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Arthritis Research & Therapy Aug 2019The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level. (Comparative Study)
Comparative Study
BACKGROUND
The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level.
METHODS
Peripheral blood mononuclear cells from healthy donors were incubated with different JAKis, levels of phosphorylated signal transducer and activator of transcription (pSTAT) were measured following cytokine stimulation, and half maximum inhibitory concentration (IC) values were calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of the in vitro analysis was assessed using calculated mean concentration-time profiles over 24 h obtained from JAKi-treated subjects. Time above IC and average daily percent inhibition of pSTAT formation were calculated for each JAKi, cytokine, and cell type.
RESULTS
Distinct JAKis displayed different in vitro pharmacologic profiles. For example, tofacitinib and upadacitinib were the most potent inhibitors of the JAK1/3-dependent cytokines tested (interleukin [IL]-2, IL-4, IL-15, and IL-21) with lower IC values and increased time above IC translating to a greater overall inhibition of STAT signaling during the dosing interval. All JAKis tested inhibited JAK1/2-dependent cytokines (e.g., IL-6 and interferon [IFN]-γ), the JAK1/tyrosine kinase 2 (TYK2)-dependent cytokines IL-10 and IFN-α, the JAK2/2-dependent cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the JAK2/TYK2-dependent cytokine granulocyte colony-stimulating factor (G-CSF), but often to significantly differing degrees.
CONCLUSIONS
Different JAKis modulated distinct cytokine pathways to varying degrees, and no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Notably, baricitinib inhibited JAK1/3 signaling to a lesser extent than upadacitinib and tofacitinib, while upadacitinib, baricitinib, and tofacitinib inhibited the signaling of JAK2/2-dependent cytokines, including GM-CSF and IL-3, as well as the signaling of the JAK2/TYK2-dependent cytokine G-CSF.
Topics: Arthritis, Rheumatoid; Azetidines; Biomarkers; Cytokines; Flow Cytometry; Heterocyclic Compounds, 3-Ring; Humans; Janus Kinase Inhibitors; Leukocytes, Mononuclear; Piperidines; Protein Kinase Inhibitors; Purines; Pyrazoles; Pyrimidines; Pyrroles; Signal Transduction; Sulfonamides
PubMed: 31375130
DOI: 10.1186/s13075-019-1964-1 -
In Vivo (Athens, Greece) 2022Although taurolidine is known to exert a wide spectrum of biological actions, its effects on immune cells have not been characterized in detail. In this study, we...
BACKGROUND/AIM
Although taurolidine is known to exert a wide spectrum of biological actions, its effects on immune cells have not been characterized in detail. In this study, we investigated the ex vivo effects of taurolidine on relevant innate and adaptive immune cell functions.
MATERIALS AND METHODS
Leukocyte functions in whole blood were assessed following treatment with various taurolidine concentrations. Viability of peripheral blood mononuclear cells (PBMCs) and granulocytes was measured using the WST-1 assay. PBMC function was assessed by measuring TNFα and IFNγ production after stimulation with lipopolysaccharide (LPS) or Candida, respectively. Reactive oxygen species (ROS) production by granulocytes was measured in whole blood using luminol-enhanced chemiluminescence. Granulocyte degranulation and activation were evaluated by membrane expression of degranulation (CD63, CD66B) and adhesion markers (CD62L, CD11b) using immunofluorescent staining followed by flow-cytometric analysis.
RESULTS
Taurolidine decreased viability of PBMCs and granulocytes: after 2 h, IC concentrations were 500 and 520 μg/ml, respectively. Following prolonged exposure (≥24 h) of PBMCs, the IC concentrations declined to 40 μg/ml. PBMC cytokine production significantly decreased at taurolidine concentrations below the cytotoxic threshold, whereas no changes in ROS production were observed. The expression of all granulocyte adhesion and degranulation markers increased at concentrations higher than 500 μg/ml (the cytotoxic level of taurolidine).
CONCLUSION
Taurolidine exhibits a dose- and time-dependent cytotoxicity toward PBMCs and granulocytes. The effects on PBMCs, as exemplified by a decrease in cytokine production, occurred below the toxic threshold, whereas granulocyte function (ROS production) remained unchanged at these taurolidine concentrations. Granulocyte activation and degranulation markers only increased at cytotoxic taurolidine concentrations.
Topics: Anti-Infective Agents, Local; Antineoplastic Agents; Cytokines; Leukocytes, Mononuclear; Reactive Oxygen Species; Taurine; Thiadiazines
PubMed: 36099103
DOI: 10.21873/invivo.12933 -
Obstetrics and Gynecology Apr 2013To estimate the associations of change in immune response with preterm delivery, omega-3 supplementation, and fish diet. (Randomized Controlled Trial)
Randomized Controlled Trial
OBJECTIVE
To estimate the associations of change in immune response with preterm delivery, omega-3 supplementation, and fish diet.
METHODS
This was an ancillary study to a randomized trial of omega-3 fatty acid supplementation for the prevention of recurrent preterm birth. In vitro maternal peripheral blood mononuclear leukocyte production of the anti-inflammatory cytokine, interleukin-10, and the proinflammatory cytokine, tumor necrosis factor-α, in response to stimulation with lipopolysaccharide, was measured at 16-22 weeks of gestation (baseline) and again at 25-28 weeks of gestation (follow-up) among women with prior spontaneous preterm birth. Changes in concentrations from baseline to follow-up ([INCREMENT]) were compared separately among groups defined by gestational age category at delivery, fish diet history, and omega-3 compared with placebo treatment assignment with Kruskal-Wallis tests.
RESULTS
Interleukin-10 [INCREMENT] differed by gestational age category among 292 women with paired assays. Concentrations increased less in women delivering between 35 and 36 6/7 weeks of gestation (48.9 pg/mL) compared with women delivering at term (159.3 pg/mL) and decreased by 65.2 pg/mL in women delivering before 35 weeks of gestation (P=.01). Tumor necrosis factor-α Δ also differed by gestational age category among 319 women, but the pattern was inconsistent. Those delivering between 35 and 36 6/7 weeks of gestation exhibited decreased concentrations of tumor necrosis factor-α at follow-up compared with baseline (-356.0 pg/mL); concentrations increased among women delivering before 35 weeks of gestation and those delivering at term, 132.1 and 86.9 pg/mL (P=.03). Interleukin-10 Δ and tumor necrosis factor-α Δ were unaffected by either omega-3 supplementation or fish diet.
CONCLUSION
Recurrent preterm birth was associated with decreased peripheral blood mononuclear leukocyte production of interleukin-10 in response to a stimulus during the second trimester.
CLINICAL TRIAL REGISTRATION
ClinicalTrials.gov, www.clinicaltrials.gov, NCT00135902.
LEVEL OF EVIDENCE
II.
Topics: Adult; Animals; Dietary Supplements; Fatty Acids, Omega-3; Female; Fishes; Humans; Interleukin-6; Leukocytes, Mononuclear; Pregnancy; Pregnancy Trimester, Second; Premature Birth; Tumor Necrosis Factor-alpha; Young Adult
PubMed: 23635681
DOI: 10.1097/AOG.0b013e3182878a80 -
Frontiers in Immunology 2022Ankylosing spondylitis (AS) is a chronic inflammatory disease with serious consequences and a high rate of morbidity and mortality, In our previous work, we reveal the...
BACKGROUND
Ankylosing spondylitis (AS) is a chronic inflammatory disease with serious consequences and a high rate of morbidity and mortality, In our previous work, we reveal the key features of proteins in new-onset ankylosing spondylitis patients.
MATERIAL AND METHODS
Ankylosing spondylitis (AS) is a chronic inflammatory condition that affects the spine, and inflammation plays an essential role in AS pathogenesis. The inflammatory process in AS, however, is still poorly understood due to its intricacy. Systematic proteomic and phosphorylation analyses of peripheral blood mononuclear cells (PBMCs) were used to investigate potential pathways involved in AS pathogenesis.
RESULTS
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed and discovered 782 differentially expressed proteins (DEPs) and 122 differentially phosphorylated proteins (DPPs) between 9 new-onset AS patients and 9 healthy controls. The DEPs were further verified using parallel reaction monitoring (PRM) analysis. PRM analysis verified that 3 proteins (HSP90AB1, HSP90AA1 and HSPA8) in the antigen processing and presentation pathway, 6 proteins (including ITPR1, MYLK and STIM1) in the platelet activation pathway and 10 proteins (including MYL12A, MYL9 and ROCK2) in the leukocyte transendothelial migration pathway were highly expressed in the PBMCs of AS patients.
CONCLUSION
The key proteins involved in antigen processing and presentation, platelet activation and leukocyte transendothelial migration revealed abnormal immune regulation in patients with new-onset AS. These proteins might be used as candidate markers for AS diagnosis and new therapeutic targets, as well as elucidating the pathophysiology of AS.
Topics: Chromatography, Liquid; Humans; Leukocytes, Mononuclear; Proteomics; Spondylitis, Ankylosing; Tandem Mass Spectrometry
PubMed: 35371008
DOI: 10.3389/fimmu.2022.838891 -
Redox Report : Communications in Free... Jul 2015Prolidase plays a major role in collagen turnover, matrix remodeling, and cell growth. Benign prostatic hyperplasia (BPH) may be associated with an increased...
OBJECTIVES
Prolidase plays a major role in collagen turnover, matrix remodeling, and cell growth. Benign prostatic hyperplasia (BPH) may be associated with an increased extracellular matrix deposition. Therefore, the present study was designed to investigate the plasma prolidase activity, oxidative status, and peripheral mononuclear leukocyte DNA damage in patients with BPH.
PATIENTS AND METHODS
Twenty-six male patients with BPH and 24 healthy male subjects were included in this study. Blood samples were collected from antecubital vein after an overnight fasting period, and the plasma was separated. Plasma prolidase activity, total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) were determined. The peripheral lymphocyte oxidative DNA damage was determined using an alkaline single cell gel electrophoresis assay (comet assay).
RESULTS
The plasma prolidase activity, TOS levels, OSI values, and peripheral mononuclear leukocyte DNA damage were significantly higher (P < 0.001), while the TAC levels were significantly lower (P < 0.001) in patients with BPH than controls. In BPH patients, the prolidase activity was significantly associated with TAC levels (r = -0.366, P < 0.05), TOS levels (r = 0.573, P < 0.001), and OSI (r = 0.618, P < 0.001) and peripheral mononuclear leukocyte DNA damage (r = 0.461, P < 0.001).
CONCLUSIONS
Our results showed that BPH might be associated with an increased oxidative stress, and also an increased plasma prolidase activity. Increased prolidase activity might play an important role in the etiopathogenesis and/or progression of BPH.
Topics: Antioxidants; Collagen; Comet Assay; DNA Damage; Dipeptidases; Extracellular Matrix; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Oxidants; Oxidative Stress; Prospective Studies; Prostatic Hyperplasia
PubMed: 25551736
DOI: 10.1179/1351000214Y.0000000121 -
Clinical Rheumatology Dec 2022To seek significant features of systemic lupus erythematosus (SLE) by utilizing bioinformatics analysis.
INTRODUCTION/OBJECTIVES
To seek significant features of systemic lupus erythematosus (SLE) by utilizing bioinformatics analysis.
METHOD
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify lysine crotonylation (Kcr) and lysine 2-hydroxyisobutyrylation (Khib) in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) patients and normal controls.
RESULTS
Seventy-six differentially modified proteins (DMPs) dually modified by Kcr and Khib were identified between SLE patients and healthy people. GO enrichment analysis prompted significant enrichment of seventy-six DMPs in MHC class II protein complex binding and leukocyte migration. KEGG pathways were enriched in antigen processing and presentation pathway and leukocyte transendothelial migration pathway. Six DMPs (CLTC, HSPA1B, HSPA8, HSP90AB1, HSPD1, and PDIA3) were identified in antigen processing and presentation pathway, of which HSPA8 was the core protein. Significant changes of Kcr and Khib in HSPA8 may increase ATP hydrolysis and promote antigen binding to MHC II molecule. In leukocyte transendothelial migration pathway, 7 DMPs (ACTN1, ACTN4, EZR, MSN, RAC1, RHOA, and VCL) were identified. MSN was the protein with the most modification sites in this pathway. In amino terminal ferm region of MSN, Kcr and Khib expression change may lead to the adhesion between leukocytes and endothelial cells, which was an important step of leukocyte migration.
CONCLUSION
Kcr and Khib may promote the antigen presentation and jointly regulate the tissue damage mediated by leukocyte migration in SLE patients, which may play key roles in the pathogenesis of SLE probably. Key Points • Antigen processing and presentation and leukocyte transendothelial migration may play key roles in the pathogenesis of SLE.
Topics: Humans; Lysine; Chromatography, Liquid; Protein Processing, Post-Translational; Proteomics; Leukocytes, Mononuclear; Endothelial Cells; Tandem Mass Spectrometry; Lupus Erythematosus, Systemic
PubMed: 35941338
DOI: 10.1007/s10067-022-06254-4 -
BMC Molecular and Cell Biology Jul 2021Research on cell-in-cell (CIC) phenomena, including entosis, emperipolesis and cannibalism, and their biological implications has increased in recent years. Homotypic...
BACKGROUND
Research on cell-in-cell (CIC) phenomena, including entosis, emperipolesis and cannibalism, and their biological implications has increased in recent years. Homotypic and heterotypic engulfment of various target cells by numerous types of host cells has been studied in vitro and in tissue sections. This work has identified proteins involved in the mechanism and uncovered evidence for CIC as a potential histopathologic predictive and prognostic marker in cancer. Our experimental study focused on non-professional phagocytosis of leukocytes.
RESULTS
We studied the engulfment of peripheral blood mononuclear cells isolated from healthy donors by counting CIC structures. Two non-tumorigenic cell lines (BEAS-2B, SBLF-9) and two tumour cell lines (BxPC3, ICNI) served as host cells. Immune cells were live-stained and either directly co-incubated or treated with irradiation or with conventional or microwave hyperthermia. Prior to co-incubation, we determined leukocyte viability for each batch via Annexin V-FITC/propidium iodide staining. All host cells engulfed their targets, with uptake rates ranging from 1.0% ± 0.5% in BxPC3 to 8.1% ± 5.0% in BEAS-2B. Engulfment rates of the cancer cell lines BxPC3 and ICNI (1.6% ± 0.2%) were similar to those of the primary fibroblasts SBLF-9 (1.4% ± 0.2%). We found a significant negative correlation between leukocyte viability and cell-in-cell formation rates. The engulfment rate rose when we increased the dose of radiotherapy and prolonged the impact time. Further, microwave hyperthermia induced higher leukocyte uptake than conventional hyperthermia. Using fluorescent immunocytochemistry to descriptively study the proteins involved, we detected ring-like formations of diverse proteins around the leukocytes, consisting, among others, of α-tubulin, integrin, myosin, F-actin, and vinculin. These results suggest the involvement of actomyosin contraction, cell-cell adhesion, and the α-tubulin cytoskeleton in the engulfment process.
CONCLUSIONS
Both non-tumorigenic and cancer cells can form heterotypic CIC structures by engulfing leukocytes. Decreased viability and changes caused by microwave and X-ray irradiation trigger non-professional phagocytosis.
Topics: Cell Line; Cell Line, Tumor; Entosis; Fibroblasts; Humans; Leukocytes, Mononuclear; Neoplasms; Phagocytosis
PubMed: 34332531
DOI: 10.1186/s12860-021-00377-3 -
Veterinary Immunology and... May 2019In mammals, the immune system is undergoing significant changes during development, which has many impacts on the individual's capacity to cope with infectious diseases... (Review)
Review
In mammals, the immune system is undergoing significant changes during development, which has many impacts on the individual's capacity to cope with infectious diseases or other pathologic conditions, where the immune system is involved. Especially in livestock, it is important to know in detail about these changes, including shifts in the composition of systemic leukocyte populations, as this knowledge may help to focus on relevant cell populations when developing novel vaccines for use in juvenile versus adult animals. In this mini-review, a synoptic comparison of published PBMC populations, which were analysed in healthy weaned piglets as well as multiparous non-gestating sows, shows remarkable shifts within leukocyte populations. γδ T cells increase by factor 1.5, plasmacytoid dendritic cells and T helper cells more than double, and cytotoxic T cells as well as regulatory T cells increase more than four fold, whereas NK cells as well as B cells in adult sows comprise only 40% and monocytes 70% of the relative population sizes in weaned piglets. In summary, these insights into age-dependent shifts of porcine leukocyte populations indicate a principal increase of acquired immunity-associated leukocyte populations, whereas primarily innate immunity-associated cell types (NK cells, monocytes) are diminished.
Topics: Aging; Animals; Leukocytes, Mononuclear; Swine
PubMed: 31084891
DOI: 10.1016/j.vetimm.2019.04.002 -
Stem Cell Reports Apr 2021ESC- and iPSC-derived retinal transplantation is a promising therapeutic approach for disease with end-stage retinal degeneration, such as retinitis pigmentosa and...
ESC- and iPSC-derived retinal transplantation is a promising therapeutic approach for disease with end-stage retinal degeneration, such as retinitis pigmentosa and age-related macular degeneration. We previously showed medium- to long-term survival, maturation, and light response of transplanted human ESC- and iPSC-retina in mouse, rat, and monkey models of end-stage retinal degeneration. Because the use of patient hiPSC-derived retina with a disease-causing gene mutation is not appropriate for therapeutic use, allogeneic transplantation using retinal tissue/cells differentiated from a stocked hESC and iPSC line would be most practical. Here, we characterize the immunological properties of hESC- and iPSC-retina and present their three major advantages: (1) hESC- and iPSC-retina expressed low levels of human leukocyte antigen (HLA) class I and little HLA class II in vitro, (2) hESC- and iPSC-retina greatly suppressed immune activation of lymphocytes in co-culture, and (3) hESC- and iPSC-retina suppressed activated immune cells partially via transforming growth factor β signaling. These results support the use of allogeneic hESC- and iPSC-retina in future clinical application.
Topics: Animals; Cell Differentiation; Histocompatibility Antigens Class I; Human Embryonic Stem Cells; Humans; Immunomodulation; Immunosuppression Therapy; Induced Pluripotent Stem Cells; Interferon-gamma; Leukocytes, Mononuclear; Primates; Recombinant Proteins; Retina; Retinal Pigment Epithelium; Transforming Growth Factor beta
PubMed: 33770500
DOI: 10.1016/j.stemcr.2021.02.021 -
PloS One 2022Vitamin D deficiency is common among postmenopausal women. Telomere length can be a potential protective mechanism for age-related diseases. The objective of our study... (Randomized Controlled Trial)
Randomized Controlled Trial
Vitamin D deficiency is common among postmenopausal women. Telomere length can be a potential protective mechanism for age-related diseases. The objective of our study is to examine the association of vitamin D supplementation on leukocyte telomere length (LTL) in healthy postmenopausal women with vitamin D deficiency. The study was designed as a placebo-controlled study to investigate the short-term effects of vitamin D supplementation and seasonal changes on vitamin D related parameters, including 25(OH)D, 1,25(OH)2D parathormone (PTH), Vitamin D binding protein (VDBP), vitamin D receptor (VDR), and telomere length in a cohort of postmenopausal women (n = 102). The group was divided as supplementation (n = 52) and placebo groups (n = 50). All parameters were measured before and after treatment. Serum VDBP levels were measured by ELISA method and VDR, GC (VDBP) gene expressions and relative telomere lengths were measured in peripheral blood mononuclear cells (PBMC) using a quantitative real-time PCR method. The results demonstrate that baseline levels were similar between the groups. After vitamin D supplementation 25(OH)D, 1,25(OH)2D, PTH and VDBP levels were changed significantly compared to the placebo group. At the end of the study period, LTL levels were significantly increased in both groups and this change was more prominent in placebo group. The change in GC expression was significant between treatment and placebo groups but VDR expression remained unchanged. Even though the study was designed to solely assess the effects of vitamin D supplementation, LTL was significantly increased in the whole study group in summer months suggesting that LTL levels are affected by sun exposure and seasonal changes rather than supplementation. The study displayed the short-term effect of Vitamin D supplementation on vitamin D, PTH levels, LTL and vitamin D associated gene expressions. The relation between Vitamin D and LTL is not linear and could be confounded by several factors such as the population differences, regional and seasonal changes in sun exposure.
Topics: Aged; Cohort Studies; Female; Humans; Leukocytes, Mononuclear; Middle Aged; Postmenopause; Receptors, Calcitriol; Telomere; Telomere Homeostasis; Transcriptome; Vitamin D; Vitamin D Deficiency
PubMed: 35202418
DOI: 10.1371/journal.pone.0264337