scholarly journals POS1135 MONOSODIUM URATE CRYSTALS REDUCE SCHWANN CELLS VIABILITY

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 847.1-847
Author(s):  
Y. Liu ◽  
Y. Huang ◽  
S. Sun ◽  
W. Deng ◽  
T. W. LI
Keyword(s):  
Flow Cytometry ◽  
Survival Rate ◽  
Peripheral Neuropathy ◽  
Schwann Cells ◽  
Rheumatic Diseases ◽  
Cell Counting ◽  
Pathological Feature ◽  
Monosodium Urate ◽  
Migration Ability ◽  
Msu Crystals

Background:The prevalence of peripheral neuropathy in patients with gout almostly reaches 25%[1]. Demyelination caused by Schwann cell (SCs) injury and apoptosis is the major pathological feature of peripheral[2]. None of study has focused on the effects of monosodium urate (MSU) crystals on SCs.Objectives:To assess the effect of MSU crystals on SCs.Methods:Mouse-derived Schwann cells (RSC96) are stimulated with different concentrations of MSU crystals (0mg/ml,0.25mg/ml,0.5mg/ml) and time (24h,48h,72h). The migration ability of Schwann cells is evaluated by acratch assay, the proliferation level is assessed by the cell counting kit-8 (CCK-8) assay, and the apoptosis rate is detected by flow cytometry.Results:The acratch assay showed that the migration ability of SCs was worsened, CCK-8 assay suggested that proliferation of SCs was reduced in a dose-dependent manner (P<0.05). According to the result of flow cytometry, the survival rate of SCs at 0.5mg/ml(78.60%±2.26%) was lower than that 0.25mg/ml(87.50%±0.95%)and 0mg/ml (98.80%±0.26%)(p<0.05) at 24h. When the stimulation time increased to 72h, the survival rate at 0.5mg/ml(47.90%±11.70%) dropped significantly, which was significantly different from the other two groups(p<0.05).Conclusion:MSU crystals can cause damage to Schwann cells. It may help to explain the reason of peripheral neuropathy in gout patients.References:[1]López-López, C.O., et al., Peripheral neuropathies in rheumatic diseases: More diverse and frequent than expected. A cross-sectional study. International journal of rheumatic diseases, 2020. 23(2): p. 226-232.[2]Liu, Y., S. Shao and H. Guo, Schwann cells apoptosis is induced by high glucose in diabetic peripheral neuropathy. Life sciences, 2020. 248: p. 117459.Figure 1.Flow cytometry assays of RSC96 on MSU crystals at 72hDisclosure of Interests:None declared.

scholarly journals POS0133 MONOSODIUM URATE CRYSTALS REDUCE HUMAN LIGAMENT CELLS VIABILITY THROUGH INCREASE OF ROS PRODUCTION

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 278.1-278
Author(s):  
Y. Huang ◽  
Y. Liu ◽  
Q. Huang ◽  
W. Deng ◽  
T. W. Li
Keyword(s):  
Cell Migration ◽  
Annexin V ◽  
Cell Counting ◽  
Ros Production ◽  
Monosodium Urate ◽  
Migration Ability ◽  
Monosodium Urate Crystals ◽  
Migration Assay ◽  
Msu Crystals ◽  
Urate Crystals

Background:Ligament destruction is a frequent complication of gout and is strongly associated with tophi. Ligament fibroblasts are important cellular mediators of ligament remodeling. None of study has paid attention to the effects of monosodium urate (MSU) crystals on ligament fibroblasts.Objectives:The study aims to investigate the effects and mechanism of MSU crystals on ligament fibroblasts.Methods:MSU crystals were added to human ligament fibroblasts(HLFs) cultures or primary ligament cells cultures. Cell counting kit-8 (CCK-8) assay, cell migration assay, Annexin V-FITC/PI assay were conducted. Reactive Oxygen Species(ROS) was tested by ROS Assay Kit.Results:The higher concentrations of MSU crystals (0.5-1mg/mL) reduced the viability of HLFs or primary ligament cells after 24 h as assessed by CCK8 assays, with a further reduction in viability observed at the 48 h time point. When observed under light microscopy, HLFs cultured with MSU crystals (0.5mg/mL) appeared unhealthy with fewer cells present. The cell migration ability of HLFs was decreased significantly on MSU crystals (0.5mg/mL). According to the result of Annexin V-FITC/PI assay, the survival rate of HLFs on MSU crystals (0.5mg/mL) was lower than that of 0.25mg/ml and 0 mg/ml at 72h. ROS assay results showed that the production of ROS increased as the concentrations of MSU crystals increased.Conclusion:MSU crystals inhibit human ligament cells viability through the increase of ROS production. It may contribute to disordered ligament remodeling in gout patients with ligament destruction.References:[1]Ashika Chhana, et al. Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state. Arthritis Res Ther. 2018, 20(1): 208.Figure 1.MSU crystals reduce human ligament fibroblasts and primary human ligament cells viability over time. A: CCK-8 assay; B: Observation of HLFs morphology; C: Annexin V-FITC/PI assay.Disclosure of Interests:None declared

scholarly journals THU0406 IDENTIFICATION OF INTRACELLULAR VACUOLES IN SYNOVIAL FLUID WITH CALCIUM PYROPHOSPHATE AND MONOSODIUM URATE CRYSTALS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 440.1-441
Author(s):  
M. L. Peral ◽  
I. Calabuig ◽  
A. Martín-Carratalá ◽  
M. Andrés ◽  
E. Pascual
Keyword(s):  
Clinical Practice ◽  
Synovial Fluid ◽  
Phase Contrast ◽  
Calcium Pyrophosphate ◽  
Monosodium Urate ◽  
Fluid Analysis ◽  
Cytoplasmic Vacuoles ◽  
Contrast Microscopy ◽  
Ordinary Light ◽  
Msu Crystals

Background:Synovial fluid analysis using polarized microscopy is the gold standard for the diagnosis of crystal-related arthritis. In our experience, we have noted that, when calcium pyrophosphate (CPP) crystals are observed, they sometimes appear within intracellular vacuoles. However, this phenomenon is not seen in those samples containing monosodium urate (MSU) crystals. This finding has been scantly reported in the literature, but may be useful in clinical practice to ensure accurate crystal identification.Objectives:Our study aims to assess whether the presence of vacuoles contributes to identifying the type of crystal, and also to gauge the frequency of their presentation.Methods:We conducted an observational study in a rheumatology unit between February and June of 2019. Synovial fluids containing CPP or MSU crystals, obtained in daily clinical practice, were consecutively included for analysis. Two observers simultaneously analyzed the presence of vacuoles by ordinary light and phase contrast microscopy in less than 24 hours after their extraction, using a microscope equipped with two viewing stations. The primary study variable was to determine whether CPP and MSU crystals are seen inside intracellular vacuoles, and to calculate the frequency of this finding for each type of crystal, estimating their 95% confidence interval (95% CI) and comparing rates using Fisher’s exact test.Results:Twenty-one samples were obtained. Data is given in the Table. MSU crystals were present in 7 (33.3%) and CPP crystals in 14 (66.6%). Interestingly, none of the MSU samples showed crystal-containing vacuoles (95% CI 0-35.4%). On the contrary, cytoplasmic vacuoles containing crystals were present in all of the CPP samples (95% CI 78.5-100%). The findings were confirmed by phase-contrast microscopy. Differences were statistically significant (p<0.001).Table.SAMPLES ACCORDING TO TYPE OF MICROCRYSTAL(n=21)SAMPLES WITH VACUOLS(UNDER ORDINARY LIGHT)SAMPLES WITH VACUOLS(UNDER PHASE CONTRAST)CPP (14; 66.6%)14 (100%)(95%CI 78.5-100%)14 (100%)(95%CI 78.5-100%)MSU (7; 33.3%)0 (0%)(95%CI 0-35.4%)0 (0%)(95%CI 0-35.4%)Conclusion:The presence of vacuoles may be a useful and easy way to differentiate MSU and CPP crystals when performing synovial fluid microscopy in clinical practice, since it appears to be a distinctive feature in CPP crystal fluids.References:[1]Kohn NN, Hughes RE, McCarty DJ Jr, Faires JS. The significance of calcium phosphate crystals in the synovial fluid of arthritic patients: the «pseudogout syndrome». II. Identification of crystals. Ann InternMed. 1962 May;56:738-45.[2]Pascual E, Sivera F, Andrés M. Synovial Fluid Analysis for Crystals. CurrOpRheumatol 2011;23:161-169.[3]McCarty DJ, Koopman WJ. Arthritis and allied conditions: A textbook of rheumatology, volumen 1. Lea &amp;Febiger. 1993.[4]Pascual E, Sivera F. Synovial fluid crystal Analysis. En Gout and other crystal arthropathies. Terkeltaub R ed. Elsevier; 2012: p.20-34.[5]Hwang HS, Yang CM, Park SJ, Kim HA. Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process. Int J Mol Sci. 2015 Dec 8;16(12):29265-77.Image 1. Microscopy with ordinary light. Cells with cytoplasmic vacuoles are observed, as well as abundant intra and extracellular CPP crystals.Image 2. Microscopy with phase contrast technique. Cells with intracellular vacuoles are observed inside which have microcrystals with parallelepiped morphology, compatible with CPP.Disclosure of Interests: :None declared

scholarly journals Lower Temperatures Exacerbate NLRP3 Inflammasome Activation by Promoting Monosodium Urate Crystallization, Causing Gout

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1919
Author(s):  
Huijeong Ahn ◽  
Gilyoung Lee ◽  
Geun-Shik Lee
Keyword(s):  
Environmental Factor ◽  
Nlrp3 Inflammasome ◽  
Inflammasome Activation ◽  
Optimal Temperature ◽  
Monosodium Urate ◽  
Nlrp3 Inflammasome Activation ◽  
Hyperthermia Therapy ◽  
Effects Of Temperature ◽  
Lower Temperature ◽  
Msu Crystals

Gout is a recurrent and chronic form of arthritis caused by the deposition of monosodium urate (MSU) crystals in the joints. Macrophages intake MSU crystals, the trigger for NLRP3 inflammasome activation, which leads to the release of interleukin (IL)-1β and results in the flaring of gout. The effects of temperature, an environmental factor for MSU crystallization, on IL-1β secretion have not been well studied. This study examined the effects of temperature on inflammasome activation. Specific triggers activated canonical inflammasomes (NLRP3, NLRC4, and AIM2) in murine macrophages at various temperatures (25, 33, 37, 39, and 42 °C). The maturation of IL-1β and caspase-1 was measured as an indicator for inflammasome activation. As expected, the optimal temperature of inflammasome activation was 37 °C. The MSU crystal-mediated activation of inflammasome increased at temperatures lower than 37 °C and decreased at higher temperatures. MSU crystals at lower temperatures enhanced IL-1β secretion via the NLRP3 inflammasome pathway. A lower temperature promoted the formation of MSU crystals without changing phagocytosis. Overall, lower temperatures form more MSU crystals and enhance NLRP3 inflammasome activation. In light of these findings, it is possible that hyperthermia therapy may reduce gout flaring.

scholarly journals 724 Evaluation of an anti-human IL-1β antibody in monosodium urate crystals-induced peritonitis model in hIL-1β HuGEMM™ mice

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A753-A753
Author(s):  
Xiaoyu An ◽  
Kaixia Lian ◽  
Jia Zheng ◽  
Fei Jian ◽  
Henry Li ◽  
...  
Keyword(s):  
Treatment Group ◽  
Murine Model ◽  
Gouty Arthritis ◽  
Chronic Inflammatory Disease ◽  
Vehicle Group ◽  
Inflammasome Activation ◽  
Monosodium Urate ◽  
Vehicle Treatment ◽  
Peritonitis Model ◽  
Msu Crystals

BackgroundGout is a chronic inflammatory disease featuring the deposition of monosodium urate (MSU) crystals in the synovial fluid of patients, followed by NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome activation and bioactive IL-1β release, which recruits neutrophils to the local inflammation sites. Blocking IL-1β function is becoming a a potent therapeutic approach for gout and gouty arthritis. Conventional MSU-induced peritonitis in C57BL/6 mice provides a simple and rapid evaluation of therapeutics targeting inflammasome activation. However, this murine model has limitations when it comes to the evaluation of human-specific antibodies, for example, anti-human IL-1β (anti-hIL-1β) monoclonal antibodies (mAb). Thus, a murine model to assess the efficacy of anti-hIL-1β mAb is needed. We have developed a hIL-1β knock-in mouse model (hIL-1β HuGEMM™), which is able to facilitate the pre-clinical evaluation of drugs targeting specific human biological molecules especially when mouse ortholog is not available. Therefore, an MSU crystals induced peritonitis model using hIL-1β HuGEMM™ mice provides a robust model to evaluate therapies targeting hIL-1β.MethodsMSU crystals were injected intraperitoneally into human IL-1β (hIL-1β) knock-in mice, where the coding sequence of mouse IL-1β was replaced by hIL-1β. Prior to MSU crystal administration, mice received treatment of either vehicle or anti-hIL-1β antibody. Six hours facilitate post MSU crystal injection, serum and lavage flushed with PBS were collected. Subsequently, cytokine protein levels in the serum were determined by MSD, and the population of polymorphonuclear leukocytes (PMNs) (live CD11b+ Ly-6GHi cells) in the lavage was analysed by flow cytometry.ResultsThe vehicle treatment group showed a dramatic increase in hIL-1β secretion and PMN leukocytes, in comparison to the group that did not receive MSU, which suggests a successful induction of acute inflammatory response in the peritoneal cavity. In contrast, mice that received a single administration of anti-hIL-1β antibody 24 hours prior to MSU injection exhibited a significantly lower level of hIL-1β when compared to the vehicle treatment group, which implies that the anti-hIL-1β mAb efficaciously neutralized hIL-1β secretion. In addition, TNF-α and IL-6, two further cytokines downstream of IL-1β, were significantly reduced in the anti-hIL-1β mAb treatment group. However, the PMN leukocyte infiltration in the anti-hIL-1β mAb treatment group did not change in comparison to the vehicle group.ConclusionsIn this study, an MSU crystals-induced peritonitis model was successfully established in hIL-1β HuGEMM mice, which has the potential to evaluate immune therapeutics with anti-hIL-1β blockades.

scholarly journals Long Non-coding RNA XIST Attenuates Diabetic Peripheral Neuropathy by Inducing Autophagy Through MicroRNA-30d-5p/sirtuin1 Axis

2021 ◽  
Vol 8 ◽  
Author(s):  
Bei-Yan Liu ◽  
Lin Li ◽  
Li-Wei Bai ◽  
Chang-Shui Xu
Keyword(s):  
Oxidative Stress ◽  
Peripheral Neuropathy ◽  
Schwann Cells ◽  
Diabetic Peripheral Neuropathy ◽  
Beclin 1 ◽  
Diabetic Mice ◽  
Non Coding Rna ◽  
Non Coding Rnas ◽  
Long Non Coding Rna

Diabetic peripheral neuropathy (DPN) is a prevalent diabetes mellitus (Feldman et al., 2017) complication and the primary reason for amputation. Meanwhile, long non-coding RNAs (lncRNAs) are a type of regulatory non-coding RNAs (ncRNAs) that broadly participate in DPN development. However, the correlation of lncRNA X-inactive specific transcript (XIST) with DPN remains unclear. In this study, we were interested in the role of XIST in the modulation of DPN progression. Significantly, our data showed that the expression of XIST and sirtuin1 (SIRT1) was inhibited, and the expression of microRNA-30d-5p (miR-30d-5p) was enhanced in the trigeminal sensory neurons of the diabetic mice compared with the normal mice. The levels of LC3II and Beclin-1 were inhibited in the diabetic mice. The treatment of high glucose (HG) reduced the XIST expression in Schwann cells. The apoptosis of Schwann cells was enhanced in the HG-treated cells, but the overexpression of XIST could block the effect in the cells. Moreover, the levels of LC3II and Beclin-1 were reduced in the HG-treated Schwann cells, while the overexpression of XIST was able to reverse this effect. The HG treatment promoted the production of oxidative stress, while the XIST overexpression could attenuate this result in the Schwann cells. Mechanically, XIST was able to sponge miR-30d-5p and miR-30d-5p-targeted SIRT1 in the Schwann cells. MiR-30d-5p inhibited autophagy and promoted oxidative stress in the HG-treated Schwann cells, and SIRT1 presented a reversed effect. MiR-30d-5p mimic or SIRT1 depletion could reverse XIST overexpression-mediated apoptosis and autophagy of the Schwann cells. Thus, we concluded that XIST attenuated DPN by inducing autophagy through miR-30d-5p/SIRT1 axis. XIST and miR-30d-5p may be applied as the potential targets for DPN therapy.

scholarly journals Therapeutic Effect of Exogenous Treg Cells on Collagen-Induced Arthritis and Rheumatoid Arthritis

2019 ◽  
Author(s):  
Shutong Li ◽  
Hongxing Wang ◽  
Hui Wu ◽  
Guoqing Zhang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Synovial Fibroblast ◽  
Treg Cells ◽  
Cell Counting ◽  
Fibroblast Cells ◽  
Collagen Induced Arthritis ◽  
Gm Csf

Abstract Background Regulatory T (Treg) cells have anti-inflammatory and anti-autoimmune functions. The proportion and functions of Treg cells are perturbed in rheumatoid arthritis (RA) patients. Methods Human Treg cells were induced to amplify in vitro and cocultured with RA synovial fibroblast cells (RASFs). The proliferation and apoptosis of RASFs were determined by the cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Human Treg cells were also injected to collagen-induced arthritis (CIA) rats via the tail vein. Changes in lymphocyte subtypes and cytokines in the peripheral blood and spleen were observed by flow cytometry. Results After coculture with the Treg cells, the proliferation of RA synovial fibroblast cells decreased (p<0.01), and the rate of apoptosis increased (p=0.037). The human Treg cells were injected into the tail veins of collagen-induced arthritis (CIA) rats. The severity of the CIA was reduced (p<0.01) following the injection, the percentages of rat endogenous Treg cells in the peripheral blood and spleen increased significantly (p=0.007 and p<0.01, respectively), and the proportion of B cells decreased (p=0.031). The levels of interleukin IL-5 and IL-6 and the Th1/Th2 ratio in the peripheral blood were significantly decreased (p=0.013, 0.009 and 0.012, respectively). The number of NK cells and the levels of IL-4, IL-13, TNF-α, IFN-γ and GM-CSF in the peripheral blood and spleen did not change significantly. Conclusion These results suggest that exogenous Treg cells play a therapeutic role in RA and CIA. Treg cell treatment could serve as a therapy for RA.

scholarly journals In vitro Induced Mitotic Polyploidy in Drosera capensis L.

2013 ◽  
Vol 46 (4) ◽  
pp. 107-110 ◽  
Author(s):  
Pavla Zahumenicka ◽  
Barbora Sysova ◽  
Ales Holik ◽  
Eloy C. Fernandez
Keyword(s):  
Flow Cytometry ◽  
Survival Rate ◽  
Exposure Time ◽  
Leaf Segments

Abstract The objective of this study was to induce mitotic polyploidization in Drosera capensis. Tetraploid plants of D. capensis were induced successfully by treating leaf segments in vitro with oryzalin solution with four different concentrations (20, 40, 60 or 80 μM) for 12, 24 or 48 hours. Three tetraploid (2n = 4x = 80) plants were obtained in three treatments (20 μM for 48 h, 60 μM for 24 h and 80 μM for 12 h). Tetraploidy was confirmed by flow cytometry. The survival rate of these plants was not significantly influenced by oryzalin concentration or exposure time.

scholarly journals Effect of Silicon, Titanium, and Zirconium Ion Implantation on NiTi Biocompatibility

2012 ◽  
Vol 2012 ◽  
pp. 1-16 ◽  
Author(s):  
L. L. Meisner ◽  
A. I. Lotkov ◽  
V. A. Matveeva ◽  
L. V. Artemieva ◽  
S. N. Meisner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Ion Implantation ◽  
Cell Counting ◽  
High Dose ◽  
Surface Layers ◽  
Test Flow ◽  
Ion Implanted

The objective of the work was to study the effect of high-dose ion implantation (HDII) of NiTi surface layers with Si Ti, or Zr, on the NiTi biocompatibility. The biocompatibility was judged from the intensity and peculiarities of proliferation of mesenchymal stem cells (MSCs) on the NiTi specimen surfaces treated by special mechanical, electrochemical, and HDII methods and differing in chemical composition, morphology, and roughness. It is shown that the ion-implanted NiTi specimens are nontoxic to rat MSCs. When cultivated with the test materials or on their surfaces, the MSCs retain the viability, adhesion, morphology, and capability for proliferationin vitro, as evidenced by cell counting in a Goryaev chamber, MTT test, flow cytometry, and light and fluorescence microscopy. The unimplanted NiTi specimens fail to stimulate MSC proliferation, and this allows the assumption of bioinertness of their surface layers. Conversely, the ion-implanted NiTi specimens reveal properties favorable for MSC proliferation on their surface.

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