Intravenous administration of bone marrow mesenchymal stem cells alleviates renal failure in diabetic mice.

OBJECTIVE: To study the protective effect and mechanism of intravenously administered bone marrow mesenchymal stem cells (BMSCs) on renal failure in diabetic mice. PATIENTS AND METHODS: BMSCs were obtained from the bone marrow of mice, identified by flow cytometry and tri-line differentiation test. Diabetic model (DM) mice were established using STZ and infused with mice BMSCs intravenously, followed by the analysis of fasting blood glucose and proteinuria levels, renal tissue damage by optical microscope and electron microscope, and PI3K/AKT signaling protein expression in kidney by Western blot and endocrine function by immunofluorescence staining. RESULTS: DM mice exhibited gradually increased albumin excretion rate with time, and the MSC-treated diabetic mice presented significantly reduced proteinuria levels. HE staining indicated that MSC administration mitigated renal damage as proved by smaller tubular dilatation, reduced glomerulosclerosis and trace protein cylinders, and recovered kidney ultrastructure as shown by improved mesangial dilatation and podocyte loss. Further, BMSCs treatment activated PI3K/AKT signaling, which was downregulated in diabetic mice. However, MSC administration failed to improve pancreatic endocrine function in DM mice. CONCLUSIONS: Intravenous administration of MSCs can effectively prevent renal failure in diabetic mice by activating PI3K/AKT signaling pathway.

Prognostic model for risk stratification of de novo metastatic nasopharyngeal carcinoma patients treated with chemotherapy followed by locoregional radiotherapy.

BACKGROUND: There is no clinically applicable prognostic model designed for patients with de novo metastatic nasopharyngeal carcinoma (mNPC) treated with chemotherapy followed by locoregional radiotherapy (LRRT). We sought to develop a predictive tool of overall survival for individualized prediction and risk stratification in this heterogeneous patient population. PATIENTS AND METHODS: A total of 244 eligible patients with de novo mNPC, who were treated with platinum-based first-line chemotherapy followed by LRRT, were included in this retrospective study. We divided patients into the training and validation sets based on the date of initial treatment, with 152 patients treated between 2008 and 2013 comprising the training set for model development and 92 patients treated at a later time (2014 to 2015) forming the validation set. We applied Cox proportional hazards model to examine factors associated with overall survival (OS). We developed and subsequently validated a prognostic model to predict OS. We assessed the performance of this prognostic model and stratified patients based on prognostic scores obtained from this proposed model. RESULTS: The median OS of the entire cohort was 60.9 months. C-creative protein, number of metastatic sites, liver metastasis, post-treatment Epstein-Barr virus DNA, and response of metastasis were significantly associated with OS. A prognostic model for individual survival prediction was developed and graphically represented as a nomogram. The model showed favorable discrimination (C-index: 0.759), predictive accuracy [time dependent area under the curve (tAUC) at 5 years: 0.800], and calibration, and was further validated in an independent dataset. A risk stratification derived from the model can stratify these patients into three prognostic subgroups with significantly different survival. CONCLUSION: We developed and validated a prognostic model that exhibited adequate performance in individualized prediction and risk stratification for patients with de novo mNPC treated with chemotherapy followed by LRRT.

Identification of potential mechanosensitive ion channels involved in texture discrimination during Drosophila suzukii egg-laying behaviour.

Drosophila suzukii (spotted wing drosophila) has become a major invasive insect pest of soft fruits in the America and Europe, causing severe yield losses every year. The female D. suzukii shows the oviposition preference for ripening or ripe fruit by cutting the hard skin with its serrated ovipositor. A recent study reported that mechanosensation is involved in the texture discrimination during egg-laying behaviour in D. suzukii. However, the underlying mechanism and molecular entity that control this behaviour are not known. The transient receptor potential (TRP) channels and degenerin/epithelial sodium channels (DEG/ENaC) are two candidate gene families of mechanically activated ion channels. Thus, we first identified TRP and DEG/ENaC genes in D. suzukii by bioinformatic analysis. Using transcriptome sequencing, we found that many TRP genes were expressed in the ovipositor in both D. suzukii and D. melanogaster, while some DEG/ENaCs showed species-specific expression patterns. Exposure to drugs targeting TRP and DEG/ENaC channels abolished the oviposition preference for harder texture in female D. suzukii. Therefore, mechanosensitive ion channels may play significant roles in the texture assessment of egg-laying behaviour in D. suzukii, which has promising implications to further research on the development of novel control measures.

LncRNA LUCAT1 promotes proliferation of ovarian cancer cells by regulating miR-199a-5p expression.

OBJECTIVE: To investigate the biological effect of long non-coding ribonucleic acid (lncRNA) lung cancer-associated transcript 1 (LUCAT1) in the development of ovarian cancer. MATERIALS AND METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to detect the expression levels of lncRNA LUCAT1 in three human ovarian cancer cell lines (CaoV-3, SK-OV-3 and HO-8910) and the normal human ovarian surface epithelial cell line (IOSE80). Small interfering RNAs against lncRNA LUCAT1 (si-LUCAT1) were transfected into SK-OV-3 cells. Transfection efficiency of si-LUCAT1 was verified via RT-qPCR. Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to test the effect of silencing lncRNA LUCAT1 on SK-OV-3 cell proliferation. The apoptosis was measured by flow cytometry. The miRcode database was searched to predict potential microRNAs (miRNAs) binding lncRNA LUCAT1. It was found that lncRNA LUCAT1 contained a highly conserved binding site of miR-199a-5p in the 3'-untranslated region (3'-UTR). Subsequently, the targeting relationship between them was determined through Dual-Luciferase reporter gene assay and RT-qPCR analysis. RESULTS: LncRNA LUCAT1 was highly expressed in three human ovarian cancer cell lines compared to that in normal ovarian surface epithelial cell line (p<0.05). The cell proliferation rate in SK-OV-3 cells with lncRNA LUCAT1 knockdown was remarkably lower in comparison to that in control group. Moreover, colony formation assay also revealed that the number of cell clones decreased significantly after knockdown of lncRNA LUCAT1 compared to that in control group (p<0.05). In addition, the apoptosis rate was distinctly elevated in the lncRNA LUCAT1 silencing group (p<0.05). Furthermore, a highly conserved binding site of miR-199a-5p was found in the 3'-UTR of lncRNA LUCAT1. Dual-Luciferase reporter gene assay exhibited that the Luciferase activity of LUCAT1-wt was significantly reduced after overexpression of miR-199a-5p (p<0.05), while that of LUCAT1-mut was unchangeable. Further analysis via RT-qPCR suggested that miR-199a-5p overexpression significantly decreased the expression level of lncRNA LUCAT1 (p<0.05). CONCLUSIONS: LncRNA LUCAT1 is overexpressed in ovarian cancer cells, which may target miR-199a-5p to exert its effects on driving the malignant development of ovarian cancer.

Functional integrity of honeybee (Apis mellifera L.) resistant to dieldrin γ-aminobutyric acid receptor channels conjugated with three fluorescent proteins.

To generate an efficient tool used in Xenopus oocyte expression for in situ investigation of channel receptor expression, distribution and function, the C-terminus of the honeybee (Apis mellifera L.) resistant to dieldrin (RDL) subunit was fused with *FP, including monomeric red, enhanced yellow or enhanced green fluorescent protein (referred to as mRFP, EYFP and EGFP, respectively). In the present study, all fused *FP-AmRDLs could be visualized using fluorescence and laser confocal microscopy in cRNA-injected oocytes. Fluorescence was distributed isotropically in the cellular membrane. The potencies of the agonist γ-aminobutyric acid (GABA), but not ß-alanine, and the test antagonists (fipronil, flufiprole, dieldrin, α-endosulfan, bifenazate and avermectin B1a) in the *FP-AmRDL receptor did not significantly differ from that of the untagged receptor with two-electrode voltage clamp detection. The half maximal effective concentrations (EC50 s) of GABA in AmRDL, EGFP-AmRDL, EYFP-AmRDL and mRFP-AmRDL receptors were 11.98, 12.61, 18.92 and 22.11 µM, respectively, and those of ß-alanine were 651.6, 629.6, 1643.0 and 2146.0 µM, respectively. Inhibition percentages of test antagonists against *FP-AmRDL and AmRDL were not significantly different from each other. Overall, the consistency in functional properties between *FP-AmRDL and AmRDL receptors makes pGH19-*FP a promising tool for further in situ investigation of GABA receptors.

Therapeutic strategy for multiple VSD combined with MVSD in infants and young children.

OBJECTIVE: To investigate the therapeutic strategy for the multiple ventricular septal defects (VSD) combined with a muscular ventricular septal defect (MVSD) in the infants and young children. PATIENTS AND METHODS: We analyzed clinical data of 63 child patients with multiple VSD who received the treatment between January 2009 and April 2013 in our hospital. There were 33 males and 30 females, the patients aged from 6 to 28 (10 ± 6) months and weighed between 5.5 and 18.0 (7.1 ± 2.9) kg. Primary repair was performed for all of the patients; the MVSD in 7 patients was not detected during the surgery and no extra treatment was taken. The surgical suture was performed for 36 patients, hybrid repair under the direct vision for 8 patients, and hybrid repair via the right ventricle for 7 patients. After surgery, we followed up the patients regularly to reexamine the X-ray image of the chest, EEG, and color Doppler echocardiography to observe the closure of MVSD and the presence of a residual shunt. RESULTS: All of the 63 enrolled patients with multiple VSD survived without perioperative death. Three patients who were undergoing hybrid repair under direct vision received delayed sternal closure. One patient who was undergoing hybrid repair under direct vision had a postoperative cardiac dysfunction. 55 patients were followed up for 1 to 24 months. 28 patients had residual shunt of varying degrees during the follow-up, and most of the MVSD of patients with residual shunt was less than 4 mm, who were receiving further follow-up and observation. CONCLUSIONS: Appropriate surgical strategies can be applied according to the specific surgical condition for the treatment of MVSD in infants with multiple VSD, and the satisfactory clinical outcome can be achieved.

Better predictive value of axillary lymph node (ALN) status after systemic therapy for operable HER2-overexpressing breast cancer: A single-institution retrospective study.

BACKGROUND: Thresholds for using Preoperative Systemic Therapy (PreST) have decreased to include early breast cancer. This study investigates the predictive value of axillary lymph node (ALN) status before and after systemic therapy and discusses whether it is better to receive PreST first in operable HER2-overexpressing breast cancer patients. METHODS: From January 2008 to June 2013 at Fudan University Shanghai Cancer Center, we identified 406 eligible female patients with stage II-IIIa, operable and pathologically confirmed HER2-overexpressing invasive ductal carcinoma. Of these patients, 269 underwent surgery first followed by chemotherapy plus trastuzumab (chemo-trastuzumab) (SurgFirst group), whereas 137 received systemic chemo-trastuzumab therapy first followed by surgery (STFirst group). Disease-free survival (DFS) and overall survival (OS) were evaluated according to different ALN statuses using the Kaplan-Meier method. Multivariate COX model analyses were also conducted. RESULTS: The median follow-up time was 47 months (IQR: 37-60). Both ALN status and overall pathological complete remission (pCR) status were shown to be significant for the prediction of DFS (p = 0.001 and p = 0.005, respectively) and OS (p = 0.009 and p = 0.027, respectively) in the STFirst group. However, patients with positive ALN(s) did not experience significantly poorer survival compared with those with negative ALN in the SurgFirst group. The adjusted hazard ratios (HRs) for positive ALN status in the STFirst and SurgFirst groups were 6.66 (p = 0.001, 95%CI: 2.18-20.38) and 2.40 (p = 0.126, 95%CI: 0.78-7.34), respectively. CONCLUSION: The ALN status after systemic chemo-trastuzumab therapy better predicts the survival outcome. We recommend the application of PreST followed by surgery in patients with operable HER2-overexpressing breast cancer.

Single-cell mechanics--An experimental-computational method for quantifying the membrane-cytoskeleton elasticity of cells.

The membrane-cytoskeleton system plays a major role in cell adhesion, growth, migration, and differentiation. F-actin filaments, cross-linkers, binding proteins that bundle F-actin filaments to form the actin cytoskeleton, and integrins that connect the actin cytoskeleton network to the cell plasma membrane and extracellular matrix are major cytoskeleton constituents. Thus, the cell cytoskeleton is a complex composite that can assume different shapes. Atomic force microscopy (AFM)-based techniques have been used to measure cytoskeleton material properties without much attention to cell shape. A recently developed surface chemical patterning method for long-term single-cell culture was used to seed individual cells on circular patterns. A continuum-based cell model, which uses as input the force-displacement response obtained with a modified AFM setup and relates the membrane-cytoskeleton elastic behavior to the cell geometry, while treating all other subcellular components suspended in the cytoplasmic liquid (gel) as an incompressible fluid, is presented and validated by experimental results. The developed analytical-experimental methodology establishes a framework for quantifying the membrane-cytoskeleton elasticity of live cells. This capability may have immense implications in cell biology, particularly in studies seeking to establish correlations between membrane-cytoskeleton elasticity and cell disease, mortality, differentiation, and migration, and provide insight into cell infiltration through nonwoven fibrous scaffolds. The present method can be further extended to analyze membrane-cytoskeleton viscoelasticity, examine the role of other subcellular components (e.g., nucleus envelope) in cell elasticity, and elucidate the effects of mechanical stimuli on cell differentiation and motility. STATEMENT OF SIGNIFICANCE: This is the first study to decouple the membrane-cytoskeleton elasticity from cell stiffness and introduce an effective approach for measuring the elastic modulus. The novelty of this study is the development of new technology for quantifying the elastic stiffness of the membrane-cytoskeleton system of cells. This capability could have immense implications in cell biology, particularly in establishing correlations between various cell diseases, mortality, and differentiation with membrane-cytoskeleton elasticity, examining through-tissue cell migration, and understanding cell infiltration in porous scaffolds. The present method can be further extended to analyze membrane-cytoskeleton viscous behavior, identify the contribution of other subcellular components (e.g., nucleus envelope) to load sharing, and elucidate mechanotransduction effects due to repetitive compressive loading and unloading on cell differentiation and motility.

Knockdown of neuropilin-1 suppresses invasion, angiogenesis, and increases the chemosensitivity to doxorubicin in osteosarcoma cells - an in vitro study.

OBJECTIVES: Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). NRP-1 expression in osteosarcoma tissues was significantly higher, and high NRP-1 expression was more frequently occurred in osteosarcoma tissues with advanced clinical stage, positive distant metastasis and poor response to chemotherapy. We tested a hypothesis that the NRP-1 gene plays a role in the invasiveness, angiogenesis and chemoresistance of human OS. MATERIALS AND METHODS: To determine the role of NRP-1 in OS, NRP-1 was stably transfected into the human OS cell line MG-63 to increase the NPR-1 level, and NRP-1 siRNA was stably transfected into the human OS cell line SaOS-2 to knockdown of NRP-1. The effect of NRP-1 on invasion and angiogenesis was assessed by Matrigel invasion assay and in vitro angiogenesis assay. Chemosensitivity to doxorubicin was assessed by MTT assay in the MG-63 and SaOS-2 cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. RESULTS: The NRP-1 transfected MG-63 cells showed a markedly higher level of invasion in Matrigel invasion assay. The capillary-like structure formation of endothelial cells was also increased by coculture with the NRP-1 transfected MG-63 cells. On the contrary, the NRP-1 siRNA transfected SaOS-2 cells showed a markedly lower level of invasion in Matrigel invasion assay. The capillary-like structure formation of endothelial cells was also repressed by coculture with the NRP-1 siRNA transfected SaOS-2 cells. NRP-1 overexpression in MG-63 cells increased survival of cells after exposure to doxorubicin. In contrast, downregulation of NRP-1 expression in SaOS-2 cells markedly increased chemosensitivity after exposure to doxorubicin. CONCLUSIONS: We suggest that NRP-1 could be used as a biomarker for OS progression and a novel therapeutic or chemopreventive target for human OS treatment.

Bioequivalence and pharmacokinetic evaluation of two tablet formulations of carvedilol 25-mg: a single-dose, randomized-sequence, open-label, two-way crossover study in healthy Chinese male volunteers.

BACKGROUND: Carvedilol is a nonselective-blocking agent and is used in the treatment of hypertension and angina pectoris. OBJECTIVE: The aim of this study was to evaluate bioequivalence of two 25-mg tablet formulations of carvedilol following single oral dose in adult male volunteers. METHODS: This was a randomized, single-dose, open-label, crossover bioequivalence study. Plasma samples were collected before dosing and at 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 12 and 24 h after dosing. Plasma concentrations of Carvedilol were determined by using a validated LC-MS/MS method. Statistical analysis of the pharmacokinetic parameters Cmax, AUC0-24, and AUC0-∞ was conducted to determine bioequivalence. METHODS: 23 healthy male Chinese volunteers were enrolled in the study. The mean (SD) Cmax, AUC0-24, and AUC0-∞ values after administration of the test and reference formulations, respectively, were as follows: 73.71 (34.04) vs. 78.93 (43.64) ng/mL, 285.1 (147.0) vs. 296.9 (176.1) ng/mL · h, and 296.5 (161.4) vs. 303.4 (177.9) ng/mL · h. The 90% CIs of the ratios (test vs. reference) for the ln-transformed Cmax, AUC0-24, and AUC0 - ∞ were 85.3% to 114.3%, 90.4% to 107.6%, and 90.9% to 108.4%, respectively, meeting the criteria of SFDA, FDA and EMEA for bioequivalence. The relative bioavailability of the test formulation to reference formulation was 100.1%. Both formulations were generally well tolerated and no serious AEs were reported in the study. CONCLUSIONS: The 90% CIs for the ratios of mean Cmax, AUC0-24, and AUC0-∞ met the regulatory criteria for bioequivalence.

A parallel design study to assess the bioequivalence of generic and branded hydroxychloroquine sulfate tablets in healthy volunteers.

Hydroxychloroquine (HCQ) is a racemic 4-aminoquinoline derivative that was first introduced as an antimalarial, and subsequently applied to the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Information on the pharmacokinetics of HCQ in healthy volunteers, especially in a Chinese population is limited, and this study was conducted to provide support for a generic product to obtain marketing authorization in China.The aim of the present study was to compare the pharmacokinetics and assess bioequivalence of a new generic test and the branded reference hydroxychloroquine sulfate tablets in healthy volunteers.This was a parallel, open-label, randomized, single-dose, 1-period fasting study. 54 healthy subjects were randomly assigned (1:1) to receive 200 mg hydroxychloroquine sulfate tablets of the test or the reference formulation. 15 blood samples were collected and whole blood concentrations of HCQ were determined by a validated liquid chromatography-isotopic dilution mass spectrometry method. Log-transformed Cmax and AUC0-24 values were used to test for bioequivalence. The 2 formulations were considered bioequivalent if 90% confidence intervals (CIs) for the log-transformed ratios of Cmax and AUC0-24 were within the predetermined bioequivalence range of 80-125%. Tolerability was evaluated throughout the study by vital signs, physical examinations, clinical laboratory tests, 12-lead electrocardiograms, and interviews with the subjects about adverse events.54 healthy subjects were enrolled and completed the study (mean [SD] age, height, body weight, and BMI were 23.9 [2.4] years, 168.9 [5.0] cm, 61.3 [5.4] kg, and 21.5 [1.7] kg/m2), 27 subjects per group. No formulation or sequence effects were observed. The mean values of Cmax and AUC0-24 for the test and reference formulations of HCQ (197.6 and 199.0 ng/mL, 2460.1 and 2468.3 ng/mL/h) were not significantly different. The 90% CIs of the ratios of Cmax and AUC0-24 were 99.3% (98.1-102.1%), 99.7% (98.9-101.4%), respectively. 4 subjects (7.41%) experienced a total of 4 mild AEs (headache and microscopic hematuria, 1 each; and increase in plasma triglycerides, 2).The results of this study suggest that the test and reference hydroxychloroquine sulfate tablets are bioequivalent. Both formulations were generally well tolerated.

Pharmacokinetics and bioequivalence of 2 tablet formulations of olanzapine in healthy Chinese volunteers: a randomized, open-label, single-dose study.

Olanzapine is a widely used agent for the treatment of schizophrenia.The aim of this study was to evaluate bioequivalence of two 10-mg tablet formulations of olanzapine following single oral dose in adult male volunteers.This was a randomized, single-dose, open-label, crossover bioequivalence study. Plasma samples were collected before dosing and at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 12.0, 24.0, 36.0, 48.0, 72.0, 96.0, 120.0 and 144.0 h after dosing. Plasma concentrations of olanzapine were determined by using a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Statistical analysis of the pharmacokinetic parameters Cmax, AUC0-144, and AUC0-∞ was conducted to determine bioequivalence. Adverse events were monitored, recorded and evaluated by investigators throughout the study.24 healthy male Chinese volunteers between the ages of 18-40 years with a body mass index (BMI) between 19 and 24 kg/m2 were enrolled in the study. The mean (SD) Cmax, AUC0-144, and AUC0-∞ values after administration of the test and reference formulations, respectively, were as follows: 18.91 (5.320) vs. 18.44 (4.758) ng/mL, 582.9 (118.23) vs. 587.3 (127.12) ng/mL · h, and 615.4 (131.39) vs. 615.8 (137.45) ng/mL · h. The 90% CIs for the ratios of AUC0-144 and Cmax were 96.9% to 102.4% and 93.7% to 110.2%, respectively. The relative bioavailability of the test formulation to reference formulation was 100.1%. Both formulations were generally well tolerated and no serious AEs were reported in the study.The 90% CIs for the ratios of mean Cmax, AUC0-144, and AUC0-∞ met the regulatory criteria for bioequivalence.

Two-dimensional crystallization of hexagonal bilayer with Moiré patterns.

Direct observation of crystallization dynamics in real space is of special interest to scientists in various disciplines. Although direct observation of transient structural transformation in a nanocrystalline system has been recently achieved using the state-of-the-art aberration-corrected transmission electron microscopy (AC-TEM), the small length scales of individual species in molecular systems still preclude routine observation of crystallization dynamics. Unidirectional packing of microbeads can serve as an experimental model system, as their dynamics can be observed and recorded readily in the laboratory due to their larger size and slower time scale. Herein, we present direct observation of a two-dimensional (2D) crystallization enabled by such a packing process. The direct imaging approach not only allows observation of the dynamics in a bead-by-bead fashion but also reveals intriguing phenomena, such as the formation of grain boundaries, disorder-order transitions, and the Moiré patterns which arise when two periodic monolayers are overlaid at certain angles. In addition, the imaging afforded by confocal microscopy facilitates a structural analysis of height-dependent polygonal tiling of the top monolayer, which has implication to the formation of 2D quasicrystals.

Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases' cascade.

Antizymes delicately regulate ornithine decarboxylase (ODC) enzyme activity and polyamine transportation. One member of the family, antizyme-1, plays vital roles in molecular and cellular functions, including developmental regulation, cell cycle, proliferation, cell death, differentiation and tumorigenesis. However, the question of how does it participate in the cell apoptotic mechanism is still unsolved. To elucidate the contribution of human antizyme-1 in haematopoietic cell death, we examine whether inducible overexpression of antizyme enhances apoptotic cell death. Antizyme reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells, acute T leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G(1) appearance, loss of mitochondrial membrane potential (Deltapsi( m )), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following conditional antizyme overexpression, all protein levels of cyclin-dependent kinases (Cdks) and cyclins are not significantly reduced, except cyclin D, before their entrance into apoptotic cell death. However, introduced cyclin D1 into Jurkat T tetracycline (Tet)-On cell system still couldn't rescue cells from apoptosis. Antizyme doesn't influence the expression of tumor suppressor p53 and its downstream p21, but it interferes in the expressions of Bcl-2 family. Inducible antizyme largely enters mitochondria resulting in cytochrome c release from mitochondria to cytosol following Bcl-xL decrease and Bax increase. According to these data, we suggest that antizyme induces apoptosis mainly through mitochondria-mediated and cell cycle-independent pathway. Furthermore, antizyme induces apoptosis not only by Bax accumulation reducing the function of the Bcl-2 family, destroying the Deltapsi( m ), and releasing cytochrome c to cytoplasm but also by the activation of apoptosomal caspase cascade.

Increased expression and secretion of interleukin-6 in human parvovirus B19 non-structural protein (NS1) transfected COS-7 epithelial cells.

Human parvovirus B19 (B19) has been associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We have demonstrated previously that B19 non-structural protein (NS1) induced apoptosis through the mitochondria cell death pathway in COS-7 epithelial cells and that B19 NS1 may play a role in the pathogenesis of autoimmune diseases. In order to examine the expression profiles of cytokines and chemokines in B19 NS1 transfected COS-7 cells, we constructed the NS1 gene in the pEGFP-C1 vector named enhanced green fluorescence protein gene (EGFP)-NS1. COS-7 cells were transfected with EGFP or EGFP-NS1 plasmid. The expression profiles of cytokines and chemokines, including interleukin (IL)-1beta, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-related oncogene alpha (GROalpha), interferon gamma-inducible protein (IP)-10, stromal cell derived factor (SDF)-1, macrophage inflammatory protein (MIP)-1beta, monocyte chemoattractant protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), Fractalkine, CX3CR1, CCR2, CCR5 and CCR11 were examined in COS-7 cells, EGFP and EGFP-NS1 transfected cells using enzyme-linked immunosorbent assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR). Increased expression and levels of IL-6 were found in EGFP-NS1 transfected cells using RT-PCR and ELISA. There were no significant increases in the expression of IL-1beta, IL-8, IP-10, SDF-1, RANTES, Fractalkine, CX3CR-1, CCR2, CCR5, CCR11, TNF-alpha, GM-CSF and TGF-beta using RT-PCR. There were no significantly increased levels of IL-5, IL-10, TNF-alpha, TGF-beta, GROalpha, MIP-1beta and MCP-1 found by ELISA in this study. Our results show that increased expression and secretion of IL-6 in B19 NS1 transfected epithelial cells may play a role in the pathogenesis of autoimmune diseases.

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: crosstalk between ODC and BCL-2.

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-alpha)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCdelta) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKdelta pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.

Overexpression of peptidylarginine deiminase IV features in apoptosis of haematopoietic cells.

Peptidylarginine deiminases (PADIs) convert peptidylarginine into citrulline via posttranslational modification. One member of the family, PADI4, plays an important role in immune cell differentiation and cell death. To elucidate the participation of PADI4 in haematopoietic cell death, we examine whether inducible overexpression of PADI4 enhances the apoptotic cell death. PADI4 reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells and human acute T leukemia Jurkat cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G1 appearance, loss of mitochondrial membrane potential (delta psi(m)), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following PADI4 overexpression, cells arrest in G1 phase significantly before their entrance into apoptotic cell death. PADI4 increases tumor suppressor p53 and its downstream p21 to control cell cycle. In the detections of protein expression and kinase activity, all protein levels of cyclin-dependent kinases (CDKs) and cyclins are not reduced except cyclin D, however, CDK2 (G1 entry S phase) and CDK1 (G2 entry M phase) enzyme activities are inhibited by conditionally inducible PADI4. p53 also expands its other downstream Bax to induce cytochrome c release from mitochondria. According to these data, we suggest that PADI4 induces apoptosis mainly through cell cycle arrest and mitochondria-mediated pathway. Furthermore, p53 features in PADI4-induced apoptosis by increasing intracellular p21 to control cell cycle and by Bax accumulation to decline Bcl-2 function, destroy delta psi(m), release cytochrome c to cytoplasm and activate the caspase cascade.

Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species.

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-alpha) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF-alpha -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential (Delta psi(m)) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained Delta psi(m) and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of Delta psi(m) and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt Delta psi(m) or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of Delta psi(m) and apoptosis when cells were treated with TNF-alpha . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained Delta psi(m) and prevented apoptosis, but could not reduce ROS until four hours after TNF-alpha treatment. According to these data, we suggest that TNF-alpha induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF-alpha -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain Delta psi(m), prevent cytochrome c release and deactivate the caspase cascade pathway.

Proteinase 3 and dihydrolipoamide dehydrogenase (E3) are major autoantigens in hepatitis C virus (HCV) infection.

Hepatitis C virus (HCV) infection has been found to be strikingly associated with autoimmune phenomena. The aim of the present study was to investigate the presence of various autoantibodies in patients with HCV infection. Anti-neutrophil cytoplamic antibody (ANCA), anti-dihydrolipoamide dehydrogenase (anti-E3), rheumatoid factor (RF), anti-dihydrolipoamide acetyltransferase (anti-E2), anti-SS-A/Ro (60 kD), anti-SS-A/Ro (52 kD), anti-SS-B/La, anti-topoisomerase II (anti-topo II), anti-cardiolipin (aCL), anti-dsDNA, anti-ssDNA, anti-nuclear antibodies (ANA), anti-proteinase 3 (anti-Pr3) and anti-myeloperoxidase (anti-MPO) were determined in sera from 516 patients with HCV infection, 11 with primary biliary cirrhosis (PBC) and 44 healthy controls. Assays employed were indirect immunofluoresence, the particle latex agglutination test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. ANCA, anti-E3 antibody and RF were positive in 278/516 (55.6%), 276/516 (53.3%) and 288/516 (56%) patients with HCV infection, respectively. Positivity for ANA was present in 15.8%, anti-ssDNA in 15.6%, anti-dsDNA in 8.5%, aCL in 5%, anti-SS-B/La in 4.1%, anti-SS-A/Ro (60 kD) in 3.9%, anti-E2 in 3.3% and anti-SSA/Ro (52 kD) in 1.2%, anti-MPO in 4.8%, anti-Topo II and anti-actinin in 0%. All sera with ANCA showed c-ANCA patterns and contained anti-PR3 specificity. HCV patients with ANCA showed a higher prevalence of skin involvement, anaemia, abnormal liver function and alpha-Fetoprotein (alpha-FP). HCV patients with anti-E3 antibodies showed a higher prevalence of liver cirrhosis, arthritis, abnormal liver function and elevated alpha-FP levels. The prevalence of autoantibodies was not affected by treatment with interferon-alpha (IFN-alpha). In conclusion, autoantibodies are commonly found in patients with HCV infection. There is a high prevalence of anti-E3, ANCA and RF in these patients. Proteinase 3 and E3 are the major target antigens in HCV infection. HCV may be regarded as a possible causative factor in ANCA-related vasculitis.