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At least 613 records · Page 34Linked to original sources
PubMed2003

Synthesis and evaluation of fluorescent probes for the detection of calpain activity.

Two new probes for the detection of calpain I activity based on fluorescence resonance energy transfer technology have been synthesized and evaluated. The probes incorporated the cleavage site present in alpha-spectrin, a naturally occurring substrate of calpain I. The design of the internally quenched substrates is such that the calpain-sensitive bond of the peptides (between the Tyr-Gly residues) is located centrally between the donor and the quencher chromophores. The calpain assay protocol is capable of detecting enzymatic activity in the nanomolar region.

Amino Acid Sequence↗
PubMed2005

Broadly expressed tumour-associated proteins as targets for cytotoxic T lymphocyte-based cancer immunotherapy.

T cell-based antigen-specific immunotherapy targeting self-proteins aberrantly expressed in many tumours offers the potential for widely applicable cancer immunotherapy, but carries the risk of autoimmunity. Immunological tolerance represents an inherent limitation of cancer vaccines targeting such broadly expressed tumour-associated proteins. Therefore, strategies to circumvent T cell tolerance have been developed and, when combined with T cell receptor (TCR) gene transfer technology, can generate highly avid tumour-reactive patient cytotoxic T lymphocytes (CTLs) specific for peptide epitopes of tumour-associated proteins. This review analyses the level of tolerance to broadly expressed tumour-associated proteins in the autologous T cell repertoire, assesses strategies that have been developed to circumvent T cell tolerance to such antigens, and evaluates the prospects for effective immunotherapy targeting broadly expressed tumour-associated proteins.

Adoptive Transfer↗
PubMed2004

Imaging and therapy of tumors induced to express somatostatin receptor by gene transfer using radiolabeled peptides and single chain antibody constructs.

The fields of radioimmunodetection and radioimmunotherapy began with an initial paradigm that a targeting molecule (eg, antibody) carrying a radioisotope had the potential of selectively imaging and delivering a therapeutic dose of radiation to tumor sites. A second paradigm was developed in which injection of the targeting molecule was separated from injection of a short-lived radioisotope-labeled ligand (so-called "pretargeting strategy"). This strategy has improved radioisotope delivery to tumors in animal models, enhanced radioimmune imaging in man, and therapeutic trials are in an early phase. We proposed a third paradigm to achieve radioisotopic localization at tumor sites by inducing tumor cells to synthesize a membrane expressed receptor with a high affinity for infused radiolabeled ligands. The use of gene transfer technology to induce expression of high affinity membrane receptors can enhance the specificity of radioligand localization, while the use of radioisotopes with the ability to deliver radiation damage across several cell diameters will compensate for less than perfect transduction efficiency. This approach was termed "Genetic Radioisotope Targeting Strategy." Using this strategy, induction of high levels of gastrin releasing peptide receptor or human somatostatin receptor subtype 2 expression and selective tumor uptake of radiolabeled peptides was achieved. The advantages of the genetic transduction approach are (1) constitutive expression of a tumor-associated antigen/receptor is not required; (2) tumor cells are altered to express a new target receptor or increased quantities of an existing receptor at levels that may significantly improve tumor targeting of radiolabeled ligands compared with normal tissues; (3) gene transfer can be achieved by intratumoral or regional injection of gene vectors; (4) it is feasible to target adenovirus vectors to receptors overexpressed on tumor cells by modifying adenoviral tropism (binding) so that the virus will be targeted specifically to the desired tumor; and (5) it is possible to coexpress the receptor gene and a therapeutic gene, such as cytosine deaminase, for molecular prodrug therapy to produce an enhanced therapeutic effect.

Animals↗
PubMed2001

[Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis]

Collagen Induced Arthritis (CIA) is a widely studied animal model to develop and test novel therapeutic approaches for treating Rheumatoid Arthritis (RA) in humans. Soluble Cytotoxic T-Lymphocyte Antigen 4 (CTLA4-Ig), which binds B7 molecule on antigen presenting cells and blocks CD28 mediated T-lymphocyte activation, has been shown to ameliorate experimental autoimmune diseases such as lupus, diabetes and CIA. Objective of our research was to investigate in vivo the effectiveness of blocking the B7/CD28 T-lymphocyte co-stimulatory pathway, utilizing a gene transfer technology, as a therapeutic strategy against CIA. Replication-deficient adenoviruses encoding a chimeric CTLA4-Ig fusion protein, or beta-galactosidase as control, have been injected intravenously once at arthritis onset. Disease activity has been monitored by the assessment of clinical score, paw thickness and type II collagen (CII) specific cellular and humoral immune responses for 21 days. The adenovirally delivered CTLA4-Ig fusion protein at a dose of 2x10^8 pfu suppressed established CIA, whereas the control beta-galactosidase did not significantly affect the disease course. CII-specific lymphocyte proliferation, IFNgamma production and anti-CII antibodies were significantly reduced by CTLA4-Ig treatment. Our results demonstrate that blockade of the B7/CD28 co-stimulatory pathway by adenovirus-mediated CTLA4-Ig gene transfer is effective in treating established CIA suggesting its potential in treating RA.

Journal Article↗
PubMed2004

Identification of ciprofloxacin-resistant Campylobacter jejuni and analysis of the gyrA gene by the LightCycler mutation assay.

A real-time PCR assay was developed to identify ciprofloxacin-resistant Campylobacter jejuni. Ciprofloxacin resistance in C. jejuni has been associated with a C-->T nucleotide point mutation occurring at the 86 codon of the gyrA gene. Other nucleotide substitutions have been identified in proximity to or at the same codon in the gyrA gene, but their role in ciprofloxacin resistance is still unknown. The LightCycler assay is based on the fluorescence resonance energy transfer technology using melting peak analysis of two fluorescent probes hybridized on PCR amplicons. This assay was used to detect the 86-codon mutation conferring ciprofloxacin resistance, as well as other nucleotides substitutions occurring within the same site in the gyrA gene. This gyrA mutation assay allows a rapid and reproducible screening method of ciprofloxacin resistant strains and was applied to C. jejuni strains isolated in Italy in 2000.

Base Sequence↗
PubMed2005

Gene therapy for the prevention of ischemia/reperfusion injury in organ transplantation.

Introduction of gene therapy into molecular medicine has been gaining increasing interest. Although treatment of various diseases e.g. monogenetic defects or cancer by using gene transfer technologies has been extensively probed, the clinical success has been limited. However, recent experimental data suggest that gene therapy may represent an attractive and powerful approach in preventing ischemia/reperfusion injury as well as organ rejection in transplant recipients. Easy and selective access to the donor organ facilitates the reduction of potentially harmful systemic side effects of gene therapy vectors. By introducing anti-apoptotic or cytoprotective genes, these studies focused on the protection of the transplant from the apoptotic cell death. In addition, down-regulation of adhesion molecules and/or blockade of gene expression in the graft itself also ameliorated ischemia/reperfusion injury. This review summarizes the current progress on gene therapy application in combating ischemia-reperfusion injury in organ transplantation. Although the use of viral vectors is emphasized, non-viral gene transfer techniques are also discussed. Future development of novel, low-immunogenic vectors should further contribute to the minimization of ischemia/reperfusion injury, and thus to the overall success of organ transplantation.

Adenoviridae↗
PubMed2001

[Hope for gene therapy. Is this the future of tumor treatment?].

Conventional antineoplastic chemotherapy is limited by the low binding specificity of cytostatic agents and the ubiquity of critical target molecules in both neoplastic and normal cells. Recent advances in molecular biology have led to the identification of ever more genetic alterations specific to tumor cells that may serve as targets for gene therapy. Since appropriately designed nucleic acids hybridize to their "target nucleic acid" with extremely high binding specificity, and can be prepared with relative ease, gene therapy is theoretically superior to conventional chemotherapy. However, the low efficacy of currently available gene transfer technologies, the redundancy of genetic control mechanisms, and, for example under antitumor treatment, the multitude of genetic alterations accumulating during tumor progression represent significant problems. With the exception of monogenic disease, therefore, most clinical studies have so far demonstrated the feasibility of gene therapy, rather than its therapeutic efficacy. Ethical considerations rule out germline therapy. In comparison with conventional treatment, the assessment of the risks of this form of therapy should include a consideration of irreversible and reversible damage.

Animals↗
PubMed1998

Strategies for activating nuclear transfer oocytes.

The technique of nuclear transfer can have enormous applications in the fields of agriculture and biomedicine. This is especially true if a cell line that has been transformed can be used as a source of nuclei for the nuclear transfer. One major aspect of the nuclear transfer procedures is that of oocyte activation. Without oocyte activation the transferred nucleus would never progress to the first interphase. It is therefore of utmost importance that the oocyte be activated in a fashion that is as normal as fertilization. The inability to obtain development after artificial activation of pig oocytes has been a limiting factor in the application of the nuclear transfer technology. Recently, a number of techniques have been developed that result in blastocyst stage embryos after oocyte maturation in vitro and artificial activation. The theories behind normal oocyte activation are reviewed as well as a number of methods of artificial oocyte activation. It is anticipated that such a review will provide the basis for the development of additional methods that are as efficient, or more efficient, at activating the unfertilized oocyte.

Animals↗
PubMed1994

Global information infrastructure.

The High Performance Computing and Communications Program (HPCC) is a multiagency federal initiative under the leadership of the White House Office of Science and Technology Policy, established by the High Performance Computing Act of 1991. It has been assigned a critical role in supporting the international collaboration essential to science and to health care. Goals of the HPCC are to extend USA leadership in high performance computing and networking technologies; to improve technology transfer for economic competitiveness, education, and national security; and to provide a key part of the foundation for the National Information Infrastructure. The first component of the National Institutes of Health to participate in the HPCC, the National Library of Medicine (NLM), recently issued a solicitation for proposals to address a range of issues, from privacy to 'testbed' networks, 'virtual reality,' and more. These efforts will build upon the NLM's extensive outreach program and other initiatives, including the Unified Medical Language System (UMLS), MEDLARS, and Grateful Med. New Internet search tools are emerging, such as Gopher and 'Knowbots'. Medicine will succeed in developing future intelligent agents to assist in utilizing computer networks. Our ability to serve patients is so often restricted by lack of information and knowledge at the time and place of medical decision-making. The new technologies, properly employed, will also greatly enhance our ability to serve the patient.

Computer Communication Networks↗
PubMed2001

Progress toward vector design for hematopoietic stem cell gene therapy.

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy because of their capacity for self renewal and the wide systemic distribution of their progeny. Sustained expression of transgenes at clinically relevant levels in the progeny of HSCs would provide novel and potentially curative treatments for a wide range of inherited and acquired blood diseases. Recent improvements in retroviral transduction protocols have resulted in the first successful amelioration of a human hematologic disease--a form of severe combined immunodeficiency--by HSC gene transfer. However, continued advances in gene transfer technology are necessary if the inherent promise of HSC gene therapy is to be fully realized. Ongoing efforts are focused on modifying oncoretroviral vector designs and pseudotyping with alternative envelope proteins. In addition, because of their ability to transduce non-divided cells, safety-modified human immunodeficiency virus-1-based lentiviral vectors have emerged as promising tools for gene modification of HSCs, which reside primarily in the G0/G1 phase of the cell cycle. Irrespective of these advances, accumulated data indicate that stably integrated transgenes are frequently subject to position-effect variegation and extinction of expression. Therefore, the extent to which genetic control elements such as chromatin domain insulators and scaffold/matrix attachment regions in conjunction with posttranscriptional regulatory elements will result in enhanced probability and level of transgene expression is under active investigation. Collectively, these developments increase the likelihood that HSC gene transfer will ultimately become an effective therapeutic strategy.

Binding Sites↗
PubMed2003

Solid dispersions: revival with greater possibilities and applications in oral drug delivery.

Improvement of oral bioavailability of poorly water-soluble drugs remains one of the most challenging aspects of drug development. Solid dispersions seem to be a viable technique for overcoming this problem. However, the practical applicability of these systems has remained limited because of difficulties in conventional methods of preparation, poor reproducibility of physicochemical properties, difficulties in dosage form development, and lack of feasibility for scale-up of manufacturing processes. This review addresses various aspects of solid dispersions and compiles some of the recent technology transfers from various fields such as the chemical, food, and polymer industries for the preparation of solid dispersions that can lead to highly efficient and controlled large-scale manufacturing. Some of the practical aspects to be considered for the preparation of solid dispersions, such as selection of carrier and methods of physicochemical characterization, along with an insight into the release mechanism of drugs are also discussed. Finally, an in-depth rationale for limited commercialization of solid dispersions and recent revival has been considered.

Administration, Oral↗
PubMed1993

Major approaches for generating and analyzing transgenic mice. An overview.

Over the past decade, the development of gene-transfer technology in whole animals has afforded unprecedented opportunities for investigators to probe complex regulatory systems in vivo. Important advances in our understanding of the mechanisms of gene expression and regulation and the development of animal models of human diseases are but two examples of how this technology has affected medical science. Transgenic animals are defined as animals in which a segment of DNA has been physically integrated into the genome of all cells, including the germ line, so that it can be transmitted to offspring as a simple Mendelian trait. The DNA segment generally consists of a whole cloned gene, cDNA, or a novel gene modified by recombinant DNA methodologies. Whole genomic clones of genes are often used to study tissue- and cell-specific expression and regulation or can be used to overexpress a gene product. Alternatively, the coding region of one gene can be fused to the transcriptional regulatory region of another gene, causing it to be expressed in a new spectrum of tissues and cell types. A number of methods can be used to introduce the DNA segment, including direct microinjection of one-cell fertilized embryos, retroviral-mediated transfer, or gene transfer in embryonic stem cells. The technique most often used to generate transgenic animals and perform "gene addition" experiments is direct microinjection. Alternatively, gene deletions or "knockouts" are performed by gene transfer in embryonic stem cells by specifically targeting the site of integration in the genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Utilizing endocrine secretory pathways in salivary glands for systemic gene therapeutics.

Mammalian salivary glands are commonly used models of exocrine secretion. However, there is substantial experimental evidence showing the physiological existence of endocrine secretory pathways in these tissues. The use of gene transfer technology in vivo has allowed the unambiguous demonstration of these endocrine pathways. We and others have exploited such findings and evaluated salivary glands as possible target tissues for systemic applications of gene therapeutics. Salivary glands present numerous advantages for this purpose, including being well encapsulated, which limits extra-glandular vector dissemination, and having the luminal membranes of almost all parenchymal cells accessible via intraoral delivery of vectors through the main excretory ducts. Existing studies suggest that clinical benefits will result from salivary gland targeted systemic gene therapeutics.

Animals↗
PubMed2004

[Medical transportation of Congolese children by the Foundation "Terre des hommes" Netherlands (1989--1998)].

We report the 10 year assessment of collaboration with the Foundation "Terre des hommes" concerning the medical transfer in the Netherlands of 41 children carrying pathologies which couldn't be treated or operated on in Brazzaville. The average age was 3 years and 6 months old (extremes: 2 months - 15 years). 33 non-cyanotic cardiopathies dominated by ventricular septal defect (VSD) (n = 10) and 11 cyanotic cardiopathies among them the tetralogy of Fallot (n = 5) were admitted. The other pathologies were respectively: osseous (n = 3), vesical (n = 2), pulmonary tumoral, ophthalmic in 1 case. The surgery consisted in a complete repair in 19 cases, palliative in 9 cases. Two children proved to be inoperable. Eight other extra-cardiac pathologies had a specific surgery for each case. The average stay in the Netherlands was 1 month 13 days (extremes: 1 - 12 months). The evolution was favourable for 35 children all pathologies included. Four deaths occurred in the Netherlands and 2 in Congo. The organization of the technology transfer would be probably a better choice in the future.

Adolescent↗
PubMed2004

Safety concerns related to hematopoietic stem cell gene transfer using retroviral vectors.

Endogenous retroviruses have developed efficient methods during their life cycle for stable integration into the host genome. Because of this ability, retroviral vectors were designed with the goal of gene transfer into hematopoietic stem cells (HSCs). The ability to genetically modify HSCs provides a vehicle for durable expression of potentially therapeutic transgenes in all lineages of mature blood cells for the lifetime of the patient. Combined with bone marrow transplant, retroviral gene transfer has many potential applications for a wide range of blood diseases. Advances in the development of oncoretroviral vectors based on murine leukemia viruses (MLV) and more recent development of human immunodeficiency virus (HIV)-based vectors have greatly increased the gene transfer efficiency. Optimization of methods for gene transfer using MLV-based vectors has substantially improved marking levels in mice, with lower levels in large animals and in human clinical trials. With advances in gene transfer technology has also come renewed concern about insertional mutagenesis and activation of oncogenes. Advanced techniques for integration site analysis combined with sequence comparison using mouse and human genome databases has now made it possible to begin to understand the spectrum of possible integration sites for both MLV- and HIV-based vectors. Furthermore, other studies have shown positive and negative dosage-dependent effects of transgene expression in mouse and human cells. Therefore, vector design and safety testing are at the forefront of the field of gene therapy and this review discusses recent developments.

Animals↗
PubMed1997

Direct gene transfer into the colon using a double-balloon catheter.

BACKGROUND AND STUDY AIMS: In spite of the many advances that have been made in understanding the molecular basis for diseases, a major obstacle to the treatment of human disorders remains the inability to express genes at specific sites in vivo. Recent progress in gene transfer technology has provided access to a variety of recombinant gene products that can be applied in clinical medicine for therapeutic purposes. MATERIALS AND METHODS: In an animal model, we describe here the way in which a marker gene can be introduced into the colon using a double-balloon catheter. Cationic liposomes were used as vehicles to introduce DNA into the living organism. RSV-LacZ plasmid coding for the enzyme beta-galactosidase was used as a marker gene. Cells expressing beta-galactosidase can be stained using the chromogen X-gal. Positive cells show a blue coloration in the cytoplasm. RESULTS: Both absorptive cells and goblet cells were successfully transduced with the marker gene. No evidence of similar staining was observed in control animals receiving a control plasmid or liposomes alone. CONCLUSIONS: The method used is a simple, safe, and nontoxic way of delivering genes of interest to specific sites in the colon. Gene transfer may offer fresh potential for endoscopic interventions in colonic disease.

Animals↗
PubMed2002

Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.

One of the goals of developmental neuroscience in the post-genomic era is to clarify functions of a huge number of anonymous genes of which only DNA sequences are identified. More convenient methods for genetic manipulation in vertebrates, especially mammals, could help us to identify functions of the novel genes. Here we introduce a novel gene transfer technology using electroporation (EP), which is a simple and powerful strategy for genetic analysis. We have applied this method to cultured mammalian embryos in order to understand the function of specific genes in the developing brain. We have also performed EP in developing fetuses in utero guided by ultrasound image. The combination of these techniques in addition to analysis of genetic mutants will clarify functions of individual genes, gene interactions, and the molecular mechanisms underlying the brain development.

Animals↗
PubMed2003

Gene therapy for chronic granulomatous disease.

Identification of gene mutations responsible for leukocyte dysfunction along with the application of gene transfer technology has made genetic correction of such disorders possible. Much of the research into molecular therapy for inherited disorders of phagocytes has been focused on chronic granulomatous disease (CGD). CGD results from mutations in any one of the four genes encoding essential subunits of respiratory burst NADPH oxidase, the enzyme complex required for the production of reactive oxygen intermediates in phagocytes. The absence of phagocyte oxidants results in a predisposition to recurrent bacterial and fungal infections and inflammatory granulomas in CGD patients, associated with significant morbidity and mortality. Allogeneic bone marrow transplantation can cure CGD, but transplant-related toxicity and the limited availability of matched donors have restricted its wider application. Because the gene defects causing CGD are known, and CGD is a stem cell disorder treatable by marrow transplantation, CGD has emerged as a promising disease for somatic gene therapy targeted at the hematopoietic system. Multiple reports have demonstrated the reconstitution of NADPH oxidase activity by gene transfer to human CGD marrow and cell lines cultured in vitro. CGD mouse models have been developed by gene disruption, and preclinical studies on these animals using recombinant retroviral vectors have demonstrated reconstitution of functionally normal neutrophils and increased resistance to pathogens such as Aspergillus fumigatus, Burkholderia cepacia and Staphylococcus aureus. Although the results of these murine studies are encouraging, human phase-I clinical studies in CGD patients have yet to produce clinically beneficial numbers of corrected neutrophils for extended periods. Efforts to improve gene transfer efficiency into human hematopoietic stem cells and to increase engraftment of transduced stem cells are ongoing.

Animals↗