Knockdown of MAGE-A6 enhanced the irradiation sensitivity of non-small cell lung cancer cells by activating the AMPK pathway.

Non-small cell lung cancer is a common respiratory tumor. The mortality rate of lung cancer patients has continued to rise in recent years. Several studies revealed that the expression of melanoma antigen 6 (MAGE-A6) promoted the development of multiple types of cancer. In addition, the suppression of AMPK pathway could restrict the radiosensitization of prostate cancer cells. Inhibition of MAGE-A6 activated the AMPK pathway in colorectal cancer cells. However, whether the MAGE-A6 could regulate the radiosensitivity of non-small cell lung cancer cells by regulating of the AMPK pathway is unclear. In this study, we established the MAGE-A6 knockdown in A549 and H1299 cells. Next, the apoptosis and proliferation of these cells were detected by the flow cytometry analysis and colony formation assay after the irradiation, respectively. Then, the expression of p-AMPKα1 and p-S6K1 in these cells was explored by the western blotting. After that, we inhibited the expression of AMPKα1 in MAGE-A6 knockdown cells. The proliferation and apoptosis of these cells were detected with colony formation assay and flow cytometry analysis. Finally, the tumor formation of these cells was detected in nude mice. Our results showed that inhibition of MAGE-A6 suppressed the proliferation and aggravated the apoptosis of A549 and H1299 cells after the irradiation. Knockdown of MAGE-A6 activated the expression of p-AMPKα1 and repressed the expression of p-S6K1 in these cells. Suppression of AMPKα1 in MAGE-A6 knockdown cells abolished these effects. Knockdown of MAGE-A6 also enhanced the radiosensitivity of these cells in vivo. These results suggested that inhibition of MAGE-A6 promoted the radiosensitivity of non-small cell lung cancer cells by activating AMPK pathway. Therefore, MAGE-6 has the potential to be explored as the therapeutic target for the treatment of non-small cell lung cancer in clinical.

New understanding of electrical activity brought by surface potential of cardiomyocytes.

Aiming at the problem encountered in the previous research: during the electrical activity of cardiomyocytes, the influent ions do not seem to be directly derived from the extracellular fluid. We chose to cut in from the colloidal properties of the cells, follow the basic principles of physical chemistry, and establish hypotheses along the derivation of the structural characteristics of cardiomyocytes. Through the surface ion adsorption experiment and patch clamp experiment of living cells, under the condition of sequentially reducing the concentration of Na+ in the extracellular fluid, we observed the exchange and diffusion of adsorbed ions on the cell surface; the changes of inflow INa, ICa-L and action potential; and correlation between results. The results showed that the hypothesis is true. The observed parameter changes were consistent with the fact that during depolarization of cardiomyocytes, the ions of influx were derived from the inference of adsorbed ions on the cell surface; at the same time, it also provided an objective and realistic explanation for the generation of electrocardiogram.

Protective Effect of Mannitol on Cisplatin-Induced Nephrotoxicity: A Systematic Review and Meta-Analysis.

INTRODUCTION: Cisplatin, a chemotherapeutic drug, is widely used for the treatment of various malignant tumors with good effects. However, cisplatin-induced nephrotoxicity is a major dose-limiting factor and a significant adverse event. Mannitol is used to reduce cisplatin-induced nephrotoxicity, which is controversial. This study aimed to evaluate the efficacy and safety of a hydration regimen containing mannitol against cisplatin-induced nephrotoxicity through a meta-analysis. METHODS: Potential records from PubMed, EMBASE, Cochrane Library, and ClinicalTrials that met the inclusion criteria were included from inception to May 2021. Cochrane Collaboration tools were used to assess the risk of bias in the included studies. Jadad's and NOS scores were applied to assess the quality of randomized controlled trials (RCTs) and case-control studies. A random-effects model or fixed-effects model was used depending on the heterogeneity. Subgroup analyses were performed to evaluate the potential study characteristics. The pooled odds ratios (ORs) and 95% confidence intervals (CIs) were evaluated. RESULTS: Four RCTs and seven case-control studies involving 4168 patients were included. Pooled results showed that mannitol use could reduce the incidence of cisplatin-induced nephrotoxicity (OR = 0.66, 95% CI [0.45-0.97], p = 0.03), especially reducing grade 3 nephrotoxicity events according to CTCAE 4.0 (OR = 0.37,95% CI [0.16-0.84]). Moreover, mannitol use was not significantly associated with creatinine clearance, serum creatine, and electrolyte disturbance (p > 0.05). Gastrointestinal cancer (OR = 0.36, 95% CI [0.15-0.83], p = 0.02) and urinary tract cancer (OR = 0.32,95% CI [0.14-0.73], p = 0.007) may be more sensitive to mannitol, although the test for overall effect was significantly different (OR = 0.66, 95% CI [0.49-0.89], p = 0.007). For patients with diabetes and hypertension, mannitol may worsen renal function (OR = 1.80, 95% CI [1.18-2.72], p = 0.006; OR = 2.19, 95% CI [1.50, 3.19], p < 0.0001, respectively). Mannitol may have a better protective effect when doses of mannitol were ≥ 25 g (OR = 0.58, 95% CI [0.39-0.88], p = 0.01) and doses of cisplatin < 75 mg/m2 (OR = 0.59, 95% CI [0.36-0.94], p = 0.03). It revealed that mannitol use was likely to cause nausea or vomiting (OR = 1.86, 95% CI [1.20-2.89], p = 0.006). CONCLUSION: Current evidence revealed that mannitol was an effective and safe drug to reduce cisplatin-induced nephrotoxicity events, especially Grade 3 events. However, it may cause more nausea/vomiting events and deteriorate renal function in patients with diabetes or hypertension. We also found that mannitol had the best effect when mannitol was ≥ 25 g in total or cisplatin was < 75 mg/m2. Meanwhile, mannitol may have a better effect on gastrointestinal and urinary tract cancers. SYSTEMATIC REVIEW REGISTRATION: crd. york. ac. uk/PROSPERO, CRD 42021253990.

Compare the Effects of Magnolol on Gastric Cancer Cells Through c-Jun N-Terminal Kinase Signaling Pathway and Gold Magnetic.

This article explores the effects and mechanisms of magnolol on the proliferation of gastric cancer cells as well as the apoptosis. First, 0 (control group), 20, 40, and 80 /x mol/L magnolol were observed on SGC-7901 cells for 24, 48, and 72 h. We use MTT method to measure the cell viability, and apoptosis and cells were detected by flow cytometry. Cell proliferation inhibition rate, apoptosis and cell cycle experiments showed that P-value < 0.05 means the difference is statistically significant. And the results which compare the control group, the 20, 40, and 80 /x mol/L show that honokiol had lower cell viability (P < 0.01), increased apoptotic rate (P < 0.01), and cell cycle stay in the G1 phase (P < 0.01), so we found that honokiol may suppress the proliferation of SGC-7901 cells and stimulate apoptosis by regulating cyclin and apoptosis-related proteins. With the development of nanomaterials synthesis technology and application in biomedicine, gold magnetic composite nanomaterials have unique properties, so they have been widely concerned in many applications. We combine gold and magnetic nanomaterials through other nanostructures to achieve the integration of diagnosis and treatment of tumors. We have synthesized two kinds of gold magnetic nanocomposites, GNR-PPy-FA nanocomposites. With the role of chemotherapy and heat and light therapy, GNR-PPy-FA nanocomposites have high light-to-heat conversion efficiency. Cell experiments verify the effect of chemotherapy and photothermal treatment of composite nanomaterials. After incubation with gold magnetic composite nanomaterials, the cell survival rate of tumor cells decreased to about 15%. In addition, both types of gold magnetic nanocomposites have the ability to dually target cancer cells, and the modification of folic acid and cancer cell membranes makes the material more biocompatible.

Follicular Helper T Cell Derived Exosomes Promote B Cell Proliferation and Differentiation in Antibody-Mediated Rejection after Renal Transplantation.

Follicular helper T cells (Tfh cells) are closely related to the occurrence and development of antibody-mediated rejection (AMR) after renal transplantation. Exosomes play a key role in the rejection after organ transplantation. However, whether Tfh-derived exosomes are involved in AMR has not been reported. We collected peripheral blood from 42 kidney transplant patients and found no significant differences in CD4+CXCR5+ and CD4+CXCR5+CXCR3+CCR6-exosomes between AMR and non-AMR groups, whereas the proportion of CD4+CXCR5+CXCR3-exosomes was significantly higher in AMR group than that in non-AMR group; CTLA-4 expression of CD4+CXCR5+exosomes was significantly lower in AMR group than that in non-AMR group. HLA-G expression was not significantly different between two groups. We further separated CD4+CXCR5+cells from patients by magnetic beads. Coculture experiments showed that Tfh cell-derived exosomes in AMR patients significantly promoted B cell proliferation and differentiation, compared with non-AMR group, the percentage of B cells and plasma cells increased by 87.52% and 110.2%, respectively. In conclusion, our study found that Tfh cell-derived exosomes could promote the proliferation and differentiation of B cells and they may play an important role in the development of AMR after renal transplantation.

Indirubin inhibits cell proliferation, migration, invasion and angiogenesis in tumor-derived endothelial cells.

PURPOSE: Hepatocellular carcinoma is one of the most predominant malignancies with high fatality rate and its incidence is rising at an alarming rate because of its resistance to radio- and chemotherapy. Indirubin is the major active anti-tumor ingredient of a traditional Chinese herbal medicine. The present study aimed to analyze the effects of indirubin on cell proliferation, migration, invasion, and angiogenesis of tumor-derived endothelial cells (Td-EC). METHODS: Td-EC were derived from human umbilical vein endothelial cells (HUVEC) by treating HUVEC with the conditioned medium of human liver cancer cell line HepG2. Cell proliferation, migration, invasion, and angiogenesis were assessed by MTT, wound healing, in vitro cell invasion, and in vitro tube formation assay. RESULTS: Td-EC were successfully obtained from HUVEC cultured with 50% culture supernatant from serum-starved HepG2 cells. Indirubin significantly inhibited Td-EC proliferation in a dose- and time-dependent manner. Indirubin also inhibited Td-EC migration, invasion, and angiogenesis. However, indirubin's effects were weaker on HUVEC than Td-EC. CONCLUSION: Indirubin significantly inhibited Td-EC proliferation, migration, invasion, and angiogenesis.

Simultaneous detection of IgG and IgM antibodies against a recombinant polyprotein PstS1-LEP for tuberculosis diagnosis.

BACKGROUND: Commercial serological tests for the diagnosis of tuberculosis (TB) show poor sensitivity and specificity, and a new approach to antigen screening is required to improve the accuracy of serodiagnosis. METHODS: Using an indirect enzyme-linked immunosorbent assay (ELISA), we evaluated the responses of IgG and IgM antibodies to the recombinant PstS1-LEP protein expressed in Escherichia coli, a polyprotein of PstS1 and line multi-epitopes polypeptide (LEP). RESULTS: The mixture of anti-human IgG and IgM added to a well [Ig(G + M)], which was different from the combination of IgG and IgM (IgG + IgM), had a stronger immunoreactivity to PstS1-LEP than the single antibody. IgG and Ig(G + M), but not IgM against the PstS1-LEP protein effectively distinguished TB patients from patients with nontuberculous pulmonary disease (NTBPD) and healthy controls (HCs). Compared with IgG, the sensitivities of Ig(G + M) and IgG + IgM varied from 71.4% to 77.6% and 72.7% in pulmonary TB (PTB) patients and from 42.1% to 64.0% and 55.8% in extrapulmonary TB (EPTB) patients, respectively. The specificity of Ig(G + M) did not decrease, and was higher than that provided by IgG + IgM in HCs with positive tuberculin skin test. CONCLUSION: These findings suggest that PstS1-LEP can act as a candidate for detecting Ig(G + M) in serum from PTB and EPTB patients.

[Expression of T lymphocyte cytokines in peripheral blood of renal transplant recipients].

OBJECTIVE: To evaluate the clinical values of T-lymphocyte cytokines in renal transplant acute rejection. METHODS: A total of 31 recipients with renal transplantation and 15 healthy volunteers from January 2010 to January 2012 were enrolled and divided into acute rejection group (n = 11) and stable renal allograft function group (n = 20) according to the inclusion criteria. Peripheral blood was collected from the patients before transplantation, 1, 7, 14, 28 day after transplantation and acute rejection onset. Cytometric bead array (CBA) was used to monitor the levels of interleukin-17 (IL-17), interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin(IL)-10, IL-6, IL-4 and IL-2. The associations of the changes and levels of cytokines in 3 groups were examined with Pearson correlation analysis. RESULTS: The levels of IL-17A, TNF, IL-10 and IL-2 in recipients before transplantation were (3.40 ± 1.29) , (1.79 ± 0.53) , (2.73 ± 0.65) and (1.79 ± 1.02) ng/L respectively and decreased significantly compared to healthy volunteers ((4.52 ± 2.01), (3.36 ± 1.09) , (3.91 ± 0.42) , (3.12 ± 1.07) ng/L respectively, all P < 0.05). However the levels of IFN-γ, IL-6 and IL-4 showed no significant changes between two groups (all P > 0.05). In acute rejection group after transplantation, the levels of IL-17A, IFN-γ, IL-10 and IL-6 were (9.47 ± 4.75) , (5.01 ± 2.23) , (12.20 ± 5.79) , (6.55 ± 3.45) ng/L respectively and increased significantly compared to the renal allograft function group ((4.39 ± 1.26), (2.90 ± 0.87),(5.68 ± 2.25) and (2.10 ± 0.70) ng/L respectively, all P < 0.05); the level of IL-17A was correlated with those of IFN-γ and IL-4 (Pearson r = 0.519, 0.395, both P < 0.01), the level of IFN-γ was correlated with those of IL-4 and IL-2 (r = 0.276, 0.335, all P < 0.05) , the level of TNF was correlated with that of IL-4 (r = 0.423, P < 0.05) and the level of IL-10 was correlated with that of IL-6 (r = 0.361, P < 0.05). CONCLUSIONS: Cytokines play an important role in acute rejection of renal transplant. And further understanding of its mechanism may provide experimental and preventive rationales.

Clinical efficacy of rituximab for acute rejection in kidney transplantation: a meta-analysis.

OBJECTIVES: This meta-analysis was undertaken to compare the efficacy and safety of pretransplant treatment with rituximab in sensitized patients receiving kidney transplantation. METHODS: PubMed, EMBASE, and Cochrane databases were searched to identify studies that used pretransplantation rituximab in eligible patients. The major outcomes included antibody-mediated rejections (AMR) after kidney transplantation and one-year graft survival rate. The meta-analysis was performed using fixed-effects model. RESULTS: Seven studies were identified including a total of 589 patients, of whom 312 were treated without rituximab, while 277 were treated with rituximab. In our meta-analysis, patients treated with rituximab had significantly fewer AMR after kidney transplantation [odds ratio (OR) 0.52, 95 % CI 0.28, 0.98, P = 0.04] and higher rate of one-year graft survival rates (OR 3.02, 95 % CI 1.14, 8.02, P = 0.03), indicating that rituximab is effective against acute rejection and enhances graft survival in kidney transplantation. No differences were noted in other efficacy and safety parameters in these two patient groups. CONCLUSIONS: We demonstrated that preinduction with rituximab could significantly improve AMR and graft survival rates in sensitized patients undergoing kidney transplantation. Future prospective controlled studies are warranted to further understand rituximab's role in kidney transplantation.

Dynamic expression changes between non-muscle-invasive bladder cancer and muscle-invasive bladder cancer.

AIMS AND BACKGROUND: Despite elaborate characterization of the risk factors, bladder cancer is still a major epidemiological problem whose incidence continues to rise each year. We aim to investigate the dynamic expression changes between non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). METHODS: The gene expression profile GSE13507 was obtained from the Gene Expression Omnibus, and the R package was used to identify gene expression signatures (GESs) between NMIBC and MIBC. Gene ontology enrichment analysis was performed for GES function analysis. We used miRTarBase and TargetScan to identify the differentially regulated microRNAs, and TfactS to identify transcription factors between NMIBC and MIBC. Bionet was used to identify the differentially expressed subnetwork. RESULTS: A total of 802 upregulated NMIBC GESs and 668 downregulated MIBC GESs were identified. Functional enrichment analysis revealed that the MIBC GESs were majorly involved in cell cycle and inflammatory response. miR-29c and miR-9 were regarded as key microRNAs in MIBC. SMAD3 in MIBC and SMAD5 and SMAD7 in NMIBC were potential activated transcription factors. In addition, a subnetwork that was considered to capture the differences between MIBC and NMIBC was identified, of which GRB2 and UBC were the hub nodes. CONCLUSIONS: Some key microRNAs, activated transcription factors and hub nodes have been identified in this study, which may be used as potential biomarkers or targets for the diagnosis, treatment and detection of bladder cancer at different stages.

[Frequency analyses of human cytomegalovirus specific CD8(+) T cell in kidney transplantation].

OBJECTIVE: To establish a new detection method for cytomegalovirus (CMV) specific cytotoxic CD8(+)T cells and examine its proportion and significance in peripheral blood from kidney transplant recipients. METHODS: A total of 30 recipients of kidney transplantation from January 2009 to December 2010 for the first time were enrolled. And 10 healthy volunteers were selected as health control group. Tetramer technology was applied. The proportions of CMV antigen specific T cells expressed in each group were detected directly by flow cytometry. RESULTS: The positive rate of CMV antigen specific CTL, CMV-pp65 specific CD8(+)T cell was between 0.16%-7.21% in kidney transplant recipients (n = 30) and health control group (n = 10). The proportions of CMV antigen specific CTL were 2.95% ± 0.62% in CMV+ group. And it was significantly higher than that in CMV-group (1.17% ± 0.45%) and health control group (0.65% ± 0.17%) (P = 0.003,0.006). In CMV+ group, the proportion of CMV antigen specific CTL was 2.95% ± 0.62% in CMV-pp65 positive phase and decreased significantly to 1.50% ± 0.32% after turning into negative phase. In CMV+ group (n = 15), the proportion of CMV antigen specific CTL was positively related with the number of CMV-pp65 positive cells (Pearson test, r = 0.871, P < 0.01). CONCLUSIONS: The proportion of specific CTL is an important guide for evaluating and judging the prognosis of CMV infection. And it may provide rationales for future targeted therapy in kidney transplant recipients.

HLA-G expression in the peripheral blood of live kidney transplant recipients.

BACKGROUND: The human leukocyte antigen-G (HLA-G) has been considered to be an important tolerogeneic molecule playing an essential role in maternal-fetal tolerance, upregulated in the context of transplantation, malignancy, and inflammation, and has been correlated with various clinical outcomes. The aim of this study was to investigate the clinical relevance of the expression of membrane HLA-G (mHLA-G), intracellular HLA-G (iHLA-G), and soluble HLA-G (sHLA-G) in the peripheral blood of live kidney transplant recipients. METHODS: We compared the expression of the three HLA-G isoforms in three groups, healthy donors (n=20), recipients with acute rejection (n=19), and functioning transplants (n=30). Flow cytometry was used to detect the expression of mHLA-G and iHLA-G in the T lymphocytes of peripheral blood from subjects in the three groups. Enzyme-linked immunosorbent assays were used to detect sHLA-G in the plasma from the three groups. RESULTS: There were no significant differences in mHLA-G and intracellular HLA-G among the three groups, but the sHLA-G plasma level was higher in the functioning group than in the acute rejection or healthy group. We found a subset of CD4(+)HLA-G(+) and CD8(+)HLA-G(+) T lymphocytes with low rates of mHLA-G expression in the peripheral blood of kidney transplantation recipients. Intracellular expression of HLA-G was detected in T lymphocytes. However, there was no correlation between acute rejection and the mHLA-G or intracellular HLA-G expression. CONCLUSION: sHLA-G was the major isoform in the peripheral blood of live kidney transplant recipients and high sHLA-G levels were associated with allograft acceptance.

[Quantitative proteomic analysis of streptomycin resistant and sensitive clinical isolates of Mycobacterium tuberculosis].

OBJECTIVE: To identify specific antigens related to streptomycin resistant (SMr) Mycobacterium tuberculosis. METHODS: Cellular proteins were extracted from SMr clinical isolate 01108, SM-sensitive clinical isolate 01105 and H37Rv. Differential expression proteins were identified with isobaric tags for relative and absolute quantitation (iTRAQ) combined with Nano LC-MS/MS technology. RESULTS: Approximately 194 and 146 differential expression proteins were identified in 01108 strain compared with the proteomic profiles of 01105 strain and H37Rv, respectively, and 121 proteins were identified in 01108 strain compared with the proteomic profiles of both 01105 strain and H37Rv. Identified proteins showed a pI (isoelectric point) variation between 3.74-12.48 and a molecular mass (M) range between 7.63 and 326.2 kDa. Differential expression proteins were mainly associated with metabolism (involved in intermediary metabolism, respiration, and lipid metabolism) and took part in catalysis and binding function. Seven ribosomal proteins (Rv0056, Rv0641, Rv0652, Rv0701, Rv1630, Rv2442c and Rv2785c) and seven proteins (the ratios > 1.20 or < 0.55) were commonly down-regulated in 01108 strain compared with both 01105 strain and H37Rv, i. e. the thiol peroxidase (Rv1932), acyl carrier protein dehydrogenase (Rv0824c), 30S ribosomal protein S15 (Rv2785c), acetone acid dehydrogenase E2 part (Rv2215), two-component transcriptional regulatory protein (Rv3133c) and Hypothetical protein (Rv2466e and Rv2626c). CONCLUSION: Differential expression proteins were found in SMr strain compared with both SM-sensitive strain and H37Rv. Further studies are needed to assess the role of these differential expression proteins in SM resistance.

Γ-aminobutyric acid B receptor improves carbon tetrachloride-induced liver fibrosis in rats.

BACKGROUND: It was well known that angiotension II can inhibit hepatic stellate cell activation. The GABAB receptor was upregulated when the hepatic stellate cell line was stimulated by angiotension II in our previous study. But the role of the GABAB receptor in liver fibrosis has never been reported. AIM: In the present study, we investigated the effects of this receptor on carbon tetrachloride-induced liver fibrosis in rats. METHODS: The rats were divided into four groups including GABAB receptor agonist, antangonist, model and control group. α-smooth muscle actin (α-SMA) and GABAB receptor expression levels were detected by immunohistochemistry and real-time polymerase chain reaction. Liver function tests were performed once blood samples was taken; Western blot analysis was used to detect protein expression level of α-SMA and TGF-ß1. RESULTS: We found baclofen ameliorated the CCl4-induced rats's liver fibrosis. The highest liver enzymes and α-SMA protein levels were found in the CGP35348 group. CONCLUSION: The GABAB receptor may have a protective role in the liver.

Association of vitamin D receptor FokI and ApaI polymorphisms with human cytomegalovirus disease in the first three months following kidney transplantation.

BACKGROUND: Untreated human cytomegalovirus (CMV) disease (CMVD) is an identified risk factor for reduced rates of patient (and graft) survival, death or retransplantation in kidney transplant recipients due to increased immunological tolerance after transplant. Vitamin D receptor (VDR) gene polymorphisms have an obvious relationship with autoimmune diseases but the relationship between VDR gene polymorphisms and CMVD are not well understood. This study investigated the relationship between VDR FokI and ApaI gene polymorphisms and CMVD, and their value for predicting risk of CMVD. METHODS: Ninety-eight kidney transplantation recipients were randomly chosen for which peripheral blood samples and case histories for the first three months after kidney transplantation were obtained. Using polymerase chain reaction-restriction fragment length polymorphisms, 30 recipients were found to be homozygous for the FokI gene (FF), 47 heterozygous (Ff), and 21 were homozygous (ff). Likewise, similar analyses determined that 12 recipients were homozygous for the ApaI gene (AA), 36 heterozygous (Aa), and 50 homozygous (aa). Factors affecting the prognosis of the kidney transplantation were compared for all genotypes by statistical analysis before operation. Infection by CMV for all recipients was detected by immunofluorescence assay to diagnose CMVD. RESULTS: No statistical significance was observed for the factors affecting the prognosis of the kidney transplantation between both genotypes; however, statistical differences in CMVD among the FokI genotypes were identified. It was determined that the risk of CMVD was significantly increased for recipients of the ff genotype than for other genotypes. There was no statistical significance observed for CMVD among ApaI genotypes. CONCLUSIONS: The recessive f allelic gene of VDR can be regarded as a risk factor of CMVD while FF recipients have lower incidence of CMVD after kidney transplantation. ApaI genotypes showed no relationship with predisposition to CMVD.

Crystallization and preliminary crystallographic study of a trypsin-resistant catalytic domain of human calcineurin.

Calcineurin, a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase, plays a key role in a number of cellular pathways, including T-cell activation, and is an important molecular target of the immunosuppressive drugs cyclosporin A and FK506. To understand the structural basis underlying the activation of calcineurin by calmodulin, X-ray crystallography was employed to solve the three-dimensional structure of the free calcineurin catalytic domain (residues 20-347 of the A subunit). To accomplish this, a bacterially expressed glutathione S-transferase (GST) fusion protein of the human calcineurin catalytic domain was first purified by GST-affinity chromatography. After limited digestion by trypsin, the catalytic domain (Cncat) was purified using anion-exchange and size-exclusion chromatography. Crystallization of Cncat was achieved by the hanging-drop vapour-diffusion method at pH 6.5 using PEG 6000 as precipitant. The diffraction results showed that the Cncat crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 161.6, b = 87.4, c = 112.0 Å. There are four Cncat molecules in the asymmetric unit, with 49.5% solvent content. An X-ray diffraction data set was collected to 2.87 Šresolution and a clear molecular-replacement solution was obtained. The active site of Cncat is open to the solvent channels in the crystal packing.

[Up-regulation of the sHLA-G5 level by human-mouse chimeric anti-IL-2R monoclonal antibody treatment in kidney transplantation].

OBJECTIVE: To find whether the up-regulation of soluble human leucocyte antigen-G5 (sHLA-G5) levels is a new function mechanism of anti-interleukin-2 receptors (anti-IL-2R) monoclonal antibody treatment in kidney transplantation. METHODS: A total of 215 recipients at our centre from January 2006 to December 2007 were divided into antibody use group (n = 141) and antibody non-use group (n = 74) and another healthy group (n = 69). The sHLA-G5 level in peripheral blood was detected by enzyme-linked immunosorbent assay (ELISA). And the expression of HLA-G5 was confirmed by Western blot and Real-time polymerase chain reaction (PCR). RESULTS: sHLA-G5 levels was (56 ± 30) µg/L in using anti-IL-2 receptor monoclonal antibody before transplantation, It was higher than that before use antibody [(34 ± 20) µg/L], also higher than healthy group [(35 ± 17) µg/L] and antibody non-use group [(36 ± 19) µg/L, P < 0.05, respectively]. At Day 1, Day 4, Week 1, Week 2 post-transplantation, the level of sHLA-G5 of recipients with antibody use was significantly higher than that of those with antibody non-use. The values were as follows: (95 ± 35) µg/L vs (54 ± 16) µg/L, (131 ± 24) µg/L vs (75 ± 22) µg/L, (167 ± 44) µg/L vs (62 ± 17) µg/L, (172 ± 35) µg/L vs (45 ± 16) µg/L (all P < 0.01). And the results of Western blot and RT-PCR corresponded to those of ELISA. CONCLUSION: The preoperative use of first dose of anti-IL-2R monoclonal antibodies results in the up-regulated level of sHLA-G5. Thus it is beneficial for protecting the kidney survival and reducing the risks of acute rejection.

Assessment of five antigens from Mycobacterium tuberculosis for serodiagnosis of tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major public health issue, particularly in developing countries, and thus effective diagnostic methods for TB remain a central theme in basic and clinical research. To evaluate five antigens (38-kDa protein [38kDa], Rv3621c, Rv3618, 38kDa-ESAT-6 [38E6], and Ag85B-HBHA [AH]) in serological tests for TB patients, we recruited 288 patients and 201 healthy controls. The median IgG reactivity to 38kDa, 38E6, and AH was higher than that to Rv3618 and Rv3621c in pulmonary TB. 38kDa and 38E6 provided high sensitivities in pulmonary TB but low sensitivities in extrapulmonary TB (EPTB). The specificities achieved by 38kDa and 38E6 ranged from 82.0% to 93.9% in patients with non-TB respiratory disease (PD) and in controls. 38kDa and 38E6 exhibited lower sensitivities and higher specificities than their combinations with Rv3618. These findings provide useful information on the relative importance of the above five antigens and suggest that combinations of Rv3618 with 38kDa and 38E6 can increase their sensitivities, but their specificities need to be further increased.

[Identification and functional study of Tim-1(+)CD19(+) regulatory B cell in kidney transplantation recipients].

OBJECTIVE: To observe the ratio of Tim-1(+)CD19(+) B cell in the peripheral blood of kidney transplantation recipients and elucidate its functions. METHODS: From December 2009 to June 2010, a total of 35 pairs of kidney transplant recipients were selected and divided into 3 groups: healthy donors as control (n = 35), pre-transplantation (n = 35) and post-transplantation (n = 35). The profiles of Tim-1(+)CD19(+) B cell in kidney transplantation donors and recipients were analyzed and sorted by flow cytometry (FCM). Mixed lymphocyte culture (MLC) was carried out between kidney transplantation donors and recipients. After the additions of Tim-1(+)CD19(+) and Tim-1(-)CD19(+) B cells, there were 3 groups: Tim-1(+), Tim-1(-) and blank. Lymphocyte proliferation and inhibition status were evaluated by propidium iodide uptake and Annexin V binding. And the cytokine levels were detected by FCM. RESULTS: The absolute values of peripheral CD19(+)B cells were (170 ± 90), (202 ± 99), (155 ± 71) cells/µl in the pre-transplantation, post-transplantation and control groups respectively, post-transplantation group were higher than control group (P = 0.0300). The Tim-1(+)CD19(+) cell ratios were (2.20 ± 0.98)%, (35.46 ± 10.66)% and (1.95 ± 0.95)% in three groups. And the differences were statistically significant (both P < 0.01). Tim-1(+)CD19(+) B and Tim-1(-)CD19(+) B cells were added into MLC respectively. The early apoptotic cells of the Tim-1(+) group were higher than those in the Tim-1(-) group [(45.31 ± 12.37)% vs (10.92 ± 2.14)%, P < 0.05] and significantly higher than the blank group [(1.93 ± 0.26)%, P < 0.01]. Late apoptotic and dead cells of the Tim-1(+) group were higher than those in the Tim-1(-) group [(21.32 ± 5.67)% vs (2.32 ± 0.31)%, P < 0.01] and the blank group [(1.27 ± 0.19)%, P < 0.05]. The interleukin 10 levels in MLC supernatant of the Tim-1(+) group were significantly higher than those in the Tim-1(-) group [(5.32 ± 0.37) pg/ml vs (2.46 ± 0.25) pg/ml, P = 0.0001]. However, the interferon-γ levels were lower than those in the Tim-1(-) group [(1.51 ± 0.22) pg/ml vs (4.69 ± 0.32) pg/ml, P = 0.0015]. CONCLUSION: Present in the peripheral blood of kidney transplantation recipients, Tim-1(+)CD19(+) B cell has the capacity of promoting lymphocytic apoptosis. As a new regulatory subset of B cells, it plays important roles in the immune responses of transplantation.

[Applications of monitoring human leukocyte antigen G and its inhibitory receptors in post-renal transplantation].

OBJECTIVE: to study the feasibility of human leucocyte antigen-G (HLA-G) as a post-transplantation prognostic biomarker and discuss the correlation of its receptor expression and the mechanisms. METHODS: a total of 215 recipients in our centre from February 2006 to June 2008 were divided into stable kidney function group (n = 173) and acute rejection group (n = 42). The soluble human leucocyte antigen-G5 (sHLA-G5) level in peripheral plasma was detected by ELISA. And the HLA-G receptor ILT-2, KIR2DL4 on T, B, NK lymphocytes were analyzed by flow cytometry (FCM). The sHLA-G5 cutoff level by ROC curve was employed to predict the events of acute post-transplantation rejection. And regression analysis was used to determine the association of sHLA-G5 with acute rejection. RESULTS: an optimal cutoff value of 139.0 microg/L could be defined for sHLA-G5 (sensitivity: 63.6%, specificity: 82.1%, AUC: 0.780). Binary regression analysis showed that sHLA-G5 played an independent role on acute rejection (P = 0.019, OR = 0.039, 95%CI: 2.091 - 5.661). The rate of HLA-G receptor ILT-2 on CD4(+)T cell, CD8(+)T cell and B cell in acute rejection group was statistically lower than that in stable kidney function group (21% ± 7% vs 52% ± 17%, 23% ± 6% vs 39% ± 16%, 21% ± 7% vs 39% ± 16%, all P < 0.05). The expression of KIR2DL4 on NK cells in acute rejection group was statistically lower than that in stable kidney function group (31% ± 10%vs 57% ± 21%, P < 0.05). CONCLUSION: sHLA-G5 level may be predicted for acute rejection with a high sensitivity and specificity. The up-regulated expression of ILT-2 and KIR2DLT may contribute to immunology tolerance in peripheral circulation.