Interaction of antiphospholipid antibodies with endothelial cells in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease with arteriovenous thrombosis and recurrent miscarriages as the main clinical manifestations. Due to the complexity of its mechanisms and the diversity of its manifestations, its diagnosis and treatment remain challenging issues. Antiphospholipid antibodies (aPL) not only serve as crucial "biomarkers" in diagnosing APS but also act as the "culprits" of the disease. Endothelial cells (ECs), as one of the core target cells of aPL, bridge the gap between the molecular level of these antibodies and the tissue and organ level of pathological changes. A more in-depth exploration of the relationship between ECs and the pathogenesis of APS holds the potential for significant advancements in the precise diagnosis, classification, and therapy of APS. Many researchers have highlighted the vital involvement of ECs in APS and the underlying mechanisms governing their functionality. Through extensive in vitro and in vivo experiments, they have identified multiple aPL receptors on the EC membrane and various intracellular pathways. This article furnishes a comprehensive overview and summary of these receptors and signaling pathways, offering prospective targets for APS therapy.

Histone H3K18 and Ezrin Lactylation Promote Renal Dysfunction in Sepsis-Associated Acute Kidney Injury.

Histone lactylation is a metabolic stress-related histone modification. However, the role of histone lactylation in the development of sepsis-associated acute kidney injury (SA-AKI) remains unclear. Here, histone H3K18 lactylation (H3K18la) is elevated in SA-AKI, which is reported in this study. Furthermore, this lactate-dependent histone modification is enriched at the promoter of Ras homolog gene family member A (RhoA) and positively correlated with the transcription. Correction of abnormal lactate levels resulted in a reversal of abnormal histone lactylation at the promoter of RhoA. Examination of related mechanism revealed that histone lactylation promoted the RhoA/Rho-associated protein kinase (ROCK) /Ezrin signaling, the activation of nuclear factor-κB (NF-κB), inflammation, cell apoptosis, and aggravated renal dysfunction. In addition, Ezrin can undergo lactylation modification. Multiple lactylation sites are identified in Ezrin and confirmed that lactylation mainly occurred at the K263 site. The role of histone lactylation is revealed in SA-AKI and reportes a novel post-translational modification in Ezrin. Its potential role in regulating inflammatory metabolic adaptation of renal proximal tubule epithelial cells is also elucidated. The results provide novel insights into the epigenetic regulation of the onset of SA-AKI.

Circ DENND4C inhibits pyroptosis and alleviates ischemia-reperfusion acute kidney injury by exosomes secreted from human urine-derived stem cells.

Acute kidney injury (AKI) is a disease characterised by acute onset, high mortality, and poor prognosis, and is mainly caused by ischemia-reperfusion (I/R). Human urine-derived stem cells (USCs) exhibit antioxidant, anti-inflammatory, and anti-apoptotic cytoprotective effects. Previously, we found that exosomes from USCs had the ability to inhibit apoptosis and protect kidneys from I/R injury. This study aimed to investigate the role of USC-derived exosomes (USC-Exos) in reducing pyroptosis and alleviating I/R-AKI. Models of HK-2 cells hypoxia-reoxygenation (H/R) and I/R kidney injury was established in Sprague Dawley rats to simulate AKI in vitro and in vivo. USC-Exos were isolated using ultracentrifugation and identified via electron microscopy and western blotting. USC-Exos were co-cultured with HK-2 cells and injected into rats via the tail vein. The expression of pyroptosis-related molecules (GSDMD, caspase-1, and NLRP-3) was verified using PCR and western blotting. Changes in renal function were reflected in the serum creatinine, urea, and cystatin C levels. The degree of renal injury was determined using haematoxylin and eosin and immunohistochemical staining. The levels of IL-1ß and IL-18 were detected using enzyme-linked immunosorbent assay (ELISA) to verify the role of USC-Exos in pyroptosis. Differentially expressed circRNAs in I/R rat kidneys were screened by transcriptome sequencing, and a dual-luciferase experiment was used to verify the interaction between upstream and downstream molecules. Ischemia-reperfusion resulted in significantly impaired renal function and expression of pyroptosis molecules, and significantly increased concentrations of inflammatory factors. These effects were reversed by injecting USC-Exos. Circ DENND4C was the most significantly decreased circRNA in I/R rat renal tissue, and knock-down of circ DENND4C can aggravate AKI in vivo and in vitro. DAVID(http://david.abcc.ncifcrf.gov) website showed that miR 138-5p/FOXO3a is a potential downstream target of circ DENND4C. Knock-down of circ DENND4C in HK-2 cells resulted in increased expression of miR 138-5p and increased miR 138-5p can reverse the regulation of FOXO3a. Dual-luciferase assay verified the reverse interaction between circ DENND4C, miR 138-5p, and FOXO3a. Exosomes promote cell proliferation and inhibit the activation of NLR family pyrin domain containing 3 through the circ DENND4C/miR 138-5p/FOXO3a pathway, thereby reducing pyroptosis and AKI. Circ DENND4C may be a potential therapeutic target for AKI.

ARID5B-mediated LINC01128 epigenetically activated pyroptosis and apoptosis by promoting the formation of the BTF3/STAT3 complex in ß2GPI/anti-ß2GPI-treated monocytes.

BACKGROUND: Alterations of the trimethylation of histone 3 lysine 4 (H3K4me3) mark in monocytes are implicated in the development of autoimmune diseases. Therefore, the purpose of our study was to elucidate the role of H3K4me3-mediated epigenetics in the pathogenesis of antiphospholipid syndrome (APS). METHODS: H3K4me3 Cleavage Under Targets and Tagmentation and Assay for Transposase-Accessible Chromatin were performed to determine the epigenetic profiles. Luciferase reporter assay, RNA immunoprecipitation, RNA pull-down, co-immunoprecipitation and chromatin immunoprecipitation were performed for mechanistic studies. Transmission electron microscopy and propidium iodide staining confirmed cell pyroptosis. Primary monocytes from patients with primary APS (PAPS) and healthy donors were utilised to test the levels of key molecules. A mouse model mimicked APS was constructed with beta2-glycoprotein I (ß2GPI) injection. Blood velocity was detected using murine Doppler ultrasound. RESULTS: H3K4me3 signal and open chromatin at the ARID5B promoter were increased in an in vitro model of APS. The epigenetic factor ARID5B directly activated LINC01128 transcription at its promoter. LINC01128 promoted the formation of the BTF3/STAT3 complex to enhance STAT3 phosphorylation. Activated STAT3 interacted with the NLRP3 promoter and subsequently stimulated pyroptosis and apoptosis. ARID5B or BTF3 depletion compensated for LINC01128-induced pyroptosis and apoptosis by inhibiting STAT3 phosphorylation. In mice with APS, ß2GPI exposure elevated the levels of key proteins of pyroptosis and apoptosis pathways in bone marrow-derived monocytes, reduced the blood velocity of the ascending aorta, increased the thrombus size of the carotid artery, and promoted the release of interleukin (IL)-18, IL-1ß and tissue factor. Patients with PAPS had the high-expressed ARID5B and LINC01128, especially those with triple positivity for antiphospholipid antibodies. Moreover, there was a positive correlation between ARID5B and LINC01128 expression. CONCLUSION: This study indicated that ARID5B/LINC01128 was synergistically upregulated in APS, and they aggravated disease pathogenesis by enhancing the formation of the BTF3/STAT3 complex and boosting p-STAT3-mediated pyroptosis and apoptosis, thereby providing candidate therapeutic targets for APS. HIGHLIGHTS: The H3K4me3 mark and chromatin accessibility at the ARID5B promoter are increased in vitro model mimicked APS. ARID5B-mediated LINC01128 induces pyroptosis and apoptosis via p-STAT3 by binding to BTF3. ARID5B is high- expressed in patients with primary APS and positively correlated with LINC01128 expression. OICR-9429 treatment mitigates pyroptosis and related inflammation in vivo and in vitro models mimicked APS.

Accurate correction model of blood potassium concentration in hemolytic specimens.

BACKGROUND AND AIMS: The results of blood potassium can be seriously affected by specimen hemolysis which may interfere with clinicians' interpretation of test results. Redrawing blood and retesting may delay treatment time and it is not feasible for critically ill patients with difficulty in specimen collection. Therefore, it is significant to establish a mathematical model that can quickly correct the blood potassium concentration of hemolytic specimens. MATERIALS AND METHODS: The residual blood samples from 107 patients at Peking University Third Hospital were collected to establish potassium correction model. Samples with different hemolysis indexes were obtained by ultrasonic crushing method. Blood potassium correction models of hemolysis specimens were established by linear regression and curve fitting using SPSS and MATLAB, respectively. In addition, blood samples from another 85 patients were used to verify the accuracy of the models and determine the optimal model. RESULTS: Variation of potassium (ΔK) was 0.003HI-0.03 (R2 = 0.9749) in linear regression model which had high correlation in ΔK and HI, and the correction formula was Kcorrection = Khemolysis-0.003 × HI + 0.03. Average rate of potassium change (αaverage) was 0.003 ± 0.0002 mmol/L in curve fitting model, and correction formula was Kcorrection = Khemolysis-0.003 × HI, and both men and women can use the same correction model. The accuracy of linear regression model was 96.5 %, and there was statistical difference between the verification results and the measured values (p < 0.05), while the accuracy of curve fitting model was 100 %, and there was no statistical difference between the verification results and the measured values (p = 0.552). The model was validated in an independent set of samples and all were within the TEa of 6 % and the accuracy of 100 %. CONCLUSIONS: Both linear regression and curve fitting models of potassium correction had high accuracy, and can effectively correct the potassium concentration of hemolytic specimens, while the curve fitting model have superior accuracy.

Performance evaluation of a novel high-sensitivity cardiac troponin T assay: analytical and clinical perspectives.

OBJECTIVES: To evaluate the analytical characteristics of a novel high-sensitivity cardiac troponin T (hs-cTnT) test on the automatic light-initiated chemiluminescent assay (LiCA®) system, and validated its diagnostic performance for non-ST-segment elevation myocardial infarction (NSTEMI). METHODS: Studies included an extensive analytical evaluation and established the 99th percentile upper reference limit (URL) from apparently healthy individuals, followed by a diagnostic performance validation for NSTEMI. RESULTS: Sex-specific 99th percentile URLs were 16.0 ng/L (1.7 % CV: coefficient of variation) for men (21-92 years) and 13.4 ng/L (2.0 % CV) for women (23-87 years) in serum, and 30.6 ng/L (0.9 % CV) for men (18-87 years) and 20.2 ng/L (1.4 % CV) for women (18-88 years) in heparin plasma. Detection rates in healthy individuals ranged from 98.9 to 100 %. An excellent agreement was identified between LiCA® and Elecsys® assays with a correlation coefficient of 0.993 and mean bias of -0.7 % (-1.8-0.4 %) across the full measuring range, while the correlation coefficient and overall bias were 0.967 and -1.1 % (-2.5-0.3 %) for the lower levels of cTnT (10-100 ng/L), respectively. At the specific medical decision levels (14.0 and 52.0 ng/L), assay difference was estimated to be <5.0 %. No significant difference was found between these two assays in terms of area under curve (AUC), sensitivity and specificity, negative predictive value (NPV) and positive predictive value (PPV) for the diagnosis of NSTEMI. CONCLUSIONS: LiCA® hs-cTnT is a reliable 3rd-generation (level 4) high-sensitivity assay for detecting cardiac troponin T. The assay is acceptable for practical use in the diagnosis of NSTEMI.

Serum exosomal microRNA-1258 may as a novel biomarker for the diagnosis of acute exacerbations of chronic obstructive pulmonary disease.

Acute exacerbation chronic obstructive pulmonary disease (AECOPD) has a high mortality rate. However, there is no efficiency biomarker for diagnosing AECOPD. The purpose of this study was to find biomarkers that can quickly and accurately diagnose AECOPD.45 normal controls (NC), 42 patients with stable COPD (SCOPD), and 66 patients with AECOPD were enrolled in our study. Serum exosomes were isolated by ultracentrifuge and verified by morphology and specific biomarkers. Fluorescent quantitation polymerase chain reaction (qRT-PCR) was used to detect the expression of micro RNAs (miRNAs), including miR-660-5p, miR-1258, miR-182-3p, miR-148a-3p, miR-27a-5p and miR-497-5p in serum exosomes and serum. Logistic regression and machine learning methods were used to constructed the diagnostic models of AECOPD. The levels of miR-1258 in the patients with AECOPD were higher than other groups (p < 0.001). The ability of exosomal miR-1258 (AUC = 0.851) to identify AECOPD from SCOPD was superior to other biomarkers, and the combination of exosomal miR-1258 and NLR can increase the AUC to 0.944, with a sensitivity of 81.82%, and specificity of 97.62%. The cross-validation of the models displayed that the logistic regression model based on exosomal miR-1258, NLR and neutrophil count had the best accuracy (0.880) in diagnosing AECOPD from SCOPD. The three most correlated biomarkers with serum exosome miR-1258 were neutrophil count (r = 0.57, p < 0.001), WBC (r = 0.50, p < 0.001) and serum miR-1258 (r = 0.33, p < 0.001). In conclusion, serum exosomal miR-1258 is associated with inflammation, and can be used as a valuable and reliable biomarker for the diagnosis of AECOPD, and the establishment of diagnostic model based on miR-1258, NLR and neutrophils count can help to improving the accuracy of AECOPD diagnosis.

Effects of Different Kinds of Hand Sanitizers on Complete Blood Count and Leukocyte Differential Count.

BACKGROUND: To explore the effects of different contents and types of hand sanitizers on the complete blood count and leukocyte differential count. METHODS: EDTA anticoagulant whole blood samples of healthy individuals were selected, and were treated with 75% alcohol, liquid hand sanitizer, and gel hand sanitizer. The samples were detected by the Sysmex automatic blood analyzer, and the capillary blood smear was prepared. The appearance of cells was examined under the microscope. RESULTS: The absolute lymphocyte count, MCV, MCHC, and HGB were significantly increased, while PLT and MCH were significantly decreased after adding 1 µL 75% alcohol (p < 0.05). By adding 1 µL liquid hand sanitizer, the absolute counts of lymphocyte, neutrophil and basophilic, MCHC, HGB, and PLT were significantly increased (p < 0.05). The results of WBC, MCHC and HGB were significantly increased when 1 µL gel hand sanitizer was added. The classification of leukocytes was obviously abnormal and could not be detected by the instrument. The effect of gel hand sanitizer on WBC was time-dependent. Pyknotic, karyorrhexis, and karyolysis of nuclei were observed under microscope. CONCLUSIONS: Three kinds of hand sanitizers had effects on complete blood counts and leukocyte differential counts, in particular, gel hand sanitizer has the greatest impact on the results of tests. Therefore, gel hand sanitizer is not suitable for hand disinfection of operators before the collection of capillary blood samples.

Analytical and clinical performance evaluation of a new high-sensitivity cardiac troponin I assay.

OBJECTIVES: To validate the analytical performance and diagnostic accuracy for non-ST-segment elevation myocardial infarction (NSTEMI) with a new high-sensitivity cardiac troponin I (hs-cTnI) assay on the automated light-initiated chemiluminescent assay (LiCA®) platform. METHODS: Comprehensive analytical validations were performed, and the 99th percentile upper reference limit (URL) from apparently healthy individuals were established. We evaluated the diagnostic performance of the assay for NSTEMI. RESULTS: The limit of quantitation (LoQ) were 1.9 ng/L (20% CV) and 5.1 ng/L (10% CV). The sex-specific 99th percentile URLs were 17.6 ng/L (4.2% CV) for men (age 20-79y) and 14.2 ng/L (4.9% CV) for women (age 19-89y) in serum, 14.4 ng/L (4.9% CV) for men (age 19-88y) and 12.9 ng/L (5.2% CV) for women (age 19-87y) in plasma, respectively. Detection rates in healthy individuals were from 98.7 to 99.1%. The correlation coefficient and median bias between LiCA and Architect were 0.985 and 0.1% (-2.0-2.9%) in full analytical range of serum specimens. In lower range (<100 ng/L), LiCA had an overall positive bias 6.7% (-1.6-13.3%), R=0.949. At the specific medical decision levels (15.2, 26.2 and 64.0 ng/L), assay difference was estimated to be <10%. No significant differences on AUC, sensitivity and specificity, NPV and PPV were found between LiCA and Architect for the diagnosis of NSTEMI. CONCLUSIONS: LiCA hs-cTnI is a precise, highly sensitive and specific assay that meets the requirement of a 3rd generation (level 4) high-sensitivity method. The diagnostic accuracy of LiCA assay for NSTEMI is comparable to the established Architect hs-cTnI assay.

Increased Peripheral NKG2A-NKG2D+CD3-CD16+CD56dim NK Cell Subset Was Positively Correlated with Antiphospholipid Antibodies in Patients of Obstetric Antiphospholipid Syndrome.

Obstetric antiphospholipid syndrome (OAPS) is an autoimmune disorder with severe life-threatening complications shown during pregnancy. It has been reported that the increase in CD16+CD56dim natural killer (NK) cells in peripheral blood are risk factors for recurrent miscarriages, but this expression of CD16+CD56dim NK cells in OAPS patients has not been reported, and the mechanism is not clearly illustrated. In this study, we compared the distributional profiles of different NK cell subsets and the expressions of NK cell-activating receptors in peripheral blood of patients with OAPS and healthy women. Our results showed significantly increased NKG2A-NKG2D+ subset and decreased NKG2A+NKG2D- subset in CD3- CD16+CD56dim NK cells, CD3-CD16-CD56bright NK cells and CD56+T cells in OAPS patients compared with those in healthy control women. The CD27-CD11b+ subset significantly increased in CD3-CD16+CD56dim NK cells in OAPS patients compared with those in healthy control women. In addition, the NKG2A-NKG2D+ subset in CD3-CD16+CD56dim NK subset in triple positivity was higher than single positivity OAPS patients. At the optimal diagnostic threshold established by ROC analysis, using the cut-off of NKG2A-NKG2D+ and CD27-CD11b+ subset in CD3-CD16+CD56dim NK cells is 10.10% and 92.75%, the sensitivity of NKG2A-NKG2D+ and CD27-CD11b+ to detect patients with OAPS compared with healthy control results was 94.1% and 60.8%, and specificity was 84.2% and 89.5%, respectively, with an area under the curve (AUC) of 0.903 and 0.829, respectively. The NKG2A-NKG2D+ subset in CD3-CD16+CD56dim NK cells was positively correlated with the antiphospholipid antibodies lg anti-aCL IgG, lg anti-aCL IgM, lg anti-aCL IgA, lg anti-ß2GP1 IgM and Complement 4(C4), while the CD27+CD11b+ subset in CD3-CD16+CD56dim NK cells was correlated with lg anti-ß2GP1 IgG and lg anti-ß2GP1 IgA. These results suggested that the NK cytotoxic function enhanced in OAPS patients and unbalanced of NK activating receptors and inhibiting receptors may contribute to the immune pathogenesis of OAPS.

Study of risk factor of urinary calculi according to the association between stone composition with urine component.

Urolithiasis is a common urinary disease with high recurrence. The risk factor for the recurrence of calculi is not very clear. The object of the present study was to evaluate the association between calculi composition and urine component and analyse the risk factor for the recurrence of urolithiasis. In this study, a total of 223 patients with calculi and healthy control were enrolled, and the components of the calculi and urina sanguinis collected before surgery were analysed. Of the 223 patients, 157 were males and 66 were females. According to the stone composition, the case group was subdivided into three groups. 129 patients had single calcium oxalate stones, 72 had calcium oxalate stones mixed with other stones and 22 had other type of stones excluding calcium oxalate stones. Urine biochemicals were analysed and the associations were found between the chemicals in each group. Multivariate logistic analysis demonstrated that reduced urinary magnesium and uric oxalic acid were independent risk factors when comparing all cases with normal controls. Only decreased urinary magnesium was found to be a risk factor comparing the single calcium oxalate group with normal control group. Low level of urinary magnesium and uric oxalic acid were found to be risk factors comparing the mixed calcium oxalate group with normal control group. No risk factor was found comparing the other stone group with normal control group. In conclusion, there were clear relationships between stone components and urine chemicals. Urine chemicals might be risk factors to predicate the occurrence of urolithiasis.

Transfer of MicroRNA-216a-5p From Exosomes Secreted by Human Urine-Derived Stem Cells Reduces Renal Ischemia/Reperfusion Injury.

Human urine-derived stem cells (USCs) protect rats against kidney ischemia/reperfusion (I/R) injury. Here we investigated the role of USCs exosomes (USCs-Exos) in protecting tubular endothelial cells and miRNA transfer in the kidney. Human USCs and USCs-Exos were isolated and verified by morphology and specific biomarkers. USC-Exos played a protective role in human proximal tubular epithelial cells (HK-2) exposed to hypoxia/reoxygenation (H/R). USCs-Exos were rich in miR-216a-5p, which targeted phosphatase and tensin homolog (PTEN) and regulated cell apoptosis through the Akt pathway. In HK-2 cells exposed to H/R, incubation with USC-Exos increased miR-216-5p, decreased PTEN levels, and stimulated Akt phosphorylation. Exposure of hypoxic HK-2 cells to USCs-Exos pretreated with anti-miR-216a-5p can prevent the increase of miR-216-5p and Akt phosphorylation levels, restore PTEN expression, and promote apoptosis. The dual-luciferase reported gene assay in HK-2 cells confirmed that miR-216a-5p targeted PTEN. In rats with I/R injury, intravenous infusion of USCs-Exos can effectively induce apoptosis suppression and functional protection, which is associated with decreased PTEN. Infusion of exosomes from anti-miR-216a-5p-transfected USCs weakened the protective effect in the I/R model. Therefore, USCs-Exos can reduce renal I/R injury by transferring miR-216a-5p targeting PTEN. Potentially, USCs-Exos rich in miR-216a-5p can serve as a promising therapeutic option for AKI.