Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

AIM： To generate an adenoviral vector specifically targeting the EphA2 receptor （EphA2R） highly expressed on pancreatic cancer cells in vivo. METHODS： YSA, a small peptide ligand that binds the EphA2R with high affinity, was inserted into the HI loop of the adenovirus serotype 5 fiber knob. To further increase the specificity of this vector, binding sites for native adenoviral receptors, the coxsackie and adenovirus receptor （CAR） and integrin, were ablated from the viral capsid. The ablated retargeted adenoviral vector was produced on 293T cells. Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines. Upon demonstrating specific in vitro targeting, in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector. RESULTS： Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold. Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor. YSA-mediated transduction was inhibited by addition of synthetic YSA peptide. The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro. In a subsequent in vivo study in a nude （nu/nu） mouse model however, no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein, nor upon injection into the peritoneum. CONCLUSION： Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.     

A rapid and efficient method to express target genes in mammalian cells by baculovirus

AIM: To investigate the modification of baculovirus vector and the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of Spodoptera frugiperta (Sf9) cells infected by recombinant baculoviruses. METHODS: Two recombinant baculoviruses (BacV-CMV-EGFPA, BacV-CMV-EGFPB) containing CMV-EGFP expression cassette were constructed. HepG2 cells were directly incubated with the culture supernatant of Sf9 cells infected by recombinant baculoviruses, and reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM). The optimal transduction conditions were investigated by FCM assay in HepG2 cells. Gene-transfer and expression efficiencies in HepG2 or CV1 cells by baculovirus vectors were compared with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Twenty different mammalian cell lines were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the culture supernatant of infected Sf9 cells. RESULTS: CMV promoter could directly express reporter genes in Sf9 cells with a relatively low efficiency. Target cells incubated with the 1:1 diluted culture supernatant (moi=50) for 12 h at 37 ℃ could achieve the highest transduction and expression efficiencies with least impairment to cell viability. Under similar conditions the baculovirus vector could achieve the highest gene-transfer and expression efficiency than lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Most mammalian cell lines could be transduced with recombinant baculovirus. In primate adherent culture cells the recombinant baculovirus could arrive the highest infection and expression efficiencies, but it was not very satisfactory in the cell lines from mice and suspended culture cells. CONCLUSION: Mammalian cells incubated with the culture supernatant of infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign genes in mammalian cells, but it might be more suitable for primate adherent culture cells.     

Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients

AIM: To determine whether gastric and enteric Helicobacter species are associated with pancreatic cancer. METHODS: Patients with exocrine pancreatic cancer (n = 40), neuroendocrine cancer (n=14), multiple endocrine neoplasia type 1 {n = 8), and chronic pancreatitis (n = 5) were studied. Other benign pancreatic diseases (n = 10) and specimens of normal pancreas (n=7) were included as controls. Pancreatic tissue specimens were analyzed by He/icobacter-specific PCR-assay and products were characterized by denaturing gradient electrophoresis and DNA-sequencing. From a subset of the pancreatic cancer patients, gastric and/or duodenal tissue as well as gallbladder and ductus choledochus tissue were analyzed. Gallbladder and choledochus samples were included as controls. Stomach and duodenum samples were investigated to analyze whether a gastric helicobacter might disseminate to the pancreas in pancreatic cancer patients. Pancreatic specimens were analyzed by Bacteroides-specific PCR for detecting the translocation of indigenous gut microbes to the diseased pancreas. RESULTS: Helicobacter DNA was detected in pancreas (tumor and/or surrounding tissue) of 75% of patients with exocrine cancer, 57% of patients with neuroendocrine cancer, 38% of patients with multiple endocrine neoplasia, and 60% of patients with chronic pancreatitis. All samples from other benign pancreatic diseases and normal pancreas were negative. Thirty-three percent of the patients were helicobacter-positive in gastroduodenal specimens. Surprisingly, H. bilis was identified in 60% of the positive gastro-duodenal samples. All gallbladder and ductus cho-ledochus specimens were negative for helicobacter. Bacteroides PCR-assay was negative for all pancreatic samples. CONCLUSION: Helicobacter DNA commonly detected in pancreatic cancer suggests a possible role of the emerging pathogens in the development of chronic pancreatitis and pancreatic cancer.     

Treating diabetes β mellitus through endogenous cell regeneration

Transplantation of islets offers an attractive strategy for the treatment of diabetes, but its wide application has been limited by donor shortage and immunological rejection after transplantation. During normal pancreatic tissue turnover, the renewal and apoptosis of β cells are in homeostasis. After the destruc-tion of existing β cells through physical or chemical injuries, β cells could be regenerated from pancreatic     

Treating diabetes β mellitus through endogenous cell regeneration

Transplantation of islets offers an attractive strategy for the treatment of diabetes, but its wide application has been limited by donor shortage and immunological rejection after transplantation. During normal pancreatic tissue turnover, the renewal and apoptosis of β cells are in homeostasis. After the destruc-tion of existing β cells through physical or chemical injuries, β cells could be regenerated from pancreatic     

Experience of limited pancreatic head resection for management of branch duct intraductal papillary mucinous neoplasm in a single center

AIM： To share our surgical experience and the outcome of limited pancreatic head resection for the management of branch duct intraductal papillary mucinous neoplasm （IPMN）.
METHODS： Between May 2005 and February 2008, nine limited pancreatic head resections （LPHR） were performed for IPMN of the pancreatic head. We reviewed the nine patients, retrospectively.
RESULTS： Tumor was located in the uncinate process of the pancreas in all nine patients. Three patients had stents inserted in the main pancreatic duct due to inju- ry. The mean size of tumor was 28.4 mm. Postoperative complications were found in five patients： 3 pancreatic leakages, a pancreatitis, and a duodenal stricture. Pancreatic leakages were improved by external drainage. No perioperative mortality was observed and all patients are recorded alive during the mean follow-up period of 17.2 mo.
CONCLUSION： In selected patients after careful evaluation, LPHR can be used for the treatment of branch duct type IPMN. In order to avoid pancreatic ductal injury, pre- and intra-operative definite localization and careful operative techniques are required.     

Antioxidant activity of chito-oligosaccharides on pancreatic islet cells in streptozotocin-induced diabetes in rats

AIM： To investigate the antioxidant activity of chitooligosaccharides （COSs） on pancreatic islet cells in diabetic rats induced by streptozotocin.
METHODS： The antioxidant effect of COSs on pancreatic islet cells was detected under optical microscopy and with colorimetric assay and gel electrophoresis. The activities of glutathione peroxidase and superoxide dismutase, total antioxidant capacity, and content of malondialdehyde in serum and tissue slices of pancreas were examined after 60 d to determine the effect of COSs in streptozotocin-induced diabetes in rats.
RESULTS： COSs can prohibit the apoptosis of pancreatic islet cells. All concentrations of COSs can improve the capability of total antioxidant capacity and activity of superoxide dismutase and decrease the content of malondialdehyde drastically. Morphological investigation in the pancreas showed that COSs have resulted in the reduction of islets, loss of pancreatic cells, and nuclear pyknosis of pancreatic cells.
CONCLUSION： COSs possess various biological activities and can be used in the treatment of diabetes mellitus.     

Viral replication modulated by synthetic peptide derived from hepatitis B virus X protein

AIM:A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx.METHODS:We synthesized the fully conserved peptide (CP) with nine residues, FVLGGCRHK. HBV-producJng 2.2.15 cells were treated with or without 3.5μM CP for 36 hours.Quantitative detection of viral DNA was performed by realtime PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence.Flow cytometry was performed to detect cell cycle and apoptosis.RESULTS: Both extracellular and intracellular copies of HBV DNA per ml were significantly increased after incubation with 3.5μM of CP.HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control ceils. CP influenced negatively the extracellular viral gene products, and 3.5μM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5μM CP could efficiently increase the level of intracellular HBeAg.Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2,15 cells with or without CP.CONCLUSION: Together with the results generated from the synthetic peptide, we address that the conserved region,a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.     

Efficient and non-toxic gene transfer to cardiomyocytes using novel generation amplicon vectors derived from HSV-1

OBJECTIVE: The feasibility of gene transfer to myocardial tissue using viral vectors was investigated over the last few years. In this study we report gene transfer using a recently described improved of Herpes simplex virus (HSV-1)-derived amplicon vectors and demonstrate that these vectors are a powerful and potentially very interesting tool for gene transfer into neonatal primary as well as in adult cardiac myocytes. METHODS AND RESULTS: Non-pathogenic HSV-1 amplicon vectors simultaneously expressing GFP and LacZ were constructed using a novel helper system that yields essentially helper-free vector particles. These vectors were used to infect either cultured primary neonatal rat cardiomyocytes or adult cardiac tissue. Transgenic expression was quantified using a FACS (GFP) or X-gal staining (LacZ). Infection of primary cardiomyocytes showed efficient transduction even at very low multiplicity of infection (MOI), and expression increased with the infectious dose. By investigating release of lactate dehydrogenase (LDH) or spontaneous beating of the cells, we failed to detect cytotoxic effects in cardiomyocytes infected at high MOI. Thin slices of adult cardiac tissue placed in medium containing vectors also showed very good levels of transduction, without any evidence of toxic effects. CONCLUSIONS: Helper-free amplicon vectors very efficiently transduce genes into cardiomyocytes. Our results indicate similar or better transduction efficiencies than those reported using other vector systems. Furthermore, the very high transgenic capacity of amplicon vectors (up to 150 kbp) makes these vectors a unique and very suitable system to transduce large genomic sequences into cardiomyocytes.     

The effect of promoter strength in adenoviral vectors in hyperplastic synovium

OBJECTIVES: To compare the activity of the CytoMegaloVirus promoter (CMV) and the Major Late promoter (MLP) in synoviocytes in vitro and in vivo. To determine the phenotype of infected cells and the induction of inflammation. To investigate the effects of the cytomegalovirus (CMV) or major late (MLP) promoter on adenovirus-mediated reporter gene transduction of synoviocytes in vitro and in vivo. METHODS: After infection with adenoviral vectors harboring CMV- and MLP-driven luciferase and lacZ genes, gene expression was examined in cultured synoviocytes and in the synovium of rhesus monkeys with collagen-induced arthritis. Immunohistochemical staining for the macrophage-marker CD68 and lacZ expression was performed. Inflammation was scored in the synovial membrane of injected and non-injected joints. RESULTS: CMV-driven reporter gene expression was found to be 6 to 10 times higher than MLP-driven gene expression in both cultured synoviocytes and monkey synovium. Both CD68 positive and CD68 negative cells were lacZ positive. Inflammation in joints injected with CMV-driven adenoviral vectors was not higher than that in MLP-driven adenoviral vectors- or non-injected joints. CONCLUSION: These experiments show that the CMV promoter induces higher gene expression in synoviocytes than the MLP promoter. Both fibroblast-like and macrophage-like synoviocytes can be infected with adenoviral vectors. No deleterious effects of the CMV-promoter driven adenoviral vectors were observed.     

A genetically modified adenoviral vector exhibits enhanced gene transfer of human smooth muscle cells

Adenoviral vector-based gene therapy is a promising approach for the treatment of restenosis postangioplasty. However, a high concentration of adenoviral vector can cause cellular activation, damage, and an enhanced immune response. One approach to solving this problem is to increase gene transfer efficiency by directing adenoviral vector entry via an alternate receptor system. We have constructed an adenoviral vector, Av9LacZ, that encodes the beta-galactosidase gene and contains a chimeric fiber protein that redirects viral vector binding to the Ad3 adenoviral receptor on the host cell. We examined the ability of Av9LacZ to transduce primary human smooth muscle cells (SMC) and found that it showed a 10- to 15-fold higher transduction efficiency when compared to the prototypic adenoviral vector currently used for preclinical and clinical studies. While both vectors were able to transduce rabbit, pig and monkey SMCs, the genetically modified vector transduced human SMC with much higher efficiency. SMC obtained from the aorta, coronary, renal, popliteal and pulmonary arteries were all efficiently transduced by Av9LacZ. Consistent with the data obtained from cultured cells, Av9LacZ also transduced fresh human arterial tissues considerably more efficiently than Av1LacZ. We conclude that the large discrepancy between transduction of animal and human cells by conventional vectors supports a cautious extrapolation of the results of in vivo animal studies to man. Furthermore, the genetically modified AV9 vector may deliver better efficacy and studies in large animal models with this vector could be more predictive of therapeutic efficacy in the treatment of human restenosis.     

A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans

Aims/hypothesis Successful gene transfer to pancreatic islets might be a powerful tool for dissecting the biological pathways involved in the functional impairment and destruction of beta cells in type 1 diabetes. In the long run, such an approach may also prove useful for promoting islet graft survival after transplantation in diabetic patients. However, efficient genetic modification of primary insulin-producing cells is limited by the specific compact structure of the pancreatic islet. We present here a whole-pancreas perfusion-based transduction procedure for genetic modification of intact pancreatic islets. Materials and methods We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion. Results In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure. Conclusions/interpretation These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research. Electronic supplementary material Supplementary material is available for this article at and is accessible to authorized users     

Preclinical gene therapy studies for hemophilia using adenoviral vectors

Hemophilia A and B are hereditary coagulation defects resulting from a deficiency of factor VIII (FVIII) and factor IX (FIX), respectively. Introducing a functional FVIII or FIX gene could potentially provide a cure for these bleeding disorders. Adenoviral vectors have been used as tools to introduce potentially therapeutic genes into mammalian cells and are by far the most efficient vectors for hepatic gene delivery. Long-term expression of both FVIII and FIX has been achieved in preclinical (hemophilic) mouse models using adenoviral vectors. Therapeutic levels of FVIII and FIX also have been achieved in hemophilic dogs using adenoviral vectors and in some cases expression was long-term. The performance of earlier generation adenoviral vectors, which retained residual viral genes, was compromised by potent acute and chronic inflammatory responses that contributed to significant toxicity and morbidity and short-term expression of FVIII and FIX. The development of improved adenoviral vectors devoid of viral genes (gutless or high-capacity adenoviral vectors) was therefore warranted, which led to a significant reduction in acute and chronic toxicity and more prolonged expression of FVIII and FIX. Strategies aimed at making these vectors safer and less immunogenic and their implications for hemophilia gene therapy are discussed in this review.     

Haematopoietic progenitor cells from the common marmoset as targets of gene transduction by retroviral and adenoviral vectors

To establish a new non-human primate model for human cytokine and gene therapy, we characterized lymphocytes and haematopoietic progenitor cells of the small New World monkey, the common marmoset. We first assessed the reactions of marmoset bone marrow (BM) and peripheral blood (PB) cells to mouse anti-human monoclonal antibodies (mAbs) for the purpose of isolating marmoset lymphocytes and haematopoietic progenitor cells. Both cell fractions stained with CD4 and CD8 mAbs were identified as lymphocytes by cell proliferation assay and morphological examination. Myeloid-specific mAbs such as CD14 and CD33 did not react with marmoset BM and PB cells. No available CD34 and c-kit mAbs could be used to purify the marmoset haematopoietic progenitor cells. Furthermore, we studied the in vitro transduction of the bacterial beta-galactosidase (LacZ) gene into CFU-GM derived from marmoset BM using retroviral and adenoviral vectors. The transduction efficiency was increased by using a mixed culture system consisting of marmoset BM stromal cells and retroviral producer cells. It was also possible to transduce LacZ gene into marmoset haematopoietic progenitor cells with adenoviral vectors as well as retroviral vectors. The percentage of adenovirally transduced LacZ-positive clusters was 15% at day 4 (multiplicity of infection=200), but only 1-2% at day 14. The differential use of viral vector systems is to be recommended in targeting different diseases. Our results suggested that marmoset BM progenitor cells were available to examine the transduction efficiency of various viral vectors in vitro.     

Targeted delivery of adenoviral vectors by cytotoxic T cells

Effective targeting of vectors to tumor cells that have metastasized to multiple different tissue sites remains a major challenge for gene therapy. Tumor-specific cytotoxic T lymphocytes (CTLs) have been shown in animal models and in humans to be able to cross tissue barriers and traffic to tumor cells. However, their capacity to eliminate malignancy has been limited by tumor immune evasion strategies. We now use a model of Epstein-Barr virus-mediated malignancy to show that human CTLs themselves may be modified to release therapeutic vectors following engagement of their antigen-specific receptors and that these vectors will effectively transduce and destroy tumor targets. We generated EBV-specific CTLs that were transgenic for the adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand (CD40L) promoter. Following transduction with E1-deficient adenoviral vectors, these CTLs produced infectious virus when exposed to HLA-matched EBV-expressing targets, but not on exposure to major histocompatibility complex (MHC)-mismatched or otherwise irrelevant cells. This approach provides a means of delivering oncolytic/therapeutic vectors not only to locally accessible macroscopic tumors as is presently the case, but also to disseminated metastatic disease, while avoiding the risks associated with systemic administration of large doses of adenoviral vectors.     

Positron emission tomography imaging of adenoviral-mediated transgene expression in liver cancer patients

BACKGROUND & AIMS: In gene-therapy protocols, imaging of gene expression is needed to evaluate the transduction efficiency of the vector, its tissue distribution, and the duration of transgene expression and to assess the feasibility of repeated vector administration. METHODS: We have used positron emission tomography with a fluorine-18-labeled penciclovir analogue to monitor thymidine kinase gene expression after intratumoral injection of a first-generation recombinant adenovirus in patients with hepatocellular carcinoma. Patients were enrolled in a pilot clinical trial and treated with escalating doses of the vector. Two days after adenovirus inoculation, transgene expression was evaluated during the first hours after administration of the radiotracer both on the treated lesion and on a whole-body basis. RESULTS: Transgene expression in the tumor was dependent on the injected dose of the adenovirus and was detectable in all patients who received > or = 10(12) viral particles. However, when the study was repeated 9 days after vector injection, no expression could be observed. It is interesting to note that no specific expression of the transgene could be detected in distant organs or in the surrounding cirrhotic tissue in any of the cases studied. CONCLUSIONS: Our findings show the real possibility of imaging transgene expression in humans by using viral vectors. We show that hepatocarcinoma is a permissive tumor for adenoviral infection and that the nontumoral cirrhotic liver is spared from transduction when the vector is administered by intratumoral injection. These results show that positron emission tomography imaging may help in the design of gene-therapy strategies and in the clinical assessment of new-generation vectors.