Publications by authors named "Richard A Lempicki"

72 Publications

Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of Multiple Species.

mBio 2020 03 3;11(2). Epub 2020 Mar 3.

Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.

, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all species characterized to date, highlighting its important role in biology. In this report, through a comprehensive and in-depth characterization of 459 genes from 7 species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual families in two rodent species, the substantial variability of the repertoires in from laboratory and wild rats, and the distinct features of the expression site for the classic genes in from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in biology. continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with pneumonia costs ∼$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.
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http://dx.doi.org/10.1128/mBio.02878-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064768PMC
March 2020

Comparative Population Genomics Analysis of the Mammalian Fungal Pathogen .

mBio 2018 05 8;9(3). Epub 2018 May 8.

Critical Care Medicine Department, NIH Clinical Center, National Institutes of Health, Bethesda, Maryland, USA

species are opportunistic mammalian pathogens that cause severe pneumonia in immunocompromised individuals. These fungi are highly host specific and uncultivable Human infections present major challenges because of a limited therapeutic arsenal and the rise of drug resistance. To investigate the diversity and demographic history of natural populations of infecting humans, rats, and mice, we performed whole-genome and large-scale multilocus sequencing of infected tissues collected in various geographic locations. Here, we detected reduced levels of recombination and variations in historical demography, which shape the global population structures. We report estimates of evolutionary rates, levels of genetic diversity, and population sizes. Molecular clock estimates indicate that species diverged before their hosts, while the asynchronous timing of population declines suggests host shifts. Our results have uncovered complex patterns of genetic variation influenced by multiple factors that shaped the adaptation of populations during their spread across mammals. Understanding how natural pathogen populations evolve and identifying the determinants of genetic variation are central issues in evolutionary biology. , a fungal pathogen which infects mammals exclusively, provides opportunities to explore these issues. In humans, can cause a life-threatening pneumonia in immunosuppressed individuals. In analysis of different species infecting humans, rats, and mice, we found that there are high infection rates and that natural populations maintain a high level of genetic variation despite low levels of recombination. We found no evidence of population structuring by geography. Our comparisons of the times of divergence of these species to their respective hosts suggest that may have undergone recent host shifts. The results demonstrate that strains are widely disseminated geographically and provide a new understanding of the evolution of these pathogens.
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http://dx.doi.org/10.1128/mBio.00381-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5941068PMC
May 2018

Distinguishing highly similar gene isoforms with a clustering-based bioinformatics analysis of PacBio single-molecule long reads.

BioData Min 2016 5;9:13. Epub 2016 Apr 5.

Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.

Background: Gene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs.

Results: We present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms.

Conclusions: PacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences.
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http://dx.doi.org/10.1186/s13040-016-0090-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820869PMC
April 2016

Towards Better Precision Medicine: PacBio Single-Molecule Long Reads Resolve the Interpretation of HIV Drug Resistant Mutation Profiles at Explicit Quasispecies (Haplotype) Level.

J Data Mining Genomics Proteomics 2016 Jan 8;7(1). Epub 2015 Nov 8.

Applied and Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, MD 21702, USA.

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient's HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only ≥ 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients' HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.
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http://dx.doi.org/10.4172/2153-0602.1000182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775093PMC
January 2016

Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts.

Nat Commun 2016 Feb 22;7:10740. Epub 2016 Feb 22.

Genome Sequencing and Analysis Program, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses.
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http://dx.doi.org/10.1038/ncomms10740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764891PMC
February 2016

Differential Specificity of Interferon-alpha Inducible Gene Expression in Association with Human Immunodeficiency Virus and Hepatitis C Virus Levels and Declines .

J AIDS Clin Res 2015;6(1)

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892, USA.

Objective: This study was aimed to correlate interferon (IFN) inducible gene (IFIG) expression and IFIG induction with viral-load (VL) and VL-kinetics of Human-Immunodeficiency-Virus (HIV) or Hepatitis-C-Virus (HCV) in HIV-positive patients treated with pegylated IFN-alpha-2a (PegIFNα).

Methods: HIV mono-infected patients (N=8) and HIV/HCV co-infected patients (N=23, without HIV-viremia) were treated with PegIFNα (180 μg/week) for 12 and 48 weeks, respectively. Blood sampling for monitoring IFIG expression occurred at day_0 and week_3, _6 and _12 for HIV mono-infected patients vs. only at day_0 and week_48 for HIV/HCV co-infected subjects. IFIG expression (N=20) was measured in peripheral blood mononuclear cells by bDNA-assay. VL levels/changes in plasma were analyzed for correlation with IFIG expression/induction at/between selected time points. Overall, P<0.05 was considered significant.

Results: None of the 20 IFIG expression profiles at day_0 correlated significantly with HIV-VL at day_0. Expression at day_0 of 3 IFIG (APOBEC3G/OAS1/OAS2) correlated significantly (r>+0.42/P<0.05) with HCV-VL at day_0. The strongest antiviral effect [measured as median viral decline per week: ΔVL/week (log10)] occurred in common against HIV and HCV between day_0 and week_3 during 12 weeks of continuous PegIFNα treatment in both cohorts. Expression at day_0 of 1 IFIG (APOBEC3A) correlated significantly (r<-0.71/P<0.05) with HIV-ΔVL/week (log10) from day_0 to week_3. No significance was reached in correlations between expression values of 20 IFIG at day_0 and HCV-ΔVL/week (log10) from day_0 to week_3. No significant correlation was detected between IFIG expression changes (ΔIFIG=induction) from day_0 to week_3 and HIV-ΔVL/week (log10) from day_0 to week_3. Interestingly, induction of 1 IFIG (ΔISG20) from day_0 to week_48 was significantly associated (P<0.05) with permanent HCV clearance.

Conclusion: This study demonstrates the differential specificity of PegIFNα mediated molecular actions by dissecting the kinetics of IFIG expression and induction, suggesting multiple, possibly non-overlapping mechanisms for antiviral effects against HCV and HIV.
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http://dx.doi.org/10.4172/2155-6113.1000410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456029PMC
January 2015

Characterization of chemokine and chemokine receptor expression during Pneumocystis infection in healthy and immunodeficient mice.

Microbes Infect 2015 Sep 5;17(9):638-50. Epub 2015 Jun 5.

Critical Care Medicine Department, NIH Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

We examined gene expression levels of multiple chemokines and chemokine receptors during Pneumocystis murina infection in wild-type and immunosuppressed mice, using microarrays and qPCR. In wild-type mice, expression of chemokines that are ligands for Ccr2, Cxcr3, Cxcr6, and Cxcr2 increased at days 32-41 post-infection, with a return to baseline by day 75-150. Concomitant increases were seen in Ccr2, Cxcr3, and Cxcr6, but not in Cxcr2 expression. Induction of these same factors also occurred in CD40-ligand and CD40 knockout mice but only at a much later time-point, during uncontrolled Pneumocystis pneumonia (PCP). Expression of CD4 Th1 markers was increased in wild-type mice during clearance of infection. Ccr2 and Cx3cr1 knockout mice cleared Pneumocystis infection with kinetics similar to wild-type mice, and all animals developed anti-Pneumocystis antibodies. Upregulation of Ccr2, Cxcr3, and Cxcr6 and their ligands supports an important role for T helper cells and mononuclear phagocytes in the clearance of Pneumocystis infection. However, based on the current and prior studies, no single chemokine receptor appears to be critical to the clearance of Pneumocystis.
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http://dx.doi.org/10.1016/j.micinf.2015.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4554965PMC
September 2015

Hepatitis C-associated mixed cryoglobulinemic vasculitis induces differential gene expression in peripheral mononuclear cells.

Front Immunol 2014 27;5:248. Epub 2014 May 27.

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services , Bethesda, MD , USA.

This study examines the distinct gene expression profile of peripheral blood mononuclear cells from patients with chronic hepatitis C infection and mixed cryoglobulinemic (MC) vasculitis. Our DNA microarray analysis indicates that hepatitis C virus (HCV)-associated MC vasculitis is characterized by compromised neutrophil function, impaired chemotaxis, and increased interferon-stimulated gene (ISG) expression, contributing to overall MC pathogenesis and end-organ damage. Increased ISG expression is suggestive of an enhanced endogenous interferon gene signature. PBMC depletion assays demonstrate that this increased expression is likely due to an activation of monocytes and not a direct result of B cell expansion. Notably, this monocyte activation of ISG expression in HCV-associated MC vasculitis suggests a poor predictor status of interferon-based treatment. Further analysis of PBMC gene expression profiles before and after in vivo B cell depletion therapy is critical to completely understanding the mechanisms of MC vasculitis pathogenesis.
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http://dx.doi.org/10.3389/fimmu.2014.00248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034044PMC
June 2014

Interferon stimulated exonuclease gene 20 kDa links psychiatric events to distinct hepatitis C virus responses in human immunodeficiency virus positive patients.

J Med Virol 2014 Aug 30;86(8):1323-31. Epub 2014 Apr 30.

Department of Gastroenterology and Hepatology, University Hospital Essen, Hufelandstrasse 55, Essen, 45147, Germany.

Hepatitis C Virus (HCV) infection occurs frequently in patients with preexisting mental illness. Treatment for chronic hepatitis C using interferon formulations often increases risk for neuro-psychiatric symptoms. Pegylated-Interferon-α (PegIFN-α) remains crucial for attaining sustained virologic response (SVR); however, PegIFN-α based treatment is associated with psychiatric adverse effects, which require dose reduction and/or interruption. This study's main objective was to identify genes induced by PegIFN-α and expressed in the central nervous system and immune system, which could mediate the development of psychiatric toxicity in association with antiviral outcome. Using peripheral blood mononuclear cells from Human Immunodeficiency Virus (HIV)/HCV co-infected donors (N = 28), DNA microarray analysis was performed and 21 differentially regulated genes were identified in patients with psychiatric toxicity versus those without. Using these 21 expression profiles a two-way-ANOVA was performed to select genes based on antiviral outcome and occurrence of neuro-psychiatric adverse events. Microarray analysis demonstrated that Interferon-stimulated-exonuclease-gene 20 kDa (ISG20) and Interferon-alpha-inducible-protein 27 (IFI27) were the most regulated genes (P < 0.05) between three groups that were built by combining antiviral outcome and neuro-psychiatric toxicity. Validation by bDNA assay confirmed that ISG20 expression levels were significantly associated with these outcomes (P < 0.035). Baseline levels and induction of ISG20 correlated independently with no occurrence of psychiatric adverse events and non-response to therapy (P < 0.001). Among the 21 genes that were associated with psychiatric adverse events and 20 Interferon-inducible genes (IFIGs) used as controls, only ISG20 expression was able to link PegIFN-α related neuro-psychiatric toxicity to distinct HCV-responses in patients co-infected with HIV and HCV in vivo.
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http://dx.doi.org/10.1002/jmv.23956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114765PMC
August 2014

Clearance of Pneumocystis murina infection is not dependent on MyD88.

Microbes Infect 2014 Jun 25;16(6):522-7. Epub 2014 Mar 25.

Critical Care Medicine Department, NIH Clinical Center, NIH, Building 10, Room 2C145, MSC 1662, Bethesda, MD 20892-1662, USA. Electronic address:

To determine if myeloid differentiation factor 88 (MyD88), which is necessary for signaling by most TLRs and IL-1Rs, is necessary for control of Pneumocystis infection, MyD88-deficient and wild-type mice were infected with Pneumocystis by exposure to infected seeder mice and were followed for up to 106 days. MyD88-deficient mice showed clearance of Pneumocystis and development of anti-Pneumocystis antibody responses with kinetics similar to wild-type mice. Based on expression levels of select genes, MyD88-deficient mice developed immune responses similar to wild-type mice. Thus, MyD88 and the upstream pathways that rely on MyD88 signaling are not required for control of Pneumocystis infection.
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http://dx.doi.org/10.1016/j.micinf.2014.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071138PMC
June 2014

Lifespan of effector memory CD4+ T cells determined by replication-incompetent integrated HIV-1 provirus.

AIDS 2014 May;28(8):1091-9

aClinical and Molecular Retrovirology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda bClinical Services Program, Applied and Development Research Directorate, Leidos Biomedical Research Incorporated, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.

Objective: Determining the precise lifespan of human T-cell is challenging due to the inability of standard techniques to distinguish between dividing and dying cells. Here, we measured the lifespan of a pool of T cells that were derived from a single cell 'naturally' labelled with a single integrated clone of a replication-incompetent HIV-1 provirus.

Design/methods: Utilizing a combination of techniques, we were able to sequence/map an integration site of a unique provirus with a stop codon at position 42 of the HIV-1 protease. In-vitro reconstruction of this provirus into an infectious clone confirmed its inability to replicate. By combining cell separation and integration site-specific PCR, we were able to follow the fate of this single provirus in multiple T-cell subsets over a 20-year period. As controls, a number of additional integrated proviruses were also sequenced.

Results: The replication-incompetent HIV-1 provirus was solely contained in the pool of effector memory CD4 T cells for 17 years. The percentage of the total effector memory CD4 T cells containing the replication-incompetent provirus peaked at 1% with a functional half-life of 11.1 months. In the process of sequencing multiple proviruses, we also observed high levels of lethal mutations in the peripheral blood pool of proviruses.

Conclusion: These data indicate that human effector memory CD4 T cells are able to persist in vivo for more than 17 years without detectably reverting to a central memory phenotype. A secondary observation is that the fraction of the pool of integrated HIV-1 proviruses capable of replicating may be considerably less than the 12% currently noted in the literature.
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http://dx.doi.org/10.1097/QAD.0000000000000223DOI Listing
May 2014

Evolution of the pygmy phenotype: evidence of positive selection fro genome-wide scans in African, Asian, and Melanesian pygmies.

Hum Biol 2013 Feb-Jun;85(1-3):251-84

Department of Anthropology, University College London, London, UK.

Human pygmy populations inhabit different regions of the world, from Africa to Melanesia. In Asia, short-statured populations are often referred to as "negritos." Their short stature has been interpreted as a consequence of thermoregulatory, nutritional, and/or locomotory adaptations to life in tropical forests. A more recent hypothesis proposes that their stature is the outcome of a life history trade-off in high-mortality environments, where early reproduction is favored and, consequently, early sexual maturation and early growth cessation have coevolved. Some serological evidence of deficiencies in the growth hormone/insulin-like growth factor axis have been previously associated with pygmies' short stature. Using genome-wide single-nucleotide polymorphism genotype data, we first tested whether different negrito groups living in the Philippines and Papua New Guinea are closely related and then investigated genomic signals of recent positive selection in African, Asian, and Papuan pygmy populations. We found that negritos in the Philippines and Papua New Guinea are genetically more similar to their nonpygmy neighbors than to one another and have experienced positive selection at different genes. These results indicate that geographically distant pygmy groups are likely to have evolved their short stature independently. We also found that selection on common height variants is unlikely to explain their short stature and that different genes associated with growth, thyroid function, and sexual development are under selection in different pygmy groups.
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http://dx.doi.org/10.3378/027.085.0313DOI Listing
April 2015

A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS.

J Data Mining Genomics Proteomics 2013 Jul;4(3)

Laboratory of Immunopathogenesis and Bioinformatics, SAIC-Frederick, Inc., Frederick National Laboratory, MD 21702, USA.

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.
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http://dx.doi.org/10.4172/2153-0602.1000136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811116PMC
July 2013

The HIV-1 envelope protein gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression.

Nat Immunol 2013 Dec 27;14(12):1256-65. Epub 2013 Oct 27.

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

The humoral immune response after acute infection with HIV-1 is delayed and ineffective. The HIV-1 envelope protein gp120 binds to and signals through integrin α4β7 on T cells. We found that gp120 also bound to and signaled through α4β7 on naive B cells, which resulted in an abortive proliferative response. In primary B cells, signaling by gp120 through α4β7 resulted in increased expression of the immunosuppressive cytokine TGF-β1 and FcRL4, an inhibitory receptor expressed on B cells. Coculture of B cells with HIV-1-infected autologous CD4(+) T cells also increased the expression of FcRL4 by B cells. Our findings indicated that in addition to mediating chronic activation of the immune system, viral proteins contributed directly to HIV-1-associated B cell dysfunction. Our studies identify a mechanism whereby the virus may subvert the early HIV-1-specific humoral immune response.
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http://dx.doi.org/10.1038/ni.2746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870659PMC
December 2013

Interleukin-2 inhibits HIV-1 replication in some human T cell lymphotrophic virus-1-infected cell lines via the induction and incorporation of APOBEC3G into the virion.

J Biol Chem 2013 Jun 2;288(24):17812-22. Epub 2013 May 2.

Laboratory of Human Retrovirology, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.

IL-2 has been used in culture of primary T cells to maintain cell proliferation. We have previously reported that IL-27 inhibits HIV-1 replication in primary T cells in the presence of IL-2. To gain a better understanding of the mechanisms involved in this inhibitory effect, we attempted to investigate in detail the effects of IL-27 and IL-2 using several cell lines. Unexpectedly, IL-27 did not inhibit HIV-1 in T cell lines, whereas IL-2 inhibited HIV-1 replication in the human T cell lymphotrophic virus (HTLV)-1-transformed T cell lines, MT-2, MT-4, SLB-1, and ATL-2. No effects were seen in HTLV-1-negative cell lines. Utilizing MT-2 cells, we demonstrated that IL-2 treatment inhibited HIV-1 syncytia-inducing ability and dose-dependently decreased supernatant p24 antigen levels by >90%. Using real time PCR and Western blot analysis, we observed that IL-2 treatment induced the host restriction factor, APOBEC3G with accumulation into the lower molecular mass active form as characterized by FPLC. Further analysis revealed that the virus recovered from IL-2-treated MT-2 cells had impaired replication competency. This was found to be due to incorporation of APOBEC3G into the virion despite the presence of Vif. These findings demonstrate a novel role for IL-2 in regulating production of infectious HIV-1 virions in HTLV-1-infected cells through the induction of APOBEC3G.
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http://dx.doi.org/10.1074/jbc.M113.468975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682580PMC
June 2013

High interferon-stimulated gene ISG-15 expression affects HCV treatment outcome in patients co-infected with HIV and HCV.

J Med Virol 2013 Jun;85(6):959-63

Department of Gastroenterology and Hepatology, University Hospital, Essen, Germany.

Increased baseline expression and lack of induction of interferon-stimulated genes (ISG) are strong negative predictors of therapeutic response to PegIFN/RBV in patients co-infected with HIV and hepatitis C virus (HCV). This study specifically addressed whether ISG-15 expression influences therapeutic responses in 20 HIV/HCV genotype-1 subjects undergoing HCV treatment. Non-responders had significantly higher baseline expression and selective induction of ISG-15 after IFN-α treatment relative to participants with sustained virological response. High baseline levels of ISG-15 were also associated with less induction of ISG with treatment. These results support a role for ISG-15 as a prognostic indicator and resistance factor to IFN-α.
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http://dx.doi.org/10.1002/jmv.23576DOI Listing
June 2013

Interleukin-27 treated human macrophages induce the expression of novel microRNAs which may mediate anti-viral properties.

Biochem Biophys Res Commun 2013 May 25;434(2):228-34. Epub 2013 Mar 25.

Applied and Developmental Research Directorate, Science Application International Corporation (SAIC)-Frederick, Inc., Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD 21702, USA.

Interleukin-27 (IL-27) is a pleiotropic cytokine which plays important and diverse roles in the immune system. We have previously demonstrated that IL-27 induces potent anti-viral effects against HIV-1, HIV-2, SIV, HSV-2, KSHV and influenza viruses in macrophages. This induction occurred in an interferon (IFN) independent manner and involved down regulation of SPTBN1. MicroRNAs (miRNAs) are critical regulators of mRNA translation and turnover. There have been reports that some miRNAs inhibit viral replication. In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity and primary monocytes were differentiated into macrophages using either M-CSF (M-Mac) or a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in these macrophages. Four of which were preferentially expressed in I-Mac (miR-SX1, -SX2, -SX3 and -SX6) whilst three were detected in both M-Mac and I-Mac (miR-SX4, -SX5 and -SX7). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. Finally, several of these novel miRNAs (miR-SX1, -SX4, -SX5, -SX6 and -SX7) were shown to target the open reading frames of a number of viruses (including HSV-1, HSV-2 and HHV-8) which may partially explain the anti-viral properties observed.
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http://dx.doi.org/10.1016/j.bbrc.2013.03.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700531PMC
May 2013

Interleukin-27 is a potent inhibitor of cis HIV-1 replication in monocyte-derived dendritic cells via a type I interferon-independent pathway.

PLoS One 2013 20;8(3):e59194. Epub 2013 Mar 20.

Applied and Developmental Research Directorate (ADD), Science Application International Corporation (SAIC)-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.

IL-27, a member of the IL-12 family of cytokines, plays an important and diverse role in the function of the immune system. Whilst generally recognized as an anti-inflammatory cytokine, in addition IL-27 has been found to have broad anti-viral effects. Recently, IL-27 has been shown to be a potent inhibitor of HIV-1 infection in CD4+ T cells and macrophages. The main objective of this study was to see whether IL-27 has a similar inhibitory effect on HIV-1 replication in dendritic cells (DCs). Monocytes were differentiated into immature DCs (iDCs) and mature DCs (mDCs) with standard techniques using a combination of GM-CSF, IL-4 and LPS. Following differentiation, iDCs were infected with HIV-1 and co-cultured in the presence or absence of IL-27. IL-27 treated DCs were shown to be highly potent inhibitors of cis HIV-1, particularly of CCR5 tropic strains. Of note, other IL-12 family members (IL-12, IL-23 and IL-35) had no effect on HIV-1 replication. Microarray studies of IL-27 treated DCs showed no up-regulation of Type I (IFN) gene expression. Neutralization of the Type-I IFN receptor had no impact on the HIV inhibition. Lastly, IL-27 mediated inhibition was shown to act post-viral entry and prior to completion of reverse transcription. These results show for the first time that IL-27 is a potent inhibitor of cis HIV-1 infection in DCs by a Type I IFN independent mechanism. IL-27 has previously been reported to inhibit HIV-1 replication in CD4+ T cells and macrophages, thus taken together, this cytokine is a potent anti-HIV agent against all major cell types targeted by the HIV-1 virus and may have a therapeutic role in the future.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059194PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3604098PMC
September 2013

IL-27 inhibits HIV-1 infection in human macrophages by down-regulating host factor SPTBN1 during monocyte to macrophage differentiation.

J Exp Med 2013 Mar 4;210(3):517-34. Epub 2013 Mar 4.

Applied and Developmental Directorate, Science Applications International Corporation-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

The susceptibility of macrophages to HIV-1 infection is modulated during monocyte differentiation. IL-27 is an anti-HIV cytokine that also modulates monocyte activation. In this study, we present new evidence that IL-27 promotes monocyte differentiation into macrophages that are nonpermissive for HIV-1 infection. Although IL-27 treatment does not affect expression of macrophage differentiation markers or macrophage biological functions, it confers HIV resistance by down-regulating spectrin β nonerythrocyte 1 (SPTBN1), a required host factor for HIV-1 infection. IL-27 down-regulates SPTBN1 through a TAK-1-mediated MAPK signaling pathway. Knockdown of SPTBN1 strongly inhibits HIV-1 infection of macrophages; conversely, overexpression of SPTBN1 markedly increases HIV susceptibility of IL-27-treated macrophages. Moreover, we demonstrate that SPTBN1 associates with HIV-1 gag proteins. Collectively, our results underscore the ability of IL-27 to protect macrophages from HIV-1 infection by down-regulating SPTBN1, thus indicating that SPTBN1 is an important host target to reduce HIV-1 replication in one major element of the viral reservoir.
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http://dx.doi.org/10.1084/jem.20120572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600911PMC
March 2013

Sequencing and characterization of the complete mitochondrial genomes of three Pneumocystis species provide new insights into divergence between human and rodent Pneumocystis.

FASEB J 2013 May 7;27(5):1962-72. Epub 2013 Feb 7.

Critical Care Medicine Department, NIH Clinical Center, 10 Center Dr., Bethesda, MD 20892, USA.

Pneumocystis jirovecii is an important opportunistic pathogen associated with AIDS and other immunodeficient conditions. Currently, very little is known about its nuclear and mitochondrial genomes. In this study, we sequenced the complete mitochondrial genome (mtDNA) of this organism and its closely related species Pneumocystis carinii and Pneumocystis murina by a combination of sequencing technologies. Our study shows that P. carinii and P. murina mtDNA share a nearly identical number and order of genes in a linear configuration, whereas P. jirovecii has a circular mtDNA containing nearly the same set of genes but in a different order. Detailed studies of the mtDNA terminal structures of P. murina and P. carinii suggest a unique replication mechanism for linear mtDNA. Phylogenetic analysis supports a close association of Pneumocystis species with Taphrina, Saitoella, and Schizosaccharomyces, and divergence within Pneumocystis species, with P. murina and P. carinii being more closely related to each other than either is to P. jirovecii. Comparative analysis of four complete P. jirovecii mtDNA sequences in this study and previously reported mtDNA sequences for diagnosing and genotyping suggests that the current diagnostic and typing methods can be improved using the complete mtDNA data. The availability of the complete P. jirovecii mtDNA also opens the possibility of identifying new therapeutic targets.
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http://dx.doi.org/10.1096/fj.12-224444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633818PMC
May 2013

Differential gene and microRNA expression between etoposide resistant and etoposide sensitive MCF7 breast cancer cell lines.

PLoS One 2012 18;7(9):e45268. Epub 2012 Sep 18.

Laboratory of Experimental Immunology, Cancer and Inflammation Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.

In order to develop targeted strategies for combating drug resistance it is essential to understand it's basic molecular mechanisms. In an exploratory study we have found several possible indicators of etoposide resistance operating in MCF7VP cells, including up-regulation of ABC transporter genes, modulation of miRNA, and alteration in copy numbers of genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045268PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445463PMC
February 2013

The CD8+ HLA-DR+ T cells expanded in HIV-1 infection are qualitatively identical to those from healthy controls.

Eur J Immunol 2012 Oct 6;42(10):2608-20. Epub 2012 Aug 6.

Clinical and Molecular Retrovirology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

HIV-induced immune activation leads to expansion of a subset of human CD8(+) T cells expressing HLA-DR antigens. Expansion of CD8(+) HLA-DR(+) T cells can be also observed in non-HIV settings including several autoimmune diseases and aging. Although these cells are felt to represent "immune exhaustion" and/or to be anergic, their precise role in host defense has remained unclear. Here, we report that this subset of cells exhibits a restricted repertoire, shows evidence of multiple rounds of division, but lacks markers of recent TCR engagement. Detailed cell cycle analysis revealed that compared with their CD8(+) HLA-DR(-) counterpart, the CD8(+) HLA-DR(+) T-cell pool contained an increased fraction of cells in S-phase with elevated levels of the G2/M regulators: cyclin A2, CDC25C, Cdc2 (CDK1), indicating that these cells are not truly anergic but rather experiencing proliferation in vivo. Together, these data support a hypothesis that antigen stimulation leads to the initial expansion of a CD8(+) pool of cells in vivo that undergo further expansion independent of ongoing TCR engagement. No qualitative differences were noted between CD8(+) HLA-DR(+) cells from HIV(+) and HIV(-) donors, indicating that the generation of CD8(+) HLA-DR(+) T cells is a part of normal immune regulation that is exaggerated in the setting of HIV-1 infection.
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http://dx.doi.org/10.1002/eji.201142046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818066PMC
October 2012

Gene expression profiles predict emergence of psychiatric adverse events in HIV/HCV-coinfected patients on interferon-based HCV therapy.

J Acquir Immune Defic Syndr 2012 Jul;60(3):273-81

Office of the Clinical Director, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.

Background: The efficacy of pegylated interferon-α and ribavirin (pegIFN/RBV) in the treatment of Hepatitis C infection is limited by psychiatric adverse effects (IFN-PE). Our study examined the ability of differential gene expression patterns before therapy to predict emergent IFN-PE among 28 HIV/HCV-coinfected patients treated with pegIFN-α2b/RBV.

Methods: Patients dually infected with HIV and HCV were evaluated at baseline and during treatment by board-certified psychiatrists who classified patients into 2 groups: those who developed IFN-PE and those who did not (IFN-NPE). Gene expression analysis (Affymetrix HG-U133A) was performed using peripheral blood mononuclear cells before and after initiation of treatment. Analysis of Variance, post hoc analysis based on pair-wise comparisons, and functional annotation analysis identified differentially expressed genes within and between groups. Prediction analysis for microarrays was used to test the predictive ability of selected genes.

Results: Twenty-four genes (16 upregulated and 8 downregulated) that were differentially expressed at baseline in patients who subsequently developed IFN-PE compared with the IFN-NPE group showed the ability to predict IFN-PE with an accuracy of 82%. In 16 patients with IFN-PE, 135 genes (117 upregulated; 18 downregulated) were significantly modulated after treatment. Of these, 10 genes have already been shown to be associated with neuropsychiatric illnesses and were significantly modulated only in patients who experienced IFN-PE.

Conclusions: We describe a novel molecular diagnostic biomarker panel to predict emergent IFN-PE in HIV/HCV-coinfected patients undergoing pegIFN/RBV treatment, which may improve the identification of patients at greatest risk for IFN-PE and suggest candidate therapeutic targets for preventing or treating IFN-PE.
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http://dx.doi.org/10.1097/QAI.0b013e31824c17c4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383605PMC
July 2012

Cytokine/chemokine patterns connect host and viral characteristics with clinics during chronic hepatitis C.

Eur J Med Res 2012 May 11;17. Epub 2012 May 11.

Department of Gastroenterology and Hepatology, University Hospital of Essen, Germany.

Background: In chronic hepatitis C virus (HCV) infection, liver tissue pathology and HCV genotype are important determinants of clinical and/or treatment-related outcome. Although consistent epidemiological and/or molecular-biological clues derived from different studies on single virus-host interactions are meanwhile published, the in vivo transcriptional responses and cellular pathways affected in >1 key aspects of the disease or treatment process are far from being understood.

Methods: Microarray analysis was performed in peripheral whole blood (PB) samples from 36 therapy-naïve HCV-infected patients with known liver histology. Linear regression analysis identified gene expression profiles significantly correlating (P < 0.015) with ≥1 out of 7 variables: sustained viral response (SVR), viral non-response (NR), end of treatment viral response (ETR), viral breakthrough (VB), HCV genotype (Gt. 1 vs. Gt. 2/3), stage of hepatic fibrosis [St. 0/1 vs. St. 2/3/4] and grade of hepatic inflammation (Gr. 0/1 vs. Gr. 2/3/4). Correlation values across all seven contrasts were considered for hierarchical clustering (HCL).

Results: A total of 1,697 genes showed ≥1 significant correlation results and genes involved in cell differentiation (183), immune response (53), and apoptosis (170) were leading fractions. HCL grouped the genes into six major clusters. Functional annotation analysis using DAVID (http://david.abcc.ncifcrf.gov) revealed that expression profiles that best linked these variables were highly enriched in cytokine/chemokine activity (Fisher-exact P < 0.0001) and specific biological module-centric algorithms finally led our focus on four out of fifty-three immune response genes: SMAD family member 3 (SMAD3), interleukin 1 receptor accessory protein (IL1RAP), tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), and chemokine 'C-C motif' receptor 5 (CCR5). Of those, TNFRSF1A and CCR5 showed significant correlation with two out of seven variables based on microarray and/or quantitative real-time polymerase chain reaction (qRT-PCR) data.

Conclusion: We identified molecular targets of the innate and adaptive immune system and validated their transcriptional specificity in vivo suggesting significant involvement in two unique outcomes during HCV treatment.
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http://dx.doi.org/10.1186/2047-783X-17-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489717PMC
May 2012

DAVID-WS: a stateful web service to facilitate gene/protein list analysis.

Bioinformatics 2012 Jul 27;28(13):1805-6. Epub 2012 Apr 27.

Laboratory of Immunopathogenesis and Bioinformatics, National Cancer Institute at Frederick, Frederick, MD 21702, USA.

Summary: The database for annotation, visualization and integrated discovery (DAVID), which can be freely accessed at http://david.abcc.ncifcrf.gov/, is a web-based online bioinformatics resource that aims to provide tools for the functional interpretation of large lists of genes/proteins. It has been used by researchers from more than 5000 institutes worldwide, with a daily submission rate of ∼1200 gene lists from ∼400 unique researchers, and has been cited by more than 6000 scientific publications. However, the current web interface does not support programmatic access to DAVID, and the uniform resource locator (URL)-based application programming interface (API) has a limit on URL size and is stateless in nature as it uses URL request and response messages to communicate with the server, without keeping any state-related details. DAVID-WS (web service) has been developed to automate user tasks by providing stateful web services to access DAVID programmatically without the need for human interactions.

Availability: The web service and sample clients (written in Java, Perl, Python and Matlab) are made freely available under the DAVID License at http://david.abcc.ncifcrf.gov/content.jsp?file=WS.html.
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http://dx.doi.org/10.1093/bioinformatics/bts251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381967PMC
July 2012

HIV infection and antiretroviral therapy have divergent effects on mitochondria in adipose tissue.

J Infect Dis 2012 Jun 3;205(12):1778-87. Epub 2012 Apr 3.

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1403, USA.

Background: Although human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) affect mitochondrial DNA (mtDNA) content and function, comprehensive evaluations of their effects on mitochondria in muscle, adipose tissue, and blood cells are limited.

Methods: Mitochondrial DNA quantification, mitochondrial genome sequencing, and gene expression analysis were performed on muscle, adipose tissue, and peripheral blood mononuclear cell (PBMC) samples from untreated HIV-positive patients, HIV-positive patients receiving nucleoside reverse transcriptase inhibitor (NRTI)-based ART, and HIV-negative controls.

Results: The adipose tissue mtDNA/nuclear DNA (nDNA) ratio was increased in untreated HIV-infected patients (ratio, 353) and decreased in those receiving ART (ratio, 162) compared with controls (ratio, 255; P < .05 for both comparisons); the difference between the 2 HIV-infected groups was also significant (P = .002). In HIV-infected participants, mtDNA/nDNA in adipose tissue correlated with the level of activation (CD38+ /HLA-DR+) for CD4+ and CD8+ lymphocytes. No significant differences in mtDNA content were noted in muscle or PMBCs among groups. Exploratory DNA microarray analysis identified differential gene expression between patient groups, including a subset of adipose tissue genes.

Conclusions: HIV infection and ART have opposing effects on mtDNA content in adipose tissue; immune activation may mediate the effects of HIV, whereas NRTIs likely mediate the effects of ART.
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http://dx.doi.org/10.1093/infdis/jis101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357134PMC
June 2012

Host gene expression changes correlating with anti-HIV-1 effects in human subjects after treatment with peginterferon Alfa-2a.

J Infect Dis 2012 May 26;205(9):1443-7. Epub 2012 Mar 26.

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

We investigated whether interferon-inducible genes (IFIGs) with known anti-human immunodeficiency virus (HIV) activity in vitro were associated with in vivo virological response in HIV infection. Nine untreated HIV-1-infected volunteers were treated for 12 weeks with peginterferon alfa-2a. A subset of IFIGs (23 of 47) increased compared with baseline through 6 weeks beyond therapy, and 10 of the 23 IFIGs significantly inversely correlated (r = -0.7; P < .05) with virological response. The strength of peginterferon alfa-2a-induced IFIG response significantly correlated with declines in HIV load during treatment (r(2) = 0.87, p = .003). This study links HIV virological response to a specific IFIG subset, a potential prognostic indicator in peginterferon alfa-2a-treated patients with HIV infection.
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http://dx.doi.org/10.1093/infdis/jis211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324397PMC
May 2012

Interleukin-27 induces interferon-inducible genes: analysis of gene expression profiles using Affymetrix microarray and DAVID.

Methods Mol Biol 2012 ;820:25-53

Laboratory of Human Retrovirology, Clinical Services Programs (CSP), Applied Developmental Directorate (ADD), Science Applications International Corporation (SAIC)-Frederick, Inc., National Cancer Institute (NCI)-Frederick, Frederick, MD 21702, USA.

We have previously demonstrated that IL-27 is a novel anti-HIV cytokine, which inhibits HIV replication in CD4 T cells and macrophages as interferon (IFN)-α does. To further understand the mechanism of the antiviral effect, we performed Affymetrix DNA microarray and gene functional annotation analysis using DAVID (the Database for Annotation, Visualization, and Integrated Discovery). DAVID is a web-based bioinformatics application that systematically identifies enriched biology associated with large gene list(s) derived from high-throughput genomic experiments, such as microarray. The enriched annotation terms identified by DAVID will give important insights into understanding the biological themes under study. Having used the DAVID bioinformatics tools, we have shown that IL-27 differentially regulates the gene expression between T cells and macrophages. IL-27 significantly induces IFN-inducible genes including antiviral genes in macrophages as does IFN-α, suggesting that IL-27 inhibits HIV replication in macrophages via a mechanism similar to that of IFN-α.
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http://dx.doi.org/10.1007/978-1-61779-439-1_3DOI Listing
March 2012

CCL5: a double-edged sword in host defense against the hepatitis C virus.

Int Rev Immunol 2011 Oct-Dec;30(5-6):366-78

Laboratory of Immunopathogenesis and Bioinformatics, SAIC-Frederick Inc., National Cancer Institute at Frederick (NCI-Frederick), Frederick, Maryland, USA.

C-C motif ligand 5 (CCL5) facilitates induction of chemotaxis in immune cells and activation of hepatic stellate cells (HSC) at sites of liver inflammation during chronic hepatitis C virus (HCV) infection. Importantly, CCL5 participates in the establishment of T-helper 1 responses crucial in controlling liver disease and HCV infection outcome and demonstrates distinct gene expression patterns between the blood and the liver, stressing the importance of immunoregulatory networks differentially functioning between these compartments. This review illustrates the significance of CCL5-dependent pathways in HCV-related immunopathogenesis by elaborating on biological mechanisms interconnecting peripheral and tissue immunology, liver pathology, HSC activation, and interferon-α immunotherapy.
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http://dx.doi.org/10.3109/08830185.2011.593105DOI Listing
March 2012

LIM kinase 1 - dependent cofilin 1 pathway and actin dynamics mediate nuclear retinoid receptor function in T lymphocytes.

BMC Mol Biol 2011 Sep 16;12:41. Epub 2011 Sep 16.

Laboratory of Molecular Cell Biology, SAIC-Frederick, National Cancer Institute, Frederick, MD 21702, USA.

Background: It is known that retinoid receptor function is attenuated during T cell activation, a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. Here we have investigated the role of actin dynamics and the effect of actin cytoskeleton modifying agents on retinoid receptor-mediated transactivation.

Results: Agents that disturb the F-actin assembly or disassembly attenuated receptor-mediated transcription indicating that actin cytoskeletal homeostasis is important for retinoid receptor function. Overexpression or siRNA-induced knockdown of cofilin-1 (CFL1), a key regulator of F-actin assembly, induced the loss of receptor function. In addition, expression of either constitutively active or inactive/dominant-negative mutants of CFL1or CFL1 kinase LIMK1 induced loss of receptor function suggesting a critical role of the LIMK1-mediated CFL1 pathway in receptor-dependent transcription. Further evidence of the role of LMK1/CFL1-mediated actin dynamics, was provided by studying the effect of Nef, an actin modifying HIV-1 protein, on receptor function. Expression of Nef induced phosphorylation of CFL1 at serine 3 and LIMK1 at threonine 508, inhibited retinoid-receptor mediated reporter activity, and the expression of a number of genes that contain retinoid receptor binding sites in their promoters. The results suggest that the Nef-mediated inhibition of receptor function encompasses deregulation of actin filament dynamics by LIMK1 activation and phosphorylation of CFL1.

Conclusion: We have identified a critical role of LIMK1-mediated CFL1 pathway and actin dynamics in modulating retinoid receptor mediated function and shown that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. We suggest that T cell activation-induced repression of nuclear receptor-dependent transactivation is in part through the modification of actin dynamics.
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http://dx.doi.org/10.1186/1471-2199-12-41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187726PMC
September 2011
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