Factors associated with nursing home placement of all patients admitted for inpatient rehabilitation in Singapore community hospitals from 1996 to 2005: A disease stratified analysis

10.1371/journal.pone.0082697PLoS ONE812-POLN

HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells

Background The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). Methods We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean$^{+}$/mCherry$^{+}$) and inactive (Cerulean$^{-}$/mCherry$^{+}$) HIV-1 promoters were characterised. Results Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Conclusion Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity

Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4(+) T cells

The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells

Preclinical Evaluation of a Novel TALEN Targeting CCR5 Confirms Efficacy and Safety in Conferring Resistance to HIV-1 Infection

Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts

Low prevalence of transmitted HIV-1 drug resistance detected by a dried blood spot (DBS)-based next-generation sequencing (NGS) method in newly diagnosed individuals in Cameroon in the years 2015-16

Objectives To determine the most recent prevalence, transmission patterns and risk factors of transmitted drug-resistance mutations (TDRMs) in Cameroon, we initiated a multicentre study monitoring HIV-1 drug resistance in newly HIV-1-diagnosed individuals using a novel next-generation sequencing (NGS) assay applicable to fingerprick dried blood spot (DBS) samples. Methods Fingerprick DBS samples and questionnaires were collected from 360 newly HIV-1-diagnosed individuals in four hospitals in urban areas in Cameroon in the years 2015-16. We developed an HIV-1 protease and reverse transcriptase drug resistance genotyping assay applicable to DBS samples and HIV-1 genomes of groups M, N and O. The WHO 2009 list of mutations for surveillance of transmitted drug-resistant HIV strains was used to analyse TDRMs. Results Applying our 'DBS-NGS-genotypic resistance test', baseline HIV-1 drug resistance data were successfully obtained from 82.8% (298/360) of newly diagnosed individuals. At nucleotide frequencies >15%, TDRMs to NRTIs were observed in 3.0% (9/298), to NNRTIs in 4.0% (12/298) and to PIs in 1.3% (3/240). The NNRTI mutation K103N was most commonly detected (2.7%). Expanding the analysis to low-abundance TDRMs, i.e. 3%-15%, 12 additional individuals (4.0%) harbouring TDRMs were identified. Having unprotected sex with a known HIV-1-positive person was significantly associated with the transmission of DRMs (adjusted OR 9.6; 95% CI 1.79-51.3). Conclusions The prevalence of transmitted HIV-1 drug resistance is currently low in the study sites in Cameroon. Evidence of some risky sexual behaviours depicts a public health problem with possible implications for the prevention of new HIV-1 infections

Spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle as revealed by a genetic-insulators-containing dual-fluorescence HIV-1-based vector

Long-lived latently HIV-1-infected cells represent a barrier to cure. We developed a dual-fluorescence HIV-1-based vector containing a pair of genetic insulators flanking a constitutive fluorescent reporter gene to study HIV-1 latency. The protective effects of these genetic insulators are demonstrated through long-term (up to 394 days) stable fluorescence profiles in transduced SUP-T1 cells. Analysis of 1,941 vector integration sites confirmed reproduction of HIV-1 integration patterns. We sorted monoclonal cells representing latent HIV-1 infections and found that both vector integration sites and integrity of the vector genomes influence the reactivation potentials of latent HIV-1 promoters. Interestingly, some latent monoclonal cells exhibited a small cell subpopulation with a spontaneously reactivated HIV-1 promoter. Higher expression levels of genes involved in cell cycle progression are observed in these cell subpopulations compared to their counterparts with HIV-1 promoters that remained latent. Consistently, larger fractions of spontaneously reactivated cells are in the S and G2 phases of the cell cycle. Furthermore, genistein and nocodazole treatments of these cell clones, which halted cells in the G2 phase, resulted in a 1.4-2.9-fold increase in spontaneous reactivation. Taken together, our HIV-1 latency model reveals that the spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle

HIV-1 integration sites in CD4+ T cells during primary, chronic, and late presentation of HIV-1 infection

HIV-1 is capable of integrating its genome into that of its host cell. We examined the influence of the activation state of CD4+ T cells, the effect of antiretroviral therapy (ART), and the clinical stage of HIV-1 infection on HIV-1 integration site features and selection. HIV-1 integration sites were sequenced from longitudinally sampled resting and activated CD4+ T cells from 12 HIV-1–infected individuals. In total, 589 unique HIV-1 integration sites were analyzed: 147, 391, and 51 during primary, chronic, and late presentation of HIV-1 infection, respectively. As early as during primary HIV-1 infection and independent of the activation state of CD4+ T cells collected on and off ART, HIV-1 integration sites were preferentially detected in recurrent integration genes, genes associated with clonal expansion of latently HIV-1–infected CD4+ T cells, cancer-related genes, and highly expressed genes. The preference for cancer-related genes was more pronounced at late stages of HIV-1 infection. Host genomic features of HIV-1 integration site selection remained stable during HIV-1 infection in both resting and activated CD4+ T cells. In summary, characteristic HIV-1 integration site features are preestablished as early as during primary HIV-1 infection and are found in both resting and activated CD4+ T cells

HIV-1 integration sites in CD4+ T cells during primary, chronic, and late presentation of HIV-1 infection

HIV-1 is capable of integrating its genome into that of its host cell. We examined the influence of the activation state of CD4+ T cells, the effect of antiretroviral therapy (ART), and the clinical stage of HIV-1 infection on HIV-1 integration site features and selection. HIV-1 integration sites were sequenced from longitudinally sampled resting and activated CD4+ T cells from 12 HIV-1–infected individuals. In total, 589 unique HIV-1 integration sites were analyzed: 147, 391, and 51 during primary, chronic, and late presentation of HIV-1 infection, respectively. As early as during primary HIV-1 infection and independent of the activation state of CD4+ T cells collected on and off ART, HIV-1 integration sites were preferentially detected in recurrent integration genes, genes associated with clonal expansion of latently HIV-1–infected CD4+ T cells, cancer-related genes, and highly expressed genes. The preference for cancer-related genes was more pronounced at late stages of HIV-1 infection. Host genomic features of HIV-1 integration site selection remained stable during HIV-1 infection in both resting and activated CD4+ T cells. In summary, characteristic HIV-1 integration site features are preestablished as early as during primary HIV-1 infection and are found in both resting and activated CD4+ T cells

Low prevalence of transmitted HIV-1 drug resistance detected by a dried blood spot (DBS)-based next-generation sequencing (NGS) method in newly diagnosed individuals in Cameroon in the years 2015-16

Objectives To determine the most recent prevalence, transmission patterns and risk factors of transmitted drug-resistance mutations (TDRMs) in Cameroon, we initiated a multicentre study monitoring HIV-1 drug resistance in newly HIV-1-diagnosed individuals using a novel next-generation sequencing (NGS) assay applicable to fingerprick dried blood spot (DBS) samples. Methods Fingerprick DBS samples and questionnaires were collected from 360 newly HIV-1-diagnosed individuals in four hospitals in urban areas in Cameroon in the years 2015-16. We developed an HIV-1 protease and reverse transcriptase drug resistance genotyping assay applicable to DBS samples and HIV-1 genomes of groups M, N and O. The WHO 2009 list of mutations for surveillance of transmitted drug-resistant HIV strains was used to analyse TDRMs. Results Applying our 'DBS-NGS-genotypic resistance test', baseline HIV-1 drug resistance data were successfully obtained from 82.8% (298/360) of newly diagnosed individuals. At nucleotide frequencies >15%, TDRMs to NRTIs were observed in 3.0% (9/298), to NNRTIs in 4.0% (12/298) and to PIs in 1.3% (3/240). The NNRTI mutation K103N was most commonly detected (2.7%). Expanding the analysis to low-abundance TDRMs, i.e. 3%-15%, 12 additional individuals (4.0%) harbouring TDRMs were identified. Having unprotected sex with a known HIV-1-positive person was significantly associated with the transmission of DRMs (adjusted OR 9.6; 95% CI 1.79-51.3). Conclusions The prevalence of transmitted HIV-1 drug resistance is currently low in the study sites in Cameroon. Evidence of some risky sexual behaviours depicts a public health problem with possible implications for the prevention of new HIV-1 infections