Olaf Kutsch - Academia.edu (original) (raw)

Papers by Olaf Kutsch

Journal of Virology, 2013

Following integration, HIV-1 in most cases produces active infection events; however, in some rar... more Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-B element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon.

Virology, 2015

Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interfer... more Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interference from neighboring host promoters has been proposed to contribute to the establishment and maintenance HIV-1 latency. To gain insights into how endogenous promoters influence HIV-1 transcription we utilized a set of inducible T cell lines and characterized whether there were correlations between expression of endogenous genes, provirus and long terminal repeat architecture. We show that neighboring promoters are active but have minimal impact on HIV-1 transcription, in particular, expression of the endogenous gene did not prevent expression of HIV-1 following induction of latent provirus. We also demonstrate that releasing paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus regardless of the integration site and orientation of the provirus suggesting that NELF-mediated RNAP II pausing is a common mechanism of maintaining HIV-1 latency.

Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association, 1997

Contact of HIV glycoprotein-expressing cells with CD4+ T lymphocytes in vitro causes cell-cell fu... more Contact of HIV glycoprotein-expressing cells with CD4+ T lymphocytes in vitro causes cell-cell fusion and/or cytopathogenicity. The question of whether this process similarly underlies the death of helper T cells in vivo has not yet been resolved. To investigate the loss of uninfected CD4+ T cells in an environment that may reflect the in vivo situation, unfractionated, unstimulated peripheral blood mononuclear cells were cocultured with HIV-1 glycoprotein-expressing cells, and early alterations of T-cell numbers were quantitated using a newly developed quantitative flow cytometric assay. The results demonstrate that a large fraction of normal-sized, regular CD4+ T cells disappeared immediately on cocultivation with envelope glycoprotein-expressing cells. In contrast, CD8+ T lymphocytes remained unaffected. Significant loss of uninfected T-helper cells required the presence of less than 1% infected cells. Moreover, memory T cells (CD45RO+, CD29 hi+) were depleted more rapidly than n...

PLoS ONE, 2012

Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molec... more Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ,0.5 and led to siRNA/polymer complexes of ,100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia. Citation: Landry B, Aliabadi HM, Samuel A, Gü l-Uludag H, Jiang X, et al. (2012) Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines. PLoS ONE 7(8): e44197.

The Journal of Gene Medicine, 2010

Background Inducible gene expression systems are powerful research tools and could be of clinical... more Background Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line-dependent-manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T-cells.

Chemistry & Biology, 2011

ASSAY and Drug Development Technologies, 2012

The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of pot... more The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the importance of developing novel antimalarial therapies. Using a previously generated stable transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a highthroughput screening assay in 384-well plates. Assay conditions, including the percentage of parasitemia and hematocrit, were optimized. Parameters of assay robustness, including Z 0 -value, coefficient variation (CV), and signal-to-background (S/B) ratio, were determined. The LOPAC 1280 small-compound library was used to validate this assay. Our results demonstrated that this assay is robust and reliable, with an average Z 0 -value of >0.7 and CV of <10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay. It is evident that this assay is suitable for large-scale screening of chemical libraries for antimalarial drug discovery.

Journal of Virology

While human immunodeficiency virus type 1 (HIV-1) infection is associated with hyperimmune activa... more While human immunodeficiency virus type 1 (HIV-1) infection is associated with hyperimmune activation and systemic depletion of CD4(+) T cells, simian immunodeficiency virus (SIV) infection in sooty mangabeys or chimpanzees does not exhibit these hallmarks. Control of immune activation is thought to be one of the major components that govern species-dependent differences in the disease pathogenesis. A previous study introduced the idea that the resistance of chimpanzees to SIVcpz infection-induced hyperimmune activation could be the result of the expression of select sialic acid-recognizing immunoglobulin (Ig)-like lectin (Siglec) superfamily members by chimpanzee T cells. Siglecs, which are absent on human T cells, were thought to control levels of T-cell activation in chimpanzees and were thus suggested as a cause for the pathogenic differences in the course of SIVcpz or HIV-1 infection. As in human models of T-cell activation, stimulation had been attempted using an anti-CD3 mono...

ABSTRACT Recent research,has,emphasized,the notion that HIV-1 latency,is controlled by a restrict... more ABSTRACT Recent research,has,emphasized,the notion that HIV-1 latency,is controlled by a restrictive histone code at, or DNA methylation of the integrated viral promoter (LTR). The present concept of HIV-1 latency has essentially been patterned from the principles of cellular 5

Antimicrobial Agents and Chemotherapy, 2015

Tuberculosis is a severe disease affecting millions worldwide. Unfortunately, treatment strategie... more Tuberculosis is a severe disease affecting millions worldwide. Unfortunately, treatment strategies are hampered both by the prohibitively long treatment regimen and the rise of drug-resistant strains. Significant effort has been expended in the search for new treatments, but few options have successfully emerged, and new treatment modalities are desperately needed. Recently, there has been growing interest in the synergistic antibacterial effects of copper ions (Cu II/I ) in combination with certain small molecular compounds, and we have previously reported development of a drug screening strategy to harness the intrinsic bactericidal properties of Cu II/I . Here, we describe the copper-dependent antimycobacterial properties of disulfiram, an FDA-approved and well-tolerated sobriety aid. Disulfiram was inhibitory to mycobacteria only in the presence of Cu II/I and exerted its bactericidal activity well below the active concentration of Cu II/I or disulfiram alone. No other physiologically relevant bivalent transition metals (e.g., Fe II , Ni II , Mn II , and Co II ) exhibited this effect. We demonstrate that the movement of the disulfiram-copper complex across the cell envelope is porin independent and can inhibit intracellular protein functions. Additionally, the complex is able to synergistically induce intracellular copper stress responses significantly more than Cu II/I alone. Our data suggest that by complexing with disulfiram, Cu II/I is likely allowed unfettered access to vulnerable intracellular components, bypassing the normally sufficient copper homeostatic machinery. Overall, the synergistic antibacterial activity of Cu II/I and disulfiram reveals the susceptibility of the copper homeostasis system of Mycobacterium tuberculosis to chemical attacks and establishes compounds that act in concert with copper as a new class of bacterial inhibitors.

Virology, Jan 5, 2005

To date, attempts to eliminate HIV-1 infection from its latent reservoirs, a prerequisite for the... more To date, attempts to eliminate HIV-1 infection from its latent reservoirs, a prerequisite for the development of a curative treatment strategy for HIV-1 infection, have been unsuccessful. We demonstrate that the FDA approved antifungal agent amphotericin B efficiently reactivates HIV-1 infection in THP89GFP cells, a model of HIV-1 latency in macrophages. Although amphotericin B does not directly reactivate latent HIV-1 infection in T cells (e.g., J89GFP), amphotericin-B-stimulated macrophages (THP89GFP cells or primary macrophages) when cocultured with J89GFP cells can induce HIV-1 reactivation in these cells in trans. Because of the close proximity of antigen presenting macrophages and T cells in the primary lymphoid organs, such interaction between antigen presenting macrophages and T cells are frequent, and it seems reasonable to assume that trans-reactivation strategies hold promise to also reactivate latent HIV-1 infection in vivo.

Virology, Jan 15, 2003

Reservoirs of latent HIV-1 in T cells and macrophages pose one of the major obstacles that hamper... more Reservoirs of latent HIV-1 in T cells and macrophages pose one of the major obstacles that hamper final eradication of HIV-1 from infected patients. Targeting costimulatory molecules expressed on cell types harboring latent HIV-1 to achieve reactivation may provide a new approach to overcome this problem. One such molecule is CD40, a member of the tumor necrosis factor (TNF)-receptor family. Using THP89GFP cells as a model for latently infected macrophages, we demonstrate that trimeric forms of recombinant CD154 allow for the direct reactivation of latent HIV-1 infection. Reactivation is augmented by the release of TNF-alpha. The presence of TNF-alpha is also crucial for the expression of late structural genes such as p24 Gag. In addition, levels of secreted TNF-alpha are sufficiently high to reactivate latent HIV-1 in a latently HIV-1-infected T-cell line (J89GFP). Taken together, our results demonstrate that costimulatory molecules may be attractive targets to reactivate latent HI...

Molecular and Cellular Biology, 2002

Among the tumor necrosis factor (TNF) family of cytokines, FasL and TNF-related apoptosis-inducin... more Among the tumor necrosis factor (TNF) family of cytokines, FasL and TNF-related apoptosis-inducing ligand (TRAIL) are known to induce cell death via caspase activation. Recently, other biological functions of these death ligands have been postulated in vitro and in vivo. It was previously shown that Fas ligation induces chemokine expression in human glioma cells. In this study, we investigated whether

Virology, 2009

Human immunodeficiency virus type 1 (HIV-1) originated in chimpanzees; yet, several previous stud... more Human immunodeficiency virus type 1 (HIV-1) originated in chimpanzees; yet, several previous studies have shown that primary HIV-1 isolates replicate poorly in chimpanzee CD4+ T lymphocytes in vitro and in vivo. The reasons for this apparent restriction are not understood. Here, we describe a new activation protocol that led to a reproducible expansion and activation of chimpanzee CD4+ T lymphocytes in vitro. Using this protocol, we uncovered species-specific differences in the activation profiles of human and chimpanzee CD4+ T cells, including HLA-DR and CD62L. Moreover, we found that improved activation facilitated the replication of both CXCR4 and CCR5tropic HIV-1 in CD4+ T cell cultures from over 30 different chimpanzees. Thus, the previously reported "replication block" of CCR5-tropic HIV-1 in chimpanzee lymphocytes appears to be due, at least in large part, to suboptimal T cell activation.

Proceedings of the National Academy of Sciences, 2004

Journal of Virology, 2009

Recent research has emphasized the notion that HIV-1 latency is controlled by a restrictive histo... more Recent research has emphasized the notion that HIV-1 latency is controlled by a restrictive histone code at, or DNA methylation of the integrated viral promoter (LTR). The present concept of HIV-1 latency has essentially been patterned from the principles of cellular 5 gene regulation. Here we introduce an experimental system that allows for the qualitative and quantitative kinetic study of latency establishment and maintenance at the population level. In this system, we find no evidence that HIV-1 latency establishment is the consequence of down regulation of initial active infection followed by the establishment of a restrictive histone code at the viral LTR. Latent infection was established following integration of the virus in the absence 10 of viral gene expression (silent integration) and was a function of the NF-κB activation level in the host cell at the time of infection. In the absence of a role for epigenetic regulation, we demonstrate that transcriptional interference, a mechanism that has recently been suggested to add to the stabilization of HIV-1 latency, is the primary mechanism to govern latency maintenance. These findings provide direct experimental evidence that the high number of viral 15 integration events (>90%) found in actively expressed genes of CD4 + memory T cells from HAART suppressed patients represent indeed latent infection events, and that transcriptional interference may be the primary mechanism to control HIV-1 latency in vivo. HIV-1 latency may thus not be governed by the principles of cellular gene regulation, and therapeutic strategies to deplete the pool of latently HIV-1 infected cells should be reconsidered. Downloaded from 45 Mullins, and A. S. Fauci. 2000. Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy. Nat Med 6:757-61.

Journal of Virology, 2000

A finding commonly observed in human immunodeficiency virus type 1 (HIV-1)-infected patients is i... more A finding commonly observed in human immunodeficiency virus type 1 (HIV-1)-infected patients is invasion of the brain by activated T cells and infected macrophages, eventually leading to the development of neurological disorders and HIV-1-associated dementia. The recruitment of T cells and macrophages into the brain is likely the result of chemokine expression. Indeed, earlier studies revealed that levels of different chemokines were increased in the cerebrospinal fluid of HIV-1-infected patients whereas possible triggers and cellular sources for chemokine expression in the brain remain widely undefined. As previous studies indicated that HIV-1 Tat, the retroviral transactivator, is capable of inducing a variety of cellular genes, we investigated its capacity to induce production of chemokines in astrocytes. Herein, we demonstrate that HIV-1 Tat 72aa is a potent inducer of MCP-1, interleukin-8 (IL-8), and IP-10 expression in astrocytes. Levels of induced IP-10 protein were sufficiently high to induce chemotaxis of peripheral blood lymphocytes. In addition, Tat 72aa induced IL-8 expression in astrocytes. IL-8 mRNA induction was seen less then 1 h after Tat 72aa stimulation, and levels remained elevated for up to 24 h, leading to IL-8 protein production. Tat 72aa -mediated MCP-1 and IL-8 mRNA induction was susceptible to inhibition by the MEK1/2 inhibitor UO126 but was only modestly decreased by the inclusion of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190. In contrast, Tat-mediated IP-10 mRNA induction was suppressed by SB202190 but not by the MEK1/2 inhibitor UO126. These findings indicate that MAPKs play a major role in Tat 72aa -mediated chemokine induction in astrocytes.

Journal of Virology, 2002

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cell... more The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replicationcompetent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.

Journal of Virology, 2008

Our results thus imply that prior to studying possible differences between human and chimpanzee T... more Our results thus imply that prior to studying possible differences between human and chimpanzee T-cell activation, a relevant model of chimpanzee T cell activation needs to be established.