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CD8In this study, we analyzed peripheral blood CD8Compared with the healthy control group, the proportion of CD8Therefore, the depletion of CD8

Tim-3 is a negative immunoregulator in anti-tumor response, but its mechanism in chronic lymphocytic leukemia (CLL) is not yet clear. The aim of this study was to understand the role of Galectin-9/Tim-3 signaling pathway in the regulation of CD4+ T cell subsets in CLL patients. Flow cytometry results showed that the number of Treg cells obviously increased, and there was a significant Treg/Th17 imbalance in CLL patients. In addition, Tim-3 overexpressed on the surface of Th1 and Treg cells in CLL patients. The levels of Galectin-9 and IL-10 were significantly elevated in patients of CLL, especially in stages of Binet B, and C. However, IFN-γ decreased. Moreover, Galectin-9 in CLL patients was positively correlated with the number of Tim-3+ Treg cells and the level of IL-10. Interestingly, when the Tim-3/Galectin-9 pathway was blocked in vitro, the level of IL-10 in the culture supernatant of CD4+ T was significantly reduced, while the levels of IFN-γ and TNF-α were increased. After co-culture with activated Th1 cells, the apoptosis of CLL cells was significantly increased, and this effect was reversed after treatment with Tim-3+ Tregs. In summary, Galectin-9/Tim-3 are elevated in CLL and associated with disease progression. By the negative regulation of CD4+ T cells, activated Galectin-9/Tim-3 suppresses Th1 effector function and also promotes Treg to be involved in immune escape of CLL. This pathway might become the potential target of immunotherapy in CLL patients.

Tim-3/Galectin-9 is involved in the immune escape of many pathogens. However, the role of Tim-3/Galectin-9 in persistent infection of Echinococcus multilocularis (Em), which is related to immune escape, is still unclear.To investigate the role of Tim-3/Galectin-9 and related cytokines in mice with persistent infection of Em.Em infection model was established by injecting the protoscoleces. Serum was collected at days 2, 8, 30, 60, 90, 180 and 270 after infection. Lymphocytes were isolated from liver tissue samples with Ficoll. Tim-3 + CD4 + T percentage was analyzed by flow cytometry. CD4 + T cells were isolated from liver tissues of Em infected mice and cultured in vitro. The mRNA levels of Tim-3, Galectin-9, IFN-γ and IL-4 were detected by qRT-PCR. Cytokine levels in serum and culture supernatant (IFN-γ and IL-4) were analyzed by cytometric bead array.The expression of Tim-3 and Galectin-9 mRNA significantly increased after 30 days of infection, reached peak on day 90, and then decreased slightly on days 180-270. The expression of IFN-γ mRNA, increased on day 2 and 8 after infection, slightly decreased on days 30-60, and obvious decreased on days 90-270, but were still higher than those of the control group. The expression of IL-4 mRNA gradually increased along with the time of infection. In serum of Em infected mice, level of IFN-γ peaked at day 30 and then gradually decreased; whereas IL-4 level peaked at day 90 and then gradually decreased. In vitro experiment found that Tim-3/Galectin-9 directly caused the changes in the levels of IFN-γ and IL-4.Tim-3/Galectin-9 signaling pathway may be involved in the development of persistent infection of Em by regulating the production of Th1 and Th2 cytokines.

To explore the application of immunophenotype resources in clinical teaching of hematologic malignancies. There were 102 students who studied the content of hematologic malignancies were divided into experimental group and control group. The experimental group introduced immunophenotype resources to assist teaching. The control group used traditional teaching mode, then we observed the teaching effects of the two groups. The scores of the examinations in the experimental group were significantly higher than those in the control group (P

Background Grape is an important fruit consumed either fresh or processed, therefore, fungicide misuse of grape has become an issue of global food safety and human health. Pyraclostrobin, and cyazofamid have been applied to grape frequently. Results Here a simple QuEChERS (quick, easy, cheap, effective, rugged, and safe) liquid chromatography mass spectrometry technique has been developed and validated for the determination of pyraclostrobin, cyazofamid and its metabolite CCIM in open field grape samples. The recoveries of these three in the range of 0.01 to 5 mg kg-1 (n = 5) ranged from 73.1% to 97.9%. The relative standard deviations (RSDs) were below 12% for all cases. The limits of quantitation of each analyte was 0.005 mg kg-1 , which was lower than maximum residue limits of not only pyraclostrobin but also cyazofamid. Not only dissipation kinetics but also residue determination was obtained in grape for those three pesticides. Furthermore, their half-lives in grapes were 10.7-30.1 days, recommending the pre-harvest intervals for these three of 14 days. The calculated hazard quotient and acute hazard index lower than 100% illustrated the safety of intake of grape for the Chinese population for not only long-term but also short-term dietary risk assessment. Conslusions The less than 30 day half-life illustrated that pyraclostrobin and cyazofamid could degrade relatively easily in the environment. The long-term and short-term dietary risk assessment also illustrated the intake safety of these three. Thus, a 14 day pre-harvest interval was safe and recommended. The results of this study contributed to environmental protection, food safety and human health. © 2019 Society of Chemical Industry.

Objective To investigate the regulation and clinical significance of T cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3)/galectin-9 signaling pathway in regulatory T cells (Tregs) and Th17 cells in patients with chronic lymphocytic leukemia (CLL). Methods The study enrolled 36 healthy individuals and 40 newly diagnosed CLL patients. The Tregs, Th17 cells, TIM-3

The interaction between Tim-3 on T cell and its ligand, Galectin-9, negatively regulates cellular immune responses. However, the role of Tim-3/Galectin-9 pathway in the immune evasion of cervical cancer remains unknown. This study is to investigate the expression, function, and regulation of Tim-3/Galectin-9 signaling pathway in human papilloma virus (HPV) positive cervical cancer. Flow cytometry showed that Tim-3 expression on T cell and Galectin-9 expression on monocytes in HPV positive cervical cancer patients were significantly higher compared to cervical intraepithelial neoplasia and benign uterine fibroids Tim-3 + CD4+ Th1 cells and Tim-3 + CD8+ T cells in HPV positive cervical cancer patients were significantly reduced after surgery. Serum TGF-β and IL-10 levels were positively correlated with Tim-3 + Treg cells, while IFN-γ and IL-2 were negatively correlated with Tim-3 + Th1 cells. Additionally, Tim-3 + CD4+ T cells were positively correlated with Galectin-9 + monocytes. Survival curve analysis showed that Tim-3 + CD4+ T cells were negatively correlated with patient survival, and closely related to FIGO stage, degree of differentiation, and lymph node metastasis of HPV positive cervical cancer. In vitro experiments showed that by blocking the Tim-3/Galectin-9 pathway, the proliferation of T cells and their ability to express IFN-γ, IL-2, perforin, and granzyme B was significantly restored. In conclusion, high levels of Tim-3 and Galectin-9 in HPV positive cervical cancer patients play roles in the progression of disease by promoting Treg cells to inhibit the cytotoxic function of Th1 and CD8+ T cells. Tim-3/Galectin-9 may serve as a new immunotherapy target for patients with HPV positive cervical cancer.

The present study was designed to investigate the effect of adipose-derived stem cells (ADSCs) on acute graft vs. host disease (aGVHD) and hematopoietic recovery after allogeneic hematopoietic stem cell transplantation. ADSCs, bone marrow-derived stem cells (BMSCs) and fibroblasts were cultured. ADSCs were cocultured with hematopoietic stem/progenitor cells. Then, ADSCs were infused into the aGVHD rat model. The survival of the rats was recorded. Livers and small intestines were obtained from sacrificed rats for pathological examinations. Expression of the Sry gene in recipient rats that survived longer than 21 days was examined by real-time PCR to detect the presence of donor Y chromosome. Expression of serum interferon (INF)-γ and interleukin (IL)-4 was detected by ELISA at 0, 7, 14, 21 and 50 days after transplantation. Transplantation of ADSCs improved the survival of aGVHD rats. Survived ADSCs participated in hematopoietic reconstitution in aGVHD rats. ADSCs decreased aGVHD severity by immunomodulation. ADSCs support the proliferation of hematopoietic stem/progenitor cells in vitro. The present study demonstrated that ADSCs may reduce aGVHD by influencing the balance of IL-4 and INF-γ and can promote long-term hematopoiesis.

An efficient method was developed and validated for simultaneous determination of tebuconazole, thiophanate-methyl and its metabolite carbendazim in grape and soil using QuEChERS procedure and liquid chromatography-tandem mass spectrometry. The average recoveries of the method were 83.2%–105.4%, the limit of detection (LOD) ranged from 2.1 × 10−5 to 8.7 × 10−4 mg L−1, and the limit of quantitation (LOQ) was 0.1 mg kg−1. The field results showed that tebuconazole and thiophanate-methyl in soil and grape were rapidly dissipated with half-lives less than 24.7 days. The terminal residues of all analytes in grape were lower than the maximum residue limit (MRL) set by China (2 mg kg−1 for tebuconazole; 3 mg kg−1 for both thiophanate-methyl and carbendazim). Risk assessment showed that tebuconazole, thiophanate-methyl and its metabolite would be unlikely to cause health problems. However, carbendazim, the higher active metabolites of thiophanate-methyl, should receive more concerns.

The influence of mild perioperative hypothermia on the immune function and incidence of postoperative wound infections has been suggested, but the specific mechanism is unclear. This study aimed to analyze the body temperature, immune function, and wound infection rates in patients receiving open surgery for gastric cancer. Body temperature was controlled in each patient using one of four different methods: wrapping limbs, head and neck; insulated blankets; warming infusion fluids and insulated blankets; and warming fluids without insulated blankets. One hundred patients were randomly divided into four groups of 25 patients each, and every group received a different intraoperative treatment for maintaining normal body temperature. Nasopharyngeal and rectal temperatures, transforming growth factor beta (TGF-β), interleukin 10 (IL-10) levels, and cluster of differentiation (CD)3+T and CD4+/CD25+ regulatory T cell (Treg) counts were measured before surgery and at 2 and 4 hours postoperatively. Patients were evaluated at one week after surgery for signs of infection. Intraoperative body temperature and measures of immune function varied significantly between the four groups, with the largest temperature changes observed in the group in which only the limbs were wrapped in cotton pads to control the body temperature. The lowest temperature change (i.e., close to normal temperature) and cytokine response after surgery were observed in the group in which infusion fluids and transfused blood (if needed) were heated to 37℃, peritoneal irrigation fluid was heated to 37℃, and an insulation blanket was heated to 39℃ and placed under the patient. No intergroup differences were found in the infection rates at one week after surgery. In conclusion, body temperature variation during surgery affects the immune function of patients, and maintaining body temperature close to normal results in the least variation of immune function.

Objectives Cystic echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus (Eg) infection. Th9 cells are reported to be involved in the immune responses in CE patients. This study aims to investigate the role of TGF-β/Smad pathway in the regulation of Th9 cells in CE patients. Methods Using Western blot analysis, flow cytometry, qPCR, immunohistochemistry, ELISA and MTT assay, we measured the expression levels of TGF-β/Smad, PU.1, IRF-4, and IL-9 in CE patients. Results The levels of TGF-β, p-Samd3, PU.1 and IL-9 were elevated in the liver of CE patients. IL-9 and IL-9R expressions were also elevated in the infected liver tissue, and IL-9 level was positively correlated with the liver inflammation. The levels of IL-9, IL-4, TGF-β and IL-10 in the supernatant were also significantly increased after stimulating hepatic lymphocytes of CE patients with Eg antigen B. After blocking the TGF-β pathway signaling in vitro, PU.1 and IL-9 were obviously reduced. Conclusions IL-9 may aggravate the inflammatory response in the liver of CE patients. The TGF-β/Smad signaling pathway is activated, and the signaling pathway may promote the differentiation of Th9 cells and IL-9 expression in active CE patients.

The present study is to measure the expression of programmed death (PD)-1 / programmed death ligand-1 (PD-L1) negative costimulatory molecules, soluble format sPD-1 in patients with immune thrombocytopenia (ITP), and to investigate their correlation with the secretion of cytokines. A total of 35 patients with ITP were included in the present study. Twenty healthy subjects who received physical examination at our hospital were included as control group. Peripheral blood was collected from all ITP patients and healthy subjects. Flow cytometry was performed to determine the percentages of PD-1+CD4+T cells and PD-L1+DCs in ITP patients and healthy subjects. Enzyme-linked immunosorbent assay was performed to measure the concentrations of interferon (IFN)-γ, interleukin (IL)-17 and sPD-1 in peripheral blood from ITP patients and healthy subjects. Percentages of PD-1+CD4+T cells and PD-L1+DCs in peripheral blood from ITP patients before treatment were significantly higher than that from healthy subjects, but were not different from those after treatment. Serum concentrations of IFN-γ, IL-17 and sPD-1 in ITP patients before treatment were significantly higher than those in healthy subjects, and these concentrations were significantly reduced after treatment. The concentration of sPD-1 was positively correlated with the concentration of IFN-γ, and negatively correlated with platelet count. Percentages of PD-1+CD4+T cells and PD-L1+DCs in ITP patients are higher than those in healthy subjects, but elevated sPD-1 concentration in the blood blocks PD-1/PD-L1 signaling pathway, leading to unaffected Th cell function. Elevated concentrations of IFN-γ and IL-17 in the blood may participate in the occurrence and development of ITP.

Objective This study is to investigate the association between the Treg/Th17 cells and prognosis of chronic lymphocytic leukemia (CLL). Methods Totally 50 CLL patients and 20 Health controls were included in this study. Regulatory T (Treg) cells and the cell subset secreting IL-17 (Th17) in peripheral blood were detected with flow cytometry. Serum levels of IL-10 and IL-17 were determined with ELISA, and expression of Foxp3 and RORγt was assessed with quantitative real-time PCR. Results Treg and Th17 cell proportions in peripheral blood in the CLL patients were significantly higher than control. Serum levels of IL-10 and IL-17, and expression of Foxp3 and RORγt, were significantly increased in the CLL patients. Ratios of Treg/Th17 and IL-10/IL-17 were significantly elevated in the CLL patients. Compared with those before treatment, Treg/Th17 and IL-10/IL-17 ratios were declined in the CLL patients in remission. Compared with the non-remission group, Treg cells were significantly decreased, while Th17 cells were significantly increased, resulting in decreased Treg/Th17 ratio, in the remission group. Moreover, the serum IL-10 level was significantly decreased, while the serum IL-17 level was significantly increased, resulting in declined IL-10/IL-17 ratio, in the remission group. Correlation analysis showed that, Treg and Th17 cell counts were significantly associated with CD38 and ZAP-70 expression in the CLL patients. Moreover, the IL-10/IL-17 ratio was also significantly associated with CLL prognostic factors. Conclusion Altered Treg/Th17 and IL-10/IL-17 ratios in CLL would be aggravated along with the disease progression, which might be used as indicators for the disease prognosis.

Objectives this study aims to identify the potential marker for hepatic cystic echinococcosis (CE) activity. Methods totally 97 CE patients and 47 health control (HC) subjects were included. Based on the ultrasound (US) images, CE patients were further categorized as active CE (ACE; 52 cases) and inactive CE (IACE; 45 cases). The sST2 and IL (interleukin)-33 levels were measured by enzyme-linked immunosorbent assay. Univariate, multivariate and subgroup analysis were performed. The relationship between sST2 and CE activity was analyzed by the smooth curve fitting. The receiver operating characteristic (ROC) curve analysis was also performed. Results the serum sST2 level in the CE patients was significantly higher than the HC subjects. Moreover, there was significant difference in the serum sST2 level between the ACE and IACE group. However, no significant difference was observed in the IL-33 level between the ACE and IACE groups. Univariate analysis showed that CE activity was significantly correlated with the sST2 level when the sST2 was greater than 1496.6 pg/mL. Multivariable analysis after adjustment for potential confounding factors, and subgroup analysis showed that sST2 had independent effect on CE activity. ROC curve showed that sST2 could differentially diagnose CE activity at the cut-off value of 1246.550 pg/mL with an AUC of 0.770. Conclusion the sST2 could be used as a biomarker for hepatic CE activity. High levels of sST2 suggest that the hepatic CE is more likely to be active. Our findings have important guiding significance for the diagnosis of CE activity and the choice of treatment methods.

Abamectin has been widely used in agriculture and animal husbandry. It has been shown that abamectin exposure could induce multiple toxic effects on non-target organisms, but the underlying mechanism is still largely unknown. In the current study, the mechanism of abamectin-induced cytotoxicity was investigated in mouse embryonic fibroblast cells. Abamectin treatment could cause oxidative stress in cells (beginning at 0.4 μg/ml, 0.5 μM) and the ROS overproduction was mainly induced by the impacts of abamectin on the activities of CAT (beginning at 4.4 μg/mL, 5 μM), SOD (beginning at 8.7 μg/mL, 10 μM), GPx (beginning at 4.4 μg/mL, 5 μM), and contents of GSH (beginning at 4.4 μg/mL, 5 μM), which are important components of the ROS elimination pathway in mammal cells. Abamectin could impair DNA integrity (as demonstrated by increased 8-OHdG/dG ratio) in cells, even at environmental level (0.4 μg/mL, NOAEL), and abamectin-induced oxidative stress was one of the main reasons for the DNA damage that occurred in cells. Moreover, pretreatment with the inhibitor of JNK and ATM/ATR signaling pathway could partially rescue the decreased cell viability, indicating that oxidative stress and DNA damage might be involved in abamectin-induced cytotoxicity. These findings could provide new insights into the mechanism of abamectin-induced cytotoxicity and should be useful for a more comprehensive assessment of the adverse effects of abamectin.

The widespread use of abamectin has recently raised safety concerns as abamectin has yielded various toxicities to non-target organisms. However, the underlying mechanisms of abamectin-induced toxicity are still largely unknown. The present study aimed to investigate the abamectin-induced cytotoxicity in mouse macrophage cells (RAW264.7) and its underlying mechanisms. Abamectin treatment caused oxidative stress as characterized by increased intensity of the ROS indicator. Abamectin also led to DNA damage as demonstrated by increased 8-OHdG/dG ratio in cells even at a relatively low dose (NOAEL). Pretreatment with catalase-PEG, a ROS inhibitor, attenuated abamectin-induced DNA damage, indicating that ROS overproduction should be the reason for abamectin-induced DNA damage. The effects of abamectin on ROS elimination and generation were also investigated, and the results showed that abamectin induced concentration-dependent alteration in the expression and activities of CAT, SOD, GPx enzymes and GSH level (ROS elimination), but had limited effects on the expression and activities of NOX, mitochondrial complex I and III (ROS production) in RAW264.7 cells. Therefore, the effects of abamectin on ROS elimination should be the main reason for abamectin-induced oxidative stress in RAW264.7 cells. Abamectin treatment activated MAPK and ATM/ATR signaling pathways as demonstrated by increased phosphorylation of JNK, ATM and ATR. In addition, inhibiting JNK and ATM/ATR signaling pathways partially rescued the decrease in cell viability, indicating that abamectin-induced ROS overproduction and DNA damage might finally lead to cytotoxicity through JNK and ATM/ATR signaling pathways. These findings should be useful for the more comprehensive assessment of the toxic effects of abamectin.

Cystic echinococcosis (CE) is a global zoonosis caused by the larvae of the parasite Echinococcus granulosus (E. granulosus). According to its life cycle and previous studies of antigen candidates for vaccines against E. granulosus, we chose two proteins expressed at different stages of the E. granulosus life cycle to design a multi-epitope vaccine. The EgA31 antigen gene is derived from the adult stage of E. granulosus, and the EgG1Y162 antigen gene is derived from the larval stage of E. granulosus. In this study, we used several bioinformatics methods to analyze various aspects of the EgA31 and EgG1Y162 proteins, including the physicochemical properties, secondary and tertiary structures, and the dominant T-cell and B-cell epitopes. The results showed that EgA31 protein was an unstable and hydrophilic protein, while EgG1Y162 was stable and hydrophobic. The secondary structure of the EgA31 protein consisted of 82.36% alpha helixes, 4.16% extended strands, 3.16% beta turns and 10.32% random coils. The secondary structure of EgG1Y162 consisted of 33.33% alpha helixes, 25.49% extended strands, 5.88% beta turns and 35.29% random coils. Moreover, our results identified 6 dominant T-cell epitopes and 5 dominant B-cell epitopes in the EgA31 protein structure and 6 dominant T-cell epitopes and 3 dominant B-cell epitopes in EgG1Y162. In conclusion, this study provides comprehensive biological information about the EgA31 and EgG1Y162 proteins, which will lay a theoretical foundation for multi-epitope vaccines against Echinococcus granulosus.

Nanosheets of graphitic carbon nitride (GCN) were used as the dispersive solid-phase extraction (d-SPE) sorbent for the first time. GCN successfully purified complex matrices of soil, wheat, and wheat straw utilising a simple QuEChERS (quick, easy, cheap, effective, rugged, and safe) method with liquid chromatography tandem mass spectrometry. Pesticides recoveries in the range of 80–110%, small matrix effect, and decreased amounts guaranteed and distinguished this new sorbent. The application of a real formulation of pesticides under field conditions during 2015 in the wheat ecosystem illustrated the clean-up effect and application potential of GCN. It is a valuable and potential substitute for C18 according to the results comparison between two sorbents, including recoveries, matrix-matched calibration, and dissipation kinetics of pesticides. The new use of GCN also enriched the knowledge of d-SPE and sample preparation furthermore.

Wheat products are consumed worldwide and every day; therefore, herbicides misuse in the wheat ecosystem has become a global food safety issue. Here, a simple and versatile method, QuEChERS (quick, easy, cheap, effective, rugged, and safe) with liquid chromatography-tandem mass spectrometry was developed to measure four herbicides in soil, wheat, and wheat straw – florasulam, carfentrazone-ethyl, fluroxypyr-meptyl, and fluroxypyr. By adjusting the amount of graphitized carbon black from 0 to 10 mg, the herbicides could be extracted with satisfactory recoveries of 80%–110%. Application of two water-dispersible granules showed first-order kinetics as well as half-lives between 1.2 and 5.1 d under open-field conditions. The dissipation kinetics of the four herbicides differed in the soil, wheat, and wheat straw, with residual concentrations in straw at 2 h after application being higher than in soil and faster degradation occurring in straw. The terminal residues in soil, wheat grain, and wheat straw...

The present study is to measure the expression of programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), as well as its clinical significance in cervical cancer patients. Our results showed that different T cell subsets in patients with cervical cancer had high expression of PD-1, and DCs had high expression of PD-L1. High expression of PD-1 on Treg cells in cervical cancer patients facilitated the production of TGF-βand IL-10 but inhibited the production of IFN-γ. Cervical cancer elevated the expression of PD-1 and PD-L1 in mRNA level. PD-1 expression in peripheral blood of cervical cancer patients was related with tumor differentiation, lymph node metastasis, and invasiveness. PD-1/PD-L1 pathway inhibited lymphocyte proliferation but enhanced the secretion of IL-10 and TGF-βin vitro. In summary, our findings demonstrate that elevated levels of PD-1/PD-L1, TGF-β, and IL-10 in peripheral blood of cervical cancer patients may negatively regulate immune response against cervical cancer cells and contribute to the progression of cervical cancer. Therefore, PD-1/PD-L1 pathway may become an immunotherapy target in the future.

Thiamethoxam is a widely used pesticide applied to different field crops. To inform risk assessment for this pesticide across relevant crops, we usually rely on field trials, which require time, costs and energy. For providing reliable data across crops and reduce experimental efforts, field trials should be complemented with dynamic modelling. In the present work, we hence focused on combining field trials with dynamic modelling to simulate mass evolutions of the pesticide-plant-system for thiamethoxam applied to wheat, lettuce and tomato as three major food crops. Field trials were conducted with QuEChERS (quick, easy, cheap, effective, rugged and safe) liquid chromatography-mass spectrometry, which gave consistent maximum residue concentrations for thiamethoxam in wheat, lettuce and tomato. We used these residues to evaluate the related dietary risk of humans consuming these food crops. Our results indicated that thiamethoxam did not provide any unacceptable dietary risk for humans across these three food crops, which is in line with findings from previous studies. Results for the studied crops could be extrapolated to other crops and with that, our study constitutes a cost- and time-efficient way of providing reliable input for risk assessment of pesticides across crops, which is relevant for both practitioners and regulators.

The present study was designed to investigate the effect of adipose-derived stem cells (ADSCs) on acute graft vs. host disease (aGVHD) and hematopoietic recovery after allogeneic hematopoietic stem cell transplantation. ADSCs, bone marrow-derived stem cells (BMSCs) and fibroblasts were cultured. ADSCs were cocultured with hematopoietic stem/progenitor cells. Then, ADSCs were infused into the aGVHD rat model. The survival of the rats was recorded. Livers and small intestines were obtained from sacrificed rats for pathological examinations. Expression of the Sry gene in recipient rats that survived longer than 21 days was examined by real-time PCR to detect the presence of donor Y chromosome. Expression of serum interferon (INF)-γ and interleukin (IL)-4 was detected by ELISA at 0, 7, 14, 21 and 50 days after transplantation. Transplantation of ADSCs improved the survival of aGVHD rats. Survived ADSCs participated in hematopoietic reconstitution in aGVHD rats. ADSCs decreased aGVHD severity by immunomodulation. ADSCs support the proliferation of hematopoietic stem/progenitor cells in vitro. The present study demonstrated that ADSCs may reduce aGVHD by influencing the balance of IL-4 and INF-γ and can promote long-term hematopoiesis.

The present study is to measure the expression of programmed death (PD)-1 / programmed death ligand-1 (PD-L1) negative costimulatory molecules, soluble format sPD-1 in patients with immune thrombocytopenia (ITP), and to investigate their correlation with the secretion of cytokines. A total of 35 patients with ITP were included in the present study. Twenty healthy subjects who received physical examination at our hospital were included as control group. Peripheral blood was collected from all ITP patients and healthy subjects. Flow cytometry was performed to determine the percentages of PD-1+CD4+T cells and PD-L1+DCs in ITP patients and healthy subjects. Enzyme-linked immunosorbent assay was performed to measure the concentrations of interferon (IFN)-γ, interleukin (IL)-17 and sPD-1 in peripheral blood from ITP patients and healthy subjects. Percentages of PD-1+CD4+T cells and PD-L1+DCs in peripheral blood from ITP patients before treatment were significantly higher than that from healthy subjects, but were not different from those after treatment. Serum concentrations of IFN-γ, IL-17 and sPD-1 in ITP patients before treatment were significantly higher than those in healthy subjects, and these concentrations were significantly reduced after treatment. The concentration of sPD-1 was positively correlated with the concentration of IFN-γ, and negatively correlated with platelet count. Percentages of PD-1+CD4+T cells and PD-L1+DCs in ITP patients are higher than those in healthy subjects, but elevated sPD-1 concentration in the blood blocks PD-1/PD-L1 signaling pathway, leading to unaffected Th cell function. Elevated concentrations of IFN-γ and IL-17 in the blood may participate in the occurrence and development of ITP.

In vitro fertilization-embryo transfer (IVF-ET) can be used by infertile couples to assist with reproduction; however, failure of the embryo to implant into the endometrial lining results in failure of the IVF treatment. The present study investigated the expression of chemokine receptor 7 (CCR7)(lo) programmed death-1(PD-1)(hi) chemokine receptor type 5 (CXCR5)+ cluster of differentiation 4 (CD4)+ T cells and associated factors in patients with repeated implantation failure (RIF). A total of 30 females with RIF and 30 healthy females were enrolled in the current study. Flow cytometry was used to detect the proportion of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells in the peripheral blood. Cytokine bead arrays were performed to detect the levels of interleukin (IL)-6, -4 and -2 in the serum. ELISAs were used to detect the level of IL-21 in the serum. Quantitative real time polymerase chain reaction analysis and immunohistochemistry were used to investigate the expression of B-cell lymphoma 6 (Bcl-6), chemokine receptor type 5 (CXCR5) and IL-21 in the endometrium. The results revealed that the percentage of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells was increased in the RIF group compared with the control group during the mid luteal phase. The mRNA and protein levels of Bcl-6, IL-21 and CXCR5 in the endometrium and the concentrations of IL-21 and IL-6 in the serum were significantly increased in the RIF group; however, no significant difference was observed between the two groups in regards to the expression of IL-4 and IL-2. Furthermore, a significant positive correlation was identified between the percentage of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells and IL-21 and IL-6 levels. The expression of IL-21 also had a positive correlation with Bcl-6 and CXCR5 expression in the RIF group. These results suggest that increased levels of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells and associated factors contribute to RIF and could therefore be a potential therapeutic target.

Cystic echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus (Eg) infection. Th9 cells are reported to be involved in the immune responses in CE patients. This study aims to investigate the role of TGF-β/Smad pathway in the regulation of Th9 cells in CE patients.Using Western blot analysis, flow cytometry, qPCR, immunohistochemistry, ELISA and MTT assay, we measured the expression levels of TGF-β/Smad, PU.1, IRF-4, and IL-9 in CE patients.The levels of TGF-β, p-Samd3, PU.1 and IL-9 were elevated in the liver of CE patients. IL-9 and IL-9R expressions were also elevated in the infected liver tissue, and IL-9 level was positively correlated with the liver inflammation. The levels of IL-9, IL-4, TGF-β and IL-10 in the supernatant were also significantly increased after stimulating hepatic lymphocytes of CE patients with Eg antigen B. After blocking the TGF-β pathway signaling in vitro, PU.1 and IL-9 were obviously reduced.IL-9 may aggravate the inflammatory response in the liver of CE patients. The TGF-β/Smad signaling pathway is activated, and the signaling pathway may promote the differentiation of Th9 cells and IL-9 expression in active CE patients.

Aims This study is to predict and purify the T-B combined epitopes of egG1Y162 antigen in Echinococcus granulosus, and to evaluate their immunogenicity in mice. Methods The bioinformatics software was used to predict the T-B combined epitopes of egG1Y162 antigen. Recombinant egG1Y161/2 peptides were constructed, expressed and purified. Mice were immunized with egG1Y161/2 peptides. The serum and spleen cells were isolated. The isolated spleen cells were stimulated with egG1Y161/2 peptides in vitro and the culture supernatant was collected. The levels of IgG in serum and levels of IL-4 and IFN-γ in the culture supernatant were measured by ELISA. The weight and number of the fresh hydatid cysts were evaluated. The serum ptotoscolicidal activity was measured by the complement dependent cytotoxicity assay. Results Peptides of 6–19aa, 64–82aa, 106–119aa were predicted as T-B combined epitopes of egG1Y162 antigen. And, recombinant protein egG1Y162-1 or egG1Y162-2, which contained T-B combined epitope(s) of the 6–19aa, or the 64–82aa and the 106–119aa in egG1Y162 antigen, respectively, was successfully expressed and purified. Serum IgG levels of mice immunized with egG1Y162-1/2 were significantly increased during the immune response to Echinococcus granulosus. The levels of IFN-γ, IL-4 and the ratio of IFN-γ/IL-4 after egG1Y162-1/2 immunization were significantly higher. Weight and number of the fresh hydatid cysts in egG1Y162-1/2 immunized mice was significantly decreased. And, the serum protoscolicidal activity after egG1Y162-1/2 immunization was enhanced. Conclusions The egG1Y162-1/2 induces production of serum IgG levels and Th1 cell immune response, which enhances the protective immunity in Echinococcus granulosus challenged mice and thus may be used as a potential vaccine candidate.

T helper type 9 (Th9) cells have recently been identified as a new effector T cell subset. This study is to analyze the reconstitution of Th9 cell after matched sibling peripheral blood hematopoietic stem cell transplantation (MS-PBSCT) and the relationship between Th9 cell and acute graft-versus-host disease (aGVHD). Flow cytometry and ELISA were used to analyze the percentages of Th9 cell, levels of IL-9, TGF-β, IFN-γ, and IL-4. The results showed that for patients without aGVHD, Th9 cells recovery started from day 60 after transplantation and reached normal level on day 90. Serum TGF-β, IL-4, and IFN-γ reached normal levels on day 60, 60, and 90 post transplantation respectively. The serum IL-9 recovery is slower than that of IFN-γ and IL-4. For patients suffering from aGVHD, they had declined Th9 cell numbers, lower IL-9 and TGF-β levels, but higher serum IFN-γ level when compared with those without aGVHD after transplantation. Serum IFN-γ/IL-9 ratios increased linearly with grades of aGVHD. In conclusion, Th9 cells recovery is delayed after MS-PBSCT in patients with aGVHD but early in patients without aGVHD, indicating quick immune reconstitution of Th9 cells and IL-9 after MS-PBSCT may promote the immune tolerance.

In vitro fertilization-embryo transfer (IVF-ET) can be used by infertile couples to assist with reproduction; however, failure of the embryo to implant into the endometrial lining results in failure of the IVF treatment. The present study investigated the expression of chemokine receptor 7 (CCR7)(lo) programmed death-1(PD-1)(hi) chemokine receptor type 5 (CXCR5)+ cluster of differentiation 4 (CD4)+ T cells and associated factors in patients with repeated implantation failure (RIF). A total of 30 females with RIF and 30 healthy females were enrolled in the current study. Flow cytometry was used to detect the proportion of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells in the peripheral blood. Cytokine bead arrays were performed to detect the levels of interleukin (IL)-6, −4 and −2 in the serum. ELISAs were used to detect the level of IL-21 in the serum. Quantitative real time polymerase chain reaction analysis and immunohistochemistry were used to investigate the expression of B-cell lymphoma 6 (Bcl-6), chemokine receptor type 5 (CXCR5) and IL-21 in the endometrium. The results revealed that the percentage of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells was increased in the RIF group compared with the control group during the mid luteal phase. The mRNA and protein levels of Bcl-6, IL-21 and CXCR5 in the endometrium and the concentrations of IL-21 and IL-6 in the serum were significantly increased in the RIF group; however, no significant difference was observed between the two groups in regards to the expression of IL-4 and IL-2. Furthermore, a significant positive correlation was identified between the percentage of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells and IL-21 and IL-6 levels. The expression of IL-21 also had a positive correlation with Bcl-6 and CXCR5 expression in the RIF group. These results suggest that increased levels of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells and associated factors contribute to RIF and could therefore be a potential therapeutic target.

Because of less functionally critical carbohydrate sectors that contributed to the stability, efforts have been made to quantify intact recombinant human erythropoietin. A simple, rapid capillary electrophoresis with laser-induced fluorescence method for the assay of recombinant human erythropoietin was developed, with a limit of detection of intact recombinant human erythropoietin at subnanomolar concentration (up to 10 ng/mL or 3 × 10(-10) M), which is among the lowest reported. High sensitivity was accomplished by precolumn derivatization with the noncovalent dye NanoOrange. Capillary electrophoresis separation and reaction conditions were carefully manipulated for avoiding microheterogeneity of glycoforms and inhomogeneity of multiple labeling products. The fluorescence signal was linear over the range of 10 ng/mL-10 μg/mL, corresponding to the detection requirement of recombinant human erythropoietin in biofluids and pharmaceutical samples, as demonstrated by a real sample analysis. Although the salt in reaction mixtures showed a detrimental effect on the fluorescence of the derivatives, this method could tolerate a certain amount of salt, extending its application in biofluid analysis. In addition, zero-order fluorescence emission kinetics was obtained, indicating that the rapid decay of recombinant human erythropoietin was derived from a self-quenching effect.

The direct separation of five acidic phytohormones including auxin (indole-3-acetic acid, indole-3-propionic acid, and indole-3-butyric acid), jasmonic acid, and abscisic acid by differential mobility spectrometry–mass spectrometry (DMS–MS) with the use of the polar gas-phase chemical modifier 2-propanol was demonstrated. The method was rapid and simple to operate with selectivity and repeatability. The influence of experimental conditions on the separation, such as modifier types and concentration, separation voltage, and temperature, was systematically investigated. The field mobilities dependence was measured as the normalized α function using the developed DMS–MS method. All the analytes behaved as type A ions and the clustering separation mechanism dominated. These results not only gave insights of ion properties but also contributed to the prediction of DMS separation by choosing targeted and appropriate conditions for the study of acidic phytohormones.

This study aims to investigate the changes of cytokines and the effect of programmed death ligand 1 (PD-L1) signaling pathway on T cell function in patients with immune thrombocytopenic purpura (ITP). Totally, 40 untreated ITP patients were recruited and 30 healthy people were recruited as the healthy control. Then whole blood of ITP patients and healthy control was collected, respectively. The sPD-L1/anti-PD-1 was used to activate or block the programmed death (PD-1)/PD-L1 signaling pathway. The expression of PD-1 and PD-L1 on peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry. PBMCs were treated with cluster of differentiation (CD3), cluster of differentiation 28 (CD28), and phytohaemagglutinin (PHA) for 48 hours. Serum levels of sPD-1, sPD-L1, and cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the healthy control group, the percentages of PD-1+CD3+CD4+ T cells and PD-L1+HLA-DR+CD11c+ DC cells were increased in ITP patients. The levels of interferon-gamma (IFN-γ), interleukin-17 (IL-17), and sPD-1 in the serum of ITP patients were increased, while IL-4 and transforming growth factor-β (TGF-β) were decreased. Additionally, the level of sPD-1 was negatively correlated with the platelet count. Consistently, after treatment with CD3, CD28, and PHA, IFN-γ and IL-17 levels in culture supernatant of PBMCs from ITP patients were significantly higher than those from healthy controls whereas IL-4 and TGF-β levels were significantly lower. Furthermore, IFN-γ and IL-17 levels secreted by PBMCs from ITP patients decreased after sPD-L1 administration, however, IL-4 and TGF-β levels were increased. The level of IFN-γ in ITP group remained higher after anti-PD-1 blockage, but the levels of IL-4, TGF-β, and IL-17 were not significantly influenced. sPD-1 may cause the dysfunction of PD-1/PD-L1 signaling pathway, and its level is related to the severity of ITP patients. Activation of PD-1/PD-L1 with sPD-L1 may restore the imbalance of Th1/Th2 and Treg/Th17 cell subtypes in ITP patients but anti-PD-1 may exacerbate disease by enhancing IFN-γ production.

A dynamic model of dynamiCROP was applied to study environmental fate and behavior of four herbicides in wheat including florasulam, carfentrazone-ethyl, fluroxypyr-meptyl, and fluroxypyr. Meantime, their residue in wheat and dissipation half-lives in plant determined by field trials using QuEChERS liquid chromatography tandem mass spectrometry were used to verify modelling results. The combination of experimental verification and modelling prediction deciphered the fate of four pesticides in wheat field ecosystem. Besides, temperature difference of 3 °C only resulted in lower than 15% half-life difference. By quantifying the contribution of temperature, the predominant role of rain on pesticide dissipation was highlighted for the first time, namely higher precipitation leaded to faster degradation and vice versa.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) with one chiral center at the carbinol is a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As tobacco specific N-nitrosamines (TSNAs), NNK and NNAL are the most pulmonary carcinogens in tobacco products and smoke. In this paper, a chiral CE method modified with highly sulfated β-cyclodextrin (S-β-CD) was developed to investigate the stereoselective formation of NNAL from NNK in vitro in normal human bronchial epithelial (NHBE) cells. Combined with solid phase extraction (SPE) of the cell samples, NNK and NNAL enantiomers were baseline separated under the proposed CE conditions, with satisfactory recoveries (72.5-113% for NNK and (±)-NNAL) and low limits of detection (LOD, 2.5-3 μg/mL for NNK and (±)-NNAL). The cytotoxicity of NNK in NHBE cells was investigated through the cell counting kit (CCK) assay and proved to be highly dependent on the NNK's concentration. The metabolic results obtained from CE analysis demonstrated that NNK was preferentially metabolized to (+)-NNAL through carbonyl reduction. Meanwhile, the ratio of [(+)-NNAL]/[(-)-NNAL] was independent of NHBE cells' incubation time with NNK, but could be changed according to the original incubation concentration of NNK. This chiral CE method could be useful for the study on toxicology and metabolic transformations of related TSNAs.

Five naturally bioactive compounds, belonging to the representative flavonoid and pentacyclic triterpenoid types, were separated by liquid chromatography and analyzed on-line with negative ion ESI–MS. Rutin, quercetin, and oleanolic acid in the extracts of Rosa multiflora Thunb. of China were identified by the comparison of their retention times and mass spectrometric data with those of authentic standards, and their contents were 0.22, 3.8, and 45 µg g−1 of dry weight in rose hips, respectively. Five standards, including rutin, quercetin, kaempferol, lysionotin, and oleanolic acid were investigated by MS3, which facilitate further understanding of their structures.

A hydrophilic basic polysaccharide, carboxymethyl chitosan (CMC) as a capillary coating is presented with a simple preparation procedure. The CMC-coated capillary showed a long lifetime of more than 100 runs, and had good tolerance to some organic solvents, 0.1 M HCl, 0.1 M NaOH, and 5 M urea. The run-to-run, day-to-day, and capillary-to-capillary RSDs for the CMC-coated capillary were all below 2.0% for the determination of EOF. Moreover, the coatings with different concentrations and molecular weights of CMC were also investigated. The CMC-coated capillary was successfully applied to separate basic proteins and recombinant human erythropoietin (rhEPO). Furthermore, several experimental parameters, such as the concentration and pH of the running buffer, temperature, and applied voltage, were optimized for the separation of rhEPO glycoforms. Comparison of an uncoated capillary with chitosan- and CMC-coated capillaries for the separation of rhEPO glycoforms was also discussed. The results demonstrated that rhEPO glycoforms can be well separated by a CMC-coated capillary within 8 min with good reproducibility and resolution. Finally, the volatile BGE HAc-NH 4 Ac was utilized to separate rhEPO for its further application with CE-MS, achieving a satisfactory result.

Related HLA-haploidentical HSCT has been applied more and more recently, but the reconstitution of T lymphocyte subsets and its clinical significance in patients received related HLA-haploidentical non T-cell depleted in vitro high-dose peripheral blood hematopoietic SCT (RHNT-PSCT) are incompletely defined. In the present study of our RHNT-PSCT, we found that in non-aGVHD group, CD3(+) T lymphocyte recovered to normal levels gradually between 60 and 90 days, and the recovery of CD4(+) T lymphocyte was retarded significantly, CD4(+)/CD8(+) ratio was apparently inverted. Whereas, the ratio of CD4(+) CD25(+) Foxp3(+) Treg cells was significantly lower in aGVHD group than in healthy control group and non-aGVHD group, and also in grade III-IV aGVHD patients than in grade I-II aGVHD patients. Meanwhile, we observed the level of interleukin-10 (IL-10) gradually increased in serum of patients without aGVHD, but decreased in III-IV aGVHD patients significantly. Spearman correlation analysis showed that serum IL-10 level was negatively correlated with the grade of aGVHD. These results suggest that the reconstitution of peripheral blood T lymphocyte subsets is good, and dynamic detection of Treg cells and serum IL-10 level might predict aGVHD in the early stage after our RHNT-PSCT.

A versatile liquid chromatography tandem mass spectrometry method with modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation was developed for the determination of rimsulfuron, mesotrione, fluroxypyr-meptyl, and fluroxypyr. By adjusting the amount of graphitized carbon black, the herbicide analytes could be quantified with satisfactory recoveries in the range of 80–110%. A dissipation kinetics study conducted under open field conditions at two sites during 2014 showed first order equations with half-lives between 0.6 d and 3.6 d, illustrating an appropriate degree of stability and safety. The dissipation kinetics were different in the different matrices. Although the herbicides had higher initial residues in straw than those in soil, they degraded faster in straw. The terminal residues for the herbicides formulated in two water dispersible granules were all below maximum residue limits. These results not only gave insights about the analytes but also contributed to environmental protection and food safety.

Alveolar echinococcosis is a zoonotic disease caused by Echinococcus multilocularis (E. multilocularis) infection. The relationship between PD-1/PD-L1 pathway and Tregs at different stages of E. multilocularis infection has rarely been reported. This study aims to investigate the role of PD-1/PD-L1 in immunosuppression of Tregs in E. multilocularis infection. Hematoxylin-eosin staining, flow cytometry, immunohistochemistry, quantitative RT-PCR analysis, cytometric bead array and MTT assay were used to analyze liver pathological changes, percentages of PD-1(+) Tregs and PD-L1(+) dendritic cells (DCs), expression levels of PD-1, PD-L1 and Foxp3, levels of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) and proliferation of lymphocytes. During middle-late stage (day 30 to day 330) the percentages of PD-1(+) Tregs and PD-L1(+) DCs together with levels of Foxp3, IL-10 and TGF-β increased significantly and maintained at high level. The expression of PD-1 and PD-L1 was increased with the enlarging erosion of E. multilocularis, and was mainly distributed in hepatic sinus, fibrous wall of alveolar hydatid and germinal layer around foci of infection. PD-1/PD-L1 promoted the secretion of IL-10 and TGF-β. Our results indicate that engagement of the PD-1 and PD-L1 correlates with inhibition of T-cell effector function, cytokine secretion and proliferation. High expression of PD-1/PD-L1 may play an important role in stimulating CD4(+)CD25(+) T cells, and maintaining peripheral tolerance and immune evasion during chronic infection of E. multilocularis.

Because of less functionally critical carbohydrate sectors that contributed to the stability, efforts have been made to quantify intact recombinant human erythropoietin. A simple, rapid capillary electrophoresis with laser-induced fluorescence method for the assay of recombinant human erythropoietin was developed, with a limit of detection of intact recombinant human erythropoietin at subnanomolar concentration (up to 10 ng/mL or 3 × 10(-10) M), which is among the lowest reported. High sensitivity was accomplished by precolumn derivatization with the noncovalent dye NanoOrange. Capillary electrophoresis separation and reaction conditions were carefully manipulated for avoiding microheterogeneity of glycoforms and inhomogeneity of multiple labeling products. The fluorescence signal was linear over the range of 10 ng/mL-10 μg/mL, corresponding to the detection requirement of recombinant human erythropoietin in biofluids and pharmaceutical samples, as demonstrated by a real sample analysis. Although the salt in reaction mixtures showed a detrimental effect on the fluorescence of the derivatives, this method could tolerate a certain amount of salt, extending its application in biofluid analysis. In addition, zero-order fluorescence emission kinetics was obtained, indicating that the rapid decay of recombinant human erythropoietin was derived from a self-quenching effect.

To explore the percentage of CD4(+);CD25(+);Foxp3(+); regulatory T cells (Treg) in peripheral blood of chronic lymphocytic leukemia (CLL) patients and the correlations with clinical prognosis.The study enrolled 30 healthy individuals and 28 CLL patients. The CD4(+);CD25(+); Treg and CD4(+);CD25(+);Foxp3(+); Treg were detected by the flow cytometry in their peripheral blood. Of the 28 CLL patients, 19 received treatment and follow-up.The number of CD4(+);CD25(+); Treg in the pre-treated cases (n = 28) was higher than that in the healthy controls (n = 30) with significant statistical difference (P0.05). The number of CD4(+);CD25(+);Foxp3(+); Treg was higher in the pre-treated cases (n = 28) than that in the treated cases (n = 19) and in the healthy controls (n = 30) (P0.05). Compared with the healthy controls, the treated cases (n = 19) had the higher level of CD4(+);CD25(+);Foxp3(+); Treg (P0.05). The CD4(+);CD25(+);Foxp3(+); Treg was positively correlated with the expressions of CD38, β2-microglobulin (β(2);-MG), zeta-associated protein 70(ZAP-70) and the clinical Binet and Rai stages.The CD4(+);CD25(+);Foxp3(+); Treg might be a valuable indicator for assessing the therapeutic efficacy, disease progression and prognosis of the CLL patients.

Alveolar echinococcosis (AE) is a severe parasitic disease caused by the infection of Echinococcus multilocularis (Em). Very little is known on the relationship between TGF-β/Smad signaling pathway and Treg/Th17 balance in the infected liver at different periods after Em infection. Using qRT-PCR, immunohistochemistry, flow cytometry and CBA assay, we measured the expression levels of TGF-β, Smad2/3/7, ROR-γt, Foxp3, IL-17, IL-10 and percentages of Th17 cells and Treg cells in mouse AE model, from day 2 to day 270 after infection. In the early stage of infection (day 2 to day 30), Smad7 was up-regulated and the TGF-β pathway was inactivated. In the middle stage of infection (day 30 to day 90), TGF-β and Smad2/3 were up-regulated. And levels of Treg cells, Foxp3, Th17 cells, RORγt, IL-17, IL-10 and IL-6 were significantly increased. In the late stage of infection (day 90 to day 270), Treg cells, Foxp3, TGF-β and IL-10 maintained at high levels whereas Th17 cells and IL-17 decreased significantly. TGF-β/Smad signaling pathway was activated during the chronic infection. Our data suggest that there were Treg/Th17 imbalance in the middle and especially in the late stage of Em infection and that Treg/Th17 imbalance may be regulated by TGF-β/Smad signaling pathway. Treg and Th17 subsets may be involved in regulating immune tolerance and tissue inflammation, and facilitating the long-term survival of Em in the host.

Background The fine balance of Th17/Treg is crucial for maintenance of immune homeostasis. The objective of this study was to investigate the balance of Th17/Treg and the expression of related cytokines in Uighur cervical cancer patients. Methods Peripheral blood was collected from 65 cases of cervical cancer patients, 42 cases of cervical CIN patients and 40 healthy people. Flow cytometry was used to detect the percentages of T cell subsets, including CD3+ T cells, CD4+ T cells, CD8+ T cells, Treg cells and Th17 cells. ELISA assay was conducted to detect expression levels of TGF-β, IL-6, IL-10, IL-17, IL-23 and IFN-γ. Results There were no significant difference in the levels of CD3+ T cells, CD4+ T cells, CD8+ T cells, and the ratio of CD4+/CD8+ among the cervical cancer group, the CIN group and the healthy control group. However, compared with the healthy control group, the percentages of CD4+ CD25+ Treg, CD4+CD25+CD127- Treg, CD4+IL17+ Th17, CD4+CD25+Foxp3+, CD4+CD25- Foxp3+, CD8+CD25+CD127-Treg and CD8+CD25+Foxp3 were significantly higher in the cervical cancer group and the CIN group. Similar results were also found in the Th17/Treg ratio and the related cytokines. There was no significant difference between the cervical cancer group and the CIN group. Additionally, Th17 cell levels were positively correlated with IL-6, IL-23 and IL-17. Also, Treg cell levels were positively correlated with TGF-β, IL-10 and IL-6. Contrarily, Treg cell levels and IFN-γ were negatively correlated. Conclusions Our data indicated that the Th17/Treg balance was broken in peripheral blood of cervical cancer patients. Analysis of Th17/Treg balance may have a significant implication in diagnosing cervical cancer. Virtual slides The virtual slide for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1813823795931511

To develop a reversed-phase HPLC method for simultaneous determination of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde in Rhizoma Gastrodia.A Kromasil C18 column (4.6 mm x 250 mm, 5 microm) was used with a methanol-water-0.1% acetic acid gradient elution system. The eluates were detected at 270 nm, the flow rate was 1.0 mL x min(-1) and the column temperature was 35 degrees C.The linear range of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde were 19.1-383 (r = 0.999 9), 0.620-12.4 (r = 0.999 9), 2.45-49.0 (r = 0.999 9), 0.280-5.63 mg x L(-1) (r = 0.999 6), respectively. The average recoveries (n = 9) of the four components were 96.7% -97.7%, RSD1.6%.The method is accurate, sensitive and reliable for determination of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde in Rhizoma Gastrodia.

A CE-MS method for rapid determination of aristolochic acid-I and aristolochic acid-II (AA-II) in traditional Chinese medicines and biological samples was described in the present paper. AA-I and AA-II can be baseline separated within 6 min by CE-MS with carboxymethyl-chitosan-coated capillary. CZE conditions including pH, concentration of buffer, applied voltage, and capillary temperature were systematically investigated, and the composition and flow rate of sheath liquid were also optimized for CE-MS. Furthermore, the CE-UV method without any additives in BGE solution was established and compared with the CE-MS method. The results showed that the two methods could achieve satisfactory separation efficiency, repeatability, and linearity, while the LOD was 0.6 microg/mL for CE-UV and 0.05 microg/mL for CE-MS. Compared with the CE-UV method, the sensitivity of CE-MS was significantly improved, in addition to the structure information provided by MS detection at the same time. As an application example, a spiked sample in human serum was analyzed by the CE-MS method, indicating that the new CE-MS method can be applied to analyze AAs in biological samples.

Recombinant human erythropoietin (rhEPO) is one of hormone dopings. In the recent years, it has been always misused in endurance competition sports. It is extremely difficult to discriminate between the natural endogenous erythropoietin (EPO) and recombinant exogenous hormone because they have identical amino acid sequences, and EPO has a relatively short half-life and very low concentration in blood or urine. In this paper, the research progress on the analysis and detection of rhEPO and its analogs by direct and indirect methods are reviewed. Future direction and prospects of the detection of rhEPO in doping control are discussed based on the research of our group.

We designed and fabricated a novel microdevice to facilitate continuous adsorption phenomena for biological sample preparation. Using the device, we also developed an online, highly integrated, multifunctional strategy, with a promise of accepting a large volume of crude tissue extracts with the end point generation of a reliable MS identification within 20 min. Under an external electric field, charged membranes can adsorb multiple layers of proteins, which exceed the capacity limit of common resins or membranes. It enlarges sample loading and trapping efficiency, thus bypasses the tradeoff between sample capacity and downstream detection sensitivity. This integrated approach, formed by synergistic utilization among electric field, membrane, and fluidic handling at the microscale, reduces the overall complexity of crude samples in one step for direct MS analysis. The sample preparation goals, including enrichment, desalting, removal of noncharged contaminants, and initial fractionation, can be rapidly performed in a single device. The strategy facilitates reproducible MS quantification by circumventing traditional laborious and time-consuming sample preparation steps. In addition, MEPD extended the ion trap linear dynamic range from 2 to at least 4 orders of magnitude by eliminating ion suppression effect, enriching target analyte(s), and decreasing sample loss during integrated sample preparation.

The enantioresolution of zolmitriptan was performed using cyclodextrin (CD)-modified capillary zone electrophoresis (CZE) with hydroxypropyl-beta-CD (HP-beta-CD) as the chiral selector. The influence of experimental conditions on the enantioseparation of zolmitriptan, such as pH, temperature, applied voltage, and concentrations of running electrolyte and CD, was systematically investigated, obtaining a baseline separation of two enantiomers by the use of a 25 mM sodium dihydrogen phosphate (SDPH) running electrolyte (pH 2.4) containing 30 mM HP-beta-CD at 15 degrees C. Binding constants for each enantiomer-HP-beta-CD pair at different temperatures, as well as thermodynamic parameters for binding, were calculated. A nonlinear van't Hoff plot was obtained, indicating that the thermodynamic parameters of complexation were temperature-dependent for zolmitriptan enantiomers. The significant contribution of the enthalpy difference to the Gibbs free energy change suggested a stereomeric barrier mechanism for chiral recognition.