Search

Glycans, particularly sialic acids (SAs), play crucial roles in diverse biological processes. Despite their significance, analyzing specific glycans, such as sialic acids, on individual small extracellular vesicles (sEVs) has remained challenging due to the limited glycan capacity and substantial heterogeneity of sEVs. To tackle this issue, we introduce a chemical modification method of surface SAs on sEVs named PALEV-nFCM, which involves periodate oxidation and aniline-catalyzed oxime ligation (PAL), in conjunction with single-particle analysis using a laboratory-built nano-flow cytometer (nFCM). The specificity of the PALEV labeling method was validated using SA-decorated liposomes, enzymatic removal of terminal SA residues, lectin preblocking, and cellular treatment with an endogenous sialyltransferase inhibitor. Comprehensive mapping of SA distributions was conducted for sEVs derived from different sources, including conditioned cell culture medium (CCCM) of various cell lines, human saliva, and human red blood cells (RBCs). Notably, treatment with the calcium ionophore substantially increases the population of SA-positive RBC sEVs and enhances the SA content on individual RBC sEVs as well. nFCM provides a sensitive and versatile platform for mapping SAs of individual sEVs, which could significantly contribute to resolving the heterogeneity of sEVs and advancing the understanding of their glycosignature.

The expression of CD73 by flow cytometry (FC) in bone marrow (BM) specimens of B-cell acute lymphoblastic leukemia (B-ALL) with or without minimal residual disease (MRD) was studied, and its advantages were evaluated using the MRD assay. This study also detected the expression profile of CD73 in hematogones and mature B cells in BM specimens of 18 healthy donors. Results showed that the mean value of CD73 expression in MRD-positive B cells was 6-fold greater than that in the MRD negative ones. Also, 41.82% MRD-positive B-ALL cases expressed high CD73 and the sensitivity of CD73-based MRD detection reached 10(-4). Since the expression of CD73 increases with the maturation of normal B cells, it is better to mix it with CD34, CD10 and CD20 in one tube to prevent the disturbance of mature B cells. CD73 is recommended as an optional MRD marker for B-ALL patients by using FC.

Background: Automated urine sediment analysis of white blood cells (WBCs) and bacteria is a promising approach for urinary tract infections (UTIs) screening. However, available data on their screening efficacy is inconsistent., Methods: English articles from Pubmed, EMBASE, and Web of Science published before December 1, 2012 were analyzed. The Quality Assessment for Studies of Diagnostic Accuracy (QUADAS) tool was used to evaluate the quality of eligible studies. Performance characteristics of WBCs and bacteria (sensitivity, specificity, and other measures of accuracy) were pooled and examined by random-effects models., Results: Nineteen studies containing 22,305 samples were included. Pooled sensitivities were 0.87 (95% confidence interval [CI], 0.86-0.89) for WBCs and 0.92 (95% CI, 0.91-0.93) for bacteria. Corresponding pooled specificities were 0.67 (95% CI, 0.66-0.68) for WBCs and 0.60 (95% CI, 0.59-0.61) for bacteria. Areas under the summary receiver operating characteristics curves were 0.87 and 0.93 for WBCs and bacteria, respectively. The major limitation of eligible studies was that enrolled subjects were often not representative of clinical patient populations in which UTI would be suspected., Conclusions: WBC and bacterial measurements by the UF-100 and UF-1000i are useful indicators in UTI screening; however, the performances of these systems should be rigorously evaluated by additional studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: To study the diagnostic value of DNA, RNA and proliferating cell nuclear antigen (PCNA) examination in malignant effusion by multiparametric flow cytometry, and therefore to provide proof for clinical application., Methods: Forty seven patients with pleural effusions in our hospital from August 2003 to February 2004 were divided into two groups: 19 suffering from benign pleural effusions and 28 from malignant effusions confirmed by pathologic examination. The cells for diagnosis were divided into four groups stained by PI (Propidium-iodide), PY (Pyronin), PCNA-FITC and PCNA-mouse-alpha-2a. The specimens were analyzed by a flow cytometer (FacS Caliber, Becton Dickinson). The sensitivity and the specificity of each examination and combined examination were calculated by statistic software SPSS 13.0., Results: (1) The expression of DI, RI, and PI in benign pleural effusion was 1.03 +/- 0.06, 10.03 +/- 0.54, and (4.86 +/- 0.72)%, respectively, and those in malignant ones was 1.26 +/- 0.17, 11.65 +/- 1.45, and (11.97 +/- 1.50)%, respectively, the difference being statistically significant. The cutoff value of DI, RI and PI was 1.10%, 10.75% and 4.56%, and the sensitivity of DI examination was 89.3%, 78.6%, 75.0%, and the specificity was 89.5%, 98.5%, 84.2%, respectively. (2) In 6 cases suffering from malignant pleural effusions, RI was positive but DI was negative, indicating that DI combined with RI examination was better than DI examination alone. (3) In 5 cases suffering from malignant pleural effusions confirmed by tissue examination, the cytology was negative, but the result of DI and RI was abnormal, indicating that flow cytometry was complementary to pathologic examination. (4) The sensitivity of DI + RI, DI + PI, RI + PI and DI + RI + PI combined examination was 98.2%, 89.3%, 89.3%, 92.9%; the specificity was 84.2%, 89.5%, 84.2%, 94.2% respectively. The results demonstrated that DI + RI + PI combined examination was the best, which showed the least false negative and false positive results. The sensitivity of DI + RI combined examination was 98.2%, but the specificity was 84.2%, the false positive rate being higher than DI + RI + PI combined examination. In none of the benign pleural effusions was the DI + RI + PI higher than the cutoff value, suggesting that combined examination can exclude benign pleural effusions., Conclusions: DNA, RNA and PCNA examinations by flow cytometry are of value in the diagnosis of malignant effusion, especially for cases which can not be diagnosed by cytological examination. DI + RI + PI combined examination showed better results with the lowest false negative and false positive rates.

Aim: To clarify the role of Eomesodermin (EOMES) to serve as a disease‐relevant biomarker and the intracellular molecules underlying the immunophenotype shifting of CD4+T subsets in amyotrophic lateral sclerosis (ALS). Methods: The derivation and validation cohorts included a total of 148 ALS patients and 101 healthy controls (HCs). Clinical data and peripheral blood were collected. T‐cell subsets and the EOMES expression were quantified using multicolor flow cytometry. Serum neurofilament light chain (NFL) was measured. In 1‐year longitudinal follow‐ups, the ALSFRS‐R scores and primary endpoint events were further recorded in the ALS patients of the validation cohort. Results: In the derivation cohort, the CD4+EOMES+T‐cell subsets were significantly increased (p < 0.001). EOMES+ subset was positively correlated with increased serum NFL levels in patients with onset longer than 12 months. In the validation cohort, the elevated CD4+EOMES+T‐cell proportions and their association with NFL levels were also identified. The longitudinal study revealed that ALS patients with higher EOMES expression were associated with higher progression rates (p =.010) and worse prognosis (p =.003). Conclusions: We demonstrated that increased CD4+EOMES+T‐cell subsets in ALS were associated with disease progression and poor prognosis. Identifying these associations may contribute to a better understanding of the immunopathological mechanism of ALS. [ABSTRACT FROM AUTHOR]

BackgroundB-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance.MethodsThe anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro.ResultsAfter the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion.ConclusionsThis preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses.

Purpose: To evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM). Methods: This study analyzed 60 Chinese MM patients. During MRD monitoring in these patients' post-therapy, clonal immunoglobulin heavy chain (IGH) rearrangements were detected via NGS using LymphoTrack assays. MRD monitoring was performed using NGS or next-generation flow cytometry (NGF), and the results were compared. Additionally, the sensitivity and reproducibility of the NGS method were assessed. Results: The MRD detection range of the NGS method was 10–6–10–1, which suggested good linearity, with a Pearson correlation coefficient of 0.985 and a limit of detection of 10–6. Intra- and inter-assay reproducibility analyses showed that NGS exhibited 100% reproducibility with low variability in clonal cells. At diagnosis, unique clones were found in 42 patients (70.0%) with clonal IGH rearrangements, which were used as clonality markers for MRD monitoring post-therapy. Comparison of NGS and NGF for MRD monitoring showed 79.1% concordance. No samples that tested MRD-positive via NGF were found negative via NGS, indicating the higher sensitivity of NGS. MRD could be detected using NGS in 6 of 7 samples before autologous hematopoietic stem-cell transplantation, and 5 of them tested negative post-transplantation. In contrast, the NGF method could detect MRD in only 1 sample pre-transplantation. Conclusion: Compared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients. [ABSTRACT FROM AUTHOR]

A systemic and local inflammatory immune imbalance is thought to be the cause of traumatic tracheal stenosis (TS). However, with CD4+ T lymphocytes being the predominant immune cells in TS, the mechanism of action and recruitment has not been described. In our research, using flow cytometry, ELISA, immunofluorescence, and Transwell chamber assays, the expression, distribution, and potential chemotactic function of CD4+ T cells in TS patients were examined before and after treatment. The results showed that the untreated group had significantly more CD4+ T cells and their secreted TGF‐β1 than the treated group. Additionally, the untreated group's CD4+ T cells showed a significant rise in CCL22 and CCL1, as well as a larger proportion of CCR4 and CCR8. CD4+ T cells and CD68+ macrophages located in TS also expressed CCL1 and CCL22. In vitro, anti‐CCL1 and anti‐CCL22 can partially block the chemoattractant effect of TS bronchoalveolar lavage (BAL) on purified CD4+ T cells. The findings of this study indicated that TS contained unbalanced CD4 immune cells that were actively recruited locally by CCR4/CCL22 and CCR8/CCL1. As a result, it is anticipated that CD4 immune rebalancing can serve as a novel treatment for TS. [ABSTRACT FROM AUTHOR]

Aim: Periodontitis (PD) is the sixth most prevalent disease around the world and is involved in the development and progression of multiple systemic diseases. Previous studies have reported that PD may aggravate liver injuries. The objective of this study was to investigate whether and how PD affects liver fibrosis. Materials and Methods: Ligature‐induced PD (LIP) was induced in male C57/B6J mice, and sub‐gingival plaques (PL) from patients with PD were applied to mouse teeth. Liver fibrosis was induced by carbon tetrachloride (CCl4) injection. The mice were randomly divided into six groups: Oil, Oil+LIP, Oil+LIP+PL, CCl4, CCl4+LIP, and CCl4+LIP+PL. Alveolar bone resorption was evaluated by methylene blue staining. Hepatic function was analysed by serum alanine aminotransferase and hepatic hydroxyproline. Picrosirius red and α‐smooth muscle actin (SMA) staining were used to evaluate the fibrotic area. RNA sequencing and quantitative RT‐PCR were used to measure gene expression. Western blotting was used to measure protein levels. Flow cytometry was used to analyse the accumulation of immune cells. Mouse microbiota were analysed using 16S rRNA gene sequencing. Results: Mice in the CCl4+LIP+PL group displayed higher serum alanine aminotransferase and hepatic hydroxyproline as well as more Picrosirius red‐positive and α‐SMA‐positive areas in liver samples than those of the CCl4 group, suggesting that PD (LIP+PL) aggravated CCl4‐induced hepatic dysfunction and liver fibrosis. Consistently, the expression of fibro‐genic genes and the protein levels of transforming growth factor β were much higher in the CCl4+LIP+PL group than in the CCl4 group. Flow cytometry revealed that PD increased the accumulation of immune cells, including Kupffer cells, B cells, and Th17 cells, in the liver of mice with CCl4 treatment. PD also increased the expression of inflammatory genes and activated pro‐inflammatory nuclear factor‐kappa B pathway in the livers of CCl4‐injected mice. Moreover, PD altered both oral and liver microbiota in CCl4‐injected mice. Conclusions: PD aggravates CCl4‐induced hepatic dysfunction and fibrosis in mice, likely through the increase of inflammation and alteration of microbiota in the liver. [ABSTRACT FROM AUTHOR]

The potential of flow cytometry for the study of changes in prodigiosin on the cell surface of Serratia marcescens is of academic and practical interest. This is because S. marcescens can produce prodigiosin, a secondary metabolite, with potential use as a cancer‐cell inhibitor. In this study, three groups of bacterial cultures with different carbon sources were compared, and the effect of the addition of cAMP to the sucrose‐based culture was studied. Both cellular morphology and DNA content were detected by flow cytometry, rendering a broad description of the bacterial behavior. It is the first use of flow cytometry to investigate the dynamics of prodigiosin on the surface of S. marcescens during growth in different media. The fluorescence intensity is related to the DNA content, the forward‐scattered light is related to cell volume, and the side‐scattered light is related to the surface morphology, especially the surface prodigiosin. These may contribute to the potential development of a bacterial metabolic monitoring strategy using both DNA content analysis and bacterial morphology based on flow cytometry technique. [ABSTRACT FROM AUTHOR]

Background: As a life-threatening respiratory syndrome, acute lung injury (ALI) is characterized by uncontrollable inflammatory activities. Semaglutide (SEM) has been identified as an effective anti-inflammatory drug in a variety of diseases. This study intended to explore the functional effect and potential mechanisms of SEM in ALI. Methods: Lipopolysaccharide (LPS) was used to construct an in vivo ALI model based on Sprague-Dawley (SD) rats and an in vitro ALI model based on human pulmonary artery endothelial cells (HPAECs). Hematoxylin & eosin (H&E) staining and ELISA were applied to evaluate the histopathological changes in pulmonary tissues and detect TNF-α and IL-6 levels. RT-qPCR and Western blotting were used to measure gene and protein expressions in pulmonary tissues and cells. HPAEC viability and apoptosis were evaluated by CCK-8 method and flow cytometry methods. Results: Semaglutide pretreatment significantly mitigated pulmonary injury, reduced TNF-α and IL-6 production, and led to a decrease in cleaved caspase-3 level and an increase in Bcl-2 level, suggesting SEM could ameliorate LPS-induced ALI in rats. In vitro, SEM increased the proliferative capability and mitigated inflammation and apoptosis in LPS-stimulated HPAECs. In addition, SEM inhibited HDAC5-mediated NF-κB signaling pathway in HPAECs. HDAC5 overexpression or NF-κB signaling activation could partly impair SEM-mediated protective effects against LPS-induced damage to HPAECs. Conclusion: Semaglutide restrains LPS-induced ALI by inhibiting HDAC5/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Objective: The conditioned medium of calcined tooth powder (CTP‐CM) is a type of biomimetic mineralized material and well contributing to bone healing and bone formation in vivo. However, little is known about the effect of CTP‐CM on human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanisms. Methods: ALP activity assay was conducted to select the concentration with the highest ALP level, which was used for the following experiments. Cell proliferation was measured by cell counting kit‐8 assay and flow cytometry analysis. Expression levels of osteogenic markers in CTP‐CM‐induced hPDLSCs were evaluated with real‐time quantitative reverse transcription polymerase chain reaction (qRT‐PCR), immunofluorescence staining, and Western blot. Mineralization of CTP‐CM‐induced hPDLSCs was evaluated by alizarin red staining. Furthermore, the involvement of NF‐κB pathway was examined by immunofluorescence staining and Western blot. Results: 20 μg/ml was selected for the further experiments. Functional studies demonstrated that CTP‐CM exerted almost no influence on the proliferation of hPDLSCs and CTP‐CM increased the osteogenic differentiation of hPDLSCs. Mechanistically, CTP‐CM leads to activation of NF‐κB signaling pathway. When treated with BMS345541, the osteogenic differentiation of CTP‐CM‐treated hPDLSCs was significantly attenuated. Conclusion: CTP‐CM can promote the osteogenic differentiation of hPDLSCs via activating NF‐κB pathway. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a widespread inflammatory disease whose clinical manifestations are joint swelling, pain, and disability, affecting approximately 1% of individuals worldwide. Conventional anti-RA drugs currently used in clinic have severe side effects. The present study is aimed at investigating the antiarthritic effects of total saponins from Nigella glandulifera seeds (TSNGS) in rats with adjuvant-induced rheumatoid arthritis (AIA). Arthritis score, paw swelling, and body weight were monitored throughout the period of TSNGS treatment. The histopathological features and levels of cytokines, including IFN-γ, TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-17A, and OPG/RANKL signaling, were measured to determine the amelioration by TSNGS and its potential mechanisms on the inflammatory response and bone erosion. The differentiation of regulatory T cells (Tregs) in serum was assessed by flow cytometry. The results demonstrate that TSNGS at 10 mg/kg, 50 mg/kg, and 250 mg/kg inhibited AIA-induced clinical score, paw swelling, and histological changes. TSNGS reduced the immune-inflammatory reaction by restoring the secretion and expression of inflammatory cytokines and elevating the proportion of CD4+ CD25+ Tregs, accompanied by an increase in transcription factor Foxp3 levels. TSNGS also displayed bone protection by upregulation of the OPG/RANKL pathway. Collectively, TSNGS inhibited arthritis in AIA rats and so represents a potential novel treatment for RA. [ABSTRACT FROM AUTHOR]

Intraperitoneal adhesion is a common complication after abdominal surgery, which seriously affects the quality of life of patients. HuoXueTongFu Formula (HXTF) plays an important role in the prevention and treatment of intraperitoneal adhesions. However, the molecular-related mechanisms are still not fully known. In this study, the model of Intrapetitoneal adhesion was established by cecum abrasion and treated with HXTF for one week. RAW264.7 cells were given LPS, IFN-γ, IL-4, HXTF-medicated serum, and PPAR-γ agonist/antagonist, respectively. Histopathology, flow cytometry, ELISA, real-time PCR, and Western blotting were used to further detect the related protein, M1/M2 polarization tendency, and PPAR-γ nuclear translocation. The deposition of collagen fibres reduced in the local area of rats after the operation with HXTF treatment. Similar to IL-4, HXTF induced a tendency for macrophages to polarize toward M2 and promoted peroxisome proliferator-activated receptor-gamma (PPAR-γ) nuclear translocation. Furthermore, the use of HXTF and PPAR-γ agonists downregulated macrophage M1 polarization-related factors IL-1, IL-6, and TNF-alpha and upregulated M2 polarization-related factors IL-4, IL-10, and TGF-beta 1. Meanwhile, the use of HXTF and PPAR-γ agonists downregulated the SOCS3/JAK2/STAT1 pathway and activated the SOCS1/STAT6/PPAR-γ pathway. These results show that HXTF may reduce intraperitoneal adhesion by inducing macrophage M2 polarization and regulating the SOCS/JAK2/STAT/PPAR-γ pathway. [ABSTRACT FROM AUTHOR]

In order to observe and ascertain the properties of a sub-group of T cells in the lymph node (LN) from seven patients who did not suffer from T cell lymphoproliferative disorders (T-LPDs), the expression levels of several pan-T markers were evaluated by multiparameter flow cytometry (FC) and the clonality of these T-cells was evaluated by both FC analysis and PCR assessment. It turned out that multiple pan-T-cell markers such as CD2, CD5 and CD7 were found to be lost in these T cells. The majority of them were positive for TCRαβ, only a minority of them being positive for TCRγδ. A subset of these T-cells were positive for CD4 or CD8 or dual-negative for CD4 and CD8. Oligoclonality was detected in one case by FC, while clonalTCRrearrangement was detected in three cases. Absence of multiple pan-T-cell markers could be found in benign T cells in LNs. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells (hiPSCs) may provide potential resource for regenerative medicine research, including generation of insulin-producing cells for diabetes research and insulin production. Testosterone (T) is an androgen hormone which promotes protein synthesis and improves the management of type 2 diabetes in clinical studies. Concurrently, co-existed hyperandrogenism and hyperinsulinism is frequently observed in polycystic ovary syndrome, congenital adrenal hyperplasia and some of Wermer's syndrome. However, the relationship among androgens, insulin and the differentiation of pancreatic β cells is still not fully clear. Here we find that T improves the differentiation efficiency of insulin-producing cells from hiPSCs. The addition of T into routine differentiation formula for pancreatic β cells increases the differentiation efficiency from 12% to 35%. The administration of T promotes the expression of key genes associated with β cells differentiation including NGN3, NEUROD1 and INS. This finding benefits the ongoing process to optimize the differentiation protocol of pancreatic β cells from hiPSCs, and provides some degree of understanding the clinical management of T for type 2 diabetes. [ABSTRACT FROM AUTHOR]

Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen. [ABSTRACT FROM AUTHOR]

Raddeanin A (RA) is an extractive from Anemone raddeana Regel, a traditional Chinese medicine. The aim of this study is to assess the efficacy of RA against human gastric cancer (GC) cells (SGC-7901) and explore its mechanism. MTT assay showed that RA inhibition of proliferation of SGC-7901 cells increased in a dose-dependent manner. Flow cytometry analysis and Hoechst 33258 staining showed that RA induced apoptosis on SGC-7901 cells. Meanwhile, it induced autophagy. Western blotting analysis showed that the RA induces apoptosis and autophagy by activating p38 MAPK pathway and inhibiting mTOR pathway. Further studies showed that autophagy inhibition could protect from RA-induced apoptosis in SGC-7901 cells. In conclusion, RA can induce SGC-7901 cell apoptosis and autophagy by activating p38 MAPK pathway. And autophagy can protect SGC-7901 cells from apoptosis induced by RA. [ABSTRACT FROM AUTHOR]

Jianpi Huayu Decoction (JHD), a Chinese medicine formula, is a typical prescription against multiple tumors in the clinical treatment, which can raise quality of life and decrease complications. The aim of this study is to assess the efficacy of JHD against human colorectal carcinoma cells (SW480) and explore its mechanism. MTT assay showed that JHD decreased the cellular viability of SW480 cells in dose-dependent and time-dependent manner. Flow cytometry analysis revealed that JHD induced G0/G1-phase cell cycle arrest in SW480 cells and had a strong apoptosis-inducing effect on SW480 cells. Meanwhile it enhanced the expression of p27, cleaved PARP, cleaved caspase-3, and Bax and decreased the levels of PARP, caspase-3, Bcl-2, CDK2, CDK4, CDK6, cyclin D1, cyclin D2, cyclin D3, and cyclin E1, which was evidenced by RT-qPCR and Western blot analysis. In conclusion, these results indicated that JHD inhibited proliferation in SW480 cells by inducing G0/G1-phase cell cycle arrest and apoptosis, providing a practicaltherapeutic strategy against colorectal cancer. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) were used widely as seed cells in tissue engineering blood vessel construction. However, the biological characteristics difference of different generation MSCsin vitroculture is unknown, which laid a foundation for appropriate generation seeded cells selection for tissue engineering blood vessel construction. In this report, MSCs were isolated from SD rat bone marrow and identified by flow cytometry; cell growth curve test, cell surface antigen expression rate detection, cryopreservation resuscitation rate test, CD31 expression rate test, cell cycle analysis, and adhesion difference on vascular scaffold test were performed. The research results indicated that the MSCs shape was spindle and uniform with vigorous growth. CD105 and CD90 factor expression rate reached 82.5 and 84.9%, respectively, and the expression rate of CD45 was only 7.3%. The proliferation capacity of the fourth generation MSCs were more exuberant, with proliferation index as 20.3%; the cell proliferation index of the eighth generation decreased to only 9.1%. The cryopreservation resuscitation rate of the second generation and fourth generation MSCs were both higher than 80%, and the cryopreservation resuscitation rate of the eighth generation MSCs was only about 60%. After the induction for 5 days, MSCs had weak CD31 expression, and with the prolonged induction time, expression increased. All generation MSCs expressed CD31 after being induced for 10 days; however, the CD31 positive expression rate of the second generation, fourth generation, and sixth generation MSCs had significant difference with the eighth generation MSCs. Adhesion rate of MSCs before sixth generation was around 40%, but the adhesion rate of eighth generation MSCs was only about 27%. In all, biological characteristics of different generation MSCs existed certain differences, and especially the eighth generation MSCs aged seriously, whose cell activity decreased significantly. The researchers believed that the MSCs before the sixth generation can maintain excellent properties of MSCs, and can be used as seed cells for vascular tissue engineering. [ABSTRACT FROM AUTHOR]

Background: Primary immune thrombocytopenia (ITP) is an autoimmune bleeding disorder with an unclear etiology. This study aims to investigate the role of IL-23/Th17 pathway in patients with ITP. Method: The gene expressions of IL-17, IL-23 and their receptors in ITP patients and healthy controls were analyzed by quantitative real-time PCR. ELISA was used to test the IL-17 and IL-23 levels in plasma. Flow cytometry was used to detect the frequency of Th17 cells. The correlation between plasma IL-23 and IL-17 levels, Th17 cells, platelets were analyzed. The level of Th17-related cytokines was measured by ELISA following stimulation with IL-23. Subsequently, the IL-23 and IL-17 levels were measured in patients post-treatment. Results: The PBMCs of ITP patients showed increased mRNA expression levels in each of the following: IL-23p19, IL-12p40, IL-23R, IL-12Rβ1, IL-17A, IL-17F, and RORC. In addition, elevated Th17 cells and plasma IL-17, IL-23 levels were also observed in these ITP patients. Furthermore, it was found that IL-23 levels in plasma are positively correlated with IL-17 levels and Th17 cells, yet negatively correlated with platelet count. Following IL-23 stimulation in vitro, IL-17 levels showed significant elevation. Furthermore, both IL-23 and IL-17 levels decreased after effective treatment. Conclusion: The IL-23/Th17 pathway may be involved in the pathogenesis of ITP through enhancement of the Th17 response. Moreover, our results suggest that the IL-23/Th17 pathway is a potential therapeutic target in future attempts of ITP treatment. [ABSTRACT FROM AUTHOR]

Gambogic acid (GA) is the major active ingredient of gamboges, a brownish to orange resin product from Garcinia hanburyi tree in Southeast Asia. This compound exhibits anti-cancer effect on solid tumors. In this study, we investigated the effects of GA on the growth and differentiation of acute myeloid leukemia cells by growth-inhibition detection, morphological changes observation, nitroblue tetrazolium reduction, and the expression of the relative cell-surface differentiation markers. The results showed that GA could inhibit cell growth and promote differentiation in U937 and HL-60 cells. In addition, GA upregulated the expression of p21waf1/cip1 in the two cell lines. Finally, downregulating the p21waf1/cip1 expression with small interfering RNA partially blocked GA-induced cell growth inhibition and differentiation. These results of this study revealed that GA may be used as one of the investigational drugs for acute myeloid leukemia. [ABSTRACT FROM AUTHOR]

Aim:Skewed cytoplasmic accumulation of NPM mutant protein (NPM1c+) is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c+ protein trafficking and induce apoptosis in NPM1c+ acute myeloid leukemia cells in vitro.Methods:OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR.Results:Oridonin (2-12 μmol/L) dose-dependently inhibited the viability of OCI-AML3 cells (the IC50 value was 3.27±0.23 μmol/L at 24 h). Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c+ protein. Oridonin did not change the expression of Crm1 (the export receptor for nuclear export signal-containing proteins), but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 (Nup98), which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53.Conclusion:In NPM1c+ leukemia cells, oridonin induces NPM1c+ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis. [ABSTRACT FROM AUTHOR]

Clavibacter michiganensis subsp. nebraskensis (Cmn) and Pantoea stewartii subsp. stewartii (Pss) are two plant pathogens that can cause tremendous agricultural economic losses. This novel method based on microsphere immunoreaction was developed for the simultaneous detection of Cmn and Pss in maize. This multiplex method was constructed based on microsphere immunodetection with fluorescent labels such as quantum dots (QDs) and R-phycoerythrin (R-PE) for the detection of Cmn and Pss. Captured QDs and R-PE serve as signal reporters for fluorescent readout. The principle of this method is based on a sandwich immunoreaction. Cmn and Pss captured by the microspheres were detected using flow cytometry. The limit of detection of this method was 10 times lower than the enzyme-linked immunosorbent assay (ELISA), and its analysis time (1 h) was much shorter compared with ELISA (6–8 h). The method, which has been proven to be an effective approach to multiplex detection of plant bacteria (Cmn and Pss as models), not only increased the varieties but also improved the sensitivity. The microsphere immunoreaction provides a universal method for the multiplex determination of microbes because of its high sensitivity, specificity, and speed. In the future, the method will be more fully validated in vivo to detect diversiform bacteria. [ABSTRACT FROM PUBLISHER]

In modern biological research, there are many analytical methods to evaluate the toxicity caused by chemical agents, such as higher plant bioassay, flow cytometry, single-cell gel electrophoresis or the comet assay. However, these methods are either tediously experimental procedures or require a high-tech platform. In this study, the effects of chromium trioxide (CrO) on the primary root growth of Vicia faba seeds over 72 h were investigated. The primary root length of seeds cultivated in different concentrations of CrO was collected and analyzed. It was indicated that the Vicia faba seeds maintained a good growing state when the CrO concentration was below 100 μmol/L, while the root growth was suppressed when they were kept in 250 μmol/L of CrO for 72 h or cultured with CrO concentrations above 500 μmol/L. In comparison with the chromosome aberration bioassay, the primary root length assay is proved in this paper to be a simple and effective alternative for water toxicity testing in areas where heavy metal pollution has become serious. [ABSTRACT FROM AUTHOR]

Aim:To investigate the effects of betulinic acid (BA) on apoptosis and autophagic flux in multiple myeloma cells and the relationship between the two processes.Methods:The proliferation of human multiple myeloma KM3 cells was measured with MTT assay. FITC/PI double-labeled flow cytometry (FCM) and Hoechst 33258 staining were used to analyze the cell apoptosis. Caspase 3, PARP, Beclin1, LC3-II, and P62 were detected using Western blotting.Results:Treatment of KM3 cells with BA (5−25 μg/mL) suppressed the cell proliferation in time- and dose-dependent manners. The IC50 values at 12, 24, and 36 h were 22.29, 17.36, and 13.06 μg/mL, respectively. BA treatment dose-dependently induced apoptosis of KM3 cells, which was associated with the activation of caspase 3. However, Z-DEVD-FMK, a specific inhibitor of caspase 3, did not decrease, but rather sensitized the cells to BA-induced apoptosis, suggesting an alternative mechanism involved. On other hand, BA treatment dose-dependently increased the accumulation of LC3-II and P62 in KM3 cells, representing the inhibition of autophagic flux. Furthermore, BA treatment dose-dependently downregulated the expression of Beclin 1, an important inducer of autophagy, in KM3 cells. In the presence of BA, Z-DEVD-FMK induced autophagy and increased the amount of LC3-II in KM3 cells, which may occur via attenuating BA-induced decrease in the level of Beclin 1. Similarly, rapamycin, an autophagy inducer, increased the amount of LC3-II in KM3 cells. In the presence of BA, rapamycin caused further increase in the amount of LC3-II. Furthermore, rapamycin sensitized BA-treated KM3 cells to apoptosis.Conclusion:The results demonstrate that BA induces apoptosis and blocks autophagic flux in KM3 cells. Furthermore, in addition to activation of caspase 3, the inhibition of autophagic flux also contributes to the BA-mediated apoptosis of KM3 cells. [ABSTRACT FROM AUTHOR]

Background: Polybrominated diphenyl ethers (PBDEs), commonly used in building materials, electronics, plastics, polyurethane foams, and textiles, are health hazards found in the environment. Objective: In this study we investigated the effects of PBDE-209, a deca-PBDE, on the regulation of growth and apoptosis of breast, ovarian, and cervical cancer cells as well as the underlying protein alterations. Methods: We used MCF-7 and MCF-7/ADR (multidrug-resistant MCF-7) breast cancer cell lines, the HeLa cervical cancer cell line, the OVCAR-3 ovarian cancer cell line, and the normal CHO (Chinese hamster ovary) cell line to assess the effects of PBDE-209 using cell viability, immunofluorescence, and flow cytometric assays. Western blot assays were used to detect changes in protein expression. To assess the effects of PBDE-209 on apoptosis, we used the protein kinase Cα (PKCα) inhibitor Gö 6976, the extracellular signal-regulated kinase (ERK) inhibitor PD98059, and tamoxifen. Results: Our data indicate that PBDE-209 increased viability and proliferation of the tumor cell lines and in CHO cells in a dose- and time-dependent manner. PBDE-209 also altered cell cycle distribution by inducing the S phase or G2/M phase. Furthermore, PBDE-209 partially suppressed tamoxifen-induced cell apoptosis in the breast cancer cell lines (MCF-7 and MCF-7/ADR) but suppressed Gö 6976- and PD98059-induced apoptosis in all cell lines. At the molecular level, PBDE-209 enhanced PKCα and ERK1/2 phosphorylation in the cell lines. Conclusions: Our data demonstrate that PBDE-209 is able to promote proliferation of various cancer cells from the female reproductive system and normal ovarian CHO cells. Furthermore, it reduced tamoxifen, PKCα, and ERK inhibition-induced apoptosis. Finally, PBDE-209 up-regulated phosphorylation of PKCα and ERK1/2 proteins in tumor cells and in CHO cells. [ABSTRACT FROM AUTHOR]

Aim:To elucidate the relationship between triptolide-induced changes in histone methylation and its antitumor effect on human multiple myeloma (MM) cells in vitro.Methods:Human multiple myeloma cell line RPMI8226 was used. Apoptosis was evaluated using Annexin-V-FITC/PI-labeled flow cytometry, Hoechst 33258 staining, and transmission electron microscopy. Flow cytometry was used to detect the cell cycle distribution of the apoptotic cells. The presence of the LSD1, JMJD2B, H3K4me2, H3K9me2, and H3K36me2 proteins was verified by Western blot analysis. Semi-quantitative real-time PCR was performed to examine the expression of LSD1 and JMJD2B.Results:Triptolide (10-160 nmol/L) suppressed the proliferation of MM cells in a dose- and time-dependent manner with an IC50 value of 99.2±9.0 nmol/L at 24 h. Triptolide (50 nmol/L) induced G0/G1 cell cycle arrest in MM cells. The agent (50-150 nmol/L) induced apoptosis of MM cells in a dose-dependent manner. The same concentrations of triptolide suppressed the expression of dimethylated H3K4, dimethylated H3K9 and dimethylated H3K36 by altering the expression of histone demethylase LSD1 and JMJD2B without affecting the expression of histone demethylase LSD1.Conclusion:Triptolide potently inhibits the growth of MM cells via regulating the expression of histone demethylase LSD1 and JMJD2B, which lead to abnormal histone methylation. [ABSTRACT FROM AUTHOR]

Aim:To explore the mechanisms underlying the oridonin-induced apoptosis and autophagy in human multiple myeloma cells in vitro.Methods:Human multiple myeloma RPMI8266 cells were used. The cell viability was assessed using MTT assay. Morphological changes of apoptosis and autophagy were observed under transmission electron microscope. TUNEL and annexin V-FITC/PI dual staining assays were used to measure apoptosis. Autophagy was analyzed using Western blot analysis and immunofluorescence staining with a QDs605 nm-Anti-LC3 fluorescent probe. Intracellular ROS was estimated with flow cytometry using DCFH-DA fluorescent probe. Protein levels of active caspase 3, Beclin 1 and SIRT1 were determined with Western blot analysis.Results:Exposure to oridonin (1-64 μmol/L) inhibited the proliferation of RPMI8266 cells in a concentration-dependent manner with an IC50 value of 6.74 μmol/L. Exposure to oridonin (7 μmol/L) simultaneously induced caspase 3-mediated apoptosis and Beclin 1-dependent autophagy of RPMI8266 cells. Both the apoptosis and autophagy were time-dependent, and apoptosis was the main effector pathway of cell death. Exposure to oridonin (7 μmol/L) increased intracellular ROS and reduced SIRT1 nuclear protein in a time-dependent manner. The blockade of intracellular generation of ROS by NAC (5 mmol/L) abrogated apoptosis, autophagy and the decrease of SIRT1 in the cells exposed to oridonin (7 μmol/L). The inhibition of autophagy by 3-MA (5 mmol/L) sensitized the cells to oridonin-induced apoptosis, which was accompanied by increased intracellular ROS and decreased SIRT1.Conclusion:Oridonin simultaneously induces apoptosis and autophagy of human multiple myeloma RPMI8266 cells via regulation of intracellular ROS generation and SIRT1 nuclear protein. The cytotoxicity of oridonin is mainly mediated through the apoptotic pathway, whereas the autophagy protects the cells from apoptosis. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that the total alkaloids of Sophora alopecuroides (TASA), which contains many different ingredients like sophocarpine, matrine, oxymatrine, sophoridine, sophoramine, aloperine and cytosine, were able to protect colon against ulcers caused by 2,4,6-trinitrobenze sulphonic acid (TNBS)/ethanol treated models. In order to elucidate the mechanisms by which TASA exerts its effect of anti-inflammation and immunoregulation on rats with colitis, DAI (disease activity index) and histological grading of colitis were evaluated in the animal model. Moreover, the expression of CD4+CD25+ regulatory T cells (Tregs) and IL-10 in rats with experimental colitis were observed by FCM, ELISA and RT-PCR in this study. Results showed that TASA (15, 30 or 60 mg/kg/day) significantly up-regulated CD4+CD25+Tregs (P = 0.02, P = 0.02, P = 0.03) and IL-10 levels (ELISA: P = 0.03, P = 0.02, P = 0.00; RT-PCR: P = 0.04, P = 0.02, P = 0.01) respectively and decreased the DAI and histological grading of colitis in the peripheral blood (PB) and colon of rat colitis models (3.44 ± 1.53, 4.25 ± 1.27, 4.42 ± 1.24 and 3.50 ± 1.42, 4.05 ± 1.32, 4.51 ± 1.55 vs. 7.18 ± 1.32 and 7.38 ± 1.52, P < 0.05, P < 0.01, respectively). Most interestingly, a negative correlation was demonstrated between the expression of CD4+CD25+ Tregs and DAI (Pearson rPB = -0.677, P < 0.01; Pearson rCOLON = -0.663, P < 0.01, n = 60), or histological grading of colitis (Pearson rPB = -0.725, P < 0.01; Pearson rCOLON = -0.623, P < 0.01, n = 60). Simultaneously, a positive correlation existed between CD4+CD25+ Tregs and IL-10 cytokine (IL-10 mRNA) in the colon and PB of rats (Pearson rPB = 0.789, P < 0.01, n = 60; Pearson rCOLON = 0.678, P < 0.01, n = 60). These results may explain to some extent the mechanisms of TASA on treating rats with experimental colitis. [ABSTRACT FROM AUTHOR]

Aim: To investigate the antiproliferative effect of triptolide on B-NHL cell line Raji cells, to study its effect on lymph node metastasis in patients with non-Hodgkin's lymphoma (NHL) in vitro, and to explore the underlying mechanism regulating SDF-1/CXCR4 axis. Methods: The effects of triptolide on the growth of Raji cells were studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium (MTT) assay. The effects of triptolide on SDF-1 mRNA expression in lymph node stromal cells from patients with NHL were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effects of triptolide on CXCR4 expression on lymphoma cells freshly isolated from the lymph nodes of these patients were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of primary lymphoma cells towards recombinant human SDF-1α (rhSDF-1α) or cultured lymph node stromal cells in vitro. Results: Triptolide inhibited the proliferation of B-NHL cell line Raji cells in a dose- and time-dependent manner with a 24-h IC50 value of 43.06 nmol/L and a 36-h IC50 value of 25.08 nmol/L. The expression of SDF-1α mRNA in lymph node stromal cells obtained from patients with NHL was decreased after treatment by triptolide at concentrations of 25 and 50 nmol/L for 24 h. Flow cytometry analysis showed that the CXCR4 expression on primary lymphoma cells were downregulated gradually in a dose-dependent manner following triptolide treatment. Chemotaxis assays revealed that the migration of freshly isolated lymphoma cells towards either rhSDF-1 or cultured lymph node stromal cells was markedly inhibited by the addition of triptolide in vitro, and the inhibition was dose-dependent. Conclusion: Triptolide can inhibit the proliferation of B-NHL cell line Raji cells. Moreover, triptolide is able to inhibit the migration of lymphoma cells via lymph nodes in vitro. The potential antitumor mechanisms of triptolide are related to the antiproliferative effect and the blockage of SDF-1/CXCR4 axis. [ABSTRACT FROM AUTHOR]

Background: Catalpol, a natural iridoid glycoside in Rehmannia glutinosa, can alleviate proteinuria associated with diabetic nephropathy (DN), however, whether catalpol has a protective effect against podocyte injury in DN remains unclear. Methods: In this study, we used a high glucose (HG)-induced podocyte injury model to evaluate the protective effect and mechanism of catalpol against HG-induced podocyte injury. Cell viability was determined by the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by commercial assay kits. Cell apoptosis and reactive oxygen species (ROS) were determined by using flow cytometry. Tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels were determined by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl2-associated x (Bax), cleaved caspase-3, nicotinamide adenine dinucleotide phosphate oxidase enzyme 4 (NOX4), toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88), p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 MAPK (p-p38 MAPK), nuclear factor kappa B inhibitor alpha (IκBα) and phosphorylated IκBα (p-IκBα) were measured by western blotting. In addition, Bcl-2, Bax, caspase-3 and nuclear factor kappa B (NF-κB) levels were determined by immunofluorescence staining. Results: Catalpol significantly increased cell viability and decreased LDH release in HG-induced podocyte injury. Catalpol significantly decreased ROS generation, apoptosis, level of MDA, levels of inflammatory cytokine TNF-α, IL-1β, and IL-6 and increased SOD activity in HG-induced podocyte injury. Moreover, catalpol significantly decreased expression of cleaved caspase-3, Bax, NOX4, TLR4, MyD88, p-p38 MAPK, p-IκBα and NF-κB nuclear translocation, as well as increased Bcl-2 expression in HG-induced podocyte injury. Conclusion: Catalpol can protect against podocyte injury by ameliorating apoptosis and inflammation. These protective effects may be attributed to the inhibition of NOX4, which alleviates ROS generation and suppression of the TLR4/MyD88 and p38 MAPK signaling pathways to prevent NF-κB activation. Therefore, catalpol could be a promising drug for the prevention of DN. [ABSTRACT FROM AUTHOR]