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At least 451 records · Page 25Linked to original sources
PubMed2004

Combined alpha tumor necrosis factor gene therapy and engineered dendritic cell vaccine in combating well-established tumors.

BACKGROUND: Although current immunotherapeutic strategies including adenovirus (AdV)-mediated gene therapy and dendritic cell (DC) vaccine can all stimulate antitumor cytotoxic T lymphocyte (CLT) responses, their therapeutic efficiency has still been limited to generation of prophylactic antitumor immunity against re-challenge with the parental tumor cells or growth inhibition of small tumors in vivo. However, it is the well-established tumors in animal models that mimic clinical patients with existing tumor burdens. Alpha tumor necrosis factor (TNF-alpha) is a multifunctional and immunoregulatory cytokine that induces antitumor activity and activates immune cells such as DCs and T cells. We hypothesized that a combined immunotherapy including gene therapy and DC vaccine would have some advantages over each modality administered as a monotherapy. METHODS: We investigated the antitumor immunotherapeutic efficiency of gene therapy by intratumoral injection of AdVTNF-alpha and DC vaccine using subcutaneous injection of TNF-alpha-gene-engineered DC(TNF-alpha) cells, and further developed a combined AdV-mediated TNF-alpha-gene therapy and TNF-alpha-gene-engineered DC(TNF-alpha) vaccine in combating well-established MO4 tumors expressing the ovalbumin (OVA) gene in an animal model. RESULTS: Our data show that vaccination of DC(TNF-alpha) cells pulsed with the OVA I peptide can (i) stimulate type 1 immune response with enhanced antitumor CTL activities, (ii) induce protective immunity against challenge of 5 x 10(5) MO4 tumor cells, and (iii) reduce growth of the small (3-4 mm in diameter), but not large, established MO4 tumors (6-8 mm in diameter). Our data also show that AdVTNF-alpha-mediated gene therapy can completely eradicate small tumors in 6 out of 8 (75%) mice due to the extensive tumor necrosis formation, but not the large tumors (0%). Interestingly, a combined AdVTNF-alpha-mediated gene therapy and TNF-alpha-gene-engineered DC(TNF-alpha) vaccine is able to cure 3 out of 8 (38%) mice bearing large MO4 tumors, indicating that the combined immunotherapy strategy is much more efficient in combating well-established tumors than monotherapy of either gene therapy or DC vaccine alone. CONCLUSIONS: This novel combined immunotherapy may become a tool of considerable conceptual interest in the implementation of future clinical objectives.

Animals↗
PubMed2005

Plasminogen kringle 5-engineered glioma cells block migration of tumor-associated macrophages and suppress tumor vascularization and progression.

Angiostatin, a well-characterized angiostatic agent, is a proteolytic cleavage product of human plasminogen encompassing the first four kringle structures. The fifth kringle domain (K5) of human plasminogen is distinct from angiostatin and has been shown, on its own, to act as a potent endothelial cell inhibitor. We propose that tumor-targeted K5 cDNA expression may act as an effective therapeutic intervention as part of a cancer gene therapy strategy. In this study, we provide evidence that eukaryotically expressed His-tagged human K5 cDNA (hK5His) is exported extracellularly and maintains predicted disulfide bridging conformation in solution. Functionally, hK5His protein produced by retrovirally engineered human U87MG glioma cells suppresses in vitro migration of both human umbilical vein endothelial cells and human macrophages. Subcutaneous implantation of Matrigel-embedded hK5His-producing glioma cells in nonobese diabetic/severe combined immunodeficient mice reveals that hK5His induces a marked reduction in blood vessel formation and significantly suppresses the recruitment of tumor-infiltrating CD45+ Mac3+ Gr1- macrophages. Therapeutically, we show in a nude mouse orthotopic brain cancer model that tumor-targeted K5 expression is capable of effectively suppressing glioma growth and promotes significant long-term survival (>120 days) of test animals. These data suggest that plasminogen K5 acts as a novel two-pronged anticancer agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages, resulting in a potent, clinically relevant antitumor effect.

Amino Acid Sequence↗
PubMed2002

Considerations for successful transplantation of encapsulated pancreatic islets.

Encapsulation of pancreatic islets allows for transplantion in the absence of immunosuppression. The technology is based on the principle that transplanted tissue is protected for the host immune system by an artificial membrane. Encapsulation offers a solution to the shortage of donors in clinical islet transplantation because it allows animal islets or insulin-producing cells engineered from stem cells to be used. During the past two decades three major approaches to encapsulation have been studied. These include intravascular macrocapsules, which are anastomosed to the vascular system as AV shunt; extravascular macrocapsules, which are mostly diffusion chambers transplanted at different sites; and extravascular microcapsules transplanted in the peritoneal cavity. The advantages and pitfalls of these three approaches are discussed and compared in the light of their applicability to clinical islet transplantation. All systems have been shown to be successful in preclinical studies but not all approaches meet the technical or physiological requirements for application in human beings. The extravascular approach has advantages over the intravascular because since it is associated with less complications such as thrombosis and infection. Microcapsules, due to their spatial characteristics, have a better diffusion capacity than macrocapsules. Recent progress in biocompatibility of microcapsules has brought this technology close to clinical application. Critical issues such as limitations in the functional performance and survival are being discussed. The latest results show that both issues can be solved by the transplantation of microencapsulated islets close to blood vessels in prevascularized solid supports.

Animals↗
PubMed2001

Chemical engineering of cell penetrating antibodies.

Antibodies, being exquisitely specific tools in biology, are routinely used to detect and identify intra-cellular structures. However, current intra-cellular application of antibodies requires that the membrane be rendered leaky, resulting in the death of cells. Here, we present a novel method to allow antibodies to penetrate the cellular membrane of living cells without affecting cell viability. A peptide (MTS, membrane transport sequence) that facilitates transport across membranes has been site-specifically attached to antibodies. MTS-antibodies enter the living cells in culture and can be detected by immunofluorescence and ELISA after extraction. Cellular structures are visualized in living cells using a specific MTS-antibody. Antibodies with membrane penetrating properties can become an important tool for the study of intra-cellular processes in living cells. Furthermore, such membrane penetrating antibodies can be used to selectively stimulate or suppress functions of the cellular machinery.

3T3 Cells↗
PubMed2005

The bioartificial pancreas: progress and challenges.

Diabetes remains a devastating disease, with tremendous cost in terms of human suffering and healthcare expenditures. A bioartificial pancreas has the potential as a promising approach to preventing or reversing complications associated with this disease. Bioartificial pancreatic constructs are based on encapsulation of islet cells with a semipermeable membrane so that cells can be protected from the host's immune system. Encapsulation of islet cells eliminates the requirement of immunosuppressive drugs, and offers a possible solution to the shortage of donors as it may allow the use of animal islets or insulin-producing cells engineered from stem cells. During the past 2 decades, several major approaches for immunoprotection of islets have been studied. The microencapsulation approach is quite promising because of its improved diffusion capacity, and technical ease of transplantation. It has the potential for providing an effective long-term treatment or cure of Type 1 diabetes.

Diabetes Mellitus, Type 1↗
PubMed1995

Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions.

CAMPATH-1H (where CAMPATH is a trade mark of Wellcome group companies), a humanized IgG antibody used in the therapy of lymphoma, leukaemia and rheumatoid arthritis, has been expressed in Chinese hamster ovary, Y0 myeloma and NS0 myeloma cell lines. These engineered cell lines were grown under different culture conditions, and the antibody isolated and purified. N-Linked oligosaccharides, on the CH2 heavy chain region of the antibody, were isolated and analysed by hydrazinolysis, high-performance anion-exchange chromatography with pulsed amperometric detection, laser-desorption mass spectrometry and sequential exoglycosidase treatment. Both the glycosylation pattern and the biological activity of CAMPATH-1H, as measured by antibody-dependent cell-mediated cytotoxicity, were markedly affected by the cell line used to express the antibody. It is concluded that glycosylation of the antibody may be important in the clinical outcome of therapy.

Alemtuzumab↗
PubMed2004

High CD46 receptor density determines preferential killing of tumor cells by oncolytic measles virus.

Live attenuated Edmonston B strain of measles virus (MV-Edm) is a potent and specific oncolytic agent, but the mechanism underlying its tumor selectivity is unknown. The virus causes cytopathic effects (CPEs) of extensive syncytial formation in tumor cells but minimal damage or cell killing in normal cells. The CPE is dependent on expression of viral proteins and the presence of CD46, the major cellular receptor of MV-Edm. Using a virally encoded soluble marker peptide to provide a quantitative readout of the level of viral gene expression, we determined that tumor cells and normal cells expressed comparable levels of viral proteins. CD46 mediates virus attachment, entry, and virus-induced cell-to-cell fusion. Using engineered cells expressing a range of CD46 densities, we determined that whereas virus entry increased progressively with CD46 density, cell fusion was minimal at low receptor densities but increased dramatically above a threshold density of CD46 receptors. It is well established that tumor cells express abundant CD46 receptors on their surfaces compared with their normal counterparts. Thus, at low CD46 densities typical of normal cells, infection occurs, but intercellular fusion is negligible. At higher densities typical of tumor cells, infection leads to extensive cell fusion. Intercellular fusion also results in enhancement of viral gene expression through recruitment of neighboring uninfected cells into the syncytium, further amplifying the CPE. Discrimination between high and low CD46 receptor density provides a compelling basis for the oncolytic specificity of MV-Edm and establishes MV-Edm as a promising CD46-targeted cancer therapeutic agent.

Animals↗
PubMed2005

Ferritin contributes to melanoma progression by modulating cell growth and sensitivity to oxidative stress.

PURPOSE: Employing an in vitro model system of human melanoma progression, we previously reported ferritin light chain (L-ferritin) gene overexpression in the metastatic phenotype. Here, we attempted to characterize the role of ferritin in the biology of human melanoma and in the progression of this disease. EXPERIMENTAL DESIGN: Starting from the LM human metastatic melanoma cell line, we engineered cell clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. These cells were then assayed for their growth capabilities, chemoinvasive properties, and sensitivity to oxidative stress. Additionally, ferritin protein content in primary and metastatic human melanomas was determined by immunohistochemistry. RESULTS: Artificial L-ferritin down-regulation in the LM cells strongly inhibited proliferation and chemoinvasion in vitro and cell growth in vivo. In addition, L-ferritin down-regulated cells displayed enhanced sensitivity to oxidative stress and to apoptosis. Concurrently, immunohistochemical analysis of a human melanoma tissue array revealed that ferritin expression level in metastatic lesions was significantly higher (P < 0.0001) than in primary melanomas. Furthermore, ferritin expression was constantly up-regulated in autologous lymph node melanoma metastases when compared with the respective primary tumors in a cohort of 11 patients. CONCLUSIONS: These data suggest that high ferritin expression can enhance cell growth and improve resistance to oxidative stress in metastatic melanoma cells by interfering with their cellular antioxidant system. The potential significance of these findings deserves to be validated in a clinical setting.

Animals↗
PubMed2002

Biologic liver support: optimal cell source and mass.

Hepatic support is indicated in acute liver failure (ALF) patients to foster liver regeneration, or until a liver becomes available for orthotopic liver transplantation (OLT), in primary non function of the transplanted liver, and hopefully in chronic liver disease patients affected by ALF episodes, in whom OLT is not a therapeutic option. The concept of bioartificial liver (BAL) is based on the assumption that only the hepatocytes can perform the whole spectrum of biotransformation functions, which are needed to prevent hepatic encephalopathy, coma and cerebral edema. Among others, two important issues are related to BAL development: 1) the choice of the cellular component; 2) the cell mass needed to perform an adequate BAL treatment. Primary hepatocytes, of human or animal origin, should be considered the first choice because they express highly differentiated functions. Accordingly, a minimal cell mass corresponding to 10% of a human adult liver, i.e. 150 grams of freshly isolated, > or = 90% viable hepatocytes should be used. When 4 degrees C cold-stored or cryopreserved hepatocytes are used, the cellular mass should be increased because of a drop in cell viability and function. In case of hepatoma-derived cells, cultured cell lines or engineered cells, an adequate functional cell mass should be used, expressing metabolic and biotransformation activities comparable to those of primary hepatocytes. Finally, the use of porcine hepatocytes or other animal cells in BAL devices should be presently directed only to ALF patients as a bridge treatment to OLT, because of potential transmission of animal retrovirus and prions which may potentially cause major pandemics.

Animals↗
PubMed2004

Interplay of glucagon-like peptide-1 and transforming growth factor-beta signaling in insulin-positive differentiation of AR42J cells.

The differentiation of pancreatic exocrine AR42J cells into insulin-expressing endocrine cells has served as an important model for both endogenous in vivo beta-cell differentiation as well as potential application to beta-cell engineering of progenitor cells. Exogenous activin, possibly working through intracellular smad 2 and/or smad 3, as well as exogenous exendin-4 (a long-acting glucagon-like peptide-1 agonist) have both been shown to induce insulin-positive/endocrine differentiation in AR42J cells. In this study, we present evidence of significant interplay and interdependence of these two pathways as well as potential synergy between the pathways. In particular, insulin-positive differentiation seems to entail an exendin-4-induced drop in smad 2 and elevation in smad 3 in RNA levels. The latter appears to be dependent on endogenous transforming growth factor (TGF)-beta isoform release by the AR42J cells and may serve as a mechanism to promote beta-cell maturation. The drop in smad 2 may mediate early endocrine commitment. The coapplication of exogenous exendin-4 and, specifically, low-dose exogenous TGF-beta1 led to a dramatic 20-fold increase in insulin mRNA levels, supporting a novel synergistic and codependent relationship between exendin-4 signaling and TGF-beta isoform signaling.

Animals↗
PubMed1994

Mammalian cell cultivation in space.

In order to study the effect of microgravity on mammalian cell growth, proliferation and biosynthesis, the human T leukemia cells, human lung adenocarcinoma cells, EVI-H1 hybridoma cells and genetic engineering cells producing human growth hormone were flown on re-entry Scientific Satellite 90105 for a duration of eight days. The preliminary results indicate that the mammalian cells are sensitive to gravity. They might adapt to microgravity conditions leading to altered growth rate and subsequently, to altered biosynthesis and molecular secretion. In addition, their morphology may be affected in microgravity.

Adenocarcinoma↗
PubMed2000

[SV40 and cell immortalization].

OBJECTIVE: To explore the SV40-mediated immortalization, the related factors and their roles in cell immortalization. METHODS: The original articles about cell immortalization and replicative senescence in recent decade were reviewed. RESULTS: Cell immortalization was a multifaceted phenomenon, it was involved in viral DNA integration, activation of telomerase, inactivation of growth suppressors, and so on, and their roles were closely related. CONCLUSION: The research on cell immortalization may be expected to provide important insights into a broad range of cellular biological phenomenon, and the immortalized cells can play important roles in the research of cell engineering and tissue engineering as standard cells.

Animals↗
PubMed2000

Antitumoral vaccination with granulocyte-macrophage colony-stimulating factor or interleukin-12-expressing DHD/K12 colon adenocarcinoma cells.

Immunomodulating gene therapy for the treatment of malignant diseases is under extensive investigation. In this study, we induced an antitumoral immune response with murine interleukin-12 (mIL-12) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cells in a model of peritoneal carcinomatosis. Intraperitoneal injection of DHD/K12 tumoral cells engineered to produce IL-12 or GM-CSF did not generate any tumors, whereas untransduced DHD/K12 cells gave rise to peritoneal carcinomatosis. IL-12-expressing DHD/K12 cells also protected against tumors derived from coinjected parental cells. To test whether cytokine-producing cells could elicit a memory antitumoral immune response, animals received a challenge with parental DHD/K12 cells 35 days after the injection of proliferating or irradiated DHD/K12 engineered cells. Under our experimental conditions, irradiated tumor cells did not generate any antitumoral immunity. In contrast, tumor development was delayed and survival increased in the animals vaccinated with cytokine-secreting proliferating cells. A specific cytotoxic T-lymphocyte response against DHD/K12 parental cells was observed after vaccination with GM-CSF-expressing cells. Our results demonstrated that intraperitoneal vaccination with IL-12- or GM-CSF-expressing adenocarcinoma cells induced a systemic immune antitumoral response that may be useful as an adjuvant therapy after surgical resection of colorectal cancer.

Adenocarcinoma↗
PubMed2002

Engineering Vero cells to secrete human insulin.

Cell therapy may have the potential for the treatment of Type I diabetes. To date, cells suitable for this purpose have not been developed. This study investigates the feasibility of modifying Vero, a cell line that may be considered safe to implant into humans, for this purpose. Stable Vero transfectants containing full-length human preproinsulin complementary deoxyribonucleic acid (cDNA) were generated using a liposomal transfection reagent. Reverse transcriptase-polymerase chain reaction, immunocytochemistry, Western blotting, and enzyme-linked immunosorbent assays were used to assess the resulting cells. Proinsulin was expressed but was not processed to insulin by these cells. Proinsulin cDNA was genetically modified, resulting in a form that is furin sensitive. The resulting stably transfected Vero clones constitutively release approximately 34%/h (32.68 +/- 2.21 to 35.62 +/- 3.14%) of the product formed, approximately 62% (59.99 +/- 6.45 to 64.64 +/- 4.57%) of which is mature insulin. These Vero transfectants did not exhibit glucose-stimulated insulin secretion. As GLUT2 and glucokinase (GCK) are not constitutively expressed by these cells, human GLUT2 cDNA and GCK cDNA were cotransfected with furin-sensitive preproinsulin cDNA into Vero cells. Insulin and GCK proteins were detected in the cytoplasmic region of the resulting cells, whereas GLUT2 was predominantly expressed in the nucleus. Coexpression of GLUT2 and GCK did not result in glucose-stimulated insulin secretion. The results from this study demonstrate the feasibility of engineering a relatively "safe" nonbeta cell line to produce human insulin. Coexpression of GLUT2 and GCK, at the levels achieved here, is not adequate enough to induce glucose-stimulated insulin secretion in such cells; the subcellular location of transfected components may be relevant.

Animals↗