Tanshinone IIA modulates cancer cell morphology and movement via Rho GTPases-mediated actin cytoskeleton remodeling.

Actin filaments form unique structures with robust actin bundles and cytoskeletal networks affixed to the extracellular matrix and interact with neighboring cells, which are crucial structures for cancer cells to acquire a motile phenotype. This study aims to investigate a novel antitumor mechanism by which Tanshinone IIA (Tan IIA) modulates the morphology and migration of liver cancer cells via actin cytoskeleton regulation. 97H and Huh7 exhibited numerous tentacle-like protrusions that interacted with neighboring cells. Following treatment with Tan IIA, 97H and Huh7 showed a complete absence of cytoplasmic protrusion and adherens junctions, thereby effectively impeding their migration capability. The fluorescence staining of F-actin and microtubules indicated that these tentacle-like protrusions and cell-cell networks were actin-based structures that led to morphological changes after Tan IIA treatment by retracting and reorganizing beneath the membrane. Tan IIA can reverse the actin depolymerization and cell morphology alterations induced by latrunculin A. Tan IIA down-regulated actin and Rho GTPases expression significantly, as opposed to inducing Rho signaling activation. Preventing the activity of proteasomes and lysosomes had no discernible impact on the modifications in cellular structure and protein expression induced by Tan IIA. However, as demonstrated by the puromycin labeling technique, the newly synthesized proteins were significantly inhibited by Tan IIA. In conclusion, Tan IIA can induce dramatic actin cytoskeleton remodeling by inhibiting the protein synthesis of actin and Rho GTPases, resulting in the suppression of tumor growth and migration. Targeting the actin cytoskeleton of Tan IIA is a promising strategy for HCC treatment.

Identification of pattern recognition receptor genes in peripheral blood mononuclear cells and monocytes as biomarkers for the diagnosis of lupus nephritis.

BACKGROUND: The study aimed to investigate the diagnostic value of lupus-related pattern recognition receptors (PRRs) genes in peripheral blood mononuclear cells (PBMCs) and monocytes (MONs) for lupus nephritis (LN). METHODS: PBMCs were isolated from a cohort with 37 LN patients and 39 healthy controls (HCs), and MONs were derived from another cohort with 70 LN patients and 66 HCs. Q-PCR was used to measure the mRNA levels of CGAS, IFNB1, AIM2, IL1Β, NLRC4, NLRP3, NLRP12 and ZBP1 in the PBMCs and MONs. The Mann-Whitney U test was used to compare the data in LN patients and HCs. Eleven GEO datasets of SLE/LN were used to perform differentially expressed genes (DEGs) analysis to these PRR genes. Receiver operating characteristic (ROC) curve analysis was employed to assess the performance of individual genes or the disease prediction model established by combining multiple genes in LN diagnosis. Spearman correlation method was done to analyze the correlation between these PRRs and other clinical characteristics. RESULTS: The mRNA levels of five genes (AIM2, NLRC4, IL1B, NLRP12 and ZBP1) in PBMCs, and seven genes (CGAS, IFNB1, AIM2, IL1B, NLRP3, NLRP12 and ZBP1) in MONs of LN patients were significantly higher than those of HCs (P < 0.05). DEGs analysis based on the GEO datasets showed that ZBP1, AIM2 and IL1B were significantly increased in several datasets. The ROC curve analysis indicated that the area under curve (AUC) of the LN prediction models derived from PBMCs or MONs were 0.82 or 0.91 respectively. In addition, the expression levels of these PRRs were correlated with other clinical features in LN patients, including Anti-Sm, ESR, serum Cr, and C3. CONCLUSION: Our study suggests that these lupus-related PRRs might be served as potential biomarkers of LN.

Triptolide inhibits the proinflammatory potential of myeloid-derived suppressor cells via reducing Arginase-1 in rheumatoid arthritis.

Triptolide (TPT) is widely used in the treatment of rheumatoid arthritis (RA). However, its regulatory mechanisms are not fully understood. This study demonstrated that Myeloid-derived suppressor cells (MDSCs) were expanded in both RA patients and arthritic mice. The frequency of MDSCs was correlated with RA disease severity and T helper 17 (Th17) responses. MDSCs from RA patients promoted the polarization of Th17 cells in vitro, which could be substantially attenuated by blocking arginase-1 (Arg-1). TPT inhibited the differentiation of MDSCs, particularly the monocytic MDSCs (M-MDSCs) subsets, as well as the expression of Arg-1 in a dose dependent manner. Alongside, TPT treatment reduced the potential of MDSCs to promote the polarization of IL-17+ T cell in vitro. Consistently, TPT immunotherapy alleviated adjuvant-induced arthritis (AIA) in a mice model, and reduced the frequency of MDSCs, M-MDSCs and IL-17+ T cells simultaneously. The presented data suggest a pathogenic role of MDSCs in RA and may function as a novel and effective therapeutic target for TPT in RA.

Microarray Expression Profile of Exosomal circRNAs from High Glucose Stimulated Human Renal Tubular Epithelial Cells.

Introduction: Circular RNA (circRNAs) are a type of non-coding RNA (ncRNAs) with a wealth of functions. Recently, circRNAs have been identified as important regulators of diabetic kidney disease (DKD), owing to their stability and enrichment in exosomes. However, the role of circRNAs in exosomes of tubular epithelial cells in DKD development has not been fully elucidated. Methods: In our study, microarray technology was used to analyze circRNA expression in cell supernatant exosomes isolated from HK-2 cells with or without high glucose (HG) treatment. The small interfering RNAs (siRNA) and plasmid overexpression were used to validate functions of differentially expressed circRNAs. Results: We found that exosome concentration was higher in HG-stimulated HK-2 cells than in controls. A total of 235 circRNAs were significantly increased and 458 circRNAs were significantly decreased in the exosomes of the HG group. In parallel with the microarray data, the qPCR results showed that the expression of circ_0009885, circ_0043753, and circ_0011760 increased, and the expression of circ_0032872, circ_0004716, and circ_0009445 decreased in the HG group. Rescue experiments showed that the effects of high glucose on regulation of CCL2, IL6, fibronetin, n cadherin, e cadherin and epcam expression can be reversed by inhibiting or overexpressing these circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses indicated that circRNA parental genes are associated with glucose metabolism, lipid metabolism, and inflammatory processes, which are important in DKD development. Further analysis of circRNA/miRNA interactions indicated that 152 differentially expressed circRNAs with fold change (FC) ≥1.5 could be paired with 43 differentially expressed miRNAs, which are associated with diabetes or DKD. Discussion: Our results indicate that exosomal circRNAs may be promising diagnostic and therapeutic biomarkers, and may play a critical role in the progression of DKD.

A simple and accurate LC­MS/MS method for monitoring cyclosporin A that is suitable for high throughput analysis.

With time, the number of samples in clinical laboratories from therapeutic drug monitoring has increased. Existing analytical methods for blood cyclosporin A (CSA) monitoring, such as high-performance liquid chromatography (HPLC) and immunoassays, have limitations including cross-reactivity, time consumption, and the complicated procedures involved. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has long been considered the reference standard owing to its high accuracy, specificity, and sensitivity. However, large numbers of blood samples, multi-step preparation procedures, and longer analytical times (2.5-20 min) are required as a consequence of the different technical strategies, to ensure good analytical performance and routine quality assurance. A stable, reliable, and high throughput detection method will save personnel time and reduce laboratory costs. Therefore, a high throughput and simple LC-MS/MS method was developed and validated for the detection of whole-blood CSA with CSA-d12 as the internal standard in the present study. Whole blood samples were prepared through a modified one-step protein precipitation method. A C18 column (50x2.1 mm, 2.7 µm) with a mobile phase flow rate of 0.5 ml/min was used for chromatographic separation with a total running time of 4.3 min to avoid the matrix effect. To protect the mass spectrometer, only part of the sample after LC separation was allowed to enter the mass spectrum, using two HPLC systems coupled to one mass spectrometry. In this way, throughput was improved with detection of two samples possible within 4.3 min using a shorter analytical time for each sample of 2.15 min. This modified LC-MS/MS method showed excellent analytical performance and demonstrated less matrix effect and a wide linear range. The design of multi-LC systems coupled with one mass spectrometry may play a notable role in the improvement of daily detection throughput, speeding up LC-MS/MS, and allowing it to be an integral part of continuous diagnostics in the near future.

A population-based characterization study of anti-mitochondrial M2 antibodies and its consistency with anti-mitochondrial antibodies.

OBJECTIVE: This study aims to estimate the prevalence of anti-mitochondrial antibody subtype M2 (AMA-M2) and assess its consistency with AMA in a general population. METHODS: A total of 8954 volunteers were included to screen AMA-M2 using enzyme-linked immunosorbent assay. Sera with AMA-M2 >50 RU/mL were further tested for AMA using an indirect immunofluorescence assay. RESULTS: The population frequency of AMA-M2 positivity was 9.67%, of which 48.04% were males and 51.96% were females. The AMA-M2 positivity in males had a peak and valley value of 7.81% and 16.88% in those aged 40 to 49 and ≥70 years, respectively, whereas it showed a balanced age distribution in females. Transferrin and immunoglobulin M were the risk factors for AMA-M2 positivity and exercise was the only protective factor. Of 155 cases with AMA-M2 >50 RU/mL, 25 cases were AMA-positive, with a female-to-male ratio of 5.25:1. Only 2 people, with very high AMA-M2 of 760 and >800 RU/mL, met the diagnostic criteria of primary biliary cholangitis (PBC), making the prevalence of PBC 223.36 per million in southern China. CONCLUSION: We found that AMA-M2 has a low coincidence rate with AMA in the general population. A new decision-making point for AMA-M2 is needed to improve consistency with AMA and diagnostic accuracy.

Metabolite profile of COVID-19 revealed by UPLC-MS/MS-based widely targeted metabolomics.

The metabolic characteristics of COVID-19 disease are still largely unknown. Here, 44 patients with COVID-19 (31 mild COVID-19 patients and 13 severe COVID-19 patients), 42 healthy controls (HC), and 42 patients with community-acquired pneumonia (CAP), were involved in the study to assess their serum metabolomic profiles. We used widely targeted metabolomics based on an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The differentially expressed metabolites in the plasma of mild and severe COVID-19 patients, CAP patients, and HC subjects were screened, and the main metabolic pathways involved were analyzed. Multiple mature machine learning algorithms confirmed that the metabolites performed excellently in discriminating COVID-19 groups from CAP and HC subjects, with an area under the curve (AUC) of 1. The specific dysregulation of AMP, dGMP, sn-glycero-3-phosphocholine, and carnitine was observed in the severe COVID-19 group. Moreover, random forest analysis suggested that these metabolites could discriminate between severe COVID-19 patients and mild COVID-19 patients, with an AUC of 0.921. This study may broaden our understanding of pathophysiological mechanisms of COVID-19 and may offer an experimental basis for developing novel treatment strategies against it.

A modified LC-MS/MS method for the detection of whole blood tacrolimus and its clinical value in Chinese kidney transplant patients.

Background: For patients who treated with tacrolimus after kidney transplant, therapeutic drug monitoring is essential to improve their prognosis. However, previous detection methods have limitations, such as the overestimation and unacceptable bias in the immunoassays. Precision medicine has been challenged. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is recognized as the gold standard due to its accuracy and specificity, but lack of throughput and complex process limits its clinical application. Therefore, an accurate, simple and high throughput method for tacrolimus monitoring is needed for clinical practice. Methods: A modified LC-MS/MS method was introduced and validated. Whole blood samples were prepared by a one-step protein precipitation method. Chromatographic separation was achieved using a Phenomenex Kinetex 2.6 µm XB-C18 2.1 × 50 mm column with a total run time of 3.5 min to avoid matrix effect. An electrospray ionization source (ESI) was used in positive ion multiple reaction monitoring (MRM) mode for mass spectrometric detection. In order to protect the mass spectrometer, only part of the sample after LC separation was allowed to enter the mass spectrum, through a two HPLC systems coupled one mass spectrometry design. In this way, the instrument throughput is also improved and realizing the detection of 2 samples within 3.5 min and carried out a shorter analyzing time for each sample of 1.75 min. Additionally, we calculated tacrolimus-intrapatient variant (Tac-IPV) based on this modified method and assessed the prognostic value of Tac-IPV in Chinese kidney transplant patients. Results: The LC-MS/MS was modified by streamlining the procedure and increasing the throughput. The method proved to be accurate and reproducible with all performance parameters suitably meeting the clinical requirements over a calibration ranged from 0.37 to 42.90 ng/mL. Parameters such as linearity, limit of quantification (LoQ) and dilution integrity were validated with a clinical reportable range from 0.37 to 343.20 ng/mL, which was particularly useful for high drug concentrations patients (rare but very serious). Both cross-contamination and matrix effects were negligible. Clinical data of 83 patients showed that Tac-IPV was associated with poor kidney transplant outcome in Chinese (Hazard Ratio (HR) = 3.96, 4.75; 95% Cl: 1.10-14.21, 1.23-18.36; P < 0.05). Conclusions: This modified LC-MS/MS method possessed high throughput and simple sample preparation, allowing it to meet daily clinical needs. At the same time, Tac-IPV based on this modified LC-MS/MS had excellent prognostic value in kidney transplantation. These advantages have great significance for the individualized treatment of Chinese kidney transplant patients and broad application of Tac-IPV.

Long Noncoding RNA RP11-732M18.3 Promotes Glioma Angiogenesis by Upregulating VEGFA.

Gliomas are the most aggressive and common type of malignant brain tumor, with limited treatment options and a dismal prognosis. Angiogenesis, a hallmarks of cancer, is one of two critical events in the progression of gliomas. Accumulating evidence has demonstrated that in glioma dysregulated molecules like long noncoding RNAs (lncRNAs), are closely linked to tumorigenesis and prognosis. However, the effects of and mechanisms of action of lncRNAs during tumor angiogenesis are poorly understood. The effect of lncRNA RP11-732M18.3 on angiogenesis was elucidated through an intracranial orthotopic glioma model, immunohistochemistry, and an in vitro angiogenesis assay. Co-culture experiments and cell migration assays were performed to investigate the function of lncRNA RP11-732M18.3 in vitro. lncRNA RP11-732M18.3 increased CD31+ microvessel density, and overexpression of lncRNA RP11-732M18.3 resulted in poor mouse survival. lncRNA RP11-732M18.3 promoted endothelial cell migration and tube formation. Nomogram and Kaplan-Meier survival analyses indicated that higher VEGFA is correlated with a poor prognosis. Mechanistically, lncRNA RP11-732M18.3 promotes angiogenesis by increasing the nuclear level of EP300 and facilitating the transcription and secretion of VEGFA. Our study contributes to the latest understanding of glioma angiogenesis and prognosis. lncRNA RP11-732M18.3 may be a potential treatment target in glioma.

Novel Plasma Proteomic Biomarkers for Early Identification of Induction Chemotherapy Beneficiaries in Locoregionally Advanced Nasopharyngeal Carcinoma.

Background: Induction chemotherapy (IC) can alleviate locoregionally advanced nasopharyngeal carcinoma (LA-NPC), but effectiveness differs between patients, toxicity is problematic, and effective blood-based IC efficacy predictors are lacking. Here, we aimed to identify biomarkers for early identification of IC beneficiaries. Methods: Sixty-four pairs of matched plasma samples collected before and after IC from LA-NPC patients including 34 responders and 30 non-responders, as well as 50 plasma samples of healthy individuals, were tested using data-independent acquisition mass spectrometry. The proteins associated with clinical traits or IC benefits were investigated by weighted gene co-expression network analysis (WGCNA) and soft cluster analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional annotations were performed to determine the potential function of the identified proteins. The area under the receiver operating characteristic curve (AUC) was used to evaluate the performance of candidate biomarkers in predicting IC beneficiaries. Results: Compared with healthy individuals, 1027 differentially expressed proteins (DEPs) were found in the plasma of LA-NPC patients. Based on feedback from IC outcomes, 463 DEPs were identified in the pre-IC plasma between responders and non-responders. A total of 1212 DEPs represented the proteomic changes before and after IC in responders, while 276 DEPs were identified in post-IC plasma between responders and non-responders. WGCNA identified nine protein co-expression modules correlated with clinical traits. Soft cluster analysis identified four IC benefits-related protein clusters. Functional enrichment analysis showed that these proteins may play a role in IC via immunity, complement, coagulation, glycosaminoglycan and serine. Four proteins differentially expressed in all group comparisons, paraoxonase/arylesterase 1 (PON1), insulin-like growth factor-binding protein 3 (IGFBP-3), rheumatoid factor D5 light chain (v-kappa-3) and RNA helicase (DDX55), were associated with clinical traits or IC benefits. A four-protein model accurately identified potential IC beneficiaries (AUC=0.95) while diagnosing LA-NPC (AUC=0.92), and the prediction performance was verified using the models to confirm the effective IC (AUC=0.97) and evaluate IC outcome (AUC=0.94). Conclusion: The plasma protein profiles among IC responders and non-responders were different. PON1, IGFBP3, v-kappa-3 and DDX55 could serve as potential biomarkers for early identification of IC beneficiaries for individualised treatment of LA-NPC.

Long non­coding RNA AL355711 promotes smooth muscle cell migration through the ABCG1/MMP3 pathway.

Atherosclerosis and related cardiovascular diseases pose severe threats to human health worldwide. There is evidence to suggest that at least 50% of foam cells in atheromas are derived from vascular smooth muscle cells (VSMCs); the first step in this process involves migration to human atherosclerotic lesions. Long non­coding RNAs (lncRNAs) have been found to play significant roles in diverse biological processes. The present study aimed to investigate the role of lncRNAs in VSMCs. The expression of lncRNAs or mRNAs was detected using reverse transcription­quantitative polymerase chain reaction. The Gene Expression Omnibus datasets in the NCBI portal were searched using the key words 'Atherosclerosis AND tissue AND Homo sapiens' and the GSE12288 dataset. Gene expression in circulating leukocytes was measured to identify patients with coronary artery disease (CAD) or controls, and used to analyze the correlation coefficient and expression profiles. The protein level of ATP­binding cassette sub­family G member 1 (ABCG1) and matrix metalloproteinase (MMP)3 was determined using immunohistochemistry and western blot analysis. The analysis of mouse aortic roots was performed using Masson's and Oil Red O staining. The expression of lncRNA AL355711, ABCG1 and MMP3 was found to be higher in human atherosclerotic plaques or in patients with atherosclerotic CAD. The correlation analysis revealed that ABCG1 may be involved in the regulation between lncRNA AL355711 and MMP3 in atherosclerotic CAD. The knockdown of lncRNA AL355711 inhibited ABCG1 transcription and smooth muscle cell migration. In addition, lncRNA AL355711 was found to regulate MMP3 expression through the ABCG1 pathway. The expression of ABCG1 and MMP3 was found to be high in an animal model of atherosclerosis. The results indicated that lncRNA AL355711 promoted VSMC migration and atherosclerosis partly via the ABCG1/MMP3 pathway. On the whole, the present study demonstrates that the inhibition of lncRNA AL355711 may serve as a novel therapeutic target for atherosclerosis. lncRNA AL355711 in circulating leukocytes may be a novel biomarker for atherosclerotic CAD.

Comparative Performance of Four Nucleic Acid Amplification Tests for SARS-CoV-2 Virus.

BACKGROUND: COVID-19 is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was discovered in 2019 and spread around the world in a short time. SARS-CoV-2 nucleic acid amplification tests (NAATs) have been rapidly developed and quickly applied to clinical testing of COVID-19. Aim of this study was to evaluate the performance of four NAAT assays. METHODS: Limit of detection (LOD), precision, accuracy, analytical specificity and analytical interference studies on four NAATs (Daan, Sansure, Hybribio, and Bioperfectus) were performed according to Clinical Laboratory Standards Institute protocols and guidelines. The four NAATs were compared using 46 clinical samples. RESULTS: The LOD of the N gene for Daan, Sansure, and Hybribio was 500 copies/mL, and that for Bioperfectus was 1,000 copies/mL. The LOD of the ORF1ab gene for Daan, Bioperfectus, and Hybribio was 3,000 copies/mL, and that for Sansure was 2,000 copies/mL. A good precision was shown at the concentration above 20% of the LOD for all four NAATs, with all individual coefficients of variation below 3.6%. Satisfactory results were also observed in the accuracy, analytical specificity, and analytical interference tests. The results of the comparison test showed that Daan, Sansure, and Hybribio NAATs could detect the samples with a specificity of 100% (30/30) and a sensitivity of 100% (16/16), whereas Bioperfectus NAAT detected the samples with a specificity of 100% (30/30) and a sensitivity of 81.25% (13/16). However, no significant difference in sensitivity was found between Bioperfectus NAAT and the three other NAATs (p > 0.05). CONCLUSIONS: The four SARS-CoV-2 NAATs showed comparable performance, with the LOD of the N gene lower than the LOD of the ORF1ab gene.

Clinical relevance of serum α-l-fucosidase activity in the SARS-CoV-2 infection.

BACKGROUND AND AIMS: The reduced fucosylation in the spike glycoprotein of SARS-CoV-2 and the IgG antibody has been observed in COVID-19. However, the clinical relevance of α-l-fucosidase, the enzyme for defucosylation has not been discovered. MATERIALS AND METHODS: 585 COVID-19 patients were included to analyze the correlations of α-l-fucosidase activity with the nucleic acid test, IgM/IgG, comorbidities, and disease progression. RESULTS: Among the COVID-19 patients, 5.75% were double-negative for nucleic acid and antibodies. All of them had increased α-l-fucosidase, while only one had abnormal serum amyloid A (SAA) and C-reactive protein (CRP). The abnormal rate of α-l-fucosidase was 81.82% before the presence of IgM, 100% in the presence of IgM, and 66.2% in the presence of IgG. 73.42% of patients with glucometabolic disorders had increased α-l-fucosidase activity and had the highest mortality of 6.33%. The increased α-l-fucosidase was observed in 55.8% of non-severe cases and 72.9% of severe cases, with an odds ratio of 2.118. The α-l-fucosidase mRNA was irrelevant to its serum activity. CONCLUSION: The change in α-l-fucosidase activity in COVID-19 preceded the IgM and SAA and showed a preferable relation with glucometabolic disorders, which may be conducive to virus invasion or invoke an immune response against SARS-CoV-2.

Performance Evaluation of Antiphospholipid Antibodies on INOVA Quanta Flash System.

BACKGROUND: The diagnosis of antiphospholipid syndrome (APS) relies predominantly on the laboratory measurement of antiphospholipid antibodies (aPLs). We attempt to verify the analytical performance of anticardiolipin antibodies (aCL) IgA/IgG/IgM and anti-ß2-glycoprotein I antibodies (aß2GPI) IgA/IgG/IgM on a high-throughput automated immunoassay platform. METHODS: Limit of blank (LOB), limit of detection (LOD), imprecision, and linearity were calculated according to the corresponding Clinical and Laboratory Standards Institute (CLSI) guidelines protocols. The biological reference intervals (RIs) were verified in healthy individuals. RESULTS: The LoB of aCL IgA/IgG/IgM and aß2GPI IgA/IgG/IgM were 0.000, 1.200, 0.200, and 0.400, 1.250, 0.100, respectively. The LoD were 0.093, 1.715, 0.337 and 0.547, 2.174, 0.185 CU, respectively. All the within-run CVs and total CVs were less than the criterion at 10%. The linear analysis showed a good correlation between the predictive values and observed values with correlation coefficients greater than 0.99. CONCLUSIONS: The BIO-FLASH automated chemiluminescent analyzer performed well in measuring aPLs.

Establishing Classifiers With Clinical Laboratory Indicators to Distinguish COVID-19 From Community-Acquired Pneumonia: Retrospective Cohort Study.

BACKGROUND: The initial symptoms of patients with COVID-19 are very much like those of patients with community-acquired pneumonia (CAP); it is difficult to distinguish COVID-19 from CAP with clinical symptoms and imaging examination. OBJECTIVE: The objective of our study was to construct an effective model for the early identification of COVID-19 that would also distinguish it from CAP. METHODS: The clinical laboratory indicators (CLIs) of 61 COVID-19 patients and 60 CAP patients were analyzed retrospectively. Random combinations of various CLIs (ie, CLI combinations) were utilized to establish COVID-19 versus CAP classifiers with machine learning algorithms, including random forest classifier (RFC), logistic regression classifier, and gradient boosting classifier (GBC). The performance of the classifiers was assessed by calculating the area under the receiver operating characteristic curve (AUROC) and recall rate in COVID-19 prediction using the test data set. RESULTS: The classifiers that were constructed with three algorithms from 43 CLI combinations showed high performance (recall rate >0.9 and AUROC >0.85) in COVID-19 prediction for the test data set. Among the high-performance classifiers, several CLIs showed a high usage rate; these included procalcitonin (PCT), mean corpuscular hemoglobin concentration (MCHC), uric acid, albumin, albumin to globulin ratio (AGR), neutrophil count, red blood cell (RBC) count, monocyte count, basophil count, and white blood cell (WBC) count. They also had high feature importance except for basophil count. The feature combination (FC) of PCT, AGR, uric acid, WBC count, neutrophil count, basophil count, RBC count, and MCHC was the representative one among the nine FCs used to construct the classifiers with an AUROC equal to 1.0 when using the RFC or GBC algorithms. Replacing any CLI in these FCs would lead to a significant reduction in the performance of the classifiers that were built with them. CONCLUSIONS: The classifiers constructed with only a few specific CLIs could efficiently distinguish COVID-19 from CAP, which could help clinicians perform early isolation and centralized management of COVID-19 patients.

Differential diagnosis and prospective grading of COVID-19 at the early stage with simple hematological and biochemical variables.

We evaluated simple laboratory variables to discriminate COVID-19 from bacterial pneumonia or influenza and for the prospective grading of COVID-19. Multivariate logistic regression and receiver operating characteristic curve were used to estimate the diagnostic performance of the significant discriminating variables. A comparative analysis was performed with different severity. The leukocytosis (P = 0.017) and eosinopenia (P = 0.001) were discriminating variables between COVID-19 and bacterial pneumonia with area under the curve (AUC) of 0.778 and 0.825. Monocytosis (P = 0.003), the decreased lymphocyte-to-monocyte ratio (P < 0.001), and the increased neutrophil-to-lymphocyte ratio (NLR) (P = 0.028) were predictive of influenza with AUC of 0.723, 0.895, and 0.783, respectively. Serum amyloid protein, lactate dehydrogenase, CD3+ cells, and the fibrinogen degradation products had a good correlation with the severity of COVID-19 graded by age (≥50) and NLR (≥3.13). Simple laboratory variables are helpful for rapid diagnosis on admission and hierarchical management of COVID-19 patients.

Clinical Correlates and Reference Intervals for Cystatin C in a Han Population from Southeast China.

BACKGROUND: Cystatin C (CysC) is an endogenous filtration marker for estimation of kidney function. This study aimed to define the reference interval (RI) for serum CysC in a southeast Chinese adult population and to explore the variables that affect serum CysC levels. METHODS: 532 reference individuals (259 male, 273 female, aged 18 - 79 years) were recruited from Guangzhou, China. Multiple regression analysis (MRA) was used to investigate the association between serum CysC levels and clinical factors including age, gender, body mass index, lifestyle, and biochemistry parameters. Reference values were defined using a parametric method according to Clinical and Laboratory Standards Institute guideline (C28A3). RESULTS: The mean serum CysC levels were significantly lower in females than in males (p < 0.001). Serum CysC levels increased with age (~0.047 mg/L increase per decade). MRA demonstrated that serum CysC levels correlated significantly with serum creatinine (Cr), high density lipoprotein (HDL-C), alkaline phosphatase (ALP), albumin (ALB), and uric acid (UA) concentrations, although their relationships were less prominent than those of gender or age. The RIs for serum CysC levels were calculated at 0.73 - 1.17 mg/L for subjects aged 18 - 49 years and at 0.73 - 1.49 mg/L for those aged 50 - 79 years. CONCLUSIONS: The RIs for serum CysC were established in a southeast Chinese population. In addition to gender and age, serum Cr, HDL-C, ALP, ALB, and UA also influenced serum CysC levels.

Peritumoral stromal neutrophils are essential for c-Met-elicited metastasis in human hepatocellular carcinoma.

Inflammation is a component of tumor progression mechanisms. Neutrophils are a common inflammatory infiltrate in many tumors, but their regulation and functions in neoplasia are not understood. Here, we showed, in detailed studies of c-Met molecule in 225 untreated patients with hepatocellular carcinoma (HCC), that high infiltration of neutrophils in HCC tissues determined malignant cell c-Met-associated clinical outcome of patients. High infiltration of neutrophils in HCCs determined malignant cell c-Met-associated clinical outcome of patients. Neutrophils were enriched predominantly in invading tumor edge of HCCs; the accumulated neutrophils were the major source of c-Met ligand HGF in HCCs. Exposure to HCC environments resulted in neutrophil activation and the following HGF production. Inhibiting the activities of Erk1/2, p38, and NF-κB, but not the phosphorylation of AKT or JNK, successfully attenuated the neutrophil HGF production induced by HCC environments. Further investigation revealed that GM-CSF was an important determinant in malignant cell-elicited neutrophil HGF production in vitro and in vivo. Moreover, we demonstrated that tumor neutrophils, via HGF/c-Met interaction, actively enhanced the metastasis of malignant cells in vitro and in vivo. These data provide direct evidence supporting the critical role of neutrophils in human tumor progression and reveal a fine-tuned collaborative action between cancer cells and immune cells in tumor milieu, which reroutes the immune activation into a tumor-promoting direction.

Performance evaluation of the Sysmex Cystatin C assay on the Roche Modular P Analyzer.

OBJECTIVES: Serum Cystatin C (CysC) is a promising endogenous marker for estimation of glomerular filtration rate. This study evaluated the analytical performance of the Sysmex CysC assay on the Roche Modular P Chemistry Analyzer (Modular P). DESIGN AND METHODS: The evaluation of imprecision, linearity, recovery, and interference were performed in accordance with the relevant Clinical and Laboratory Standards Institute guidelines protocols, using quality controls and patient samples. Comparison studies were conducted against the Siemens and Roche assay for CysC. RESULTS: At CysC concentrations of 0.45 and 1.80mg/L, the within-run coefficient of variations (CVs) were 2.81% and 2.04%, respectively, and the between-run CVs were 3.14% and 3.26%. The CysC assay was proven throughout the measuring range from 0.10 to 8.00mg/L. Good correlations were achieved among the 3 CysC assays. The Sysmex assay appeared to report higher results than the Siemens assay. No interference was detected from hemoglobin 5g/L, bilirubin (free or conjugated) 200mg/L, or chyle 1500 formazin turbidity units. CONCLUSIONS: The Sysmex CysC assay performed well with regard to basic performance, including precision, dilution linearity, and effects of interfering substances, and is an acceptable assay for the determination of CysC on the Modular P.

[Expression characteristics of differentiation antigens on granulocytes in patients with megaloblastic anemia].

This study was aimed to explore the change characteristics of cell differentiation antigen (CD) on bone marrow (BM) granulocytes in patients,with megaloblastic anemia (MA). In combination with BM cell morphology, hemogram, level of blood serum folic acid, level of Vit B(12), cell genetics and biological examination data, the BM granulocytes differentiation antigens in 13 patients with MA were detected by flow cyto metry and analyzed retrospectively, in order to summarize the variation characteristics of CD13, CD33 and CD15 expressed on myeloid cells in patient with MA, including forward scatter light (FSC) and side scatter light (SSC) signal intensity, then these findings were compared with that in normal healthy persons. The results showed that the expression rates of CD13, CD15 and CD33 on granulocytic in patients with MA and normal healthy persons were (44.53 ± 16)%, (96.16 ± 2.67)%, (80.81 ± 14.71)% and (62.33 ± 11.02)%, (99.53 ± 0.46)%, (70.00 ± 7.81)% respectively, in which the expression rate of CD13 and CD15 in patients with MA decreased (P < 0.01), while the expression rate of CD33 increased (P < 0.01). The mean fluorescence intensity (MFI) of CD13, CD15, CD33, SSC and FSC in MA patients and normal healthy persons were 3.39 ± 1.41, 14.29 ± 6.59, 1.95 ± 0.94, 478.78 ± 70.43, 633.46 ± 75.53 and 5.12 ± 1.15, 20.67 ± 5.13, 1.04 ± 0.17, 332.00 ± 38.16, 537.00 ± 16.70 respectively, in which the MFI of CD13 and CD15 on granulocytes in MA patients decreased (P < 0.01),while the MFI of FSC,SSC and CD33 increased (P < 0.01 and P < 0.05). It is concluded that not only the morphology of BM granulocytes in patents with MA shows dysmaturity, but the expressing feature of differentiation antigens on BM granulocytes in MA patients also displays dysmaturity.These findings will contribute to the clinical diagnosis of MA patients.