scholarly journals Function of miR-24 and miR-27 in Pediatric Patients With Idiopathic Nephrotic Syndrome

2021 ◽  
Vol 9 ◽  
Author(s):  
Fen-fen Ni ◽  
Guang-lei Liu ◽  
Shi-lei Jia ◽  
Ran-ran Chen ◽  
Li-bing Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nephrotic Syndrome ◽  
Idiopathic Nephrotic Syndrome ◽  
Gradient Centrifugation ◽  
Th2 Cells ◽  
Healthy Controls ◽  
Rt Pcr ◽  
Critical Function ◽  
Significant Difference ◽  
Ficoll Density Gradient Centrifugation

Purpose: We investigated the pathogenesis of idiopathic nephrotic syndrome (INS) by measuring the effects two specific miRNAs on Th2 cells in children with this disease.Methods: After informed consent, we enrolled 20 children with active INS before steroid initiation, 20 children with INS in remission after steroid therapy, and 20 age-matched healthy controls. Flow cytometry was used to measure the levels of Th2 cells and a cytometric bead array was used to measure the levels of IgE, interleukin (IL)−4, and IL-13. RT-PCR was used to measure the levels of miR-24 and miR-27 in CD4+TCD25− cells. PBMCs were isolated using Ficoll density gradient centrifugation, and transfected with different mimic or inhibitor miRNAs. RT-PCR was used to measure the expression of different RNAs, and flow cytometry was used to determine the percentage of Th2 cells.Results: Relative to healthy controls, children with active INS had higher percentages of Th2 cells (P < 0.05), but there was no significant difference in controls and children in remission. The plasma levels of IgE, IL-4, and IL-13 were significantly increased in children with active INS (P < 0.05). There were lower levels of miR-24 and miR-27 in children with active non-atopic INS (P < 0.05). Transfection experiments indicated that upregulation of each miRNA decreased the percentage of Th2 cells and the level of IL-4 (P < 0.05), and down-regulation of each miRNA had the opposite effects (P < 0.05).Conclusion: Children with active INS, with or without atopy, had higher levels of IgE, possibly related to their higher levels of IL-13 and IL-4 due to a drift toward Th2 cells. miR-24 and miR-27 suppressed the expression of Th2 cells and have a critical function regulating Th2 cell expression in INS.

scholarly journals Function of miR-24 and miR-27 in Pediatric Patients With Idiopathic Nephrotic Syndrome

2021 ◽  
Author(s):  
Fen-fen Ni ◽  
Guang-lei Liu ◽  
shi-lei Jia ◽  
Ran-ran Chen ◽  
Li-bing Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nephrotic Syndrome ◽  
Idiopathic Nephrotic Syndrome ◽  
Gradient Centrifugation ◽  
Th2 Cells ◽  
Rt Pcr ◽  
Critical Function ◽  
Significant Difference ◽  
Ficoll Density Gradient Centrifugation ◽  
Ficoll Density Gradient

Abstract Background: The specific etiology and mechanism of idiopathic nephrotic syndrome (INS) in children remain unclear, so we investigated the pathogenesis of INS by measuring the effects two specific miRNAs on Th2 cells in children with this disease. Methods: Flow cytometry was used to measure the levels of Th2 cells and a cytometric bead array was used to measure the levels of IgE, interleukin (IL) -4, and IL-13. RT-PCR was used to measure the levels of miR-24 and miR-27 in CD4+TCD25− cells. PBMCs were isolated using Ficoll density gradient centrifugation, and transfected with different mimic or inhibitor miRNAs. RT-PCR was used to measure the expression of different RNAs, and flow cytometry was used to determine the percentages of Th2 cells. Results: Children with active INS had higher percentages of Th2 cells than healthy controls (P<0.05), but there was no significant difference for children in remission. The plasma levels of IgE, IL-4, and IL-13 were significantly increased in children with active INS (P<0.05). miR-24 and miR-27 were at lower levels in children with active non-atopic INS (P<0.05). Transfection experiments indicated that upregulation of each miRNA decreased the percentage of Th2 cells and the level of IL-4 (P<0.05), and down-regulation of each miRNA had the opposite effects (P<0.05). Conclusion: Children with active INS, with or without atopy, had higher levels of IgE, possibly related to their higher levels of IL-13 and IL-4 due to drift toward Th2 cells. miR-24 and miR-27 suppress the expression of Th2 cells and have a critical function in Th2 expression in INS.

RNA expression as a prognostic tool in idiopathic nephrotic syndrome

2007 ◽  
Vol 148 (23) ◽  
pp. 1067-1075
Author(s):  
Krisztina Fischer ◽  
Orsolya Galamb ◽  
Béla Molnár ◽  
Zsolt Tulassay ◽  
András Szabó
Keyword(s):  
Nephrotic Syndrome ◽  
Northern Blot ◽  
Idiopathic Nephrotic Syndrome ◽  
Minimal Change ◽  
Ribonuclease Protection Assay ◽  
Rna Expression ◽  
Rt Pcr ◽  
Protection Assay ◽  
Ribonuclease Protection

A gyermekkori nephrosis 90%-a idiopathiás nephrosis szindróma. Az idetartozó három kórkép, a minimal change betegség, a mesangialis proliferatio és a focalis sclerosis hasonló klinikai képpel jelentkező, eltérő prognózisú és terápiás válaszú betegség. Dolgozatunk célja az idiopathiás nephrosis szindrómába tartozó kórképek kialakulásával, progressziójával összefüggő genetikai ismeretek, génexpressziós változások áttekintése és funkcionális csoportosítása. A génexpressziós változások meghatározásának eszközeként, dolgozatunk röviden összefoglalja a northern blot, a ribonuclease protection assay, az in situ RNS-hibridizáció, a kvantitatív RT-PCR és a microarray módszerek lényegét. Az eddig elvégzett vizsgálatok a DNS-szintézis és repair gének, növekedési faktorok, extracelluláris mátrix, extracelluláris ligandreceptorok, extracelluláris jelátvitel zavarai mellett kiemelik a metabolikus és transzporter gének, illetve az immunszabályozó gének molekuláris eltéréseit, amelyek összefüggésben vannak az idiopathiás nephrosis szindróma eddig megismert molekuláris hátterével. A chiptechnológia fejlődésével és elterjedésével ezek a markerek és a hagyományos vizsgálati módszerek párhuzamos alkalmazása rutindiagnosztikai szempontból is fontossá válhat.

scholarly journals Evidence for a role of angiotensin converting enzyme 2 in proteinuria of idiopathic nephrotic syndrome

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Roberta da Silva Filha ◽  
Sérgio Veloso Brant Pinheiro ◽  
Thiago Macedo e Cordeiro ◽  
Victor Feracin ◽  
Érica Leandro Marciano Vieira ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Idiopathic Nephrotic Syndrome ◽  
Renin Angiotensin System ◽  
Cross Sectional Study ◽  
Healthy Controls ◽  
Ang Ii ◽  
Cross Sectional ◽  
Angiotensin Converting Enzyme 2 ◽  
Inflammatory Molecules

AbstractIntroduction: Renin angiotensin system (RAS) plays a role in idiopathic nephrotic syndrome (INS). Most studies investigated only the classical RAS axis. Therefore, the aims of the present study were to evaluate urinary levels of RAS molecules related to classical and to counter-regulatory axes in pediatric patients with INS, to compare the measurements with levels in healthy controls and to search for associations with inflammatory molecules, proteinuria and disease treatment. Subjects and methods: This cross-sectional study included 31 patients with INS and 19 healthy controls, matched for age and sex. Patients and controls were submitted to urine collection for measurement of RAS molecules [Ang II, Ang-(1-7), ACE and ACE2] by enzyme immunoassay and cytokines by Cytometric Bead Array. Findings in INS patients were compared according to proteinuria: absent (<150 mg/dl, n = 15) and present (≥150 mg/dl, n = 16). Results: In comparison to controls, INS patients had increased Ang II, Ang-(1-7) and ACE, levels while ACE2 was reduced. INS patients with proteinuria had lower levels of ACE2 than those without proteinuria. ACE2 levels were negatively correlated with 24-h-proteinuria. Urinary concentrations of MCP-1/CCL2 were significantly higher in INS patients, positively correlated with Ang II and negatively with Ang-(1-7). ACE2 concentrations were negatively correlated with IP-10/CXCL-10 levels, which, in turn, were positively correlated with 24-h-proteinuria. Conclusion: INS patients exhibited changes in RAS molecules and in chemokines. Proteinuria was associated with low levels of ACE2 and high levels of inflammatory molecules.

scholarly journals A Flow Cytometry-Based Assay for the Measurement of Total Complement Activity in the Serum and Clinical Practice

2022 ◽  
Vol 2022 ◽  
pp. 1-12
Author(s):  
Xuewei Ding ◽  
Shijun Li ◽  
Hui Liu
Keyword(s):  
Flow Cytometry ◽  
Nephrotic Syndrome ◽  
Linear Relationship ◽  
High Volume ◽  
Healthy Individuals ◽  
Serum Samples ◽  
Complement Activity ◽  
Significant Difference ◽  
Cell Groups ◽  
Total Complement

Objective. To develop a novel sensitive and accurate assay suitable for high-volume testing of the total complement activity in the serum for clinical laboratories. Methods. The total complement activity (TCA) to be measured was quantified by detecting the number of fragments produced by erythrocyte lysis and the erythrocyte fragmentation index (EFI), indicating TCA. EFI = M × M 2 / M 1 + M 2 , where M is the number of erythrocyte fragments (removed from the background), M 1 is the number of unagglutinated red cells, M 2 is the number of agglutinated red cell groups, and M 2 / M 1 + M 2 is the agglutination coefficient indicating the degree of erythrocyte agglutination. Mild changes in hemolysin and erythrocyte concentrations were made to optimize the testing conditions. The same serum samples were tested for 10 consecutive days to determine the stability of the experimental results. Serum EFI was detected in both nephrotic syndrome patients and healthy subjects. Results. There was a linear relationship between hemolysin and erythrocyte agglutination ( r = 0.999 , P < 0.001 ). A good linear relationship existed between EFI and TCA ( r = 0.991 , P < 0.001 ). The results were not affected by slight fluctuations in the concentrations of hemolysin or erythrocytes. The interbatch CV = 8.6 % of the test results showed good stability. There was a significant difference in the EFI between nephrotic syndrome patients and healthy individuals, P < 0.001 , and EFI was reduced in nephrotic syndrome patients compared to healthy individuals. Conclusion. The flow cytometry-based assay for TCA was sensitive and accurate and had potential value for clinical application.

scholarly journals SLAM/SAP Decreased Follicular Regulatory T Cells in Patients With Graves’ Disease 

2021 ◽  
Author(s):  
Lina Geng ◽  
Jun Yang ◽  
Xinyi Tang ◽  
Huiyong Peng ◽  
Jie Tian ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Negative Correlation ◽  
Real Time Pcr ◽  
Graves Disease ◽  
Healthy Controls ◽  
Significant Difference ◽  
Ifn Γ ◽  
Signaling Lymphocytic Activation Molecule

Abstract Background: Signaling lymphocytic activation molecule (SLAM) and SLAM-associated protein (SAP) play important role in inflammatory and autoimmune diseases. Our study aimed to detect the expression of SLAM and SAP in patients with Graves’ disease (GD) and analyze the effect of SLAM/SAP on circulating blood CD4+CXCR5+ Foxp3+ follicular regulatory T (Tfr) cells.Methods: The expression of SLAM and SAP was assessed by flow cytometry and real-time PCR. The percentages of IFN-γ+ cells, IL-4+ cells, IL-17+ cells and Foxp3+ cells in CD4+CXCR5+ T cells and circulating CD4+CXCR5+ Foxp3+ Tfr cells after treatment with anti-SLAM and anti-CD3 antibodies were also assessed by flow cytometry. The correlations between the percentages of Tfr cells and the levels of autoantibodies as well as SAP were analyzed.Results: The level of SAP in CD4+CXCR5+ T cells and the level of SLAM on CD19+ B cells were significantly increased in the patients with GD, but no significant difference in the level of SLAM on CD4+CXCR5+ T cells was observed between the patients with GD and the healthy controls. A decrease in the percentage of Foxp3+ cells in CD4+CXCR5+ T cells was observed following anti-SLAM treatment, but the percentages of IFN-γ+ cells, IL-4+ cells and IL-17+ cells showed no obvious differences. The proportion of circulating Tfr cells was decreased in the patients with GD, and the proportion of circulating Tfr cells had a negative correlation with the level of SAP in CD4+CXCR5+ T cells and the levels of autoantibodies in the serum of the patients with GD.Conclusions: Our results indicate that the SLAM/SAP signaling pathway regulates Tfr cells, which may be involved in the pathogenesis of Graves’ disease.

scholarly journals MiR-24 and miR-27 negatively regulate the expression of Th2 cells in children with idiopathic nephrotic syndrome

Authorea ◽  
2020 ◽  
Author(s):  
Cheng rong Li ◽  
Fen Ni ◽  
Guang lei Liu ◽  
Jun Yang ◽  
Shi lei Jia ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Idiopathic Nephrotic Syndrome ◽  
Th2 Cells

scholarly journals Effect of calcium and vitamin D supplementation on serum calcium level in children with idiopathic nephrotic syndrome

2014 ◽  
Vol 54 (3) ◽  
pp. 162 ◽  
Author(s):  
Vaya Dasitania ◽  
Alex Chairulfatah ◽  
Dedi Rachmadi
Keyword(s):  
Vitamin D ◽  
Nephrotic Syndrome ◽  
Serum Calcium ◽  
Idiopathic Nephrotic Syndrome ◽  
Controlled Trial ◽  
Vitamin D Supplementation ◽  
Significant Difference ◽  
Low Levels ◽  
Single Blind ◽  
Elemental Calcium

Background Patients with idiopathic nephrotic syndrome (NS) maydevelop hypocalcemia caused by low levels of albumin and vitaminD -binding protein, which subsequently decreases calcium absorptionin the intestine. Hypocalcemia may result in neuromuscularmanifestations, such as Chvostek's and Trosseau's signs.Objectives To evaluate the effect of calcium and vitamin Dsupplementation on hypocalcemia and its clinical manifestationsin idiopathic NS children.Methods A randomized, single-blind, controlled trial wasperformed in idiopathic NS patients aged 1-14 years. Subjectswere divided into treatment and placebo groups. Subjects inthe treatment group received 800 mg elemental calcium and400 IU vitamin D supplementation, while they in control groupreceived placebo syrup, both for 8 weeks. Serum calcium andmanifestations of hypocalcemia were examined before and aftersupplementation.Results Thirty subjects completed the study (15 in each group).Seventeen subjects experienced hypocalcemia. Chvostek's andTrosseau's signs were observed in 6 subjects in the treatment groupand 2 subjects in the placebo group (P= 0.427). After 8 weeks ofintervention, Chvostek's and Trosseau's signs disappeared in bothgroups, and calcium levels were significantly increased in bothgroups compared to the levels before intervention. However, therewas no significant difference in serum calcium levels after 8 weeksbetween the treatment and placebo groups (P =0.707).Conclusion Normalization of serum calcium levels and improvedclinical manifestations ofhypocalcemia occur both in NS patientswho receive calcium and vitamin D supplementation and thosewho do not.

Leukotriene Release from Neutrophils of Patients on Hemodialysis with Cellulose Membranes

1992 ◽  
Vol 15 (2) ◽  
pp. 84-88 ◽  
Author(s):  
A. Jörres ◽  
D. Jörres ◽  
G.M. Gahl ◽  
E. Schulz ◽  
A. Mahiout
Keyword(s):  
Gradient Centrifugation ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dialysis Patients ◽  
Healthy Donors ◽  
Significant Difference ◽  
Before And After ◽  
Ficoll Density Gradient Centrifugation ◽  
Cellulose Membranes

The role of cytokines in patients with chronic renal failure is currently under investigation. We therefore studied the release of leukotriene B4 (LTB4) from polymorphonuclear leukocytes (PMN) in stable dialysis patients treated with two different cellulose membranes, Cuprophan and Hemophan®, a modified cellulose with less complement activation. Six patients were treated for four weeks with Cuprophan then switched to Hemophan® for another four weeks. Before and after the last treatment of each period, PMN were separated from 20 ml heparinized blood by FICOLL density gradient centrifugation. Portions of 5x106 PMN were resuspended in Hanks' buffer and stimulated for 5 minutes with calcium ionophore A23187 (5 /μg/ml). LTB4 in cell supernatants was determined by specific radioimmunoassay. PMN from dialysis patients before HD released significantly (p<0.01) more LTB4 than healthy donors. No significant difference between pre- and post-dialysis values was observed with Cuprophan or Hemophan® dialyzers. Our data suggest that the acute effects of blood membrane interaction with either complement activating or non-activating dialyzers do not lead to changes in post-dialysis leukotriene metabolism, but leukotriene production is enhanced chronically in dialysis patients.

scholarly journals Transcription Factor Assay of Peripheral Blood T cells in Different Groups of Rheumatoid Arthritis Patients

2020 ◽  
Vol 8 (4) ◽  
pp. 153-162
Author(s):  
Saeid Taghiloo ◽  
◽  
Abolghasem Ajami ◽  
Mohsen Tehrani ◽  
Arezou Abbasi ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Transcription Factors ◽  
Mrna Expression ◽  
Peripheral Blood ◽  
Treg Cells ◽  
The Body ◽  
Th2 Cells ◽  
Healthy Controls ◽  
Relative Expression ◽  
Significant Difference

Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and other tissues and organs of the body. Previous reports have demonstrated the imbalance of T helper (Th) subsets and Treg activity in the development, progression, and remission of RA. Here, we investigated the mRNA expression of four major transcription factors T-bet (Th1), GATA (Th2), RORc (Th17), and Foxp3 (Treg) in peripheral blood of different groups of RA patients. Materials and methods: In this case-control study, 60 patients with RA, including 20 newly diagnosed, 20 under treatment, and 20 in remission, as well as 20 patients with osteoarthritis, and 20 age- and the sex-matched healthy individual were enrolled. Diagnosis and classification of patients were done according to the American College of Rheumatology criteria. The relative mRNA expression of transcription factors, including T-bet, GATA, RORc, and Foxp3, was measured using qRT-PCR. Results: The relative expression of T-bet in RA patients was significantly increased in healthy controls (P = 0.002), while the relative expression of Foxp3 in RA patients was significantly decreased in healthy controls (P < 0.0001). There was no significant difference in the expression of GATA3 or RORc among RA patients, healthy controls, and osteoarthritis group. Conclusions: The results indicate the importance of Th1 and Treg cells in RA; however, the role of Th17 cells appear to be of little importance in these patients. It seems that Th2 cells do not interfere with RA development.

scholarly journals Pulmonary manifestations in Behçet disease: impaired natural killer cells activity

2013 ◽  
Vol 8 ◽  
Author(s):  
Kamel Hamzaoui ◽  
Anissa Berraies ◽  
Wajih Kaabachi ◽  
Jamel Ammar ◽  
Agnès Hamzaoui
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Natural Killer ◽  
Systemic Vasculitis ◽  
Orphan Receptor ◽  
Healthy Controls ◽  
Rt Pcr ◽  
Foxp3 Mrna ◽  
Pulmonary Manifestations ◽  
Lak Activity

Background: Behçet’s disease (BD) is a systemic vasculitis with unknown aetiology, where, besides genetic predisposition, an immune dysregulation involving T and B lymphocytes and hyperactive neutrophils contribute to disease pathogenesis. The aim of this study was to determine the cytotoxicity of natural killer (NK) cells in bronchoalveolar lavage (BAL) from BD patients with pulmonary manifestations. Methods: BAL was performed in 27 patients with BD and pulmonary manifestations, 14 patients with Rheumatoid Arthritis (RA) and 23 healthy controls (HC). Related orphan receptor C (RORC) and forkheadbox P3 (FOXP3) mRNA transcript were determined in BAL by reverse transcription–polymerase chain reaction (RT-PCR). NK cells, NK cell cytotoxicity, and lymphokine-activated killer (LAK) activity against K562 cells were measured by flow cytometry. Proportions of NK precursors and expression of genes for IL-2 receptor β (IL-2Rβ; CD122), perforin, and granzyme in NK cells were measured by flow cytometry or RT-PCR. Results: The analysis of transcription factors revealed an increase in the RORC/FOXP3 ratio (Th17/Treg cells) in BAL from BD patients. Percentages of NK were significantly lower in BD than in RA patients and healthy controls. Purified NK cells derived from BD patients were found to have lower cytotoxicity and LAK activity than those from controls. This defect of NK cells in BD patients was related to down-regulation of perforin and granzyme expression in NK cells. Conclusion: In BD patients, the increased RORC/FOXP3 ratio indicated an inflammatory state of the lung. NK cells were decreased together with an impairment of their activity due to a defective expression of granzyme and perforin. These abnormalities possibly contribute to immune system dysregulation found in BAL of BD patients with pulmonary manifestations.

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