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Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions insilico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with anintegration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.

Motivation: The relative abundance of retroviral insertions in a host genome is important in understanding the persistence and pathogenesis of both natural retroviral infections and retroviral gene therapy vectors. It could be estimated from a sample of cells if only the host genomic sites of retroviral insertions could be directly counted. When host genomic DNA is randomly broken via sonication and then amplified, amplicons of varying lengths are produced. The number of unique lengths of amplicons of an insertion site tends to increase according to its abundance, providing a basis for estimating relative abundance. However, as abundance increases amplicons of the same length arise by chance leading to a non-linear relation between the number of unique lengths and relative abundance. The difficulty in calibrating this relation is compounded by sample-specific variations in the relative frequencies of clones of each length. Results: A likelihood function is proposed for the discrete lengths observed in each of a collection of insertion sites and is maximized with a hybrid expectation–maximization algorithm. Patient data illustrate the method and simulations show that relative abundance can be estimated with little bias, but that variation in highly abundant sites can be large. In replicated patient samples, variation exceeds what the model implies—requiring adjustment as in Efron (2004) or using jackknife standard errors. Consequently, it is advantageous to collect replicate samples to strengthen inferences about relative abundance. Availability: An R package implements the algorithm described here. It is available at http://soniclength.r-forge.r-project.org/ Contact: ccberry@ucsd.edu Supplementary information: Supplementary data are available at at Bioinformatics online.

Human T-lymphotropic virus type 1 (HTLV-1) persists by driving clonal proliferation of infected T lymphocytes. A high proviral load predisposes to HTLV-1–associated diseases. Yet the reasons for the variation within and between persons in the abundance of HTLV-1–infected clones remain unknown. We devised a high-throughput protocol to map the genomic location and quantify the abundance of > 91 000 unique insertion sites of the provirus from 61 HTLV-1+ persons and > 2100 sites from in vitro infection. We show that a typical HTLV-1–infected host carries between 500 and 5000 unique insertion sites. We demonstrate that negative selection dominates during chronic infection, favoring establishment of proviruses integrated in transcriptionally silenced DNA: this selection is significantly stronger in asymptomatic carriers. We define a parameter, the oligoclonality index, to quantify clonality. The high proviral load characteristic of HTLV-1–associated inflammatory disease results from a larger number of unique insertion sites than in asymptomatic carriers and not, as previously thought, from a difference in clonality. The abundance of established HTLV-1 clones is determined by genomic features of the host DNA flanking the provirus. HTLV-1 clonal expansion in vivo is favored by orientation of the provirus in the same sense as the nearest host gene.

Background The outcomes of gene therapy to correct congenital immunodeficiencies are unknown. We reviewed long-term outcomes after gene therapy in nine patients with X-linked severe combined immunodeficiency (SCID-X1), which is characterized by the absence of the cytokine receptor common γ chain. Methods The nine patients, who lacked an HLA-identical donor, underwent ex vivo retrovirus-mediated transfer of γ chain to autologous CD34+ bone marrow cells between 1999 and 2002. We assessed clinical events and immune function on long-term follow-up. Results Eight patients were alive after a median follow-up period of 9 years (range, 8 to 11). Gene therapy was initially successful at correcting immune dysfunction in eight of the nine patients. However, acute leukemia developed in four patients, and one died. Transduced T cells were detected for up to 10.7 years after gene therapy. Seven patients, including the three survivors of leukemia, had sustained immune reconstitution; three patients required immunoglobulin-replacement therapy. Sustained thymopoiesis was established by the persistent presence of naive T cells, even after chemotherapy in three patients. The T-cell−receptor repertoire was diverse in all patients. Transduced B cells were not detected. Correction of the immunodeficiency improved the patients’ health. Conclusions After nearly 10 years of follow-up, gene therapy was shown to have corrected the immunodeficiency associated with SCID-X1. Gene therapy may be an option for patients who do not have an HLA-identical donor for hematopoietic stem-cell transplantation and for whom the risks are deemed acceptable. This treatment is associated with a risk of acute leukemia. (Funded by INSERM and others.)

The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting.Primary resting or activated CD4 T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined.We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions.HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes.These data indicate that integration site distributions show modest but significant differences between resting and activated CD4 T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression.

Lentiviral vector-based gene therapy has been used to target the human immunodeficiency virus (HIV) using an antisense env payload. We have analyzed lentiviral-vector integration sites from three treated individuals. We compared integration sites from the ex vivo vector-transduced CD4+ cell products to sites from cells recovered at several times after infusion. Integration sites were analyzed using 454 pyrosequencing, yielding a total of 7,782 unique integration sites from the ex vivo product and 237 unique sites from cells recovered after infusion. Integrated vector copies in both data sets were found to be strongly enriched within active genes and near epigenetic marks associated with active transcription units. Analysis of integration relative to nucleosome structure on target DNA indicated favoring of integration in outward facing DNA major grooves on the nucleosome surface. There was no indication that growth of transduced cells after infusion resulted in enrichment for integration sites near proto-oncogene 5'-ends or within tumor suppressor genes. Thus, this first look at the longitudinal evolution of cells transduced with a lentiviral vector after infusion of gene modified CD4+ cells provided no evidence for abnormal expansions of cells due to vector-mediated insertional activation of proto-oncogenes.

The question of where retroviral DNA becomes integrated in chromosomes is important for understanding (i) the mechanisms of viral growth, (ii) devising new anti-retroviral therapy, (iii) understanding how genomes evolve, and (iv) developing safer methods for gene therapy. With the completion of genome sequences for many organisms, it has become possible to study integration targeting by cloning and sequencing large numbers of host-virus DNA junctions, then mapping the host DNA segments back onto the genomic sequence. This allows statistical analysis of the distribution of integration sites relative to the myriad types of genomic features that are also being mapped onto the sequence scaffold. Here we present methods for recovering and analyzing integration site sequences.

SummaryDeciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

Integration of retroviral DNA into host cell DNA is a defining feature of retroviral replication. HIV integration is known to be favored in active transcription units, which promotes efficient transcription of the viral genes, but the molecular mechanisms responsible for targeting are not fully clarified. Here we used pyrosequencing to map 40,569 unique sites of HIV integration. Computational prediction of nucleosome positions in target DNA indicated that integration sites are periodically distributed on the nucleosome surface, consistent with favored integration into outward-facing DNA major grooves in chromatin. Analysis of integration site positions in the densely annotated ENCODE regions revealed a wealth of new associations between integration frequency and genomic features. Integration was particularly favored near transcription-associated histone modifications, including H3 acetylation, H4 acetylation, and H3 K4 methylation, but was disfavored in regions rich in transcription-inhibiting modifications, which include H3 K27 trimethylation and DNA CpG methylation. Statistical modeling indicated that effects of histone modification on HIV integration were partially independent of other genomic features influencing integration. The pyrosequencing and bioinformatic methods described here should be useful for investigating many aspects of retroviral DNA integration.

The goal of this study was to identify genes consistently differentially expressed in multiple pairs of isogenic cisplatin (DDP)-sensitive and resistant human ovarian carcinoma cell lines using microarray-based expression profiling. Expression profiling was carried out on six pairs of ovarian carcinoma cells lines growing under identical conditions; each cell expression profile was independently replicated six times. No genes were differentially expressed in all six pairs of cells or even in even in any five of the six pairs. Eighteen genes and 1 EST were upregulated, and four genes and 1 EST were downregulated, in at least four cell pairs. Of these, only metallothionein 2A has previously been implicated in DDP resistance. Among the genes identified on the basis of six replicates, an average of 24.8% would have been missed if only five replicates had been performed, and 38.3% would have been missed with only four replicates. The genes did not identify a dominant biochemical pathway or ontology category as being linked to DDP resistance; however, hierarchical clustering provided evidence for two classes DDP-resistant phenotypes within which there are additional cell pair-specific alterations. Many of the genes identified in this study play important roles in cell surface interactions and trafficking pathways not previously linked to DDP resistance. The genes discovered by this extensively replicated analysis are candidates for prediction of DDP responsiveness in ovarian cancer patients.

In order to identify genes whose expression is associated with resistance to the chemotherapeutic agent oxaliplatin, transcripts differentially expressed between an oxaliplatin sensitive and a stably resistant subline were compared in six independent replicates using Stanford cDNA microarrays for five cell lines. "Significance analysis of microarrays" (SAM) was used to identify genes whose expression was statistically significantly different in the sensitive versus resistant members of each cell line pair. The biochemical pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were searched to identify those pathways in which the number of SAM-identified genes exceeded the number expected. This identified four pathways in which upregulated genes were significantly associated with resistance in two of the cell line pairs, and two pathways in which the association was found in three cell line pairs. The search also identified 12 pathways in which downregulated genes were associated with resistance in two cell line pairs and one pathway in which the association reached statistical significance in three cell line pairs. Pathways identified included the ribosome pathway, the Huntington's disease pathway that includes caspase 8, and the ATP synthesis pathways. Determination of the chromosomal location of each SAM-identified gene revealed several locales within which genes lay in close proximity, including three genes (APACD, IF-2, and REV1L) located on chromosome 2 that lie immediately adjacent to each other and were significantly upregulated in three of five cell line pairs. Biochemical pathway and chromosomal mapping of genes identified by SAM as differentially expressed in related cell line pairs points to mechanisms and chromosomal sites not previously suspected of association with the oxaliplatin-resistant phenotype.

Genetic analysis of natural variation in ecotypes of Arabidopsis thaliana can facilitate the discovery of new genes or of allelic variants of previously identified genes controlling physiological processes in plants. We mapped quantitative trait loci (QTL) for light response in recombinant inbred lines (RILs) derived from the Columbia and Kashmir accessions via two methods: composite interval mapping and eXtreme array mapping (XAM). After measuring seedling hypocotyl lengths in blue, red, far-red, and white light, and in darkness, eight QTL were identified by composite interval mapping and five localized near photoreceptor loci. Two QTL in blue light were associated with CRY1 and CRY2, two in red light were near PHYB and PHYC, and one in far-red light localized near PHYA. The RED2 and RED5 QTL were verified in segregating lines. XAM was tested for the identification of QTL in red light with pools of RILs selected for extreme phenotypes. Thousands of single feature polymorphisms detected by differential DNA hybridized to high-density oligo-nucleotide arrays were used to estimate allele frequency differences between the pools. The RED2 QTL was identified clearly; differences exceeded a threshold of significance determined by simulations. The sensitivities of XAM to population type and size and genetic models were also determined by simulation analysis.

Modulation of epitope-specific immune responses would represent a major addition to available therapeutic options for many autoimmune diseases. The objective of this work was to induce immune deviation by mucosal peptide-specific immunotherapy in rheumatoid arthritis (RA) patients, and to dissect the related immunological mechanisms by using a technology for the detection of low-affinity class II-restricted peptide-specific T cells. A group of patients with early RA was treated for 6 months orally with dnaJP1, a peptide that induces proinflammatory T cell responses in naive RA patients. Immunological analysis at initial, intermediate and end treatment points showed an intriguing change from proinflammatory to regulatory T cell function. In fact, dnaJP1-induced T cell production of IL-4 and IL-10 increased significantly when initial and end treatment points were compared, whereas dnaJP1-induced T cell proliferation and production of IL-2, IFN-γ, and tumor necrosis factor-α decreased significantly. The total number of dnaJP1-specific cells did not change over time, whereas expression of foxP3 by CD4+CD25brightcells increased, suggesting that the treatment affected regulatory T cell function. Thus, rather than clonal deletion, the observed change in immune reactivity to dnaJP1 was the outcome of treatment-induced emergence of T cells with a different functional phenotype. This study contributes to our knowledge of mechanisms and tools needed for antigen-specific immune modulation in humans, thus laying the foundation for exploitation of this approach for therapeutic purposes.

We have examined the changes in cellular transcription resulting from infection with HIV-based vectors. Previous work suggested that the incoming viral genome may under some circumstances be detected as DNA damage, so to explore this possibility, we compared the transcriptional response to infection with an HIV-based vector to the response to treatment with the DNA-damaging agent etoposide. Expression levels of about 12,000 cellular RNA transcripts were determined in a human B-cell line at different times after either treatment. Statistical analysis revealed that the infection with the lentivirus vector resulted in quite modest changes in gene expression. Treatment with etoposide, in contrast, caused drastic changes in expression of genes known or inferred to be involved in apoptosis. Statistically significant though subtle parallels in the cellular transcriptional responses to etoposide treatment and HIV-vector infection could be detected. Several further data sets analyzing infections with HIV-based vectors or wild-type HIV-1 showed similar modest effects on cellular transcription and very modest parallels among different data sets. These findings establish that HIV-vector or HIV-1 infection has remarkably little effect on cellular transcription. The statistical methods described here may be of wide use in mining microarray data sets. Our observations support the idea that gene therapy with HIV-based vectors should not be particularly toxic to cells due to disruption of cellular transcription.

Purpose To quantify factors affecting test-retest variability of threshold measurements over a series of 3 serial visual fields (VF). Design Prospective comparative observational study. Participants Forty-one normals, 10 suspects and 35 stable glaucoma patients. Methods All subjects performed 3 standard and 3 short-wavelength automated perimetry (SWAP) VFs. At each VF location, severity (defined as age-corrected total deviation) and test-retest variability (TRV), defined as the standard deviation of 3 serial threshold values, were calculated. A multiple regression model (constructed separately for standard VF and SWAP) incorporated 13 factors: severity, location, eccentricity, study group, diagnosis, superior versus inferior hemifield, nasal versus temporal hemifield, one-versus-two thresholds, age, mean pupil size, pupil size variability, between-subject variation, and residual variation. Main outcome measures Variability in threshold sensitivity VF values. Results Mean TRV (± standard deviation) for normal, suspect and glaucoma eyes, respectively, was: 1.28 ± 0.87, 1.53 ± 1.04 and 2.20 ± 1.79 dB for standard VF, and 1.87 ± 1.35, 1.86 ± 1.24 and 2.68 ± 1.85 dB for SWAP. The contribution of each factor to the model for standard VF and SWAP (SWAP in parentheses) were: severity 15.5% (6.9%); location 2.7% (4.1%); eccentricity 1.1% (0.64%); diagnosis 2.9% (5.9%); “superior versus inferior” hemifield 0.17% (1.7%); “nasal versus temporal” hemifield 0.06% (0.02%); one-versus-two thresholds 0.04% (0.16%); age 0.1% (0.06%); mean pupil size 0.59% (0.1%); pupil size variability 3.2% (2.8%); between-subject 8.0% (13.5%) and residual variation 61.0% (66.6%). Excluding between-subject and residual variation, the 11-factor model was able to account for less than one third of the variability seen in both standard VF and SWAP. Conclusions Severity of defect and between subject variation exerted the largest effect on TRV. However, even if all 11 factors could be adjusted for, it would reduce the magnitude of TRV by only 30%. More work is needed to reduce the remaining variability inherent in psychophysical testing and to better understand the intrinsic physiological variability present both in healthy and diseased eyes. It is possible that a larger number of VFs used for the calculation of TRV might further reduce the magnitude of the remaining variability found in this study.

This study examined the effects of hypertension and an acute psychological stressor on white blood cells and their expression of CD62L and CD11a. Seventeen mild hypertensive and 23 normotensive volunteers were studied prior to and following a standardized laboratory public speech. In response to the speech, all subjects increased the number of circulating leukocyte populations (p's.01). Patients with hypertension increased the number of circulating white blood cells more than normotensives (p.01). Hypertensives also showed a greater increase in the number of circulating CD3(+)CD8(+) T cells (p.02) in response to the speech. Only hypertensives increased the number of circulating CD8(+)CD62L(high) T cells (p=.001). The density of CD11a on lymphocytes was increased in all subjects following the speech (p.001). Hypertensives showed a greater mean density of CD11a on lymphocytes (p.01). Coupled with observations of increased expression of the endothelial CD11a ligand ICAM-1 in hypertension, these findings are consistent with the notion that patients with hypertension exhibit a circulatory environment conducive to increased leukocyte adhesion. Exposure to repeated psychological stressors may further augment this potentially adverse circulatory environment.

We have developed a high-throughput genotyping platform by hybridizing genomic DNA from Arabidopsis thaliana accessions to an RNA expression GeneChip (AtGenome1). Using newly developed analytical tools, a large number of single-feature polymorphisms (SFPs) were identified. A comparison of two accessions, the reference strain Columbia (Col) and the strain Landsberg erecta (Ler), identified nearly 4000 SFPs, which could be reliably scored at a 5% error rate. Ler sequence was used to confirm 117 of 121 SFPs and to determine the sensitivity of array hybridization. Features containing sequence repeats, as well as those from high copy genes, showed greater polymorphism rates. A linear clustering algorithm was developed to identify clusters of SFPs representing potential deletions in 111 genes at a 5% false discovery rate (FDR). Among the potential deletions were transposons, disease resistance genes, and genes involved in secondary metabolism. The applicability of this technique was demonstrated by genotyping a recombinant inbred line. Recombination break points could be clearly defined, and in one case delimited to an interval of 29 kb. We further demonstrate that array hybridization can be combined with bulk segregant analysis to quickly map mutations. The extension of these tools to organisms with complex genomes, such as Arabidopsis, will greatly increase our ability to map and clone quantitative trait loci (QTL).[Supplemental material is available online atwww.genome.org.]

By promoting atherosclerosis and thrombosis, a blood-clotting diathesis could contribute to excess cardiovascular morbidity and mortality in patients with systemic hypertension and/or obstructive sleep apnoea. Since psychological states affect haemostatic activity, we wondered about the contribution of behavioural factors to a hypercoagulable state in subjects with increased risk of cardiovascular disease. To tease apart the potential additive nature of cardiovascular disease risk, we examined four patient groups–hypertensives and normotensives, with and without sleep apnoea. The procoagulant molecules thrombin–antithrombin III complex, fibrin D-dimer and von Willebrand factor antigen were measured in 88 subjects (mean age 47 years; range 32–64 years) who underwent full polysomnography. Subjects completed the Center for Epidemiological Studies–Depression (CES-D) Scale, the Cook–Medley (CM) Hostility Scale, and the Profile of Mood States (POMS). Sleep apnoea, hypertension status, age, body mass index and psychological variables (CES-D, CM Stress, and POMS Vigour–Activity) together explained 29% of the variance in D-dimer, a marker of fibrin turnover (r2 = 0.29, P = 0.001). CES-D, CM Stress and POMS Vigour–Activity explained 17% of this variance even after controlling for sleep apnoea, hypertension status, age and body mass index (Δr2 = 0.17, P = 0.001). Thrombin–antithrombin III complex and von Willebrand factor were not significantly related to psychological variables, but this may reflect limited statistical power. Thus psychological factors are independently associated with D-dimer and explain as much of its variance as do traditional correlates (hypertension, sleep apnoea, age and body mass index). These results may provide a rationale for linking behavioural aspects with cardiovascular events.

Identifying correlates of children's television (TV) watching could help identify strategies to reduce children's TV watching and impact children's weight status. Children's TV time and home environment factors were assessed longitudinally among 169 families with children followed from 6 to 12 years of age. TV watching increased with age, as did the number of in-home TVs and the presence of videocassette recorders (VCRs), the frequency of meal eating while watching TV, and the percentage of children with bedroom TVs. Greater meal eating while watching TV was consistently related to overall TV watching and increases in TV watching over time. The number of in-home TVs and the presence of a bedroom TV were less consistent correlates. TV watching was related to weight status when children were younger, and when children were older, more than 2 hours of TV daily was a risk factor for higher weight. Modifying TV access, particularly by reducing the frequency of meals eaten while watching TV, could promote lower children's TV watching and adiposity.

Objective To evaluate the effects of the repeatability criteria on the detection of change in visual fields by six progression algorithms used in standard automated perimetry. Design Retrospective, observational case series Participants Fifty-one glaucoma patients, each with multiple visual fields performed between May 1990 and December 1998, were included. Methods Each patient's set of visual fields were analyzed using the glaucoma change probability, the Early Manifest Glaucoma Trial (EMGT) algorithm, a modified glaucoma change probability score, a modified EMGT score, the Advanced Glaucoma Intervention Study algorithm, and the Collaborative Initial Glaucoma Treatment Study algorithm. Main outcome measures The effects of repeatability on the detection of field change, the level of agreement among algorithms, as well as the number of eyes identified as changed with each algorithm, were assessed. Results Mean follow-up was 34 months (range, 12–87 months). The average percentage of eyes with change based on three consecutive follow-up fields was 8.2% (4.0%–12.5%). However, of those showing change on the initial follow-up, this change from baseline was observed in subsequent examinations on average in 23% (18%–33%), depending on the algorithm. When change was based on just one field, four of the six algorithms noted a significantly greater number of eyes with change. The algorithms, however, did not differ significantly when confirmation of field change required two versus three consecutive follow-up visual fields. Conclusions Although current algorithms may help identify change, there are inconsistencies among them. We found that requiring repeatable change from baseline significantly reduces the number of changed eyes identified with each subsequent follow-up field. Identification and confirmation of change in visual fields plays an important role in helping to identify true glaucoma progression; however, the specific methods to do so have yet to be determined.

BACKGROUND In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus–based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancermediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS All patients received bone marrow–derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.)

Motivation: Gene therapy with retroviral vectors can induce adverse effects when those vectors integrate in sensitive genomic regions. Retroviral vectors are preferred that target sensitive regions less frequently, motivating the search for localized clusters of integration sites and comparison of the clusters formed by integration of different vectors. Scan statistics allow the discovery of spatial differences in clustering and calculation of false discovery rates providing statistical methods for comparing retroviral vectors. Results: A scan statistic for comparing two vectors using multiple window widths is proposed with software to detect clustering differentials and compute false discovery rates. Application to several sets of experimentally determined HIV integration sites demonstrates the software. Simulated datasets of various sizes and signal strengths are used to determine the power to discover clusters and evaluate a convenient lower bound. This provides a toolkit for planning evaluations of new gene therapy vectors. Availability and implementation: The geneRxCluster R package containing a simple tutorial and usage hints is available from http://www.bioconductor.org. Contact: ccberry@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Because plants depend on light for growth, their development and physiology must suit the particular light environment. Plants native to different environments show heritable, apparently adaptive, changes in their response to light. As a first step in unraveling the genetic and molecular basis of these naturally occurring differences, we have characterized intraspecific variation in a light-dependent developmental process-seedling emergence. We examined 141 Arabidopsis thaliana accessions for their response to four light conditions, two hormone conditions and darkness. There was significant variation in all conditions, confirming that Arabidopsis is a rich source of natural genetic diversity. Hierarchical clustering revealed that some accessions had response patterns similar to known photoreceptor mutants, suggesting changes in specific signaling pathways. We found that the unusual far-red response of the Lm-2 accession is due to a single amino-acid change in the phytochrome A (PHYA) protein. This change stabilizes the light-labile PHYA protein in light and causes a 100-fold shift in the threshold for far-red light sensitivity. Purified recombinant Lm-2 PHYA also shows subtle photochemical differences and has a reduced capacity for autophosphorylation. These biochemical changes contrast with previously characterized natural alleles in loci controlling plant development, which result in altered gene expression or loss of gene function.

Objective To determine whether 1-week continuous positive airway pressure (CPAP) treatment, compared with placebo CPAP, improves cognitive functioning in patients with obstructive sleep apnea (OSA). Methods 36 OSA patients (aged 32–60 years, respiratory disturbance index [RDI]> 15) were monitored 2 nights with polysomnography, then randomized for 1-week treatment to CPAP or placebo (CPAP at 2 cm H2O with holes in mask). Participants completed Wechsler Adult Intelligence Scale—Revised Digit Symbol and Digit Span, Trailmaking A/B, Digit Vigilance, Stroop Color-Word, Digit Ordering, and Word Fluency tests pre- and posttreatment. These produced 22 scores per participant, which were analyzed by use of repeated-measures analysis of variance (ANOVA) and a rank-sum test. Results In ANOVA, only 1 of the 22 scores showed significant changes specific to CPAP treatment, a number that could be expected by chance alone: Digit Vigilance-Time (p = .035). The CPAP group improved their time (from 7.5 to 6.9 minutes, p = .013). The rank-sum test revealed that the CPAP group had significantly better overall cognitive functioning posttreatment than the placebo group (mean ranks of 17.8 vs. 20.2, respectively;p = .022). Conclusions Although results suggest overall cognitive improvement due to CPAP, no beneficial effects in any specific cognitive domain were found. Future studies of neuropsychological effects of CPAP treatment should include a placebo CPAP control group. Placebo studies that use longer-term treatment might demonstrate additional effects. It is also possible that, even at 2 cm H2O, CPAP conveys some beneficial neuropsychological effects.

The historical model of hematopoiesis, mainly derived from murine studies, is based on the existence of a single hematopoietic stem cell (HSC) capable of generating all blood cell lineages. However this model has been challenged by the proposal of four types of murine HSC, which differ by their relative contribution to the myeloid and lymphoid lineages. Here we have used data from a gene therapy trial to treat Wiskott-Aldrich syndrome (WAS) to explore hematopoiesis in humans. In the trial, the therapeutic vector (lentivirus) integrates into the genome at unique positions in each hematopoietic stem and progenitor cell (HSPC) and is consequently transmitted to all its progeny. Thus hematopoietic ontogeny in humans can be inferred by tracking the appearance of unique integration sites in fractionated blood cell populations. This provides a unique opportunity to model the developmental complexity of the human haematological system. Considerable effort over the last 15 years has been devoted to optimizing retroviral integration sites (RIS) analysis using ligation mediated PCR (LM-PCR), combined with acoustic shearing and high-throughput Illumina sequencing. Acoustic shearing enables more precise quantification of RIS abundance through the enumeration of the various sizes of shear fragments (SonicAbundance) containing a given RIS, which correspond to individual ancestor HSPC and its blood progeny. In four WAS patients treated by gene therapy, we have sorted peripheral blood samples for 5 cell types: myeloid (granulocytes and monocytes) and lymphoid subpopulations (T, B and NK cells), and analysed their RIS profile. Each RIS corresponds to a particular stem/progenitor cell clone, with a particular pattern defined by its presence or absence in each of the 5 lineages. These data are then use to reconstruct aspects of the hematopoietic hierarchy. In order to face the challenging issue of cell sorting contamination we have been using a stringent sort precision mode and we treat residual contamination explicitly in downstream statistical models. Using these approaches, we have characterized up to tens of thousands RIS per patients with a follow up of 4 years. In order to minimize biases due to sparse sampling, we concentrate on the more abundant RIS clones that can be more easily caught and are therefore analysed more reliably. We showed that a significant fraction of RIS clones are detected in a single lineage, while other RIS clones are characterized by different levels of contribution to the myeloid and lymphoid lineages, highlighting the heterogeneity of human HSC. Clones contributing to all 5 lineages are readily recovered but this study also unravels a diversity of inferred hematopoietic programs with various potentials contributing to human blood homeostasis. Longitudinal analysis of clonal dynamics is ongoing, with preliminary results highlighting the maintenance of this heterogeneity of HSPC over time. These new findings provide unique data on human hematopoiesis based on gene corrected WAS patients. Disclosures No relevant conflicts of interest to declare.

Objective To evaluate the reproducibility of optical coherence tomograph (OCT) retinal nerve fiber layer (RNFL) measurements in normal and glaucomatous eyes by means of the commercially available OCT 2000 instrument (Humphrey Systems, Dublin, CA). Design Prospective instrument validation study. Participants One eye each from 10 normal subjects and 10 glaucoma patients. Methods Twenty subjects underwent a total of eight scanning sessions during two independent visits. In each session, five circular scans centered on the optic nerve head were performed. The first two sessions were performed by two experienced technicians. Followed by a 30-minute break, a third and a fourth session was completed by the same technicians. This sequence was duplicated on a second visit. Intrasession, intersession, intervisit, and interoperator reproducibility of quadrant and global RNFL measurements were calculated by use of a components of variance model. Main outcome measures RNFL thickness. Results The coefficient of variation for the mean RNFL thickness was significantly smaller ( P = 0.02) in normal eyes (6.9%) than in glaucoma eyes (11.8%). The estimated root mean squared error based on the statistical model using three scans per patient was 5.8 and 8.0 μm for normal and glaucoma eyes, respectively. A components of variance model showed most of the variance (79%) to be due to differences between patients. Only a modest contribution to variability was found for session (1%), visit (5%), and operator (2%). Conclusion With the commercially available OCT, our results indicate that the RNFL measurements are reproducible for both normal and glaucomatous eyes.

Purpose To compare optical coherence tomography (OCT) retinal nerve fiber layer (RNFL) thickness measurements with established methods for assessment of glaucomatous damage using RNFL photography and visual field testing. Design Cross-sectional study. Participants Fifty-eight eyes of 58 healthy volunteer ocular hypertensive patients, glaucoma suspect patients, and glaucoma patients were included. Methods Optical coherence tomography 3.4-mm diameter circular scans were obtained within 3 months of RNFL photography and standard achromatic visual field testing. Three independent observers graded RNFL photographs using two standardized protocols. For each method, superior and inferior arcuate bundles were scored separately, and interobserver and intraobserver variation was measured. Standard achromatic visual field mean deviation in the superior and inferior hemifields was compared with RNFL damage as assessed by photography and OCT RNFL thickness measurements. Main outcome measures Visual field mean deviation and severity of glaucomatous RNFL damage as assessed by photography. Results Optical coherence tomography RNFL thickness decreased with increasing RNFL damage as assessed by photography using both methods of photographic assessment. Standard achromatic perimetry mean deviation was significantly associated with OCT RNFL thickness ( R 2 = 35%–43%) and RNFL photography severity score ( R 2 = 18%–29%). Conclusions These results suggest that the OCT shows promise for providing quantitative measures of RNFL thickness for diagnosing and monitoring glaucoma.

The accuracy and variability of burn size calculations using four Lund and Browder charts currently in clinical use and two Rule of Nine's diagrams were evaluated. The study showed that variability in estimation increased with burn size initially, plateaued in large burns and then decreased slightly in extensive burns. The Rule of Nine's technique often overestimates the burn size and is more variable, but can be performed somewhat faster than the Lund and Browder method. More burn experience leads to less variability in burn area chart drawing estimates. Irregularly shaped burns and burns on the trunk and thighs had greater variability than less irregularly shaped burns or burns on more defined anatomical parts of the body.

The purpose of this study is to look for associations between the following: the frequency of television advertising for certain categories of food products; the number of times they are requested by children; and subsequent purchases by parents. The study design consisted of a cross-analysis of two sets of existing and published databases, one being an independent study of television advertising where data were collected in 1993, and the other being the Study of Children's Activity and Nutrition, with data collected in 1991. The results showed that the correlation coefficient between foods advertised on television during common child viewing hours and foods children requested was 0.91 (p

The goal of this study was to identify genes whose mRNA levels are differentially expressed in human cells with acquired cisplatin (cDDP) resistance. Using the parental UMSCC10b head and neck carcinoma cell line and the 5.9-fold cDDP-resistant subline, UMSCC10b/Pt-S15, two suppressive subtraction hybridization (SSH) cDNA libraries were prepared. One library represented mRNAs whose levels were increased in the cDDP resistant variant (the UP library), the other one represented mRNAs whose levels were decreased in the resistant cells (the DOWN library). Arrays constructed with inserts recovered from these libraries were hybridized with SSH products to identify truly differentially expressed elements. A total of 51 cDNA fragments present in the UP library and 16 in the DOWN library met the criteria established for differential expression. The sequences of 87% of these cDNA fragments were identified in Genbank. Among the mRNAs in the UP library that were frequently isolated and that showed high levels of differential expression were cytochrome oxidase I, ribosomal protein 28S, elongation factor 1α, α-enolase, stathmin, and HSP70. The approach taken in this study permitted identification of many genes never before linked to the cDDP-resistant phenotype. © 2000 Cancer Research Campaign

Purpose: To provide daily estimates of first experimentation and attainment of established smoking in adolescents 11–17 years of age. Methods: The 1989 and 1993 Teenage Attitudes and Practices Surveys (TAPS) (16,954 observations) was used to estimate rates in 1991 for: (a) first experimentation (even a few puffs), and (b) established smoking (reaching a lifetime level of at least 100 cigarettes). The 1992 and 1993 Current Population Surveys (CPS) (82,279 adults) allowed calculation of year-specific initiation rates (1980–1989) from the age respondents reported as having started smoking "fairly regularly." Rates were applied to Census data for the appropriate years to yield numbers accruing each day. Estimates were calculated for adolescents (11- to 17-year-olds) and youth (11- to 20-year-olds). Results: For TAPS, in 1991, 4824 adolescents first tried cigarettes and 1975 became established smokers each day. Considering all youth, these estimates increase to 5497/day for first experimentation and to 2933/day for established smokers. For CPS, from 1980 to 1989, around 2300 adolescents initiated fairly regular smoking each day. For all youth, the estimate has increased to about 3400/day. Conclusions: Because approximately 4800 adolescents and 5500 youth appear to be experimenting with cigarettes for the first time each day, and close to 3000 youth become established smokers daily, increased prevention efforts are clearly justified.

Purpose To determine the effect of pupil dilation on topographic optic disc parameters measured with confocal scanning laser ophthalmoscopy. Design Prospective, comparative, observational case series. Participants Thirty-eight subjects (25 glaucoma patients, 5 glaucoma suspects, and 8 normal subjects) representing a range of lens opacities participated in this study. Methods Topographic optic disc parameter measurements were obtained before and after pupil dilation with a confocal scanning laser ophthalmoscope (Heidelberg Retina Tomograph). Cataract severity was measured with the LOCS III grading system. Image quality was assessed using the standard deviation of the mean topography image and an image quality score. Multiple regression techniques were used to evaluate the effect of image quality, cataract density, and pupil size on the change in topographic parameters after dilation. Main outcome measures Topographic optic disc parameter measurements. Results With dilation, mean change in most topographic optic disc parameters was not significantly different from zero. Changes in topographic optic nerve parameters tended to be relatively small when undilated images were of good quality and larger in images of relatively poor quality. The strongest predictor of the absolute value of change in topographic optic disc parameters was the standard deviation of the undilated mean topography image. Conclusions When confocal scanning laser ophthalmoscope images are of good quality, topographic optic disc parameter measurements obtained with a dilated pupil are similar to those obtained with an undilated pupil. However, caution should be exercised when interpreting information from poor quality confocal scanning laser ophthalmoscopic images.

Previous studies have not used both self-report and objective measures to assess sex and ethnic differences in children’s physical activity. In the present study, 187 Mexican American and Anglo American children, aged 11 to 12 years, were assessed by two 7-day physical activity recall interviews and up to 8 days of accelerometer (Caltrac) monitoring over a 6-month period. Compared to Anglo American boys, accelerometer data showed Mexican American boys, Anglo American girls, and Mexican American girls to be 95,81, and 75% as active, respectively. Activity recall data showed that, compared to Anglo American boys, Mexican American boys, Anglo American girls, and Mexican American girls were 95,95, and 90% as active, respectively. The extent of sex and ethnic differences in children’s physical activity depend on the measure used.

Hospitalization routinely lowers blood pressure (BP). This study examined the effects of race and psychologic characteristics on this phenomenon. Data are reported from two separate cohorts of hypertensive and normotensive black and white men and women who were studied following a stay at a clinical research center where sodium intake was held constant. Blacks (N = 88), as compared to whites (N = 77), showed consistently smaller declines in systolic BP (P.01) following hospitalization (-11.6 mm Hg SBP v -19.5 mm Hg SBP, respectively). A multiple regression model that treated BP as a function of physiologic and psychologic attributes indicated that preadmission BP level, body mass index, stress level, and anger expression were related to the drop in systolic (r2 = 65%) and diastolic (r2 = 45%) BP brought about by hospitalization (P.0001). In blacks, high environmental stress ratings were unrelated to the change in BP with hospitalization. In contrast, whites with high environmental stress ratings lowered their BP noticeably with hospitalization. Given that the reduction in BP with hospitalization can be similar to that attained with pharmacologic therapy, these findings may have a bearing on studies examining BP in the hospital.

Introduction This study examined the adolescent smoking uptake process, specifically, the progression from experimentation to established smoking. Although adolescent smoking uptake has been described as consisting of five stages (preparation, initial trying, experimentation, regular smoking, and addiction), there is no accepted method of identifying which experimenters will proceed to become addicted. Methods Using a nationally representative sample of adolescents between the ages of 12 and 18 at baseline, we examined their transition from experimentation (had at least a puff but has not smoked 100 cigarettes) to established smoking (smoked at least 100 cigarettes in lifetime) four years later. Results At follow-up, 31% of the experimenters at baseline had progressed to established smoking. Baseline level of smoking experience was the strongest independent predictor of established smoking, with current experimenters (smoked in the past 30 days but less than 100 cigarettes in lifetime) having the greatest risk of progressing to established smoking compared to puffers (puffed but have not smoked a whole cigarette). Furthermore, this effect was modified by age; older current experimenters at baseline had more than double the risk of younger current experimenters of progressing to established smoking at follow-up. Absence of a firm commitment not to smoke was a significant predictor among older experimenters but not in younger experimenters. Other important predictors of the transition from experimentation to established smoking were exposure to other smokers and perceived school performance. Conclusions We found that, even among experimenters, there is an identifiable group of adolescents who are at higher risk of progressing to established smoking that can be targeted for intervention.

Purpose To evaluate the decrease in intraocular pressure associated with cidofovir (1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine dihydrate; HPMPC) intravitreal injections. Methods We followed up 97 eyes of 63 patients with acquired immunodeficiency syndrome (AIDS) who had cytomegalovirus retinitis and had been treated with up to nine 20-μg intravitreal cidofovir injections. Measurements were taken at baseline, between 2 and 3 weeks, and at 5 to 6 weeks after injections. Anterior chamber fluorophotometry was studied in seven eyes (four patients) before and after injections. Ciliary body anatomy was evaluated in two patients. Results After the first intravitreal injection, mean intraocular pressure was 2.2 mm Hg lower than that at baseline at 2 to 3 weeks (P Conclusions Intraocular pressure decreases after the initial 20-μg cidofovir intravitreal injection. However, eyes stabilize (pressure plateaus) after three injections. Effects on the ciliary body are the main cause of the decrease after cidofovir injections.

To obtain an early estimate of the effectiveness of the American Stop Smoking Intervention Study (ASSIST).Seventeen American states funded through ASSIST are compared with 32 others regarding per capita cigarette consumption from 1989 to 1995. California, which already had an extensive tobacco control programme, was omitted. ASSIST states were selected competitively (not randomly) based on their proposals' merit, state smoking prevalence, and geographical distribution.Comprehensive tobacco control programmes, emphasising policy interventions, were implemented in the ASSIST states beginning in 1993.Trends in aggregated per capita cigarette consumption and inflation-adjusted average price/pack of cigarettes in the intervention states were compared. Percentage change in per capita consumption is also compared with percentage change in inflation-adjusted cigarette price by state in each group from 1992 to 1994.Per capita consumption and inflation-adjusted cigarette price were nearly identical in both groups of states before 1993, when full funding for the ASSIST interventions began. However, by 1996 smokers in the intervention states were consuming about 7% less cigarettes per capita (P0.05, beginning in 1994), and in 1994 the average price was over $0.12/pack higher in the intervention states. All but three states (all intervention) showed decreases in cigarette price. Nonetheless, 76% of the intervention and 55% of the comparison states showed some decrease in consumption despite decreases in price. The relationship between changes in price and consumption was considerably diminished in the intervention group.These interim results suggest that the ASSIST programme is associated with a substantial difference in tobacco consumption in a third of the United States, and that increased price from taxation may not be the only programme influence.

Prospective data from the California Tobacco Surveys (n = 2066) were used to perform a critical test of the Prochaska et al. (1991) stages of change model. When the stages of change model was used as a stand alone predictor, smokers in preparation at baseline were more likely to be in cessation at follow-up than smokers in pre-contemplation at baseline (ORadj = 1.9). When stage membership was combined with baseline measures of addiction including smoking behaviors and quitting history, it was not a significant predictor of future cessation. A prediction equation that combined daily vs. occasional smoking, cigarettes per day smoked, life-time quits of at least a year, and quits of more than 5 days in the previous year discriminated smokers in cessation at follow-up of 1 to 2 years better than did the stages of change model. The area under the ROC curve for the equation based on addiction measures was 69.3% vs. 55.1% for the stages of change. Cessation rates ranged from 7.7% to 35.7% for the four-category addiction equation compared with 15.1% to 24.9% for stages of change model.

We compared two stage-of-change models that differentiate smokers by their level in the quitting process. The original 1983 model by Prochaska and DiClemente (1) divided smokers first by relapse status and then by intention to quit; their revised 1991 model (2) reversed the primacy of these factors. No published data justify whether the revision improves prediction of cessation. We used data from a population-based panel of 1,921 smokers interviewed in 1990 and 1992 for the California Tobacco Surveys. Model variables (quitting intention and recent quitting history) were used in a logistic regression to predict 30-day or longer cessation at follow-up and quit attempts made during the year preceding the survey. Predictive power of the revised model was not better than predictive power of the original model. New approaches to differentiating smokers on likelihood to quit should emphasize quitting behavior rather than intention to quit.