (PKC), here we evaluated SVCV replication in host cells by treated with a strong activator of PKC phorbol-12-myristate-13-acetate (PMA) and an inhibitor staurosporine. Our results showed that PMA significantly repressed SVCV replication and viral-induced apoptosis in Epithelioma papulosum cyprini (EPC) cell, suggesting that PKC may exhibit an anti-SVCV effect. Likewise, PMA resulted in a higher phosphorylation levels of PKCε rather than PKCα/β to participate in the activation of Nrf2, mainly involved in the activation of Nrf2 phosphorylation of Ser40 to favor Nrf2 translocation to nucleus. Furthermore, the data revealed that PMA up-regulated an antiviral response heme oxygenase-1 (HO1) gene expression that was confirmed as the key player against SVCV infection by HO1 specific siRNA. Overall, this study provided a new therapeutic target for the treatment of SVCV infection, and modulating PKC activity could be used for the prevention and treatment of SVCV., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4 - CD8 - double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion of Robnr, an inducible lncRNA located downstream of the anti-apoptotic gene Bcl2, demonstrated a critical role of the Robnr locus in the induction of Bcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation.

The Tyr705 STAT3 constitutive activation, besides promoting PEL cell survival, contributes to the maintenance of viral latency. We found indeed that its de-phosphorylation by AG490 induced KSHV lytic cycle. Moreover, Tyr705 STAT3 de-phosphorylation, mediated by the activation of tyrosine phosphatases, together with the increase of Ser727 STAT3 phosphorylation contributed to KSHV lytic cycle induction by TPA. We then observed that p53-p21 axis, essential for the induction of KSHV replication, was activated by the inhibition of Tyr705 and by the increase of Ser727 STAT3 phosphorylation. As a possible link between STAT3, p53-p21 and KSHV lytic cycle, we found that TPA and AG490 reduced the expression of KAP-1, promoting p53 stability, p21 transcription and KSHV lytic cycle activation in PEL cells., (Copyright © 2018. Published by Elsevier Inc.)

Environmental exposure to arsenic is known to induce immunotoxicity. Macrophages are the professional phagocytes that are important in the immune system. In this study, we utilized the macrophages derived from the THP-1 human monocyte cell line as the experimental model to study the functional suppression induced by arsenite (As +3 ), one of the most prevalent forms of inorganic arsenic, at environmentally-relevant concentrations. Apoptosis was observed in the THP-1 derived macrophages treated with 500 nM As +3 for 18 h. Suppression of phagocytosis was induced by 18 h As +3 treatment starting from 100 nM. Suppressive effects on the production of two pro-inflammatory cytokines, IL-1β and TNF-α, were also found with the treatment of low to moderate doses of As +3 in lipopolysaccharides-stimulated THP-1 derived macrophages. The nitric oxide production was also inhibited by As +3 treatments, which was negatively correlated with the production of superoxide. Collectively, the results from the study demonstrated that environmentally-relevant concentrations of As +3 induced cytotoxicity and suppressed the major cellular functions in THP-1 derived macrophages. The macrophages were showed to be relatively sensitive to As +3 , and could be the essential target of the toxicity induced by environmental arsenic exposures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Up to this day, the roles of PEA15 expression and its phosphorylation in seizure-related events have not been still unclear. In the present study, we found that PEA15 was distinctly phosphorylated in reactive astrocytes and apoptotic astrocytes in the rat hippocampus following LiCl-pilocarpine-induced status epilepticus (SE, a prolonged seizure activity). PEA15-serine (S) 104 phosphorylation was up-regulated in reactive astrocytes following SE, although PEA15 expression and its S116 phosphorylation were unaltered. Bisindolylmaleimide (BIM), a protein kinase C (PKC) inhibitor, attenuated SE-induced reactive astrogliosis, but phorbol 12-myristate 13-acetate (PMA, a PKC activator) aggravated it. Unlike reactive astrocytes, PEA15-S116 phosphorylation was reduced in apoptotic astrocytes. However, PEA15 expression and its S104 phosphorylation were unchanged in apoptotic astrocyte. Neither BIM nor PMA affected SE-induced astroglial apoptosis. PEA15 expression and its phosphorylations were not relevant to SE-induced CA1 neuronal death. These findings indicate that PEA15-S104 and S116 phosphorylations may play a role in reactive astrogliosis and prevention of astroglial apoptosis, respectively. Therefore, we suggest that the selective manipulation of PEA15 phosphorylations may regulate apoptotic and/or proliferative signals in astrocytes., (Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Therapeutic targeting of specific genetic changes in cancer has proven to be an effective therapy and the concept of synthetic lethality has emerged. CCAAT/enhancer-binding protein-β (C/EBPβ), a basic leucine zipper transcription factor, has important roles in cellular processes including differentiation, inflammation, survival, and energy metabolism. Using a genetically engineered mouse model, we report that the deletion C/EBPβ in pre-existing oncogenic Ha-Ras mouse skin tumors in vivo resulted in rapid tumor regression. Regressing tumors exhibited elevated levels of apoptosis and p53 protein/activity, while adjacent C/EBPβ-deleted skin did not. These results indicate that the deletion of C/EBPβ de-represses p53 in oncogenic Ras tumors but not in normal wild-type Ras keratinocytes, and that C/EBPβ is essential for survival of oncogenic Ras tumors. Co-deletion of C/EBPβ and p53 in oncogenic Ras tumors showed p53 is required for tumor regression and elevated apoptosis. In tumors, loss of a pathway that confers adaptability to a stress phenotype of cancer/tumorigenesis, such as DNA damage, could result in selective tumor cell killing. Our results show that oncogenic Ras tumors display a significant DNA damage/replicative stress phenotype and these tumors have acquired a dependence on C/EBPβ for their survival. RNAseq data analysis of regressing tumors deleted of C/EBPβ indicates a novel interface between p53, type-1 interferon response, and death receptor pathways, which function in concert to produce activation of extrinsic apoptosis pathways. In summary, the deletion of C/EBPβ in oncogenic Ras skin tumors is a synthetic lethal event, making it a promising target for future potential anticancer therapies.

Background: Geraniol is a monoterpene alcohol that has anti-fungal, anti-cancer and anti-nociceptive properties, but its anti-allergic rhinitis (AR) property is unclear., Methods: In this study, the anti-inflammatory role and its possible mechanisms of geraniol in human mast cell line (HMC-1) cells stimulated by inflammatory trigger phorbol 12-myristate 13-acetate plus A23187 (PMACI), as well as in ovalbumin (OVA)-induced AR mice models were investigated., Results: PMACI results in a significant increase in the production of proinflammatory cytokines, such as TNF-α, IL-1β, MCP-1, IL-6 and as well as histamine. Geraniol was found to inhibit both TNF-α, IL-1β and IL-6 protein and mRNA expressions at concentrations of 40, 80, 160 μM. In OVA-induced AR models, geraniol treatment was able to suppress AR biomarkers (OVA-specific IgE and IL-1β as well as histamine) and nasal rub scores. Interestingly, p38, a member of the mitogen-activated protein kinase (MAPK) signaling family, was found to be increasingly hypophosphorylated as geraniol dose was increased. Similar decreases in the nuclear level of p65, a member of the nuclear factor kappa B (NF-κB) signaling pathway, were also observed., Conclusion: Our data highlights that the anti-inflammatory properties of geraniol on AR-related markers in activated HCM-1 cells and OVA-induced AR models may be mediated through the regulation of the MAPK/NF-κB signaling pathway., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The cytokines secreted by immune cells have a large impact on the tissue, surrounding a fracture, e.g., by attraction of osteoprogenitor cells. However, the underlying mechanisms are not yet fully understood. Thus, this study aims at investigating molecular mechanisms of the immune cell-mediated migration of immature primary human osteoblasts (phOBs), with transforming growth factor beta (TGF-β), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 ( NOX4 ) and focal adhesion kinase (FAK) as possible regulators. Monocyte- and macrophage (THP-1 cells ± phorbol 12-myristate 13-acetate (PMA) treatment)-conditioned media, other than the granulocyte-conditioned medium (HL-60 cells + dimethyl sulfoxide (DMSO) treatment), induce migration of phOBs. Monocyte- and macrophage (THP-1 cells)-conditioned media activate Smad3-dependent TGF-β signaling in the phOBs. Stimulation with TGF-β promotes migration of phOBs. Furthermore, TGF-β treatment strongly induces NOX4 expression on both mRNA and protein levels. The associated reactive oxygen species (ROS) accumulation results in phosphorylation (Y397) of FAK. Blocking TGF-β signaling, NOX4 activity and FAK signaling effectively inhibits the migration of phOBs towards TGF-β. In summary, our data suggest that monocytic- and macrophage-like cells induce migration of phOBs in a TGF-β-dependent manner, with TGF-β-dependent induction of NOX4 , associated production of ROS and resulting activation of FAK as key mediators.

N-Acylethanolamine acid amidase (NAAA) is a cysteine enzyme that catalyzes the hydrolysis of palmitoylethanolamide (PEA). Pharmacological blockage of NAAA elevates PEA levels and exerts powerful anti-inflammatory activities. We have recently identified a highly potent NAAA inhibitor F215. Here, we demonstrated that F215 was an unusual inflammation-restricted NAAA inhibitor. In lipopolysaccharides (LPS) induced acute lung injury (ALI) model, F215 markedly accelerated inflammation resolution, promoted clearance of neutrophils infiltration and alveolar repair in the lungs. F215 efficiently inhibited NAAA and protected endogenous PEA from degradation in ALI model, but it cannot readily suppress the NAAA activity in naïve mice. The inflammation-restricted effect of F215 was further confirmed in the alveolar macrophage, F215 only increased PEA levels and exerted anti-inflammatory effects in activated macrophages, but not in unstimulated macrophages. Moreover, we also showed that the pharmacological effects of F215 were restricted to the local inflamed skin elicited by 12-o-tetradecanoylphorbol-13-acetate (TPA), but not the normal tissues. We believe that F215 could be a useful probe to investigate the function of NAAA, as well as a potent anti-inflammatory agent, and its inflammation-restricted feature might offer a new approach to prevent potential side effects of systemic enzyme inhibition., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cross-sectional studies have shown that exposure to indoor moisture damage and mold may be associated with subclinical inflammation. Our aim was to determine whether early age exposure to moisture damage or mold is prospectively associated with subclinical systemic inflammation or with immune responsiveness in later childhood. Home inspections were performed in children's homes in the first year of life. At age 6 years, subclinical systemic inflammation was measured by serum C-reactive protein (CRP) and blood leukocytes and immune responsiveness by ex vivo production of interleukin 1-beta (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) in whole blood cultures without stimulation or after 24 hours stimulation with phorbol 12-myristate 13-acetate and ionomycin (PI), lipopolysaccharide (LPS), or peptidoglycan (PPG) in 251-270 children. Moisture damage in child's main living areas in infancy was not significantly associated with elevated levels of CRP or leukocytes at 6 years. In contrast, there was some suggestion for an effect on immune responsiveness, as moisture damage with visible mold was positively associated with LPS-stimulated production of TNF-α and minor moisture damage was inversely associated with PI-stimulated IL-1β. While early life exposure to mold damage may have some influence on later immune responsiveness, it does not seem to increase subclinical systemic inflammation in later life., (© 2018 National Institute for Health and Welfare, Finland Indoor Air published by John Wiley & Sons Ltd.)

T cell-mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti-porcine CD69 mAb-producing mouse hybridomas, 01-14-22-51 (IgG2b-κ) and 01-22-44-102 (IgG2a-κ), both showing fine reactivity with phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti-interferon-γ mAb and anti-tumor necrosis factor-α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs., (© 2018 Japanese Society of Animal Science.)

Neutrophils operate at the site of injury or inflammation in the periodontal pocket to ensure periodontal health and clearance of bacterial pathogens. Filifactor alocis is recently identified as a potential periodontal pathogen, and in this study, we assessed the formation of neutrophil extracellular traps (NETs), in response to the presence of the organism . NET formation by human neutrophils was not induced when challenged with F. alocis, independent of opsonization, viability, time, or bacterial dose. F. alocis also failed to induce NETs from TNF-α-primed neutrophils and did not induce the release of extracellular neutrophil elastase. However, significant NET induction was observed when neutrophils were challenged with Streptococcus gordonii or Peptoanaerobacter stomatis, In addition, co-infection studies revealed that the presence of F. alocis with S. gordonii or P. stomatis does not enhance or reduce NETs. Additionally, F. alocis failed to impact pre-formed NETs induced by either S. gordonii or P. stomatis. Pretreatment with F. alocis prior to stimulation with phorbol 12-myristate 13-acetate (PMA), S. gordonii, or P. stomatis revealed that the bacterium is capable of reducing only PMA but not S. gordonii or P. stomatis NET formation. These results indicate that F. alocis manipulates neutrophils, inhibiting the triggering of NET induction.

A disintegrin and metalloproteinase (ADAM) family are crucial enzymes for ectodomain shedding of multiple substrates and are involved in diverse biologic and pathologic processes. However, the molecular mechanism underlying substrate selectivity of ADAMs is poorly understood. In this study, we observed that disruption of actin polymerization by pharmacological inhibitors, latrunculin A (LatA) and cytochalasin D (CyD), induced ectodomain shedding of epidermal growth factor (EGF) family ligands. Induced shedding activity by LatA or CyD was suppressed by a metalloprotease inhibitor KB-R7785, indicating that ADAMs-mediated shedding is tightly controlled by actin cytoskeleton. We also investigated roles of cullin family, a component of cullin-RING based E3 ubiquitin ligases, in ectodomain shedding, since cullin family is implicated in the regulation of cytoskeletal dynamics. Knockdown of cullin 3 (Cul3) by a specific siRNA inhibited ectodomain shedding of amphiregulin (AREG), a member of EGF family, and responses were associated with activation of RhoA GTPase and induction of stress fiber formation. On the other hand, the RhoA inhibitor C3 transferase rescued AREG shedding reduced by Cul3 knockdown. These results describe a novel molecular mechanism of Cul3 to regulate AREG shedding by modulating cytoskeletal dynamics in a RhoA dependent manner., (Copyright © 2018. Published by Elsevier Inc.)

Macrophages are accepted as cells that initially contact with the pathogens and initiate the innate immune response. They play effective roles in innate immune and inflammatory responses by intercellular relations and inflammatory mediator secretion. Human THP-1 leukemia cells are frequently used for the in vitro determination of the signal pathways, and the functions of macrophages. Phorbol-12-Myristate-13-Acetate (PMA) is commonly used to induce macrophage differentiation of monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. Midkine acts as a cytokine and growth factor which organizes proliferation, differentiation, survival, adhesion and migration of immune cells. The aim of this study was to observe the differences in the secretion of midkine, TNF-α, IL-10 and IFN-γ of macrophages differentiated from monocytes when stimulated with different doses of PMA for different durations. For this purpose, THP-1 monocytic cells were proliferated by PMA at 24, 48 and 72 hours by using the concentrations of 10 ng/ml, 20 ng/ml, 40 ng/ml and 60 ng/ ml. Midkine, TNF-α, IL-10 and IFN-γ cytokine levels were determined by ELISA in the supernatants of the cells collected at the end of incubation times. PMA stimuli initiated changes that were indicative of differentiation in the cell morphology. Differentiation of cells by PMA induced midkine, TNF-α, IL-10 and IFN-γ secretions in monocytic cells even at the lowest dosage (10 ng/ml). PMA caused cytotoxicity in the cells when the dosages were increased (> 20 ng/ml). THP-1 cells have a basal secretion of midkine and are increased by dosage dependent with PMA stimulation. Midkine secretion has shown changes dependent with dosage and time. A difference was also observed in the cytokine profile of PMA stimulated cells at different doses. The results indicated that the differentiation of THP-1 monocytes to macrophages requires optimization to ensure that this in vitro macrophage model more precisely reflects real in vivo physiologic conditions. As a conclusion the results have shown that a modified PMA differentiation protocol (20 ng/ml and 48 hours incubation) might enhance macrophage differentiation of THP-1 cells without induced cell death (viability 92.2%) and cytokine secretion and midkine responses were the important discriminators of the level of macrophage differentiation.

The effector function of natural killer, lymphokine--activated killer cells and T lymphocytes is commonly evaluated by radioactive chromium‑release cytotoxicity assays. In addition to this indirect method, fluorescence assays have been described for the assessment of in vitro cell‑mediated cytotoxicity. In the present study, target cells were stained with 5‑(and‑6)‑carboxyfluorescein diacetate succinimidyl ester (CFSE), which is a stable integrated fluorescent probe that allows target and effector cells to be distinguished from one another. Staining of target THP‑1 cells with 8 µM CFSE revealed high and stable loading of fluorescence and no effect of the viability of cells. After 4 h of in vitro co‑culture between γδ T cells and CFSE‑labeled infected or uninfected THP‑1 cells, staining with propidium iodide (PI) was performed to distinguish between vital and dead cells. During sample acquisition, target cells were gated on the CFSE positivity and examined for cell death based on the uptake of PI. CFSE and PI double positive cells were recognized as the dead target cells. The percentage of cytotoxicity in the CFSE‑gated cell population was calculated by subtracting the value obtained for non‑specific PI‑positive target cells, which was measured in a control group that did not contain effector cells. The present study describes a simple and convenient assay that is based on the direct quantitative and qualitative analysis of cell damage at a single cell level utilizing a two‑color flow cytometric assay. In conclusion, the flow cytometric‑based assay described in the current study is a simple, sensitive and reliable tool to determine the cytolytic activity of γδ T lymphocytes against mycobacteria. Therefore, the present study may provide valuable information concerning the methods employed to investigate the function of γδ T cells and potentially other lymphocyte subsets.

Rapamycin and its derivative possess anti-atherosclerosis activity, but its effects on adhesion molecule expression and macrophage adhesion to endothelial cells during atherosclerosis remain unclear. In this study we explored the effects of rapamycin on ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells in vitro and the underlying mechanisms. Ox-LDL (6-48 μg/mL) dose-dependently increased the protein levels of two adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and E-selectin, in human umbilical vein endothelial cells (HUVECs), whereas pretreatment with rapamycin (1-10 μmol/L) dose-dependently inhibited ox-LDL-induced increase in the adhesion molecule expression and macrophage adhesion to endothelial cells. Knockdown of mTOR or rictor, rather than raptor, mimicked the effects of rapamycin. Ox-LDL (100 μg/mL) time-dependently increased PKC phosphorylation in HUVECs, which was abolished by rapamycin or rictor siRNA. Pretreatment with PKC inhibitor staurosporine significantly reduced ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells, whereas pretreatment with PKC activator PMA/TPA attenuated the inhibitory effect of rapamycin on adhesion molecule expression. Ox-LDL (100 μg/mL) time-dependently increased c-Fos levels in HUVECs, and pretreatment with rapamycin or rictor siRNA significantly decreased expression of c-Fos. Knockdown of c-Fos antagonized ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells. Our results demonstrate that rapamycin reduces ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells by inhibiting mTORC2, but not mTORC1, and mTORC2 acts through the PKC/c-Fos signaling pathway.

Adherent cells such as mouse RAW cells or human cancer U2OS cells are beneficial to DNA transfection, with 20%-60% transfection efficiency. However, this DNA transfection is rarely used on suspension cells due to its low transfection efficiency (≤5%). We recently found a new DNA transfection method to increase the efficiency up to 13.5% in suspension cells without PMA treatment. We also found that DNA transfection of human TNFAIP1 or CXCL1 recombinant plasmid DNA in THP-1 cells induces a high level of TNF-α protein. Overall, this new method is simple yet efficient and can be used for the overexpression of DNA in suspension cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Superoxide dismutase (SOD) 3, a copper (Cu)-containing anti-oxidative enzyme, plays a key role in extracellular redox homeostasis. Cu chaperone antioxidant-1 (Atox-1) not only delivers Cu ions to SOD3 at the trans-Golgi network, it also functions as a transcription factor of SOD3; however, the role of Atox-1 in the regulation of SOD3 during the monocytic differentiation of THP-1 cells has not yet been elucidated. A treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the expression of the Cu transport protein ATP7A in THP-1 cells. On the other hand, the nuclear translocation of Atox-1 was detected in TPA-treated THP-1 cells, and was suppressed in the presence of the Cu chelator, bathocuproinedisulfonic acid. Furthermore, Atox-1 bound to the SOD3 promoter region in TPA-treated THP-1 cells. The overexpression of Atox-1 in THP-1 cells significantly enhanced TPA-elicited SOD3 expression, whereas its knockdown suppressed this induction. The present results demonstrate that Atox-1 functions as a key molecule in TPA-elicited SOD3 expression.

Monocytes are recruited to injured tissue sites and differentiate into tissue macrophages or dendritic cells to protect against pathogens and repair the damaged tissues. Phorbol-12-myristate-13-acetate (PMA) is a well-known stimulus commonly used for differentiation of monocytes into macrophage-like cells (MϕLC). Here, we report the effect of Cold Atmospheric Plasma (CAP) on PMA-induced U937 differentiation into MϕLC. Treatment of U937 cells with PMA for 3 days and resting for 4 days increased the size of cytoplasm as compared with nucleus, and exposure to CAP before addition of PMA led to further increase in cytoplasm indicating the ability of CAP to modulate the differentiation of monocytes. Exposure of U937 cells to CAP or PMA increased cellular reactive oxygen species (ROS) level and the combination led to further augmentation of ROS. Treatment of U937 cells with PMA displayed a biphasic activation of proinflammatory transcription factor NF-κB, which plays an important role in differentiation and pretreatment with CAP further increased PMA induced NF-κB-DNA-binding activity. CAP also increased lipopolysaccharide induced secretion of TNF-α and IL-6 in MϕLC. Further investigation revealed that MϕLC or CAP-treated MϕLC were more resistant to anticancer drugs such as doxorubicin and 5-fluorouracil (5-FU) than U937 cells. Our present studies suggest an alternate protocol to modulate the differentiation of U937 cells into MϕLC by combining CAP and PMA.

Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)