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Cytometry. Part a : the Journal of the... Feb 2023This 25-parameter, 22-color full spectrum flow cytometry panel was designed and optimized for the comprehensive enumeration and functional characterization of innate...
This 25-parameter, 22-color full spectrum flow cytometry panel was designed and optimized for the comprehensive enumeration and functional characterization of innate lymphoid cell (ILC) subsets in mouse tissues. The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR ILC3, NCR ILC3, CCR6 lymphoid tissue-inducer (LTi)-like ILC3 and mature natural killer (NK) cell populations. Further characterization of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimization of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin, and pro-ILC activating cytokines.
Topics: Mice; Animals; Lymphocytes; Immunity, Innate; Immunophenotyping; Flow Cytometry; Killer Cells, Natural; Cytokines
PubMed: 36331092
DOI: 10.1002/cyto.a.24702 -
Biomedicine & Pharmacotherapy =... Oct 2019Enterovirus 71 (EV71) brainstem encephalitis (BE) is divided into-uncomplicated BE, autonomic nervous system (ANS) dysregulation, and pulmonary edema (PE)-based on...
Enterovirus 71 (EV71) brainstem encephalitis (BE) is divided into-uncomplicated BE, autonomic nervous system (ANS) dysregulation, and pulmonary edema (PE)-based on cytokine-mediated severe systemic and central nervous system (CNS) inflammatory responses. Minocycline has been found to have anti-inflammatory and immunomodulatory properties in infectious and inflammatory neurological disease models. The effects of minocycline on EV71 infection were studied in vitro and in vivo experiments. The minocycline treatment (100-300 μg/mL) on cytokine expressions and viral replications were investigated in rhabdomyosarcoma (RD), U-87MG, and THP-1 cells. The mouse-adapted-EV71 strain (MP4)-infected 7-day-old ICR mice model was used to explore the anti-inflammatory and antiviral effects of minocycline (1 and 5 μg/g) for the treatment of EV71 infection. In in vitro, minocycline reduced cytopathic effects (CPEs), viral protein expressions, viral titers, the levels of interleukin (IL)-6 and IL-8 and relative mRNA expressions of IL-12p40, IL-1β, and tumor necrosis factor (TNF) after EV71 infection. The levels of TNF, IL-1β, IL-6, and IL-8 decreased with a single dose of minocycline in EV71-infected THP-1 cells. Double-dose minocycline treatment demonstrated more effective reduction in cytokines. In the MP4-infected animal model, clinical scores, mortality rates and viral titers in various brain tissues were decreased evidently after double-dose minocycline treatment. Minocycline inhibited IL-6 and granulocyte colony-stimulating factor (G-CSF) in plasma and TNF in the cerebellum. Minocycline has properties that enable it to function both as an anti-inflammatory and antiviral agent in EV71 infection. These results evidence its potential usefulness in clinical treatment.
Topics: Animals; Anti-Inflammatory Agents; Antiviral Agents; Brain; Cell Line, Tumor; Cytokines; Cytopathogenic Effect, Viral; Disease Models, Animal; Enterovirus A, Human; Humans; Mice, Inbred ICR; Minocycline; RNA, Messenger; RNA, Viral; Viral Load; Viral Proteins; Virus Replication
PubMed: 31377467
DOI: 10.1016/j.biopha.2019.109271 -
Scientific Reports Jul 2022Sirtuin 6 (SIRT6) regulation is involved in carcinogenesis. However, its role in breast cancer (BC) metastasis remains unclear. We investigated the effects of SIRT6 on...
Sirtuin 6 (SIRT6) regulation is involved in carcinogenesis. However, its role in breast cancer (BC) metastasis remains unclear. We investigated the effects of SIRT6 on protein kinase C activator- and cytokine-mediated cancer cell invasion and migration in MCF-7 and MDA-MB-231 cells and the association between SIRT6 and matrix metalloproteinase-9 (MMP-9) expression. To assess MMP-9 and SIRT6 expression in patients, protein levels in BC tissues were analyzed. MCF-7 and MDA-MB-231 cell viability was analyzed using MTT assays. SIRT6 was silenced in both cell lines and protein secretion, expression, and mRNA levels were analyzed. Transcription factor DNA activity was investigated using luciferase assays. Matrigel invasion assays were used to assess the effects of SIRT6 in both cell lines. SIRT6 and MMP-9 expression in cancer tissues was significantly higher than in paired normal breast tissues. 12-O-tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-α (TNF-α) increased MMP-9 expression and cell invasion and migration, but SIRT6 knockdown abolished these effects. SIRT6 overexpression additively increased TPA- and TNF-α-induced MMP-9 expression. SIRT6 knockdown suppressed the mitogen-activated protein kinase (MAPK) signaling pathway and thus TPA- and TNF-α-induced MMP-9 expression. SIRT6 silencing suppressed TPA- and TNF-α-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) expressions in both cell lines, and treatment with MAPK, NF-κB, and AP-1 inhibitors reduced MMP-9 expression. The anti-invasive effects of SIRT6 in BC cells might be mediated by suppression of MAPK phosphorylation and reduction in NF-κB and AP-1 DNA activities, leading to MMP-9 downregulation, suggesting that SIRT6 modulation has the potential to target BC metastasis.
Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; MCF-7 Cells; Matrix Metalloproteinase 9; NF-kappa B; Neoplasm Invasiveness; Sirtuins; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Tumor Necrosis Factor-alpha
PubMed: 35840633
DOI: 10.1038/s41598-022-16405-x -
Journal of Veterinary Diagnostic... Sep 2021Canine parvoviral enteritis (CPE) is a severe disease characterized by systemic inflammation and immunosuppression. The function of circulating phagocytes (neutrophils...
Canine parvoviral enteritis (CPE) is a severe disease characterized by systemic inflammation and immunosuppression. The function of circulating phagocytes (neutrophils and monocytes) in affected dogs has not been fully investigated. We characterized the functional capacity of canine phagocytes in CPE by determining their oxidative burst and phagocytic activities using flow cytometry. Blood was collected from 28 dogs with CPE and 11 healthy, age-matched, control dogs. Oxidative burst activity was assessed by stimulating phagocytes with opsonized or phorbol 12-myristate 13-acetate (PMA) and measuring the percentage of phagocytes producing reactive oxygen species and the magnitude of this production. Phagocytosis was measured by incubating phagocytes with opsonized and measuring the percentage of phagocytes containing and the number of bacteria per cell. Complete blood counts and serum C-reactive protein (CRP) concentrations were also determined. Serum CRP concentration was negatively and positively correlated with segmented and band neutrophil concentrations, respectively. Overall, no differences in phagocyte function were found between dogs with CPE and healthy control dogs. However, infected dogs with neutropenia or circulating band neutrophils had decreased PMA-stimulated oxidative burst activity compared to healthy controls. Additionally, CPE dogs with neutropenia or circulating band neutrophils had decreased PMA- and -stimulated oxidative burst activity and decreased phagocytosis of compared to CPE dogs without neutropenia or band neutrophils. We conclude that phagocytes have decreased oxidative burst and phagocytic activity in neutropenic CPE dogs and in CPE dogs with circulating band neutrophils.
Topics: Animals; Dog Diseases; Dogs; Enteritis; Escherichia coli; Neutrophils; Parvovirus, Canine; Phagocytes; Respiratory Burst
PubMed: 34148453
DOI: 10.1177/10406387211025513 -
Frontiers in Immunology 2022Although recent studies have highlighted the link of TIPE2 and asthma airway inflammation, its roles and molecular mechanisms in different asthma inflammatory phenotypes...
PURPOSE
Although recent studies have highlighted the link of TIPE2 and asthma airway inflammation, its roles and molecular mechanisms in different asthma inflammatory phenotypes remain largely unknown. We evaluated sputum TIPE2 expression level and its correlation with different asthma phenotypes. Additionally, we explored the roles and mechanism of TIPE2 in M1 polarization of macrophages.
METHODS
A total of 102 asthma patients who underwent sputum induction were enrolled to evaluate the expression level of TIPE2 and its association with different asthma phenotypes. To explore the roles and mechanism of TIPE2 in M1 polarization of macrophages, THP-1 monocytes stimulated with phorbol-12-myristate-13-acetate, were used as a model of undifferentiated (M0) macrophages, and M0 macrophages were treated with lipopolysaccharide to induce M1 macrophages.
RESULTS
The sputum TIPE2 level was significantly lower in patients with neutrophilic asthma (NA) and higher in patients with eosinophilic asthma (EA) compared with patients with paucigranulocytic asthma. The levels of IL-1β, TNF-α and IL-6 were highest in NA compared with other groups. TIPE2 levels in sputum negatively correlated with IL-1β and TNF-α levels but positively correlated with IL-4, IL-5, IL-13, and IL-10 levels ( < 0.05). , TIPE2 enhanced Nrf2/HO-1 pathway activation in macrophages and inhibited LPS-induced M1 macrophage differentiation and related cytokine release. Further analysis showed that the Nrf2 inhibitor ML385 weakened TIPE2-induced activation of the Nrf2/HO-1 pathway, as well as TIPE2-induced suppression in M1 polarization of macrophage and inflammatory cytokines secretion.
CONCLUSIONS
TIPE2 expression level was highly down-regulated in NA and was negatively correlated with inflammatory factors (IL-1β and TNF-α). Aberrant expression of TIPE2 may target the Nrf2/HO-1 pathway to inhibit M1 macrophage-related neutrophilic inflammation in asthma.
Topics: Asthma; Heme Oxygenase-1; Humans; Inflammation; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; Macrophages; NF-E2-Related Factor 2; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha
PubMed: 35572500
DOI: 10.3389/fimmu.2022.883885 -
Blood Advances Oct 2023Activated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell's DNA covered with granule-derived antimicrobial...
Activated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell's DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.
Topics: Humans; Extracellular Traps; Neutrophils; Histones; DNA; Cell Membrane
PubMed: 37428870
DOI: 10.1182/bloodadvances.2022009432 -
Frontiers in Immunology 2021We collected peripheral blood from thirty-nine elite male endurance runners at rest (24 hours after the last exercise session) and used the Allergy Questionnaire for... (Comparative Study)
Comparative Study
We collected peripheral blood from thirty-nine elite male endurance runners at rest (24 hours after the last exercise session) and used the Allergy Questionnaire for Athletes score and plasma specific IgE level to separate them into atopic and non-atopic athletes. Neutrophils obtained from atopic and non-atopic athletes were subsequently stimulated with fMLP (N-formyl-methionyl-leucyl-phenylalanine), LPS (lipopolysaccharide), or PMA (phorbol 12-myristate 13-acetate). Neutrophils from non-atopic runners responded appropriately to LPS, as evidenced by the production of pro (IL-8, TNF-α, and IL-6) and anti-inflammatory (IL-10) cytokines. Neutrophils from atopic elite runners exhibited lower responses to LPS stimulus as indicated by no increase in IL-1β, TNF-α, and IL-6 production. Neutrophils from non-atopic and atopic runners responded similarly to fMLP stimulation, indicating that migration function remained unaltered. Both groups were unresponsive to PMA induced reactive oxygen species (ROS) production. Training hours and training volume were not associated with neutrophil IgE receptor gene expression or any evaluated neutrophil function. Since non-atopic runners normally responded to LPS stimulation, the reduced neutrophil response to the stimuli was most likely due to the atopic state and not exercise training. The findings reported are of clinical relevance because atopic runners exhibit a constant decline in competition performance and are more susceptible to invading microorganisms.
Topics: Adult; Cells, Cultured; Cytokines; Disease Susceptibility; Humans; Hypersensitivity, Immediate; Immunoglobulin E; Infections; Lipopolysaccharides; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Physical Endurance; Running; Surveys and Questionnaires
PubMed: 34177910
DOI: 10.3389/fimmu.2021.670763 -
The Biochemical Journal Aug 2022The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations;...
The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell-cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAFV600E or NRASQ61K mutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC-PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer.
Topics: Humans; Melanoma; Phorbol Esters; Phosphorylation; Protein Kinases; p21-Activated Kinases
PubMed: 35969127
DOI: 10.1042/BCJ20220184 -
Frontiers in Immunology 2022Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune...
Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.
Topics: Animals; Antibodies, Monoclonal; Cattle; Cross Reactions; Dolphins; Enzyme-Linked Immunosorbent Assay; Goats; Interferon-gamma; Interleukin-17; Ionomycin; Leukocytes, Mononuclear; Recombinant Proteins; Swine; T-Lymphocytes; Tetradecanoylphorbol Acetate; Zebrafish
PubMed: 35185884
DOI: 10.3389/fimmu.2022.786396 -
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