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At least 145 records · Page 8Linked to original sources
PubMed1997

Engineered cell lines for insulin replacement in diabetes: current status and future prospects.

The recently completed diabetes complications and control trial has highlighted the need for improvement of insulin delivery systems for treatment of insulin-dependent diabetes mellitus. Despite steady improvement in methods for islet and whole pancreas transplantation over the past three decades, the broad-scale applicability of these approaches remains uncertain due in part to the difficulty and expense associated with procurement of functional tissue. To address this concern, we and others have been using the tools of molecular biology to develop cell lines with regulated insulin secretion that might serve as a surrogate for primary islets or pancreas tissue in transplantation therapy. This article seeks to provide a brief summary of the current status of this growing field, with a particular emphasis on progress in producing cell lines with appropriate glucose-stimulated insulin secretion.

Cell Line↗
PubMed2005

Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development.

Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.

Adipocytes↗
PubMed2004

Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas.

PURPOSE: To evaluate the feasibility and safety of intratumoral injection of autologous dendritic cells (DCs) transfected with an adenovirus encoding interleukin-12 genes (AFIL-12) for patients with metastatic gastrointestinal carcinomas. Secondarily, we have evaluated biologic effects and antitumoral activity. PATIENTS AND METHODS: Seventeen patients with metastatic pancreatic (n = 3), colorectal (n = 5), or primary liver (n = 9) malignancies entered the study. DCs were generated from CD14+ monocytes from leukapheresis, cultured and transfected with AFIL-12 before administration. Doses from 10 x 10(6) to 50 x 10(6) cells were escalated in three cohorts of patients. Patients received up to three doses at 21-day intervals. RESULTS: Fifteen (88%) and 11 of 17 (65%) patients were assessable for toxicity and response, respectively. Intratumoral DC injections were mainly guided by ultrasound. Treatment was well tolerated. The most common side effects were lymphopenia, fever, and malaise. Interferon gamma and interleukin-6 serum concentrations were increased in 15 patients after each treatment, as well as peripheral blood natural killer activity in five patients. DC transfected with AFIL-12 stimulated a potent antibody response against adenoviral capsides. DC treatment induced a marked increase of infiltrating CD8+ T lymphocytes in three of 11 tumor biopsies analyzed. A partial response was observed in one patient with pancreatic carcinoma. Stable disease was observed in two patients and progression in eight patients, with two of the cases fast-progressing during treatment. CONCLUSION: Intratumoral injection of DC transfected with an adenovirus encoding interleukin-12 to patients with metastatic gastrointestinal malignancies is feasible and well tolerated. Further studies are necessary to define and increase clinical efficacy.

Adenoviridae↗
PubMed2004

Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

Erythrocytes↗
PubMed1989

A genetically engineered cell line that produces empty capsids of B19 (human) parvovirus.

B19 parvovirus is pathogenic in humans, causing the common childhood exanthem fifth disease and bone-marrow failure, both acute (transient aplastic crisis of hemolysis) and chronic (pure erythrocyte aplasia in immunodeficiency). The virus is tropic for a human red cell progenitor cell, and failure to culture B19 in a cell line has limited its clinical study. We cotransfected the right half of the cloned B19 genome and a minigene derived from the human dihydrofolate reductase gene (DHFR) into dhfr--Chinese hamster ovary cells and screened selected clones by RNA analysis; after amplification in methotrexate, clones were tested for capsid protein expression. A cell line, designated 3-11-5, stably expressed nearly full-length transcripts for the two capsid proteins. These cells produced the major and minor structural protein species in natural proportions that self-assembled into virion capsids. Capsids from 3-11-5 cells could be separated from virions by sucrose gradient sedimentation and had the density on cesium chloride isopycnic sedimentation of empty parvovirus capsids. Capsid protein was present in both nuclei and cytoplasm on immunofluorescence study but fractionated with the cytosol on purification. Empty capsid production was equal to or greater than virion production by infected bone-marrow cells, 1000-2000 capsids per cell, but cell growth was not diminished by capsid production. This cell line will be useful in developing practical assays for B19 parvovirus antibody and a vaccine for the virus, as well as potentially serving as a packaging cell line for gene therapy.

Animals↗
PubMed1999

Anti-tumor immunity generated by tumor cells engineered to express B7-1 via retroviral or adenoviral gene transfer.

We engineered B7-1 retroviral and adenoviral gene transfer systems and studied them in four immunogenic tumor models. M-MSV tumor cells, but not K-Balb, 38.2 and 205 tumor cells, when expressing B7-1 by retroviral transduction were rejected and conferred protection against a tumor challenge. Transient expression of B7-1 after transduction with adenoviruses was less efficient. We observed enhanced cytotoxic T-lymphocyte activity accompanied by increased secretion of IL-6, IFNgamma and GM-CSF. GM-CSF secretion correlated with tumor rejection. Enhanced IFNgamma but unchanged IL-4 secretion suggested a T-helper 1-mediated anti-tumor immune response.

3T3 Cells↗
PubMedDate not supplied

[Biophysical aspects of reconstruction of a single cell by the methods of cell engineering].

The problems of a low efficiency of mammalian cloning are discussed with emphasis on the necessity of the expertise of each step of single cell reconstruction, beginning with microsurgical manipulations. The fact of cell content leakage when the cell is held during microsurgery or microinjections with the help of the conventional method using negative pressure in the holding micropipette was demonstrated in experiments on murine embryos. It was shown that the rate of cell content efflux depends on the value of negative pressure generated in the holding micropipette, and is directly proportional to the dimensions of its orifice and the duration of micromanipulations. An alternative method of cell fixation using the capillary forces of the holding micropipette was proposed. The method optimizes the process of cell fixation, reducing the holding effort by two orders of magnitude. As a result, 92% of embryos remain viable after fixation of embryo, as compared with 39% in the conventional technique. In order to diminish the cell damage produced by the tip of a microinstrument, a new technique of fabricating micropipettes was proposed. The improved method of filling the micropipette with viscous liquids, including DNA, which is described in details in the paper, enabled constant (non-stop) microinjection of more than 1000 cells by hand, without any special automatic device.

Animals↗
PubMed2005

Sequential immunogene therapy with interleukin-12- and interleukin-15-engineered neuroblastoma cells cures metastatic disease in syngeneic mice.

PURPOSE: To investigate the potential synergistic effects of Neuro2a neuroblastoma cells engineered with IL-12 and/or IL-15 genes in improving survival of syngeneic mice bearing neuroblastoma metastatic disease. EXPERIMENTAL DESIGN: Neuro2a cells engineered with interleukin (IL)-12 (Neuro2a/IL-12), IL-15 (Neuro2a/IL-15), or both cytokines (Neuro2a/IL-12/IL-15) were injected s.c. in syngeneic A/J mice challenged i.v. with Neuro2a parental cells (Neuro2apc) using different schedules of administration in either preventive or therapeutic settings. RESULTS: A single injection of Neuro2a/IL-12 or Neuro2a/IL-15 cells induced resistance to a subsequent i.v. Neuro2apc challenge in 45% and 28% of mice, respectively. Neuro2a/IL-12/IL-15 cells protected 28% of mice, showing no synergistic effect. However, sequential vaccination with Neuro2a/IL-12 (day -30) followed by Neuro2a/IL-15 (day -15) protected 71% of mice from subsequent challenge with Neuro2apc. A single dose of Neuro2a/IL-12 prolonged the mean survival time of mice bearing established metastatic neuroblastoma from 21 +/- 3 to 46 +/- 27 days but failed to cure mice, whereas Neuro2a/IL-15 or Neuro2a/IL-12/IL-15 were ineffective. However, sequential vaccination with Neuro2a/IL-12 (day +3) followed by Neuro2a/IL-15 (day +13) cured 43% of mice as assessed by histologic analysis of different organs from long-term surviving mice. CTL activity against Neuro2apc cells was observed in splenocytes from treated mice, and CD8(+) T-cell depletion abrogated the therapeutic effect of vaccination. CONCLUSIONS: Sequential vaccination with IL-12- and IL-15-engineered neuroblastoma cells induced optimal preventive and therapeutic effects, which may be related to the Th1 priming effect of IL-12 followed by the enhancement of CD8(+) T-cell responses and their maintenance mediated by IL-15.

Animals↗
PubMed1999

Tumour cells engineered to secrete interleukin-15 augment anti-tumour immune responses in vivo.

We examined the effect of interleukin-15 (IL-15) gene transfer into tumour cells on the host's anti-tumour response. In BALB/c mice IL-15 producing Meth-A cells (Meth-A/IL-15) underwent complete rejection, in a response characterized by massive infiltration of CD4+ T-cells and neutrophils. In contrast, Meth-A cells transfected with vector alone (Meth-A/Neo) grew rapidly. Moreover, rechallenged parental cells also were rejected in association with CD8* T-cell infiltration. However, in nude mice there was no drastic difference between Meth-A/IL-15 and Meth-A/Neo cells. These results demonstrate that IL-15-secreting tumour cells can stimulate local and systemic T-cell-dependent immunity and therefore may have a potential role in cancer therapy.

Animals↗
PubMed2007

Thermally associating polypeptides designed for drug delivery produced by genetically engineered cells.

Thermally associating polymers, including gelatin, cellulose ethers (e.g., Methocels and poloxamers (e.g., Pluronics) have a long history of use in pharmacy. Over the past 20 years, significant advances in genetic engineering and the understanding of protein secondary and tertiary structures have been made. This has led to the development of a variety of polypeptides that do not occur naturally but can be expressed in recombinant cells and have useful properties that lend themselves to novel applications where current materials cannot perform. The most intensively studied motifs are derived from the consensus repeats of elastin and silk, as well as coiled-coil helices. Many of these designed polypeptides or 'artificial proteins' are thermally associating materials. This property can be exploited to develop solid dosage forms, injectable drug delivery systems, micro- or nanoparticle drug carriers, triggered or targeted release systems, or as a means of simplifying the purification process and thus reducing costs of production of these materials. This review focuses on the development and characterization of this novel class of biomaterials and examines their potential for pharmaceutical applications.

Amino Acid Sequence↗
PubMedDate not supplied

Strategies for cell engineering in tissue repair.

Cellular and tissue engineering are new areas of research, currently attracting considerable interest because of the remarkable potential they have for clinical application. Some claims have indeed been dramatic, including the possibility of growing complete, artificial organs, such as the liver. However, amid such long-term aspirations there is the very real possibility that small tissues (artificial grafts) may be fabricated in the near future for use in reconstructive surgery. Logically, we should focus on how it is possible to produce modest, engineered tissues for tissue repair. It is evident that strategies to date either depend on innate information within implanted cells, to reform the target tissue or aim to provide appropriate environmental cues or guidance to direct cell behavior. It is argued here that present knowledge of tissue repair biology points us toward the latter approach, providing external cues which will direct how cells should organize the new tissue. This will be particularly true where we need to reproduce microscopic and ultrastructural features of the original tissue architecture. A number of such cues have been identified, and methods are already available, including substrate chemistry, substrate contact guidance, mechanical loading, and biochemical mediators to provide these cues. Examples of these are already being used with some success to control the formation of tissue structures.

Journal Article↗
PubMed1999

Encapsulation in hollow fibres of xenogeneic cells engineered to secrete IL-4 or IL-13 ameliorates murine collagen-induced arthritis (CIA).

A strategy of gene therapy using IL-4 or IL-13 xenogeneic transfected cells encapsulated into permeable hollow fibres (HF) was used to treat CIA. Hydrogel-based hollow fibres were obtained from AN-69 copolymer, already known for its biocompatibility and tolerance in rodents. Permeability to IL-4 and lack of cell leakage from the fibres were ascertained in vitro and in vivo. Chinese hamster ovary (CHO) fibroblasts transfected with mouse IL-4 gene were encapsulated in HF (6.25 x 105 cells/HF). IL-4 was detected in vitro in the culture supernatant of filled fibres for at least 19 days. IL-4 or IL-13 transfected CHO cells encapsulated in HF were implanted in the peritoneum of mice on days 11-13 after immunization with type II collagen. Control mice were treated with fibre containing CHO cells transfected with beta-galactosidase (betagal) gene; a positive control group consisted of mice treated by subcutaneous injection of 106 cells on days 10 and 25. Mice were monitored for signs of arthritis by observers unaware of the status of animals. Results of these experiments indicate that severity of the articular disease was significantly reduced in the groups of mice treated with CHO/IL-4 or CHO/IL-13 cells encapsulated in HF, compared with control groups receiving CHO/betagal cells encapsulated in HF. Histological analysis confirmed these data and extended them to a better inhibitory effect of encapsulated cells compared with free cells on inflammatory and destructive joint disease. Moreover, such long-term treatment with HF was well tolerated; macroscopic and histological aspects of peritoneal cavity were moderately inflammatory. Thus, our results may have important implications for clinical use of gene transfected cells as therapeutic agents in the treatment of autoimmune diseases.

Acrylic Resins↗
PubMedDate not supplied

Eradication of melanoma pulmonary metastases by immunotherapy with tumor cells engineered to secrete interleukin-2 or gamma interferon.

This study was undertaken to investigate the effectiveness of interleukin-2 (IL-2) and gamma interferon (gammaIFN)-modified B16 melanoma cells in the immunotherapy of established melanoma pulmonary metastases. The genes for IL-2 and gammaIFN were introduced retrovirally into B16 melanoma cells. Transduction with the gammaIFN, but not the IL-2, gene caused significant increases in the expression of major histocompatibility complex (MHC) antigens on B16-gammaIFN cells. The in vivo tumor-forming capacity of both IL-2- and gammaIFN-transduced B16 cells was drastically reduced when the cells were inoculated subcutaneously (SC) in syngeneic C57BL/6 mice. After intravenous (IV) inoculation, most of the B16-gammaIFN cells were rejected, but B16-IL-2 cells were relatively tumorigenic and formed pulmonary metastases. C57BL/6 mice bearing 4-day established parental B16 lung metastases were treated with B16 parental (B16P) unmodified cells, IL-2- or gammaIFN-modified B16 cells, or a combination of both transduced cells. Treatment consisted of a weekly intraperitoneal (IP) injection of one million irradiated (10,000 rad) tumor cells alone or in combination with exogenous IL-2 for a total of three to four injections. Immunotherapy with B16 parental or B16-IL-2 secreting cells caused a moderate reduction in the number of lung metastases. However, mice treated with gammaIFN-secreting B16 cells showed a significant reduction or complete elimination of lung metastases. There was no additive effect for combining both IL-2- and gammaIFN-modified tumor cells in the immunotherapy. Exogenous IL-2 (50,000-100,000 U/day for 3 days) caused a significant enhancement of the immunotherapeutic benefit of the vaccines. Moreover, mice treated with gammaIFN-modified B16 cells survived longer than the other groups. Twenty-five percent of these mice were tumor free and remained alive for an observation period of 4 months. The in vitro cytolytic activity of splenocytes in chromium release assays did not correlate in every case with the in vivo antitumor effect of the treatment. Our findings have implications for the use of cytokine-modified cells for immunotherapy and for evaluating the therapeutic benefit of this novel treatment.

Animals↗
PubMed1998

5-Fluorocytosine-mediated apoptosis and DNA damage in glioma cells engineered to express cytosine deaminase and their enhancement with interferon.

To explore the antitumor mechanism of bacterial cytosine deaminase plus 5-fluorocytosine (CD/5-FCyt) in combination with interferons (IFNs), glioma cells were transduced with recombinant retroviruses expressing CD. The transduced glioma cells become sensitive to the nontoxic prodrug 5-FCyt. Apoptosis, DNA damage, bystander effect, and inhibition of thymidylate synthase (TS) and DNA synthesis are associated with CD/5-FCyt-mediated glioma cell killing. Furthermore, IFNs enhance this effect by increasing DNA damage and further inhibiting TS activity. The bystander effect is mediated by the release of cytotoxic metabolites of 5-FCyt into the extracellular milieu triggering apoptosis and DNA damage. Our data indicate that the use of CD/5-FCyt in combination with IFNs may provide a more effective approach for the treatment of brain tumors.

3T3 Cells↗
PubMed2000

Spleen-derived dendritic cells engineered to enhance interleukin-12 production elicit therapeutic antitumor immune responses.

The major goal in cancer immunotherapy is the induction of tumor-specific T lymphocytes capable of killing tumor cells. As both dendritic cells (DCs) and interleukin-12 (IL-12) can play immunostimulatory roles in vivo, the use of a combination of these has become a promising approach. In the present study, we used a murine tumor model to examine whether spleen-derived DCs transduced with the IL-12 gene could elicit tumor-specific immune responses. BALB/c mice injected peritumorally with adenovirus-mediated IL-12 gene-transduced antigen-unpulsed DCs inhibited the growth of day 5-established subcutaneous CT26 tumors. Splenocytes from treated mice responded specifically to parental tumor cells and showed increased production of interferon gamma (IFN-gamma) and antitumor cytotoxic T-lymphocyte (CTL) activity. Increased numbers of both CD4(+) and CD8(+) T cells were detected in the treated tumors. The inhibition of tumor growth was significantly greater in mice injected with IL-12 gene-transduced DCs than in those injected with IL-12 gene-transduced fibroblasts or the IL-12 gene-encoding adenovirus itself. Taken together, these results indicate that DCs transduced with the IL-12 gene by a recombinant adenovirus are effective in inducing tumor-specific Th1 and CTL responses that inhibit the growth of established subcutaneous tumors.

Adenocarcinoma↗