Imbalance of T follicular helper cell subsets trigger the differentiation of pathogenic B cells in idiopathic membranous nephropathy.

OBJECTIVE: This study aims to elucidate the role of T follicular helper (Tfh) cells and their subsets in idiopathic membranous nephropathy (IMN). METHODS: The frequencies of Tfh cell subsets and B cell subsets in peripheral blood (PB) were detected in both IMN patients and healthy controls (HCs). The involvement of Tfh cells in the disease pathogenesis was examined by coculturing human Tfh cells with B cells. The dynamic changes of Tfh cells in PB or spleen were monitored in passive Heymann nephritis (PHN) rats. RESULTS: The frequencies of circulating Tfh (cTfh) cells, cTfh2 cells, and plasmablasts were enriched in the PB of patients with IMN. cTfh cells expressed higher ICOS, and lower BTLA than healthy counterparts. The frequency of ICOS + cTfh2 was associated with the severity of IMN, including 24h urine protein, IgG4 concentration and the IgG4: IgG ratio. Positive correlations were also observed between the frequency of cTfh2 cells with plasmablasts, serum IL-21 and IL-4 levels. Importantly, cTfh cells isolated from IMN patients were able to induce the differentiation of B cells to memory B cells (MBC) and plasmablasts, this process could be substantially attenuated by blocking the IL-21. Similar increases of ICOS + cTfh cells were also detected in spleen of PHN rats, concomitant with elevated urine protein levels. CONCLUSIONS: Collectively, our results demonstrate that the imbalance of cTfh cell subsets play a crucial pathogenic role in IMN by inducing the differentiation of B cells through IL-21, and cTfh2 cells might serve as useful markers to evaluate the progression of IMN.

Development of simultaneous quantitation method for 20 free advanced glycation end products using UPLC-MS/MS and clinical application in kidney injury.

Advanced glycation end products (AGEs), derived from the non-enzymatic glycation reaction, are defined as glycotoxins in various diseases including aging, diabetes and kidney injury. Exploring AGEs as potential biomarkers for these diseases holds paramount significance. Nevertheless, the high chemical structural similarity and great heterogeneity among AGEs present a formidable challenge when it comes to the comprehensive, simultaneous, and accurate detection of multiple AGEs in biological samples. In this study, an UPLC/MS/MS method for simultaneous quantification of 20 free AGEs in human serum was firstly established and applied to quantification of clinical samples from individuals with kidney injury. Simple sample preparation method through protein precipitation without derivatization was used. Method performances including imprecision, accuracy, sensitivity, linearity, and carryover were systematically validated. Intra- and inter- imprecision of 20 free AGEs were 1.93-5.94 % and 2.30-8.55 %, respectively. The method accuracy was confirmed with good recoveries ranging from 96.40 % to 103.25 %. The LOD and LOQ were 0.1-3.13 ng/mL and 0.5-6.25 ng/mL, respectively. Additionally, the 20 free AGEs displayed excellent linearity (R2 >0.9974) across a wide linear range (1.56-400 ng/mL). Finally, through simultaneous quantitation of 20 Free AGEs in 100 participants including kidney injury patient and healthy controls, we identified six free AGEs, including N6-carboxyethyl-L-arginine (CEA), N6-carboxymethyl-L-lysine (CML), methylglyoxal-derived hydroimidazolones (MG-H), N6-formyl-lysine, N6-carboxymethyl-L-arginine (CMA), and glyoxal-derived hydroimidazolone (G-H), could well distinguish kidney injury patients and healthy individuals. Among them, the levels of four free AGEs including CML, CEA, MG-H, and G-H strongly correlate with traditionally clinical markers of kidney disease. The high area under the curve (AUC) values (AUC=0.965) in receiver operating characteristic (ROC) curve indicated that these four free AGEs can be served as combined diagnostic biomarkers for the diagnosis of kidney disease.

Elevated circulating CD19+CD24hiCD38hi B cells display pro-inflammatory phenotype in idiopathic membranous nephropathy.

CD19+CD24hiCD38hi regulatory B cells exert immunosuppressive functions by producing IL-10, but their role in idiopathic membranous nephropathy (IMN) remains elusive. Here, we investigated the frequency and functional changes of circulating CD19+CD24hiCD38hi B cells and evaluated the correlation of CD19+CD24hiCD38hi B cells with clinical features and T helper cell subsets in IMN patients. Compared with healthy controls (HCs), IMN patients showed an increased frequency of CD19+CD24hiCD38hi B cells, but a significant reduction in the percentage of CD19+CD24hiCD38hi B cells was observed 4 weeks after cyclophosphamide treatment. The frequency of CD19+CD24hiCD38hi B cells was positively correlated with the levels of 24h urinary protein, but negatively correlated with serum total protein and serum albumin, respectively. CD19+CD24hiCD38hi B cells in IMN patients displayed a skewed pro-inflammatory cytokine profile with a higher level of IL-6 and IL-12, but a lower concentration of IL-10 than their healthy counterparts. Accompanied by upregulation of Th2 and Th17 cells in IMN patients, the percentage of CD19+CD24hiCD38hi B cell subset was positively associated with Th17 cell frequency. In conclusion, CD19+CD24hiCD38hi B cells were expanded but functionally impaired in IMN patients. Their altered pro-inflammatory cytokine profile may contribute to the pathogenesis of IMN.

Development of matrix-based reference materials for 17 beta-estradiol by the recommended reference method of ID-LC-MS/MS.

We developed and evaluated two-level, namely 2017011 and 2017012, serum-based reference materials (RMs) for 17 beta-estradiol (17 ß-E2) by the reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) from the remaining serum samples after routine clinical tests, to help improve clinical routine testing and provide the traceability of results. This paper describes the development process of these RMs. The National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 6004-a was used as the primary RM for the measurement of 17 ß-E2. These serum-based RMs showed satisfactory homogeneity and stability. They also assessed the commutability between the reference method and the three routine clinical immunoassay systems. Besides, a collaborative study was carried out in five reference laboratories, all of which had been accredited by the China National Accreditation Service for Conformity Assessment (CNAS) in accordance with ISO/WD 15725-1. Statistical analysis of raw results and uncertainty assessment obtained certified values: 2017011 was 445.2 ± 39.0 pmol/L, and 2017012 was 761.9 ± 35.5 pmol/L.

Maintenance of cathepsin D-dependent autophagy-lysosomal function protects against cardiac ischemia/reperfusion injury.

Cardiac ischemia/reperfusion(I/R) induced-cardiac vascular endothelial injury is an important pathological process that appears in the early stage of cardiac I/R injury. The autophagy-lysosomal pathway is essential for the maintenance of cellular homeostasis. However, in cardiac I/R injury, the role of the autophagy-lysosomal pathway is controversial. The present study aimed to use oxygen-glucose deprivation/oxygen-glucose resupply(OGD/OGR) in human coronary artery endothelial cells(HCAECs) with I/R injury to assess the role of the autophagy-lysosomal pathway in I/R-induced endothelial injury. The results revealed lysosomal dysfunction and impaired autophagic flux in endothelial cells exposed to OGD/OGR. Meanwhile, our data showed that the levels of cathepsin D(CTSD) decreased time-dependently. Knockdown of CTSD caused lysosomal dysfunction and impaired autophagic flux. Conversely, restoration of CTSD levels protected HCAECs against OGD/OGR induced-defects in autophagy-lysosomal function and cellular damage. Our findings indicated that I/R induced-impaired autophagic flux, rather than excessive autophagic initiation, mediates endothelial cells injury. The maintenance of autophagy-lysosomal function is critical to protect endothelial cells against I/R injury, and CTSD is a key regulator. Thus, strategies focused on restoring CTSD function are potentially novel treatments for cardiac reperfusion injury.

Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1highCD11b+ cells elimination in hepatocellular carcinoma.

Cryoablation (CRA) and microwave ablation (MWA) are two main local treatments for hepatocellular carcinoma (HCC). However, which one is more curative and suitable for combining with immunotherapy is still controversial. Herein, CRA induced higher tumoral PD-L1 expression and more T cells infiltration, but less PD-L1highCD11b+ myeloid cells infiltration than MWA in HCC. Furthermore, CRA had better curative effect than MWA for anti-PD-L1 combination therapy in mouse models. Mechanistically, anti-PD-L1 antibody facilitated infiltration of CD8+ T cells by enhancing the secretion of CXCL9 from cDC1 cells after CRA therapy. On the other hand, anti-PD-L1 antibody promoted the infiltration of NK cells to eliminate PD-L1highCD11b+ myeloid cells by antibody-dependent cell-mediated cytotoxicity (ADCC) effect after CRA therapy. Both aspects relieved the immunosuppressive microenvironment after CRA therapy. Notably, the wild-type PD-L1 Avelumab (Bavencio), compared to the mutant PD-L1 atezolizumab (Tecentriq), was better at inducing the ADCC effect to target PD-L1highCD11b+ myeloid cells. Collectively, our study uncovered the novel insights that CRA showed superior curative effect than MWA in combining with anti-PD-L1 antibody by strengthening CTL/NK cell immune responses, which provided a strong rationale for combining CRA and PD-L1 blockade in the clinical treatment for HCC.

TREM2+ macrophages suppress CD8+ T-cell infiltration after transarterial chemoembolisation in hepatocellular carcinoma.

BACKGROUND & AIMS: The immune landscape of hepatocellular carcinoma (HCC) following transarterial chemoembolisation (TACE) remains to be clarified. This study aimed to characterise the immune landscape following TACE and the underlying mechanism of HCC progression. METHODS: Tumour samples from five patients with treatment-naive HCC and five patients who received TACE therapy were collected and subjected to single-cell RNA sequencing. Another 22 paired samples were validated using immunofluorescence staining and flow cytometry. To clarify the underlying mechanisms, in vitro co-culture experiments and two types of TREM2-KO/WT mouse models, namely, an HCC cell orthotopic injection model and a spontaneous HCC model, were used. RESULTS: A reduced number of CD8+ T cells and an increased number of tumour-associated macrophages (TAMs) were observed in the post-TACE microenvironment. TACE therapy reduced the cluster CD8_C4, which was highly enriched with tumour-specific CD8+ T cells of pre-exhausted phenotype. TREM2 was found to be highly expressed in TAMs following TACE, which was associated with a poor prognosis. TREM2+ TAMs secreted less CXCL9 but more galectin-1 than did TREM2- TAMs. Galectin-1 promoted PD-L1 overexpression in vessel endothelial cells, impeding CD8+ T cell recruitment. TREM2 deficiency also increased CD8+ T cell infiltration, which inhibited tumour growth in both in vivo HCC models. More importantly, TREM2 deficiency enhanced the therapeutic effect of anti-PD-L1 blockade. CONCLUSIONS: This study shows that TREM2+ TAMs play an important role in suppressing CD8+ T cells. TREM2 deficiency increased the therapeutic effect of anti-PD-L1 blockade by enhancing antitumour activity of CD8+ T cells. These findings explain the reasons for recurrence and progression after TACE and provide a new target for HCC immunotherapy after TACE. IMPACT AND IMPLICATIONS: Studying the immune landscape in post-TACE HCC is important to uncover the mechanisms of HCC progression. By using scRNA sequencing and functional assays, we discovered that both the number and function of CD8+ T cells are compromised, whereas the number of TREM2+ TAMs is increased in post-TACE HCC, correlating with worse prognosis. Moreover, TREM2 deficiency dramatically increases CD8+ T cell infiltration and augments the therapeutic efficacy of anti-PD-L1 blockade. Mechanistically, TREM2+ TAMs display lower CXCL9 and increased Gal-1 secretion than do TREM2- TAMs, with Gal-1 mediating the overexpression of PD-L1 in vessel endothelial cells. These results suggest that TREM2 could be a novel immunotherapeutic target for patients treated with TACE in HCC. This provides an opportunity to break the plateau of limited therapeutic effect. This study has the value of understanding the tumour microenvironment of post-TACE HCC and thinking a new strategy of immunotherapy in the field of HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

Quantitative Detection of 15 Serum Bile Acid Metabolic Products by LC/MS/MS in the Diagnosis of Primary Biliary Cholangitis.

To determine 15 bile acid metabolic products in human serum by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and value their diagnostic outcome in primary biliary cholangitis (PBC). Serum from 20 healthy controls and 26 patients with PBC were collected and went LC/MS/MS analysis of 15 bile acid metabolic products. The test results were analyzed by bile acid metabolomics, and the potential biomarkers were screened and their diagnostic performance was judged by statistical methods such as principal component and partial least squares discriminant analysis and area under curve (AUC). 8 differential metabolites can be screened out: Deoxycholic acid (DCA), Glycine deoxycholic acid (GDCA), Lithocholic acid (LCA), Glycine ursodeoxycholic acid (GUDCA), Taurolithocholic acid (TLCA), Tauroursodeoxycholic acid (TUDCA), Taurodeoxycholic acid (TDCA), Glycine chenodeoxycholic acid (GCDCA). The performance of the biomarkers was evaluated by the AUC, specificity and sensitivity. In conclusion, DCA, GDCA, LCA, GUDCA, TLCA, TUDCA, TDCA and GCDCA were identified as eight potential biomarkers to distinguish between healthy people and PBC patients by multivariate statistical analysis, which provided reliable experimental basis for clinical practice.

Serum 25-hydroxyvitamin D status of a large Chinese population from 30 provinces by LC-MS/MS measurement for consecutive 3 years: differences by age, sex, season and province.

PURPOSE: We aimed to describe the vitamin D status and its distribution in different age groups, sexes, seasons, and provinces of a large Chinese population. METHODS: This study retrospectively analyzed 1,528,685 results of serum 25-hydroxyvitamin D (25(OH)D) in the central laboratory of KingMed Diagnostics. The samples were from the individuals aged 0-119 years old in 30 provinces of China. Serum 25(OH)D was measured by an accurate commercial liquid chromatography-tandem mass spectrometry (LC-MS/MS) method from January 2017 to December 2019. The subjects were stratified by age, sex, the season of blood collection, and the province of residence. RESULTS: The median 25(OH)D concentration was 25.5 ng/mL (interquartile range (IQR) 18.7-32.7 ng/mL) in males and 20.8 ng/mL (IQR 14.4-28.2 ng/mL) in females. Overall, the median 25(OH)D concentration decreased with age in both males and females. Males had a 0.2-2.4 ng/mL higher median 25(OH)D concentration than females in different age groups. Vitamin D deficiency (25(OH)D < 15 ng/mL for the individuals under 14 years old; < 20 ng/mL for the individuals over 14 years old) was found in 21.3% of males and 43.6% of females. Significant seasonal variation of serum 25(OH)D concentrations was repeatedly observed in 3 years, with median concentration higher in summer (25.3 ng/mL (IQR 19.3-31.9 ng/mL)) and lower in winter (18.5 ng/mL (IQR 12.3-26.6 ng/mL)). Vitamin D status varied by province. The median 25(OH)D concentration was the highest in Hainan (31.0 ng/mL (IQR 24.9-39.2 ng/mL)) and the lowest in Qinghai (14.4 ng/mL (IQR 9.6-20.0 ng/mL)). 25(OH)D2 was detected in 12.2% of the results, and no significant seasonal variation was observed. CONCLUSION: In China, vitamin D deficiency is prevalent in the population participating in clinical vitamin D measurement. Age and sex differences in vitamin D levels were observed in our study. Seasonal variation and provincial differences are important aspects of serum vitamin D status. 25(OH)D2 cannot be ignored entirely in clinical measurement practice in China.

Multidimensional analysis of the essential elements in pregnant women's whole blood and characterization of maternal status by elemental pattern.

BACKGROUND: During pregnancy, the fetus needs to obtain a lot of nutrients from the mother, but the micronutrient deficiencies in pregnancy are not clear at present, and there is no reliable basis for nutrient intake and supplement. The purpose of this study was to understand the levels of essential elements in whole blood of pregnant women during various pregnancy stages at different ages and in different regions, to evaluate the deficiency of essential elements in Chinese pregnant women, and to explore the feasibility of using the elemental pattern to characterize maternal status. METHODS: Whole blood samples of 11222 healthy pregnant women enrolled in different areas of China from Jan-Dec 2019, were analyzed for concentrations of six essential elements including Mn, Cu, Zn, Ca, Mg, and Fe, using the inductively coupled plasma mass spectrometer. A retrospective comparative study during different pregnancy periods at different ages and in different regions in whole blood essential elements content from non-pregnant normal women and pregnant normal women was developed using multivariate statistical analysis. Principal component analysis evaluation elemental pattern was used to characterize pregnancy status of pregnant women. RESULTS: In general, the levels of six essential elements in whole blood of pregnant women can satisfy the needs of normal physiological activities. With the development of pregnancy, the contents of Cu and Mn increased, while the contents of Fe and Mg decreased, and the contents of Zn and Ca have no noteworthy change. At the same gestation stage, the Cu content in whole blood of elderly pregnant women was higher. There were some differences in whole blood essential elements content of pregnant women in different regions. Principal component analysis and heat map analysis showed the feasibility of using bioinformatics research strategies to identify different pregnancies. CONCLUSIONS: There are differences in the content of whole blood essential elements of women at different stages of pregnancy in different regions. It was found that there was no obvious deficiency in whole blood essential elements levels of pregnant women in recent years. The pattern of essential elements has a certain application potential in the evaluation of pregnancy and pregnant women's health status.

Light-initiated chemiluminescent assay of 17ß-estradiol metrological traceability system established by manufacturer according to ISO17511:2020 and basic performance evaluation performed by clinical end-users.

BACKGROUND: In order to ensure the accuracy of the product, we established 1st model of metrological traceability hierarchy for light-initiated chemiluminescent assay (LICA) of 17ß-estradiol (E2 ) at the manufacturer, based on International Organization for Standardization (ISO) 17511:2020. Moreover, we verified/validated the basic performance (such as matrix effect and long-term stability of end-user IVD MD calibrator, precision, linearity interval, accuracy/ trueness, and detection capability) at the clinical end-user. METHODS: Human serum samples were used in this study. E2 was detected by mass spectrometry (MS) and LICA. The metrological traceability of LICA for E2 was established according to ISO 17511: 2020 standards, and pools of human samples were used as the m.3. secondary calibrator. Precision was validated according to Clinical and Laboratory Standards Institute (CLSI) EP05-A3. The linear interval was verified according to CLSI EP06-ED2. Comparison of accuracy and trueness of E2 with MS and Roche according to CLSI EP09-A3. The detection capability was validated according to EP17-A2. Matrix effect and long-term stability evaluation of end-user IVD MD calibrator were carried out according to CLSI EP14-A2, EP25-A. Statistical software was used for data analyses. RESULTS: The use of pools of human samples and fine adjusting calibrators ensured the accuracy of end-user test results. The metrological traceability of LICA for E2 was established. It showed excellent precision, meeting the requirements of allowable imprecision (7.5%). The allowable deviation from linearity (ADL) of 5% was allowed to show a good linear interval (12.52-4167.25 pg/ml). The accuracy/ trueness was verified, and relative deviation in the medical decision level met the performance specification of 10.03% compared with MS or Roche. The validated limit of blank, limit of detection, and limit of quantitation of E2 were 4.95 pg/ml, 8.93 pg/ml, and 9.88 pg/ml, respectively (the allowed imprecision is 20.00%). The interference rate of E2 ranged from -5.5% to 6.6%. CONCLUSION: LICA showed high sensitivity, high specificity, excellent precision, wide linearity interval, IVD MD calibrator has long-term stability, and no matrix effect. The metrological traceability of E2 established by using pools of human samples as M.3. can deliver accuracy to the end-user IVD MD and show good consistency with MS and Roche.

Discovery of a Novel Stilbene Derivative as a Microtubule Targeting Agent Capable of Inducing Cell Ferroptosis.

Microtubule targeting agents (MTAs) are used as clinically effective chemotherapies for cancer treatment but might be limited by the acquired or intrinsic resistance of cancer cells to apoptosis. The vulnerability of therapy-resistant cancers to ferroptosis provides an alternative way to overcome drug resistance. In this study, on the basis of the MTAs obtained in our previous studies, a series of MTAs were synthesized, and detailed structure-activity relationships were obtained through extensive molecular dynamics studies. Among them, a diphenylethene derivative, compound 33, displayed the most potent activity in vitro and in vivo, with IC50 values of 10-50 nM toward six cancer cell lines and a 78.6% tumor growth inhibition in vivo. Interestingly, although it acted as the MTA, compound 33 triggered cell death mainly through cell ferroptosis rather than apoptosis, which might provide an alternative way to eradicate apoptosis-related drug resistance.

Development of human serum matrix-based unconjugated estriol-certified reference material by recommended ID-LC-MS/MS reference method.

To solve long-term lack of traceability of commercial calibrator kits and standardize clinical routine assays, we developed a human serum matrix-based unconjugated estriol (uE3) reference material (RM) with five concentration gradients. The RMs of uE3 were certified by the National Institute of Metrology (NIM) with the codes of GBW (E) 091048, GBW (E) 091049, GBW (E) 091050, GBW (E) 091051, and GBW (E) 091052. The RMs were determined by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method which was developed in our group and recommended by the Joint Committee on Traceability on Laboratory Medicine (JCTLM). GBW09224 is intended for use as a primary reference material to enable the SI-traceable measurement of uE3. This study describes the development process of these certified RMs. The candidate material was prepared by collecting from the remaining serum samples after routine clinical testing. Satisfactory homogeneity and stability were shown in these RMs. They are also commutable between the reference method and the three routine clinical immunoassay systems. To improve the accuracy of value assignment, a collaborative study in nine reference laboratories was conducted which was performed according to ISO/WD 15725-1 and all of the reference laboratories have been confirmed by China National Accreditation Service for Conformity Assessment (CNAS). The raw results were statistically analyzed and processed, coupled with uncertainty evaluation, to obtain the certified value: GBW (E) 091048 is 22.1 ± 1.3 nmol/L, GBW (E) 091049 is 33.6 ± 1.6 nmol/L, GBW (E) 091050 is 10.4 ± 0.8 nmol/L, GBW (E) 091051 is 15.5 ± 1.0 nmol/L, GBW (E) 091052 is 47.0 ± 2.0 nmol/L. The preparation process of human serum matrix-based reference material and the lack of these type of secondary (commutable) reference material of unconjugated estriol lead to the interruption of its traceability chain, which is a problem to be solved in its standardization as mentioned in the metrological traceability in ISO 17511, 2020.

Performances Evaluation of Four Systems for Homocysteine Determination by LC-MS/MS Reference Method.

BACKGROUND: The aim of this study is to verify the analytical performance of four homocysteine detection systems made in China and to explore the comparability of homocysteine detection systems by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method. METHODS: The intra-batch precision, inter-batch precision, accuracy, and linear range of four homocysteine detection systems were evaluated. The ID-LC-MS/MS reference method was used to evaluate the comparability and accuracy of fresh frozen serum samples in four different detection systems of homocysteine. The ID-LC-MS/MS reference method is used to assign samples as calibrators to calibrate each system. The variation and deviation of fresh serum samples between different systems before and after calibration were compared. RESULTS: The intra-batch imprecision of the four detection systems was less than 5%, and the coefficient of variation of inter-batch imprecision was less than 6.7%. The precision met the clinical requirements. Before calibration, the results measured by detection system 2 are consistent with the ID-LC-MS/MS reference method, which meets the requirements of accuracy verification. The regression equation of R² ≥ 0.975 in the regression equation of linear analysis of the four systems, the linearity of the four detection systems is good in the range of evaluation concentration, and all of them can meet the declared linear range. The absolute average bias of fresh serum measured by the four detection systems after calibration decreased from 3.76 µmol/L, 0.96 µmol/L, 1.30 µmol/L, -1.56 µmol/L to 0.31 µmol/L, 0.28 µmol/L, 0.4 µmol/L, 0.40 µmol/L, respectively. The relative average bias decreased from 22.6%, 7.50%, 11.0% and -8.50% to 1.98%, 1.78%, 2.59%, 2.34%, respectively. After calibration, the slope and intercept of the regression curve of the fresh serum measured by the four detection systems and the reference method are closer to 1 and 0 than before calibration. CONCLUSIONS: The precision, reference interval, and linear evaluation of the four detection systems are good. The ID-LC-MS/MS reference method assigning fresh frozen serum samples as calibrators can improve the accuracy and comparability of the results of different detection systems.

Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry.

Isotope-labeled proteins are expected to be used as internal standard proteins in the field of protein quantification by isotope dilution mass spectrometry (ID/MS). To achieve the absolute quantification of Cystatin C (Cys C) based on ID/MS, we aims to obtain 15N isotope-labeled recombinant Cys C (15N-Cys C) protein. Firstly, the Cys C gene was optimized based on the preferred codons of Escherichia coli, and inserted into the pET-28a(+) expression plasmid. Then, the plasmid was transformed into TOP10 and BL21 strains, and 15N-Cys C was expressed in M9 medium using 15N as the only nitrogen source. 15N-Cys C was detected by SDS-PAGE, protein immunoblotting and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The characteristic peptides obtained from 15N-Cys C were analyzed by a Q Exactive Plus MS system. Results showed that 53.06% of the codons were optimized. The codon adaptation index of the Cys C genes increased from 0.31 to 0.95, and the GC content was adjusted from 64.85% to 54.88%. The purity of 15N-Cys C was higher than 95%. MALDI-TOF MS analysis showed that the m/z of 15N-Cys C had changed from 13 449 to 14 850. The characteristic peptides showed that 619.79 m/z (M+2H)2+ was the parent ion of 15N-Cys C and that the secondary ions of 15N-labeled peptides from y+5 to y+9 were 616.27 m/z, 716.33 m/z, 788.39 m/z, 936.43 m/z, and 1052.46 m/z, respectively. In conclusion, we successfully expressed, purified and identified of 15N-Cys C protein in Escheichia coli intended for absolute quantification using ID/MS.

Combination of Serum Amyloid A and C-Reactive Protein Exhibit Synergistic Effect in Angiogenesis by Inducing Inflammation and Vascular Network.

The role of angiogenesis in tumor progression has been recognized as one of the hallmarks of cancer, but the mechanism of its action remains unclear. Inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) are proposed to play causal roles in the development of various disorders, including malignancies. Previously, we identified the complex of CRP and SAA (CRP-SAA) with diagnostic and prognostic value better than either one of them in the serum of lung cancer patients. In this study, we further explored the stimulation function of CRP-SAA on angiogenesis and inflammation. To explore possible mechanisms, microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database and multi-bioinformatics analysis revealed that THP-1 and human umbilical vein endothelial cells (HUVECs) responded to SAA stimulation with upregulation of two pro-angiogenic cytokines in common, i.e., C-X-C motif ligand 6 (CXCL6) and CXCL8, which were validated by subsequent experiments in vitro. CRP had weak effects as a single stimulus, but it can efficiently potentiate the SAA induction of cytokines, which was stronger than the sum of the both (P < 0.001). The synergistical effect of the combination of CRP and SAA enhanced HUVECs transwell and constricted morphology by upregulating the pro-angiogenic genes. These results indicated that the binding of CRP and SAA acted synergistically in pro-angiogenesis by increasing inflammation and inducing vascular network.

Biotin Interference in Susceptible CanAg Ca242 Immunoassays and Elimination Method.

BACKGROUND: High-dose biotin therapy is beneficial in progressive multiple sclerosis (MS). Biotin, as dietary supplement or therapy, may lead to analytical interference in biotin-streptavidin immunoassay. METHODS: Seven concentration gradients of biotin solutions were spiked to three different levels of Ca242 serum samples. All samples were tested by CanAg Ca242 ELISA kit to evaluate the interference from biotin. Serum samples with biotin concentration at 1,000 ng/mL were retested after absorption by streptavidin microparticles or direct analysis on the Mindray CL2000i platform. RESULTS: Our study found that CanAg Ca242 is vulnerable to interference when a sample that contains biotin exceeds 15.63 ng/mL. Biotin interference can result in falsely low results in CanAg Ca242. The effect and extent of biotin interference are, to some extent, dependent on the concentration of serum Ca242 and the concentration of biotin. CONCLUSIONS: CanAg Ca242 is vulnerable to biotin interference. The laboratory can overcome biotin interference on CanAg Ca242 by using a non-biotin streptavidin method or by absorbing biotin with streptavidin-coated microparticles before testing. Clinicians should use caution in interpreting abnormal results in patients who ingest biotin.

Prognostic value of serum soluble interleukin-23 receptor and related T-helper 17 cell cytokines in non-small cell lung carcinoma.

The signaling of interleukin (IL)-23 and its receptor (IL-23R) play a crucial role in the development of cancers. However, the clinical significance of human serum soluble IL-23R (sIL-23R) and its relationship with IL-23 are still not explored in non-small cell lung cancer (NSCLC). In our study, sIL-23R was first identified in the serum of NSCLC patients, but not in healthy controls, by proteomics. The IL-23R mRNA and protein were upregulated in NSCLC cell lines and tissues tested by quantitative PCR, western blot analysis and immunohistochemistry. The levels of sIL-23R, IL-23, and IL-17 in 195 NSCLC patients' serum were determined by ELISA, and high levels of sIL-23R were significantly associated with advanced N stage (P = .039), clinical stage (P = .007), and poor 5-year survival rate. In vitro, sIL-23R was shown binding to IL-23 and the balance could affect patients' N and T stage, overall survival, and downstream cytokine IL-17 in a potential antagonistic relationship. Although sIL-23R, IL-23, and IL-17 were all associated with poor prognosis, only the sIL-23R/IL-23 ratio (hazard ratio, 1.945; 95% confidence interval, 1.147-3.299; P = .014) was found to be an independent factor for prognosis. Therefore, we identified fragments of soluble cytokine receptor of IL-23R with affinity ability to its natural ligand IL-23 in NSCLC patients' serum. The balance between the 2 antagonists can work as a potential prognostic serum marker.

Evaluation of a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for 17α-hydroxyprogesterone in human plasma.

A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated for the quantification of 17α-hydroxyprogesterone (17-OHP) in human plasma. 17-OHP spiked with a deuterium-labeled internal standard was extracted from plasma by liquid-liquid extraction with 1 mL n-hexane/ethyl acetate (3:2, v/v). Reversed-phase chromatography and positive electrospray ionization were used in the ID-LC-MS/MS. Gradient elution coupled with use of a C18-packed ultrahigh-performance liquid chromatography column allowed complete baseline resolution of 17-OHP from its structural analogue desoxycorticosterone in 6 min. To determine the 17-OHP level in human plasma, a bracketing calibration method was used to give higher accuracy and precision. The limit of detection and the lower limit of the measuring interval for the candidate RMP were 2.1 pg/mL (6.4 pmol/L) and 4.6 pg/mL (13.9 pmol/L), respectively. Extraction recovery was determined to be (96.08 ± 3.03)% (n = 3). Imprecision (intra-assay and interassay) was 4.03% or less at 0.83, 15.19, 64.22, and 313.46 ng/mL (2.51, 45.97, 194.34, and 948.56 nmol/L, respectively). Recoveries ranged from 98.05% to 102.24%. When comparing our RMP results with those obtained with an established RMP via International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine (RELA) samples, we found that the biases ranged from -1.99% to 3.08% against the targets. No interference was observed, and the linear response ranged from 0.47 to 958.63 ng/mL (1.42 to 2900.90 nmol/L). Moreover, the candidate RMP was used to measure the concentration of 17-OHP in human plasma and was compared with an immunoassay using 40 plasma samples. The performance of the method meets the needs of an RMP (total coefficient of variation of 5% or less and bias of 3.08% or less). This method can be used for reference material value assignment of 17-OHP in human plasma matrix. It could also serve as an accurate reference baseline for routine methods to increase the accuracy and precision of certain clinical laboratory measurements. Graphical abstract Selected ion chromatograms obtained by liquid chromatography-tandem mass spectrometry with a C18 column for 17α-hydroxyprogesterone (17-OHP) from a plasma sample.

Optimization of an enzyme-coupling method by spectrophotometer for serum adenosine deaminase: As a candidate reference method.

Adenosine deaminase (ADA) is a key enzyme of adenosine metabolism. There are currently various kits and systems available for ADA measurement, and all yield variable results. This study optimized a reference measurement procedure (RMP) for serum ADA for the standardization of routine methods. ADA coupled with purine-nucleoside-phosphorylase, xanthine-oxidase and peroxidase was selected as the basic method and was optimized using Response Surface Methodology. Then the performance was validated and the results were compared after replication by 3 other reference laboratories. A reference interval was also developed. In addition, this optimized method was applied to calibrate a routine system. The intra-assay precision was 0.44% at both concentrations of 29.8 and 100.4 U/L, and inter-assay precision was 1.01% and 0.95% at 30.1 and 100.3 U/L, respectively. The linearity was up to 351.9 U/L (R2 = 0.9998), with no significant interference or carryover (<5%). A Comparison among 4 reference laboratories showed good reproducibility (R2 ≥ 0.9975). The procedure proved valid for a reference interval of 11.7-38.5 U/L. The mean relative deviation for a routine system was -55.9% and -3.7% before and after calibration. This candidate RMP for serum ADA can potentially be used for standardization of clinical systems.