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PubMed2001

An evidence-based vector control strategy for military deployments: the British Army experience.

We describe the British Army's current strategy for controlling arthropod vectors of disease during overseas deployments. Military commanders and medical officers have different, but complementary responsibilities in achieving vector control. In this paper we define a hierarchy of evidence-based vector control guidelines. Field guidelines must be based on the best available research evidence, preferably that derived from pragmatic randomised controlled trials (RCTs), and from systematic reviews of trials. Assessing the effectiveness of different vector control measures involves a trade-off between the relative benefits and harm of different technology options. There is compelling scientific evidence that bed nets and screens treated with a pyrethroid insecticide are highly effective in protecting against nocturnally active, anthropophilic arthropods (including ectoparasites), and will reduce the incidence of malaria, leishmaniasis, lymphatic filariasis and Chagas' disease. Etofenprox and deltamethrin are the safest pyrethroids, and permethrin the least safe. Vector control strategies of probable effectiveness are the use of insecticide-treated clothing, the wearing of protective clothing, and the correct use of DEET-based topical insect repellents. Aerosol insecticides are of debatable effectiveness. Other effective vector control measures, of limited usefulness during deployments, include electric fans, mosquito coils/vaporising mats, and smoke. "Biological" vector control measures, and insect buzzers/electrocuters are ineffective. Practical insect avoidance measures, based on an understanding of vector biology, complete the military vector-control arsenal. We conclude that practical insect avoidance measures, combined with pyrethroid-treated nets and clothing, and DEET-based topical repellents, can achieve almost 100% protection against biting arthropods.

Animals↗
PubMed2001

[Adenovirus vectors and their clinical application in gene therapy].

The potential therapeutic application of the gene transfer technology with adenovirus vectors seems to be enormous. Adenovirus vectors offer several advantages over other vectors, but several important limitations of adenovirus mediated gene transfer are also known. Great number of studies in inherited diseases and in different cancer therapy clinical trials have provided information of critical importance for design of efficient clinical protocols. Clinical trials have been extended to the treatment of many other diseases, too. There are about thirty currently active gene therapy protocols for the treatment only of HIV-1 infection in the USA. These programs aim to confer protective immunity against HIV-1 transmission to individuals who are in risk of infection, to develop preventive or therapeutic vaccines for patients with AIDS and other infectious diseases. Gene therapy represents one of the most important developments in oncology, however, before this can be realised as standard treatment the technical problems of gene delivery and higher safety must be overcome. The early--first and second generation--adenovirus vectors are now likely to be phased out for most diseases, and further experiments seem to be necessary. It might be change to the third generation or other, more modern vector application in clinical trials, as the helper dependent vectors. Almost all transcriptional unit is removed from the DNA of these vectors ("gutless vectors"), therefore they cannot reproduce, give higher gene expression and far less inflammatory. Despite the latest achievement reported in vector design it is not possible to predict yet to what extent and when gene therapy will be effective.

Adenoviridae↗
PubMedDate not supplied

Encapsulated cells producing retroviral vectors for in vivo gene transfer.

BACKGROUND: Because gene therapy of the future will primarily take an in vivo approach, a number of problems associated with its current implementation exist. Currently, repeated delivery of a vector in vivo is necessary to ensure adequate transfer of the therapeutic gene. This may lead to the development of an immune response against the vector, thus interfering with gene delivery. To circumvent this problem, retroviral vector packaging cells that permanently produce recombinant retroviral vector particles have been encapsulated. METHODS: Vector (pBAG)-producing amphotropic cells were encapsulated in beads composed of polymerized cellulose sulphate. These capsules were analysed in vitro for expression of the vector construct using X-gal staining, as well as for the release of particles by performing RT-PCR from culture supernatant. Infectivity studies were performed in vitro and in vivo. The latter was assayed using histological sections of the microcapsule and the surrounding area stained for beta-galactosidase activity and by RT-PCR. RESULTS: In culture, the virus-producing cells inside the capsules remained viable and released virus into the culture medium for at least 6 weeks. To test whether these capsules, upon implantation into mice, also release vector virions that infect the surrounding cells, two different models were used. In the first, capsules were implanted in the fat pad of the mammary gland of Balb/c mice. The capsules were well tolerated for at least 6 weeks and a self-limiting inflammatory reaction without any other gross immune response was observed during this period. Furthermore, the virus-producing cells remained viable. In the second model, SCID mice were immunologically reconstituted by subcutaneous implantation of thymus lobes from MHC-identical Balb/c newborn mice and gene transfer into lymphoid cells was achieved by retroviral vectors released by co-implanted capsules. CONCLUSION: The implantation of such capsules containing cells that continually produce retroviral vector particles may be of use for in vivo gene therapy strategies. The data presented demonstrate the feasibility of the concept.

3T3 Cells↗
PubMed2002

[Development of gene-viral vector and its anti-tumor effect, a primary study].

OBJECTIVE: To develop a new kind of vector system, named as gene-viral vector, which combines the advantages of the gene therapy and virus therapy. METHOD: An anti-tumor gene was inserted into the genome of the replicative virus specific for the tumor cells by virus recombination technology. The killing effect, report gene expression of the green fluorescence protein, expression of the anti-tumor gene of mouse IL12, and the replication of the virus were observed respectively by cell pathology, fluorescence microscopy, ELISA and electron microscopy. RESULTS: A new kind of gene-viral vector system, in which the E1b-55 000 gene is deleted but the E1a gene of adenovirus is preserved, was constructed. The vector system possessed the same characteristics as the replicative virus ONYX-015, replication and proliferation in the tumor cells but not in the normal cells, thus specifically killing the tumor cells. Besides, it carried many kinds of anti-tumor genes. When carrying the report gene of the green fluorescence protein it made the expression of this gene in tumor cells far more effectively than the adenovirus vector employed in the traditional gene therapy did. However in the normal cells the expression of green fluorescence protein caused by this vector system was as little as or even less than that by the traditional adenovirus system. The similar result was also observed in the experiments of this vector system carrying the anti-tumor gene, gene of mouse IL12. The replication and proliferation of the virus carrying the gene of mouse IL12 in the tumor cells were confirmed by electron microscopy. CONCLUSION: Gene-viral vector is a new kind of vector in which the anti-tumor gene is inserted into the genome of the replicative virus specific for the tumor cells. It increases the expression of the anti-tumor gene by hundreds even tens of thousand times. It posseses all the advantages of gene therapy and virus therapy, thus further enhancing the curative effect and it overcomes such disadvantages as low transfer rate, low expression, lack of target tropism and low anti-tumor activity. It will become one of the most promising means in tumor treatment.

Adenoviridae↗
PubMed2003

Baculovirus vectors: novel mammalian cell gene-delivery vehicles and their applications.

Various methods have been developed to transfer and express genes in mammalian cells. Each method, whether virally, non-virally, or physically-based, has unique favorable features, but also drawbacks with respect to meeting desired and specific needs. Baculoviruses have been used since 1983 to express recombinant genes controlled by strong insect-virus promoters in their natural host (insect) cells. Today this is a well-established and easy to handle system for producing large quantities of recombinant proteins for numerous purposes. In 1995 it was first published that recombinant baculoviruses are able to deliver genes into mammalian cells. These genes are expressed provided that they are controlled by a promoter which is active in mammalian cells. Since then, various vector variants have been developed and numerous potential and meaningful applications have been described. It is not surprising that the use of baculovirus vectors as mammalian cell gene delivery vectors is constantly increasing and that the system is undergoing permanent improvements. Based on the convenience of the system to transfer genes into mammalian cells, baculoviruses can be applied in cell-based assays for drug screening to overcome the long periods of time required to generate stable cell lines. Baculovirus vectors are able to deliver very large DNA sequences into mammalian cells and vectors for toxic gene products can also be generated. In addition, baculoviruses are valuable tools for launching viral infection in cases where there is no appropriate cell culture system. Moreover, recent research has shown that the vectors can be applied in vivo. Depending on the design of the study, baculovirus vectors allow for sustained gene expression or are able to induce an immune response directed against the delivered and/or displayed gene product. The latter offers the opportunity to generate monoclonal antibodies against certain proteins that have failed by other means. In addition, it points to the potential usefulness of baculovirus vectors as new kinds of vaccines. Baculovirus vectors are therefore considered an enabling technology for various product opportunities.

Animals↗
PubMed2003

[Construction and verification of the effectiveness of pMBL: a cloning vector of exported proteins encoding genes].

The beta-lactamase was used as the reporter of expression and transmembrane secretion in this paper. A fragment of Amp resistance gene encoding the mature part of beta-lactamase (delta P delta SP Amp, i.e. without promoter and signal peptide coding sequences) was amplified from pUC18 vector. The upstream primer has BglII, BclI, BamHI in three reading frame respectively, in order to in frame fuse and express target genes together with the downstream reporter in finally constructed vector. Meanwhile, pET-28 was digested with the restriction enzymes BglII and Bst1107 I. The 2.8 kb fragment with replication origin, Kan resistance gene and MCS was recovered, filled, self-ligated and resulted in a plasmid pKan-B. The Bgl II site on pKan-B was then filled and the plasmid pKan was obtained. The delta P delta SP Amp gene, which was first cloned into pGEM-T-EASY vector, was inserted into pKan between EcoR I and XbaI sites. A plasmid pMBL-E was selected, with which the bacteria host could grew on Kan plate but not on plate with both Amp and Kan. An EcoRI site beside HindIII on the plasmid pMBL-E was then filled, and the plasmid pMBL, a cloning vector of the exported proteins encoding genes was finally obtained. Both results of the restriction enzyme digestion and sequencing demonstrated the correctness of the construction. The Tet resistance gene, a transmemebrane protein encoding gene, was applied to verify the effectiveness of the reporter in the vector. Cut with EcoRI and BamHI, a 375 bp fragment including promoter and 96 animo acids coding sequence (including signal peptide) of Tet was obtained from pBR322 vector. The fragment was then ligated to the vector pMBL which had been cut with both enzymes of EcoRI and BglI, or EcoRI and BclI, or EcoRI and BamHI (as 0, +1, +2 respectively of the beta-lactamase gene reading frame). Kan and Amp double resistant colonies only grew with the EcoRI and BglII combination (0 position). Restriction enzyme digestion and sequencing results of the recombinant plasmid showed that Tet resistance gene, which promoted the expression and transmembrane secretion of downstream beta-lactamase, was inserted in a correct reading frame into the vector. Thus, the results verified the effectiveness of the constructed vector pMBL, which may be used effectively to clone genes encoding exported proteins with promoters and signal peptide sequences.

Bacterial Proteins↗
PubMed2006

Malaria vector control and personal protection.

Malaria transmission rates and risks can be greatly reduced by vector control, mitigating high malaria incidence and prevalence rates. Methods and strategies for malaria vector control (MVC) have been well documented by WHO, although its implementation varies widely. Technical guidelines for MVC strategies and materials are readily available, but the status and role of MVC have not been reviewed and redefined in terms of programme management and resource allocation. There are huge changes since November 1993 when the last WHO Study Group reviewed vector control for malaria and other mosquito-borne diseases, following the 1992 adoption of the Global Malaria Control Strategy. Operationally, with reform of the health sector in many countries, the centrally managed and vertically structured malaria control programme (MCP) has been superseded by a community-based and decentralized one. This poses challenges for effective implementation of MVC strategies. Therefore it became evident that the role of vector control in malaria control needs to be reconsidered to develop a strategic framework for MVC implementation by national malaria control programmes and other partners. This report of a WHO Study Group on Malaria Vector Control and Personal Protection reviewed the current vector control strategies and their effectiveness in various operational and eco-epidemiological settings, and identified challenges for implementation in different health systems. An outline strategic framework for strengthening malaria vector control implementation was developed. The process of deciding about which mosquito control method is appropriate in a given situation should be guided by an analysis of the level of malaria endemicity and vector bionomics, the eco-epidemiological setting, the health management system and an estimate of the programme sustainability. This report also provides a basis for the development of a strategic framework for strengthening malaria vector control implementation.

Animals↗
PubMedDate not supplied

Enhancement of antiangiogenic effects of human canstatin with a hypoxia-regulated transgene vector in lung cancer model.

UNLABELLED: Canstatin, a newly identified antiangiogenesis protein, has a potent inhibitory effect on the proliferation and growth of endothelial cells. To enhance the expression and antiangiogenic effects of canstatin in solid tumors, we constructed a eukaryotic expression vector that encodes human canstatin cDNA downstream from nine copies of the hypoxia-response element. METHODS: Canstatin complementary DNA from adult liver tissues was cloned into the mammalian expression vector pCMV-Script. Nine copies of the hypoxia-response element were ligated upstream from the canstatin gene near the cytomegalovirus promoter. The recombinant vector, pCMV9Cans, was transformed into A549 cells by cationic liposomes. The transformed cells were cultured under oxic and anoxic conditions. We detected canstatin messenger ribonucleic acid and protein expression in transformed cells by TaqMan polymerase chain reaction and Western blot analysis, respectively. Human umbilical vein endothelial cells were cocultured with recombinant vector transformed A549 cells using Transwell plates under oxic and anoxic conditions. The proliferation and apoptosis of the cocultured endothelial cells were evaluated with 3H-thymidine incorporation and terminal deoxynucleotidyl-mediated biotinylated deoxyuridine triphosphate nick end-labeling methods (TUNEL), respectively. A canstatin-encoding vector with no hypoxia-response element, pCMVCans, was used as the positive control, and naked plasmid-transformed and singly cultured parental cells were used as negative controls. The biologic activity of the vector in tumor tissues of lung cancer-bearing nude mice was evaluated by microvessel counts. Canstatin protein expression was assessed by Western blot analysis in tumor tissues. pCMVCans and empty vector were used as controls in the in vivo assays. RESULTS: Canstatin messenger RNA and protein were detected in both pCMV9Cans- and pCMVCans-transformed A549 clones. Under oxic conditions, canstatin expression was not significantly different in clones stably transformed with pCMV9Cans or pCMVCans. However, under anoxic conditions canstatin expression was significantly higher in pCMV9Cans-transformed cells than in pCMVCans-transformed cells. Moreover, the 3H-thymidine uptake rate of the human umbilical vein endothelial cells was markedly lower than that of the pCMVCans-transformed cells, and many endothelial cells underwent apoptosis when cocultured with pCMV9Cans-transformed A549 cells, especially under anoxic conditions. We detected canstatin expression in tumor tissues; the expression level in pCMV9Cans-transformed tumors was significantly higher than that in pCMVCans-transformed tumors. An in vivo assay showed that tumors transformed with pCMV9Cans remained small, and microvessels in those tumors were much fewer than those in pCMVCans-transformed tumors. CONCLUSION: The hypoxia-regulated vector pCMV9Cans increases the expression of canstatin, thereby enhancing its biological effects. We believe that the hypoxia-inducible canstatin-expressing vector is a promising gene therapy tool for antiangiogenesis research.

Angiogenesis Inhibitors↗
PubMed1989

A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation.

A group of efficient cDNA cloning strategies employs vector-primers where cDNA synthesis starts from the oligo(dT)-primer tail, which is conventionally attached to cloning vectors by use of terminal deoxynucleotidyl transferase. An alternative, efficient and more versatile method of vector-primer preparation is to directly ligate, by use of T4 DNA ligase, a double-digested vector, e.g., pTZ18R/Pst I/Bam HI, to a synthetic (Bam HI)-adapter-end-primer, 5'-pGATCC-Tn or 5'-pGATCC-site-specific sequence. The use of a utility-vector containing a sizable spacer between the two selected restriction sites enables unambiguous separation on agarose gels of the double-digested vector precursors from single-digested ones, further simplifying the vector preparation. The adapter-end-primer ligation method can be applied to any suitable vectors with multiple cloning sites for the preparation of not only oligo(dT)-tailed, but also site-specific sequence-tailed vectors. Thus, the method enables the cDNA cloning of total poly (A+)-mRNAs, as well as specific RNA or mRNA species with or without poly(A)-tail.

Biotechnology↗
PubMedDate not supplied

[Vectors--derivatives of phage lambda for construction and analysis of genome libraries].

Basic features of lambda phage derived, cosmid and plasmid vectors are described. Plasmid vectors combine the most useful features of phage and plasmid vectors. Plasmids can exist in vivo as a plasmid or as a phage. Plasmid vectors are similar to large capacity phage vectors, but can be maintained in vivo without a stuffer fragment. That is why plasmids are easier in preparation for cloning than phage vectors. The yield of recombinants is higher with plasmid vectors (up to 3.10(6)) and the background of non-recombinants in the library is lower. Analysis of recombinant plasmids is more simple and effective than analysis of recombinant phages and plasmids. Probably plasmid vectors will soon be widely used instead of phage or cosmid vectors for genomic libraries construction and analysis.

Bacteriophage lambda↗
PubMed1995

Enhanced transduction efficiency of retroviral vectors coprecipitated with calcium phosphate.

Retroviral vectors are being used increasingly in clinical gene therapy protocols but low transduction frequencies are presenting a significant obstacle to progress. In this paper we report a simple method to enhance the efficiency of ex vivo retroviral gene transfer. Calcium chloride is added to the vector stock and calcium phosphate precipitates out of solution in complex with the retroviral vectors. When such coprecipitated vectors were used for gene transfer, the vector titres were increased at least five-fold and as much as 50-fold compared with the titres obtained in standard polybrene-enhanced infection protocols. Titre enhancement was not dependent on the starting concentration of vector, was equally effective for ecotropic and amphotropic vectors on a variety of mouse and human cells, and was not associated with any alteration in vector host range properties. The method may be of value to concentrate retroviral vectors and to enhance the efficiency of gene transfer in selected human gene therapy protocols.

Animals↗
PubMed1994

Retroviral vector design for long-term expression in murine hematopoietic cells in vivo.

A series of retroviral vectors containing the human glucocerebrosidase (GC) cDNA driven by various promoters have been constructed in an attempt to discover which vector design can most efficiently transduce murine hematopoietic stem cells (HSCs) and drive expression of the transferred gene in hematopoietic cells of mice reconstituted with the transduced stem cells. The simplest vector, LG, in which the GC gene is driven by the viral LTR, was the most efficient vector at infecting HSCs, with an average viral copy number in hematopoietic tissues of 3 copies/cell in recipient mice. In general, the viral vectors that contained any additional promoters or enhancers to drive expression of either the GC gene or a selectable marker gene (Neo) had lower titers and/or transduced HSCs at a lower efficiency. This was seen most markedly when the human phosphoglycerate (PGK) promoter was used to drive the human GC cDNA. Despite repeated attempts to obtain a high titer producer clone, this virus consistently produced low titers and subsequently resulted in the lowest proviral copy numbers in long-term reconstituted mice. Only the viral LTR and PGK promoter were capable of driving significant levels of human GC RNA in hematopoietic cells of long-term reconstituted mice, with a much lower level of RNA generated by an internal herpes TK or SV40 immediate early promoter. Insertion of the internal transcription unit in the opposite orientation relative to the viral LTRs had a detrimental effect on gene expression. The levels of RNA generated by a hybrid LTR containing the myeloproliferative sarcoma virus enhancer were higher in bone marrow-derived macrophages than in nonadherent cells of the bone marrow when compared with the LG vector. The presence of an internal promoter to drive expression of the human GC cDNA did not seem to have a detrimental effect on expression levels from the viral LTR. In fact, in the presence of an internal TK or PGK promoter expression from the LTR was increased despite the presence of lower proviral copy numbers. Insertion of a second gene (Neo) into the vector had a negative impact on long-term expression in hematopoietic cells in vivo; however, this seems to be due solely to the lower transduction efficiency of this vector. Overall, the highest levels of GC activity in macrophages of long-term reconstituted mice were generated by the LG vector; however, these levels were variable.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Long-term gene expression from autonomously replicating vectors in mammalian cells.

We tested the longevity of gene expression provided by autonomously replicating vectors. The vectors contain segments of human genomic DNA that provide efficient replication initiation and sequences derived from Epstein-Barr virus that provide nuclear retention. In order to monitor gene expression, the vectors also carry an expression cassette containing the chloramphenicol acetyl transferase gene. Replicating and control vectors were transfected into rapidly dividing human 293 cells, and gene expression and DNA retention were monitored. Gene expression decreased rapidly from a control vector lacking replication and retention functions, reaching near background levels by 10 days. Vectors having both replication and retention showed greatly prolonged gene expression, with vector DNA still detectable after 2 months. Autonomously replicating vectors also prolonged gene expression in rodent cells, in human lung epithelial cells, and in slowly dividing cell cultures. These results demonstrate the utility of this autonomous replication system for long-term retention and expression of introduced genes in mammalian cells. Such vectors could be useful for gene therapy, in combination with any of several methods for introduction of DNA.

Animals↗
PubMed1997

Lipidic vector systems for gene transfer.

Clinical application of gene therapy depends on the development of suitable gene transfer vehicles (vectors). Although generally not as efficient as viral vectors, nonviral systems such as lipidic vectors have the potential advantages of being less toxic, nonrestrictive in cargo DNA size, potentially targetable, and easy to produce in relatively large amounts. More important, lipidic vectors generally lack immunogenicity, allowing repeated in vivo transfection using the same vector. In this paper, we will attempt to summarize some of the recent advances in lipidic gene delivery vectors. Three types of lipidic gene transfer vectors are described: 1) DNA/cationic liposome complexes, 2) DNA encapsulated in neutral or anionic liposomes, and 3) liposome-entrapped, polycation-condensed DNA (LPDI and LPDII). We review the various factors affecting vector structure and gene delivery efficiency, and we discuss the possible mechanisms of gene transfer and their implications in vector design.

Animals↗
PubMedDate not supplied

Local inflammatory response and vector spread after direct intraprostatic injection of a recombinant adenovirus containing the herpes simplex virus thymidine kinase gene and ganciclovir therapy in mice.

We have evaluated the safety and potential toxicity of an adenoviral vector containing the herpes simplex virus thymidine kinase gene (adenovirus/Rous sarcoma virus thymidine kinase in a preclinical model for prostate cancer. Clinical grade vector prepared for human trials was injected directly into the dorsolateral prostate of C57Bl/6 mice in a volume of 5 microL at doses of 2.5 x 10(6), 2.5 x 10(7), or 2.5 x 10(8). The mice received intraperitoneal injections of either ganciclovir or saline twice daily for 6 days, beginning 12 hours after vector injection. Representative tissues and fluids were collected for evaluation the day after the final dose. Microscopic pathologic evaluation revealed inflammatory infiltration without necrosis within the dorsolateral and ventral prostate, but no necrosis or leukocyte infiltration was observed in sample tissues from lung, liver, large intestine, bladder, seminal vesicle, testis, or epididymis. DNA was extracted from the above tissues as well as pelvic lymph nodes, blood, seminal fluid, urine, and sperm and analyzed by polymerase chain reaction for the presence of vector sequences. The vector was readily detected in the dorsolateral prostate, the site of injection. The amount of vector detected was reduced in some samples from ganciclovir-treated animals. At the highest dose, vector spread was observed in the ventral prostate, seminal vesicle, testis, pelvic lymph nodes, gut, and liver. Spread to the testis was observed in only one animal. Vector DNA was not detected in urine, seminal fluid, or sperm but was detected in the blood of one animal. This adenoviral vector, therefore, appears to have minimal spread to sites distant from the site of injection and no detectable pathological sequelae within this dose range in this preclinical model for prostate cancer, which may be generalizable to other solid tumors.

Adenoviridae↗
PubMed1998

Production of high-titer retroviral vectors and detection of replication-competent retroviruses.

Retroviral infection or calcium phosphate-mediated DNA transfection has been used for the generation of retrovirus producing cell lines through the introduction of vector DNA into the chromosomes of packaging cells. To compare the ability of the methods for DNA delivery to produce high-titer virus, we generated stable retroviral vector producing cell lines by the transfection or infection of a LN-based vector DNA into PA317 cells and assayed individual clones for production of virus. Of eight randomly chosen G418-resistant clones generated by transfection, only one clone produced the vector at up to > 10(7) cfu/ml. Two of the five clones generated by infection yielded higher-titer viruses in the absence of helper virus--up to 5 x 10(7) more than the transfected clones. The titer of retroviral vectors can be increased by multiple rounds of infection through long-term incubation of amphotropic virus producing cells with ecotropic virus vectors. Such amplification of vector copy number resulted in increase in vector titer of up to 20-fold. For the experiments presented here, we have used an improved vector/packaging system designed for minimizing the possibilities for the generation of an replication-competent retrovirus (RCR). However, the potential of RCR generation was detected in the culture medium harvested from the highest-titer virus producing PA317 clonal cells generated by amplification of the vector through the modified cocultivation technique, although the generation of RCR is very infrequent in the system.

3T3 Cells↗
PubMed1998

Direct synovial gene transfer with retroviral vectors in rat adjuvant arthritis.

OBJECTIVE: To evaluate the feasibility of direct in vivo gene transfer in an animal model of arthritis using a retroviral vector. METHODS: The timing and dose of retroviral vector was examined using very high titer retroviral vector (> or = 10(9) CFU) in rat adjuvant arthritis. Retroviral vector expressing beta-galactosidase (beta-gal) or vehicle alone was injected into the right ankle of rats with adjuvant arthritis. Ankles were injected either on Day 7 (pre-arthritis), Day 10 (early arthritis), Day 15 (accelerating arthritis), or Day 28 (chronic arthritis) after adjuvant immunization. Joints were harvested 3 days later and extracts were assayed for beta-gal activity. RESULTS: Synovial beta-gal expression was minimal in the Day 7 group and elevated in the Day 10, Day 15, and Day 28 groups. Gene transfer with retroviral vector did not exacerbate the local inflammatory response. Minimal or no beta-gal expression was observed in the contralateral uninjected paw or in the spleen, lung, liver, and kidneys. Frozen sections of retroviral vector injected joints were stained with X-gal and revealed transduced cells in the lining and superficial sublining layers. To determine the longevity of gene expression, ankle joints were injected with vector on Day 15 post-adjuvant, harvested, and assayed for beta-gal activity for up to 49 days after injection. Expression of the enzyme peaked from Day 3 to 7 and was still readily detected up to 49 days after retrovirus infection. CONCLUSION: This is the first report of successful direct in vivo gene transfer in the rat adjuvant arthritis model using a retroviral vector. Appropriate timing of administration and very high titer retroviral vector preparations are key determinants of adequate gene transduction.

Animals↗
PubMed2004

T vector and loop characteristics in coronary artery disease and during acute ischemia.

OBJECTIVE: Three-dimensional characterization of the ventricular repolarization by the T vector and T vector loop morphology in coronary artery disease (CAD), and their response to short-term (no flow) ischemia induced by coronary occlusion during a percutaneous intervention (PCI). BACKGROUND: The risk for sudden cardiac death is increased in conditions of acute or permanently heterogeneous ventricular repolarization, for which ischemia is a risk factor. METHODS: Fifty-six CAD patients without visible collateral circulation were studied during an elective single-vessel PCI, and 10 healthy controls twice at rest. T vector parameters (Televation, Tazimuth, and QRS-T angle), and T loop parameters (Tarea, Tavplan, and Teigenv) were measured by vectorcardiography. ST vector magnitude (ST-VM) and its change (STC-VM) were used for reference. RESULTS: At rest, T vector loop morphology (Tarea, Teigenv) was significantly different in CAD patients and controls, while T vector angles did not separate the groups. Ischemia induced significant changes in T loop parameters in the entire CAD group, whereas in the LAD subgroup significant changes were seen also in T vector angle. The T loop morphology was significantly different at baseline and a more pronounced response to ischemia (Tarea) was seen in patients with, than in those without, a history of hypertension. CONCLUSION: T loop morphology, rather than the T vector angle, separated CAD patients from healthy controls. Coronary occlusion had significant impact on ventricular repolarization, as assessed by T vector and morphology analysis, and most prominently in the LAD group. Hypertensive patients appeared especially vulnerable to ischemia.

Acute Disease↗