ORCID Profile
0000-0002-2531-794X
Current Organisation
UNSW Sydney
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Publisher: Japanese Society of Veterinary Science
Date: 1997
DOI: 10.1292/JVMS.59.841
Abstract: Quantitative measurement of reverse transcriptase-inhibiting (RTI) antibodies in Japanese household cats naturally infected with feline immunodeficiency virus (FIV) was performed by poly A-linked colorimetric reverse transcriptase assay (PAC-RTA). Eight FIV-seropositive plasma s les were diluted twofold from 1:10 to 1:160 and incubated with FIV RT. Fifty percent RTI activity (RTI50) was calculated from a dose response PAC-RTA curve. The plasma of FIV-seropositive cats showed different RTI activities against two Japanese isolates and Petaluma strain. Six of eight plasma s les showed RTI activities against the Japanese isolates (subtype B), but only one showed RTI activity against Petaluma strain (subtype A). It is important to use the appropriate strain as a source of RT for detection of RTI antibody in cats.
Publisher: Elsevier BV
Date: 1993
DOI: 10.1016/0166-0934(93)90159-O
Abstract: A colorimetric assay for reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) was developed using a double labelled (biotin and digoxigenin) deoxyuridine triphosphate mixture instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from oligodeoxythymidylic acid (oligo-dT) contained both biotin and digoxigenin labels. This nucleotide was bound to streptavidin-magnetic beads by the reaction to biotin. At the detection step, an alkaline phosphatase conjugated antibody to digoxigenin was added, followed by the reaction of a colorimetric substrate for this enzyme. This RT assay was comparable to the isotopic RT assay using purified AMV-RT and two strains of HIV grown in cell lines. In addition it was equivalent to the isotopic RT assay for analysis of the time course of in vitro infection of human peripheral blood lymphocytes by HIV-1. The total assay time after the RT reaction step was less than one hour.
Publisher: Elsevier BV
Date: 10-2016
DOI: 10.1016/J.VACCINE.2016.09.009
Abstract: Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2021
Publisher: Mary Ann Liebert Inc
Date: 10-2013
Publisher: Oxford University Press (OUP)
Date: 07-11-2011
DOI: 10.1093/NAR/GKR891
Publisher: American Society for Microbiology
Date: 15-10-2017
DOI: 10.1128/JVI.00744-17
Abstract: Viruses manipulate the complex interferon and interferon-stimulated gene (ISG) system in different ways. We have previously shown that HIV inhibits type I and III interferons in its key target cells but directly stimulates a subset of ISGs in macrophages and dendritic cells, many of which are antiviral. Here, we examine the mechanism of induction of ISGs and show this occurs in two phases. The first phase was transient (0 to 24 h postinfection [hpi]), induced mainly by extracellular vesicles and one of its component proteins, HSP90α, contained within the HIV inoculum. The second, dominant, and persistent phase ( hpi) was induced via newly transcribed HIV RNA and sensed via RIGI, as shown by the reduction in ISG expression after the knockdown of the RIGI adaptor, MAVS, by small interfering RNA (siRNA) and the inhibition of both the initiation and elongation of HIV transcription by short hairpin RNA (shRNA) transcriptional silencing. We further define the induction pathway, showing sequential HIV RNA stimulation via Tat, RIGI, MAVS, IRF1, and IRF7, also identified by siRNA knockdown. IRF1 also plays a key role in the first phase. We also show that the ISGs IFIT1 to -3 inhibit HIV production, measured as extracellular infectious virus. All induced antiviral ISGs probably lead to restriction of HIV replication in macrophages, contributing to a persistent, noncytopathic infection, while the inhibition of interferon facilitates spread to adjacent cells. Both may influence the size of macrophage HIV reservoirs in vivo . Elucidating the mechanisms of ISG induction may help in devising immunotherapeutic strategies to limit the size of these reservoirs. IMPORTANCE HIV, like other viruses, manipulates the antiviral interferon and interferon-stimulated gene (ISG) system to facilitate its initial infection and establishment of viral reservoirs. HIV specifically inhibits all type I and III interferons in its target cells, including macrophages, dendritic cells, and T cells. It also induces a subset of over 20 ISGs of differing compositions in each cell target. This occurs in two temporal phases in macrophages. Extracellular vesicles contained within the inoculum induce the first, transient phase of ISGs. Newly transcribed HIV RNA induce the second, dominant ISG phase, and here, the full induction pathway is defined. Therefore, HIV nucleic acids, which are potent inducers of interferon and ISGs, are initially concealed, and antiviral ISGs are not fully induced until replication is well established. These antiviral ISGs may contribute to persistent infection in macrophages and to the establishment of viral reservoirs in vivo .
Publisher: Springer Science and Business Media LLC
Date: 10-2009
Publisher: The American Association of Immunologists
Date: 2011
Abstract: The CTL response in HLA-B*27+ HIV-infected in iduals is characterized by an immunodominant response to a conserved epitope in gag p24 (aa 263–272, KRWIILGLNK KK10). Mutations resulting in substitution of the arginine (R264) at position 2 of this epitope have been identified as escape mutations. Nineteen HLA-B*27+ long-term nonprogressors were identified from an Australian cohort with an average follow-up of 16 y following infection. Viral and host genetic factors impacting on disease progression were determined at multiple time points. Twelve of 19 had wild-type sequences at codon 264 at all time points 7 of 19 carried CTL escape variants. Median viral load and CD4+ T cell counts were not significantly different between these groups at enrollment. Viral load, as judged by levels at their last visit (1,700 and 21,000 RNA copies/ml, respectively p = 0.01) or by time-weighted area under the curve was higher in the escape group (p = 0.02). Escape mutants at other HLA-B*27–restricted epitopes were uncommon. Moreover, host polymorphisms, such as CCR5Δ32, CCR2-64I, and SDF1-3′A, or breadth of TCR repertoire responding to KK10 did not segregate to wild-type or escape groups. Host and viral factors were examined for a relationship to viral load. The only factor to affect viral load was the presence of the R264 escape mutations at the immunodominant epitope. CTL escape at R264 in the KK10 epitope is a major determinant of subsequent viral load in these HLA-B*27+ in iduals.
Publisher: Elsevier BV
Date: 03-2008
Abstract: RNA interference is a conserved process by which sequence-specific double-stranded RNA is converted into small interfering double-stranded RNAs (siRNAs) that can induce gene silencing via two pathways: post-transcriptional gene silencing and transcriptional gene silencing (TGS). We previously reported TGS of human immunodeficiency virus-1 (HIV-1) could be induced by siRNAs targeting regions within its 5'-long-terminal repeat (5'LTR) promoter region. Here we show that promoter-targeted siRNAs can also induce silencing of simian immunodeficiency virus (SIV) replication by similar mechanisms. Suppression of productive infection was achieved in two different cell lines: a CD4, CCR5, CXCR4 expressing HeLa cell line (MAGIC-5) and in a human lymphoid cell line (CEMx174). HpaII digestion demonstrated induction of methylation at a CpG site within the SIV promoter region following siRNA-induced suppression. Both 5-azacytidine (5-AzaC) and trichostatin A (TSA), inhibitors of DNA methyltransferases (DNMTs) and histone deacetylation, respectively, partially reversed the silencing effect. Furthermore, using chromatin immunoprecipitation (ChIP) assays we found enrichment in the region of the LTR of heterochromatin markers dimethylated histone 3 lysine 9 (H3K9) and trimethylated histone 3 lysine 27 (H3K27) in the siRNA silenced cultures. Together, these results strongly suggest certain siRNAs targeting the promoter region of SIV can effect viral silencing through the induction of epigenetic changes.
Publisher: American Society for Microbiology
Date: 15-10-2006
DOI: 10.1128/JVI.00249-06
Abstract: We recently found that human immunodeficiency virus (HIV)-specific CD4 + T cells express coreceptor CCR5 and activation antigen CD38 during early primary HIV-1 infection (PHI) but then rapidly disappear from the circulation. This cell loss may be due to susceptibility to infection with HIV-1 but could also be due to inappropriate apoptosis, an expansion of T regulatory cells, trafficking out of the circulation, or dysfunction. We purified CD38 +++ CD4 + T cells from peripheral blood mononuclear cells, measured their level of HIV-1 DNA by PCR, and found that about 10% of this population was infected. However, a small subset of HIV-specific CD4 + T cells also expressed CD127, a marker of long-term memory cells. Purified CD127 + CD4 + lymphocytes contained fivefold more copies of HIV-1 DNA per cell than did CD127-negative CD4 + cells, suggesting preferential infection of long-term memory cells. We observed no apoptosis of antigen-specific CD4 + T cells in vitro and only a small increase in CD45RO + CD25 + CD127dimCD4 + T regulatory cells during PHI. However, 40% of CCR5 + CD38 +++ CD4 + T cells expressed gut-homing integrins, suggesting trafficking through gut-associated lymphoid tissue (GALT). Furthermore, 80% of HIV-specific CD4 + T cells expressed high levels of the negative regulator CTLA-4 in response to antigen stimulation in vitro, which was probably contributing to their inability to produce interleukin-2 and proliferate. Taken together, the loss of HIV-specific CD4 + T cells is associated with a combination of an infection of CCR5 + CD127 + memory CD4 + T cells, possibly in GALT, and a high expression of the inhibitory receptor CTLA-4.
Publisher: Elsevier BV
Date: 09-2007
Publisher: Elsevier BV
Date: 02-2003
DOI: 10.1016/S1386-6532(02)00114-2
Abstract: It is clear that transmission of drug resistant HIV-1 is possible and occurs regularly. However, there is a lack of clarity concerning the true rate of this transmission in a given population, the impact of combination therapies on this rate, and the contribution of transmitted resistant virus to treatment failure either in an in idual or on a population basis. To provide a review of our current understanding of rates of transmission of drug resistant HIV-1 in various populations and to report the results of a study conducted to determine this rate in Sydney, Australia in the years 1992-2000. A review of the literature combined with a prospective study of antiretroviral drug resistance in 130 in iduals who were diagnosed with symptomatic primary infection at St. Vincent's Hospital, Sydney, Australia between 1992 and 2000. Sequencing of reverse transcriptase (RT) and protease (PR) was performed by the TruGene HIV-1 genotyping kit (Visible Genetics Inc.). The results found in the Sydney population contrast with much of the literature. The prevalence of mutations that conferred primary resistance to protease inhibitors (PIs) was only 0.8% at position V82I. Secondary mutations olymorphisms were seen in the PR at position L10I/V, K20R, M36I, L63P, A71T/V, or V77I in 60%. L63P was the most frequently found mutation (46.3%). The incidence of protease-resistant strains of HIV in primary HIV-1 infection did not change after the introduction of PIs in 1996. The distribution of the most common resistance mutations in the RT was as follows M41L (8.5%) and T215Y (8.5%) and K70R (4.8%). The frequency of mutations associated with NRTI resistance was significantly lower in the post 1995 s les (43.9 vs. 19.1%, P < 0.05). Moreover, both M41L and K70R, but not T215Y, occurred with significantly decreased frequency in the post 1995 s les. In contrast to other studies we found no increase in the rate of PR resistance and a decrease in the rate of RT resistance in recently transmitted virus over the period 1992-2000. The reasons for the differences between these results and those reported from elsewhere may relate to treatment regimens used in the transmitting population and may have implications for treatment policies in this country.
Publisher: Scientific Archives LLC
Date: 22-11-2019
DOI: 10.33696/AIDS.1.010
Publisher: Elsevier BV
Date: 11-1995
DOI: 10.1016/0166-0934(95)00073-5
Abstract: A colorimetric reverse transcriptase assay (cRT assay) was developed for quantitative detection of HIV-1. In this format, reverse transcriptase incorporates biotin-labeled dUTP onto oligo-dT primers hybridized to poly A templates. The templates are covalently bound to the surface of microtiter wells. The amount of incorporated biotin-labeled dUTP is measured by binding horseradish peroxidase conjugated streptavidin, washing away unbound peroxidase, adding colorimetric substrate and then reading with a standard colorimetric reader. The sensitivity of the assay is very good. As little as 3 x 10(5) molecules of recombinant HIV-RT can be detected after 20 h of reaction time. Direct comparison using 3 cultured clinical isolates indicates that this level of detection is equivalent to the commercially available p24 antigen capture assay and the HIV-RNA assay based on branched DNA signal lification. Other retroviruses, such as HIV-2 and feline immunodeficiency virus (FIV), can also be detected in this format. This non-isotopic assay is easy to perform and could provide a convenient and quantitative method for HIV study by monitoring reverse transcriptase, an essential activity in the infection process.
Publisher: American Society of Hematology
Date: 14-05-2009
Publisher: Elsevier BV
Date: 2005
DOI: 10.1111/J.1524-4733.2005.03068.X
Abstract: To investigate the impact of imputing EQ-5D values to allow for informative dropout and nonresponse in a longitudinal assessment of the health-related quality of life (HRQL) of liver transplant recipients. The EQ-5D was administered at defined time intervals pre- and post-transplantation to all adults who were listed to receive liver transplants as National Health Service (NHS) treatment at each of the six Department of Health designated centers in England and Wales over a time-period of 36 months (12 month recruitment period and 24 month follow-up period). During the course of the study missing data arose for two main reasons, informative dropout and nonresponse. Informative dropout was accounted for by giving those patients who died an EQ-5D score of 0 and those patients who were too ill to respond to an EQ-5D score equivalent to the 5th percentile of respondents for each time point pretransplantation. Nonresponse was accounted for using relatively naive approaches (last value carried forward, and upper/lower 95% confidence interval around the mean) and contrasted with a more sophisticated multiple imputation method. Adjusting for informative dropout in isolation resulted in a marked deterioration in mean scores over time pretransplant relative to the base case situation in which no such adjustments were made. Nevertheless, adjusting for informative dropout and/or nonresponders did not alter the base case conclusion of no statistically significant differences in mean EQ-5D scores over time pretransplant. In contrast, post-transplant data indicated highly statistically significant improvements in quality of life over time for the base case (P < 0.001) whereas no statistically significant improvements over time were found when informative dropout was allowed for in isolation (P = 0.402) or when informative dropout and nonresponse were allowed for simultaneously (P = 0.105-0.185). It is important that future studies which purport to assess the HRQL over time of patients, such as these with end-stage liver disease, include an allowance for informative dropout and nonresponse within the analysis.
Publisher: Elsevier BV
Date: 2013
DOI: 10.1038/MTNA.2013.64
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.MICINF.2009.02.003
Abstract: We previously reported prolonged HIV-1 transcriptional gene silencing by an RNA duplex targeting a sequence located within the NF-kappaB binding motif of the HIV-1 promoter in a susceptible HeLa cell line. Here we report extremely prolonged suppression of productive HIV-1 infection in a T-cell line (Molt-4) by a retrovirally delivered short-hairpin RNA (shRNA) targeting the same region (shkappaB). Following retroviral delivery of an shRNA we established shRNA-expressing CD4(+) T-cell lines. HIV-1 gene expression was profoundly suppressed for 1 year. Results of nuclear run-on assays and HIV-1 LTR-luciferase reporter assays revealed that shkappaB acted by inhibition of HIV-1 transcription. The effect was reversed by a histone deacetylase inhibitor, trichostatin-A (TSA), but not by a DNA methyltransferase inhibitor, 5-azacytidine (5-AzaC). Furthermore, chromatin immunoprecipitation assays (ChIP) demonstrated rapid, sustained induction of heterochromatin structures within the HIV-1 promoter region, with enrichment of histone 3 lysine 27 tri-methylation (H3K27me3) and H3K9 methylation. H3K27me3 enrichment was the most pronounced. This prolonged suppression could not be recapitulated by either retrovirally delivered anti-sense or sense strands alone or in combination. Our data strongly suggest that shkappaB induces high level, sustained transcriptional gene silencing of HIV-1 and offers the possibility of new therapeutic strategies.
Publisher: Elsevier BV
Date: 05-1993
Abstract: A chemiluminescent assay for reverse transcriptase (RT) of the human immunodeficiency virus 1 was developed using biotin-labeled oligodeoxythymidylic acid (biotin oligo-dT) and digoxigenin-deoxyuridine triphosphate instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from biotin oligo-dT contained digoxigenin labels. This nucleotide was bound to a streptavidin-coated microtiter plate by the reaction to biotin. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the reaction of a chemiluminescent substrate for this enzyme. This method shows very close correlation with the isotopic assay using purified avian myeloblastosis virus reverse transcriptase (RT). This assay was also compared with the isotopic RT assay using lymphocytes infected in vitro with HTLV-IIIB and again demonstrated a close correlation. The total assay time after the RT reaction step was less than 100 min.
Publisher: Elsevier BV
Date: 08-2008
Publisher: No publisher found
Date: 1993
DOI: 10.1016/0166-0934(93)90023-K
Abstract: A rapid and nonradioactive detection method for polymerase chain reaction (PCR) lified HIV-1 DNA was developed using a colorimetric detection system. Hybridization between biotin-labeled lified targets and digoxigenin-capture probes occurs in solution followed by efficient and rapid capture onto streptavidin-magnetic beads. The presence of the digoxigenin-capture probe hybridized with biotin-labeled targets is then detected by antidigoxigenin-alkaline phosphatase conjugates using a colorimetric substrate. This approach is highly sensitive and can detect less than 10 HIV targets prior to PCR in approximately 50 min.
Publisher: American Society for Microbiology
Date: 15-03-2014
DOI: 10.1128/JVI.03331-13
Abstract: The latent HIV reservoir is a major impediment to curing HIV infection. The contribution of CD4 + T cell activation status to the establishment and maintenance of the latent reservoir was investigated by enumerating viral DNA components in a cohort of 12 in iduals commencing antiretroviral therapy (ART) containing raltegravir, an integrase inhibitor. Prior to ART, the levels of total HIV DNA were similar across HLA-DR + and HLA-DR − (HLA-DR ± ) CD38 ± memory CD4 + T cell phenotypes episomal two-long terminal repeat (2-LTR) HIV DNA levels were higher in resting (HLA-DR − CD38 − ) cells, and this phenotype exhibited a significantly higher ratio of 2-LTR to integrated HIV DNA ( P = 0.002). After 1 year of ART, there were no significant differences across each of the memory phenotypes of any HIV DNA component. The decay dynamics of integrated HIV DNA were slow within each subset, and integrated HIV DNA in the resting HLA-DR − CD38 − subset per mm 3 of peripheral blood exhibited no significant decay (half-life of 25 years). Episomal 2-LTR HIV DNA decayed relative to integrated HIV DNA in resting cells with a half-life of 134 days. Surprisingly, from week 12 on, the decay rates of both total and episomal HIV DNA were lower in activated CD38 + cells. By weeks 24 and 52, HIV RNA levels in plasma were most significantly correlated with the numbers of resting cells containing integrated HIV DNA. On the other hand, total HIV DNA levels in all subsets were significantly correlated with the numbers of HLA-DR + CD38 − cells containing integrated HIV DNA. These results provide insights into the interrelatedness of cell activation and reservoir maintenance, with implications for the design of therapeutic strategies targeting HIV persistence. IMPORTANCE It is generally believed that HIV is not cleared by extensive antiretroviral therapy (ART) due to the difficulty in eradicating the latent reservoir in resting CD4 + T cells. New therapies that attempt to activate this reservoir so that immune or viral cytopathic mechanisms can remove those infected cells are currently being investigated. However, results obtained in this research indicate that activation, at least on some level, already occurs within this reservoir. Furthermore, we are the first to describe the dynamics of different HIV DNA species in resting and activated memory CD4+ T cell subsets that point to the role different levels of activation play in maintaining the HIV reservoir.
Publisher: The American Association of Immunologists
Date: 15-06-2012
Abstract: MicroRNAs (miRNAs) are ∼22-nt small RNAs that are important regulators of mRNA turnover and translation. Recent studies have shown the importance of the miRNA pathway in HIV-1 infection, particularly in maintaining latency. Our initial in vitro studies demonstrated that HIV-1–infected HUT78 cells expressed significantly higher IL-10 levels compared with uninfected cultures. IL-10 plays an important role in the dysregulated cytotoxic T cell response to HIV-1, and in silico algorithms suggested that let-7 miRNAs target IL10 mRNA. In a time course experiment, we demonstrated that let-7 miRNAs fall rapidly following HIV-1 infection in HUT78 cells with concomitant rises in IL-10. To show a direct link between let-7 and IL-10, forced overexpression of let-7 miRNAs resulted in significantly reduced IL-10 levels, whereas inhibition of the function of these miRNAs increased IL-10. To demonstrate the relevance of these results, we focused our attention on CD4+ T cells from uninfected healthy controls, chronic HIV-1–infected patients, and long-term nonprogressors. We characterized miRNA changes in CD4+ T cells from these three groups and demonstrated that let-7 miRNAs were highly expressed in CD4+ T cells from healthy controls and let-7 miRNAs were significantly decreased in chronic HIV-1 infected compared with both healthy controls and long-term nonprogressors. We describe a novel mechanism whereby IL-10 levels can be potentially modulated by changes to let-7 miRNAs. In HIV-1 infection, the decrease in let-7 miRNAs may result in an increase in IL-10 from CD4+ T cells and provide the virus with an important survival advantage by manipulating the host immune response.
Publisher: Japanese Society of Veterinary Science
Date: 1997
DOI: 10.1292/JVMS.59.425
Abstract: The method of the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) was developed and evaluated for the measurement of Mg(2+)-dependent reverse transcriptase (RT) activity of feline immunodeficiency virus (FIV). PAC-RTA was first evaluated for the detection of RT activity in the culture supernatant of FIV Petaluma strain. The detection limit of RT activity by PAC-RTA was about 10-fold better than that by the conventional non-radioisotopic RT assay kit. Then, PAC-RTA was evaluated for the indication of FIV isolation from cats naturally infected with FIV. FIV was isolated from peripheral blood mononuclear cells of 9 FIV-seropositive cats. The time course appearance of RT activity measured by PAC-RTA corresponded with the analysis of FIV antigen expression by indirect immunofluorescence. Finally, PAC-RTA evaluated the drug susceptibility of FIV. MYA-1 cells (feline T-lymphoblastoid cells) were infected with FIV and were cultured in the presence of various concentrations of anti-human immunodeficiency virus agents such as azidothymidine (AZT) or dextran sulfate. An inverse relationship between the RT activities and the concentrations of these agents in the culture supernatant was confirmed by PAC-RTA. PAC-RTA is easy to perform without using radioactive materials, and one plate can handle 96 s les at one time. By monitoring the RT activity, this assay is a useful method for FIV studies such as viral replication and drug susceptibility.
Publisher: The Japanese Association for Infectious Diseases
Date: 1995
DOI: 10.11150/KANSENSHOGAKUZASSHI1970.69.851
Abstract: Reverse transcriptase (RT) inhibiting antibody in a series of plasma of HIV-1-seropositive subjects was quantitatively measured by poly A-linked colorimetric microtiter plate assay. The plasma were obtained from 6 asymptomatic carrier (AC)s and from 3 patients who progressed to AIDS. They had been followed 29-51 months. RT inhibiting antibody levels in the plasma were measured by inhibition assay against HTLV-IIIB RT activity. In five of the 6 AC cases, RT inhibiting antibodies in the serial plasma maintained high levels, and 50% inhibiting titers of the serial plasma did not decrease throughout the observation periods (45-51 months). HIV isolation from peripheral blood mononuclear cell (PBMC) of these 5 ACs did not succeed, and HIV p24 antigens were not detected in the plasma. In one AC case (046) RT inhibiting antibody levels gradually decreased after 48 months. In this case, HIV p24 antigen was not detected in the serial plasma throughout the observation period (48 months), but HIV was isolated from PBMC after 27 months. On the other hand, RT inhibiting antibody levels in the serial plasma of all 3 patients who progressed to AIDS gradually decreased in observation periods (29-35 months). HIV strains were isolated from these 3 cases. These results suggest that reduction of RT inhibiting antibody levels correlate well with the success of HIV isolation and with progression of clinical manifestation.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2017
Publisher: Oxford University Press (OUP)
Date: 03-2001
DOI: 10.1086/318827
Abstract: CCR5 is the major coreceptor for human immunodeficiency virus (HIV) type 1 during primary infection. CCR5+ CD4 T lymphocytes were studied in subjects with primary HIV-1 infection (PHI) or acute Epstein-Barr virus (EBV) infection and in HIV-uninfected controls. The early decline of CD4 T lymphocytes during PHI resulted from depletion of CCR5- CD4 T lymphocytes. After antiretroviral therapy, Ki-67- CCR5- CD4 T cell counts rapidly increased in the circulation, which suggests that the initial decrease was due to an alteration in trafficking and/or sequestration. In the CCR5+ subset of CD4 T cells, there was an elevation in the proliferative (Ki-67+) fraction during PHI, yet their total number remained in the normal range. In contrast, in acute EBV infection, proliferating CCR5+ CD4 T cells accumulated to very high levels, suggesting they have an important role in the early antiviral response, which may be impaired in HIV-1 infection.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 2003
DOI: 10.1097/00002030-200301240-00020
Abstract: Rates of antiretroviral resistance in recently transmitted virus in Sydney, Australia fluctuated over the past decade, influenced by treatment trends. Current rates of drug resistance are not high in historical terms or compared with those reported. Rates of resistance to reverse transcriptase inhibitors peaked in the mid-1990s, fell dramatically with the introduction of combination therapy and appear to have plateaued at 10-15% over the past 3 years. Primary resistance mutations in the protease gene are still rare.
Publisher: American Society for Microbiology
Date: 2000
DOI: 10.1128/JCM.38.9.3450-3452.2000
Abstract: Four of 107 s les obtained from hepatitis C virus (HCV) carriers showed lower HCV core antigen levels in a fluorescence enzyme immunoassay (FEIA) than expected from corresponding HCV RNA levels. Nucleotide sequencing revealed a mutation in the HCV core region (Thr49Pro) that appears to have reduced the FEIA sensitivity.
Publisher: Springer Science and Business Media LLC
Date: 07-09-2017
DOI: 10.1038/S41598-017-11405-8
Abstract: Liver disease is one of the main contributors to the increased levels of morbidity and mortality seen in the HIV-1-infected, ART-treated population. Circulating miRNAs, particularly those located inside extracellular vesicles, are seen as promising biomarkers for a number of human disease conditions, including liver-related diseases. Here, we show that serum levels of miR-122 and miR-200a are greater in HIV/HCV co-infected in iduals compared to HIV-1 mono-infected in iduals. We also show that miR-122 and miR-200a are elevated in ART-treated, HIV-1-infected in iduals prior to the development of fatal liver disease, suggesting that these miRNA may have some potential clinical utility as biomarkers. While this study is hypothesis generating, it shows clearly that both miR-122 and miR-200a are promising novel biomarkers for liver disease in the ART-treated, HIV-1-infected population.
Publisher: Elsevier BV
Date: 10-1993
DOI: 10.1016/0166-0934(93)90054-U
Abstract: An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.
Publisher: Wiley
Date: 13-07-2018
DOI: 10.1111/HIV.12532
Abstract: The Maraviroc Switch (MARCH) study week 48 data demonstrated that maraviroc, a chemokine receptor-5 (CCR5) inhibitor, was a safe and effective switch for the ritonavir-boosted protease inhibitor (PI/r) component of a two nucleos(t)ide reverse transcriptase inhibitor [N(t)RTI] plus PI/r-based antiretroviral regimen in patients with R5-tropic virus. Here we report the durability of this finding. MARCH, an international, multicentre, randomized, 96-week open-label switch study, enrolled HIV-1-infected adults with R5-tropic virus who were stable (> 24 weeks) and virologically suppressed [plasma viral load (pVL) < 50 HIV-1 RNA copies/mL]. Participants were randomized to continue their current PI/r-based regimen (PI/r) or to switch to MVC plus two N(t)RTIs (MVC) (1:2 randomization). The primary endpoint was the difference in the proportion with pVL < 200 copies/mL at 96 weeks. The switch arm was defined as noninferior if the lower limit of the 95% confidence interval (CI) for the difference was < -12% in the intention-to-treat (ITT) population. Safety endpoints (the difference in the mean change from baseline or a comparison of proportions) were analysed as key secondary endpoints. Eighty-two (PI/r) and 156 (MVC) participants were randomized and included in the ITT analysis 71 (87%) and 130 (83%) were in follow-up and on therapy at week 96. At week 96, 89.0% and 90.4% in the PI/r and MVC arms, respectively, had pVL < 50 copies/mL (95% CI -6.6, 10.2). Moreover, in those switching away from PI/r, there were significant reductions in mean total cholesterol (differences 0.31 mmol/L P = 0.02) and triglycerides (difference 0.44 mmol/L P < 0.001). Changes in CD4 T-cell count, renal function, and serious and nonserious adverse events were similar in the two arms. MVC as a switch for a PI/r is safe and effective at maintaining virological suppression while having significant lipid benefits over 96 weeks.
Publisher: Public Library of Science (PLoS)
Date: 16-02-2011
Publisher: Elsevier BV
Date: 07-1992
DOI: 10.1016/0166-0934(92)90174-C
Abstract: A rapid and sensitive method for detecting HIV-1 DNA sequences lified by polymerase chain reaction (PCR) is described. The method uses solution phase hybridization for rapid and efficient annealing between digoxigenin-labeled targets and biotinylated capture probes. Hybrids containing biotin are captured onto streptavidin coated microwells and all other PCR components are washed away, including spurious lification products. The presence of the digoxigenin-labeled lified HIV target is then detected by anti-digoxigenin-alkaline phosphatase conjugates using the chemiluminescent substrate PPD. This approach maintains high specificity by nucleic acid dependent capture, and high sensitivity by efficient solution hybridization. The method is rapid (2 hours), and capable of detecting 10 HIV targets.
Publisher: Public Library of Science (PLoS)
Date: 10-02-2017
Publisher: Bentham Science Publishers Ltd.
Date: 08-2009
DOI: 10.2174/156802609789630875
Abstract: Small RNA molecules, including small interfering RNA (siRNA) and micro RNA (miRNA), have rapidly emerged as important regulators of gene expression. Recent articles have demonstrated RNA mediated complex induced transcriptional gene silencing (TGS) occurring in the nucleus. Originally the small RNA mediated TGS pathway has been reported in yeast and plants, currently a number of articles strongly suggest that this newly established gene silencing mechanism is present in mammals. RNA mediated TGS has been reported for various human promoters including inhibition of tumor susceptibility genes, X-chromosome inactivation and suppression of human chemokine receptor. Small RNAs can inhibit human viral infection through the TGS pathway. Prolonged HIV-1 transcriptional gene silencing by an RNA duplex targeting a sequence located within the HIV-1 promoter has been reported initially using a susceptible adherent cell line model and recently prolonged suppression of productive HIV-1 infection in a T-cell line model has been demonstrated by a retrovirally delivered short-hairpin RNA (shRNA) targeting the same region. RNA mediated gene silencing in HIV-1 infection can induce heterochromatin (closed) structure in the promoter regions, which is consistent to those changes seen in studies of various RNA directed TGS in various human promoter regions. More recent observations suggest transcriptional activation can be induced through RNA duplexes targeting the human promoter of E-cadherin, p21 and the progesterone receptor. Although the precise mechanisms of how RNA mediated transcriptional gene silencing or activation functions has yet to be elucidated, this review describes linkage of small RNA mediated gene regulation and induction of epigenetic regulation in the promoter region in mammals.
Publisher: Elsevier BV
Date: 2015
DOI: 10.1038/MTNA.2014.67
Publisher: Elsevier BV
Date: 2015
DOI: 10.1038/MTNA.2015.31
Publisher: Future Medicine Ltd
Date: 03-2010
DOI: 10.2217/HIV.10.1
Abstract: Despite prolonged and intensive application, currently available combined antiretroviral therapy cannot eradicate HIV-1. It has little impact on provirus harbored within resting CD4 + T cells, which survive for long periods of time. One approach to clear this reservoir has been to administer either T cell-activating cytokines or histone deacetylase inhibitors to HIV-1 infected in iduals in order to reactivate latent virus from the cellular compartment while continuing cART to avoid reseeding of the reservoir. These approaches have had limited success. Strategies for the eradication of HIV need to be refined. Rational design of these approaches requires a clear understanding of the determinants of viral latency, which is controlled, at least in part, by epigenetic modifications in histones and recruitment of suppressive proteins to form heterochromatin in the promoter region of the virus. Reactivation of virus correlates with dissociation of repressive modifications, including acetylation of histone tails. siRNA has been found to be capable of inducing heterochromatin formation. Prolonged HIV-1 transcriptional gene silencing induced by either RNA duplexes or retrovirally delivered shRNA targeting a sequence located within the HIV-1 promoter is associated with sustained RNA induction and maintenance of heterochromatin (closed) structure in the viral promoter. These changes resemble those described in the latent infection. Observations regarding epigenetic control of viral transcription may provide important insights into mechanisms to manipulate latent viral reservoirs.
Publisher: Frontiers Media SA
Date: 16-09-2015
Publisher: Cold Spring Harbor Laboratory
Date: 27-12-2021
DOI: 10.1101/2021.12.22.21268288
Abstract: Despite effective antiretroviral therapy (ART), brain injury remains prevalent in people living with HIV-1 infection (PLHIV) possibly due to ART’s lack of direct inhibition of transcription with continued local production of viral transcripts and neurotoxic proteins, such as Tat, rather than cell-free whole virion toxicity. We quantified cell-associated (CA) HIV-1 RNA-transcripts in CSF and blood, in relation to proton Magnetic Resonance Spectroscopy ( 1 H MRS) of major brain metabolites, in well characterised PLHIV. RNA was extracted from cells in 16 paired s les of CSF and blood, from PLHIV on fully suppressive ART. HIV-1 CA-RNA copies were measured using the highly sensitive Double-R assay and normalized /10 6 CD4+ T cells. 18-colour flow cytometry was used to count and analyse CD4+ T cells and monocytes in CSF and blood. The concentrations of major brain metabolites from 1 H MRS in frontal white matter (FWM), posterior cingulate cortex (PCC), and caudate areas were measured. Brain injury in each voxel was defined using a composite score derived by principal component analysis. 14/16 CSF cell s les had quantifiable HIV-1 CA-RNA transcripts, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /10 6 CD4+ T cells p .0001). Higher levels of CSF transcripts were associated with greater brain injury in the FWM (Std β=-0.73 p=0.007) and PCC (Std β=-0.61 p=0.03). CSF cells were 91% memory T cells, equally CD4+ (median 3,605) cells and CD8+ T cells (3,632), but contained much fewer B cells (0.4 %), NK cells (2.0%) and monocytes (3.1% 378 cells % CD14+CD16+ phenotype). CXCR3+CD49d+integrin ß7-negative, CCR5+ CD4+ T cells were significantly enriched in CSF, compared with PBMC (p .001). Transcriptional activity in CSF cells was highly correlated with levels of transcriptional activity in CD4+ T cells in PBMC (r=0.76 p=0.002). In contrast, HIV-1 RNA in highly purified monocytes from PBMC was detected in only 6/16 s les. Elevated HIV-1 transcripts in CSF cells were associated with in vivo brain injury, despite suppressive ART. The cellular source is most likely the predominant CXCR3+ CD49d+ integrinß7-CCR5+ memory CD4+ T cells, not monocytes. Inhibitors of transcription to reduce local production of potentially neurotoxic proteins, should be developed.
Publisher: American Society for Microbiology
Date: 04-2013
DOI: 10.1128/JVI.02497-12
Abstract: T follicular helper (Tfh) cells are a specialized subset of memory CD4 + T cells that are found exclusively within the germinal centers of secondary lymphoid tissues and are important for adaptive antibody responses and B cell memory. Tfh cells do not express CCR5, the primary entry coreceptor for both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), and therefore, we hypothesized that these cells would avoid infection. We studied lymph nodes and spleens from pigtail macaques infected with pathogenic strain SIVmac239 or SIVmac251, to investigate the susceptibility of Tfh cells to SIV infection. Pigtail macaque PD-1 high CD127 low memory CD4 + T cells have a phenotype comparable to that of human Tfh cells, expressing high levels of CXCR5, interleukin-21 (IL-21), Bcl-6, and inducible T cell costimulator (ICOS). As judged by either proviral DNA or cell-associated viral RNA measurements, macaque Tfh cells were infected with SIV at levels comparable to those in other CD4 + memory T cells. Infection of macaque Tfh cells was evident within weeks of inoculation, yet we confirmed that Tfh cells do not express CCR5 or either of the well-known alternative SIV coreceptors, CXCR6 and GPR15. Mutations in the SIV envelope gp120 region occurred in chronically infected macaques but were uniform across each T cell subset investigated, indicating that the viruses used the same coreceptors to enter different cell subsets. Early infection of Tfh cells represents an unexpected focus of viral infection. Infection of Tfh cells does not interrupt antibody production but may be a factor that limits the quality of antibody responses and has implications for assessing the size of the viral reservoir.
Publisher: Proceedings of the National Academy of Sciences
Date: 21-11-2022
Abstract: Antiretroviral therapy (ART) can attain prolonged undetectable HIV-1 in plasma and cerebrospinal fluid (CSF), but brain injury remains prevalent in people living with HIV-1 infection (PLHIV). We investigated cell-associated (CA)-HIV-1 RNA transcripts in cells in CSF and blood, using the highly sensitive Double-R assay, together with proton Magnetic Resonance Spectroscopy (
Publisher: Mary Ann Liebert Inc
Date: 05-2002
DOI: 10.1089/088922202317406664
Abstract: Truncations of the cytoplasmic tail of the HIV-1 transmembrane (TM) protein are rare and almost always markedly reduce virus infectivity. We describe a truncation of the gp41 cytoplasmic tail in the commonly used early HIV-1 reference strain RF. This truncation apparently arose after continuous passage in H9 cells. We detected the truncation by Western blot as a size decrease in RF gp41 from 46 to approximately 34 kDa. The reduced size of RF gp41 observed was not due to differences in glycosylation. Viral DNA sequencing confirmed that a point mutation at Env residue 740 (Trp) introduced a premature stop codon, resulting in a 100-amino acid (13-kDa) truncation of the gp41 C terminus. This truncated RF species, termed RF(gp34), was characterized phenotypically by growth in Hut78 cells. Compared with other B clade HIV strains (IIIB, SF2, and NL4.3), RF(gp34) induced massive syncytia. Importantly, RF(gp34) also productively infected peripheral blood mononuclear cells in vitro.
Publisher: Walter de Gruyter GmbH
Date: 10-2010
DOI: 10.1515/BMC.2010.021
Abstract: Transcriptional regulation by small RNA molecules, including small interfering RNA and microRNA, has emerged as an important gene expression modulator. The regulatory pathways controlling gene expression, post-transcriptional gene silencing and transcriptional gene silencing (TGS) have been demonstrated in yeast, plants and more recently in human cells. In this review, we discuss the currents models of transcriptional regulation and the main components of the RNA-induced silencing complex and RNA-induced transcriptional silencing complex machinery, as well as confounding off-target effects and gene activation. We also discuss RNA-mediated TGS within the NF-κB motif of the human immunodeficiency virus type 1 5′ long tandem repeat promoter region and the associated epigenetic modifications. Finally, we outline the current RNA interference (RNAi) delivery methods and describe the current status of human trials investigating potential RNAi therapeutics for several human diseases.
Publisher: Frontiers Media SA
Date: 21-04-2017
Publisher: Informa UK Limited
Date: 11-2011
Publisher: Elsevier BV
Date: 09-2018
Publisher: Wiley
Date: 05-04-1991
Abstract: A simple and effective technique of electrofusion of the mouse myeloma cells and lymphocytes induced by AC pulses was investigated. Instead of pearl chain formation, we used a mild centrifugation of high-density cell suspensions to enhance the cell-cell contacts. The optimal frequency of the AC field used for cell fusion was 10 kHz. We confirmed the production of monoclonal antibodies against human serum albumin (HSA) by the hybridoma obtained by this method. Under the conditions used in the present work, the efficiency of hybridoma formation is several times higher than those obtained by the chemical method using polyethylene glycol (PEG). Hybridoma colonies were detected in as high as 95% of the wells and 33% of them proved positive against HSA.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 13-11-2011
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 13-03-2012
Publisher: Elsevier BV
Date: 2016
DOI: 10.1038/MTM.2016.66
Publisher: Springer Science and Business Media LLC
Date: 23-02-2018
DOI: 10.1038/S41598-018-21942-5
Abstract: HIV-1 latent reservoirs harbouring silenced but replication-competent proviruses are a major obstacle against viral eradication in infected patients. The “shock and kill” strategy aims to reactivate latent provirus with latency reversing agents (LRAs) in the presence of antiretroviral drugs, necessitating the development of effective and efficient LRAs. We screened a chemical library for potential LRAs and identified two dual Polo-like kinase (PLK)/bromodomain inhibitors, BI-2536 and BI-6727 (volasertib), which are currently undergoing clinical trials against various cancers. BI-2536 and BI-6727 significantly reactivated silenced HIV-1 provirus at both the mRNA and protein level in two latently infected model cell lines (ACH2 and U1). BI-2536 dramatically reactivated transcription of latent HIV-1 provirus in peripheral blood mononuclear cells derived from infected patients. Long terminal repeat activation by the inhibitors was associated with bromodomain rather than PLK inhibition. We also found that BI-2536 synergistically activates the latent provirus in combination with SAHA, a histone deacetylase inhibitor, or the non-tumour-promoting phorbol ester prostratin. Our findings strongly suggest that BI-2536 and BI-6727 are potent LRAs for the “shock and kill” HIV-1 eradication strategy.
Publisher: Mary Ann Liebert Inc
Date: 07-2017
Abstract: HIV-1 reservoirs are most often studied in peripheral blood (PB), but not all lymphocytes recirculate, particularly T follicular helper (Tfh) CD4
Location: United States of America
No related grants have been discovered for Kazuo Suzuki.