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1.  17β-estradiol attenuates conduit pulmonary artery mechanical property changes with pulmonary arterial hypertension 
Hypertension  2015;66(5):1082-1088.
Pulmonary arterial hypertension (PAH), a rapidly fatal vascular disease, strikes women more often than men. Paradoxically, female PAH patients have better prognosis and survival rates than males. The female sex hormone 17β-estradiol has been linked to the better outcome of PAH in females; however, the mechanisms by which 17β-estradiol alters PAH progression and outcomes remain unclear. Since proximal PA stiffness, one hallmark of PAH, is a powerful predictor of mortality and morbidity, we hypothesized that 17β-estradiol attenuates PAH-induced changes in mechanical properties in conduit proximal PAs, which imparts hemodynamic and energetic benefits to RV function. To test this hypothesis, female mice were ovariectomized and treated with 17β-estradiol or placebo. PAH was induced in mice using SU5416 and chronic hypoxia (SuHx). Extra-lobar left PAs were isolated and mechanically tested ex vivo to study both static and frequency-dependent mechanical behaviors in the presence or absence of SMC activation. Our static mechanical test showed significant stiffening of large PAs with PAH (P < 0.05). 17β-estradiol restored PA compliance to control levels. The dynamic mechanical test demonstrated that 17β-estradiol protected the arterial wall from the PAH-induced frequency-dependent decline in dynamic stiffness and loss of viscosity with PAH (P<0.05). As demonstrated by the in vivo measurement of PA hemodynamics via RV catheterization, modulation by 17β-estradiol of mechanical proximal PAs reduced pulsatile loading, which contributed to improved ventricular-vascular coupling. This study provides a mechanical mechanism for delayed disease progression and better outcome in female PAH patients and underscores the therapeutic potential of 17β-estradiol in PAH.
doi:10.1161/HYPERTENSIONAHA.115.05843
PMCID: PMC4600044  PMID: 26418020
Pulmonary arterial hypertension; 17β-estradiol; sex difference; arterial stiffness; viscoelasticity
2.  Proton density fat-fraction is an accurate biomarker of hepatic steatosis in adolescent girls and young women 
European radiology  2015;25(10):2921-2930.
Objectives
To compare complex quantitative magnetic resonance imaging (MRI) with MR spectroscopy (MRS) for quantification of hepatic steatosis (HS) and determine clinically significant MRI-based thresholds of HS in female youths.
Methods
This prospective, cross-sectional study was conducted in 132 healthy females (11–22 years, mean 13.3 ± 2). Proton density fat-fraction (PDFF) was measured using complex quantitative MRI and MRS. Body mass index (BMI), fasting labs [glucose, insulin, alanine aminotransferase (ALT), and other metabolic markers] were obtained. Outcomes were measured using regression analysis, Spearman-rank correlation, and receiver operator characteristics (ROC) analysis. HS was defined as MRI-PDFF >5.6 %.
Results
HS was detected by MRI-PDFF in 15 % of all subjects. Linear regression demonstrated excellent correlation and agreement [r2 = 0.96, slope = 0.97 (95 %CI: 0.94–1.00), intercept = 0.78 % (95 %CI: 0.58–0.98 %)] between MRI-PDFF and MRS-PDFF. MRI-PDFF had a sensitivity of 100 % (95 %CI: 0.79–1.00), specificity of 96.6 % (95 %CI: 0.91–0.99), and a kappa index of 87 % (95 %CI: 0.75-0.99) for identifying HS. In overweight subjects with HS, MRI-PDFF correlated with ALT (r = 0.84, p < 0.0001) and insulin (r = 0.833, p < 0.001), but not with BMI or WC. ROC analysis ascertained an optimal MRI-PDFF threshold of 3.5 % for predicting metabolic syndrome (sensitivity = 76 %, specificity = 83 %).
Conclusion
Complex quantitative MRI demonstrates strong correlation and agreement with MRS to quantify hepatic triglyceride content in adolescent girls and young women. A low PDFF threshold is predictive of metabolic syndrome in this population.
doi:10.1007/s00330-015-3724-1
PMCID: PMC4564339  PMID: 25916386
Magnetic resonance imaging; Magnetic resonance spectroscopy; Pediatrics; Obesity; Fatty liver
3.  In non-obese girls waist circumference predicts insulin resistance is comparably to MRI fat measures and superior to BMI 
Hormone research in paediatrics  2015;84(4):258-265.
Objective
To investigate the degree to which waist circumference (WC), BMI, and MRI measured abdominal fat deposition predict insulin resistance (IR) in non-obese girls of diverse racial and ethnic backgrounds.
Methods
Fifty-seven non-obese girls (12 African-American, 16 Hispanic White and 29 non-Hispanic White girls), aged 11–14 years old were assessed for WC, MRI hepatic proton density fat fraction, visceral and subcutaneous adipose tissue volume, BMI Z-score, fasting insulin, HOMA-IR, adiponectin, leptin, sex hormone binding globulin, HDL cholesterol, and triglycerides.
Results
Univariate and multivariate analyses adjusted for race and ethnicity indicated that only WC and visceral adipose tissue volume were independent predictors of fasting insulin and HOMA-IR, while dependent predictors were hepatic proton density fat fraction, BMI Z-score, and subcutaneous adipose tissue volume. Hispanic White girls showed significantly higher mean fasting insulin, HOMA-IR, and lower sex hormone binding globulin than non-Hispanic White girls (p-value <0.01).
Conclusions
In non-obese girls of diverse racial and ethnic backgrounds, WC, particularly when adjusted for race or ethnicity, is an independent predictor of IR comparable to MRI-derived measurements of fat and superior to BMI Z-score.
doi:10.1159/000439130
PMCID: PMC4644098  PMID: 26352642
Ethnicity; Race; Non-obese; HOMA-IR; Waist Circumference; MRI
4.  Pulmonary Gas Exchange and Exercise Capacity in Adults Born Preterm 
Rationale: Preterm birth, and its often-required medical interventions, can result in respiratory and gas exchange deficits into childhood. However, the long-term sequelae into adulthood are not well understood.
Objectives: To determine exercise capacity and pulmonary gas exchange efficiency during exercise in adult survivors of preterm birth.
Methods: Preterm (n = 14), very low birth weight (<1,500 g) adults (20–23 yr) and term-born, age-matched control subjects (n = 16) performed incremental exercise on a cycle ergometer to volitional exhaustion while breathing one of two oxygen concentrations: normoxia (fraction of inspired oxygen, 0.21) or hypoxia (fraction of inspired oxygen, 0.12).
Measurements and Main Results: Ventilation, mixed expired gases, arterial blood gases, power output, and oxygen consumption were measured during rest and exercise. We calculated the alveolar-to-arterial oxygen difference to determine pulmonary gas exchange efficiency. Preterm subjects had lower power output at volitional exhaustion than did control subjects in normoxia (150 ± 10 vs. 180 ± 10 W; P = 0.01), despite similar normoxic oxygen consumption. However, during hypoxic exercise, there was no difference in power output at volitional exhaustion between the two groups (116 ± 10 vs. 135 ± 10 W; P = 0.11). Preterm subjects also exhibited a more acidotic, acid–base balance throughout exercise compared with control subjects. In contrast to other studies, adults born preterm, as a group developed a wider alveolar-to-arterial oxygen difference and lower PaO2 than did control subjects during normoxic but not hypoxic exercise.
Conclusions: This study demonstrates that pulmonary gas exchange efficiency is lower in some adult survivors of preterm birth during exercise compared with control subjects. The gas exchange inefficiency, when present, is accompanied by low arterial blood oxygen tension. Preterm subjects also exhibit reduced power output. Overall, our findings suggest potential long-term consequences of extreme preterm birth and very low birth weight on cardiopulmonary function.
doi:10.1513/AnnalsATS.201410-470OC
PMCID: PMC4566409  PMID: 26053283
prematurity; pulmonary gas exchange; alveolar–arterial oxygen difference
5.  Expression of Spermidine/Spermine N1-Acetyl Transferase (SSAT) in Human Prostate Tissues is Related to Prostate Cancer Progression and Metastasis 
The Prostate  2015;75(11):1150-1159.
Introduction
PCa in many patients remains indolent for the rest of their lives, but in some patients it progresses to lethal metastatic disease. Gleason Score (GS) is the current clinical method for PCa prognosis. It cannot reliably identify aggressive PCa, when GS is ≤7. It is shown that oxidative stress plays a key role in PCa progression. We have shown that in cultured human PCa cells, an activation of Spermidine/Spermine N1-acetyl transferase (EC 2.3.1.57) enzyme initiates a polyamine oxidation pathway and generates copious amounts of reactive oxygen species (ROS) in polyamine-rich PCa cells.
Method
We used RNA in situ hybridization (RNA-ISH) and immunohistochemistry (IHC) methods to detect SSAT mRNA and protein expression in two tissue microarrays (TMA) created from patient prostate tissues. We analyzed 423 patient prostate tissues in the two TMAs.
Results
Our data show that there is a significant increase in both SSAT mRNA and the enzyme protein in the PCa cells as compared to their benign counterpart. This increase is even more pronounced in metastatic PCa tissues as compared to the PCa localized in the prostate. In the prostatectomy tissues from early-stage patients, the SSAT protein level is also high in the tissues obtained from the patients who ultimately progress to advanced metastatic disease.
Discussion
Based on these results combined with published data from our and other laboratories we propose an activation of an autocrine feed-forward loop of PCa cell proliferation in the absence of androgen as a possible mechanism of castrate-resistant prostate cancer (CRPCa) growth.
doi:10.1002/pros.22996
PMCID: PMC4475436  PMID: 25893668
6.  Pharmacodynamic study of axitinib in patients with advanced malignancies assessed with 18F-3′deoxy-3′fluoro-l-thymidine positron emission tomography/computed tomography 
Purpose
Rapid disease progression associated with increased tumor proliferation has been observed during withdrawal of anti-angiogenic therapy. We characterize the dynamics of withdrawal flare for axitinib.
Methods
Thirty patients with metastatic solid malignancies received axitinib for 2 weeks, followed by a 1-week drug holiday. Twenty patients suitable for PET imaging received scans with 18F-3′deoxy-3′fluoro-l-thymidine (FLT), a marker of proliferation. Plasma VEGF and axitinib pharmacokinetic levels were also assessed at specified time points.
Results
During axitinib withdrawal, significant increases in both SUVmax (+22 %; p = 0.006) and SUVmean (+20 %; p = 0.001) were observed. Significant increases relative to peak axitinib concentration were observed at day 2 withdrawal for SUVmax and SUVmean, with no further significant increase from day 2 to day 7 of withdrawal. No significant change in SUVmax or SUVmean was observed during the treatment period, relative to baseline. VEGF concentration significantly increased when on drug (p < 0.001) and decreased back to a level indistinguishable from baseline by day 7 of drug washout (p = 0.448). No correlation between change in VEGF and change in imaging metrics was observed.
Conclusions
A significant increase in tumor proliferation was observed during withdrawal of axitinib therapy, and this flare occurred within 2 days of axitinib withdrawal. An exploratory analysis indicated that this flare may be associated with poor clinical outcome.
doi:10.1007/s00280-015-2779-7
PMCID: PMC4499265  PMID: 26021741
Axitinib; Pharmacodynamic; FLT PET/CT; Angiogenesis inhibitors; Tyrosine kinase inhibitors
7.  Refinement and Validation of the Student Perceptions of Physician-Pharmacist Interprofessional Clinical Education Instrument 
Objective. To refine the Student Perceptions of Physician-Pharmacist Interprofessional Clinical Education (SPICE) instrument to address deficiencies observed in previous studies and to demonstrate external validity and reliability of the refined instrument in a broad population of medical and pharmacy students.
Methods. The original SPICE instrument plus four pilot items generated via cognitive interviewing of students was administered to 1708 medical and pharmacy students at five academic institutions. Exploratory factor analysis was used to identify candidate model structures and evaluate their psychometric properties.
Results. An improved version of the instrument was created (SPICE 2) by incorporating three pilot items and removing three original items. Validity and reliability were demonstrated.
Conclusion. The SPICE 2 instrument addresses the limitations observed to date in model structure while increasing its utility. The authors recommend use of the SPICE 2 instrument moving forward.
doi:10.5688/ajpe80347
PMCID: PMC4857642  PMID: 27170818
interprofessional education; assessment; instrument validation; exploratory factor analysis
8.  The Effectiveness of a Bundled Intervention to Improve Resident Progress Notes in an Electronic Health Record (EHR) 
Journal of hospital medicine  2014;10(2):104-107.
Providers nationally have observed a decline in the quality of documentation after implementing EHRs. In this pilot study, we examined the effectiveness of an intervention bundle designed to improve resident progress notes written in an EHR and to establish the reliability of an audit tool used to evaluate notes. The bundle consisted of establishing note-writing guidelines, developing an aligned note template, and educating interns about the guidelines and using the template. Twenty-five progress notes written by pediatric interns before and after this intervention were examined using an audit tool. Reliability of the tool was evaluated using the intra-class correlation coefficient (ICC). The total score of the audit tool was summarized in terms of means and standard deviation. Individual item responses were summarized using percentages and compared between the pre- and post-intervention assessment using Fisher’s exact test. The ICC for the audit tool was 0.96 (95% CI: 0.91–0.98). A significant improvement in the total note score and in questions related to note clutter was seen. No significant improvement was seen for questions related to copy-paste. The study suggests that an intervention bundle can lead to some improvements in note writing.
doi:10.1002/jhm.2283
PMCID: PMC4498456  PMID: 25425386
9.  CN-05DO PEDIATRIC BRAIN TUMOR SURVIVORS SUFFER AN INCREASED RISK OF OVARIAN/ENDOCRINE DYSFUNCTION AS COMPARED TO NON-BRAIN TUMOR SURVIVORS? 
Neuro-Oncology  2014;16(Suppl 5):v47.
OBJECTIVE: Various treatments of brain tumors may affect ovarian and endocrine function, resulting in a plethora of disorders. The purpose of this study was to analyze the impact of brain tumors on ovarian health and endocrine function in female childhood survivors. STUDY DESIGN: Ovarian and endocrine function in 222 childhood cancer survivors diagnosed with brain tumors at a tertiary comprehensive cancer center between 1997-2008 was retrospectively analyzed and compared. RESULTS: An equal proportion of childhood survivors of brain tumors (N = 39) and survivors of non-brain tumors (N = 183) developed ovarian dysfunction. Of those children older than 13 years, menstrual cycle irregularity (36% of brain tumor survivors vs. 21% of non-brain survivors, p = 0.3), amenorrhea (22% vs. 22% respectively, p = 0.9), and elevated FSH levels (40% vs. 50%, p = 0.9) were not statistically significant between the two groups. Brain tumor survivors experienced increased risk of endocrine disorders, such as precocious puberty (13% vs. 2%, P = 0.005), delayed puberty (8% vs. 1%, P = 0.0178), and hypopituitarism (10% vs. 1%, P = 0.01). Furthermore, children with brain tumors were significantly more likely to develop multiple endocrine disorders at the same time (31% vs. 7%, P = 0.001 for two endocrine disorders and 23% vs. 2%, P= 0.0001 for three or more endocrine disorders) and were more likely to visit with an endocrinologist (46% vs. 14%, p = 0.0001), when compared to non-brain tumor cancer survivors. While all brain tumor patients were seen at the multi-specialty neuroncology clinic, they were less likely to attend survivor clinic (4% vs. 28%, p = <0.003), and more likely to receive assistance from survivor clinic staff (97% vs. 57%, p = 0.001), when compared to non-brain tumor cancer survivors. CONCLUSIONS: Female pediatric brain tumor survivors are equally likely to experience ovarian insufficiency when compared to non-brain tumor survivors, but were significantly more likely to suffer coexistence of multiple endocrine problems.
doi:10.1093/neuonc/nou243.5
PMCID: PMC4218019
10.  Tumor-selective anti-cancer effects of the synthetic alkyl phosphocholine analog CLR1404 in neuroblastoma 
American Journal of Cancer Research  2015;5(11):3422-3435.
Neuroblastoma (NB) is the most common extracranial solid tumor in children and is associated with high mortality in advanced stages. Survivors suffer from long-term treatment-related sequelae. Thus, new targeted treatment options are urgently needed. 18-(p-[127I] iodophenyl) octadecyl phosphocholine (CLR1404) is a novel, broadly tumor targeted small molecule drug suitable for intravenous injection with highly selective tumor uptake. As a carrier molecule for radioactive iodine, CLR1404 is in clinical trials as cancer imaging agent and radiotherapeutic drug. Chemically, CLR1404 belongs to the anti-tumor alkyl phospholipids, a class of drugs known to have intrinsic cytotoxic effects on cancer cells. Therefore, we hypothesized that CLR1404 could be a tumor-targeted anti-cancer agent for neuroblastoma, and investigated its effect in vitro and in vivo. CLR1404 was taken up by NB cells in a highly tumor-selective manner both in vitro and in vivo, confirmed by flow cytometry and PET/CT imaging of mouse flank xenografts with 124I-CLR1404, respectively. Using flow cytometry, MTT assay, Western blotting and caspase 3/7 assay, we confirm that in vitro treatment with CLR1404 leads to robust apoptosis and cell death in multiple NB cell lines and is associated with Akt inhibition, while sparing normal cells. Treatment with CLR1404 in doses of 10 or 30 mg/kg administered by intravenous injection once weekly for 7 weeks significantly inhibited the tumor growth rate in a mouse flank xenograft model of NB (P<0.001) when compared to control cohorts, without causing drug-related hematotoxicity or other noticeable adverse effects, which was determined by serial tumor volume measurements, complete blood counts, and monitoring of animal-specific health parameters. We conclude that CLR1404 warrants clinical exploration as a novel, tumor selective anticancer agent in NB and potentially other cancers.
PMCID: PMC4697688  PMID: 26807322
Neuroblastoma; CLR1404; alkyl phospholipid; alkyl phosphocholine analog; targeted therapy; Akt; pediatric cancer
11.  A Bayesian adaptive design with biomarkers for targeted therapies 
Background
Targeted therapies are becoming increasingly important for the treatment of various diseases. Biomarkers are a critical component of a targeted therapy as they can be used to identify patients who are more likely to benefit from a treatment. Targeted therapies, however, have created major challenges in the design, conduct, and analysis of clinical trials. In traditional clinical trials, treatment effects for various biomarkers are typically evaluated in an exploratory fashion and only limited information about the predictive values of biomarkers obtained.
Purpose
New study designs are required, which effectively evaluate both the diagnostic and the therapeutic implication of biomarkers.
Methods
The Bayesian approach provides a useful framework for optimizing the clinical trial design by directly integrating information about biomarkers and clinical outcomes as they become available. We propose a Bayesian covariate-adjusted response-adaptive randomization design, which utilizes individual biomarker profiles and patient's clinical outcomes as they become available during the course of the trial, to assign the most efficacious treatment to individual patients. Predictive biomarker subgroups are determined adaptively using a partial least squares regression approach.
Results
A series of simulation studies were conducted to examine the operating characteristics of the proposed study design. The simulation studies show that the proposed design efficiently identifies patients who benefit most from a targeted therapy and that there are substantial savings in the sample size requirements when compared to alternative designs.
Limitations
The design does not control for the type I error in the traditional sense and a positive result should be confirmed by conducting an independent phase III study focusing on the selected biomarker profile groups.
Conclusions
We conclude that the proposed design may serve a useful role in the early efficacy phase of targeted therapy development.
doi:10.1177/1740774510372657
PMCID: PMC3788617  PMID: 20571131
12.  CSF and Blood Levels of GFAP in Alexander Disease 
eNeuro  2015;2(5):ENEURO.0080-15.2015.
Abstract
Alexander disease is a rare, progressive, and generally fatal neurological disorder that results from dominant mutations affecting the coding region of GFAP, the gene encoding glial fibrillary acidic protein, the major intermediate filament protein of astrocytes in the CNS. A key step in pathogenesis appears to be the accumulation of GFAP within astrocytes to excessive levels. Studies using mouse models indicate that the severity of the phenotype correlates with the level of expression, and suppression of GFAP expression and/or accumulation is one strategy that is being pursued as a potential treatment. With the goal of identifying biomarkers that indirectly reflect the levels of GFAP in brain parenchyma, we have assayed GFAP levels in two body fluids in humans that are readily accessible as biopsy sites: CSF and blood. We find that GFAP levels are consistently elevated in the CSF of patients with Alexander disease, but only occasionally and modestly elevated in blood. These results provide the foundation for future studies that will explore whether GFAP levels can serve as a convenient means to monitor the progression of disease and the response to treatment.
doi:10.1523/ENEURO.0080-15.2015
PMCID: PMC4603256  PMID: 26478912
astrocyte; biomarker; GFAP
13.  Predicting hepatic steatosis in a racially and ethnically diverse cohort of adolescent girls 
The Journal of pediatrics  2014;165(2):319-325.e1.
Objectives
To develop a risk assessment model for early detection of hepatic steatosis using common anthropometric and metabolic markers.
Study design
Cross-sectional study of 134 girls, age 11–22 years (mean 13.3±2), Ethnicity: 27% Hispanic, 73% Non-Hispanic; Race: 64% Caucasian, 31% African-American, 5% Asian, from a middle school and clinics (Madison, WI). Fasting glucose, fasting insulin, alanine aminotransferase (ALT), body mass index (BMI), waist circumference (WC) and other metabolic markers were assessed. Hepatic fat was quantified using magnetic resonance proton density fat fraction (MR-PDFF). Hepatic steatosis was defined as MR-PDFF >5.5%. Outcome measures were sensitivity, specificity, and positive predictive value (PPV) of BMI, WC, ALT, fasting insulin and ethnicity as predictors of hepatic steatosis, individually and combined, in a risk assessment model. Classification and regression tree methodology constructed a decision tree for predicting hepatic steatosis.
Results
MR-PDFF revealed hepatic steatosis in 16% of subjects (27% overweight, 3% non-overweight). Hispanic ethnicity conferred an odds ratio of 4.26 (CI 1.65–11.04, p=0.003) for hepatic steatosis. BMI and ALT did not independently predict hepatic steatosis. A BMI > 85% combined with ALT > 65 U/L had 9% sensitivity, 100% specificity and 100% PPV. Lowering ALT to 24 U/L increased sensitivity to 68%, but reduced PPV to 47%. A risk assessment model incorporating fasting insulin, total cholesterol, WC, and ethnicity increased sensitivity to 64%, specificity to 99% and PPV to 93%.
Conclusions
A risk assessment model can increase specificity, sensitivity, and PPV for identifying risk of hepatic steatosis and guide efficient use of biopsy or imaging for early detection and intervention.
doi:10.1016/j.jpeds.2014.04.019
PMCID: PMC4131842  PMID: 24857521
Hepatic Steatosis; Nonalcoholic hepatic steatosis; Metabolic Syndrome; Child and Adolescent
14.  Real-Time Immune Monitoring to Guide Plasmid DNA Vaccination Schedule Targeting Prostatic Acid Phosphatase (PAP) in Patients with Castration-Resistant Prostate Cancer 
BACKGROUND
We have previously reported that a DNA vaccine encoding prostatic acid phosphatase (PAP) could elicit PAP-specific T cells in patients with early recurrent prostate cancer. In the current pilot trial we sought to evaluate whether prolonged immunization with regular booster immunizations, or “personalized” schedules of immunization determined using real-time immune monitoring, could elicit persistent, antigen-specific T cells, and whether treatment was associated with changes in PSA doubling time (PSA DT).
METHODS
16 patients with castration-resistant, non-metastatic prostate cancer received six immunizations at two-week intervals, and then either quarterly (Arm 1) or as determined by multi-parameter immune monitoring (Arm 2).
RESULTS
Patients were on study a median of 16 months; four received 24 vaccinations. Only one event associated with treatment > grade 2 was observed. 6/16 (38%) remained metastasis-free at 2 years. PAP-specific T cells were elicited in 12/16 (75%), predominantly of a Th1 phenotype, which persisted in frequency and phenotype for at least one year. IFNγ-secreting T-cell responses measured by ELISPOT were detectable in 5/13 individuals at one year, and this was not statistically different between study arms. The overall median fold change in PSA DT from pre-treatment to post-treatment was 1.6 (range 0.6–7.0, p=0.036).
CONCLUSIONS
Repetitive immunization with a plasmid DNA vaccine was safe and elicited Th1-biased antigen-specific T cells that persisted over time. Modifications in the immunization schedule based on real-time immune monitoring did not increase the frequency of patients developing effector and memory T-cell responses with this DNA vaccine.
doi:10.1158/1078-0432.CCR-14-0169
PMCID: PMC4102643  PMID: 24850844
DNA vaccine; prostate cancer; prostatic acid phosphatase; clinical trial; immune monitoring
15.  Distinct Innate Immune Phagocyte Responses to Aspergillus fumigatus Conidia and Hyphae in Zebrafish Larvae 
Eukaryotic Cell  2014;13(10):1266-1277.
Aspergillus fumigatus is the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense against A. fumigatus; however, innate phagocyte responses to A. fumigatus in an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafish A. fumigatus infection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection with A. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain of A. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses to A. fumigatus that reveals distinct roles for neutrophils and macrophages in mediating host defense against IA.
doi:10.1128/EC.00080-14
PMCID: PMC4187654  PMID: 24879123
16.  Safety and Immunological Efficacy of a DNA Vaccine Encoding Prostatic Acid Phosphatase in Patients With Stage D0 Prostate Cancer 
Journal of Clinical Oncology  2009;27(25):4047-4054.
Purpose
Prostatic acid phosphatase (PAP) is a prostate tumor antigen. We have previously demonstrated that a DNA vaccine encoding PAP can elicit antigen-specific CD8+ T cells in rodents. We report here the results of a phase I/IIa trial conducted with a DNA vaccine encoding human PAP in patients with stage D0 prostate cancer.
Patients and Methods
Twenty-two patients were treated in a dose-escalation trial with 100 μg, 500 μg, or 1,500 μg plasmid DNA, coadministered intradermally with 200 μg granulocyte-macrophage colony-stimulating factor as a vaccine adjuvant, six times at 14-day intervals. All patients were observed for 1 year after treatment.
Results
No significant adverse events were observed. Three (14%) of 22 patients developed PAP-specific IFNγ-secreting CD8+ T-cells immediately after the treatment course, as determined by enzyme-linked immunospot. Nine (41%) of 22 patients developed PAP-specific CD4+ and/or CD8+ T-cell proliferation. Antibody responses to PAP were not detected. Overall, the prostate-specific antigen (PSA) doubling time was observed to increase from a median 6.5 months pretreatment to 8.5 months on-treatment (P = .033), and 9.3 months in the 1-year post-treatment period (P = .054).
Conclusion
The demonstration that a DNA vaccine encoding PAP is safe, elicits an antigen-specific T-cell response, and may be associated with an increased PSA doubling time suggests that a multi-institutional phase II trial designed to evaluate clinical efficacy is warranted.
doi:10.1200/JCO.2008.19.9968
PMCID: PMC2734418  PMID: 19636017
17.  A Randomized Phase II Trial Evaluating Different Schedules of Zoledronic Acid on Bone Mineral Density in Patients With Prostate Cancer Beginning Androgen Deprivation Therapy 
Clinical genitourinary cancer  2013;11(4):10.1016/j.clgc.2013.04.029.
Although androgen deprivation therapy for prostate cancer is associated with an increased risk of osteoporosis, the optimal timing and schedule of zoledronic acid has not been identified. This phase II trial randomized 44 men beginning androgen deprivation therapy to 3 schedules of zoledronic acid administration. Earlier or more frequent administration of zoledronic acid was found to stabilize and improve bone mineral density in men treated with androgen deprivation therapy.
Objective
To assess the effects of timing and schedule of zoledronic acid (ZA) administration on bone mineral density (BMD) in patients beginning androgen deprivation therapy (ADT) for the treatment of recurrent prostate cancer.
Patients and Methods
In this randomized, 3-arm trial, we evaluated changes in BMD after 3 different ZA administration schedules in men with recurrent prostate cancer who were beginning ADT. Forty-four patients were enrolled and randomized to receive a single dose of ZA given 1 week before beginning ADT (arm 1), a single dose of ZA given 6 months after beginning ADT (arm 2), or monthly administration of ZA starting 6 months after beginning ADT, for a total of 6 doses (arm 3).
Results
Patients who received ZA before ADT had a significant improvement in BMD at the total proximal femur and trochanter after 6 months compared with the other groups. In addition, only patients in the arm that received multiple doses improved lumbar spine BMD while on ADT, with these findings persisting to 24 months. However, this group also experienced more grade 1 adverse events.
Conclusions
Analysis of these data suggests that ZA administration before initiation of ADT was superior to treatment 6 months after starting ADT in maintaining BMD. In addition, monthly ZA administration can increase BMD above baseline but is associated with more adverse events. Further study is needed to examine whether the timing and frequency of ZA therapy in patients on ADT can reduce fracture risk.
doi:10.1016/j.clgc.2013.04.029
PMCID: PMC3836858  PMID: 23835291
Androgen deprivation; Bone mineral density; Gamma delta T cells; Prostate cancer; Zoledronic acid
18.  Effects of collagen deposition on passive and active mechanical properties of large pulmonary arteries in hypoxic pulmonary hypertension 
Proximal pulmonary artery (PA) stiffening is a strong predictor of mortality in pulmonary hypertension. Collagen accumulation is mainly responsible for PA stiffening in hypoxia-induced pulmonary hypertension (HPH) in mouse models. We hypothesized that collagen cross-linking and the type I isoform are the main determinants of large PA mechanical changes during HPH, which we tested by exposing mice that resist type I collagen degradation (Col1a1R/R) and littermate controls (Col1a1+/+) to hypoxia for 10 days with or without β-aminopropionitrile (BAPN) treatment to prevent cross-link formation. Static and dynamic mechanical tests were performed on isolated PAs with smooth muscle cells (SMC) in passive and active states. Percentages of type I and III collagen were quantified by histology; total collagen content and cross-linking were measured biochemically. In the SMC passive state, for both genotypes, hypoxia tended to increase PA stiffness and damping capacity, and BAPN treatment limited these increases. These changes were correlated with collagen cross-linking (p<0.05). In the SMC active state, hypoxia increased PA dynamic stiffness and BAPN had no effect in Col1a1+/+ mice (p<0.05). PA stiffness did not change in Col1a1R/R mice. Similarly, damping capacity did not change for either genotype. Type I collagen accumulated more in Col1a1+/+ mice whereas type III collagen increased more in Col1a1R/R mice during HPH. In summary, PA passive mechanical properties (both static and dynamic) are related to collagen cross-linking. Type I collagen turnover is critical to large PA remodeling during HPH when collagen metabolism is not mutated and type III collagen may serve as a reserve.
doi:10.1007/s10237-012-0467-7
PMCID: PMC3745811  PMID: 23377784
cross-linking; type I and III collagen; extralobar pulmonary artery stiffening; pulmonary hypertension; viscoelasticity
19.  Multiphoton Microscopy of Endogenous Fluorescence Differentiates Normal, Precancerous, and Cancerous Squamous Epithelial Tissues 
Cancer research  2005;65(4):1180-1186.
This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multi-photon fluorescence endoscopy in a clinical setting.
doi:10.1158/0008-5472.CAN-04-3031
PMCID: PMC4189807  PMID: 15735001
20.  Using a Bayesian Latent Class Model to Evaluate the Utility of Investigating Persons with Negative Polymerase Chain Reaction Results for Pertussis 
American Journal of Epidemiology  2013;178(2):309-318.
Pertussis remains difficult to control. Imperfect sensitivity of diagnostic tests and lack of specific guidance regarding interpretation of negative test results among patients with compatible symptoms may contribute to its spread. In this study, we examined whether additional pertussis cases could be identified if persons with negative pertussis test results were routinely investigated. We conducted interviews among 250 subjects aged ≤18 years with pertussis polymerase chain reaction (PCR) results reported from 2 reference laboratories in Wisconsin during July–September 2010 to determine whether their illnesses met the Centers for Disease Control and Prevention's clinical case definition (CCD) for pertussis. PCR validity measures were calculated using the CCD as the standard for pertussis disease. Two Bayesian latent class models were used to adjust the validity measures for pertussis detectable by 1) culture alone and 2) culture and/or more sensitive measures such as serology. Among 190 PCR-negative subjects, 54 (28%) had illnesses meeting the CCD. In adjusted analyses, PCR sensitivity and the negative predictive value were 1) 94% and 99% and 2) 43% and 87% in the 2 types of models, respectively. The models suggested that public health follow-up of reported pertussis patients with PCR-negative results leads to the detection of more true pertussis cases than follow-up of PCR-positive persons alone. The results also suggest a need for a more specific pertussis CCD.
doi:10.1093/aje/kws465
PMCID: PMC3968301  PMID: 23735308
Bayesian analysis; Bordetella pertussis; pertussis; polymerase chain reaction; predictive value of tests; public health surveillance; sensitivity; whooping cough
21.  Ethnic Differences in the Effects of Hepatic Fat Deposition on Insulin Resistance in Non-Obese Middle School Girls 
Obesity (Silver Spring, Md.)  2013;22(1):243-248.
Objective
In non-obese youth, to investigate whether hepatic fat deposition and its metabolic consequences vary between ethnic groups.
Design Methods
Thirty-two non-obese girls (12 Hispanic White [H] and 20 non-Hispanic White [NHW] girls), aged 11–14 years old were recruited. Outcome measures were MRI measured hepatic proton density fat fraction (hepatic PDFF), BMI Z-score, waist circumference, fasting insulin, glucose, adiponectin, sex hormone binding globulin [SHBG], ALT, AST, and triglycerides, and HOMA-IR.
Results
There were no significant differences in mean BMI Z-scores (p=0.546) or hepatic PDFF (p=0.275) between H and NHW girls; however, H girls showed significant correlations between hepatic PDFF and markers of IR (fasting insulin, HOMA-IR, adiponectin, SHBG, triglycerides; all p<0.05), while NHW girls showed no significant correlations. Matched by hepatic PDFF or BMI-Z score, H girls had more evidence of IR for a given hepatic PDFF (mean insulin, HOMA-IR, and SHBG; all p<0.05) or BMI-Z score (mean insulin and HOMA-IR; all p<0.01) than NHW girls.
Conclusions
In non-obese female youth, ethnicity-related differences in effects of hepatic fat on IR are evident, so that in H girls, a given amount of hepatic fat appears to result in a more predictable and greater degree of IR than in NHW girls.
doi:10.1002/oby.20521
PMCID: PMC3946962  PMID: 23804504
Ethnicity; Non-obese; Insulin Resistance; Hepatic Steatosis
22.  Changes in Large Pulmonary Arterial Viscoelasticity in Chronic Pulmonary Hypertension 
PLoS ONE  2013;8(11):e78569.
Conduit pulmonary artery (PA) stiffening is characteristic of pulmonary arterial hypertension (PAH) and is an excellent predictor of mortality due to right ventricular (RV) overload. To better understand the impact of conduit PA stiffening on RV afterload, it is critical to examine the arterial viscoelastic properties, which require measurements of elasticity (energy storage behavior) and viscosity (energy dissipation behavior). Here we hypothesize that PAH leads to frequency-dependent changes in arterial stiffness (related to elasticity) and damping ratio (related to viscosity) in large PAs. To test our hypothesis, PAH was induced by the combination of chronic hypoxia and an antiangiogenic compound (SU5416) treatment in mice. Static and sinusoidal pressure-inflation tests were performed on isolated conduit PAs at various frequencies (0.01–20 Hz) to obtain the mechanical properties in the absence of smooth muscle contraction. Static mechanical tests showed significant stiffening of large PAs with PAH, as expected. In dynamic mechanical tests, structural stiffness (κ) increased and damping ratio (D) decreased at a physiologically relevant frequency (10 Hz) in hypertensive PAs. The dynamic elastic modulus (E), a material stiffness, did not increase significantly with PAH. All dynamic mechanical properties were strong functions of frequency. In particular, κ, E and D increased with increasing frequency in control PAs. While this behavior remained for D in hypertensive PAs, it reversed for κ and E. Since these novel dynamic mechanical property changes were found in the absence of changes in smooth muscle cell content or contraction, changes in collagen and proteoglycans and their interactions are likely critical to arterial viscoelasticity in a way that has not been previously described. The impact of these changes in PA viscoelasticity on RV afterload in PAH awaits further investigation.
doi:10.1371/journal.pone.0078569
PMCID: PMC3819365  PMID: 24223157
23.  Phase I trial of tremelimumab in combination with short-term androgen deprivation in patients with PSA-recurrent prostate cancer 
Cancer Immunology, Immunotherapy  2011;61(7):1137-1147.
CTLA-4 blockade has demonstrated antitumor efficacy in human clinical trials. The antitumor mechanism is presumably mediated in part by the expansion of tumor-specific T cells. Androgen deprivation, the cornerstone of treatment for patients with metastatic prostate cancer, has been shown to elicit prostate tissue apoptosis and lymphocytic inflammation. We hypothesized that treatment with androgen deprivation, followed by an anti-CTLA-4 antibody, could augment a tumor-specific immune response elicited by androgen deprivation. We report here the results of a phase I trial evaluating a humanized monoclonal antibody targeting CTLA-4, CP-675,206 (tremelimumab), in combination with androgen deprivation using an antiandrogen. Eligible patients were those with PSA-recurrent prostate cancer after primary surgery and/or radiation therapy, not previously treated with androgen deprivation, and without radiographic evidence of metastatic disease. Subjects were treated in two cycles, 3 months apart, in which they received bicalutamide 150 mg daily days 1–28 and tremelimumab on day 29. The primary endpoint of the trial was safety. Secondary endpoints included measures of PSA kinetics and identification of a maximum tolerated dose. Eleven patients were enrolled and completed at least 1 year of follow-up. Dose-limiting toxicities included grade 3 diarrhea and skin rash. No favorable changes in PSA doubling time were observed in a period shortly after completing treatment; however, three patients experienced a prolongation in PSA doubling time detectable several months after completing treatment. The identification of delayed, prolonged favorable changes in serum PSA suggests that future studies could explore this combination in studies evaluating time to disease progression.
doi:10.1007/s00262-011-1193-1
PMCID: PMC3349783  PMID: 22210552
Tremelimumab; Anti-CTLA-4 monoclonal antibody; Bicalutamide; Prostate cancer; Clinical trial
24.  Inpatient Hospitalization of Oncology Patients: Are We Missing an Opportunity for End-of-Life Care? 
Journal of Oncology Practice  2012;9(1):51-54.
The authors conclude that the inpatient oncology service predominantly cares for a population nearing the end of life; therefore, goals should include symptom control and end-of-life planning.
Introduction:
Despite advances in the care of patients with cancer over the last 10 years, cancer remains the second leading cause of death in the United States. Many patients receive aggressive, in-hospital end-of-life care at high cost. There are few data on outcomes after unplanned hospitalization of patients with metastatic cancer.
Methods:
In 2000 and 2010, data were collected on admissions, interventions, and survival for patients admitted to an academic inpatient medical oncology service.
Results:
The 2000 survey included 191 admissions of 151 unique patients. The 2010 survey assessed 149 admissions of 119 patients. Lung, GI, and breast cancers were the most common cancer diagnoses. In the 2010 assessment, pain was the most common chief complaint, accounting for 28%. Although symptoms were the dominant reason for admission in 2010, procedures and imaging were common in both surveys. The median survival of patients after discharge was 4.7 months in 2000 and 3.4 months in 2010. Despite poor survival in this patient population, hospice was recommended in only 23% and 24% of patients in 2000 and 2010, respectively. Seventy percent of patients were discharged home without additional services.
Conclusion:
On the basis of our data, an unscheduled hospitalization for a patient with advanced cancer strongly predicts a median survival of fewer than 6 months. We believe that hospital admission represents an opportunity to commence and/or consolidate appropriate palliative care services and end-of-life care.
doi:10.1200/JOP.2012.000698
PMCID: PMC3545663  PMID: 23633971
25.  Maintenance rituximab following induction chemo-immunotherapy for mantle cell lymphoma: long-term follow-up of a pilot study from the Wisconsin Oncology Network 
Leukemia & lymphoma  2011;52(9):1675-1680.
Mantle cell lymphoma (MCL) is challenging to manage, with a median survival of 3–5 years. While intensive strategies are often appropriate for younger patients, these approaches are often not appropriate for older patients. In 2006, we reported our initial results using modified R-hyperCVAD (rituximab with hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) with maintenance rituximab. The complete response rate was 64%, and median progression-free survival (PFS) 37 months. Herein, we update our results, now with a median follow-up of 62 months. The median PFS is unchanged and the median overall survival (OS) is 70 months. The proportion of patients surviving at 5 years is 62%, comparable to studies using intensive strategies in similar patient populations. No late toxicities were noted in our cohort. These long-term results suggest that the modified R-hyperCVAD regimen with maintenance rituximab is an excellent option for older patients with newly diagnosed mantle cell lymphoma.
doi:10.3109/10428194.2011.580404
PMCID: PMC3489168  PMID: 21864042
Mantle cell lymphoma; non-Hodgkin’s lymphoma; rituximab maintenance; modified R-hyperCVAD

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