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1.  Measuring the Physiologic Properties of Oral Lesions Receiving Fractionated Photodynamic Therapy 
Photochemistry and photobiology  2015;91(5):1210-1218.
Photodynamic therapy (PDT) can treat superficial, early-stage disease with minimal damage to underlying tissues and without cumulative dose-limiting toxicity. Treatment efficacy is affected by disease physiologic properties, but these properties are not routinely measured. We assessed diffuse reflectance spectroscopy (DRS) for the noninvasive, contact measurement of tissue hemoglobin oxygen saturation (StO2) and total hemoglobin concentration ([tHb]) in the pre-malignant or superficial microinvasive oral lesions of patients treated with 5-aminolevulinic acid (ALA)-PDT. Patients were enrolled on a Phase 1 study of ALA-PDT that evaluated fluences of 50, 100, 150 or 200 J/cm2 delivered at 100 mW/cm2. In order to test the feasibility of incorporating DRS measurements within the illumination period, studies were performed in patients who received fractionated (two-part) illumination that included a dark interval of 90–180 seconds. Using DRS, tissue oxygenation at different depths within the lesion could also be assessed. DRS could be performed concurrently with contact measurements of photosensitizer levels by fluorescence spectroscopy, but a separate noncontact fluorescence spectroscopy system provided continuous assessment of photobleaching during illumination to greater tissue depths. Results establish that the integration of DRS into PDT of early-stage oral disease is feasible, and motivates further studies to evaluate its predictive and dosimetric value.
Graphical Abstract
doi:10.1111/php.12475
PMCID: PMC4560604  PMID: 26037487
2.  Antibiotic Exposure and Juvenile Idiopathic Arthritis: A Case–Control Study 
Pediatrics  2015;136(2):e333-e343.
BACKGROUND AND OBJECTIVE:
Recent evidence has linked childhood antibiotic use and microbiome disturbance to autoimmune conditions. This study tested the hypothesis that antibiotic exposure was associated with newly diagnosed juvenile idiopathic arthritis (JIA).
METHODS:
We performed a nested case–control study in a population-representative medical records database from the United Kingdom. Children with newly diagnosed JIA were compared with age- and gender-matched control subjects randomly selected from general practices containing at least 1 case, excluding those with inflammatory bowel disease, immunodeficiency, or other systemic rheumatic diseases. Conditional logistic regression was used to examine the association between antibacterial antibiotics (including number of antibiotic courses and timing) and JIA after adjusting for significant confounders.
RESULTS:
Any antibiotic exposure was associated with an increased rate of developing JIA (adjusted odds ratio: 2.1 [95% confidence interval: 1.2–3.5]). This relationship was dose dependent (adjusted odds ratio over 5 antibiotic courses: 3.0 [95% confidence interval: 1.6–5.6]), strongest for exposures within 1 year of diagnosis, and did not substantively change when adjusting for number or type of infections. In contrast, nonbacterial antimicrobial agents (eg, antifungal, antiviral) were not associated with JIA. In addition, antibiotic-treated upper respiratory tract infections were more strongly associated with JIA than untreated upper respiratory tract infections.
CONCLUSIONS:
Antibiotics were associated with newly diagnosed JIA in a dose- and time-dependent fashion in a large pediatric population. Antibiotic exposure may play a role in JIA pathogenesis, perhaps mediated through alterations in the microbiome.
doi:10.1542/peds.2015-0036
PMCID: PMC4516942  PMID: 26195533
3.  Erlotinib pretreatment improves photodynamic therapy of non-small cell lung carcinoma xenografts via multiple mechanisms 
Cancer research  2015;75(15):3118-3126.
Aberrant expression of the epidermal growth factor receptor (EGFR) is a common characteristic of many cancers including non-small cell lung carcinoma (NSCLC), head and neck squamous cell carcinoma, and ovarian cancer. While EGFR is currently a favorite molecular target for treatment of these cancers, inhibition of the receptor with small molecule inhibitors (i.e.- erlotinib) or monoclonal antibodies (i.e.- cetuximab) does not provide long-term therapeutic benefit as standalone treatment. Interestingly, we have found that addition of erlotinib to photodynamic therapy (PDT) can improve treatment response in typically erlotinib-resistant NSCLC tumor xenografts. Ninety-day complete response rates of 63% are achieved when erlotinib is administered in three doses before PDT of H460 human tumor xenografts, compared to 16% after PDT-alone. Similar benefit is found when erlotinib is added to PDT of A549 NCSLC xenografts. Improved response is accompanied by increased vascular shutdown, and erlotinib increases the in vitro cytotoxicity of PDT to endothelial cells. Tumor uptake of the photosensitizer (benzoporphyrin derivative monoacid ring A; BPD) is increased by the in vivo administration of erlotinib; nevertheless, this elevation of BPD levels only partially accounts for the benefit of erlotinib to PDT. Thus, pretreatment with erlotinib augments multiple mechanisms of PDT effect that collectively lead to large improvements in therapeutic efficacy. These data demonstrate that short-duration administration of erlotinib before PDT can greatly improve the responsiveness of even erlotinib-resistant tumors to treatment. Results will inform clinical investigation of EGFR-targeting therapeutics in conjunction with PDT.
doi:10.1158/0008-5472.CAN-14-3304
PMCID: PMC4526424  PMID: 26054596
Erlotinib; photodynamic therapy; non-small cell lung cancer; EGFR; VEGF
4.  Comparison of the Lonidamine Potentiated Effect of Nitrogen Mustard Alkylating Agents on the Systemic Treatment of DB-1 Human Melanoma Xenografts in Mice 
PLoS ONE  2016;11(6):e0157125.
Previous NMR studies demonstrated that lonidamine (LND) selectively diminishes the intracellular pH (pHi) of DB-1 melanoma and mouse xenografts of a variety of other prevalent human cancers while decreasing their bioenergetic status (tumor βNTP/Pi ratio) and enhancing the activities of melphalan and doxorubicin in these cancer models. Since melphalan and doxorubicin are highly toxic agents, we have examined three other nitrogen (N)-mustards, chlorambucil, cyclophosphamide and bendamustine, to determine if they exhibit similar potentiation by LND. As single agents LND, melphalan and these N-mustards exhibited the following activities in DB-1 melanoma xenografts; LND: 100% tumor surviving fraction (SF); chlorambucil: 100% SF; cyclophosphamide: 100% SF; bendamustine: 79% SF; melphalan: 41% SF. When combined with LND administered 40 min prior to administration of the N-mustard (to maximize intracellular acidification) the following responses were obtained; chlorambucil: 62% SF; cyclophosphamide: 42% SF; bendamustine: 36% SF; melphalan: 10% SF. The effect of LND on the activities of these N-mustards is generally attributed to acid stabilization of the aziridinium active intermediate, acid inhibition of glutathione-S-transferase, which acts as a scavenger of aziridinium, and acid inhibition of DNA repair by O6-alkyltransferase. Depletion of ATP by LND may also decrease multidrug resistance and increase tumor response. At similar maximum tolerated doses, our data indicate that melphalan is the most effective N-mustard in combination with LND when treating DB-1 melanoma in mice, but the choice of N-mustard for coadministration with LND will also depend on the relative toxicities of these agents, and remains to be determined.
doi:10.1371/journal.pone.0157125
PMCID: PMC4902256  PMID: 27285585
5.  Fluence Rate Differences in Photodynamic Therapy Efficacy and Activation of Epidermal Growth Factor Receptor after Treatment of the Tumor-Involved Murine Thoracic Cavity 
Photodynamic therapy (PDT) of the thoracic cavity can be performed in conjunction with surgery to treat cancers of the lung and its pleura. However, illumination of the cavity results in tissue exposure to a broad range of fluence rates. In a murine model of intrathoracic PDT, we studied the efficacy of 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH; Photochlor®)-mediated PDT in reducing the burden of non-small cell lung cancer for treatments performed at different incident fluence rates (75 versus 150 mW/cm). To better understand a role for growth factor signaling in disease progression after intrathoracic PDT, the expression and activation of epidermal growth factor receptor (EGFR) was evaluated in areas of post-treatment proliferation. The low fluence rate of 75 mW/cm produced the largest reductions in tumor burden. Bioluminescent imaging and histological staining for cell proliferation (anti-Ki-67) identified areas of disease progression at both fluence rates after PDT. However, increased EGFR activation in proliferative areas was detected only after treatment at the higher fluence rate of 150 mW/cm. These data suggest that fluence rate may affect the activation of survival factors, such as EGFR, and weaker activation at lower fluence rate could contribute to a smaller tumor burden after PDT at 75 mW/cm.
doi:10.3390/ijms17010101
PMCID: PMC4730343  PMID: 26784170
photodynamic therapy; fluence rate; lung; HPPH; epidermal growth factor receptor; optical imaging; proliferation; thoracic cavity; non-small cell lung carcinoma
6.  Simultaneous Assessment of Unprocessed ProBNP1-108 in Addition to Processed BNP32 Improves Identification of High Risk Ambulatory Patients with Heart Failure 
Circulation. Heart failure  2010;3(2):220-227.
Background
B-type natriuretic peptide (BNP) is produced as a biologically inactive prohormone (proBNP1-108), processed, and released as an inactive amino-terminal fragment (NT-proBNP1-76) and a biologically active carboxyl-terminal fragment (proBNP77-108 or BNP32). We hypothesized that simultaneous assessment of proBNP1-108 and active BNP32, as an index of natriuretic peptide processing efficiency, would improve risk stratification in patients with chronic systolic heart failure.
Methods and Results
We quantified plasma proBNP1-108 and BNP32 in 756 subjects in the Penn Heart Failure Study, a prospective cohort of outpatients with predominantly systolic heart failure. Cox models were used to determine the association between biomarker level at time of study entry and incident risk of adverse cardiovascular outcomes. A significant amount of unprocessed proBNP1-108 circulates in patients with systolic heart failure (median 271 pg/ml, interquartile range 65 to 825). Higher levels of proBNP1-108 were associated with an increased risk of all-cause death or cardiac transplantation (adjusted HR 4.9, 95% CI 2.5–9.7, p<0.001 comparing 3rd versus 1st proBNP1-108 tertile). ProBNP1-108 provided additive information to BNP32 risk assessment, particularly in patients with BNP32 less than the median of 125 pg/ml (adjusted HR 1.4, 95% CI 1.2–1.8, p<0.001 per doubling of proBNP1-108).
Conclusions
Circulating proBNP1-108 is independently associated with an increased risk of adverse cardiovascular outcomes in ambulatory patients with chronic systolic heart failure. The combined assessment of BNP32 and proBNP1-108 provides additional information in determining risk of adverse clinical outcomes, particularly in patients with low BNP32 values that might otherwise be reassuring to the clinician.
doi:10.1161/CIRCHEARTFAILURE.109.903153
PMCID: PMC4667169  PMID: 20107190
cardiomyopathy; congestive heart failure; natriuretic peptides
7.  Macroscopic optical physiological parameters correlate with microscopic proliferation and vessel area breast cancer signatures 
Introduction
Non-invasive diffuse optical tomography (DOT) and diffuse correlation spectroscopy (DCS) can detect and characterize breast cancer and predict tumor responses to neoadjuvant chemotherapy, even in patients with radiographically dense breasts. However, the relationship between measured optical parameters and pathological biomarker information needs to be further studied to connect information from optics to traditional clinical cancer biology. Thus we investigate how optically measured physiological parameters in malignant tumors such as oxy-, deoxy-hemoglobin concentration, tissue blood oxygenation, and metabolic rate of oxygen correlate with microscopic histopathological biomarkers from the same malignant tumors, e.g., Ki67 proliferation markers, CD34 stained vasculature markers and nuclear morphology.
Methods
In this pilot study, we investigate correlations of macroscopic physiological parameters of malignant tumors measured by diffuse optical technologies with microscopic histopathological biomarkers of the same tumors, i.e., the Ki67 proliferation marker, the CD34 stained vascular properties marker, and nuclear morphology.
Results
The tumor-to-normal relative ratio of Ki67-positive nuclei is positively correlated with DOT-measured relative tissue blood oxygen saturation (R = 0.89, p-value: 0.001), and lower tumor-to-normal deoxy-hemoglobin concentration is associated with higher expression level of Ki67 nuclei (p-value: 0.01). In a subset of the Ki67-negative group (defined by the 15 % threshold), an inverse correlation between Ki67 expression level and mammary metabolic rate of oxygen was observed (R = −0.95, p-value: 0.014). Further, CD34 stained mean-vessel-area in tumor is positively correlated with tumor-to-normal total-hemoglobin and oxy-hemoglobin concentration. Finally, we find that cell nuclei tend to have more elongated shapes in less oxygenated DOT-measured environments.
Conclusions
Collectively, the pilot data are consistent with the notion that increased blood is supplied to breast cancers, and it also suggests that less conversion of oxy- to deoxy-hemoglobin occurs in more proliferative cancers. Overall, the observations corroborate expectations that macroscopic measurements of breast cancer physiology using DOT and DCS can reveal microscopic pathological properties of breast cancer and hold potential to complement pathological biomarker information.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-015-0578-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-015-0578-z
PMCID: PMC4487833  PMID: 26013572
8.  Impact of the 2010 resuscitation guidelines training on layperson chest compressions 
BACKGROUND:
Survival from cardiac arrest is sensitive to the quality of delivered CPR. In 2010, updated international resuscitation guidelines emphasized deeper chest compressions and faster rates, yet it is unknown whether training laypersons using updated guidelines resulted in changed CPR performance. We hypothesized that laypersons taught CPR using the 2010 guidelines performed deeper and faster compressions than those taught using the 2005 materials.
METHODS:
This work represents a secondary analysis of a study conducted at eight hospitals where family members of hospitalized cardiac patients were trained in CPR. An initial cohort was trained using the 2005 guidelines, and a subsequent cohort was trained using the 2010 guideline materials. Post training, CPR skills were quantified using a recording manikin.
RESULTS:
Between May 2009 to August 2013, 338 subjects completed the assessment. Among the subjects, 176 received 2005 training and 162 underwent 2010 training. The mean compression rate in the 2005 cohort was 87 (95%CI 83–90) per minute, and in the 2010 cohort was 86 (95%CI 83–90) per minute (P=ns), while the mean compression depth was 34 (95%CI 32–35) mm in the 2005 cohort and 46 (95%CI 44–47) mm in the 2010 cohort (P<0.01).
CONCLUSIONS:
Training with the 2010 CPR guidelines resulted in a statistically significant increase in trainees’ compression depth but there was no change in compression rate. Nevertheless, the majority of CPR performed by trainees in both cohorts was below the guideline recommendation, highlighting an important gap between training goals and trainee performance.
doi:10.5847/wjem.j.1920-8642.2015.04.004
PMCID: PMC4677069  PMID: 26693261
Cardiopulmonary resuscitation; Cardiac arrest; Sudden death; Education; Guidelines
9.  A Method for Choosing the Smoothing Parameter in a Semi-parametric Model for Detecting Change-points in Blood Flow 
Journal of applied statistics  2013;41(1):26-45.
In a smoothing spline model with unknown change-points, the choice of the smoothing parameter strongly influences the estimation of the change-point locations, and the function at the change-points. In a tumor biology example, where change-points in blood flow in response to treatment were of interest, choosing the smoothing parameter based on minimizing generalized cross validation, GCV, gave unsatisfactory estimates of the change-points. We propose a new method, aGCV, that re-weights the residual sum of squares and generalized degrees of freedom terms from GCV. The weight is chosen to maximize the decrease in the generalized degrees of freedom as a function of the weight value, while simultaneously minimizing aGCV as a function of the smoothing parameter and the change-points. Compared to GCV, simulation studies suggest that the aGCV method yields improved estimates of the change-point and the value of the function at the change-point.
doi:10.1080/02664763.2013.830085
PMCID: PMC3896242  PMID: 24459325
Change-points; Smoothing spline; Partial spline; Generalized Cross Validation; Generalized Degrees of Freedom
10.  Noninvasive Optical Quantification of Cerebral Venous Oxygen Saturation in Humans 
Academic radiology  2014;21(2):162-167.
Rationale and Objectives
Cerebral oxygen extraction, defined as the difference between arterial and venous oxygen saturations (SaO2 and SvO2), is a critical parameter for managing intensive care patients at risk for neurological collapse. Although quantification of SaO2 is easily performed with pulse oximetry or moderately invasive arterial blood draws in peripheral vessels, cerebral SvO2 is frequently not monitored because of the invasiveness and risk associated with obtaining jugular bulb or super vena cava (SVC) blood samples.
Materials and Methods
In this study, near-infrared spectroscopy (NIRS) was used to noninvasively measure cerebral SvO2 in anesthetized and mechanically ventilated pediatric patients (n = 10). To quantify SvO2, the NIRS signal component that fluctuates at the respiration frequency is isolated. This respiratory component is dominated by the venous portion of the interrogated vasculature. The NIRS measurements of SvO2 were validated against the clinical gold standard: invasively measured oxygen saturations from SVC blood samples. This technique was also applied in healthy volunteers (n = 5) without mechanical ventilation to illustrate its potential for use in healthy populations with natural airways.
Results
Ten pediatric patients with pulmonary hypertension were studied. In these patients, SvO2 in the SVC exhibited good agreement with NIRS-measured SvO2 (R2 = 0.80, P = .001, slope = 1.16 ± 0.48). Furthermore, in the healthy adult volunteers, mean (standard deviation) NIRS-measured SvO2 was 79.4 (6.8)%. This value is in good agreement with the expected average central venous saturation reported in literature.
Conclusion
Respiration frequency-selected NIRS can noninvasively quantify cerebral SvO2. This bedside technique can be used to help assess brain health in neurologically unstable patients.
doi:10.1016/j.acra.2013.10.013
PMCID: PMC4126245  PMID: 24439329
Near-infrared spectroscopy; cerebral venous oxygenation; pediatrics; validation; noninvasive
11.  Optically Measured Microvascular Blood Flow Contrast of Malignant Breast Tumors 
PLoS ONE  2014;9(6):e99683.
Microvascular blood flow contrast is an important hemodynamic and metabolic parameter with potential to enhance in vivo breast cancer detection and therapy monitoring. Here we report on non-invasive line-scan measurements of malignant breast tumors with a hand-held optical probe in the remission geometry. The probe employs diffuse correlation spectroscopy (DCS), a near-infrared optical method that quantifies deep tissue microvascular blood flow. Tumor-to-normal perfusion ratios are derived from thirty-two human subjects. Mean (95% confidence interval) tumor-to-normal ratio using surrounding normal tissue was 2.25 (1.92–2.63); tumor-to-normal ratio using normal tissues at the corresponding tumor location in the contralateral breast was 2.27 (1.94–2.66), and using normal tissue in the contralateral breast was 2.27 (1.90–2.70). Thus, the mean tumor-to-normal ratios were significantly different from unity irrespective of the normal tissue chosen, implying that tumors have significantly higher blood flow than normal tissues. Therefore, the study demonstrates existence of breast cancer contrast in blood flow measured by DCS. The new, optically accessible cancer contrast holds potential for cancer detection and therapy monitoring applications, and it is likely to be especially useful when combined with diffuse optical spectroscopy/tomography.
doi:10.1371/journal.pone.0099683
PMCID: PMC4072684  PMID: 24967878
12.  Prevalence and Prognostic Importance of Changes in Renal Function After Mechanical Circulatory Support 
Circulation. Heart failure  2013;7(1):68-75.
Background
The long-term durability and prognostic significance of improvement in renal function after mechanical circulatory support (MCS) has yet to be characterized in a large multicenter population. The primary goals of this analysis were to describe serial post-MCS changes in estimated glomerular filtration rate (eGFR) and determine their association with all-cause mortality.
Methods and Results
Adult patients enrolled in the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) with serial creatinine levels available (n=3363) were studied. Early post-MCS, eGFR improved substantially (median improvement, 48.9%; P<0.001) with 22.3% of the population improving their eGFR by ≥100% within the first few weeks. However, in the majority of patients, this improvement was transient, and by 1 year, eGFR was only 6.7% above the pre-MCS value (P<0.001). This pattern of early improvement followed by deterioration in eGFR was observed with both pulsatile and continuous-flow devices. Interestingly, poor survival was associated with both marked improvement (adjusted hazard ratio [HR], 1.64; 95% confidence interval [CI], 1.19–2.26; P=0.002) and worsening in eGFR (adjusted HR, 1.63; 95% CI, 1.15–2.13; P=0.004).
Conclusions
Post-MCS, early improvement in renal function is common but seems to be largely transient and not necessarily indicative of an improved prognosis. This pattern was observed with both pulsatile and continuous-flow devices. Additional research is necessary to better understand the mechanistic basis for these complex post-MCS changes in renal function and their associated survival disadvantage.
Clinical Trial Registration
URL: http://www.clinicaltrials.gov. Unique identifier: NCT00119834.
doi:10.1161/CIRCHEARTFAILURE.113.000507
PMCID: PMC4067252  PMID: 24214901
cardio-renal syndrome; heart failure; heart-assist devices; transplantation
13.  A Latent Class Model for Testing for Linkage and Classifying Families when the Sample May Contain Segregating and Non-Segregating Families 
Human Heredity  2010;70(2):75-91.
In a quantitative trait locus (QTL) study, it is usually not feasible to select families with offspring that simultaneously display variability in more than one phenotype. When multiple phenotypes are of interest, the sample will, with high probability, contain ‘non-segregating’ families, i.e. families with both parents homozygous at the QTL. These families potentially reduce the power of regression-based methods to detect linkage. Moreover, follow-up studies in individual families will be inefficient, and potentially even misleading, if non-segregating families are selected for the study. Our work extends Haseman-Elston regression using a latent class model to account for the mixture of segregating and non-segregating families. We provide theoretical motivation for the method using an additive genetic model with two distinct functions of the phenotypic outcome, squared difference (SqD) and mean-corrected product (MCP). A permutation procedure is developed to test for linkage; simulation shows that the test is valid for both phenotypic functions. For rare alleles, the method provides increased power compared to a ‘marginal’ approach that ignores the two types of families; for more common alleles, the marginal approach has better power. These results appear to reflect the ability of the algorithm to accurately assign families to the two classes and the relative weights of segregating and non-segregating families to the test of linkage. An application of Bayes rule is used to estimate the family-specific probability of segregating. High predictive value positive values for segregating families, particularly for MCP, suggest that the method has considerable value for identifying segregating families. The method is illustrated for gene expression phenotypes measured on 27 candidate genes previously demonstrated to show linkage in a sample of 14 families.
doi:10.1159/000312819
PMCID: PMC2952184  PMID: 20558995
Linkage; Heterogeneity; Haseman-Elston regression; Latent class analysis; Gene expression
14.  Neurovascular coupling varies with level of global cerebral ischemia in a rat model 
In this study, cerebral blood flow, oxygenation, metabolic, and electrical functional responses to forepaw stimulation were monitored in rats at different levels of global cerebral ischemia from mild to severe. Laser speckle contrast imaging and optical imaging of intrinsic signals were used to measure changes in blood flow and oxygenation, respectively, along with a compartmental model to calculate changes in oxygen metabolism from these measured changes. To characterize the electrical response to functional stimulation, we measured somatosensory evoked potentials (SEPs). Global graded ischemia was induced through unilateral carotid artery occlusion, bilateral carotid artery occlusion, bilateral carotid and right subclavian artery (SCA) occlusion, or carotid and SCA occlusion with negative lower body pressure. We found that the amplitude of the functional metabolic response remained tightly coupled to the amplitude of the SEP at all levels of ischemia observed. However, as the level of ischemia became more severe, the flow response was more strongly attenuated than the electrical response, suggesting that global ischemia was associated with an uncoupling between the functional flow and electrical responses.
doi:10.1038/jcbfm.2012.137
PMCID: PMC3597370  PMID: 23032485
cerebral hemodynamics; evoked potentials; global ischemia; intrinsic optical imaging; neurovascular coupling
15.  Protracted Postnatal Development of Sparse, Specific Dentate Granule Cell Activation in the Mouse Hippocampus 
The dentate gyrus (DG) is a critical entry point regulating function of the hippocampus. Integral to this role are the sparse, selective activation characteristics of the principal cells of the DG, dentate granule cells (DGCs). This sparse activation is important both in cognitive processing, and in regulation of pathological activity in disease states. Using a novel, combined dynamic imaging approach capable of resolving sequentially both synaptic potentials and action potential firing in large populations of DGCs, we characterized the postnatal development of firing properties of DG neurons in response to afferent activation in mouse hippocampal-entorhinal cortical slices. During postnatal development, there was a protracted, progressive sparsification of responses, accompanied by increased temporal precision of activation. Both of these phenomena were primarily mediated by changes in local circuit inhibition, and not by alterations in afferent innervation of DGCs, since GABAA antagonists normalized developmental differences. There was significant theta and gamma frequency-dependent synaptic recruitment of DGC activation in adult, but not developing, animals. Finally, we found that the decision to fire or not fire by individual DGCs was robust and repeatable at all stages of development. The protracted postnatal development of sparse, selective firing properties, increased temporal precision and frequency dependence of activation, and the fidelity with which the decision to fire is made are all fundamental circuit determinants of DGC excitation, critical in both normal and pathological function of the DG.
doi:10.1523/JNEUROSCI.1868-12.2013
PMCID: PMC3711669  PMID: 23407953
Calcium Imaging; Two-Photon Microscopy; Voltage Sensitive Dye; Confocal Microscopy; Hippocampus; Dentate; Granule Cells
16.  Sodium bicarbonate causes dose-dependent increases in cerebral blood flow in infants and children with single ventricle physiology 
Pediatric research  2013;73(5):668-673.
Background
Sodium bicarbonate (NaHCO3) is a common treatment for metabolic acidemia, however little definitive information exists regarding its treatment efficacy and cerebral hemodynamic effects. This pilot observational study quantifies relative changes in cerebral blood flow (rCBF) and oxy and deoxy-hemoglobin concentrations (ΔHbO2 and ΔHb) due to bolus administration of NaHCO3 in patients with mild base deficits.
Methods
Infants and children with hypoplastic left heart syndrome (HLHS) were recruited prior to cardiac surgery. NaHCO3 was given as needed for treatment of base deficit. Diffuse optical spectroscopies were employed for 15 minutes post-injection to non-invasively monitor ΔHb, ΔHbO2 and rCBF relative to baseline prior to NaHCO3 administration.
Results
Twenty-two anesthetized and mechanically ventilated HLHS patients (1 day to 4 years old) received a median (interquartile range) dose of 1.1 (0.8, 1.8) mEq/kg NaHCO3 administered intravenously over 10–20 seconds to treat a base deficit of −4 (−6, −3) mEq/l. NaHCO3 caused significant dose-dependent increases in rCBF, however population averaged ΔHb or Δ4HbO2 compared to controls were not significant.
Conclusions
Dose-dependent increases in cerebral blood flow (CBF) caused by bolus NaHCO3 are an important consideration in vulnerable populations wherein risk of rapid CBF fluctuations does not outweigh the benefit of treating a base deficit.
doi:10.1038/pr.2013.25
PMCID: PMC3724528  PMID: 23403802
17.  In Vitro Stretch Injury Induces Time- and Severity-Dependent Alterations of STEP Phosphorylation and Proteolysis in Neurons 
Journal of Neurotrauma  2012;29(10):1982-1998.
Abstract
Striatal-enriched tyrosine phosphatase (STEP) has been identified as a component of physiological and pathophysiological signaling pathways mediated by N-methyl-d-aspartate (NMDA) receptor/calcineurin/calpain activation. Activation of these pathways produces a subsequent change in STEP isoform expression or activation via dephosphorylation. In this study, we evaluated changes in STEP phosphorylation and proteolysis in dissociated cortical neurons after sublethal and lethal mechanical injury using an in vitro stretch injury device. Sublethal stretch injury produces minimal changes in STEP phosphorylation at early time points, and increased STEP phosphorylation at 24 h that is blocked by the NMDA-receptor antagonist APV, the calcineurin-inhibitor FK506, and the sodium channel blocker tetrodotoxin. Lethal stretch injury produces rapid STEP dephosphorylation via NR2B-containing NMDA receptors, but not calcineurin, and a subsequent biphasic phosphorylation pattern. STEP61 expression progressively increases after sublethal stretch with no change in calpain-mediated STEP33 formation, while lethal stretch injury results in STEP33 formation via a NR2B-containing NMDA receptor pathway within 1 h of injury. Blocking calpain activation in the initial 30 min after stretch injury increases the ratio of active STEP in cells and blocks STEP33 formation, suggesting that STEP is an early substrate of calpain after mechanical injury. There is a strong correlation between the amount of STEP33 formed and the degree of cell death observed after lethal stretch injury. In summary, these data demonstrate that previously characterized pathways of STEP regulation via the NMDA receptor are generally conserved in mechanical injury, and suggest that calpain-mediated cleavage of STEP33 should be further examined as an early marker of neuronal fate after stretch injury.
doi:10.1089/neu.2011.2253
PMCID: PMC3390986  PMID: 22435660
altered signal transduction; in vitro studies; neural injury; traumatic brain injury
18.  Photodynamic Therapy of Disseminated Non-Small Cell Lung Carcinoma in a Murine Model 
Lasers in surgery and medicine  2011;43(7):663-675.
Background and Objective
Photodynamic therapy (PDT) of thoracic malignancies involving the pleural surfaces is an active area of clinical investigation. The present report aims to characterize a model for PDT of disseminated non-small cell lung carcinoma grown orthotopically in nude mice, and to evaluate PDT effect on tumor and normal tissues.
Study Design
H460 human non-small cell lung carcinoma (NSCLC) cells were injected percutaneously into the thoracic cavity of nude mice. HPPH-PDT (1 mg/kg, 24 h) was performed via the interstitial delivery (150 mW/cm) of 661 nm light to the thoracic cavity at fluences of 25-200 J/cm.
Results
H460 tumors exhibited exponential growth within the thoracic cavity consisting of diffuse, gross nodular disease within 9 days after intrathoracic injection. Tumor volume, measured by magnetic resonance imaging (MRI), was highly correlated with the aggregate tumor mass extracted from the corresponding animal. Intrathoracic PDT at fluences of ≥ 50 J/cm produced significant decreases in tumor burden as compared to untreated controls, however mortality increased with rising fluence. Accordingly, 50 J/cm was selected for MRI studies to measure intra-animal PDT effects. Tumor distribution favored the ventral (vs. dorsal), caudal (vs. cranial), and right (vs. left) sides of the thoracic cavity by MRI; PDT did not change this spatial pattern despite an overall effect on tumor burden. Histopathology revealed edema and fibrin deposition within the pulmonary interstitium and alveoli of the PDT-treated thoracic cavity, as well as occasional evidence of vascular disruption. Prominent neutrophil infiltration with a concomitant decline in the lymphocyte compartment was also noted in the lung parenchyma within 24 hours after PDT.
Conclusion
HPPH-PDT of an orthotopic model of disseminated NSCLC is both feasible and effective using intracavitary light delivery. We establish this animal model, together with the treatment and monitoring approaches, as novel and valuable methods for the pre-clinical investigation of intrathoracic PDT of disseminated pleural malignancies.
doi:10.1002/lsm.21102
PMCID: PMC3676899  PMID: 22057494
HPPH; Photochlor®; 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a; interstitial illumination; magnetic resonance imaging; non-small cell lung carcinoma; photodynamic therapy; pleural malignancy
20.  Differential effects of a polyalanine tract expansion in Arx on neural development and gene expression 
Human Molecular Genetics  2011;21(5):1090-1098.
Polyalanine (poly-A) tracts exist in 494 annotated proteins; to date, expansions in these tracts have been associated with nine human diseases. The pathogenetic mechanism by which a poly-A tract results in these various human disorders remains uncertain. To understand the role of this mutation type, we investigated the change in functional properties of the transcription factor Arx when it has an expanded poly-A tract (ArxE), a mutation associated with infantile spasms and intellectual disabilities in humans. We found that although ArxE functions normally in the dorsal brain, its function in subpallial-derived populations of neurons is compromised. These contrasting functions are associated with the misregulation of Arx targets through the loss of the ability of ArxE to interact with the Arx cofactor Tle1. Our data demonstrate a novel mechanism for poly-A expansion diseases: the misregulation of a subset of target genes normally regulated by a transcription factor.
doi:10.1093/hmg/ddr538
PMCID: PMC3277309  PMID: 22108177
21.  SERUM CYTOKINE PROFILES ASSOCIATED WITH EARLY ALLOGRAFT DYSFUNCTION IN PATIENTS UNDERGOING LIVER TRANSPLANTATION 
Liver Transplantation  2012;18(2):166-176.
Early allograft dysfunction (EAD) occurring in the first week post-liver transplantation is associated with increased graft failure and mortality and is believed to be largely due to ischemia/reperfusion injury. We anticipated that the presence of EAD would be reflected by alterations in expression of serum proteins associated with an inflammatory response in the peri-operative period, and hypothesized that a specific pattern of expression might correlate with the development of EAD. The serum levels of 25 cytokines, chemokines, and immunoreceptors were measured by Luminex multiplex assays pre- and post-liver transplantation. Levels of each cytokine biomarker were compared in adult recipients with or without EAD at serial time points using samples collected pre-operatively and at 1, 7, 14, and 30 days post-transplant. EAD was defined according to standard criteria as maximum alanine transferase (ALT) or aspartate transferase (AST) levels on days 1–7 of >2000 U/ml, day 7 bilirubin level ≥10 mg/dl, or a day 7 international normalized ratio (INR) ≥1.7. Multivariable analyses showed that patients experiencing EAD had lower pre-operative IL-6 and higher IL-2R levels. Patients with EAD also showed higher MCP-1 (CCL2), IL-8 (CXCL8), and RANTES (CCL5) chemokine levels in the early post-operative period, suggesting up-regulation of the NF-κB pathway, in addition to higher levels of chemokines and cytokines associated with T cell immunity, including Mig (CXCL9), IP-10 (CXCL10) and IL-2R. These findings identify several possible biomarkers and pathways associated with EAD, that may guide future validation studies and investigation of specific cellular and molecular mechanisms of graft dysfunction. Furthermore, if validated, our findings may contribute to perioperative prediction of the occurrence of EAD and ultimately lead to identification of potential interventional therapies.
doi:10.1002/lt.22451
PMCID: PMC3266982  PMID: 22006860
immune monitoring; multiplex analysis; chemokines; immunobiology
22.  Deterministic and Stochastic Neuronal Contributions to Distinct Synchronous CA3 Network Bursts 
The Journal of Neuroscience  2012;32(14):4743-4754.
Computational studies have suggested that stochastic, deterministic, and mixed processes all could be possible determinants of spontaneous, synchronous network bursts. In the present study, utilizing multicellular calcium imaging coupled with fast confocal microscopy, we describe neuronal behavior underlying spontaneous network bursts in developing rat and mouse hippocampal area CA3 networks. Two primary burst types were studied: giant depolarizing potentials (GDPs) and spontaneous interictal bursts recorded in bicuculline, a GABAA receptor antagonist. Analysis of the simultaneous behavior of multiple CA3 neurons during synchronous GDPs revealed a repeatable activation order from burst to burst. This was validated using several statistical methods, including high Kendall’s W values for firing order during GDPs, high Pearson’s correlations of cellular activation times between burst pairs, and latent class analysis, which revealed a population of 5-6% of CA3 neurons reliably firing very early during GDPs. In contrast, neuronal firing order during interictal bursts appeared homogenous, with no particular cells repeatedly leading or lagging during these synchronous events. We conclude that GDPs activate via a deterministic mechanism, with distinct, repeatable roles for subsets of neurons during burst generation, while interictal bursts appear to be stochastic events with cells assuming interchangeable roles in the generation of these events.
doi:10.1523/JNEUROSCI.4277-11.2012
PMCID: PMC3328771  PMID: 22492030
Calcium Imaging; Fast Confocal Microscopy; Network Bursts; Hippocampus; CA3 Neurons; Latent Class Model Analysis
23.  Tumor Blood Flow Differs between Mouse Strains: Consequences for Vasoresponse to Photodynamic Therapy 
PLoS ONE  2012;7(5):e37322.
Fluctuations in tumor blood flow are common and attributed to factors such as vasomotion or local vascular structure, yet, because vessel structure and physiology are host-derived, animal strain of tumor propagation may further determine blood flow characteristics. In the present report, baseline and stress-altered tumor hemodynamics as a function of murine strain were studied using radiation-induced fibrosacomas (RIF) grown in C3H or nude mice. Fluctuations in tumor blood flow during one hour of baseline monitoring or during vascular stress induced by photodynamic therapy (PDT) were measured by diffuse correlation spectroscopy. Baseline monitoring revealed fluctuating tumor blood flow highly correlated with heart rate and with similar median periods (i.e., ∼9 and 14 min in C3H and nudes, respectively). However, tumor blood flow in C3H animals was more sensitive to physiologic or stress-induced perturbations. Specifically, PDT-induced vascular insults produced greater decreases in blood flow in the tumors of C3H versus nude mice; similarly, during baseline monitoring, fluctuations in blood flow were more regular and more prevalent within the tumors of C3H mice versus nude mice; finally, the vasoconstrictor L-NNA reduced tumor blood flow in C3H mice but did not affect tumor blood flow in nudes. Underlying differences in vascular structure, such as smaller tumor blood vessels in C3H versus nude animals, may contribute to strain-dependent variation in vascular function. These data thus identify clear effects of mouse strain on tumor hemodynamics with consequences to PDT and potentially other vascular-mediated therapies.
doi:10.1371/journal.pone.0037322
PMCID: PMC3356280  PMID: 22624014
24.  Fractalkine Is a Novel Human Adipochemokine Associated With Type 2 Diabetes 
Diabetes  2011;60(5):1512-1518.
OBJECTIVE
Leukocyte infiltration of adipose is a critical determinant of obesity-related metabolic diseases. Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in atherosclerosis, but its role in adipose inflammation and type 2 diabetes is unknown.
RESEARCH DESIGN AND METHODS
CX3CL1 mRNA and protein were quantified in subcutaneous adipose and blood during experimental human endotoxemia and in lean and obese human adipose. CX3CL1 cellular source was probed in human adipocytes, monocytes, and macrophages, and CX3CL1-blocking antibodies were used to assess its role in monocyte-adipocyte adhesion. The association of genetic variation in CX3CR1 with metabolic traits was determined in a community-based sample. Finally, plasma CX3CL1 levels were measured in a case-control study of type 2 diabetes.
RESULTS
Endotoxemia induced adipose CX3CL1 mRNA (32.7-fold, P < 1 × 10−5) and protein (43-fold, P = 0.006). Obese subjects had higher CX3CL1 levels in subcutaneous adipose compared with lean (0.420 ± 0.387 vs. 0.228 ± 0.187 ng/mL, P = 0.04). CX3CL1 was expressed and secreted by human adipocytes and stromal vascular cells. Inflammatory cytokine induction of CX3CL1 in human adipocytes (27.5-fold mRNA and threefold protein) was completely attenuated by pretreatment with a peroxisome proliferator–activated receptor-γ agonist. A putative functional nonsynonymous single nucleotide polymorphism (rs3732378) in CX3CR1 was associated with adipose and metabolic traits, and plasma CX3CL1 levels were increased in patients with type 2 diabetes vs. nondiabetics (0.506 ± 0.262 vs. 0.422 ± 0.210 ng/mL, P < 0.0001).
CONCLUSIONS
CX3CL1-CX3CR1 is a novel inflammatory adipose chemokine system that modulates monocyte adhesion to adipocytes and is associated with obesity, insulin resistance, and type 2 diabetes. These data provide support for CX3CL1 as a diagnostic and therapeutic target in cardiometabolic disease.
doi:10.2337/db10-0956
PMCID: PMC3292325  PMID: 21525510
25.  Evidence for co-regulation of myocardial gene expression by MEF2 and NFAT in human heart failure 
Background
Pathologic stresses induce heart failure in animal models through activation of multiple cardiac transcription factors (TFs) working cooperatively. However, interactions among TFs in human heart failure are less well understood. Here we use genomic data to examine the evidence that five candidate TF families co-regulate gene expression in human heart failure.
Methods and Results
RNA isolates from failing (n=86) and non-failing (n=16) human hearts were hybridized with Affymetrix HU133A arrays. For each gene on the array, we determined conserved MEF2, NFAT, NKX, GATA, and FOX binding motifs within the −1 kb promoter region using human-murine sequence alignments and the TRANSFAC database. Across 9,076 genes expressed in the heart, TF binding motifs tended to cluster together in nonrandom patterns within promoters of specific genes (P-values ranging from 10−2 to 10−21), suggesting co-regulation. We then modeled differential expression as a function of TF combinations present in promoter regions. Several combinations predicted increased odds of differential expression in the failing heart, with highest odds ratios noted for genes containing both MEF2 and NFAT bindings motifs together in the same promoter (peak OR 3.47, P=0.005).
Conclusions
These findings provide genomic evidence for co-regulation of myocardial gene expression by MEF2 and NFAT in human heart failure. In doing so, they extend the paradigm of combinatorial regulation of gene expression to the human heart and identify new target genes for mechanistic study. More broadly, we demonstrate how integrating diverse sources of genomic data yields novel insights into human cardiovascular disorders.
doi:10.1161/CIRCGENETICS.108.816686
PMCID: PMC3157251  PMID: 20031589
heart failure; hypertrophy; remodeling; genes; transcription factors

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