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1.  The effect of rituximab therapy on immunoglobulin levels in patients with multisystem autoimmune disease 
Rituximab is a B cell depleting anti-CD20 monoclonal antibody. CD20 is not expressed on mature plasma cells and accordingly rituximab does not have immediate effects on immunoglobulin levels. However, after rituximab some patients develop hypogammaglobulinaemia.
We performed a single centre retrospective review of 177 patients with multisystem autoimmune disease receiving rituximab between 2002 and 2010. The incidence, severity and complications of hypogammaglobulinaemia were investigated.
Median rituximab dose was 6 g (1–20.2) and total follow-up was 8012 patient-months. At first rituximab, the proportion of patients with IgG <6 g/L was 13% and remained stable at 17% at 24 months and 14% at 60 months. Following rituximab, 61/177 patients (34%) had IgG <6 g/L for at least three consecutive months, of whom 7/177 (4%) had IgG <3 g/L. Low immunoglobulin levels were associated with higher glucocorticoid doses during follow up and there was a trend for median IgG levels to fall after ≥ 6 g rituximab. 45/115 (39%) with IgG ≥6 g/L versus 26/62 (42%) with IgG <6 g/L experienced severe infections (p = 0.750). 6/177 patients (3%) received intravenous immunoglobulin replacement therapy, all with IgG <5 g/L and recurrent infection.
In multi-system autoimmune disease, prior cyclophosphamide exposure and glucocorticoid therapy but not cumulative rituximab dose was associated with an increased incidence of hypogammaglobulinaemia. Severe infections were common but were not associated with immunoglobulin levels. Repeat dose rituximab therapy appears safe with judicious monitoring.
PMCID: PMC4038057  PMID: 24884562
Rituximab; Hypogammaglobulinaemia; B cell; Vasculitis; Systemic lupus erythematosus (SLE); IgG; Infection; Autoimmune
2.  A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus 
PLoS ONE  2014;9(5):e97826.
PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50–80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications.
PMCID: PMC4022673  PMID: 24831802
3.  High Resolution 13C MRI With Hyperpolarized Urea: In Vivo T2 Mapping and 15N Labeling Effects 
13C steady state free precession (SSFP) magnetic resonance imaging and effective spin-spin relaxation time (T2) mapping were performed using hyperpolarized [13C] urea and [13C, 15N2] urea injected intravenously in rats. 15N labeling gave large T2 increases both in solution and in vivo due to the elimination of a strong scalar relaxation pathway. The T2 increase was pronounced in the kidney, with [13C, 15N2] urea giving T2 values of 6.3±1.3 s in the cortex and medulla, and 11±2 s in the renal pelvis. The measured T2 in the aorta was 1.3±0.3 s. [13C] urea showed shortened T2 values in the kidney of 0.23±0.03 s compared to 0.28±0.03 s measured in the aorta. The enhanced T2 of [13C, 15N2] urea was utilized to generate large signal enhancement by SSFP acquisitions with flip angles approaching the fully refocused regime. Projection images at 0.94 mm in-plane resolution were acquired with both urea isotopes, with [13C, 15N2] urea giving a greater than four-fold increase in signal-to-noise ratio [13C] over urea.
PMCID: PMC4011557  PMID: 24235273
Angiography; dynamic nuclear polarization; hyperpolarized; steady state free precession (SSFP); urea
4.  Fully Human Monoclonal Antibodies from Antibody Secreting Cells after Vaccination with Pneumovax®23 are Serotype Specific and Facilitate Opsonophagocytosis 
Immunobiology  2012;218(5):745-754.
B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax23.
PMCID: PMC3556204  PMID: 23084371
Antibody secreting cells; B cell memory; human monoclonal antibodies; Pneumovax; Streptococcus pneumoniae
5.  Measurement of Muscle Protein Fractional Synthetic Rate by Capillary Gas Chromatography/Combustion Isotope Ratio Mass Spectrometry 
Biological mass spectrometry  1992;21(10):486-490.
The measurement of skeletal muscle protein fractional synthetic rate using an infusion of (1-13C)leucine and measuring the isotopic abundance of the tracer in skeletal muscle protein by preparative gas chromatography (GC)/ninhydrin isotope ratio mass spectrometry (IRMS) is laborious and subject to errors owing to contamination by 12C. The purpose of this study was to compare muscle (13C)leucine enrichment measured with the conventional preparative GC/ninhydrin IRMS approach to a new, continuous-flow technique using capillary GC/combustion IRMS. Quadriceps muscles were removed from four Sprague–Dawley rats after each was infused at a different rate with (1-13C)leucine for 6–8 h. Muscle leucine enrichment (at.% excess) measured by both methods differed by less than 4%, except at low (13C)leucine enrichments (<0.03 at.% excess). In addition, capillary GC/combustion IRMS was used to assess muscle (13C)leucine enrichment and fractional muscle protein synthesis rate in ten normal young men and women infused with (1,2-13C2)leucine for 12–14 h. This approach reduced the variability of the isotope abundance measure and gave estimates of muscle protein synthesis rate (0.050 ± 0.011% h−1 (mean ± SEM); range = 0.023–0.147% h−1) that agree with published values determined using the standard analytical approach. The measurement of (13C)leucine enrichment from skeletal muscle protein by capillary GC/combustion IRMS provides a simple, acceptable and practical alternative to preparative GC/ninhydrin IRMS.
PMCID: PMC4003887  PMID: 1420371
6.  Modeling of Cost Effectiveness of Pneumococcal Conjugate Vaccination Strategies in U.S. Older Adults 
The 13-valent pneumococcal conjugate vaccine (PCV13) is approved by the U.S. Food and Drug Administration for adults, but its role in older adults is unclear.
To compare PCV13 strategies to currently recommended vaccination strategies in adults aged ≥65 years.
Using a Markov model, the cost effectiveness of PCV13 and the 23-valent pneumococcal polysaccharide vaccine (PPSV23), alone or in combination, was estimated, in adults aged either 65 years or 75 years. No prior vaccination, prior vaccination, and vaccine hyporesponsiveness scenarios were examined. Pneumococcal disease rates, indirect childhood PCV13 effects, and costs were estimated using CDC Active Bacterial Core surveillance data and U.S. national databases. An expert panel estimated vaccine-related protection. A societal perspective was taken and outcomes were discounted 3% per year.
In those aged 65 years, single-dose PCV13 cost $11,300 per quality-adjusted life-year (QALY) gained compared to no vaccination; at ages 65 and 80 years, PCV13 cost $83,000/QALY. In those aged 75 years, single-dose PCV13 cost $62,800/QALY gained. PPSV23 cost more and was less effective than PCV13. Results were sensitive to varying vaccine effectiveness and indirect effect estimates. In hyporesponsiveness scenarios, cost-effectiveness ratios increased by 37%–78% for single-dose strategies and 29%–35% for multiple-dose strategies.
Single-dose PCV13 strategies are likely to be economically reasonable in older adults.
PMCID: PMC3601581  PMID: 23498103
7.  Microtomographic Analysis of Lower Urinary Tract Obstruction 
Prenatal obstruction of the lower urinary tract may result in megacystis, with subsequent development of hydro-ureter, hydronephrosis, and renal damage. Oligo- or anhydramnios, pulmonary hypoplasia, and prune belly syndrome are lethal consequences. Causes and mechanisms responsible for obstruction remain unclear but might be clarified by anatomic study at autopsy. To this end, we employed 2 methods of tomographic imaging—optical projection tomography and contrast-enhanced microCT scanning—to elucidate the anatomy of the intact urinary bladder and urethra in 10 male fetuses with lower urinary tract obstruction. Images were compared with those from 9 age-matched controls. Three-dimensional images, rotated and sectioned digitally in multiple planes, permitted thorough examination while preserving specimens for later study. Both external and internal features of the bladder and urethra were demonstrated; small structures (ie, urethral crest, verumontanum, prostatic utricle, ejaculatory ducts) were seen in detail. Types of obstruction consisted of urethral atresia (n = 5), severe urethral stenosis (n = 2), urethral diaphragm (n = 2), or physical kinking (n = 1); classic (Young type I) posterior urethral valves were not encountered. Traditional light microscopy was then used to verify tomographic findings. The prostate gland was hypoplastic or absent in all cases; in 1, prostatic tissue was displaced inferior to the verumontanum. Findings support previous views that dissection may produce valve-like artifacts (eg, bisection of an obstructing diaphragm) and that deformation of an otherwise normal urethra may result in megacystis. The designation “posterior urethral valves” should not be used as a generic expression of urethral obstruction unless actual valves are demonstrated.
PMCID: PMC3965709  PMID: 23977847
lower urinary tract obstruction; microCT scanning; optical projection tomography; posterior urethral valves; prune belly syndrome; urethral atresia
8.  Cost-Effectiveness of a Program to Eliminate Disparities in Pneumococcal Vaccination Rates in Elderly Minority Populations: An Exploratory Analysis 
Invasive pneumococcal disease is a major cause of preventable morbidity and mortality in the United States, particularly among the elderly (>65 years). There are large racial disparities in pneumococcal vaccination rates in this population. Here, we estimate the cost-effectiveness of a hypothetical national vaccination intervention program designed to eliminate racial disparities in pneumococcal vaccination in the elderly.
In an exploratory analysis, a Markov decision-analysis model was developed, taking a societal perspective and assuming a 1-year cycle length, 10-year vaccination program duration, and lifetime time horizon. In the base-case analysis, it was conservatively assumed that vaccination program promotion costs were $10 per targeted minority elder per year, regardless of prior vaccination status and resulted in the elderly African American and Hispanic pneumococcal vaccination rate matching the elderly Caucasian vaccination rate (65%) in year 10 of the program.
The incremental cost-effectiveness of the vaccination program relative to no program was $45,161 per quality-adjusted life-year gained in the base-case analysis. In probabilistic sensitivity analyses, the likelihood of the vaccination program being cost-effective at willingness-to-pay thresholds of $50,000 and $100,000 per quality-adjusted life-year gained was 64% and 100%, respectively.
In a conservative analysis biased against the vaccination program, a national vaccination intervention program to ameliorate racial disparities in pneumococcal vaccination would be cost-effective.
PMCID: PMC3733787  PMID: 23538183
cost-effectiveness; disparities; invasive pneumococcal disease; vaccination
9.  Acute Post-Exercise Myofibrillar Protein Synthesis Is Not Correlated with Resistance Training-Induced Muscle Hypertrophy in Young Men 
PLoS ONE  2014;9(2):e89431.
Muscle hypertrophy following resistance training (RT) involves activation of myofibrillar protein synthesis (MPS) to expand the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable and thus we sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE) in untrained men (n = 23) and then examined the relation between MPS with magnetic resonance image determined hypertrophy. To measure MPS, young men (24±1 yr; body mass index  = 26.4±0.9 kg•m2) underwent a primed constant infusion of L-[ring-13C6] phenylalanine to measure MPS at rest, and acutely following their first bout of RE prior to 16 wk of RT. Rates of MPS were increased 235±38% (P<0.001) above rest 60–180 min post-exercise and 184±28% (P = 0.037) 180–360 min post exercise. Quadriceps volume increased 7.9±1.6% (−1.9–24.7%) (P<0.001) after training. There was no correlation between changes in quadriceps muscle volume and acute rates of MPS measured over 1–3 h (r = 0.02), 3–6 h (r = 0.16) or the aggregate 1–6 h post-exercise period (r = 0.10). Hypertrophy after chronic RT was correlated (r = 0.42, P = 0.05) with phosphorylation of 4E-BP1Thr37/46 at 1 hour post RE. We conclude that acute measures of MPS following an initial exposure to RE in novices are not correlated with muscle hypertrophy following chronic RT.
PMCID: PMC3933567  PMID: 24586775
10.  Cost-effectiveness of adult vaccination strategies using pneumococcal conjugate vaccine compared with pneumococcal polysaccharide vaccine 
The cost-effectiveness of 13-valent pneumococcal conjugate vaccine (PCV13) compared with 23-valent pneumococcal polysaccharide vaccine (PPSV23) among US adults is unclear.
To estimate the cost-effectiveness of PCV13 vaccination strategies in adults to assist vaccination policy decision-making.
Design, Setting, and Population
A Markov state-transition model, lifetime time horizon, societal perspective. Simulations were performed in hypothetical cohorts of US 50-year-olds. Vaccination strategies and effectiveness estimates were developed by a Delphi expert panel; indirect (herd immunity) effects resulting from childhood PCV13 vaccination were extrapolated based on observed PCV7 effects. Data sources for model parameters included CDC Active Bacterial Core surveillance, National Hospital Discharge Survey and Nationwide Inpatient Sample data, and the National Health Interview Survey.
Main Outcome Measures
Pneumococcal disease cases prevented and incremental costs per quality-adjusted life year (QALY) gained.
In the base case scenario, PCV13 given as a substitute for PPSV23 in current recommendations (i.e., vaccination at 65 years and at younger ages if comorbidities are present) cost $28,900/QALY gained compared with no vaccination and was more cost-effective than the currently recommended PPSV23 strategy. Routine PCV13 at ages 50 and 65 years cost $45,100/QALY compared with PCV13 substituted in current recommendations. Adding PPSV23 at age 75 to PCV13 at ages 50 and 65 years gained 0.00002 QALYs, costing $496,000/QALY gained. Results were robust in sensitivity analyses and alternative scenarios, except when low PCV13 effectiveness against nonbacteremic pneumococcal pneumonia was assumed or when greater childhood vaccination indirect effects were modeled. In these cases, PPSV23 as currently recommended was favored.
Overall, PCV13 vaccination was favored compared to PPSV23, but the analysis is sensitive to assumptions about PCV13 effectiveness against NPP and the magnitude of potential indirect effects from childhood PCV13 on pneumococcal serotype distribution.
PMCID: PMC3924773  PMID: 22357831
11.  Cost-Effectiveness of VTE Pharmacological Prophylaxis in Total Hip and Knee Replacement: A Systematic Review 
PharmacoEconomics  2010;28(7):521-538.
Total hip and knee replacement (THR and TKR) are high risk settings for venous thromboembolism (VTE).
(1) Summarize cost-effectiveness of VTE prophylaxis regimens for THR and TKR a
Data Sources
Medline (from January 1997 to October 2009), EMBASE (January 1997 until June 2009), and the Economic Evaluation Database[12] (1997- October 2009)
We identified recent cost-effectiveness studies examining five categories of comparisons: (1) anticoagulants (warfarin, low molecular weight heparin - LMWH, or fondaparinux) vs. aspirin; (2) LMWH vs. warfarin; (3) fondaparinux vs. LMWH; (4) comparisons with new oral anticoagulants; and (5) extended (≥3 weeks) vs. short duration prophylaxis (< 3 weeks). We abstracted information on cost and effectiveness for each prophylaxis regimen in order to calculate an incremental cost-effectiveness ratio. Because of variations in effectiveness units reported and horizon length analyzed, we calculate two cost-effectiveness ratios, one for the number of symptomatic, proximal VTE events avoided at 90 days and the other for quality adjusted life-years (QALYs) at the one year mark or beyond.
We identified 33 studies with 67 comparisons. After standardization, comparisons between LMWH and warfarin were inconclusive whereas fondaparinux dominated LMWH in nearly every comparison. The latter results were derived from radiographic VTE rates. Extended duration prophylaxis after THR was generally cost-effective. Small numbers prohibit conclusions about aspirin, new oral anticoagulants, or extended duration prophylaxis after TKR.
Fondaparinux after both THR and TKR and Extended duration LMWH after THR appear to be cost cost-effective prophylaxis regimens. Small numbers for other comparisons and absence of trials reporting symptomatic endpoints prohibit comprehensive conclusions.
PMCID: PMC3916183  PMID: 20550220
thrombosis; total joint replacement; antithrombotic therapy; preventive medicine
12.  Cost-Effectiveness of Medicare Drug Plans in Schizophrenia and Bipolar Disorder 
Medicare Part D has a drug coverage gap, imposing risks for discontinuing medications, particularly in mental health disorders where drug costs are high. However, some beneficiaries have generic drug coverage in the gap. We examine the health outcomes and cost-effectiveness of generic drug coverage compared to no gap coverage in patients with bipolar disorder and schizophrenia.
Study Design
Markov model-based cost-effectiveness analysis using identical hypothetical cohorts to examine drug coverage strategies.
The incremental cost-effectiveness of Part D coverage strategies was estimated, using differences in medical costs and quality adjusted life years between plans. Coverage strategy-specific costs and hospitalization rates were obtained from 2007 Medicare data, adjusted for age, sex, race, and health status.
When comparing generic and no gap coverage, generic-only coverage cost less and was more effective than plans with no gap coverage, due mainly to lower hospitalization rates. In sensitivity analyses, generic-only coverage continued to be favored over no gap coverage, unless generic coverage costs increased ≥3% in bipolar disorder and ≥5% in schizophrenia; generic coverage in the gap was also favored in probabilistic sensitivity analyses.
In Medicare Part D, generic drug coverage was cost saving compared to no coverage in bipolar disorder and schizophrenia while improving health outcomes. Policy makers and insurers might consider generic-only coverage, rather than no gap coverage plans, to both conserve health care resources and improve health.
PMCID: PMC3589737  PMID: 23448115
13.  Impulse Conduction Increases Mitochondrial Transport in Adult Mammalian Peripheral Nerves In Vivo 
PLoS Biology  2013;11(12):e1001754.
Observations of nerve axons in vivo reveal that electrical activity increases the number and speed of transported mitochondria, showing how sudden increases in energy demand may be satisfied.
Matching energy supply and demand is critical in the bioenergetic homeostasis of all cells. This is a special problem in neurons where high levels of energy expenditure may occur at sites remote from the cell body, given the remarkable length of axons and enormous variability of impulse activity over time. Positioning mitochondria at areas with high energy requirements is an essential solution to this problem, but it is not known how this is related to impulse conduction in vivo. Therefore, to study mitochondrial trafficking along resting and electrically active adult axons in vivo, confocal imaging of saphenous nerves in anaesthetised mice was combined with electrical and pharmacological stimulation of myelinated and unmyelinated axons, respectively. We show that low frequency activity induced by electrical stimulation significantly increases anterograde and retrograde mitochondrial traffic in comparison with silent axons. Higher frequency conduction within a physiological range (50 Hz) dramatically further increased anterograde, but not retrograde, mitochondrial traffic, by rapidly increasing the number of mobile mitochondria and gradually increasing their velocity. Similarly, topical application of capsaicin to skin innervated by the saphenous nerve increased mitochondrial traffic in both myelinated and unmyelinated axons. In addition, stationary mitochondria in axons conducting at higher frequency become shorter, thus supplying additional mitochondria to the trafficking population, presumably through enhanced fission. Mitochondria recruited to the mobile population do not accumulate near Nodes of Ranvier, but continue to travel anterogradely. This pattern of mitochondrial redistribution suggests that the peripheral terminals of sensory axons represent sites of particularly high metabolic demand during physiological high frequency conduction. As the majority of mitochondrial biogenesis occurs at the cell body, increased anterograde mitochondrial traffic may represent a mechanism that ensures a uniform increase in mitochondrial density along the length of axons during high impulse load, supporting the increased metabolic demand imposed by sustained conduction.
Author Summary
As mitochondria are the main power source for most cells, their correct localization is vital for cellular homeostasis. The morphological and functional complexity of neurons, and the unpredictability of energy demand resulting from sustained impulse activity, means that correct mitochondrial function and location is particularly important for these cells. However, it is unclear how mitochondrial transport responds to the wide range of physiological conduction frequencies experienced by an intact nervous system. Here we image mitochondria moving along mouse saphenous nerve axons in vivo and observe the effects of sustained trains of impulses, evoked either by electrical stimulation of myelinated axons at physiological frequencies (1 and 50 Hz), or chemical stimulation (cutaneous capsaicin) in unmyelinated axons. We find that the number and speed of mitochondria moving towards the peripheral sensory terminals dramatically increases in active axons. The increased numbers of mobile mitochondria seem to originate from “pinched off” segments of stationary mitochondria (mitochondrial fission), and from recruitment of small stationary mitochondria. The transported mitochondria appear to accumulate in sensory nerve terminals in the skin. This study suggests that mitochondrial fission is involved in rapidly supplying mobile mitochondria to aid their re-distribution to regions of increased metabolic demand along axons.
PMCID: PMC3876979  PMID: 24391474
14.  Feeding Preferences of Abyssal Macrofauna Inferred from In Situ Pulse Chase Experiments 
PLoS ONE  2013;8(11):e80510.
Climatic fluctuations may significantly alter the taxonomic and biochemical composition of phytoplankton blooms and subsequently phytodetritus, the food source for the majority of deep-sea communities. To examine the response of abyssal benthic communities to different food resources we simulated a food sedimentation event containing diatoms and coccolithophorids at Station M in the NE Pacific. In one set of experiments we measured incorporation of diatomC and coccoN into the macrofauna using isotopically enriched 13C-diatoms and 15N-coccolithophores. In a second experiment we measured incorporation of C and N from dual-labelled (13C and 15N) diatoms. The second experiment was repeated 2 months later to assess the effect of seasonality. The simulated food pulses represented additions of 650 – 800 mg C m−2 and 120 mg N m−2 to the seafloor. In all cases rapid incorporation of tracer was observed within 4 days, with between 20% and 52% of the macrofauna displaying evidence of enrichment. However, incorporation levels of both diatomC and coccoN were low (<0.05% and 0.005% of the added C and N). Incorporation of labelled diatoms was similar during both June and September suggesting that the community was not food limited during either period. We found no evidence for selective ingestion of the different food types in the metazoan fauna suggesting that macrofauna do not have strong preferences for diatom vs. coccolithophore dominated phytodetrital pulses. C∶N ratios from both experiments suggest that the metazoan macrofauna community appear to have higher C demands and/or assimilation efficiencies compared to N. Concomitantly, the foraminifera preferentially selected for diatomN over coccoN, and we suggest that this may be related to foraminiferal requirements for intracellular nitrate. These experiments provide evidence that abyssal faunal feeding strategies are in part driven by an organism's internal stoichiometric budgets and biochemical requirements.
PMCID: PMC3841197  PMID: 24303022
16.  Development of a new Sonovue™ contrast-enhanced ultrasound approach reveals temporal and age-related features of muscle microvascular responses to feeding 
Physiological Reports  2013;1(5):e00119.
Compromised limb blood flow in aging may contribute to the development of sarcopenia, frailty, and the metabolic syndrome. We developed a novel contrast-enhanced ultrasound technique using Sonovue™ to characterize muscle microvasculature responses to an oral feeding stimulus (15 g essential amino acids) in young (∼20 years) and older (∼70 years) men. Intensity-time replenishment curves were made via an ultrasound probe “fixed” over the quadriceps, with intermittent high mechanical index destruction of microbubbles within muscle vasculature. This permitted real-time measures of microvascular blood volume (MBV), microvascular flow velocity (MFV) and their product, microvascular blood flow (MBF). Leg blood flow (LBF) was measured by Doppler and insulin by enzyme-linked immunosorbent assay. Steady-state contrast concentrations needed for comparison between different physiological states were achieved <150 sec from commencing Sonovue™ infusion, and MFV and MBV measurements were completed <120 sec thereafter. Interindividual coefficients of variation in MBV and MFV were 35–40%, (N = 36). Younger men (N = 6) exhibited biphasic vascular responses to feeding with early increases in MBV (+36%, P < 0.008 45 min post feed) reflecting capillary recruitment, and late increases in MFV (+77%, P < 0.008) and MBF (+130%, P < 0.007 195 min post feed) reflecting more proximal vessel dilatation. Early MBV responses were synchronized with peak insulin but not increased LBF, while later changes in MFV and MBF occurred with insulin at post absorptive values but alongside increased LBF. All circulatory responses were absent in old men (N = 7). Thus, impaired postprandial circulation could impact age-related declines in muscle glucose disposal, protein anabolism, and muscle mass.
PMCID: PMC3841050  PMID: 24303186
Aging; CEUS; microcirculation; muscle; nutrition
17.  Human SNP Links Differential Outcomes in Inflammatory and Infectious Disease to a FOXO3-Regulated Pathway 
Cell  2013;155(1):57-69.
The clinical course and eventual outcome, or prognosis, of complex diseases varies enormously between affected individuals. This variability critically determines the impact a disease has on a patient’s life but is very poorly understood. Here, we exploit existing genome-wide association study data to gain insight into the role of genetics in prognosis. We identify a noncoding polymorphism in FOXO3A (rs12212067: T > G) at which the minor (G) allele, despite not being associated with disease susceptibility, is associated with a milder course of Crohn’s disease and rheumatoid arthritis and with increased risk of severe malaria. Minor allele carriage is shown to limit inflammatory responses in monocytes via a FOXO3-driven pathway, which through TGFβ1 reduces production of proinflammatory cytokines, including TNFα, and increases production of anti-inflammatory cytokines, including IL-10. Thus, we uncover a shared genetic contribution to prognosis in distinct diseases that operates via a FOXO3-driven pathway modulating inflammatory responses.
Graphical Abstract
•Reanalysis of GWAS data identifies a SNP associated with outcome in Crohn’s disease•This SNP modulates inflammatory responses in monocytes via a FOXO3-driven pathway•The mild disease-associated allele reduces TNFα and increases IL-10 production•Prognosis in RA and malaria (also TNFα-related diseases) is also linked to this SNP
An analysis of GWAS data of patients with Crohn’s disease distinguishes SNPs associated with disease risk from those associated with outcome. Functional studies indicate that a noncoding SNP associated with FOXO3A influences a cytokine inflammatory pathway that impacts outcome, not only in Crohn’s but also other diseases such as rheumatoid arthritis and malaria.
PMCID: PMC3790457  PMID: 24035192
18.  Genetically Distinct Subsets within ANCA-Associated Vasculitis 
The New England journal of medicine  2012;367(3):214-223.
Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis.
A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria.
We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti–proteinase 3 ANCA was associated with HLA-DP and the genes encoding α1-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P = 6.2×10−89, P = 5.6×10−12, and P = 2.6×10−7, respectively). Anti–myeloperoxidase ANCA was associated with HLA-DQ (P = 2.1×10−8).
This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA–associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA–associated vasculitis and myeloperoxidase ANCA–associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.)
PMCID: PMC3773907  PMID: 22808956
19.  Economic Model for Emergency Use Authorization of Intravenous Peramivir 
To develop 3 computer simulation models to determine the potential economic effect of using intravenous (IV) antiviral agents to treat hospitalized patients with influenza-like illness, as well as different testing and treatment strategies.
Study Design
Stochastic decision analytic computer simulation model.
During the 2009 influenza A(H1N1) pandemic, the Food and Drug Administration granted emergency use authorization of IV neuraminidase inhibitors for hospitalized patients with influenza, creating a need for rapid decision analyses to help guide use. We compared the economic value from the societal and third-party payer perspectives of the following 4 strategies for a patient hospitalized with influenza-like illness and unable to take oral antiviral agents: Strategy 1: Administration of IV antiviral agents without polymerase chain reaction influenza testing. Strategy 2: Initiation of IV antiviral treatment, followed by polymerase chain reaction testing to determine whether the treatment should be continued. Strategy 3: Performance of polymerase chain reaction testing, followed by initiation of IV antiviral treatment if the test results are positive. Strategy 4: Administration of no IV antiviral agents. Sensitivity analyses varied the probability of having influenza (baseline, 10%; range, 10%–30%), IV antiviral efficacy (baseline, oral oseltamivir phosphate; range, 25%–75%), IV antiviral daily cost (range, $20–$1000), IV antiviral reduction of illness duration (baseline, 1 day; range, 1–2 days), and ventilated vs nonventilated status of the patient.
When the cost of IV antiviral agents was no more than $500 per day, the incremental cost-effectiveness ratio for most of the IV antiviral treatment strategies was less than $10,000 per quality-adjusted life-year compared with no treatment. When the cost was no more than $100 per day, all 3 IV antiviral strategies were even more cost-effective. The order of cost-effectiveness from most to least was strategies 3, 1, and 2. The findings were robust to changing risk of influenza, influenza mortality, IV antiviral efficacy, IV antiviral daily cost, IV antiviral reduction of illness duration, and ventilated vs nonventilated status of the patient for both societal and third-party payer perspectives.
Our study supports the use of IV antiviral treatment for hospitalized patients with influenza-like illness.
PMCID: PMC3763185  PMID: 21485418
20.  Screening Cardiac Surgery Patients for MRSA: An Economic Computer Model 
The American journal of managed care  2010;16(7):e163-e173.
To estimate the economic value of preoperative methicillin-resistant Staphylococcus aureus (MRSA) screening and decolonization for cardiac surgery patients.
Study Design
Monte Carlo decision-analytic computer simulation model.
We developed a computer simulation model representing the decision of whether to perform preoperative MRSA screening and decolonizing those patients with a positive MRSA culture. Sensitivity analyses varied key input parameters including MRSA colonization prevalence, decolonization success rates, the number of surveillance sites, and screening/decolonization costs. Separate analyses estimated the incremental cost-effectiveness ratio (ICER) of the screening and decolonization strategy from the third-party payer and hospital perspectives.
Even when MRSA colonization prevalence and decolonization success rate were as low as 1% and 25%, respectively, the ICER of implementing routine surveillance was well under $15,000 per quality-adjusted life-year from both the third-party payer and hospital perspectives. The surveillance strategy was economically dominant (less costly and more effective than no testing) for most scenarios explored.
Our results suggest that routine preoperative MRSA screening of cardiac surgery patients could provide substantial economic value to third-party payers and hospitals over a wide range of MRSA colonization prevalence levels, decolonization success rates, and surveillance costs. Healthcare administrators, infection control specialists, and surgeons can compare their local conditions with our study’s benchmarks to make decisions about whether to implement preoperative MRSA testing. Third-party payers may want to consider covering such a strategy.
PMCID: PMC3763192  PMID: 20645662
21.  Economics of Influenza Vaccine Administration Timing for Children 
To determine how much should be invested each year to encourage and operationalize the administration of influenza vaccine to children before November and how late the vaccine should be offered each year.
Study Design
Monte Carlo decision analytic computer simulation models.
The children's influenza vaccination timing model quantified the incremental economic value of vaccinating a child earlier in the influenza season and the incremental cost of delaying vaccination. The children's monthly influenza vaccination decision model evaluated the cost-effectiveness of vaccinating versus not vaccinating for every month of the influenza season.
Getting children vaccinated by the end of October rather than when they are currently getting vaccinated could save society between $6.4 million and $9.2 million plus 653 and 926 quality-adjusted life-years (QALYs) and third-party payers between $4.1 million and $6.1 million plus 647 to 942 QALYs each year. Decision makers may want to continue offering influenza vaccination to children at least through the end of December. Vaccinating with trivalent inactivated virus vaccine was more cost-effective than vaccinating with live attenuated influenza vaccine for every month.
Policymakers could invest up to $6 million to $9 million a year to get children vaccinated in September or October without expending any net costs.
PMCID: PMC3763213  PMID: 20205492
22.  The Effect of the Medicare Part D Coverage Gaps on Medication Use Among Beneficiaries with Depression and Heart Failure 
Medication use among Medicare beneficiaries has increased and adherence has improved since the implementation of the Medicare Part D prescription drug benefit in 2006. However, the structure of the benefit, particularly, the coverage gap, is still problematic. It is critical to understand how beneficiaries with coexisting conditions respond to the coverage gap and whether their response differs by type of medications.
Aims of the Study
The paper aims to evaluate the effects of Medicare Part D’s coverage gap on drug regimens among beneficiaries with coexisting depression and heart failure (HF).
Drug utilization patterns of a 5% random sample of Medicare Part D beneficiaries with depression and HF in 2007 were observed. We compared drug use pattern pre and post coverage gap among three groups: no coverage, generic coverage, and full coverage due to low-income subsidies (LIS) and used propensity score weighting to adjust for difference across groups.
Beneficiaries with some drug coverage in the gap were more likely to enter the gap: 82% for LIS, 79% for generic-only and 58% for no coverage. Beneficiaries without drug coverage reduced their use of antidepressants by 5.0% (95% CI 1.7%-8.2%), and HF drugs by 9.4% (95% CI 7.2%-11.5%) after they entered the coverage gap. Those with generic coverage cut their brand-name drugs more than generic drugs but did not shift to generic drugs. However, adherence to antidepressants did not change; adherence to HF drugs reduced slightly, 2.5% (95% CI 1.2%-3.7%) in the no-coverage group and 2.6% (95% CI 1.3%-3.9%) in the generic-coverage group.
The coverage gap was associated with a modest reduction in number of prescriptions filled for depression and HF but it was not associated with a significant effect on adherence.
Implications for Health Policy
We found that beneficiaries with coexisting depression and HF were less likely to reduce their drug use than beneficiaries in general. In addition, the gap was not associated with a large reduction in adherence. It suggests that concerns about the coverage gap’s harmful effects on medication adherence, or comorbidities might be overstated.
Implications for Further Research
Further studies on how people make medication use decisions in the face of changes in benefits and how the coverage affects non-drug medical outcomes are warranted.
PMCID: PMC3471664  PMID: 23001279
23.  Differential Targeting of Viral Components by CD4+ versus CD8+ T Lymphocytes in Dengue Virus Infection 
Journal of Virology  2013;87(5):2693-2706.
Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4+ and CD8+ T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4+ and 21 are CD8+ T cell epitopes. We observe that whereas CD8+ T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4+ epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4+ T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4+ and CD8+ T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.
PMCID: PMC3571409  PMID: 23255803
24.  Regulation of allograft survival by inhibitory FcgammaRIIb signaling 
Fc gamma receptors (FcγR) provide important immunoregulation. Targeting inhibitory FcγRIIb may therefore prolong allograft survival, but its role in transplantation has not been addressed. FcγRIIb signaling was examined in murine models of acute or chronic cardiac allograft rejection by transplanting recipients that either lacked FcγRIIb expression (FcγRIIb−/− ) or over-expressed FcγRIIb on B cells (BTG). Acute heart allograft rejection occurred at the same tempo in FcγRIIb−/− C57Bl/6 (B6) recipients as WT recipients, with similar IgG alloantibody responses. In contrast, chronic rejection of MHC class II-mismatched bm12 cardiac allografts was accelerated in FcγRIIb−/− mice, with development of more severe transplant arteriopathy and markedly augmented effector autoantibody production. Autoantibody production was inhibited, and rejection delayed, in BTG recipients. Similarly, whereas MHC class I-mismatched B6.Kd hearts survived indefinitely and remained disease-free in B6 mice, much stronger alloantibody responses and progressive graft arteriopathy developed in FcγRIIb−/− recipients. Notably, FcγRIIb-mediated inhibition of B6.Kd heart graft rejection was abrogated by increasing T cell help through transfer of additional H2.Kd-specific CD4 T cells.
Thus, inhibitory FcγRIIb signaling regulates chronic but not acute rejection, most likely because the supra-optimal helper CD4 T cell response in acute rejection overcomes FcγRIIb-mediated inhibition of the effector B cell population. Immunomodulation of FcγRIIb in clinical transplantation may hold potential for inhibiting progression of transplant arteriopathy and prolonging heart transplant survival.
PMCID: PMC3605772  PMID: 23150718
25.  Human Monoclonal Antibodies Generated Following Vaccination with AVA Provide Neutralization by Blocking Furin Cleavage but not by Preventing Oligomerization 
Vaccine  2012;30(28):4276-4283.
In order to identify the combination of antibody-mediated mechanisms of neutralization that result from vaccination with anthrax vaccine adsorbed (AVA), we isolated antibody secreting cells from a single donor seven days after booster vaccination with AVA and generated nine fully human monoclonal antibodies (hmAb) with high specificity for protective antigen (PA). Two of the antibodies were able to neutralize lethal toxin in vitro at low concentrations (IC50: p6C01, 0.12 µg/ml and p6F01, 0.45 µg/ml). Passive transfer of either of these hmAbs to A/J mice prior to challenge with lethal toxin conferred 80–90% protection. We demonstrate that hmAb p6C01 is neutralizing by preventing furin cleavage of PA in a dose-dependent manner, but the mechanism of p6F01 is unclear. Three additional antibodies were found to bind to domain 3 of PA and prevent oligomerization, although they did not confer significant protection in vivo and showed a significant prozone-like effect in vitro. These fully human antibodies provide insight into the neutralizing response to AVA for future subunit vaccine and passive immunotherapeutic cocktail design.
PMCID: PMC3367042  PMID: 22425791
anthrax; Anthrax Vaccine Adsorbed; human monoclonal antibodies; passive immunotherapeutics; protective antigen

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