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1.  A type I interferon transcriptional signature precedes autoimmunity in children genetically at-risk of type 1 diabetes 
Diabetes  2014;63(7):2538-2550.
Diagnosis of the autoimmune disease type 1 diabetes (T1D) is preceded by the appearance of circulating autoantibodies to pancreatic islets. However, almost nothing is known about events leading to this islet autoimmunity. Previous epidemiological and genetic data have associated viral infections and anti-viral type I interferon (IFN) immune response genes with T1D. Here, we first used DNA microarray analysis to identify IFN-β inducible genes in vitro and then used this set of genes to define an IFN-inducible transcriptional signature in peripheral blood mononuclear cells from a group of active systemic lupus erythematosus patients (N=25). Using this predefined set of 225 IFN signature genes, we investigated expression of the signature in cohorts of healthy controls (N=87), T1D patients (N=64) and a large longitudinal birth cohort of children genetically predisposed to T1D (N=109; 454 microarrayed samples). Expression of the IFN signature was increased in genetically-predisposed children prior to the development of autoantibodies (P=0.0012), but not in established T1D patients. Upregulation of IFN-inducible genes was transient, temporally associated with a recent history of upper respiratory tract infections (P=0.0064) and marked by increased expression of SIGLEC-1 (CD169), a lectin-like receptor expressed on CD14+ monocytes. DNA variation in IFN-inducible genes altered T1D risk (P=0.007), as exemplified by IFIH1, one of the genes in our IFN signature and for which increased expression is a known disease risk factor. These findings identify transient increased expression of type I IFN genes in pre-clinical diabetes as a risk factor for autoimmunity in children with a genetic predisposition to T1D.
PMCID: PMC4066333  PMID: 24561305
2.  A Dose- rather than Delivery Profile–Dependent Mechanism Regulates the “Muscle-Full” Effect in Response to Oral Essential Amino Acid Intake in Young Men12 
The Journal of Nutrition  2014;145(2):207-214.
Background: The anabolic response of skeletal muscle to essential amino acids (EAAs) is dose dependent, maximal at modest doses, and short lived, even with continued EAA availability, a phenomenon termed “muscle-full.” However, the effect of EAA ingestion profile on muscle metabolism remains undefined.
Objective: We determined the effect of Bolus vs. Spread EAA feeding in young men and hypothesized that muscle-full is regulated by a dose-, not delivery profile–, dependent mechanism.
Methods: We provided 16 young healthy men with 15 g mixed-EAA, either as a single dose (“Bolus”; n = 8) or in 4 fractions at 45-min intervals (“Spread”; n = 8). Plasma insulin and EAA concentrations were assayed by ELISA and ion-exchange chromatography, respectively. Limb blood flow by was determined by Doppler ultrasound, muscle microvascular flow by Sonovue (Bracco) contrast-enhanced ultrasound, and phosphorylation of mammalian target of rapamycin complex 1 substrates by immunoblotting. Intermittent muscle biopsies were taken to quantify myofibrillar-bound 13C6-phenylalanine to determine muscle protein synthesis (MPS).
Results: Bolus feeding achieved rapid insulinemia (13.6 μIU · mL−1, 25 min after commencement of feeding), aminoacidemia (∼2500 μM at 45 min), and capillary recruitment (+45% at 45 min), whereas Spread feeding achieved attenuated insulin responses, gradual low-amplitude aminoacidemia (peak: ∼1500 μM at 135 min), and no detectable capillary recruitment (all P < 0.01 vs. Bolus). Despite these differences, identical anabolic responses were observed; fasting fractional synthetic rates of 0.054% · h−1 (Bolus) and 0.066% · h−1 (Spread) increased to 0.095% and 0.104% · h−1 (no difference in increment or final values between regimens). With both Spread and Bolus feeding strategies, a latency of at least 90 min was observed before an upswing in MPS was evident. Similarly with both feeding strategies, MPS returned to fasting rates by 180 min despite elevated circulating EAAs.
Conclusion: These data do not support EAA delivery profile as an important determinant of anabolism in young men at rest, nor rapid aminoacidemia/leucinemia as being a key factor in maximizing MPS. This trial was registered at as NCT01735539.
PMCID: PMC4304023  PMID: 25644339
muscle protein synthesis; nutrition; essential amino acids; skeletal muscle; blood flow; anabolic signaling; muscle-full
3.  Potential unintended pregnancies averted and cost savings associated with a revised Medicaid sterilization policy 
Contraception  2013;88(6):10.1016/j.contraception.2013.08.004.
Medicaid sterilization policy, which includes a mandatory 30-day waiting period between consent and the sterilization procedure, poses significant logistical barriers for many women who desire publicly-funded sterilization. Our goal was to estimate the number of unintended pregnancies and the associated costs resulting from unfulfilled sterilization requests due to Medicaid policy barriers.
Study design
We constructed a cost effectiveness model from the health care payer perspective to determine the incremental cost over a 1-year time horizon of the current Medicaid sterilization policy compared to a hypothetical, revised policy in which women who desire a post-partum sterilization would face significantly reduced barriers. Probability estimates for potential outcomes in the model were based on published sources; costs of Medicaid-funded sterilizations and Medicaid-covered births were based on data from the Medicaid Statistical Information System and The Guttmacher Institute, respectively.
With the implementation of a revised Medicaid sterilization policy, we estimated that the number of fulfilled sterilization requests would increase by 45%, from 53.3% of all women having their sterilization requests fulfilled to 77.5%. Annually, this increase could potentially lead to over 29,000 unintended pregnancies averted and $215 million saved.
A revised Medicaid sterilization policy could potentially honor women's reproductive decisions, reduce the number of unintended pregnancies, and save a significant amount of public funds.
Compared to the current federal Medicaid sterilization policy, a hypothetical, revised policy that reduces logistical barriers for women who desire publicly-funded, post-partum sterilization could potentially avert over 29,000 unintended pregnancies annually and therefore lead to a cost savings of $215 million each year.
PMCID: PMC3830666  PMID: 24028751
Tubal sterilization; unfulfilled sterilization; cost analysis
4.  Regulatory T cells and control of the germinal centre response 
Germinal centres (GCs) are specialised lymphoid microenvironments that form in secondary B-cell follicles upon exposure to T-dependent antigens. In the GC, clonal expansion, selection and differentiation of GC B cells result in the production of high-affinity plasma cells and memory B cells that provide protection against subsequent infection. The GC is carefully regulated to fulfil its critical role in defence against infection and to ensure that immunological tolerance is not broken in the process. The GC response can be controlled by a number of mechanisms, one of which is by forkhead box p3 expressing regulatory T (Treg) cells, a suppressive population of CD4+ T cells. A specialised subset of Treg cells – follicular regulatory T (Tfr) cells – form after immunisation and are able to access the GC, where they control the size and output of the response. Our knowledge of Treg cell control of the GC is expanding. In this review we will discuss recent advances in the field, with a particular emphasis on the differentiation and function of Tfr cells in the GC.
PMCID: PMC4289055  PMID: 25606598
5.  CD28 expression is required after T cell priming for helper T cell responses and protective immunity to infection 
eLife  2014;3:e03180.
The co-stimulatory molecule CD28 is essential for activation of helper T cells. Despite this critical role, it is not known whether CD28 has functions in maintaining T cell responses following activation. To determine the role for CD28 after T cell priming, we generated a strain of mice where CD28 is removed from CD4+ T cells after priming. We show that continued CD28 expression is important for effector CD4+ T cells following infection; maintained CD28 is required for the expansion of T helper type 1 cells, and for the differentiation and maintenance of T follicular helper cells during viral infection. Persistent CD28 is also required for clearance of the bacterium Citrobacter rodentium from the gastrointestinal tract. Together, this study demonstrates that CD28 persistence is required for helper T cell polarization in response to infection, describing a novel function for CD28 that is distinct from its role in T cell priming.
eLife digest
Invasion by a bacterium or virus typically activates a mammalian host's immune system to eliminate the pathogen. The cells of the so-called ‘innate immune system’ are the body's first line of defense against infection, and these cells patrol the organs and tissues in an effort to locate and eliminate pathogens quickly. The innate immune response is rapid and non-specific, but often cannot completely clear an infection. When necessary, innate immune cells will escalate the immune response by activating the second branch of the immune system, called the ‘adaptive immune system’. This specifically targets and eradicates an invading pathogen.
T cells are essential components of the adaptive immune system, and these cells can be readily distinguished from other types of cell by proteins called T cell receptors (or TCRs) found on their surface. There are also different types of T cell, each with a specific function. T helper cells, for example, help other adaptive immune cells to mature and activate, which involves these immune cells proliferating and developing into more specialized cells.
For a T cell to activate, two events must occur at the same time. First, the TCR must recognize and bind to a fragment of the pathogen that is presented to it by an innate immune cell. And second, ‘co-stimulatory molecules’ present on the surfaces of both the T cell and the same innate immune cell must interact. Using these two signals to activate a T cell helps to ensure the adaptive immune response is not ‘unleashed‘ unnecessarily.
Co-stimulatory molecules have become popular targets for therapies aimed at treating autoimmune disorders—where the immune system attacks and destroys the body's own tissues. One of the most well studied co-stimulatory molecules expressed by T cells is called CD28; however, it remained unknown whether CD28 is involved in any processes after T cell activation.
Now, Linterman et al. reveal that the CD28 co-stimulatory molecule plays a number of roles in addition to T cell activation. For example, a newly developed mouse model showed that CD28 must remain on the surface of T helper cells after they have been activated for these cells to effectively specialize. Linterman et al. also discovered that CD28 helps different T helper cell subtypes to develop.
Linterman et al. demonstrate that CD28 is critical throughout a host's response to infection, and suggest that if CD28 is lost on activated T cells (which happens during aging, HIV infection and autoimmune diseases) the responses of T helper cells become limited. Furthermore, these findings reveal that treatments that target the CD28 co-stimulatory molecule will also affect on-going immune responses.
PMCID: PMC4241536  PMID: 25347065
CD28; helper T cells; infection; mouse
6.  Modules, networks and systems medicine for understanding disease and aiding diagnosis 
Genome Medicine  2014;6(10):82.
Many common diseases, such as asthma, diabetes or obesity, involve altered interactions between thousands of genes. High-throughput techniques (omics) allow identification of such genes and their products, but functional understanding is a formidable challenge. Network-based analyses of omics data have identified modules of disease-associated genes that have been used to obtain both a systems level and a molecular understanding of disease mechanisms. For example, in allergy a module was used to find a novel candidate gene that was validated by functional and clinical studies. Such analyses play important roles in systems medicine. This is an emerging discipline that aims to gain a translational understanding of the complex mechanisms underlying common diseases. In this review, we will explain and provide examples of how network-based analyses of omics data, in combination with functional and clinical studies, are aiding our understanding of disease, as well as helping to prioritize diagnostic markers or therapeutic candidate genes. Such analyses involve significant problems and limitations, which will be discussed. We also highlight the steps needed for clinical implementation.
PMCID: PMC4254417  PMID: 25473422
7.  MiR-210 is induced by Oct-2, regulates B-cells and inhibits autoantibody production1 
MicroRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally. Here, we show that miR-210 is induced by Oct-2, a key transcriptional mediator of B-cell activation. Germline deletion of miR-210 results in the development of autoantibodies from 5 months of age. Overexpression of miR-210 in vivo resulted in cell autonomous expansion of the B1 lineage and impaired fitness of B2 cells. Mice over-expressing miR-210 exhibited impaired class-switched antibody responses, a finding confirmed in wild-type B-cells transfected with a miR-210 mimic. In vitro studies demonstrated a defect in cellular proliferation and cell-cycle entry, which was consistent with the transcriptomic analysis demonstrating down-regulation of genes involved in cellular proliferation and B cell activation. These findings indicate that Oct-2 induction of miR-210 provides a novel inhibitory mechanism for the control of B cells and autoantibody production.
PMCID: PMC4162006  PMID: 23960236
8.  Cost-Effectiveness of Procalcitonin-Guided Antibiotic Use in Community Acquired Pneumonia 
Journal of General Internal Medicine  2013;28(9):1157-1164.
Although prior randomized trials have demonstrated that procalcitonin-guided antibiotic therapy effectively reduces antibiotic use in patients with community-acquired pneumonia (CAP), uncertainties remain regarding use of procalcitonin protocols in practice.
To estimate the cost-effectiveness of procalcitonin protocols in CAP.
Decision analysis using published observational and clinical trial data, with variation of all parameter values in sensitivity analyses.
Hypothetical patient cohorts who were hospitalized for CAP.
Procalcitonin protocols vs. usual care.
Costs and cost per quality adjusted life year gained.
When no differences in clinical outcomes were assumed, consistent with clinical trials and observational data, procalcitonin protocols cost $10–$54 more per patient than usual care in CAP patients. Under these assumptions, results were most sensitive to variations in: antibiotic cost, the likelihood that antibiotic therapy was initiated less frequently or over shorter durations, and the likelihood that physicians were nonadherent to procalcitonin protocols. Probabilistic sensitivity analyses, incorporating procalcitonin protocol-related changes in quality of life, found that protocol use was unlikely to be economically reasonable if physician protocol nonadherence was high, as observational study data suggest. However, procalcitonin protocols were favored if they decreased hospital length of stay.
Procalcitonin protocol use in hospitalized CAP patients, although promising, lacks physician nonadherence and resource use data in routine care settings, which are needed to evaluate its potential role in patient care.
PMCID: PMC3744292  PMID: 23463457
cost-effectiveness analysis; procalcitonin; community-acquired pneumonia
9.  FcγRIIB in autoimmunity and infection: evolutionary and therapeutic implications 
Nature reviews. Immunology  2010;10(5):328-343.
FcγRIIB is the only inhibitory Fc receptor. It controls many aspects of immune and inflammatory responses, and variation in the gene encoding this protein has long been associated with susceptibility to autoimmune disease, particularly systemic lupus erythematosus (SLE). FcγRIIB is also involved in the complex regulation of defence against infection. A loss-of-function polymorphism in FcγRIIB protects against severe malaria, the investigation of which is beginning to clarify the evolutionary pressures that drive ethnic variation in autoimmunity. Our increased understanding of the function of FcγRIIB also has potentially far-reaching therapeutic implications, being involved in the mechanism of action of intravenous immunoglobulin, controlling the efficacy of monoclonal antibody therapy and providing a direct therapeutic target.
PMCID: PMC4148599  PMID: 20414206
10.  Defunctioning Polymorphism in the Immunoglobulin G Inhibitory Receptor (FcγRIIB-T/T232) Does not Impact on Kidney Transplant or Recipient Survival 
Transplantation  2014;98(3):285-291.
There is an increasing appreciation of the deleterious effects of antibody and B cells on acute and chronic transplant outcomes. Many effector functions of antibody are mediated by a family of receptors (FcγRs) that are expressed on most immune cells, including neutrophils, natural killer cells, and B cells. Most FcγRs are activating and controlled by a single inhibitory receptor, FcγRIIB (CD32B), which also regulates some aspects of B-cell activation and antibody production. FcγRIIB-deficient mice develop severe chronic arteriopathy in a murine cardiac allograft model. A single nucleotide polymorphism in human FcγRIIB (rs1050501) results in profound receptor dysfunction and is associated with systemic lupus erythematosus. The frequency of this FcγRIIB-I/T232 polymorphism also shows significant racial variation.
In the present study, we sought to determine whether the FcγRIIB-I/T232 single nucleotide polymorphism rs1050501 affected susceptibility to renal allograft rejection or loss and transplant recipient survival. FcγRIIB-I/T232 genotype was determined in 2,851 Caucasian and 570 Afro-Caribbean renal transplant recipients, and in 236 transplant recipients with a primary diagnosis of systemic lupus erythematosus, all of whom were enrolled into the Collaborative Transplant Study.
We found no significant difference in pretransplant panel reactive antibodies, acute rejection at 1-year nor in 10-year transplant or patient survival in individuals with differing FcγRIIB-I/T232 genotype.
This negative result is surprising, given the importance of this receptor in modulating antibody effector function.
PMCID: PMC4148707  PMID: 25022320
Antibodies; IgG; Fcγ receptors; FcγRIIB; CD32B; Renal transplantation; Chronic antibody-mediated rejection
12.  Cost-effectiveness of pneumococcal conjugate vaccination in immunocompromised adults 
Vaccine  2013;31(37):3950-3956.
Pneumococcal disease is a significant problem in immunocompromised persons, particularly in HIV-infected individuals. The CDC recently updated pneumococcal vaccination recommendations for immunocompromised adults, adding the 13-valent pneumococcal conjugate vaccine (PCV13) to the previously recommended 23-valent pneumococcal polysaccharide vaccine (PPSV23). This analysis estimates the cost-effectiveness of pneumococcal vaccination strategies in HIV-infected individuals and in the broader immunocompromised adult group.
Markov model-based cost-effectiveness analysis
The model considered immunocompromised persons aged 18–64 years and accounted for childhood PCV13 herd immunity; in a separate analysis, an HIV-infected subgroup was considered. PCV13 effectiveness was estimated by an expert panel; PPSV23 protection was modeled relative to PCV13 effectiveness. We assumed that both vaccines prevented invasive pneumococcal disease, but only PCV13 prevented nonbacteremic pneumonia.
In all immunocompromised individuals, a single PCV13 cost $70,937 per quality adjusted life year (QALY) gained compared to no vaccination; current recommendations cost $136,724/QALY. In HIV patients, with a longer life expectancy (22.5 years), current recommendations cost $89,391/QALY compared to a single PCV13. Results were sensitive to variation of life expectancy and vaccine effectiveness. The prior recommendation was not favored in any scenario.
One dose of PCV13 is more cost-effective for immunocompromised individuals than previous vaccination recommendations and may be more economically reasonable than current recommendations, depending on life expectancy and vaccine effectiveness in the immunocompromised.
PMCID: PMC3742552  PMID: 23806240
13.  microRNA-155 Regulates the Generation of Immunoglobulin Class-Switched Plasma Cells 
Immunity  2007;27(6):847-859.
MicroRNA-155 (miR-155) is expressed by cells of the immune system following activation and has been shown to be required for antibody production following vaccination with attenuated Salmonella. Here we show the intrinsic requirement for miR-155 in B cell responses to thymus-dependent and independent antigens. B cells lacking miR-155 generated reduced extra-follicular and germinal center responses and failed to produce high affinity IgG1 antibodies. Gene expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function-many of which are predicted targets of miR-155. The transcription factor Pu.1 is validated as a direct target of miR155 mediated inhibition. When Pu.1 is over-expressed in wild type B cells fewer IgG1 cells are produced, indicating that loss of Pu.1 regulation is a contributing factor to the miR-155 deficient phenotype. Our results implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.
PMCID: PMC4135426  PMID: 18055230
14.  Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation 
BMC Genomics  2014;15(1):649.
Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings. The recent development of a multiplexed, digital technology for nucleic acid measurement enables counting of individual RNA molecules without amplification and, for the first time, permits such a study.
Using a set of human leukocyte subset RNA samples, we compared previously acquired microarray expression values with RNA molecule counts determined by the nCounter Analysis System (NanoString Technologies) in selected genes. We found that gene measurements across samples correlated well between the two platforms, particularly for high-variance genes, while genes deemed unexpressed by the nCounter generally had both low expression and low variance on the microarray. Confirming previous findings from spike-in and dilution datasets, this “gold-standard” comparison demonstrated signal compression that varied dramatically by expression level and, to a lesser extent, by dataset. Most importantly, examination of three different cell types revealed that noise levels differed across tissues.
Microarray measurements generally correlate with relative RNA molecule counts within optimal ranges but suffer from expression-dependent accuracy bias and precision that varies across datasets. We urge microarray users to consider expression-level effects in signal interpretation and to evaluate noise properties in each dataset independently.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-649) contains supplementary material, which is available to authorized users.
PMCID: PMC4143561  PMID: 25091430
Microarray; NanoString; nCounter; Gene expression
15.  Protective Antigen-Specific Memory B Cells Persist Years after Anthrax Vaccination and Correlate with Humoral Immunity 
Toxins  2014;6(8):2424-2431.
Anthrax Vaccine Adsorbed (AVA) generates short-lived protective antigen (PA) specific IgG that correlates with in vitro toxin neutralization and protection from Bacillus anthracis challenge. Animal studies suggest that when PA-specific IgG has waned, survival after spore challenge correlates with an activation of PA-specific memory B cells. Here, we characterize the quantity and the longevity of AVA-induced memory B cell responses in humans. Peripheral blood mononuclear cells (PBMCs) from individuals vaccinated ≥3 times with AVA (n = 50) were collected early (3–6 months, n = 27) or late after their last vaccination (2–5 years, n = 23), pan-stimulated, and assayed by ELISPOT for total and PA-specific memory B cells differentiated into antibody secreting cells (ASCs). PA-specific ASC percentages ranged from 0.02% to 6.25% (median: 1.57%) and did not differ between early and late post-vaccination individuals. PA-specific ASC percentages correlated with plasma PA-specific IgG (r = 0.42, p = 0.03) and toxin neutralization (r = 0.52, p = 0.003) early post vaccination. PA-specific ASC percentages correlated with supernatant anti-PA both early (r = 0.60, p = 0.001) and late post vaccination (r = 0.71, p < 0.0001). These data suggest PA-specific memory B cell responses are long-lived and can be estimated after recent vaccination by the magnitude and neutralization capacity of the humoral response.
PMCID: PMC4147590  PMID: 25123559
Anthrax Vaccine Adsorbed; cellular immunity; lethal toxin neutralization; protective antigen
16.  The immunopathology of ANCA-associated vasculitis 
Seminars in Immunopathology  2014;36(4):461-478.
The small-vessel vasculitides are a group of disorders characterised by variable patterns of small blood vessel inflammation producing a markedly heterogeneous clinical phenotype. While any vessel in any organ may be involved, distinct but often overlapping sets of clinical features have allowed the description of three subtypes associated with the presence of circulating anti-neutrophil cytoplasmic antibodies (ANCA), namely granulomatosis with polyangiitis (GPA, formerly known as Wegener’s Granulomatosis), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (eGPA, formerly known as Churg-Strauss syndrome). Together, these conditions are called the ANCA-associated vasculitidies (AAV). Both formal nomenclature and classification criteria for the syndromes have changed repeatedly since their description over 100 years ago and may conceivably do so again following recent reports showing distinct genetic associations of patients with detectable ANCA of distinct specificities. ANCA are not only useful in classifying the syndromes but substantial evidence implicates them in driving disease pathogenesis although the mechanism by which they develop and tolerance is broken remains controversial. Advances in our understanding of the pathogenesis of the syndromes have been accompanied by some progress in treatment, although much remains to be done to improve the chronic morbidity associated with the immunosuppression required for disease control.
PMCID: PMC4118034  PMID: 25056155
ANCA; Vasculitis; Anti-neutrophil cytoplasmic antibody; PR3; MPO
17.  Cost-effectiveness of programs to eliminate disparities in elderly vaccination rates in the United States 
BMC Public Health  2014;14:718.
There are disparities in influenza and pneumococcal vaccination rates among elderly minority groups and little guidance as to which intervention or combination of interventions to eliminate these disparities is likely to be most cost-effective. Here, we evaluate the cost-effectiveness of four hypothetical vaccination programs designed to eliminate disparities in elderly vaccination rates and differing in the number of interventions.
We developed a Markov model in which we assumed a healthcare system perspective, 10-year vaccination program and lifetime time horizon. The cohort was the combined African-American and Hispanic 65 year-old birth cohort in the United States in 2009. We evaluated five different vaccination strategies: no vaccination program and four vaccination programs that varied from “low intensity” to “very high intensity” based on the number of interventions deployed in each program, their cumulative cost and their cumulative impact on elderly minority influenza and pneumococcal vaccination rates.
The very high intensity vaccination program ($24,479/quality-adjusted life year; QALY) was preferred at willingness-to-pay-thresholds of $50,000 and $100,000/QALY and prevented 37,178 influenza cases, 342 influenza deaths, 1,158 invasive pneumococcal disease (IPD) cases and 174 IPD deaths over the birth cohort’s lifetime. In one-way sensitivity analyses, the very high intensity program only became cost-prohibitive (>$100,000/QALY) at less likely values for the influenza vaccination rates achieved in year 10 of the high intensity (>73.5%) or very high intensity (<76.8%) vaccination programs.
A practice-based vaccination program designed to eliminate disparities in elderly minority vaccination rates and including four interventions would be cost-effective.
PMCID: PMC4223514  PMID: 25023889
Vaccination; Elderly; Disparities; Cost-effectiveness
18.  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
19.  Prognosis in autoimmune and infectious disease: new insights from genetics 
A well-recognised feature of autoimmune and infectious diseases is that their clinical course and eventual outcome can vary substantially between affected individuals. This variability in disease prognosis critically determines patient well-being, and yet is relatively poorly understood and largely understudied—with many investigators opting instead to study what causes disease development in the first place. Better understanding of what determines prognosis could provide unique insights into disease biology, potentially revealing new therapeutic targets, and will also be essential if prognosis-based ‘personalised medicine' is ever to become a reality. Here, we highlight the previously under-appreciated role that genetics has in determining prognosis in autoimmune and infectious disease, and the common role that FOXO3 has been shown to have as a modulator of inflammatory responses, and thereby of outcome, across several distinct diseases.
PMCID: PMC4232069  PMID: 25505963
autoimmunity; FOXO3; genetics; infection; prognosis
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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