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International Journal of Implant... May 2022The preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to...
BACKGROUND
The preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to search for suitable substitutes, such as silica-coated plastic tubes. However, we have previously demonstrated the cytotoxicity of silica microparticles (MPs) used in plastic tubes to cultured human periosteal cells. To further establish the effects of silica MPs on inflammation, we examined silica MP-induced changes in a human promyelocytic cell model in vitro.
METHODS
Human promyelocytic HL60 cells were used either without chemical induction or after differentiation induced using phorbol myristate acetate (PMA) or dimethyl sulfoxide. HL60 cells, osteoblastic MG63, and Balb/c mouse cells were treated with silica MPs, and their surface ultrastructure and numbers were examined using a scanning electron microscope and an automated cell counter, respectively. Differentiation markers, such as acid phosphatase, non-specific esterase, and CD11b, were visualized by cytochemical and immunofluorescent staining, and superoxide dismutase (SOD) activity was quantified.
RESULTS
Regardless of SOD activity, silica cytotoxicity was observed in MG63 and Balb/c cells. At sub-toxic doses, silica MPs slightly or moderately upregulated the differentiation markers of the control, PMA-induced monocytic, and dimethyl sulfoxide-induced granulocytic HL60 cells. Although SOD activity was the highest (P < 0.05) in PMA-induced cells, a silica-induced reduction in cell adhesion was observed only in those cells (P < 0.05).
CONCLUSIONS
Silica MP contamination of PRF preparations can potentially exacerbate inflammation at implantation sites. Consequently, unless biomedical advantages can be identified, silica-coated plastic blood collection tubes should not be routinely used for PRF preparations.
Topics: Animals; Cell Lineage; Dimethyl Sulfoxide; HL-60 Cells; Humans; Inflammation; Leukemia, Promyelocytic, Acute; Mice; Plastics; Silicon Dioxide; Superoxide Dismutase; Tetradecanoylphorbol Acetate
PubMed: 35567654
DOI: 10.1186/s40729-022-00424-4 -
BMC Veterinary Research May 2022Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune...
BACKGROUND
Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4 and CD8 T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days.
RESULTS
The proportions of CD4 T lymphocytes (18.70 ± 4.21%) and CD8 T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8 T lymphocytes was significantly (p < 0.001) higher than that in CD4 T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4 T lymphocytes was significantly (p < 0.001) higher than that in CD8 T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4 (2.83 ± 0.98%) and CD8 T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period.
CONCLUSIONS
Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.
Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Flow Cytometry; Interleukin-17; Interleukin-4; Ionomycin; Leukocytes, Mononuclear; Lymphocyte Activation; Sheep; Tetradecanoylphorbol Acetate
PubMed: 35513847
DOI: 10.1186/s12917-022-03268-7 -
Brazilian Journal of Biology = Revista... 2022Neutrophil extracellular traps (NETs) were first reported as a microbicidal strategy for activated neutrophils. Through an immunologic response against several stimuli,...
Quercetin inhibits neutrophil extracellular traps release and their cytotoxic effects on A549 cells, as well the release and enzymatic activity of elastase and myeloperoxidase.
Neutrophil extracellular traps (NETs) were first reported as a microbicidal strategy for activated neutrophils. Through an immunologic response against several stimuli, neutrophils release their DNA together with proteins from granules, nucleus, and cytoplasm (e.g., elastase and myeloperoxidase). To date, NETs have been implicated in tissue damage during intense inflammatory processes, mainly when their release is dependent on oxygen radical generation. Flavonoids are antioxidant and anti-inflammatory agents; of these, quercetin is commonly found in our daily diet. Therefore, quercetin could exert some protective activity against tissue damage induced by NETs. In our in vitro assays, quercetin reduced NETs, myeloperoxidase (MPO), and elastase release from neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). The activity of these enzymes also decreased in the presence of quercetin. Quercetin also reduced the cytotoxic effect of NETs on alveolar cells (A549 cell line). Further, in silico assays indicated favorable interactions between quercetin and NET proteins (MPO and elastase). Overall, our results demonstrate that quercetin decreases deleterious cellular effects of NETs by reducing their release from activated neutrophils, and diminishing the enzymatic activity of MPO and elastase, possibly through direct interaction.
Topics: A549 Cells; Extracellular Traps; Humans; Pancreatic Elastase; Peroxidase; Quercetin; Tetradecanoylphorbol Acetate
PubMed: 35475990
DOI: 10.1590/1519-6984.252936 -
Journal of Immunology (Baltimore, Md. :... May 2022Respiratory viruses stimulate the release of antiviral IFNs from the airway epithelium. Previous studies have shown that asthmatic patients show diminished release of...
Respiratory viruses stimulate the release of antiviral IFNs from the airway epithelium. Previous studies have shown that asthmatic patients show diminished release of type I and type III IFNs from bronchial epithelia. However, the mechanism of this suppression is not understood. In this study, we report that extracellular nucleotides and histamine, which are elevated in asthmatic airways, strongly inhibit release of type I and type III IFNs from human bronchial airway epithelial cells (AECs). Specifically, ATP, UTP, and histamine all inhibited the release of type I and type III IFNs from AECs induced by activation of TLR3, retinoic acid-inducible gene I (RIG-I), or cyclic GMP-AMP synthase-STING. This inhibition was at least partly mediated by Gq signaling through purinergic P2Y and H receptors, but it did not involve store-operated calcium entry. Pharmacological blockade of protein kinase C partially reversed inhibition of IFN production. Conversely, direct activation of protein kinase C with phorbol esters strongly inhibited TLR3- and RIG-I-mediated IFN production. Inhibition of type I and type III IFNs by ATP, UTP, histamine, and the proteinase-activated receptor 2 (PAR2) receptor agonist SLIGKV also occurred in differentiated AECs grown at an air-liquid interface, indicating that the suppression is conserved following mucociliary differentiation. Importantly, histamine and, more strikingly, ATP inhibited type I IFN release from human airway cells infected with live influenza A virus or rhinovirus 1B. These results reveal an important role for extracellular nucleotides and histamine in attenuating the induction of type I and III IFNs from AECs and help explain the molecular basis of the suppression of IFN responses in asthmatic patients.
Topics: Adenosine Triphosphate; DEAD Box Protein 58; Epithelial Cells; Histamine; Humans; Interferons; Nucleotides; Protein Kinase C; Receptors, Immunologic; Respiratory Mucosa; Toll-Like Receptor 3; Uridine Triphosphate
PubMed: 35459743
DOI: 10.4049/jimmunol.2101085 -
International Journal of Molecular... Apr 2022Megakaryocytes are large hematopoietic cells present in the bone marrow cavity, comprising less than 0.1% of all bone marrow cells. Despite their small number,...
Megakaryocytes are large hematopoietic cells present in the bone marrow cavity, comprising less than 0.1% of all bone marrow cells. Despite their small number, megakaryocytes play important roles in blood coagulation, inflammatory responses, and platelet production. However, little is known about changes in gene expression during megakaryocyte maturation. Here we identified the genes whose expression was changed during K562 leukemia cell differentiation into megakaryocytes using an Affymetrix GeneChip microarray to determine the multifunctionality of megakaryocytes. K562 cells were differentiated into mature megakaryocytes by treatment for 7 days with phorbol 12-myristate 13-acetate, and a microarray was performed using RNA obtained from both types of cells. The expression of 44,629 genes was compared between K562 cells and mature megakaryocytes, and 954 differentially expressed genes (DEGs) were selected based on a p-value < 0.05 and a fold change >2. The DEGs was further functionally classified using five major megakaryocyte function-associated clusters—inflammatory response, angiogenesis, cell migration, extracellular matrix, and secretion. Furthermore, interaction analysis based on the STRING database was used to generate interactions between the proteins translated from the DEGs. This study provides information on the bioinformatics of the DEGs in mature megakaryocytes after K562 cell differentiation.
Topics: Acetates; Cell Differentiation; Computational Biology; Humans; K562 Cells; Megakaryocytes; Microarray Analysis; Myristic Acid; Phorbols; Tetradecanoylphorbol Acetate; Thrombopoiesis
PubMed: 35457039
DOI: 10.3390/ijms23084221 -
Cells Mar 2022Nociceptin and the nociceptin receptor (NOP) have been described as targets for treatment of pain and inflammation, whereas toll-like receptors (TLRs) play key roles in...
Nociceptin and the nociceptin receptor (NOP) have been described as targets for treatment of pain and inflammation, whereas toll-like receptors (TLRs) play key roles in inflammation and impact opioid receptors and endogenous opioids expression. In this study, interactions between the nociceptin and TLR systems were investigated. Human THP-1 cells were cultured with or without phorbol myristate acetate (PMA 5 ng/mL), agonists specific for TLR2 (lipoteichoic acid, LTA 10 µg/mL), TLR4 (lipopolysaccharide, LPS 100 ng/mL), TLR7 (imiquimod, IMQ 10 µg/mL), TLR9 (oligonucleotide (ODN) 2216 1 µM), PMA+TLR agonists, or nociceptin (0.01−100 nM). Prepronociceptin (ppNOC), NOP, and TLR mRNAs were quantified by RT-qPCR. Proteins were measured using flow cytometry. PMA upregulated ppNOC mRNA, intracellular nociceptin, and cell membrane NOP proteins (all p < 0.05). LTA and LPS prevented PMA’s upregulating effects on ppNOC mRNA and nociceptin protein (both p < 0.05). IMQ and ODN 2216 attenuated PMA’s effects on ppNOC mRNA. PMA, LPS, IMQ, and ODN 2216 increased NOP protein levels (all p < 0.05). PMA+TLR agonists had no effects on NOP compared to PMA controls. Nociceptin dose-dependently suppressed TLR2, TLR4, TLR7, and TLR9 proteins (all p < 0.01). Antagonistic effects observed between the nociceptin and TLR systems suggest that the nociceptin system plays an anti-inflammatory role in monocytes under inflammatory conditions.
Topics: Humans; Inflammation; Lipopolysaccharides; Opioid Peptides; RNA, Messenger; Tetradecanoylphorbol Acetate; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 7; Toll-Like Receptor 9; Toll-Like Receptors; Nociceptin
PubMed: 35406649
DOI: 10.3390/cells11071085 -
Theranostics 2022Insulin-like growth factor 1 receptor (IGF-1R) expression and signaling play important roles in promotion of skin cancer progression. Identification of signaling...
Insulin-like growth factor 1 receptor (IGF-1R) expression and signaling play important roles in promotion of skin cancer progression. Identification of signaling pathways that regulate IGF-1R is crucial for understanding the pathogenesis and therapeutic treatment of skin cancer. Molecular, cellular and genetic approaches were used to investigate the function of PINCH-1 in regulation of IGF-1R expression and skin cell behavior. Furthermore, conditional PINCH-1 knockout mouse and carcinogen (7, 12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA))-induced skin cancer model were employed to determine the function of PINCH-1 in regulation of IGF-1R expression and skin carcinogenesis . Knockdown of PINCH-1 from HaCaT keratinocytes or A431 squamous carcinoma cells diminished IGF-1R levels, suppressed cell proliferation and increased apoptosis. Re-expression of PINCH-1 in PINCH-1 knockdown cells restored IGF-1R expression, cell proliferation and survival. Furthermore, depletion of NEDD4 effectively reversed PINCH-1 deficiency-induced down-regulation of IGF-1R expression, cell proliferation and survival. Conditional knockout of PINCH-1 from keratin 5 (K5) positive keratinocytes in mice, like depletion of PINCH-1 from keratinocytes in culture, reduced the IGF-1R level. Using a mouse model of DMBA/TPA-induced skin cancer, we show that the levels of both PINCH-1 and IGF-1R were significantly increased in response to treatment with the carcinogens. Genetic ablation of PINCH-1 from the epidermis markedly reduced the IGF-1R expression and cell proliferation despite stimulation with DMBA/TPA, resulting in resistance to chemical carcinogen-induced skin cancer initiation and progression. Our results reveal a PINCH-1-NEDD4-IGF-1R signaling axis that is critical for promotion of skin tumorigenesis and suggest a new strategy for therapeutic control of skin cancer progression.
Topics: Animals; Carcinogenesis; Carcinogens; Cell Proliferation; GRB10 Adaptor Protein; Keratinocytes; Mice; Receptor, IGF Type 1; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate
PubMed: 35401828
DOI: 10.7150/thno.70744 -
SLAS Discovery : Advancing Life... Jul 2022The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that...
The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that suppresses T cell receptor (TCR) signaling. We developed a phenotypic assay to screen for small molecules that interfere with this pathway using TALL-104 human leukemic cytotoxic T lymphocytes pretreated with prostaglandin E2 to elevate cAMP. Beads coated with anti-CD3 antibodies stimulate lytic granule exocytosis, which is detected via binding of an antibody against lysosome associated membrane protein 1 (LAMP-1) measured with flow cytometry. Confirming that the assay can find compounds with desired activity, treating cells with a phorbol ester restores exocytosis. The assay behaves well in 96-well format and we screened a collection of compounds expected to have effects on epigenetic regulatory proteins. Compounds in this collection affected lytic granule exocytosis after 24-hour treatment, but none prevented cAMP from suppressing lytic granule exocytosis. We used a fully automated 384-well version of the assay to screen the Prestwick Compound Library but obtained no confirmed hits. Analyzing this assay's performance reveals two points of interest. First, cytometry offers multiple ways to quantify signals. Z' was higher using percent positive cells than mean fluorescence because the relationship between the two measures saturates, but using percent positive could make it harder to find hits in some assays. Second, variance was higher in positive controls than in negative controls in this assay, which degrades assay performance less than if variance was higher in negative controls.
Topics: Cyclic AMP; Exocytosis; Flow Cytometry; Humans; Signal Transduction; T-Lymphocytes, Cytotoxic
PubMed: 35385793
DOI: 10.1016/j.slasd.2022.03.008 -
Chemical & Pharmaceutical Bulletin 2022Five new crotofolanes, named crotocascarins R-V (1-5), one rearranged trinorcrotofolane, crotocascarin δ, and one phorbol derivative were isolated from the...
Five new crotofolanes, named crotocascarins R-V (1-5), one rearranged trinorcrotofolane, crotocascarin δ, and one phorbol derivative were isolated from the EtOAc-soluble fraction of the MeOH extract of the leaves of Croton cascarilloides. Crotocascarins R (1), T (3), and U (4) possessed isobutyric acid as an acyl moiety and crotocascarin B (2) an acetyl group, whereas crotocascarin V (5) was elucidated to be a hydroxylated compound of crotocascarin K at the 9-position. Crotocascarin δ (6) was a trinor rearranged crotofolane with a tertiary hemiketal functional group at the 8-position. The absolute configuration of the 8-position was determined by the comparison of the experimental electronic circular dichroism (ECD) spectrum and calculated ECD spectra. Compound 7 was a phorbol ester derivative with a peroxide functional group. The fatty acid attached at the 12-position was found to be a single species-i.e., lauric acid (C-12)-from the evidence of the mass spectral data.
Topics: Circular Dichroism; Croton; Diterpenes; Phorbols; Plant Leaves
PubMed: 35370206
DOI: 10.1248/cpb.c21-01034 -
International Journal of Molecular... Mar 2022The Bhas 42 cell transformation assay (Bhas 42 CTA) is the first Organization for Economic Cooperation and Development (OECD)-certificated method used as a specific tool...
The Bhas 42 cell transformation assay (Bhas 42 CTA) is the first Organization for Economic Cooperation and Development (OECD)-certificated method used as a specific tool for the detection of the cell-transformation potential of tumor-promoting compounds, including non-genotoxic carcinogens (NGTxCs), as separate from genotoxic carcinogens. This assay offers the great advantage of enabling the phenotypic detection of oncotransformation. A key benefit of using the Bhas 42 CTA in the study of the cell-transformation mechanisms of tumor-promoting compounds, including non-genotoxic carcinogens, is that the cell-transformation potential of the chemical can be detected directly without treatment with a tumor-initiating compound since Bhas 42 cell line was established by transfecting the v-Ha- gene into a mouse fibroblast cloned cell line. Here, we analyzed the gene expression over time, using DNA microarrays, in Bhas 42 cells treated with the tumor-promoting compound 12--tetradecanoylphorbol-13-acetate (TPA), and NGTxC, with a total of three repeat experiments. This is the first paper to report on gene expression over time during the process of cell transformation with only a tumor-promoting compound. Pathways that were activated or inactivated during the process of cell transformation in the Bhas 42 cells treated with TPA were related not only directly to but also to various pathways in the hallmarks of cancer.
Topics: Animals; BALB 3T3 Cells; Butylated Hydroxyanisole; Carcinogenicity Tests; Carcinogens; Cell Transformation, Neoplastic; Gene Expression; Mice; Tetradecanoylphorbol Acetate
PubMed: 35328637
DOI: 10.3390/ijms23063216