Immunofluoresence Staining Research Articles

Octaarginine mediated delivery of fluorescent cargo to human smooth muscle cells

The high incidence and severity of diseases involving smooth muscle dysfunction, which include cardiovascular diseases and premature labour, dictates the need for our continued search for novel therapeutic strategies to treat these conditions. Cell penetrating peptides (CPP) are a class of non-viral vectors that show considerable promise for drug delivery purposes yet their suitability for uptake, and delivery of biologically active cargo, to human native cells and tissues remains unresolved. For any new drug delivery strategy, including the use of CPPs, to reach fruition this needs to be elucidated. We have begun to explore this issue for CPPs applied to human uterine cells and tissues (including myometrium and blood vessels) obtained from biopsies collected, following LREC-approved written informed consent, from patients undergoing elective Caesarean section at the end of pregnancy. Primary cultured human myometrial cells were prepared on glass-bottomed culture dishes, grown to 80–90% confluence and exposed to serum-reduced conditions overnight before exposure to CPP (or, separately, were methanol-fixed for subsequent immunofluoresence staining of protein localisation). Cellular uptake of fluorescently labelled (Alexa 488) d-Octaarginine (R8, 2 μM) was assessed in the first series of experiments for 24, 48 and 72 hours (n = 2). At each time point, z-section confocal microscopy revealed punctate intracellular fluorescence (indicative of vesicular compartmentalisation) particularly dense in the perinuclear area. A second series of experiments assessed the time-course of intracellular delivery up to 24 hours. Punctate intracellular loading was observed by 4 hours. More dense perinuclear and plasma membrane-localised fluorescence was observed at later time points. Immunofluoresence labelling revealed that human myometrial cells possessed expected cytoskeletal (α-smooth muscle actin, tubulin), plasma membranous and perinuclear localised components of endocytotic pathways (Caveolin-1, Clathrin Heavy Chain, Early Endosomal Antigen-1, Lysosomal Associated Membrane Protein-1 and 2 and Flotillins). Next, small segments of native (non-cultured), human uterine tissue were incubated with 2 μM D-R8 and nuclear dye Hoechst 33342 (1 μM) for 4 hours. Confocal microscopic examination revealed peptide entry into smooth muscle cells of both the myometrium and uterine blood vessels with homogenous intracellular fluorescence in many cells but some with more punctate perinuclear/nuclear fluorescence. In uterine tissues incubated with a similar, putatively cell-impermeant, Alexa488 control peptide (GS)4GC, no intracellular fluorescence was observed. These preliminary investigations illustrate that an octameric cationic CPP can successfully enter primary cultured and native human smooth muscle cells and tissues. This opens up a new avenue for targeted delivery of cellular therapeutics in human tissues and in particular to human smooth muscles.

Hematopoietic Cells Undergo Homotypic Cell Fusion: A Novel Platform to Study Genomic Instability and Leukemogenesis.

AbstractAbstract 1196Gene copy number variation (CNV) and translocation-derived gene fusion products are commonly observed in hematological malignancies. Mechanisms precipitating CNV include failed cytokinesis or spontaneous cell fusion. Heterotypic fusion of hematopoietic cells with non-hematopoeitic cells has been reported following injury in diverse tissues. Cell fusion involves blending membranes, intracellular material, and nuclear material from two parental cells to form a genetically and immunophenotypically distinct (often hyperdiploid) single daughter cell. We therefore hypothesized that if we could generate genetically chimeric, hematopoietic cells via cell-cell fusion, they might provide an instructive model for determining the role CNV plays in leukemogenesis. To generate genetically abnormal hematopoietic cells in vivo, a transplantation model with radiation induction was employed in which donor and host mice possess numerous unique cell surface and genomic markers. Fusion was determined by co-expression of donor and host markers among hematopoietic cells isolated from recipient BM, spleen, and thymus by serial fluorescence activated cell sorting. The presence of ‘bona fide’ fused cells was confirmed by performing rigorous immunophenotypic and genotypic analyses of individual cells, including immunofluoresence staining and z-stack analysis, single nucleotide polymorphism (SNP) PCR, and fluorescent in situ hybridization (FISH). Fused cell populations were detected in both mature myeloid and lymphoid lineages. Consistent with the proposed model of studying early, initiating leukemogenic events, cell fusion was also detected in the progenitor cell populations including colony forming unit (CFU-C) myeloid progenitors, and c-kit+, sca-1+, lin- (KSL) cells. Moreover, transplanted fused hematopoietic cells could reconstitute lethally irradiated secondary hosts >16 weeks after transplant. Thus, hematopoietic progenitor cells are capable of homotypic cell fusion, and subsequent to cell fusion, these cells are further able to divide, differentiate, and are competent to contribute to functional hematopoiesis. Additional assays are underway to more definitively address the functional significance of hematopoietic cell fusion as a repair mechanism, or during homeostasis. Importantly, we detected evidence of genomic instability in fused hematopoietic cells using a variety of assays. Fused cells with loss of specific donor and/or host marker genes were identified by immunofluorescence and genomic DNA analysis. DNA strand breakage, evaluated by single cell gel electrophoresis (comet assay) showed the presence of a significantly larger tail moment in fused cells suggesting a higher degree of DNA damage in these cells. Studies for evaluation of p53 status and histone H2AX activation are underway. In summary, our data not only provide novel evidence that homotypic cell fusion occurs between hematopoietic cells following injury, but also that fused cells retain their replicative potential in vitro and in vivo and exhibit greater genomic instability compared to non-fused counterparts. Because most existing murine models of leukemogenesis focus on events in the promotion of disease, we believe fusion and reductive division of hematopoietic cells shown here will serve as a novel platform to study the role of CNV as a leukemia-initiating event. Disclosures:No relevant conflicts of interest to declare.

Focused ultrasound-mediated bbb disruption is associated with an increase in activation of AKT: experimental study in rats

BackgroundThe Blood Brain Barrier (BBB) maintains the homeostasis of central nervous system by preventing the free passage of macromolecules from the systemic circulation into the brain. This normal physiological function of the BBB presents a challenge for delivery of therapeutic compounds into the brain. Recent studies have shown that the application of focused ultrasound together with ultrasound contrast agent (microbubbles) temporarily increases the permeability of the BBB. This effect is associated with breakdown of tight junctions, the structures that regulate the paracellular permeability of the endothelial cell layer. The influence of this ultrasound effect on the activation of intracellular signaling proteins is currently not well understood. Therefore, the aim of this study was to investigate the activation of cell survival signaling molecules in response to ultrasound-mediated BBB opening;MethodsThe BBB was disrupted in two four-spot lines (1-1.5 mm spacing) along the right hemisphere of rat brain with ultrasound beams (0.3 MPa, 120 s, 10 ms bursts, repetition frequency = 1 Hz) in the presence Definity microbubbles. Contrast-enhanced MRI images were acquired to assess the extent of BBB opening upon which the animals were sacrificed and the brains removed and processed for biochemical and immunohistochemical analyses;ResultsImmunoblotting of sonicated brain lysates resolved by SDS-PAGE demonstrated an increase in phosphorylation of Akt and its downstream signaling molecule, GSK3β, while the phosphorylation of MAPK remained unchanged. The elevated levels of pAkt and pGSK3β are still evident after 24 hours post-sonication, a time point where the integrity of the BBB is known to be re-established. Furthermore, immunofluoresence staining localized this increase in pAkt and pGSK3β levels to neuronal cells flanking the region of the disrupted BBB;ConclusionsOur data demonstrates that ultrasound-mediated BBB disruption causes an activation of the Akt signaling pathway in neuronal cells surrounding the disrupted BBB.

Antisense microRNA-21 enhances U251 human glioma cell chemo-sensitivity to 5-Fluorouracil

Objective To study the effect of as - miR - 21 enhance U251 human glioma cell chemo-sensitivity to 5 - FU. Method 5 - FU/PAMAM complex was prepared by dialysis method. As - miR - 21 was incubated with 5 - FU/PAMAM to form 5 - FU/PAMAM/as - miR - 21. Transmission electronic microscopy (TEM) was performed to observe the morphology of the nanoparticles. The drug loading efficiency and encapsulation efficiency was determined by ultraviolet spectroscopy ( UV ). The transfection of PAMAM dendrimer was detected by flow cytometry assay. MTT assay was carried out to determine U251 cell growth survival rate. Cell apoptosis was analyzed by flow - cytometry assay. Immunofluoresence staining was employed to determine Ki67 and Bcl -2 expression in U251 cells after combination treatment Transwell assay was performed to detect cell invasion ability. Results The morphology of the nanoparticle was sphere observed by TEM, and the diameter was less than 100 nm. The encapsulation efficiency and loading efficiency of drug were determined by ultraviolet spectroscopy to be 66. 21% and 31. 77% ,respectively. The results of flow cytometry assay showed that PAMAM dendrimer transfection efficiency was 70. 53%. The survival rate of U251 cells with as - miR - 21 and 5 - FU co - delivery treatment was significantly suppressed(F=273.345,P=0.000). The increased apoptosis nuclei percentage in co - delivery treated U251 cell was detected ( F =43. 21, P =0. 000) by flow - cytometry assay and the expression of Ki67 and Bcl -2 were down regulated simultaneously analyzed by immunofluorescence. Transwell result also demonstrated the cell invasion ability of U251 cells treated with 5 - FU combine with as - miR - 21 was decreased. Conclusions PAMAM dendrimer could effectively deliver as - miR - 21 and 5 - FU simultaneously;combination therapy can suppress U251 cell line growth effectively in vitro and can enhance the chemo -sensitivity of glioma cells to 5 - FU chemotherapy. Key words: PAMAM; Co-delivery; As-miR-21; Fluorouracil; Glioma

441 EMBRYONIC SMOOTH MUSCLE INDUCES BLADDER SMOOTH MUSCLE DIFFERENTIATION

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2010441 EMBRYONIC SMOOTH MUSCLE INDUCES BLADDER SMOOTH MUSCLE DIFFERENTIATION Gregory Tasian, Mei Cao, Gerald Cunha, and Laurence Baskin Gregory TasianGregory Tasian More articles by this author , Mei CaoMei Cao More articles by this author , Gerald CunhaGerald Cunha More articles by this author , and Laurence BaskinLaurence Baskin More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.512AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urothelium-derived signals are necessary to induce smooth muscle differentiation from mesenchyme during murine bladder development. We tested the hypothesis that fetal bladder smooth muscle without urothelium can induce smooth muscle differentiation from bladder mesenchyme. METHODS Fetal intact bladders (IB) were harvested from timed pregnant FVB and transgenic GFP mice at the time points shown below. Bladder mesenchyme (BLM) was isolated by incubating IB in 0.02M EDTA and then removing the urothelium by microdissection. Embryonic day (E) 12 BLM from GFP mice was recombined with E12 BLM, E12 IB, and E16 BLM from FVB mice and incubated overnight on agar at 37°C. The recombined tissues were grafted beneath the kidney capsule of male nude mice, maintained for two weeks, and then were harvested and processed. H&E staining was performed and immunohistochemistry and immunofluoresence staining was used to detect smooth muscle α-actin, uroplakin and GFP. RESULTS At the time of harvest, E12 BLM does not express α-actin; E16 BLM does express α-actin. E12 BLM (GFP) recombined with E12 BLM (FVB) did not survive. E12 BLM (GFP) expressed smooth muscle α-actin when recombined with E12 IB (FVB). The α-actin positive cells derived from E12 BLM (GFP) diffusely migrated through the smooth muscle of the FVB bladder wall. When recombined with E16 BLM (FVB), E12 BLM (GFP) also demonstrated smooth muscle differentiation, as shown by the expression of smooth muscle α-actin; however, these cells did not migrate (see Figure). CONCLUSIONS Urothelium is necessary for initial induction of smooth muscle differentiation from bladder mesenchyme. After smooth muscle has formed, BLM alone can induce further smooth muscle differentiation. This may be due to an intrinsic property of smooth muscle, presence of a diffusible factor within the mesenchyme that induces smooth muscle differentiation, or initiation of signaling pathways by the urothelium prior to its removal that result in mesenchymal smooth muscle differentiation. San Francisco, CA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e174 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gregory Tasian More articles by this author Mei Cao More articles by this author Gerald Cunha More articles by this author Laurence Baskin More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of avastin combined with sorafenib in a highly metastatic murine model of human hepatocellular carcinoma

Objective To investigate the effect of combination of sorafenib and avastin,a vascular endothelial cell growth factor (VEGF) neutralizing antibody on tumor growth, lung metastasis and survival of tumor-bear nude mice in a highly metastatic xenograft murine model of human hepatocellular carcinoma (HCC). Methods Xenograft of a highly metastatic human HCC tumor (LCI-D20) was used to evaluate survival, primary tumor growth and lung metastasis after treatment with avastin alone or in combination with sorafenib. Tumor angiogenesis was identified by immunohistochemistry staining for CD31 and calculated as microvessel density (MVD). Apoptosis of tumor endothelial cells were determined by double immanofluor-esence staining for CD31 and the terminal deoxynucleotidyl-transferase mediated dUTP nick-end labeling assay (TUNEL). Results Tumor volumes were (5.70±0.17 ), (1.10±0.18), (0.60±0.12) and (2.10±0.28) mm~3 in control,serafenib,sorafenib plus avastin and avastin group. Lung metastasis was (191±23), (98±18), (31±19) and (98±20) in four groups, and median survival were 70,87,112 and 80 days,respectively. MVD were (3.77±0.44)%, (1.28±0.15)%, (0.56±0.08)%, (1.32± 0.18)% ,and indexes for apoptotic endothelial cells were (12.6±1.8)%, (32.6±8.7)%, (54.3± 11.9) %, (26.8±6.5) % respectively in four groups. Combination of avastin and serafenib significantly reduced plasma VEGF (P<0.05) ,decreased tumor volume (P<0.05) ,inhibited numbers of lung metas-tasis (P<0.01), and prolonged survival compared to mice treated with serafenib alone (P<0.05). Immu-nofluoresence staining shows reduced MVD (P<0.05) and increased apoptosis of tumor endothelial cells (P<0.05) by combination of avastin and sorafenib. Conclusion VEGF may contribute to resistance of sorafenib, avastin significantly inhibited tumor angiogenesis by inducing apoptosis of tumor endothelial cells. The study implies that it is promising to combine avastin with sorafenib for patients with advanced hepatocellular carcinoma. Key words: Sorafenib; Avastin; TUNEL

Garlic constituent diallyl trisulfide induced apoptosis in MCF7 human breast cancer cells

Identification of agents that are nontoxic but can delay onset and/or progression of breast cancer, which is the main leading cause of cancer-related deaths among women, is highly desirable. Garlic-derived organosulfur compounds (OSCs) have highly effective antitumor effects, but the mechanism has yet to be investigated. The aim of the present study was undertaken to examine the effect of diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, on growth of two cell lines respectively, MCF-7 human breast cancer cells and nontumorigenic MCF-12a mammary epithelial cells. The effects of DATS were examined by MTT assay, clonogenic survival assay, ELISA based apoptotic assay, TUNEL assay, immunofluoresence staining, flow cytometry, RT-PCR, and Western blot analysis. Garlic constituent diallyl trisulfide (DATS) suppresses viability of cultured MCF-7 and MCF-12a cells respectively by decreasing the percent of cells in G2/M and inducing apoptotic cell death. DATS-induced apoptosis was markedly elevated in MCF-7 cells compared with MCF-12a cells and this was correlated with elevated levels of cyclin B1. The results from semi-quantitative and real-time RT-PCR indicated that DATS-enhanced the expression levels of FAS and cyclin D1, but in contrast, down-regulated the expression levels of Akt and Bcl-2. Furthermore, the DATS-induced apoptosis was correlated with induction of pro-apoptotic Bax protein and p53 protein expression was up-regulated and translocation to nucleus in MCF-7 cells. Together, the results of the present study show, for the first time, that DATS administration might offer a novel strategy for the treatment of human breast cancer.

Enhanced MDR1 Expression and Chemoresistance of Cancer Stem Cells Derived from Glioblastoma

ABSTRACTWe established a cancer stem (CS) cell line, U87CS, by means of spheroid culture of U87MG cells derived from glioblastoma (GBM) in neuronal stem cell medium. U87CS cells presented positive immunohistochemical staining for multidrug resistance (MDR)1 and CD133, a marker for a subset of leukemia and GBM CS cells. The gene expression of MDR1 and CD133 on U87CS cells increased by an average of 8.51 and 47.18 times, respectively, compared to the levels on U87MG cells by real-time quantitative RT-PCR. U87CS cells possessed stronger drug-resistance to conventional anti-cancer drugs, such as doxorubicin (Dox), etoposide (VP-16), carboplastin, and BCNU than U87MG cells. Double immunofluoresence staining showed co-expression of MDR1 and CD133 on U87CS cells transplanted into nude mice brains. In addition, we identified the crossreactivity of CD133 and MDR1 in a surgical specimen of GBM. Our results suggest that CS cells may be resistant to current chemotherapy and represent a novel target for GBM therapeutics.

C-Met-targeted RNA interference inhibits growth and metastasis of glioma U251 cells in vitro

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. c-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, invasion, and angiogenesis. The present study was designed to determine the role of c-Met in growth and metastasis of glioma U251 cells using RNA interference (RNAi) technology in vitro. We constructed three kinds of shRNA expression vectors aiming at the c-Met gene, then transfected them into glioma U251 cells by lipofectamine(TM) 2000. The level of c-Met mRNA was investigated by real-time polymerse chain reaction (RT-PCR). The protein expression of c-Met was observed by immunofluoresence staining and western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and in vitro angiogenesis assays of U251 cells were done. We got three kinds of c-Met specific shRNA expression vectors which could efficiently inhibit the growth and metastasis of U251 cells and the expression of c-Met in U251 cells. RT-PCR, immunofluoresence staining and western blotting showed that inhibition rate for c-Met expression was up to 90%, 79% and 85%, respectively. The expression of c-Met can be inhibited by RNA interference in U251 cells, which can inhibit the growth and metastasis of U251 cell and induce cell apoptosis. These results indicate that RNAi of c-Met can be an effective antiangiogenic strategy for glioma.

Is Immunofluoresence Staining for Eosinophil Derived Neurotoxin Useful in the Diagnosis of Eosinophilic Esophagitis?

Purpose: We have shown that patients with low level esophageal eosinophilia (1–20 eos/hpf) have similar demographics, dysphagia history, and endoscopic findings as patients with classic eosinophilic esophagitis (EoE) with 20 eos/hpf. Some of the low-level eosinophilia patients will have eosinophil counts >20 /hpf at a second endoscopy and will have resolution of their dysphagia with topical steroid therapy. Other low-level eosinophilia patients will have resolution of dysphagia with PPI therapy. Esophageal eosinophil counts can fluctuate with time, and maximum eosinophil counts in esophageal mucosa might not be the best diagnostic tool for the diagnosis of EoE. We performed immunofluorescence staining of esophageal biopsy specimens for Eosinophil Derived Neurotoxin (EDN) in several groups of patients to evaluate its potential use as a marker for EoE. Methods: We performed staining for EDN with qualitative and semiquantitative analysis of esophageal biopsy specimens from four groups of patients: A) normals, B) low level eosinophilia –GERD patients (LL-GERD) (LA grade B-D esophagitis and dysphagia responding to PPI therapy), C) low level eosinophilia – emerging EoE patients (LL-EE) (>20 eos/hpf on second endoscopy and dysphagia response to topical steroid therapy), and D) classical EoE patients (25 to 100 eos/hpf). Specimens were scored on a 0–3 scale for intracellular (IC) and extracellular (EC) EDN deposition by two authors, and the scores were averaged. Results: The semiquantitative results are displayed in Table 1. IC and EC EDN scores were negligible in the normals and elevated in the other groups. The EC EDN score and percentage of patients with an abnormal score (>1.3) trended to be higher in the EoE and LL- EoE groups. Qualitatively, intense extracellular EDN deposition was typically observed in the papillae and on the luminal surface of the epithelium in biopsies from the EoE patients, while deposition in the LL-GERD patients, with one exception, was scattered throughout the mucosa with relatively little uptake on the luminal surface and in the papillae. The LL-EoE group had a very similar pattern to the EoE group.Table 1Conclusion: This pilot study suggests: 1) Patients with low level eosinophil density in esophageal biopsies have significant EDN immunofluorescence staining. 2) EDN staining particularly extracellular staining on the luminal mucosa has potential to be a better marker for EoE than maximum eosinophil count and should be further evaluated.

Radiation-Enhanced Vascular Targeting of Human Lung Cancers in Mice with a Monoclonal Antibody That Binds Anionic Phospholipids

New treatment strategies aimed at damaging tumor vasculature could potentially improve tumor response to radiation therapy. We recently showed that anionic phospholipids, principally phosphatidylserine, are specifically exposed on the luminal surface of tumor blood vessels. Here we tested the hypothesis that radiation therapy can increase phosphatidylserine exposure on lung tumor vasculature, thereby enhancing the antitumor properties of the anti-phosphatidylserine antibody 2aG4. The therapeutic efficacy of radiation therapy plus 2aG4 was tested in nude mice bearing radiation-resistant A549 human lung tumors. Radiation-induced phosphatidylserine exposure on endothelial cells and A549 tumor cells was analyzed by immunofluoresence staining. The mechanism of the enhanced antitumor effect was examined by histology and antibody-dependent cell-mediated cytotoxicity experiments. Focal irradiation of A549 human lung cancer xenografts increased the percentage of tumor vessels with exposed phosphatidylserine from 4% to 26%. Treatment of mice bearing A549 tumors with 2aG4 plus focal radiation therapy inhibited tumor growth by 80% and was superior to radiation therapy or 2aG4 alone (P < 0.01). Combination therapy reduced blood vessel density and enhanced monocyte infiltration into the tumor mass beyond that observed with individual treatments. In vitro, 2aG4 enhanced the ability of macrophages to kill endothelial cells with exposed phosphatidylserine in an Fc'-dependent manner. These results suggest that 2aG4 enhances the antitumor effects of radiation therapy by increasing antibody-dependent cell-mediated cytotoxicity toward tumor vessels with externalized phosphatidylserine. Bavituximab, a chimeric version of 2aG4 in clinical trials, has the potential to enhance the therapeutic efficacy of radiation therapy in lung cancer patients.

RNA interference-mediated gene silencing of vascular endothelial growth factor in colon cancer cells

To inhibit the expression of vascular endothelial growth factor (VEGF) in colon cancer cell line by RNA interference (RNAi). Followed the service of E-RNAi, we designed and constructed two kinds of shRNA expression vectors aiming at the VEGF gene, then transfected them into colon cancer HT29 cells by lipofectamine(TM) 2000. The level of VEGF mRNA was investigated by RT-PCR and Northern blotting. The protein expression of VEGF was observed by immunofluoresence staining and Western blotting. We got two kinds of VEGF specific shRNA expression vectors which could efficiently inhibit the expression of VEGF in HT29 cells. RT-PCR, Northern blotting, immunofluoresence staining and Western blotting showed that inhibition rate for VEGF expression was up to 42%, 89%, 73% and 82%, respectively. The expression of VEGF can be inhibited by RNA interference in HT29 cells.

Relations of ulcerative colitis to anti-beta2-glycoprotein antibodies and to platelet activation status

To explore the relations of ulcerative colitis (UC) to anti-beta2-glycoproteinI antibodies (abeta(2)GPI) and platelet activation status. Peripheral blood samples were collected from 56 UC patients, 34 males and 22 females, aged 43.5 (21 - 66), including 36 in active stage and 20 in remission stage, and 25 sex- and age-matched controls. The level of abeta(2)GPI was detected by ELISA. The platelet activation markers, platelet activation complex-1 (PAC-1) and P-selectin (CD62P) were detected by immunofluorescence staining and flow cytometry. The A value of IgG abeta(2)GPI of the active UC group was 0.61 +/- 0.13, significantly higher than those of the remittent UC group and the control group (0.50 +/- 0.13 and 0.22 +/- 0.14, both P < 0.01), and there was a significant difference between the remittent UC group and the control group (P < 0.01). The A value of IgM abeta(2)GPI of the active and remittent UC groups were 0.43 +/- 0.13 and 0.38 +/- 0.12, both significantly higher than that of the control group (0.20 +/- 0.12, both P < 0.01), however, there was no significant difference between the active UC and remittent UC groups (P > 0.05). The PAC-I positive rate of the active UC and remittent UC groups were 30.6% +/- 7.6% and 19.6% +/- 7.8%, both significantly higher than that of the control group (6.3% +/- 1.7%, both P < 0.01), and there was a significant difference between the active and remittent groups too (P < 0.01). The CD62P positive rates of the active UC and remittent UC groups were 45.0% +/- 8.8% and 31.9% +/- 7.8% respectively, both significantly higher than that of the control group (9.2% +/- 2.7%, both P < 0.01), and there was a significant difference between the active and remittent groups too (P < 0.01). In the active UC group, more severe the state of illness, the higher the A value of IgG abeta(2)GPI, and the positive rates of PAC-I and CD62P, and these values were all positively correlated with the state of illness (F(abeta2GPI) = 3.679, P < 0.05, F(PAC-I(%)) = 5.346, P < 0.01, and F(CD62P(%)) = 5.418, P < 0.01 respectively). The abeta(2)GPI level and the platelet activation state are closely correlated with the pathogenesis and development of UC.

P-207: Viability testing of cryopreserved ovarian tissue

P-207: Viability testing of cryopreserved ovarian tissue

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO 3− permeability of bovine corneal endothelium

Corneal endothelium secretes HCO 3 − from basolateral (stroma) to apical (anterior chamber) compartments. Apical HCO 3 − permeability can be enhanced by increasing [Ca 2+] i. We hypothesized that the bovine calcium-activated chloride channel 1 (bCLCA1), shown previously by PCR screening to be expressed in corneal endothelium, is involved in Ca 2+ activated apical HCO 3 − permeability. bCLCA1 expression in cultured bovine corneal endothelial cells (CBCEC) was examined by in situ hybridization analysis, immunoblotting, immunofluorescence and confocal microscopy. Rabbit polyclonal antibodies were generated using a 14 aa polypeptide (417–430) from the predicted sequence of bCLCA1. The small interference RNA (siRNA) knock down technique was used to evaluate the functional involvement of bCLCA1 in apical HCO 3 − permeability. In situ hybridization confirmed prominent bCLCA1-specific mRNA expression in CBCEC. bCLCA1 antiserum detected the heterologously expressed bCLCA1 in HEK293 cells and a 90 kDa band in CBCEC, which was absent when using the pre-immune serum or antigen absorption of serum. Immunofluoresence staining with anti-bCLCA1 antibody and confocal microscopy indicates an apical membrane location in CBCEC. In CBCEC transfected with bCLCA1 specific siRNA, bCLCA1 expression was reduced by 80%, while transfection with siControl scrambled sequence had no effect. Increasing [Ca 2+ i] by application of ATPγS or cyclopiazonic acid (CPA) increased apical HCO 3 − permeability in siControl transfected CBCEC, while having no effect on apical HCO 3 − permeability in bCLCA1 specific siRNA transfected cells. Baseline HCO 3 − permeability, however, was not different between controls and siRNA treated cells. We conclude that the calcium-activated chloride channel (bCLCA1) is expressed in bovine corneal endothelial cells and can contribute to Ca 2+ dependent apical HCO 3 − permeability, but not resting permeability, across the corneal endothelium.

Effects of different doses of levodopa on paraquat-induced neurotoxicity: an experiment with mice

To investigate the effects and mechanisms of different doses of levodopa on paraquat-induced neuro-toxicity. 72 C57BL mice were divided into 2 equal groups: acute experiment group and chronic experiment groups. The acute experiment group was re-divided into 2 subgroup: subgroup A to be injected with levodopa of the doses of 0 (distilled water instead), 10 mg/kg, or 100 mg/kg and then paraquat 30 mg/kg (levodopa + paraquat), and then killed 90 minutes after; and subgroup B, to be injected with paraquat 30 mg/kg and then levodopa 0, 10 mg/kg, or 100 mg/kg (paraquat + levodopa), and then killed 2 hours after. The chronic experiment group was re-divided into 2 subgroups to be injected with levodopa + paraquat or paraquat + levodopa once a week for 3 weeks, and then killed 24 hours after the injection. Fluorescent microscopy was used to observe the fluorescent staining of paraquat in the substantia nigra in the acute experiment group and the fluorescent staining of tyrosine hydroxylase (TH) in the substantia nigra in the chronic experiment group. in the chronic experiment group Western blotting was used to examine the protein expression of TH; thioflavine double labeling was used to observe the alpha-synuclein aggregation by immunofluorescence staining and Western blotting. The slices of substantia nigra of the mice in the chronic experiment group treated with distilled water + paraquat were inoculated with or without 250 micromol/L levodopa and then underwent thioflavine staining to observe the alpha-Syn aggregation. The paraquat staining was strongly positive in the substantia nigra of the mice in Group A-1, and was decreased gradually in the group A-2 and A-3. The paraquat staining was strongly positive in the substantia nigra of Group B-1 without a significant difference between Group A-1 and Group B-1, and was not remarkable in Group B-2 and B-3. The TH staining and protein expression in the substantia nigra of Group A-2 were significantly stronger than that of Group A-1 (P < 0.05), and the TH staining was remarkably weaker in Group A-3 (P < 0.05), as shown by immunofluorescence staining and Western blotting. There was no significant difference in TH staining and protein expression in the substantia nigra among Group A-1, Group B-1, and Group B-2 (all P > 0.05). However, the TH staining was remarkably weaker in Group B-3 (P < 0.05). The thioflavine and alpha-Syn double staining was significantly weaker in Groups A-2 and A-3 in comparison with Group A-1. There was no significant difference in the double staining among Group A-1, Group B-1, and Group B-2 (all P > 0.05). However, the double staining was remarkably weaker in Group B-3 (P < 0.05). The thioflavine positive staining in the tissue slices inoculated with levodopa was significantly weaker in comparison with those un-inoculated. Pre-treatment with lower dose L-dopa before the paraquat administration is neuroprotective by preventing paraquat from access into central nervous system through a blood-brain barrier competitive uptake mechanism, while higher dose L-dopa shows neurotoxicity through disaggregating alpha-synuclein deposits in Parkinsonian mice.

Blockade of chemokine-induced signalling inhibits CCR5-dependent HIV infection in vitro without blocking gp120/CCR5 interaction.

BackgroundCellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.ResultsIn this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.ConclusionThese observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.

Expression of Dlk/Pref-1 defines a subpopulation in the oval cell compartment of rat liver

We previously showed that Dlk, a transmembrane protein containing six epidermal growth factor like repeats in its extracellular domain, is strongly expressed in hepatoblasts in murine fetal liver. Here, we examined the expression of Dlk in oval cells, which are adult hepatic progenitors, in the rat 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) model. Reverse transcription polymerase chain reaction analysis showed that Dlk expression was significantly induced in the regenerating liver at day 12 and 14 after PH, when many oval cells were present in periportal areas. Immunofluoresence staining analysis revealed that Dlk + cells expressed oval cell markers, cytokeratin 19 (CK19) and α-fetoprotein, indicating that Dlk is expressed in oval cells. However, Dlk + cells accounted for only about 20% of total CK19 + oval cells. Dlk + cells were localized more distantly from the portal vein than Dlk − cells, and were adjacent to mature hepatocytes, though Dlk + cells were surrounded by the basal membrane as other oval cells. Furthermore, at day 12 after PH, only 3% of Dlk + oval cells expressed Ki67, whereas about 13% of total oval cells expressed Ki67, indicating that Dlk + oval cells are less proliferative than Dlk − oval cells. Taken together, these results demonstrate that Dlk is expressed in a subpopulation of oval cells and that Dlk + cells represent intermediate cells between Dlk − oval cells and mature hepatocytes.

Prevalent human coxsackie B-5 virus infects porcine islet cells primarily using the coxsackie-adenovirus receptor.

We have previously demonstrated that transplanting porcine encephalomyocarditis virus (EMCV)-infected porcine islet cells (PICs) results in transmission of the virus to recipient mice, which is manifested by acute fatal infection within 5 to 8 days. Here, we determined PIC susceptibility to a related and highly prevalent human picornavirus, coxsackie B-5 virus (CVB-5). PICs were inoculated with CVB-5 in vitro for up to 96 hours and infectivity, level of virus replication, and cellular function determined. Subsequently, monoclonal and polyclonal antibody blocking experiments were used to investigate the receptor CVB-5 uses to enter PICs, and the ability of CVB-5-infected islets to reverse diabetes analyzed in mice. Adult pig islets inoculated with CVB-5 in vitro showed a typical picornaviral replication cycle with a 2-h lag phase followed by a 4-h exponential phase during which the virus titer increased by 4 logs. However, CVB-5 was less cytolytic to PICs than EMCV, resulting in a persistent productive infection lasting for up to 96 h, with minimal evidence of cell lysis. Double immunostaining confirmed the presence of CVB-5 antigens in insulin-producing islets. Infection of PICs in the presence of antibodies against human coxsackie-adenovirus receptor (CAR) resulted in near complete blockage in production of infectious virus particles whereas blocking with anti-porcine decay-accelerating factor (DAF, also called CD55) or anti-porcine membrane cofactor protein (MCP, also called CD46) only slightly decreased the number of infectious CVB-5 particles produced. Immunofluoresence staining showed CAR and MCP expression on the islet surface, but not DAF. Transplanting CVB-5-infected PICs into diabetic C57BL/6 mice resulted in reversal of diabetes. Although PICs are susceptible to human CVB-5, the infection does not appear to affect xenograft function in vitro or in vivo in the short term.

Relationship between the expression of connexin43 and bystander effect of suicide gene therapy in ovarian cancer.

The relationship of connexin43 (Cx43) and bystander effect in ovarian tumor cells in herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in vitro was explored and the effect of all-trans retinoic acid (RA) on the expression of Cx43 and bystander effect investigated. The Cx43 expression was detected by flowcytometry, Western blot, and immunofluorescence in two ovarian tumor cell lines OVCAR3, CaOV3 before and after RA treatment. Bystander effect was determined by the cells growth inhibitory rate with methyl thiazolyl tetrazolium. Following exposure to ganciclovir, there was much greater bystander killing in OVCAR3 than that in CaOV3 (P<0.05). The expression of Cx43 was detected in OVCAR3 by flowcytometry and Western blot, but it could not be detected in CaOV3. The expression of Cx43 in both cell lines could be induced by RA. Immunofluoresence staining showed that Cx43 protein of OVCAR3 was located on membrane surface, whereas CaOV3 in cytoplasm. RA could not change the location of Cx43 protein in both cell lines. There is relationship between Cx43 expression and HSV-TK/GCV bystander effect. HSV-TK/GCV bystander effect can be enhanced by RA in ovarian cancer.