Sustained high-level polyclonal hematopoietic marking and transgene expression 4 years after autologous transplantation of rhesus macaques with SIV lentiviral vector-transduced CD34+ cells.

We previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus hematopoietic repopulating cells. To evaluate the persistence of vector-containing and -expressing cells long term, and the safety implications of SIV lentiviral vector-mediated gene transfer, we followed 3 rhesus macaques for more than 4 years after transplantation with transduced CD34+ cells. All 3 animals demonstrated significant vector marking and expression of the GFP transgene in T cells, B cells, and granulocytes, with mean GFP+ levels of 6.7% (range, 3.3%-13.0%), 7.4% (4.2%-13.4%), and 5.6% (3.1%-10.5%), respectively. There was no vector silencing in hematopoietic cells over time. Vector insertion site analysis of granulocytes demonstrated sustained highly polyclonal reconstitution, with no evidence for progression to oligoclonality. A significant number of clones were found to contribute at both 1-year and 3- or 4-year time points. No vector integrations were detected in the MDS1/EVI1 region, in contrast to our previous findings with a gamma-retroviral vector. These data show that lentiviral vectors can mediate stable and efficient long-term expression in the progeny of transduced hematopoietic stem cells, with an integration profile that may be safer than that of standard Moloney murine leukemia virus (MLV)-derived retroviral vectors.

A microarray analysis for differential gene expression in the soybean genome using Bioconductor and R.

This article describes specific procedures for conducting quality assessment of Affymetrix GeneChip(R) soybean genome data and for performing analyses to determine differential gene expression using the open-source R programming environment in conjunction with the open-source Bioconductor software. We describe procedures for extracting those Affymetrix probe set IDs related specifically to the soybean genome on the Affymetrix soybean chip and demonstrate the use of exploratory plots including images of raw probe-level data, boxplots, density plots and M versus A plots. RNA degradation and recommended procedures from Affymetrix for quality control are discussed. An appropriate probe-level model provides an excellent quality assessment tool. To demonstrate this, we discuss and display chip pseudo-images of weights, residuals and signed residuals and additional probe-level modeling plots that may be used to identify aberrant chips. The Robust Multichip Averaging (RMA) procedure was used for background correction, normalization and summarization of the AffyBatch probe-level data to obtain expression level data and to discover differentially expressed genes. Examples of boxplots and MA plots are presented for the expression level data. Volcano plots and heatmaps are used to demonstrate the use of (log) fold changes in conjunction with ordinary and moderated t-statistics for determining interesting genes. We show, with real data, how implementation of functions in R and Bioconductor successfully identified differentially expressed genes that may play a role in soybean resistance to a fungal pathogen, Phakopsora pachyrhizi. Complete source code for performing all quality assessment and statistical procedures may be downloaded from our web source: http://css.ncifcrf.gov/services/download/MicroarraySoybean.zip.

Live attenuated or inactivated influenza vaccines and medical encounters for respiratory illnesses among US military personnel.

CONTEXT: Since 2004, increasing numbers of military personnel have been immunized with the intranasal live attenuated influenza vaccine (LAIV) while most others received the trivalent inactivated vaccine (TIV). However, data about live virus vaccine effectiveness among healthy adults are limited. OBJECTIVE: To monitor the effectiveness of vaccines to better inform military vaccination policy. DESIGN, SETTING, AND PARTICIPANTS: Surveillance of population-based, propensity-matched, and/or vaccine-naive cohorts of more than a million active-duty, nonrecruit military service members aged 17 to 49 years stationed in the United States during the 2004-2005, 2005-2006, or 2006-2007 influenza season. MAIN OUTCOME MEASURES: Incidence of health care encounters resulting in a primary diagnostic code consistent with pneumonia or influenza. Incident hospitalizations was a secondary outcome. RESULTS: In all 3 seasons, immunization with TIV was associated with lower incidence rates of health care encounters for pneumonia and influenza when compared with no immunization: 8.6 vs 19.4 for 2004-2005, 7.8 vs 10.9 for 2005-2006, and 8.0 vs. 11.7 per 1000 person-years for 2006-2007 (all P < .001). Similar estimates were obtained from propensity-matched and/or vaccine-naive cohorts. Consistently lower vaccine effect following LAIV immunization was only seen during the 2006-2007 influenza season in the total (10.7; 95% confidence interval [CI], 2.72 to 18.1; P = .03) and propensity-matched cohorts (11.8; 95% CI, 0.85 to 21.5; P = .04), and was less than effect from TIV (TIV vs LAIV, 19.8; 95% CI, 13.6 to 25.5; P < .001). Among vaccine-naive service members, however, estimates for LAIV effect were more robust for both the 2005-2006 and 2006-2007 seasons (P = .01) and were comparable with TIV (eg, LAIV, 30.2; 95% CI, 11.2 to 45.2; vs TIV, 35.3; 95% CI, 25.9 to 43.6; in 2005-2006). CONCLUSIONS: Vaccination with TIV was associated with fewer medical encounters related to pneumonia and influenza compared with LAIV or no immunization. In this annually immunized population, this effect was less apparent in those vaccinated with LAIV.

An Integrated Genetic-Genomic Approach for the Identification of Novel Cancer Loci in Mice Sensitized to c-Myc-Induced Apoptosis.

Deregulated c-Myc is associated with a wide range of human cancers. In many cell types, overexpression of c-Myc potently promotes cell growth and proliferation concomitant with the induction of apoptosis. Secondary genetic events that shift this balance either by increasing growth and proliferation or limiting apoptosis are likely to cooperate with c-Myc in tumorigenesis. Here, the authors have performed large-scale insertional mutagenesis in Eµ-c-myc mice that, through mdm2 loss of function mutations, are sensitized to apoptosis. The authors chose to use this genetic background based on the hypothesis that the high level of apoptosis induced by c-Myc overexpression in MDM2-deficient mice would act as a rate-limiting barrier for lymphoma development. As a result, it was predicted that the spectrum of retroviral insertions would be shifted toward loci that harbor antiapoptotic genes. Nine novel common insertion sites (CISs) specific to mice with this sensitized genetic background were identified, suggesting the presence of novel antiapoptotic cancer genes. Moreover, cross-comparing the data to the Retroviral Tagged Cancer Gene Database, the authors identified an additional 23 novel CISs. Here, evidence is presented that 2 genes, ppp1r16b and hdac6, identified at CISs, are bona fide cellular oncogenes. This study highlights the power of combining unique sensitized genetic backgrounds with large-scale mutagenesis as an approach for identifying novel cancer genes.

DNA hypomethylation caused by Lsh deletion promotes erythroleukemia development.

Hematopoietic malignancies are frequently associated with DNA hypomethylation but the molecular mechanisms involved in tumor formation remain poorly understood. Here we report that mice lacking Lsh develop leukemia associated with DNA hypomethylation and oncogene activation. Lsh is a member of the SNF2 chromatin remodeling family and is required for de novo methylation of genomic DNA. Mice that received Lsh deficient hematopoietic progenitors showed severe impairment of hematopoiesis, suggesting that Lsh is necessary for normal hematopoiesis. A subset of mice developed erythroleukemia, a tumor that does not spontaneously occur in mice. Tumor tissues were CpG hypomethylated and showed a modest elevation of the transcription factor PU.1, an oncogene that is crucial for Friend virus induced erythroleukemia. Analysis of Lsh(-/-) hematopoietic progenitors revealed widespread DNA hypomethylation at repetitive sequences and hypomethylation at specific retroviral elements within the PU.1 gene. Wild type cells showed Lsh and Dnmt3b binding at the retroviral elements located within the PU.1 gene. On the other hand, Lsh deficient cells had no detectable Dnmt3b association suggesting that Lsh is necessary for recruitment of Dnmt3b to its target. Furthermore, Lsh(-/-) hematopoietic precursors showed impaired suppression of retroviral elements in the PU.1 gene, an increase of PU.1 transcripts and protein levels. Thus DNA hypomethylation caused by Lsh depletion is linked to transcriptional upregulation of retroviral elements and oncogenes such as PU.1 which in turn may promote the development of erythroleukemia in mice.

Evaluation of a single-platform technology for lymphocyte immunophenotyping.

An accurate and reproducible CD4 count is a fundamental clinical tool for monitoring and treating human immunodeficiency virus infection and its complications. Two methods exist for calculating absolute CD4 counts: dual-platform technology (DPT) and single-platform technology (SPT). Numerous studies have documented the unacceptably wide range of variation in absolute CD4 counts between laboratories. SPT was introduced in 1996 to reduce the interlaboratory variation in absolute CD4 counts. The aim of this study was to compare DPT with the BD Biosciences Trucount method (an SPT method). Both the percentages of CD4 (r = 0.986; P = 0.0541) and the absolute CD4 counts (r = 0.960; P = 0.0001) had very good correlation between the two methods. However, poor correlation was observed for the CD8(+) RO(-) (r = 0.314; P = 0.0002), CD8(+) DR(+) (r = 0.666; P = 0.0138), CD3(+) CD38(+) (r = 0.8000; P = 0.0004), CD3(+) CD25(+) (r = 0.464; P = 0.0082), and CD4(+) CD38(+) (r = 0.357; P = 0.0127) measurements.

The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists.

The DAVID Gene Functional Classification Tool http://david.abcc.ncifcrf.gov uses a novel agglomeration algorithm to condense a list of genes or associated biological terms into organized classes of related genes or biology, called biological modules. This organization is accomplished by mining the complex biological co-occurrences found in multiple sources of functional annotation. It is a powerful method to group functionally related genes and terms into a manageable number of biological modules for efficient interpretation of gene lists in a network context.