Deep vein thrombosis after open hepatectomy or other major upper abdominal surgery in Taiwan: A prospective and cross-sectional study relevant to the issue of pharmacological thromboprophylaxis.

BACKGROUD/PURPOSE: Venous thromboembolism, including deep vein thrombosis (DVT) and pulmonary embolism (PE), is an important complication in patients who underwent open hepatic surgery as well as other major upper abdominal surgery. This study aims to investigate the occurrence of postoperative DVT without pharmacological thromboprophylaxis in such cohorts in Taiwan. METHODS: This is a prospective, cross-sectional cohort study conducted from March 2010 to December 2011. Patients who underwent major upper abdominal surgery, including open hepatectomy, were enrolled. Color duplex compression ultrasonography (CUS) was used to detect DVT. Symptomatic PE was excluded if there were no suggestive respiratory symptoms or sudden death. Relevant clinicopathological and surgical information of each patient was collected and analyzed. RESULTS: 195 patients (118 male and 77 female) were enrolled, with a median age of 63.6 years. The majority (169/195, 88.7%) were treated for active malignancy. Totally 147 patients received open hepatectomy. Only one asymptomatic and distal postoperative DVT event was identified by CUS, which occurred on a 73-year-old female patient who received a left lateral segmental hepatectomy for removing the advanced hepatocellular carcinoma (pathologic stage, T3aN0M0). No cases of symptomatic PE or sudden death were observed. No correlation between DVT and precipitating factor was demonstrated in our cohort. CONCLUSION: Without pharmacological thromboprophylaxis, a low rate of postoperative DVT among patients undergoing open hepatectomy (0.7%, 1/147) or major upper abdominal surgery (0.5%, 1/195) in Taiwan was reported. A distinctively regional role of pharmacological thromboprophylaxis for hepatic surgery was also suggested by our data.

Deep vein thrombosis after major orthopedic surgery in Taiwan: A prospective cross-sectional study and literature review.

BACKGROUND/PURPOSE: Postoperative venous thromboembolism is an important complication in Taiwan. We prospectively investigated the occurrence of deep vein thrombosis (DVT) after major orthopedic surgery without pharmacologic thromboprophylaxis in a cohort of 120 patients (46 males, 74 females, median age 71 years) at our institute. METHODS: Color duplex compression ultrasonography (CUS) was used to detect DVT before and after the operation, while contrast venography was performed postoperatively for comparison and validation. RESULTS: Total knee arthroplasty (TKA, 57 cases) and total hip arthroplasty (23 cases) were the most commonly performed operations. The rate of postoperative DVT was 7.5% (9/120), including five with proximal DVT and four with distal DVT. All were detected in the limbs on the operated side. Four of them were symptomatic DVT cases. Venography was performed in 19 patients and confirmed most findings of CUS, indicating the effectiveness of CUS for detecting DVT. The type of surgery (TKA) was significantly correlated with postoperative DVT. No clinically symptomatic pulmonary embolism or sudden death events were noted. CONCLUSION: Nine out of 120 (7.5%) orthopedic patients without pharmacologic thromboprophylaxis developed postoperative sonographic DVT in our study. The DVT rate is consistent with other reports from various Asian countries and evidence from meta-analyses.

Modulation of the anticancer activities of paclitaxel by Cremophor micelles.

The objective of this study was to determine the effect of Cremophor (CrEL) on the antineoplastic effect induced by paclitaxel (PTX). Fluorescence spectroscopy, employing pyrene as a probe, was used to determine the critical micelle concentration (CMC) of CrEL. EL4 murine thymoma cells and MDA-MB-231 human breast cancer cells were treated with PTX in different concentrations of CrEL. G2 arrest with 8 N polyploidy was observed in PTX-treated EL4 cells but not in MDA-MB-231 cells. Cell cycle analysis via propidium iodide (PI) staining showed that the frequency of G2 arrest decreased as the CrEL concentration exceeded 0.02% (w/v), demonstrating the effect of CrEL micelle formation on the antimitotic activity of PTX. CrEL was also shown to enhance PTX-induced cell death in vitro by Annexin V/PI staining. Treatment of C57BL/6 mice with PTX in a lower concentration of CrEL resulted in higher myelosuppression, decreased both Ki-67 expression and survival rate, suggesting that CrEL micelle formation above the CMC may lower the cytotoxic activity of PTX in vivo. The data obtained in this study demonstrate CrEL micelle-mediated modulation of the cell cycle and cell death induced by PTX in vitro and the antineoplastic efficacy of PTX in vivo.

Recruitment of bone marrow CD11b+Gr-1+ cells by polymeric nanoparticles for antigen cross-presentation.

The objective of this study was to investigate the function of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on the activation of antigen-specific CD8+ T cell responses via the CD11b+Gr-1+ myeloid subpopulations in murine bone marrow (BM). PLGA NPs containing ovalbumin (OVA) were fabricated by the double-emulsion method. The CD11b+Gr-1lowLy-6Chigh and CD11b+Gr-1highLy-6Clow subsets from mice bone marrow were sorted and treated with the PLGA/OVA NPs, followed by co-culture with the carboxyfluorescein succinimidyl ester (CFSE)-labelled OT-I CD8+ cells. Co-culture of OT-I CD8+ T cells with PLGA/OVA NPs-primed CD11b+Gr-1+ subsets upregulated the expression of IL-2, TNF-α, INF-γ, granzyme B, and perforin, resulting in proliferation of CD8+ T cells and differentiation into effector cytotoxic T lymphocytes (CTLs). In vivo proliferation of CFSE-labelled OT-I CD8+ cells in response to OVA was also obtained in the animals immunized with PLGA/OVA NPs. The results presented in this study demonstrate the ability of polymeric NPs to recruit two CD11b+Gr-1+ myeloid subsets for effective presentation of exogenous antigen to OT-I CD8+ T cells in the context of major histocompatibility complex (MHC) class I, leading to an induction of antigen-specific cell proliferation and differentiation into effector cells.

Cellular biodistribution of polymeric nanoparticles in the immune system.

The biodistribution of polymeric nanoparticles (NPs) is of crucial importance in the development of nanoparticle-based vaccine delivery or immunotherapy for cancer. The purpose of this study was to investigate the kinetics of cellular biodistribution of polymeric NPs in the immune system. Polystyrene (PS) yellow-green nanoparticles (YG-NPs) 500nm in diameter were intravenously (i.v.) injected into the tail veins of mice, and the kinetics of YG-NP biodistribution was followed by harvesting cells at pre-determined time points from various immune organs, including blood, bone marrow, spleen, and lymph nodes and analyzing them by polychromatic flow cytometry. To observe the location of YG-NPs in the spleen after i.v. administration, spleens of mice were isolated at 6h post-injection (p.i.), cryosectioned, immunostained, and examined by confocal microscopy. Our data show that the major phagocytosing cells included granulocytes (B220¯CD11b(+)Gr-1(high)Ly-6C(low)) in the blood and bone marrow and B cells (CD11b¯B220(+)) in the spleen. The kinetics of the phenotypic analysis suggest the potential trans-differentiation of the B220¯CD11b(+)Gr-1(low)Ly-6C(high) subset into B220¯CD11b(+)Gr-1¯Ly-6C¯ double-negative (DN) cells expressing the F4/80 macrophage phenotype in the blood and CD115 in the bone marrow after treatment with YG-NPs. Based on the microscopic analysis of spleen cryosections, the majority of YG-NPs were located in the marginal zones (MZ) and red pulp of the spleen at 6h post-injection (p.i.), allowing further interaction with MZ macrophages and granulocytes. The data obtained in this study demonstrate the kinetics of biodistribution of polymeric nanoparticles in the blood, bone marrow, and spleen at the cellular level.

Activation of Antigen-Specific CD8(+) T Cells by Poly-DL-Lactide/Glycolide (PLGA) Nanoparticle-Primed Gr-1(high) Cells.

PURPOSE: The aim of this study was to investigate the induction of antigen-specific T cell activation and cell cycle modulation by a poly-DL-lactide/glycolide (PLGA) nanoparticle (NP)-primed CD11b(+)Gr-1(high) subset isolated from mouse bone marrow. METHODS: PLGA NPs containing the ovalbumin (OVA) antigen were prepared using the double emulsion and solvent evaporation method, and protein release rate and cell viability were determined. The Lin2(¯)CD11b(+)Gr-1(high)Ly6c(low) (Gr-1(high)) subset was sorted from the bone marrow of C57BL/6 J mice by fluorescence-activated cell sorting (FACS) and co-cultured with OT-I CD8(+) splenic T cells. Proliferation of OT-I CD8(+) T cells was monitored, and cell cycles were determined by 5-bromo-2'-deoxyuridine (BrdU) labeling. RESULTS: Treatment of Gr-1(high) cells with PLGA/OVA NPs upregulated expression of the SIINFEKL-H2K(b) complex in the context of MHC I. Co-cultures of OT-I CD8(+) T cells with the PLGA/OVA NP-primed Gr-1(high) cells induced the proliferation of T cells in vitro and modulated cell division and morphology. Treatment of Gr-1(high) cells with PLGA/OVA NPs also induced cell apoptosis and necrosis. CONCLUSION: This study demonstrated the function of PLGA/OVA NPs in the activation of OT-I CD8(+) T cells and the capability of cross-presentation via the Gr-1(high) polymorphonuclear subset from mouse bone marrow.

The role of tomatine adjuvant in antigen delivery for cross- presentation.

The aim of this study was to investigate the use of tomatine adjuvant to deliver soluble antigen for crosspresentation by bone marrow-derived dendritic cells (BMDCs). BMDCs were incubated with tomatine adjuvantovalbumin (OVA) complex and analyzed for antigen uptake by flow cytometry. Adjuvant-induced cell death was examined in situ by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. To elucidate the effect of antigen internalization on tomatine adjuvant-mediated antigen presentation, BMDCs were treated with several endocytosis inhibitors, and antigen presentation was analyzed by B3Z activity assay. Our data indicated that tomatine adjuvant enhanced antigen internalization by antigen presenting cells (APCs) and induced significant cell death and leukocyte infiltration at the injection sites. In vitro tomatine adjuvant treatment of BMDCs activated Ova/K(b) restricted B3Z T cell hybridomas, whereas this activation was impaired by pretreatment with brefeldin A, cytochalasin B, wortmannin, or ZnCl2. Our results demonstrated the role of tomatine adjuvant in antigen delivery to antigen presenting cells (APCs) and suggested the involvement of phagocytosis and PI3K signaling during the delivery of soluble antigens in the context of MHC class I.

Dynamics of antigen delivery and the functional roles of L121-adjuvant.

This study investigates the intracellular transport of protein antigens facilitated by L121-adjuvants and examines the associated cytotoxic T lymphocyte (CTL) effect. EL4 mouse thymoma cells were treated with L121-adjuvant and stained with AnnexinV-propidium iodide (PI) followed by flow cytometric analysis. The intracellular trafficking dynamics of bovine serum albumin (BSA)-FITC in the J774.A.1 macrophages, influenced by the L121-adjuvant, was visualized by confocal microscopy. The antigen-specific cytotoxic T lymphocyte (CTL) effect induced by the L121-adjuvant was determined by the cleavage-specific fluorogenic caspase substrate. The trafficking of BSA-FITC in the J774A.1 cells by confocal microscopy illustrated that the L121-adjuvant facilitated the intracellular transport of proteins to the subcellular compartments, including the lysosome, endoplasmic reticulum (ER), and the cis-Golgi apparatus. The L121-adjuvant also facilitated antigen delivery to the dendritic cells in the lymph nodes. Immunization of mice with the L121-adjuvant resulted in cell-mediated cytotoxic responses in the target cells, as detected by PhiPhiLux, a fluorogenic caspase substrate. Taken together, the L121-adjuvant improved the dynamics of protein delivery to antigen presenting cells, and also induced caspase activation, thereby illustrating the mechanism of antigen-specific CTL effects.

Gene delivery via the hybrid vector of recombinant adeno-associated virus and polyethylenimine.

The aim of this study was to investigate the cellular delivery mechanism of the hybrid vector comprising the recombinant adeno-associated virus (rAAV) and polyethylenimine (PEI). The rAAV vector, rAAV-rIns1-hInsM2-ΔEGFP, was fluorescently labeled with Cy3, a cyanine dye, and complexed with PEI. The interaction of the hybrid vector with the Huh7 hepatoma cells was monitored by confocal microscopy. Complexation of rAAV with PEI enhanced the transduction efficiency, which was decreased by pretreatment of the cells with sodium chlorate, an inhibitor of glycosaminoglycan sulfation, suggesting the roles of heparan sulfate proteoglycans (HSPG) in the uptake of the hybrid vector by the cells. Examination by flow cytometry and confocal microscopy demonstrated an enhanced interaction between the cells and the virus when complexed with PEI. Pretreatment with wortmannin or cytochalasin B significantly reduced the virus uptake by the cells, suggesting the involvement of phosphatidylinositol 3-kinase (PI3K) signaling and phagocytosis in the interaction between the cells and the hybrid vectors. Treatment of cells with the antioxidants, including l-ascorbic acid, δ-tocotrienol, or N-acetylcysteine (NAC), impaired the rAAV-PEI-mediated transduction. Results obtained in this study illustrated the involvement of PI3K/Akt signaling and the ROS production in gene delivery via the rAAV-PEI hybrid vector.

Delivery of DNA-based cancer vaccine with polyethylenimine.

DNA-based vaccine directed to tumor-specific antigens is an attractive strategy in cancer prevention and therapy. In view of the poor immunogenicity of most tumor-associated antigens, studies were carried out here to examine the adjuvant effect of polyethylenimine (PEI), a cationic polymer widely used in non-viral gene delivery, on the efficacy of cancer vaccine strategy. Treatment of animals with the DNA/PEI complexes resulted in antigen-specific cell lysis and activation of B3Z cells, an ovalbumin/K(b)-specific cytotoxic clone that recognizes the target cells through the class I major histocompatibility complex (MHC) molecules. Immunohistochemical examination showed that PEI-mediated DNA vaccination induced cell death and significant lymphocyte infiltration at the injection sites. Immunization of C57BL/6J mice with the DNA/PEI complexes either preceded or after tumor cell inoculation suppressed tumor growth and prolonged the survival rate of the animals. Results obtained in this study illustrated the potential use of PEI as an adjuvant in DNA-based cancer vaccination for induction of protective and therapeutic immunity.

The effect of poly(D,L-lactide-co-glycolide) microparticles with polyelectrolyte self-assembled multilayer surfaces on the cross-presentation of exogenous antigens.

A surface-engineered particulate delivery system for exogenous antigens was developed in this study. Poly(d,l-lactide-co-glycolide) (PLGA) microparticles containing ovalbumin (OVA) or fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) were fabricated by the double emulsion and solvent evaporation method. Encapsulation of the PLGA microparticles was performed by physisorption of multilayers of oppositely charged polyelectrolytes, including polyethylenimine (PEI) and dextran sulfate. Surface charges of the particles after layer-by-layer (LbL) adsorption were determined by the zeta potential measurements. The uptake of these particles by the J774A.1 murine macrophages was examined by fluorescence microscopy. Generation of reactive oxygen species (ROS) in J774A.1 cells was determined by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and hydroethidine (HE). Antigen presentation assays were performed in B3Z cells, a hybridoma of OVA-specific CD8(+) T cells. Results obtained in this study demonstrated an effective ingestion of the PLGA microparticles and enhanced production of ROS in J774A.1 murine macrophages. Treatment of murine bone marrow-derived dendritic cells (BMDCs) with polyelectrolyte-encapsulated PLGA microparticles resulted in an in vitro activation of B3Z cells, demonstrating the feasibility of induction of adaptive immunity for class I major histocompatibility complex (MHC) by surface engineering of microparticulate vaccine delivery.

Glucose- and metabolically regulated hepatic insulin gene therapy for diabetes.

PURPOSE: The purpose of this study was to examine glucose- and metabolically modulation of insulin secretion by rAAV-mediated gene delivery in vitro and in vivo. MATERIALS AND METHODS: A recombinant adeno-associated virus vector (rAAV) containing a furin-mutated human insulin gene, driven by the rat insulin I promoter, was used in this study. Glucose-responsive secretion of human insulin was determined by treating rAAV-transduced Huh7 human hepatoma cells with varying concentrations of glucose, with or without insulin secretagogues. Glucose- and metabolically modulated secretion of human insulin in the streptozotocin (STZ)-induced diabetic mice was assessed by intrahepatic administration of rAAV-polyethylenimine (PEI) complexes, followed by intraperitoneal glucose tolerance test (IPGTT), with or without theophylline. RESULTS: Glucose- and metabolically controlled human insulin secretion was obtained in the rAAV-transduced Huh7 cells. Treatment of STZ-induced diabetic animals with rAAV-polyethylenimine (rAAV-PEI) complexes resulted in production of human insulin and amelioration of hyperglycemia. Co-administration of glucose and theophylline in these animals augmented the secretion of human insulin, demonstrating metabolic modulation of insulin secretion in vivo. Immunohistochemical examination of the liver sections of rAAV-treated mice confirmed the production of human insulin. CONCLUSIONS: Glucose- and metabolically controlled hepatic insulin gene therapy was obtained both in vitro and in vivo.

Enhanced antigen delivery via cell death induced by the vaccine adjuvants.

The objective of this study was to examine the effect of cell death induced by the emulsion adjuvants on the in vitro delivery of antigens into the antigen-presenting cells (APCs). J774A.1 murine macrophage-like cells, serving as the APCs, were pulsed with various vaccine adjuvants, and incubated with fluorescein isothiocyanate-conjugated bovine serum albumin (BSA-FITC), with or without adjuvants-pretreated EL4 murine thymoma cells, followed by analysis by flow cytometry and fluorescence microscopy. To assess the potential oxidative burst in the macrophages after adjuvant treatment, cells were probed with 2',7'-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE), and analyzed by flow cytometry. Treatment of macrophages with the emulsion adjuvants, followed by co-incubation with the adjuvant-induced dead EL4 cells, resulted in a substantial uptake of soluble antigens into the APCs and generation of a considerable amount of reactive oxygen species (ROS), including hydrogen peroxide and superoxide. Pre-treatment of J774A.1 cells with several endocytosis inhibitors, including amiloride, brefeldin A or cytochalasin B, on the other hand, reduced internalization of soluble antigens. It was concluded that co-treatment of macrophages with the emulsion adjuvants and the adjuvant-induced dead cells facilitated delivery of soluble antigens, via both phagocytosis and macropinocytosis, into the antigen-presenting cells.

Effect of polyethylenimine on recombinant adeno-associated virus mediated insulin gene therapy.

BACKGROUND: Recombinant adeno-associated virus (rAAV) is becoming a promising vector for gene therapy for type I diabetes. The objective of this study was to investigate the effect of incorporation of polyethylenimine (PEI) on rAAV-mediated insulin gene therapy in vitro and in vivo. METHODS: Recombinant AAV vector, harboring the furin-mutated human insulin and enhanced green fluorescent protein (EGFP) genes, was constructed. The effect of complexation with PEI on rAAV-mediated gene transfer was examined in Huh7 human hepatoma cells. The transgene expression was also examined in streptozotocin (STZ)-induced diabetic C57BL/6J mice by direct administration of rAAV into the livers of the animals, followed by monitoring changes in body weight and blood glucose levels. Secretion of human insulin was determined by radioimmunoassay (RIA) and immunohistochemical staining in the livers. RESULTS: Complexation with PEI was shown to enhance rAAV-mediated transgene expression in Huh7 cells, resulting in higher transduction efficiency and enhanced production of immunoreactive human insulin. Heparin competition assay demonstrated that endocytosis of rAAV-PEI was partially inhibited by heparin. The enhancement of rAAV-mediated transgene expression was also demonstrated in the animals, showing lowering of blood glucose and longer duration of normoglycemia. Immunofluorescent staining of the liver sections demonstrated that PEI increased the uptake of rAAV and enhanced insulin secretion. The enhancement of PEI on rAAV-mediated insulin gene therapy was further confirmed by glucose challenge and a 10-h fasting blood glucose test. CONCLUSIONS: Results obtained in this study demonstrated that incorporation of PEI augmented rAAV-mediated insulin gene transfer and enhanced amelioration of hyperglycemia in the STZ-induced diabetic animals.

The apoptotic and necrotic effects of tomatine adjuvant.

Tomatine adjuvant, consisting of tomatine, n-octyl-beta-d-glucopyranoside (OGP), phosphatidylethanolamine and cholesterol is unique in that when combined with soluble protein antigen it elicits a cytotoxic T lymphocyte (CTL) response in immunized animals. The mechanisms underlying this property are unknown. In an attempt to understand how tomatine activates cellular immunity, we examined its potential to induce apoptosis. Thus in the present study, cell death of EL4 thymoma cells induced by whole adjuvant and the surface-active components in the formulation was examined. Cytotoxicity was monitored using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and lactate dehydrogenase release assays, apoptosis and necrosis were quantified by flow cytometry using Annexin V and propidium iodide staining, and morphology was examined by Hoechst 33342 staining. Flow cytometric analysis demonstrated the appearance of the sub-G1 phase in cells treated with these agents and Annexin V/PI staining showed that all three agents induced both apoptosis and necrosis in EL4 cells in a concentration-dependent manner. Tomatine was effective at much lower concentrations than OGP, suggesting that the majority of the effect of whole adjuvant could be attributed to this component. Microscopic examination of EL4 cells after treatment with these agents revealed morphological features of apoptosis, including chromatin condensation and DNA fragmentation. Pretreatment with zVAD-fmk did not block cell death induced by these agents, showing that tomatine adjuvant-induced EL4 cell apoptosis is caspase-independent.

Cell death induced by vaccine adjuvants containing surfactants.

Many vaccine adjuvants contain surface-active agents, but the immunological roles played by these components have been essentially ignored. The objective of this study was to examine possible apoptotic and necrotic effects of the surface-active agents, Pluronic L121 and Tween 80, which are components of L121-adjuvant (a formulation we synthesized with the aim of representing several commercially produced adjuvants), on EL4 lymphoma cells. Cell viability and cytolytic effects were analyzed using the MTT and LDH release assays, and the distribution of cells in different stages of the cell cycle after treatment with these agents was analyzed by propidium iodide (PI) staining and flow cytometry. L121-adjuvant was shown to induce cell cycle arrest and inhibit cell proliferation. Treatment of EL4 cells with surface-active agents resulted in a concentration-dependent increase in the apoptotic/necrotic cell populations. Fluorescence microscopy using Hoechst 33342 staining demonstrated chromosome condensation and DNA fragmentation in cells treated with surfactants or adjuvant. The apoptotic and necrotic effects of vaccine adjuvant containing surface-active agents were confirmed by Annexin V/propidium iodide staining and flow cytometric analysis. Pretreatment of EL4 cells with zVAD-fmk, a broad range caspase inhibitor, partially prevented apoptosis induced by Pluronic L121, but did not prevent the cell death induced by Tween 80 or L121-adjuvant. These findings suggested that Tween 80 and L121-adjuvant induced apoptosis in EL4 cells via a "non-classical" caspase-independent pathway. Results presented in this study suggest mechanisms of elicitation of CD8(+), class I-restricted CTL response by soluble antigens mediated by the vaccine adjuvant containing surface-active agents.

Induction of cell death by saponin and antigen delivery.

PURPOSE: Saponin is the major component in the formation of immune stimulating complex (ISCOM), a potent adjuvant able to induce both humoral and cellular immune reactions. The immunogenicity induced by saponin, however, has been unclear. The objective of this study was to investigate the apoptotic and necrotic effects induced by saponin in ELA mouse lymphoma cells, expected to be a possible mechanism of the cytotoxic T-lymphocyte (CTL) effect elicited by the ISCOM. METHODS: EL4 cells were treated with saponin, and viability of the cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release assays. Fluorescence microscopy was used to detect the morphological changes by staining saponin-treated cells with Hoechst 33342. Extent of apoptosis and necrosis was determined by Annexin V-FITC/propidium iodide staining, followed by flow cytometric analysis. Dendritic cells were cultured with either saponin-protein complexes or saponin-treated cells and analyzed by flow cytometry. RESULTS: Treatment of EL4 cells with saponin resulted in concentration-dependent cytotoxicity and the appearance of the hypodiploid DNA peak. Cells treated with saponin showed highly condensed chromatin when stained with fluorescent DNA-binding dye Hoechst 33342. Analysis of EL4 cells by flow cytometry after Annexin V/propidium iodide staining demonstrated that saponin induced both apoptosis and necrosis. Pretreatment of EL4 cells with zVAD-fmk, a broad-range caspase inhibitor, did not prevent cell death induced by saponin, indicating the non-caspase-dependent cell death. Dendritic cells were shown to phagocytose both the antigen-saponin complexes and the saponin-induced dead cells. CONCLUSIONS: Results obtained in this study demonstrated that saponin induced both apoptosis and necrosis in ELA cells. These events are critical for antigen processing and presentation.

Differential requirements for CTL generation by novel immunostimulants: APC tropism, use of the TAP-independent processing pathway, and dependency on CD80/CD86 costimulation.

A major drawback of subunit vaccines is their inability to generate cytolytic T lymphocytes (CTL), a deficit attributed to segregation of the class I and class II antigen-processing pathways. We sought to understand processes involved in CTL induction by three proprietary adjuvants: Tomatine, PROVAX, and a synthesized glycolipid (Glc-N-(8/16), Glycolipid). We used in vivo models to investigate antigen uptake, macrophage involvement, TAP-independent processing, and costimulatory molecule dependencies. Glycolipid required splenic and lymph node macrophages, whereas Tomatine generated CTL independently of either macrophage population. In contrast, PROVAX showed partial macrophage requirements. Immunized TAP knockout mice revealed that ovalbumin (OVA)-Tomatine and OVA-PROVAX, but not OVA-Glycolipid, generate class I-peptide complexes. All three immunostimulants also elicited CD86-dependent TH1 cytokine responses.

Incorporation of calcium phosphate enhances recombinant adeno-associated virus-mediated gene therapy in diabetic mice.

BACKGROUND: Increased efficiency of transgene expression is desired for virus-mediated gene delivery. In the present study, we examined the effect of calcium phosphate (CaPi) on recombinant adeno-associated virus (rAAV)-mediated insulin therapy in diabetic animals. METHODS: The rAAV vector, rAAV.PEPCK.Ins.EGFP, containing the human insulin gene under control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter and the enhanced green fluorescence protein (EGFP) gene driven by the cytomegalovirus (CMV) IE promoter, was employed in this study. C57BL/6J mice were made diabetic with streptozotocin (STZ), followed by injection into the livers with either rAAV alone, or noncovalent complexes with calcium phosphate. Body weight and blood glucose levels of the animals were routinely monitored after 6 h fasting. Secretion of human insulin in the rAAV-transduced animals was determined by radioimmunoassay (RIA). Expression of human insulin in the livers of the animals was detected by immunohistochemical staining. RESULTS: Compared with the STZ-treated control mice, administration of rAAV containing the human insulin gene significantly decreased blood glucose levels and maintained body weight of the diabetic animals. Complexation of rAAV with calcium phosphate enhanced the hypoglycemic effect of rAAV-mediated gene transfer. Results obtained from both RIA and immunohistochemical staining demonstrated that incorporation of calcium phosphate enhanced rAAV-mediated gene transfer in vivo, leading to higher expression and secretion of human insulin. CONCLUSIONS: Administration of rAAV harboring the human insulin gene into livers of the STZ-diabetic mice improved blood glucose levels, maintained body weight of the diabetic animals, and resulted in human insulin secretion. Complexation of rAAV with calcium phosphate significantly potentiated the efficiency of rAAV-mediated diabetic gene therapy.

Glucose-modulated transgene expression via recombinant adeno-associated virus.

PURPOSE: The objective of this study was to examine glucose modulated reporter gene expression via recombinant adeno associated viral vectors both in vitro and in vivo. METHODS: Huh7 human hepatoma cells were transduced by recombi nant adeno-associated virus (rAAV) vectors containing the luciferase gene under control of the rat insulin I gene promoter and a cytomegalovirus immediate-early promoter driving-enhanced green fluores cence protein gene. The reporter gene expression was evaluated by glucose stimulation either in the absence or presence of insulin se cretagogues, including phorbol-12-myristate-13-acetate, dibutyryl cy clic AMP, and forskolin. In vivo studies were performed by injecting rAAV into the livers of streptozotocin-induced diabetic C57BL/6J mice followed by measurements of blood glucose concentration and luciferase activity assays 2 weeks after rAAV injection. RESULTS: At a multiplicity of infection of 500, approximately 66-69% of cells expressed enhanced green fluorescence protein at 48 h post transduction. Luciferase activities, driven by the insulin gene promoter, in the rAAV-transduced hepatoma cells responded to milli molars of glucose. The addition of phorbol-12-myristate-13-acetate dibutyryl cyclic AMP, and forskolin increased luciferase expression in the presence of either 1 mM or 25 mM glucose. The stimulation of luciferase activities by these substances was inhibited by the presence of 100 nM staurosporine. Exposure to increments of exogenous in sulin up to 10(-7) M inhibited luciferase gene expression in rAAV transduced Huh7 cells. The in vivo experiments demonstrated good correlation between luciferase activities and blood glucose levels in streptozotocin-induced diabetic animals. CONCLUSION: rAAV is a promising vector for hepatic gene therapy for diabetes. Glucose and insulin secretagogues modulated transgene ex pression in rAAV-transduced hepatoma cells, suggesting that condi tions affecting insulin gene promoter function in pancreatic islet beta cells also affect transgene expression in human hepatoma cells con ferred with insulin gene promoter. Results obtained from in viv experiments demonstrated that glucose modulated transgene expres sion can be obtained in rAAV-treated diabetic C57BL16J mice.