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At least 775 records · Page 43Linked to original sources
PubMed2002

Durability of transgene expression and vector integration: recombinant SV40-derived gene therapy vectors.

Many applications of gene delivery require long-term transgene expression. In dividing cells, this result necessitates vector genome persistence, usually by integrating into cellular DNA. Since recombinant gene delivery vectors derived from tag-deleted, replication-incompetent simian virus-40 (SV40) provide for long-term transgene expression in resting and dividing cells, we tested whether such enduring transgene expression reflected integration into cellular genomes. Several lines of evidence suggested this likelihood. After transduction in vitro, continuously dividing cell lines and continuously stimulated primary cells uniformly showed transgene expression for many months. Mice whose livers were transduced in vivo, partially resected, and allowed to regenerate showed comparable levels of transgene expression in regenerated and preoperative livers. Thus, replicationincompetent SV40 vectors (rSV40) persist in vitro and in vivo despite extensive cell division. We tested the possibility that this persistence reflected integration directly. Southern blot analyses of genomic DNA from transduced 293 cells showed that vector genome incorporation into cell DNA happened within days of transduction. Episomal vector DNA was barely detectable 96 hours post-transduction. Inverted PCR, used to characterize vector integration points, showed vector DNA integrated randomly into the cell genome. The circular rSV40 genome opened at different points in each integrand. A significant proportion of the integrands did not contain the entire vector sequence, but rather only portions thereof. Quantitative Southern blot analysis showed approximately 3.05 transgene copies per cell. Therefore, recombinant SV40 gene delivery vectors integrate into the cellular DNA of both resting and dividing cells, and do so randomly and within days of transduction. This integration may explain long-term transgene expression.

Animals↗
PubMed1996

Target-sequence preferences of HIV-1 integration complexes in vitro.

Integration of reverse transcribed retroviral cDNA is not restricted to particular host DNA sequences. However, the frequency of integration into a particular phosphodiester bond is influenced by the local sequence. Here we examine the target-sequence preferences of purified HIV integrase and viral nucleoprotein complexes (preintegration complexes) isolated from freshly infected cells. We find that the three-base sequence including the integration site is not the major factor determining the frequency of integration, since identical triplets embedded in different sequences are used with very different efficiencies. However, there is a statistically significant bias against integration upstream of a pyrimidine nucleotide. The target-sequence preferences of purified integrase and preintegration complexes are very different. Strong integration sites on opposite DNA strands occur in pairs separated by five residues when preintegration complexes are used but not with purified integrase. These studies highlight the difference between the two sources of HIV integration activity and may provide the basis for a simple assay for the correct assembly of viral nucleoprotein complexes.

Binding Sites↗
PubMed2003

The roles of cellular factors in retroviral integration.

A key early step in the retroviral life cycle is the integration of reverse-transcribed viral cDNA into a chromosome of an infected cell. The key protein player in retroviral integration is the viral integrase, which enters the cell as part of the virus. Although purified integrase protein is necessary and sufficient to perform the basic catalytic DNA breakage and joining steps of retroviral integration, a variety of normal cellular proteins have been implicated as playing important roles in establishing the integrated provirus in cells. This chapter reviews the roles of host cell factors that function during integrase catalysis, during the repair of the resulting DNA recombination intermediate, and by potentially guiding viral preintegration complexes to their chromosomal locations for cDNA integration. The potential to interfere with proper integration by blocking either integrase catalysis or the function of cellular integration cofactors is also discussed.

Animals↗
PubMed2002

Integration site selection by lentiviruses: biology and possible control.

Retroviruses integrate into naked DNA in a generally sequence nonspecific fashion, but closer study reveals a variety of forces that influence target site selection. Primary sequence of the target plays a small but detectable role. Proteins bound to target DNA can inhibit integration by blocking access of integration complexes or stimulate integration by distorting DNA. An important example of the latter is DNA distortion in nucleosomal DNA. In vivo integration has not yet been convincingly shown to be biased in favor of any identifiable sequence features, though this could still change in future studies. Many applications of retroviral vectors could be facilitated by targeting integration in vivo to predetermined sites. Towards this end, several groups have studied the properties of fusions of integrase proteins to sequence-specific DNA-binding domains. To date such studies establish that targeting can work well in reactions in vitro, but a variety of obstacles complicate applications in vivo. However, naturally occurring retrotransposons do carry out highly targeted integration using retrovirus-like integrase proteins, fueling long-term hopes for targeting with retroviral integrases as well.

DNA, Viral↗
PubMedDate not supplied

Target integration by a chimeric Sp1 zinc finger domain-Moloney murine leukemia virus integrase in vivo.

A specificity protein 1 (Sp1) zinc finger domain containing two tandem zinc fingers was fused to the C terminus of the integrase (IN) protein of the Moloney murine leukemia virus (MuLV). The integrity of the MuLV IN was completely preserved, since the fusion was conducted at the last amino acid residue of the protein. The vector pMIN-Sp1, which carried the fused MuLV IN-Sp1 zinc finger domain gene, was cotransfected with a wild-type MuLV vector pMLV-K to NIH/3T3 cells. A nonradioactive reverse transcriptase assay was performed on culture supernatants collected from the cotransfected cells to confirm the production of recombinant viruses. The expression of the fusion protein and the integration of the MuLV genome by the fusion protein were confirmed by a Northern and then a Southern hybridization analysis on the total RNA or genomic DNA extracted from cells infected by viruses collected from the supernatants of the cotransfected cells. Regions of the host chromosome that were selected by the fusion protein as the integration targets were sequenced using the TOPO(TM) cloning method on a series of PCR products generated with a nested set of primers. The percentage of positive clones screened that contained the DNA-binding sequence of the fused Sp1 zinc finger domain was around 13% (5 out of 39 clones). It was found that the Sp1 DNA-binding sequence was only present in regions that were proximal to one of the long terminal repeats of the integrated viral genome, suggesting that the fusion protein could select a target sequence for integration. The host flanking sequences determined for all the positive clones were also used as queries to perform a BLAST search on the GenBank mouse EST entries. Although matching scores for sequences of some of the clones computed were more significant than others, it was difficult to judge whether or not the integration in these clones had been targeted to some gene sequences. Most of the integration sites might exist in the introns, since we found that the probability of the gene sequences containing an Sp1 DNA-binding site was low.

3' Flanking Region↗
PubMed1997

Woodchuck hepatitis virus DNA integration in a common chromosomal region of the woodchuck genome in two independent hepatocellular carcinomas.

In woodchuck hepatocellular carcinoma (HCC) the myc-oncogene family (particularly N-myc2) and the win locus of cellular genome have been reported as frequent targets for integration of woodchuck hepatitis virus (WHV) DNA. In this paper a further cellular locus, b3n, is reported as recurrent target for WHV integration in woodchuck HCC. Cloning and sequencing of a WHV-DNA integration and its cellular flanking regions showed that viral DNA was inserted in a chromosomal region already described for WHV integration in another single HCC. The two integration sites are only 0.5 kb apart. A link between WHV integration in b3n and HCC development may be postulated. Careful analysis of the sequence of the unoccupied locus revealed that, in addition to Alu-like repeats and a gag-like coding region, already described, several features of Matrix Attachment Region (MAR) sequences are present. Thus (part of) b3n might be a previously unrecognized MAR. Organization of the chromatin in functional domains and regulation of gene expression are some functions attributed to MAR sequences. The occurrence of WHV-DNA integration close to the same putative MAR in two different HCCs suggests that a mechanism of deregulation of MAR functions by WHV insertion might act in some liver tumors.

Animals↗
PubMed1996

The NUCLEUS integrated electronic patient dossier breakthrough and concepts of an open solution.

This paper addresses the requirements of healthcare providers and hospital managers vis-à-vis electronic patient records that can be integrated. It starts from some critical failure factors, found with previous attempts to standardise the electronic health record. Standardisation appears to be the key issue: the subject of standardisation requires delicate positioning. Technology must provide us with the means to obtain the standardised foundation for an integrated health record concept, which can be completely configured and customised to meet the requirements of health professionals and institutions involved. NUCLEUS, project A2025 in the AIM programme, has taken on this endeavour, and with good success. This paper summarises the benefits of this approach for various categories of people interested in using electronic patient records. Moreover, it illustrates NUCLEUS' contribution to achieving seamless integration of care. Furthermore, this paper explains the conceptual innovations that have been achieved in the NUCLEUS project. It consolidates the main concept of Act Management, structuring the professional primary process as well as the interprofessional communication. These concepts are subsequently expanded to include the key elements of the NUCLEUS integrated electronic patient record. Next, the paper reflects on what has appeared to be one of the critical success factors of the electronic patient record: its configuration and customisation facilities. These facilities make it possible to access the patient record at various intuitive aggregation levels and to make the integrated patient record 'look like' the individually specialised record of the respective healthcare professionals. Finally, the paper addresses various topics required to facilitate the successful implementation and operation of the NUCLEUS integrated electronic patient record like security, integrity, message communication, distribution, heterogeneity and the context of the hospital information system.

Communication↗
PubMed1991

Chromosome deletions associated with hepatitis B virus integration.

Integrated hepatitis B virus (HBV) DNA is often found in hepatocellular carcinomas which develop in HBV carriers. We previously found that chromosomal abnormalities such as translocations and inversions are often associated with HBV integration. Here we report deletions that were found with three integrants, by comparing the physical maps of the flanking cellular DNA of the integrants with those of the unoccupied cellular sequences. The sizes of the deletions were 25, 12, and 11 kb, respectively. Each integrant carried only a small fragment of the viral genome, from which the cohesive end region, the preferred site of integration, was deleted. We speculate that these deletions were made from primary integrants by recombination of regions between the viral DNA and the flanking cellular sequence.

Base Sequence↗
PubMed1995

A PCR-based strategy for rapid mapping of hepadnavirus integrated sequences in hepatocellular carcinomas.

A methodology based on polymerase chain reaction (PCR) and restriction analysis for rapid mapping of woodchuck hepatitis virus (WHV) integrations in hepatocellular carcinoma (HCC) tissues is described. Conventional PCR with viral primer pairs is not suitable for mapping WHV-integrated regions because the presence of minimum amounts of non-integrated (PCR amplifiable) WHV genome and replicative intermediates cannot be excluded. The first relevant part of the strategy is the identification of the cellular sequences flanking the WHV integration in order to select one (or more) integration-specific primer. The cellular flanking sequence can be rapidly obtained by means of inverse-PCR amplification of the viral/cellular junction and sequencing of the product. Mapping of the integrated regions is carried out by fixed flanking primer PCR (FFP-PCR) using the cellular primer as a 'fixed' primer in PCR association with each of an available set of WHV primers. Amplification of episomal WHV sequences is thus avoided. PCR products can also undergo restriction analysis. PCR-positive viral primers and specific WHV restriction sites are assembled into a map, based on the size and restriction pattern of the PCR products. The results of WHV integration mapping in a woodchuck HCC are described.

Animals↗
PubMed2003

Demethylation of host-cell DNA at the site of avian retrovirus integration.

The transcriptional activity of an integrated retroviral copy strongly depends on the adjacent host-cell DNA at the site of integration. Transcribed DNA loci as well as cis-acting sequences like enhancers or CpG islands usually permit expression of nearby integrated proviruses. In contrast, proviruses residing close to cellular silencers tend to transcriptional silencing and CpG methylation. Little is known, however, about the influence of provirus integration on the target sequence in the host genome. Here, we report interesting features of a simplified Rous sarcoma virus integrated into a non-transcribed hypermethylated DNA sequence in the Syrian hamster genome. After integration, CpG methylation of this sequence has been lost almost completely and hypomethylated DNA permits proviral transcription and hamster cell transformation by the proviral v-src oncogene. This, however, is not a stable state, and non-transformed revertants bearing transcriptionally silenced proviruses segregate with a high rate. The provirus silencing is followed by DNA methylation of both provirus regulatory regions and adjacent cellular sequences. This CpG methylation is very dense and resistant to the demethylation effects of 5-aza-2(')-deoxycytidine and/or trichostatin A. Our description exemplifies the capacity of retroviruses/retroviral vectors to overcome, at least transiently, negative position effects of DNA methylation at the site of integration.

Agar↗
PubMed2006

Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis.

Chronic liver disease associated with long term hepatitis B virus (HBV) infection contributes importantly to the development of hepatocellular carcinoma (HCC). A salient feature of these chronic infections is the integration of subgenomic HBV DNA fragments into many different locations within the host DNA, suggesting that integration is random. Although this may promote genetic instability during liver regeneration which accompanies a bout of chronic liver disease, the actual role of integrated HBV DNA in hepatocarcinogenesis is uncertain. Importantly, most integration events retain the HBV open reading frame encoding the HBx antigen (HBxAg), which is the virus contribution to HCC. In addition, many integration events reported in the literature occur near or within fragile sites or other cancer associated regions of the human genome that are prone to instability in tumor development and progression. Genetic instability associated with integration potentially alters the expression of oncogenes, tumor suppressor genes, and microRNAs (miRNAs) that may contribute importantly to tumorigenesis. If so, then selected integration events may alter pathways that are rate limiting in hepatocarcinogenesis, thereby providing targets with diagnostic/prognostic potential and for therapeutic intervention.

Cell Transformation, Neoplastic↗
PubMed2006

Semantic integration in healthcare networks.

A seamless support of information flow for increasingly distributed healthcare processes requires to integrate heterogeneous IT systems into a comprehensive distributed information system. Different standards contribute to ease this integration. In a research project focussing on the development of a reference architecture for inter-institutional health information systems, we identified concurring standards currently in use. We therefore categorized these integration standards by distinguishing between technical and semantic integration on the one hand, and data and functional integration on the other hand. In addition, standards for semantic integration are roughly categorized according to their scope. By placing standards into a corresponding matrix a "semantic gap" is revealed, which cannot be covered by standards as it contains volatile medical concepts. As a conclusion, it is recommended to conceptually consider the necessity of system evolution in system architectures and also in future integration standards.

Hospital Information Systems↗
PubMed2006

Data integration and genomic medicine.

Genomic medicine aims to revolutionize health care by applying our growing understanding of the molecular basis of disease. Research in this arena is data intensive, which means data sets are large and highly heterogeneous. To create knowledge from data, researchers must integrate these large and diverse data sets. This presents daunting informatic challenges such as representation of data that is suitable for computational inference (knowledge representation), and linking heterogeneous data sets (data integration). Fortunately, many of these challenges can be classified as data integration problems, and technologies exist in the area of data integration that may be applied to these challenges. In this paper, we discuss the opportunities of genomic medicine as well as identify the informatics challenges in this domain. We also review concepts and methodologies in the field of data integration. These data integration concepts and methodologies are then aligned with informatics challenges in genomic medicine and presented as potential solutions. We conclude this paper with challenges still not addressed in genomic medicine and gaps that remain in data integration research to facilitate genomic medicine.

Biomedical Research↗
PubMed2005

Resistance against Friend leukemia virus-induced leukemogenesis in DNA-dependent protein kinase (DNA-PK)-deficient scid mice associated with defective viral integration at the Spi-1 and Fli-1 site.

Retroviral DNA integration is mediated by the viral protein integrase. However, elements of the host DNA repair machinery such as the phosphatidylinositol 3-kinase (PI-3K)-related protein kinase family system would play a role in the integration of viral DNA into the host DNA. Here, we show that a host PI-3K-related protein kinase, DNA-dependent protein kinase (DNA-PK), plays a role in the specific integration of retroviral DNA and induction of retroviral diseases in vivo. DNA-PK-deficient scid mice inoculated with Friend leukemia virus (FLV) exhibited a random integration into their genomic DNA and expressed the viral envelope protein gp70. However, the specific integration of FLV at Spi-1 or Fli-1 sites did not occur in association with the significant resistance of scid mice to FLV-induced leukemogenesis. In contrast, the knockout of another member of the PI-3K-related protein kinase family, encoded by the ataxia telangiectasia mutated (ATM) gene, resulted in mice as sensitive to FLV-induced leukemogenesis as the wild type mice. FLV was specifically integrated into the DNA at Spi-1 and Fli-1 sites with significant expression of these transcription factors. These findings indicated that DNA-PK would be essential for controlling the in vivo integration of FLV at specific sites as well as the susceptibility to FLV-induced leukemogenesis.

Animals↗
PubMed2005

Uncharted ground: patterns of professional interaction among complementary/alternative and biomedical practitioners in integrative health care settings.

The development of "integrative health care" (IHC) settings combining various aspects of Western biomedicine and complementary/alternative medicine (CAM) is a relatively recent phenomenon among biomedical and CAM professions. While IHC is recognised internationally and occurs in many different contexts (e.g. clinic or hospital), patterns of interaction between biomedical and CAM practitioners, and the nature of IHC settings, are largely unknown. This paper presents findings from a research study of two newly established IHC settings in Canada. The main research question was: how are biomedical and CAM practitioners integrating or not integrating with each other at the level of professional interaction in IHC settings? Using a case study design, in-depth interviews were conducted with 13 biomedical and eight CAM practitioners during 2002-2003, and ethnographic observation and document analysis was conducted at each site. Drawing from closure theory of the professions, comparative analysis of the sites revealed that biomedical practitioners enact patterns of exclusionary and demarcationary closure, in addition to the use of "esoteric knowledge", by: (a) dominating patient charting, referrals and diagnostic tests; (b) regulating CAM practitioners to a specific "sphere of competence"; (c) appropriating certain CAM techniques from less powerful CAM professions; and (d) using biomedical language as the primary mode of communication. CAM practitioners, in turn, perform usurpationary closure strategies, by: (a) employing their own "esoteric knowledge" in relation to biomedicine and other CAM professions; (b) appropriating biomedical language and terminology; (c) increasing their professional status by working with biomedicine; and (d) referring among CAM practitioners to increase patient flow. The findings suggest that when attempts are made to integrate biomedicine and CAM, dominant biomedical patterns of professional interaction continue to exist. Despite continued patterns of social closure, biomedical and CAM practitioners continue to provide a certain form of integrative care that may be of benefit to patients, albeit not as integrative as current models of integration would prefer.

Anthropology, Cultural↗
PubMed2006

Variations in provider conceptions of integrative medicine.

Consumers often turn to complementary and alternative medicine (CAM) and use it concurrently with conventional medicine to treat illnesses and promote wellness. However, prior studies demonstrate that these two paradigms are often not combined effectively. Consumers often do not tell physicians about CAM treatments or CAM practitioners about conventional treatments that they are using. This can lead to inefficient care and/or adverse interactions. There is also a lack of consensus about the structure and practice of integrative medicine among the various types of practitioners. This qualitative study aimed to identify key domains and develop a conceptual model of integrative medicine at the provider level, using a grounded theory approach. Purposive sampling was used to select 50 practitioners, including acupuncturists, chiropractors, internists/family practitioners, and physician acupuncturists in private practice and at academic medical centers in Los Angeles. We conducted semi-structured, in-depth interviews with practitioners and then identified core statements that describe practitioners' attitudes and behaviors toward integrative medicine. Core statements were free pile sorted to ascertain key domains of integrative medicine. Four key domains of integrative medicine were identified at the provider level: attitudes, knowledge, referral, and practice. Provider age, training, and practice setting also emerged as important factors in determining clinicians' "orientation" toward integrative medicine. "Dual-trained" practitioners, such as physician acupuncturists, exemplified clinicians with a greater orientation toward integrative medicine. They advocated an open-minded perspective about other healing traditions, promoting co-management with and making referrals to practitioners of other paradigms, and treating patients with both CAM and conventional healing modalities.

Academic Medical Centers↗
PubMed2002

HIV-1 integration in the human genome favors active genes and local hotspots.

A defining feature of HIV replication is integration of the proviral cDNA into human DNA. The selection of chromosomal targets for integration is crucial for efficient viral replication, but the mechanism is poorly understood. Here we describe mapping of 524 sites of HIV cDNA integration on the human genome sequence. Genes were found to be strongly favored as integration acceptor sites. Global analysis of cellular transcription indicated that active genes were preferential integration targets, particularly genes that were activated in cells after infection by HIV-1. Regional hotspots for integration were also found, including a 2.4 kb region containing 1% of sites. These data document unexpectedly strong biases in integration site selection and suggest how selective targeting promotes aggressive HIV replication.

Base Sequence↗
PubMed2002

Simultaneous mapping of human papillomavirus integration sites and molecular karyotyping in short-term cultures of cervical carcinomas by using 49-color combined binary ratio labeling fluorescence in situ hybridization.

Infection with high-risk type human papillomavirus (HPV) is a necessary causal factor in the pathogenesis of cervical carcinoma. In most invasive cervical cancers, HPV is integrated in the host cell genome, and additional genetic aberrations are observed among which are chromosomal aberrations. To analyze in detail such often complex chromosomal changes and simultaneously map HPV integration sites, we extended the multiplicity of the combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) technique to 49 by inclusion of a large Stokes' shift fluorochrome as the third binary label. The technique allows mapping of the integrated HPV genome in the context of p- and q-arm COBRA-FISH, with a sensitivity of one copy of the HPV genome as tested for HPV 16 in SiHa cells. We investigated the molecular karyotypes and integration patterns of HPV types 16 and 18 in metaphase spreads from short-term cultures of primary cervical carcinomas (n=5). Of the tested cervical carcinomas, two contained integrated HPV at 8q24, one of which in addition harbored the integrated virus near a translocation breakpoint. Two carcinomas had integrated HPV at 17q21 through 23 in a morphologically normal chromosome 17. One carcinoma contained HPV at 1q42 in a morphologically normal chromosome 1. Our data illustrate the efficacy of 49-color COBRA-FISH to resolve complex karyotypes and simultaneously map specific sequences in metaphases obtained from short-term solid tumor cultures.

Color↗