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PubMed1998

Differential selection of cells with proviral c-myc and c-erbB integrations after avian leukosis virus infection.

Avian leukosis virus (ALV) infection induces bursal lymphomas in chickens after proviral integration within the c-myc proto-oncogene and induces erythroblastosis after integration within the c-erbB proto-oncogene. A nested PCR assay was used to analyze the appearance of these integrations at an early stage of tumor induction after infection of embryos. Five to eight distinct proviral c-myc integration events were amplified from bursas of infected 35-day-old birds, in good agreement with the number of transformed bursal follicles arising with these integrations. Cells containing these integrations are remarkably common, with an estimated 1 in 350 bursal cells having proviral c-myc integrations. These integrations were clustered within the 3' half of c-myc intron 1, in a pattern similar to that observed in bursal lymphomas. Bone marrow and spleen showed a similar number and pattern of integrations clustered within 3' c-myc intron 1, indicating that this region is a common integration target whether or not that tissue undergoes tumor induction. While all tissues showed equivalent levels of viral infection, cells with c-myc integrations were much more abundant in the bursa than in other tissues, indicating that cells with proviral c-myc integrations are preferentially expanded within the bursal environment. Proviral integration within the c-erbB gene was also analyzed, to detect clustered c-erbB intron 14 integrations associated with erythroblastosis. Proviral c-erbB integrations were equally abundant in the bone marrow, spleen, and bursa. These integrations were randomly situated upstream of c-erbB exon 15, indicating that cells carrying 3' intron 14 integrations must be selected during induction of erythroblastosis.

Animals↗
PubMed2003

Integrating leprosy control into general health service in a war situation: the level after 5 years in eastern Congo.

South Kivu Province of the Democratic Republic of Congo, plagued by a turbulent civil war, started a process of integrating leprosy into general health services in 1995. A questionnaire survey was carried out in September 2000 to assess the level of structural and functional integration, after 5 years of the integration process, in nine of its 14 health districts. The survey revealed that a total of 76 clinic nurses remained of those trained in leprosy since 1993. In all, 33-6% of the total 226 health facilities had a trained nurse, but according to the district supervisors who filled the questionnaires, nurses in only 28.3% of health facilities could diagnose leprosy. Less than 40% of the total 226 health facilities were structurally integrated with MDT and other leprosy services. Functionally, the clinic nurses were involved in dispensing MDT drugs and keeping leprosy records in 90.8 and 81.6%, respectively, of the integrated facilities, and diagnostic activities in 43.7%. The degree of involvement put health facilities into four grades of functional integration: 1) fully-functional integrated, 2) semi-functional integrated, 3) semi-integrated (structural but not functional), 4) not integrated (vertical). On this scale, 80% of 107 health facilities reported by the supervisors had some form of integration and 20% were not integrated. Treatment activities were significantly more functionally integrated than the diagnostic and POD activities, which require more skills. The presence of a trained nurse in a health facility made no significant difference to the involvement of clinic nurses in dispensing MDT drugs and performing POD activities, but significantly affected their performance of diagnostic activities and records keeping. The endemic districts had higher levels of structural integration, were not more likely to be functionally integrated. The levels of structural integration after 5 years are considered low in South Kivu Province, and reflect the significant negative effect of civil conflicts on integration of leprosy programmes in Africa.

Delivery of Health Care, Integrated↗
PubMed1996

Concerted integration of linear retroviral DNA by the avian sarcoma virus integrase in vitro: dependence on both long terminal repeat termini.

We have reconstituted integration reactions in vitro with specially designed donor DNAs, a supercoiled plasmid acceptor, purified bacterium-derived Rous sarcoma virus integrase (IN), and a host cell DNA-bending protein, HMG1. The duplex donor DNAs are approximately 300 deoxynucleotides in length and contain only 15 bp of the RSV U3 and U5 termini at the respective ends. The donor has blunt U3 and U5 termini which end with the sequence 5'CATT. Joining of the donor DNA to the acceptor DNA is detected by using a simple biochemical assay. Integration was found to be dependent on both U3 and U5 termini; mutations in either result in a significant decrease in the level of integration in vitro. Restriction digestion of the products is consistent with most integrants representing a concerted integration in which both long terminal repeat termini come from the same donor molecule. The U5 and U3 sequences in the substrate flank a supF tRNA gene, permitting biological selection of integrants. Many integrants have been sequenced, and have all of the hallmarks of authentic viral integration, including the removal of a terminal TT dinucleotide from each donor DNA end, and duplication of acceptor sequences at the integration site without introducing deletions into the acceptor. Target site selection in the acceptor plasmid was random except that the orientation of integrants selected was apparently influenced by supF transcription. Mutations which substituted the conserved CA dinucleotide with a GA pair led to a decreased rate of integration. In 2 of 14 mutant integrants sequenced, deoxynucleotides were deleted from either the U5 or U3 terminus. In one instance, an internal CA dinucleotide was used, which resulted in a 10-bp U5 donor deletion. In the other, an internal GA dinucleotide was used, which produced a 5-bp U3 donor deletion. Both of these integrants provide further evidence that concerted integration in this reconstituted system requires interactions between IN and the U3 and U5 termini from the same donor molecule.

Avian Sarcoma Viruses↗
PubMed1997

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.

Base Sequence↗
PubMed2004

Adeno-associated virus site-specific integration and AAVS1 disruption.

Adeno-associated virus (AAV) is a single-stranded DNA virus with a unique biphasic lifestyle consisting of both a productive and a latent phase. Typically, the productive phase requires coinfection with a helper virus, for instance adenovirus, while the latent phase dominates in healthy cells. In the latent state, AAV is found integrated site specifically into the host genome at chromosome 19q13.4 qtr (AAVS1), the only animal virus known to integrate in a defined location. In this study we investigated the latent phase of serotype 2 AAV, focusing on three areas: AAV infection, rescue, and integration efficiency as a function of viral multiplicity of infection (MOI); efficiency of site-specific integration; and disruption of the AAVS1 locus. As expected, increasing the AAV MOI resulted in an increase in the percentage of cells infected, with 80% of cells infected at an MOI of 10. Additional MOI only marginally effected a further increase in percentage of infected cells. In contrast to infection, we found very low levels of integration at MOIs of less than 10. At an MOI of 10, at which 80% of cells are infected, less than 5% of clonal cell lines contained integrated AAV DNA. At an MOI of 100 or greater, however, 35 to 40% of clonal cell lines contained integrated AAV DNA. Integration and the ability to rescue viral genomes were highly correlated. Analysis of integrated AAV indicated that essentially all integrants were AAVS1 site specific. Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 ( approximately 100% of infected cells). Rep expression by itself and in the presence of a plasmid integration substrate was able to mediate this disruption of the AAVS1 site. We further characterized the disruption event and demonstrated that it resulted in amplification of the AAVS1 locus. The data are consistent with a revised model of AAV integration that includes preliminary expansion of a defined region in AAVS1.

Chromosome Mapping↗
PubMed1996

Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line.

Adeno-associated virus-2 (AAV) can integrate in a site-specific manner to human chromosome 19 and is currently in phase I clinical trials for cystic fibrosis (CF) at Johns Hopkins Hospital. The goal of this study was to determine the fate of recombinant AAV containing the CFTR cDNA (AAV-CFTR) in an immortalized pseudotetraploid CF bronchial epithelial cell line (IB3-1) established from a patient with CF. Fluorescence in situ hybridization (FISH) and Southern blotting of DNA from IB3-1 cells infected with wild-type (wt) or recombinant AAV-CFTR were performed. CFRH2, an IB3-1 cell line with an estimated 15-20 integrated copies of CFTR cDNA, was used to test FISH sensitivity. All metaphase spreads had integrated copies: a single site in 36 of 56 (64.3%) and two sites within the same metaphase spread in 20 of 56 (35.7%). 3-CF-8, an IB3-1 cell line with integration of a partial CFTR cDNA (3.9 kb) was also analyzed by FISH. Integration was observed in 56 of 157 (35.7%) metaphase spreads examined. IB3-1 cells infected with wild-type AAV showed integration in 51 of 86 (59%) metaphase spreads examined. Of 51 integrations, 48 (94%) were to chromosome 19. Examination of 67 metaphase chromosome spreads of IB3-1 cells infected with AAV-CFTR vector (Azero) identified four integrations (6%) to different chromosomes. No integration was to chromosome 19 which differs significantly (P < 0.0001) from wild-type AAV. We then analyzed the A35 cell line, a clone of Azero selected for stable CFTR expression. Genomic DNA from A35 cells did not show a single site of integration; however episomal AAV-CFTR sequences were abundant in the low molecular weight DNA fraction. Examination of 68 metaphase chromosome preparations identified eight distinct integrations, none to chromosome 19. These studies show that FISH is sensitive for the detection of a partial CFTR cDNA integration. Wild-type AAV integrates in a predominantly site-specific fashion. Recombinant AAV-CFTR integrates at low frequency in a nonspecific manner and persists in episomal form in this epithelial cell line.

Bronchi↗
PubMed2005

Researchers' experiences, positive and negative, in integrative landscape projects.

Integrative (interdisciplinary and transdisciplinary) landscape research projects are becoming increasingly common. As a result, researchers are spending a larger proportion of their professional careers doing integrative work, participating in shifting interdisciplinary teams, and cooperating directly with non-academic participants. Despite the growing importance of integrative research, few studies have investigated researchers' experiences in these projects. How do researchers perceive the outcomes of integrative projects, or career effects? Do they view the projects generally as successes or failures? This study analyses researchers' experiences in integrative landscape studies and investigates what factors shape these experiences. The data stems from 19 semi-structured qualitative interviews and a Web-based survey among 207 participants in integrative landscape research projects. It finds that researchers experience participation in integrative projects as positive, in particular discussions among participants, networking, teamwork, and gaining new insights and skills. Furthermore, most researchers perceive the projects as successful and as having a positive effect on their careers. Less positive aspects of integration relate to publications and merit points. Factors found to contribute to positive experiences include reaching a high degree of integration amongst the involved disciplines, common definitions of integrative research concepts, and projects that include a large share of fundamental research as well as projects with many project outcomes. Based on these findings, we advise future projects to plan for integration, facilitate discussions, and reach agreement on integrative concepts. We suggest that aspects of fundamental research be included in integrative projects. We also suggest that planning be done at an early stage for peer-reviewed publications, to ensure that participants gain merit points from their participation in integrative research efforts.

Conservation of Natural Resources↗
PubMed1988

Microdeletion associated with the integration process of hepatitis B virus DNA.

Hepatitis B virus (HBV) DNA is often found in integrated form in hepatocellular carcinomas (HCC) and in non-cancerous liver cells of chronic carriers of HBV. However, the process of integration has not been well understood. Analyses of integrant DNA was expected to give clues. However, the majority of the integrants are products of multistep rearrangements following integrations, and analysis of randomly selected samples do not give clues for understanding the process of primary integrant formation. Therefore, one must select an appropriate integrant(s) that has a simple structure. We surveyed a collection of integrants prepared from many HCC's, and found one integrant that has the simplest structure so far studied: The viral genome is almost complete, is joined to cellular DNA using the cohesive end of the viral DNA, and furthermore, the "left" and "right" flanking cellular DNA's are almost contiguous. Analysis of the unoccupied sites in cellular DNA showed that, although almost contiguous, it has generated a microdeletion (15 base pairs) in the target sequence. This target sequence has a short region of homology to the sequence in the viral genome located close to the junction. One integrant with strikingly similar features has been reported independently. Two similar, but not identical cases from literatures could be added to this category. Therefore, the integrants with these properties may represent a unique category among those prepared from hepatocellular carcinomas. Based on these findings, we propose that this integrant represents the primary product of integration, and discuss the intermediate acting in the process of integration.

Base Sequence↗
PubMed2005

Integration site selection by HIV-based vectors in dividing and growth-arrested IMR-90 lung fibroblasts.

DNA integration is a defining step in the retroviral life cycle and the basis of stable gene transfer in retrovirus-based gene therapy. Previous studies of integration by HIV-based vectors have shown that integration is not random, but favored in active transcription units. Studies to date have focused on HIV integration in dividing cells, leaving open the question of whether integration target site selection might differ in nondividing cells. According to one idea, division of the host cell might be required for favored integration in transcription units, possibly as a result of chromatin remodeling during DNA replication. Here we have investigated this issue by comparing integration in dividing IMR-90 primary lung fibroblasts to integration in nondividing IMR-90 cells arrested in G1 by serum starvation and contact inhibition. We identified several differences in integration site selection in arrested versus dividing cells, including the frequency of integration in transcription units and in gene-rich regions. However, integration in nondividing cells was in fact more favored in transcription units, contrary to the idea that cell division was important for this bias. These data provide the first view of lentiviral integration in nondividing cells and help constrain models for the mechanism of favored integration in genes.

Cell Division↗
PubMed2006

Genomic sites of human immunodeficiency virus type 2 (HIV-2) integration: similarities to HIV-1 in vitro and possible differences in vivo.

Retroviruses have distinct preferences in integration site selection in the host cell genome during in vitro infection, with human immunodeficiency virus type 1 (HIV-1) integration strongly favoring transcriptional units. Additionally, studies with HIV-1 have shown that the genomic site of proviral integration may impact viral replication, with integration in heterochromatin associated with a block in viral transcription. HIV-2 is less pathogenic than HIV-1 and is believed to have a lower replication rate in vivo. Although differences in integration site selection between HIV-2 and HIV-1 could potentially explain the attenuated pathogenicity of HIV-2, no studies have characterized integration site selection by HIV-2. In this study, we mapped 202 HIV-2 integration sites during in vitro infection of peripheral blood mononuclear cells with a primary HIV-2 isolate. In addition, we assayed for in vivo proviral integration within heterochromatin in 21 HIV-1-infected subjects and 23 HIV-2-infected subjects, using an alphoid repeat PCR assay. During in vitro infection, HIV-2 displayed integration site preferences similar to those previously reported for HIV-1. Notably, 82% of HIV-2 integrations mapped to Refseq genes, and integration strongly favored regions of the genome with high gene density and high GC content. Though rare, the proportion of HIV-2 subjects with evidence of proviral integration within heterochromatin in vivo was higher than that of HIV-1-infected subjects. It is therefore possible that integration site selection may play a role in the differences in HIV-1 and HIV-2 in vivo pathogenesis.

Base Composition↗
PubMed2006

A helper-dependent capsid-modified adenovirus vector expressing adeno-associated virus rep78 mediates site-specific integration of a 27-kilobase transgene cassette.

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.

Adenoviridae↗
PubMed1997

Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.

The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.

Base Sequence↗
PubMed1998

Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid repeats are a disfavored target.

Integration of retroviral cDNA into host chromosomal DNA is an essential and distinctive step in viral replication. Despite considerable study, the host determinants of sites for integration have not been fully clarified. To investigate integration site selection in vivo, we used two approaches. (i) We have analyzed the host sequences flanking 61 human immunodeficiency virus type 1 (HIV-1) integration sites made by experimental infection and compared them to a library of 104 control sequences. (ii) We have also analyzed HIV-1 integration frequencies near several human repeated-sequence DNA families, using a repeat-specific PCR-based assay. At odds with previous reports from smaller-scale studies, we found no strong biases either for or against integration near repetitive sequences such as Alu or LINE-1 elements. We also did not find a clear bias for integration in transcription units as proposed previously, although transcription units were found somewhat more frequently near integration sites than near controls. However, we did find that centromeric alphoid repeats were selectively absent at integration sites. The repeat-specific PCR-based assay also indicated that alphoid repeats were disfavored for integration in vivo but not as naked DNA in vitro. Evidently the distinctive DNA organization at centromeres disfavors cDNA integration. We also found a weak consensus sequence for host DNA at integration sites, and assays of integration in vitro indicated that this sequence is favored as naked DNA, revealing in addition an influence of target primary sequence.

Binding Sites↗
PubMed1999

Double-stranded linear duck hepatitis B virus (DHBV) stably integrates at a higher frequency than wild-type DHBV in LMH chicken hepatoma cells.

Integration of hepadnavirus DNAs into host chromosomes can have oncogenic consequences. Analysis of host-viral DNA junctions of DHBV identified the terminally duplicated r region of the viral genome as a hotspot for integration. Since the r region is present on the 5' and 3' ends of double-stranded linear (DSL) hepadnavirus DNAs, these molecules have been implicated as integration precursors. We have produced a LMH chicken hepatoma cell line (LMH 66-1 DSL) which replicates exclusively DSL duck hepatitis B virus (DHBV) DNA. To test whether linear DHBV DNAs integrate more frequently than the wild type open circular DHBV DNAs, we have characterized the integration frequency in LMH 66-1 DSL cells by using a subcloning approach. This approach revealed that 83% of the LMH 66-1 DSL subclones contained new integrations, compared to only 16% of subclones from LMH-D2 cells replicating wild-type open circular DHBV DNA. Also, a higher percentage of the LMH 66-1 DSL subclones contained two or more new integrations. Mathematical analysis suggests that the DSL DHBV DNAs integrated stably once every three generations during subcloning whereas wild-type DHBV integrated only once every four to five generations. Cloning and sequencing of new integrations confirmed the r region as a preferred integration site for linear DHBV DNA molecules. One DHBV integrant was associated with a small deletion of chromosomal DNA, and another DHBV integrant occurred in a telomeric repeat sequence.

Animals↗
PubMed2005

Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.

Recombinant adeno-associated virus (rAAV) vector holds promise for gene therapy. Despite a low frequency of chromosomal integration of vector genomes, recent studies have raised concerns about the risk of rAAV integration because integration occurs preferentially in genes and accompanies chromosomal deletions, which may lead to loss-of-function insertional mutagenesis. Here, by analyzing 347 rAAV integrations in mice, we elucidate novel features of rAAV integration: the presence of hot spots for integration and a strong preference for integrating near gene regulatory sequences. The most prominent hot spot was a harmless chromosomal niche in the rRNA gene repeats, whereas nearly half of the integrations landed near transcription start sites or CpG islands, suggesting the possibility of activating flanking cellular disease genes by vector integration, similar to retroviral gain-of-function insertional mutagenesis. Possible cancer-related genes were hit by rAAV integration at a frequency of 3.5%. In addition, the information about chromosomal changes at 218 integration sites and 602 breakpoints of vector genomes have provided a clue to how vector terminal repeats and host chromosomal DNA are joined in the integration process. Thus, the present study provides new insights into the risk of rAAV-mediated insertional mutagenesis and the mechanisms of rAAV integration.

Animals↗
PubMed2005

Alteration of gene expression in human hepatocellular carcinoma with integrated hepatitis B virus DNA.

PURPOSE: Integration of hepatitis B virus (HBV) DNA into the human genome is one of the most important steps in HBV-related carcinogenesis. This study attempted to find the link between HBV DNA, the adjoining cellular sequence, and altered gene expression in hepatocellular carcinoma (HCC) with integrated HBV DNA. EXPERIMENTAL DESIGN: We examined 15 cases of HCC infected with HBV by cassette ligation-mediated PCR. The human DNA adjacent to the integrated HBV DNA was sequenced. Protein coding sequences were searched for in the human sequence. In five cases with HBV DNA integration, from which good quality RNA was extracted, gene expression was examined by cDNA microarray analysis. RESULTS: The human DNA sequence successive to integrated HBV DNA was determined in the 15 HCCs. Eight protein-coding regions were involved: ras-responsive element binding protein 1, calmodulin 1, mixed lineage leukemia 2 (MLL2), FLJ333655, LOC220272, LOC255345, LOC220220, and LOC168991. The MLL2 gene was expressed in three cases with HBV DNA integrated into exon 3 of MLL2 and in one case with HBV DNA integrated into intron 3 of MLL2. Gene expression analysis suggested that two HCCs with HBV integrated into MLL2 had similar patterns of gene expression compared with three HCCs with HBV integrated into other loci of human chromosomes. CONCLUSIONS: HBV DNA was integrated at random sites of human DNA, and the MLL2 gene was one of the targets for integration. Our results suggest that HBV DNA might modulate human genes near integration sites, followed by integration site-specific expression of such genes during hepatocarcinogenesis.

Adult↗
PubMed2002

Integration of service systems for homeless persons with serious mental illness through the ACCESS program. Access to Community Care and Effective Services and Supports.

OBJECTIVE: The aim of this study was to evaluate the first of the two core questions around which the ACCESS (Access to Community Care and Effective Services and Supports) evaluation was designed: Does implementation of system-change strategies lead to better integration of service systems? METHODS: The study was part of the five-year federal ACCESS service demonstration program, which sought to enhance integration of service delivery systems for homeless persons with serious mental illness. Data were gathered from nine randomly selected experimental sites and nine comparison sites in 15 of the nation's largest cities on the degree to which each site implemented a set of systems integration strategies and the degree of systems integration that ensued among community agencies across five service sectors: mental health, substance abuse, primary care, housing, and social welfare and entitlement services. Integration was measured across all interorganizational relationships in the local service networks (overall systems integration) and across relationships involving only the primary ACCESS grantee organization (project-centered integration). RESULTS: Contrary to expectations, the nine experimental sites did not demonstrate significantly greater overall systems integration than the nine comparison sites. However, the experimental sites demonstrated better project-centered integration than the comparison sites. Moreover, more extensive implementation of strategies for system change was associated with higher levels of overall systems integration as well as project-centered integration at both the experimental sites and the comparison sites. CONCLUSIONS: The ACCESS demonstration was successful in terms of project-centered integration but not overall system integration.

Community Mental Health Services↗
PubMed1996

Integrated care for asthma: matching care to the patient.

The purpose of the present study was to investigate whether criteria associated with assignment of asthma patients between general practice (GP) care alone, integrated care (shared between GP care and hospital clinic) or conventional specialist review could be identified, and whether outcomes for these patients differed over the next 12 months. Seven hundred and sixty four patients with a diagnosis of asthma and previously assigned to either integrated care or clinic care were reviewed after 1 year and reassigned. These patients were then followed for another 12 months and clinical data were collected over this time. After 12 months in clinic care or integrated care, assignment to integrated care was predicted by previous participation in integrated care (OR 2.94), patient preference for integrated care (OR 3.7), no admission (OR 1.56), fewer steroid courses during the previous year (OR 0.88) and nonattendance at review (OR 0.43) in the previous 12 months. Patient discharge to GP care was predicted by higher level of forced expiratory volume in one second (FEV1) (OR 1.49), lower number of GP consultations for troublesome asthma (OR 0.78), and nonattendance for review in the preceding year (OR 2.15). In the following 12 months, the three groups of patients differed significantly in hospital admissions (Discharged = 0.008; Integrated = 0.12; Clinic = 0.31), bronchodilators prescribed (Discharged = 8.5; Integrated = 10.2; Clinic = 13.9), GP consultations (Discharged = 1.3; Integrated = 3.0; Clinic = 4.1) and oral steroid courses (Discharged = 0.62; Integrated = 1.7; Clinic = 2.4). Patients assigned to integrated care, clinic care or discharged to general practice care form three distinct patient populations differing retrospectively and prospectively in morbidity and admission risk. In particular, patients assigned to integrated care fall midway in risk and morbidity between those discharged or those retained in clinic care. These results suggest that integrated care provides general practitioners with a system of management for asthma patients, for whom they do not wish frequent specialist review but who they do not believe can safely be discharged to general practice care only.

Adolescent↗