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Journal of Visualized Experiments : JoVE May 2021Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form...
Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.
Topics: Actin Cytoskeleton; Cell Membrane; Cell Surface Extensions; Microscopy, Electron, Scanning; Pinocytosis
PubMed: 34125102
DOI: 10.3791/62658 -
International Journal of Molecular... Jul 2022Reactive oxygen species (ROS) represent a group of molecules with a signaling role that are involved in regulating human cell proliferation and differentiation....
Reactive oxygen species (ROS) represent a group of molecules with a signaling role that are involved in regulating human cell proliferation and differentiation. Increased ROS concentrations are often associated with the local nonspecific oxidation of biological macromolecules, especially proteins and lipids. Free radicals, in general, may randomly damage protein molecules through the formation of protein-centered radicals as intermediates that, in turn, decay into several end oxidation products. Malondialdehyde (MDA), a marker of free-radical-mediated lipid oxidation and cell membrane damage, forms adducts with proteins in a nonspecific manner, leading to the loss of their function. In our study, we utilized U-937 cells as a model system to unveil the effect of four selected bioactive compounds (chlorogenic acid, oleuropein, tomatine, and tyrosol) to reduce oxidative stress associated with adduct formation in differentiating cells. The purity of the compounds under study was confirmed by an HPLC analysis. The cellular integrity and changes in the morphology of differentiated U-937 cells were confirmed with confocal microscopy, and no significant toxicity was found in the presence of bioactive compounds. From the Western blot analysis, a reduction in the MDA adduct formation was observed in cells treated with compounds that underlaid the beneficial effects of the compounds tested.
Topics: Free Radicals; Humans; Malondialdehyde; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species
PubMed: 35806429
DOI: 10.3390/ijms23137424 -
Translational Oncology Jan 2021Human stanniocalcin-1 (STC1) is a paracrine factor associated with inflammation and carcinogenesis. The role of STC1 in the pro- and anti-inflammatory functions of...
Human stanniocalcin-1 (STC1) is a paracrine factor associated with inflammation and carcinogenesis. The role of STC1 in the pro- and anti-inflammatory functions of differentiating macrophage, however, is not clear. In this study, our data showed that phorbol 12-myristate 13-acetate (PMA) treatment induced human leukemia monocytic cells (ThP-1) differentiation to M0 macrophages. The differentiation was accompanied by a significant increase in the mRNA expression levels of STC1, the pro-inflammatory cytokine TNFα, and anti-inflammatory markers, CD163 & CD206. An intermitted removal of PMA treatment reduced the mRNA levels of STC1 and TNFα but had no noticeable effects on the anti-inflammatory markers. The correlation in the expression of STC1 and pro-inflammatory markers in differentiating macrophages was investigated, using siRNA-transfected PMA-induced cells. Consistently, the transcripts levels of TNFα and IL-6 were significantly reduced. Moreover, LPS/IFNγ-induced M1-polarization showed remarkably higher expression levels of STC1 than IL-4/IL-13-induced M2-macrophages and PMA-induced M0-macrophages. Transcriptomic analysis of siRNA-transfected M1-polarized cells revealed an upregulation of TBC1 domain family member 3 (TBC1D3G). The gene regulates the payload of macrophage-released extracellular vesicles to mediate inflammation. The conditioned media from siRNA-transfected M1-polarized cells were found to reduce Hep3B cell motility. The data suggest that the expression of STC1 were associated with macrophage differentiation, but preferentially to M1 polarization.
PubMed: 33074126
DOI: 10.1016/j.tranon.2020.100881 -
Scientific Reports Feb 2023Macrophages (MQs) pro-inflammatory phenotype is triggered by gliadin peptides. Akkermansia muciniphila (A. muciniphila) showed to enhance the anti-inflammatory phenotype...
Macrophages (MQs) pro-inflammatory phenotype is triggered by gliadin peptides. Akkermansia muciniphila (A. muciniphila) showed to enhance the anti-inflammatory phenotype of MQs. This study aimed to investigate the anti-inflammatory effects of A. muciniphila, on gliadin stimulated THP-1 derived macrophages. THP-1 cell line monocytes were differentiated into MQs by phorbol 12-myristate 13-acetate (PMA). MQs were treated with A. muciniphila before and after stimulation with gliadin (pre- and post-treat). CD11b, as a marker of macrophage differentiation, and CD206 and CD80, as M1 and M2 markers, were evaluated by flow cytometry technique. The mRNA expression of TGF-β, IL-6, and IL-10 and protein levels of IL-10 and TNF-α were measured by RT-PCR and ELISA techniques, respectively. Results show an increased percentage of M1 phenotype and release of proinflammatory cytokines (like TNF-α and IL-6) by macrophages upon incubation with gliadin. Pre- and post-treatment of gliadin-stimulated macrophages with A. muciniphila induced M2 phenotype associated with decreased proinflammatory (IL-6, TNF-α) and increased anti-inflammatory (IL-10, TGF-β) cytokines expression relative to the group that was treated with gliadin alone. This study suggests the potential beneficial effect of A. muciniphila on gliadin-stimulated MQs and the importance of future studies focusing on their exact mechanism of action on these cells.
Topics: Interleukin-10; Gliadin; Tumor Necrosis Factor-alpha; Interleukin-6; Macrophages; Cytokines; Anti-Inflammatory Agents; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta
PubMed: 36828897
DOI: 10.1038/s41598-023-30266-y -
Biochemistry Apr 2021Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been...
Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain. The objective of this study is to identify the structural determinants of ligand binding activity of the Munc13-1 C1 domain. Molecular docking suggested that residues Trp-588, Ile-590, and Arg-592 of Munc13-1 are involved in ligand interactions. To elucidate the role of these three residues in ligand binding, we generated W588A, I590A, and R592A mutants in full-length Munc13-1, expressed them as GFP-tagged proteins in HT22 cells, and measured their ligand-induced membrane translocation by confocal microscopy and immunoblotting. The extent of 1,2-dioctanoyl--glycerol (DOG)- and phorbol ester-induced membrane translocation decreased in the following order: wild type > I590A > W588A > R592A and wild type > W588A > I590A > R592A, respectively. To understand the effect of the mutations on ligand binding, we also measured the DOG binding affinity of the isolated wild-type C1 domain and its mutants in membrane-mimicking micelles using nuclear magnetic resonance methods. The DOG binding affinity decreased in the following order: wild type > I590A > R592A. No binding was detected for W588A with DOG in micelles. This study shows that Trp-588, Ile-590, and Arg-592 are essential determinants for the activity of Munc13-1 and the effects of the three residues on the activity are ligand-dependent. This study bears significance for the development of selective modulators of Munc13-1.
Topics: Binding Sites; Cell Line; Diglycerides; Humans; Models, Molecular; Nerve Tissue Proteins; Protein Binding; Protein Conformation
PubMed: 33818064
DOI: 10.1021/acs.biochem.1c00165 -
Veterinary Sciences Sep 2021L. has gained importance as a source of seed oil for biodiesel production. The meal contained about 60% protein with a good balance of essential amino acids, containing... (Review)
Review
L. has gained importance as a source of seed oil for biodiesel production. The meal contained about 60% protein with a good balance of essential amino acids, containing various bioactive compounds, including saponins, phytic acids, trypsin inhibitors, lectins, phenolics, and flavonoids, which render it as a potential biofeed for animal production. The meal demonstrated various biological activities, including antioxidant, antibacterial, and anti-inflammatory effects which enhance its property as a bio-feed. The levels of these bioactive compounds in the seeds are dependent on the genotypes. The possessed different varieties which are either toxic or non-toxic according to the presence of phorbol esters. The presence of phorbol esters in the meal confirmed the toxic variety of resulting in the limited application of meal as a biofeed. The meal devoid of phorbol esters could be applied as a biofeed in the animal production industry, and for the toxic varieties, various techniques such as physicochemical and biological treatments have been introduced to the industry to remove the phorbol esters from meal. Several studies employing various cells and animals confirmed the toxicity of the phorbol esters. The molecular mechanism of action of phorbol esters is through up-regulation of PKC-β II gene, overexpression of down-stream proto-oncogenes resulted in inflammation and oxidative stress ending by apoptotic cell death. Despite the presence of valuable bioactive compounds in the meal, its nutritional application is not recommended unless the phorbol esters are completely removed.
PubMed: 34564573
DOI: 10.3390/vetsci8090179 -
Clinical Interventions in Aging 2022The aim of the paper is to establish and quantify the relation between healthy ageing and the innate and adaptive immune parameters as indicators of age-related diseases.
PURPOSE
The aim of the paper is to establish and quantify the relation between healthy ageing and the innate and adaptive immune parameters as indicators of age-related diseases.
PATIENTS
In order to observe the immunological changes that occur according to age, several humoral and cellular immune parameters were investigated for 288 healthy donors (30-80 years). Subjects' selection was done using clinical, biochemical and immunological parameters of inclusion/exclusion criteria from SENIEUR protocol.
RESULTS
Age-related changes were observed for both humoral and cellular immune parameters. Lymphocyte immunophenotyping revealed several significant differences in the distribution of cells, both intra- and inter-age groups, namely decreased values of T-CD3, T-CD8 and NK cells, and elevated values for T-CD4, T-CD4/T-CD8 ratio and B cells. The percentages of unstimulated neutrophils that show basal oxidative activity and the intensity of this activity had an increasing tendency age-related. The percentage of N-Formyl-Methionyl-Leucyl-Phenylalanine stimulated neutrophils clearly decreases with age, and is associated with an increasing intensity of oxidative activity. Our data also have shown an increased percentage of oxidative neutrophils after phorbol 12-myristate 13-acetate stimulation and an elevated oxidative activity with age.
CONCLUSION
Overall healthy ageing is governed by some immune-related deregulations that account for immune exhaustion due to numerous developed immune processes during a life-time and the age-related diseases.
Topics: Acetates; Aged; Aged, 80 and over; Healthy Aging; Humans; Myristates; N-Formylmethionine Leucyl-Phenylalanine; Tetradecanoylphorbol Acetate
PubMed: 36247200
DOI: 10.2147/CIA.S375926 -
Frontiers in Immunology 2020Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein (NLRP) inflammasomes are involved in the molecular pathogenesis of many... (Review)
Review
Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein (NLRP) inflammasomes are involved in the molecular pathogenesis of many diseases and disorders. Among NLRPs, the NLRP3 (in humans encoded by the gene) is expressed predominantly in macrophages as a component of the inflammasome and is associated with many diseases, including gout, type 2 diabetes, multiple sclerosis, atherosclerosis, and neurological diseases and disorders. Diterpenes containing repeated isoprenoid units in their structure are a member of some essential oils that possess diverse biological activities and are becoming a landmark in the field of drug discovery and development. This review sketches a current scenario of diterpenes or their derivatives acting through NLRPs, especially NLRP3-associated pathways with anti-inflammatory effects. For this, a literature survey on the subject has been undertaken using a number of known databases with specific keywords. Findings from the aforementioned databases suggest that diterpenes and their derivatives can exert anti-inflammatory effects NLRPs-related pathways. Andrographolide, triptolide, kaurenoic acid, carnosic acid, oridonin, teuvincenone F, and some derivatives of tanshinone IIA and phorbol have been found to act through NLRP3 inflammasome pathways. In conclusion, diterpenes and their derivatives could be one of the promising compounds for the treatment of NLRP3-mediated inflammatory diseases and disorders.
Topics: Animals; Diterpenes; Humans; Immunomodulation; Inflammasomes; Inflammation; Mitochondria; NLR Family, Pyrin Domain-Containing 3 Protein; Signal Transduction
PubMed: 33101293
DOI: 10.3389/fimmu.2020.572136 -
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 -
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