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Frontiers in Immunology 2022Cell-based functional immune-assays may allow for risk stratification of patients with complex, heterogeneous immune disorders such as sepsis. Given the heterogeneity of... (Observational Study)
Observational Study
BACKGROUND
Cell-based functional immune-assays may allow for risk stratification of patients with complex, heterogeneous immune disorders such as sepsis. Given the heterogeneity of patient responses and the uncertain immune pathogenesis of sepsis, these assays must first be defined and calibrated in the healthy population.
OBJECTIVE
Our objective was to compare the internal consistency and practicality of two immune assays that may provide data on surrogate markers of the innate and adaptive immune response. We hypothesized that a rapid turnaround, microfluidic-based immune assay (ELLA) would be comparable to a dual-color, enzyme-linked immunospot (ELISpot) assay in identifying tumor necrosis factor (TNF) and interferon (IFN)γ production following whole blood stimulation.
DESIGN
This was a prospective, observational cohort analysis. Whole blood samples from ten healthy, immune-competent volunteers were stimulated for either 4 hours or 18 hours with lipopolysaccharide, anti-CD3/anti-CD28 antibodies, or phorbol 12-myristate 13-acetate with ionomycin to interrogate innate and adaptive immune responses, respectively.
MEASUREMENTS AND MAIN RESULTS
ELLA analysis produced more precise measurement of TNF and IFNγ concentrations as compared with ELISpot, as well as a four- to five-log dynamic range for TNF and IFNγ concentrations, as compared with a two-log dynamic range with ELISpot. Unsupervised clustering accurately predicted the immune stimulant used for 90% of samples analyzed ELLA, as compared with 72% of samples analyzed ELISpot.
CONCLUSIONS
We describe, for the first time, a rapid and precise assay for functional interrogation of the innate and adaptive arms of the immune system in healthy volunteers. The advantages of the ELLA microfluidic platform may represent a step forward in generating a point-of-care test with clinical utility, for identifying deranged immune phenotypes in septic patients.
Topics: Cytokines; Enzyme-Linked Immunospot Assay; Humans; Interferon-gamma; Prospective Studies; Sepsis; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha
PubMed: 35860253
DOI: 10.3389/fimmu.2022.940030 -
International Journal of Molecular... Jul 2020Osteoarthritis (OA) is the most common type of arthritis that occurs in an aged population. It affects any joints in the body and degenerates the articular cartilage and... (Review)
Review
Osteoarthritis (OA) is the most common type of arthritis that occurs in an aged population. It affects any joints in the body and degenerates the articular cartilage and the subchondral bone. Despite the pathophysiology of OA being different, cartilage resorption is still a symbol of osteoarthritis. Matrix metalloproteinases (MMPs) are important proteolytic enzymes that degrade extra-cellular matrix proteins (ECM) in the body. MMPs contribute to the turnover of cartilage and its break down; their levels have increased in the joint tissues of OA patients. Application of chondroprotective drugs neutralize the activities of MMPs. Natural products derived from herbs and plants developed as traditional medicine have been paid attention to, due to their potential biological effects. The therapeutic value of natural products in OA has increased in reputation due to their clinical impact and insignificant side effects. Several MMPs inhibitor have been used as therapeutic drugs, for a long time. Recently, different types of compounds were reviewed for their biological activities. In this review, we summarize numerous natural products for the development of MMPs inhibitors in arthritic diseases and describe the major signaling targets that were involved for the treatments of these destructive joint diseases.
Topics: Animals; Biological Products; Cartilage, Articular; Chondrocytes; Cytokines; Drug Evaluation, Preclinical; Extracellular Matrix Proteins; Forecasting; Humans; Iodoacetic Acid; Matrix Metalloproteinase Inhibitors; Models, Animal; NF-kappa B; Osteoarthritis; Rats; Self Medication; Tetradecanoylphorbol Acetate
PubMed: 32668590
DOI: 10.3390/ijms21144931 -
Journal of Cellular Physiology Feb 2022Mechanical forces can modulate the immune response, mostly described as promoting the activation of immune cells, but the role and mechanism of pathological levels of...
Mechanical forces can modulate the immune response, mostly described as promoting the activation of immune cells, but the role and mechanism of pathological levels of mechanical stress in lymphocyte activation have not been focused on before. By an ex vivo experimental approach, we observed that mechanical stressing of murine spleen lymphocytes with 50 mmHg for 3 h induced the nuclear localization of NFAT1, increased C-Jun, and increased the expression of early activation marker CD69 in resting CD8+ cells. Interestingly, 50 mmHg mechanical stressing induced the nuclear localization of NFAT1; but conversely decreased C-Jun and inhibited the expression of CD69 in lymphocytes under lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin stimulation. Additionally, we observed similar changes trends when comparing RNA-seq data of hypertensive and normotensive COVID-19 patients. Our results indicate a biphasic effect of mechanical stress on lymphocyte activation, which provides insight into the variety of immune responses in pathologies involving elevated mechanical stress.
Topics: Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Biomarkers; CD8-Positive T-Lymphocytes; COVID-19; Cell Nucleus; Comorbidity; Gene Expression Regulation; Humans; Hypertension; Hypoxia-Inducible Factor 1, alpha Subunit; Ion Channels; Lectins, C-Type; Lipopolysaccharides; Lymphocyte Activation; Male; Mice, Inbred C57BL; NFATC Transcription Factors; Protein Transport; Proto-Oncogene Proteins c-jun; Signal Transduction; Stress, Mechanical; Tetradecanoylphorbol Acetate; Mice
PubMed: 34724217
DOI: 10.1002/jcp.30623 -
Frontiers in Immunology 2020Neutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of...
BACKGROUND
Neutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of decondensed chromatin and about 30 enzymes and peptides. Some components, such as neutrophil elastase (NE) and myeloperoxidase (MPO), present antimicrobial but also cytotoxic properties, leading to tissue injury. Many inflammatory diseases are associated with NETs, and their final role has not been identified. Pulmonary surfactant is known to have immunoregulatory abilities that alter the function of adaptive and innate immune cells. The aim of this study was to investigate the hypothesis that natural surfactant preparations inhibit the formation of NETs.
METHODS
The effect of two natural surfactants (Alveofact and Curosurf) on spontaneous and phorbol-12-myristate-13-acetate-induced NET formation by neutrophils isolated by magnetic cell sorting from healthy individuals was examined. NETs were quantitatively detected by absorption and fluorometric-based assays for the NET-specific proteins (NE, MPO) and cell-free DNA. Immunofluorescence microscopy images were used for visualization.
RESULTS
Both surfactant preparations exerted a dose-dependent inhibitory effect on NET formation. Samples treated with higher concentrations and with 30 min pre-incubation prior to stimulation with phorbol-12-myristate-13-acetate had significantly lower levels of NET-specific proteins and cell-free DNA compared to untreated samples. Immunofluorescence microscopy confirmed these findings.
CONCLUSIONS
The described dose-dependent modulation of NET formation ex vivo suggests an interaction between exogenous surfactant supplementation and neutrophil granulocytes. The immunoregulatory effects of surfactant preparations should be considered for further examination of inflammatory diseases.
Topics: Biological Products; Cells, Cultured; Dose-Response Relationship, Drug; Extracellular Traps; Granulocytes; Humans; Immunomodulation; Leukocyte Elastase; Neutrophil Activation; Neutrophils; Phospholipids; Pulmonary Surfactants; Tetradecanoylphorbol Acetate
PubMed: 33574811
DOI: 10.3389/fimmu.2020.582895 -
International Journal of Molecular... Oct 2022We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide...
We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.
Topics: Mice; Animals; Vasoactive Intestinal Peptide; Cyclic AMP Response Element-Binding Protein; Suprachiasmatic Nucleus Neurons; Suprachiasmatic Nucleus; Protein Kinase C; Luciferases; Phorbol Esters
PubMed: 36293078
DOI: 10.3390/ijms232012226 -
Archives of Biochemistry and Biophysics Jul 2020Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in...
Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 μM), and the Ca chelator BAPTA-AM (150 μM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 μM), and the MEK1/2 inhibitor U0126 (10 μM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 μM. The IL-1β autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation.
Topics: Amino Acid Sequence; Butadienes; Cell Line, Tumor; Egtazic Acid; Humans; Indoles; Isoquinolines; Maleimides; Nitriles; Protein Conformation, alpha-Helical; Protein Kinase C; Protein Structure, Tertiary; RNA, Messenger; Receptors, G-Protein-Coupled; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Up-Regulation
PubMed: 32339486
DOI: 10.1016/j.abb.2020.108375 -
Journal of Natural Products May 2023Three new phenanthrene derivatives (, , ), one new fluorenone (), and four known compounds (-) were isolated from the ethyl acetate extract of Sw. stems using column...
Three new phenanthrene derivatives (, , ), one new fluorenone (), and four known compounds (-) were isolated from the ethyl acetate extract of Sw. stems using column chromatography. The chemical structures were elucidated by analysis of spectroscopic data. The absolute configuration of was determined by electronic circular dichroism calculation. We also evaluated the immunomodulatory effects of compounds isolated from in human peripheral blood mononuclear cells from healthy individuals and those from patients with multiple sclerosis in vitro. Dendrocrumenol B () and dendrocrumenol D () showed strong immunomodulatory effects on both CD3 T cells and CD14 monocytes. Compounds and could reduce IL-2 and TNF production in T cells and monocytes that were treated with phorbol-12-myristate-13-acetate and ionomycin (PMA/Iono). Deep immune profiling using high-dimensional single-cell mass cytometry could confirm immunomodulatory effects of , quantified by the reduction of activated T cell population under PMA/Iono stimulation, in comparison to the stimulated T cells without treatment.
Topics: Humans; Dendrobium; Leukocytes, Mononuclear; Monocytes; Phenanthrenes; T-Lymphocytes; Tetradecanoylphorbol Acetate; Fluorenes
PubMed: 37140218
DOI: 10.1021/acs.jnatprod.3c00107 -
International Journal of Molecular... Aug 2022Cell-cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the... (Review)
Review
Cell-cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca channel blockers nor 3-isobutyl-1-methylxanthine (IBMX), forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester or H7 had significant effects on gating by anesthetics. In our publication, we concluded that neither cytosolic Ca nor pH were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca concentration ([Ca]) and consequential activation of calmodulin linked to gap junction proteins.
Topics: Anesthetics; Anesthetics, Inhalation; Caffeine; Calcium; Calmodulin; Cell Communication; Connexins; Gap Junctions; Halothane; Heptanol; Ion Channels; Isoflurane; Theophylline
PubMed: 36012286
DOI: 10.3390/ijms23169017 -
Clinical and Experimental Immunology Nov 2021The induction of immune tolerance without the use of immunosuppressive drugs is a crucial problem in organ transplantation. The use of myeloid-derived suppressor cells...
The induction of immune tolerance without the use of immunosuppressive drugs is a crucial problem in organ transplantation. The use of myeloid-derived suppressor cells (MDSCs) as a cell-based adjuvant immunosuppressive therapy is a bright clinical prospect in organ transplantation. MDSCs with stable immunosuppressive activities can be used to treat immune-related diseases. In this study, macrophage colony-stimulating factor (M-CSF) was used to promote myeloid progenitor cell differentiation, and phorbol 12-myristate 13-acetate (PMA) was added to induce MDSCs at the later stage of induction in vitro. Cell phenotypes were detected by flow cytometry and mRNA was detected by real-time-polymerase chain reaction (RT-PCR). A mouse skin transplantation model was used to investigate the cell inhibitory function. The combination of PMA and M-CSF induced the differentiation of myeloid-derived monocytes into MDSCs. MDSCs were found to induce immune tolerance by inhibiting the proliferation and activation of T cells, promoting cytokine secretion and inducing T cell transformation to regulatory T cells (T ). PMA significantly up-regulated the expression of Arg-1 and the Arg-1 protein expression in MDSCs and arginase 1 (Arg-1) inhibitor nor-NOHA reversed the MDSC immunosuppressive activity, indicating the involvement of the Arg-1 pathway in MDSC-mediated immunosuppression. M-CSF + PMA-induced MDSCs also significantly prolonged the survival time of skin grafts in mice, showing that MDSCs exert immunosuppressive effects in vivo. We describe a novel scheme to induce immunosuppressive MDSCs in vitro. MDSCs induced by M-CSF with PMA showed stable immunosuppression. MDSCs induced by this protocol may benefit patients with organ transplantation through immune regulation.
Topics: Animals; Cell Differentiation; Cell Proliferation; Immune Tolerance; Mice; Monocytes; Myeloid-Derived Suppressor Cells; T-Lymphocytes, Regulatory; Tetradecanoylphorbol Acetate
PubMed: 34453319
DOI: 10.1111/cei.13657 -
Bulletin of Experimental Biology and... Dec 2023NETosis, i.e., the formation of neutrophil extracellular traps (NET), and neutrophil autophagy are important elements in the pathogenesis and the development of...
NETosis, i.e., the formation of neutrophil extracellular traps (NET), and neutrophil autophagy are important elements in the pathogenesis and the development of complications of type 2 diabetes mellitus (T2DM). Therefore, the search of drugs that can regulate the level of NETosis and autophagy in T2DM is relevant. Here we studied an ex vivo NET formation and neutrophil death in whole blood from healthy subjects upon the addition of glucose up to a high concentration of 15 mM or/and the phorbol ester PMA (phorbol-12-myristate-13-acetate). Their individual and combined action caused neutrophil death and an increase in NET content. It can be hypothesized that this resulted from activation of NETosis and autophagy. It was also shown that this activation of NETosis and autophagy is completely prevented by daily intake of 1000 IU vitamin D3 for 14 days. Therefore, vitamin D3 supplementation can be considered as a preventive measure against the development of T2DM complications.
Topics: Humans; Extracellular Traps; Diabetes Mellitus, Type 2; Neutrophils; Glucose
PubMed: 38189871
DOI: 10.1007/s10517-024-05983-7