Dongseok Choi

Mycobacterial diseases and antitumour necrosis factor therapy in USA

Objective In North America, tuberculosis and nontuberculous mycobacterial (NTM) disease rates associated with antitumour necrosis factor α (anti-TNFα) therapy are unknown. Methods At Kaiser Permanente Northern California, the authors searched automated pharmacy records to identify inflammatory disease patients who received anti-TNF therapy during 2000–2008 and used validated electronic search algorithms to identify NTM and tuberculosis cases occurring during anti-TNF drug exposure. Results Of 8418 anti-TNF users identified, 60% had rheumatoid arthritis (RA). Among anti-TNF users, 18 developed NTM and 16 tuberculosis after drug start. Anti-TNF associated rates of NTM and tuberculosis were 74 (95% CI: 37 to 111) and 49 (95% CI: 18 to 79) per 100 000 person-years, respectively. Rates (per 100, 000 person-years) for NTM and tuberculosis respectively for etanercept were 35 (95% CI: 1 to 69) and 17 (95% CI: 0 to 41); infliximab, 116 (95% CI: 30 to 203) and 83 (95% CI: 10 to 156); and adalimumab, 122 (95% CI: 3 to 241) and 91 (95% CI: 19 to 267). Background rates for NTM and tuberculosis in unexposed RA-patients were 19.2 (14.2 to 25.0) and 8.7 (5.3 to 13.2), and in the general population were 4.1 (95% CI 3.9 to 4.4) and 2.8 (95% CI 2.6 to 3.0) per 100, 000 person-years. Among anti-TNF users, compared with uninfected individuals, NTM case-patients were older (median age 68 vs 50 years, p<0.01) and more likely to have RA (100% vs 60%, p<0.01); whereas, tuberculosis case-patients were more likely to have diabetes (37% vs 16%, p=0.02) or chronic renal disease (25% vs 6%, p=0.02). Conclusions Among anti-TNF users in USA, mycobacterial disease rates are elevated, and NTM is associated with RA.

Incidence and prevalence of uveitis in Veterans Affairs Medical Centers of the Pacific Northwest.

PURPOSE To ascertain the frequency of uveitis in Veterans Affairs (VA) patients in the Pacific Northwest and to compare disease rates with those in previously published epidemiologic studies. DESIGN Cross-sectional, population based-study. METHODS The medical records of 152,267 patients seen at six VA Medical Centers in Oregon and Washington during fiscal year 2004 were searched for uveitis-related International Classification of Diseases 9th edition codes. Cases were reviewed and classified anatomically, by associated systemic disease, and as incident or prevalent. Only definite cases were used for disease rate calculations. RESULTS This study found a crude incidence of 25.6 cases/100,000 person-years and a crude prevalence of 69 cases/100,000 persons. The most common anatomic location for uveitis was anterior. Approximately half of cases were idiopathic, with human leukocyte antigen-B27-related diseases being the most common identified cause. There was no statistical evidence of increased or decreased incidence with age, although uveitis seemed to be more prevalent in the younger age groups. CONCLUSIONS Our data are consistent with those of most published population-based studies on the epidemiologic features of uveitis, but we detected significantly lower incidence and prevalence than those reported in a recently published study from Kaiser Permanente. The significance of and possible explanations for the differences between our data and that published by the Kaiser group are discussed.

Molecular architecture of the chick vestibular hair bundle

Hair bundles of the inner ear have a specialized structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1,100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin cross-linkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.

Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

BackgroundPeripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system.ResultsWe find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples.ConclusionRNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles.

Replication and sustainability of improved access and retention within the Network for the Improvement of Addiction Treatment

The Network for the Improvement of Addiction Treatment (NIATx) applies process improvement strategies to enhance the quality of care for the treatment of alcohol and drug disorders. A prior analysis reported significant reductions in days to treatment and significant increases in retention in care [McCarty, D., Gustafson, D. H., Wisdom, J. P., Ford, J., Choi, D., Molfenter, T., Capoccia, V., Cotter, F. 2007. The Network for the Improvement of Addiction Treatment (NIATx): enhancing access and retention. Drug Alcohol Depend. 88, 138-145]. A second cohort of outpatient (n=10) and intensive outpatient (n=4) treatment centers tested the replicability of the NIATx model. An additional 20 months of data from the original cohort (7 outpatient, 4 intensive outpatient, and 4 residential treatment centers) assessed long-term sustainability. The replication analysis found a 38% reduction in days to treatment (30.7 to 19.4 days) during an 18-month intervention. Retention in care improved 13% from the first to second session of care (from 75.4% to 85.0%), 12% between the first and third session of care (69.2-77.7%), and 18% between the first and fourth session of care (57.1-67.5%). The sustainability analysis suggested that treatment centers maintained the reductions in days to treatment and the enhanced retention in care. Replication of the NIATx improvements in a second cohort of treatment centers increases confidence in the application of process improvements to treatment for alcohol and drug disorders. The ability to sustain the gains after project awards were exhausted suggests that participating programs institutionalized the organizational changes that led to the enhanced performance.

Role of the retinal vascular endothelial cell in ocular disease

Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell.

Accurate label-free protein quantitation with high- and low-resolution mass spectrometers.

Label-free quantitation of proteins analyzed by tandem mass spectrometry uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), such as spectral counts or summed fragment-ion intensity. We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into Escherichia coli extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well-correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10(-3) or lower were detected, with a strong falloff below 10(-4) mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate but should be limited to quantitation of proteins of intermediate to higher fractional abundance.

Hypothesis: Sarcoidosis is a STAT1-mediated disease

Immunologic pathways involved in sarcoidosis pathogenesis are largely unknown. We hypothesized that patients with sarcoidosis have characteristic mRNA profiles. Microarray analysis of gene expression was done on peripheral blood (12 patients, 12 controls), lung (6 patients, 6 controls) and lymph node (8 patients, 5 controls). Comparing peripheral blood from patients with sarcoidosis to controls, 872 transcripts were upregulated and 1039 were downregulated at >1.5-fold change and a significant q value. Several transcripts associated with interferon and STAT1 were upregulated. Lung and lymph node analyses also showed dramatic increases in STAT1 and STAT1-regulated chemokines. Granulomas in lymph nodes of patients with sarcoidosis expressed abundant STAT1 and phosphorylated STAT1. STAT1 might play an important role in sarcoidosis. This novel hypothesis unites seemingly disparate observations with regard to sarcoidosis including implication of a casual role for interferons, a suspected infectious trigger, T(H)1 predominating lymphocytes in bronchoalveolar lavage, and the association with hypercalcemia.

Rituximab for treatment of ocular inflammatory disease: a series of four cases

Rituximab may be effective in the treatment of ocular inflammatory disease. Dosing is less frequent than many medications currently available. Four cases are reported, each of which appeared to have responded well to treatment with rituximab, although patient 2 was able to remain on a low dose of prednisone for only 2 months. The ongoing pilot study will hopefully provide additional insight into the benefit of rituximab for treatment of scleritis and idiopathic orbital inflammatory disease.

Selective thinning of the perifoveal inner retina as an early sign of hydroxychloroquine retinal toxicity.

PurposeTo evaluate macular thickness profiles using spectral-domain optical coherence tomography (SDOCT) and image segmentation in patients with chronic exposure to hydroxychloroquine.MethodsThis study included eight patients with chronic exposure to hydroxychloroquine (group 1) and eight controls (group 2). Group 1 patients had no clinically evident retinal toxicity. All subjects underwent SDOCT imaging of the macula. An image segmentation technique was used to measure thickness of six retinal layers at 200 μm intervals. A mixed-effects model was used for multivariate analysis.ResultsBy measuring total retinal thickness either at the central macular (2800 μm in diameter), the perifoveal region 1200-μm-width ring surrounding the central macula), or the overall macular area (5200 μm in diameter), there were no significant differences in the thickness between groups 1 and 2. On an image segmentation analysis, selective thinning of the inner plexiform+ganglion cell layers (P=0.021) was observed only in the perifoveal area of the patients in group 1 compared with that of group 2 by using the mixed-effects model analysis.ConclusionOur study results suggest that chronic exposure to hydroxychloroquine is associated with thinning of the perifoveal inner retinal layers, especially in the ganglion cell and inner plexiform layers, even in the absence of functional or structural clinical changes involving the photoreceptor or retinal pigment epithelial cell layers. This may be a contributing factor as the reason most patients who have early detectable signs of drug toxicity present with paracentral or pericentral scotomas.