Epigenetic associations in HPA axis genes related to bronchopulmonary dysplasia and antenatal steroids.

BACKGROUND: Bronchopulmonary dysplasia (BPD), a common morbidity among very preterm infants, is associated with chronic disease and neurodevelopmental impairments. A hypothesized mechanism for these outcomes lies in altered glucocorticoid (GC) activity. We hypothesized that BPD and its treatments may result in epigenetic differences in the hypothalamic-pituitary-adrenal (HPA) axis, which is modulated by GC, and could be ascertained using an established GC risk score and DNA methylation (DNAm) of HPA axis genes. METHODS: DNAm was quantified from buccal tissue (ECHO-NOVI) and from neonatal blood spots (ELGAN ECHO) via the EPIC microarray. Prenatal maternal characteristics, pregnancy complication, and neonatal medical complication data were collected from medical record review and maternal interviews. RESULTS: The GC score was not associated with steroid exposure or BPD. However, six HPA genes involved in stress response regulation demonstrated differential methylation with antenatal steroid exposure; two CpGs within FKBP5 and POMC were differentially methylated with BPD severity. These findings were sex-specific in both cohorts; males had greater magnitude of differential methylation within these genes. CONCLUSIONS: These findings suggest that BPD severity and antenatal steroids are associated with DNAm at some HPA genes in very preterm infants and the effects appear to be sex-, tissue-, and age-specific. IMPACT: This study addresses bronchopulmonary dysplasia (BPD), an important health outcome among preterm neonates, and interrogates a commonly studied pathway, the hypothalamic-pituitary-adrenal (HPA) axis. The combination of BPD, the HPA axis, and epigenetic markers has not been previously reported. In this study, we found that BPD itself was not associated with epigenetic responses in the HPA axis in infants born very preterm; however, antenatal treatment with steroids was associated with epigenetic responses.

Epigenetic age acceleration, neonatal morbidities, and neurobehavioral profiles in infants born very preterm.

Epigenetic age acceleration is a risk factor for chronic diseases of ageing and may reflect aspects of biological ageing. However, few studies have examined epigenetic ageing during the early neonatal period in preterm infants, who are at heightened risk of developmental problems. We examined relationships between neonatal age acceleration, neonatal morbidities, and neurobehavioral domains among very preterm (<30 weeks gestation) infants to characterize whether infants with early morbidities or different neurobehavioral characteristics had accelerated or decelerated epigenetic ageing. This study uses data from the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) study, restricted to infants with data on variables assessed (n = 519). We used generalized estimating equations to test for differences in age acceleration associated with severe neonatal medical morbidities and neurobehavioral characteristics. We found that infants with neonatal morbidities, in particular, bronchopulmonary dysplasia (BPD), had accelerated epigenetic age - and some evidence that infants with hypertonicity and asymmetric reflexes had increased and decreased age acceleration, respectively. Adjustment for gestational age attenuated some associations, suggesting that the relationships observed may be driven by the duration of gestation. Our most robust finding shows that very preterm infants with neonatal morbidities (BPD in particular) exhibit age acceleration, but most neonatal neurobehavioral characteristics and morbidities are not associated with early life age acceleration. Lower gestational age at birth may be an upstream factor driving these associations.

Risk Factors for Postpartum Depression and Severe Distress among Mothers of Very Preterm Infants at NICU Discharge.

OBJECTIVE: To identify psychological, medical, and socioenvironmental risk factors for maternal postpartum depression (PPD) and severe psychological distress (SPD) at intensive care nursery discharge among mothers of very preterm infants. STUDY DESIGN: We studied 562 self-identified mothers of 641 infants born <30 weeks who were enrolled in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants Study (NOVI) conducted in nine university-affiliated intensive care nurseries. Enrollment interviews collected socioenvironmental data, depression, and anxiety diagnoses prior to and during the study pregnancy. Standardized medical record reviews ascertained prenatal substance use, maternal and neonatal medical complications. The Edinburgh Postnatal Depression Scale and Brief Symptom Inventory were administered at nursery discharge to screen for PPD and SPD symptoms, respectively. RESULTS: Unadjusted analyses indicated mothers with positive screens for depression (n = 76, 13.5%) or severe distress (n = 102, 18.1%) had more prevalent prepregnancy/prenatal depression/anxiety, and their infants were born at younger gestational ages, with more prevalent bronchopulmonary dysplasia, and discharge after 40 weeks postmenstrual age. In multivariable analyses, prior depression or anxiety was associated with positive screens for PPD (risk ratio [RR]: 1.6, 95% confidence interval [CI]: 1.1-2.2) and severe distress (RR: 1.6, 95% CI: 1.1-2.2). Mothers of male infants had more prevalent depression risk (RR: 1.7, 95% CI: 1.1-2.4), and prenatal marijuana use was associated with severe distress risk (RR: 1.9, 95% CI: 1.1-2.9). Socioenvironmental and obstetric adversities were not significant after accounting for prior depression/anxiety, marijuana use, and infant medical complications. CONCLUSION: Among mothers of very preterm newborns, these multicenter findings extend others' previous work by identifying additional indicators of risk for PPD and SPD associated with a history of depression, anxiety, prenatal marijuana use, and severe neonatal illness. Findings could inform designs for continuous screening and targeted interventions for PPD and distress risk indicators from the preconception period onward. KEY POINTS: · Preconceptional and prenatal screening for postpartum depression and severe distress may inform care.. · Prior depression, anxiety, and neonatal complications predicted severe distress and depression symptoms at NICU discharge.. · Readily identifiable risk factors warrant continuous NICU screening and targeted interventions to improve outcomes..

Prenatal and perinatal factors associated with neonatal neurobehavioral profiles in the ECHO Program.

BACKGROUND: Single-cohort studies have identified distinct neurobehavioral profiles that are associated with prenatal and neonatal factors based on the NICU Network Neurobehavioral Scale (NNNS). We examined socioeconomic, medical, and substance use variables as predictors of NNNS profiles in a multi-cohort study of preterm and term-born infants with different perinatal exposures. METHODS: We studied 1112 infants with a neonatal NNNS exam from the Environmental influences on Child Health Outcomes (ECHO) consortium. We used latent profile analysis to characterize infant neurobehavioral profiles and generalized estimating equations to determine predictors of NNNS profiles. RESULTS: Six distinct neonatal neurobehavioral profiles were identified, including two dysregulated profiles: a hypo-aroused profile (16%) characterized by lethargy, hypotonicity, and nonoptimal reflexes; and a hyper-aroused profile (6%) characterized by high arousal, excitability, and stress, with low regulation and poor movement quality. Infants in the hypo-aroused profile were more likely to be male, have younger mothers, and have mothers who were depressed prenatally. Infants in the hyper-aroused profile were more likely to be Hispanic/Latino and have mothers who were depressed or used tobacco prenatally. CONCLUSIONS: We identified two dysregulated neurobehavioral profiles with distinct perinatal antecedents. Further understanding of their etiology could inform targeted interventions to promote positive developmental outcomes. IMPACT: Prior research on predictors of neonatal neurobehavior have included single-cohort studies, which limits generalizability of findings. In a multi-cohort study of preterm and term-born infants, we found six distinct neonatal neurobehavioral profiles, with two profiles being identified as dysregulated. Hypo- and hyper-aroused neurobehavioral profiles had distinct perinatal antecedents. Understanding perinatal factors associated with dysregulated neurobehavior could help promote positive developmental outcomes.

Bronchopulmonary dysplasia and neurobehavioural outcomes at birth and 2 years in infants born before 30 weeks.

OBJECTIVE: To identify neurobehavioural risks in preterm infants with bronchopulmonary dysplasia (BPD) prior to hospital discharge. DESIGN AND PATIENTS: Longitudinal study of 676 newborns born before 30 weeks of gestation. SETTING: Nine university NICUs affiliated with six universities. All were Vermont Oxford Network (VON) participants. PATIENTS AND INTERVENTIONS: Infants were enrolled in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants Study from April 2014 to June 2016. Prospective medical record reviews, VON definitions and criteria, and maternal interviews were used to collect maternal and neonatal medical variables and socioenvironmental data. MAIN OUTCOME MEASURES: NICU Network Neurobehavioral Scale (NNNS) at the time of hospital discharge; Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) and Gross Motor Function Classification System at 2 years' corrected age. RESULTS: Infants with moderate/severe BPD were less attentive (Wald χ2 9.68, p=0.008), more lethargic (Wald χ2 9.91, p=0.007), with increased non-optimal reflexes (Wald χ2 7.37, p=0.025). Infants with moderate/severe BPD were more likely to have Bayley-III language and motor scores <85 (adjusted OR (aOR) 1.74, 95% CI 1.06 to 2.85, and aOR 2.06, 95% CI 1.10 to 3.85). Infants with both moderate/severe and mild BPD were more likely to have a cerebral palsy diagnosis (aOR 2.96, 95% CI 1.34 to 6.54, and aOR 2.81, 95% CI 1.32 to 5.99). CONCLUSIONS: BPD severity presents risks for poor neurodevelopment at NICU discharge and at age 2 years. Early identification of poorly regulated behaviour can provide critical information for early preventive and targeted interventions with potential to improve long-term outcomes.

Serious neonatal morbidities are associated with differences in DNA methylation among very preterm infants.

BACKGROUND: Infants born very preterm are more likely to experience neonatal morbidities compared to their term peers. Variations in DNA methylation (DNAm) associated with these morbidities may yield novel information about the processes impacted by these morbidities. METHODS: This study included 532 infants born < 30 weeks gestation, participating in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants study. We used a neonatal morbidity risk score, which was an additive index of the number of morbidities experienced during the NICU stay, including bronchopulmonary dysplasia (BPD), severe brain injury, serious neonatal infections, and severe retinopathy of prematurity. DNA was collected from buccal cells at discharge from the NICU, and DNAm was measured using the Illumina MethylationEPIC. We tested for differential methylation in association with the neonatal morbidity risk score then tested for differentially methylated regions (DMRs) and overrepresentation of biological pathways. RESULTS: We identified ten differentially methylated CpGs (α Bonferroni-adjusted for 706,278 tests) that were associated with increasing neonatal morbidity risk scores at three intergenic regions and at HPS4, SRRD, FGFR1OP, TNS3, TMEM266, LRRC3B, ZNF780A, and TENM2. These mostly followed dose-response patterns, for 8 CpGs increasing DNAm associated with increased numbers of morbidities, while for 2 CpGs the risk score was associated with decreasing DNAm. BPD was the most substantial contributor to differential methylation. We also identified seven potential DMRs and over-representation of genes involved in Wnt signaling; however, these results were not significant after Bonferroni adjustment for multiple testing. CONCLUSIONS: Neonatal DNAm, within genes involved in fibroblast growth factor activities, cellular invasion and migration, and neuronal signaling and development, are sensitive to the neonatal health complications of prematurity. We hypothesize that these epigenetic features may be representative of an integrated marker of neonatal health and development and are promising candidates to integrate with clinical information for studying developmental impairments in childhood.

Single-Arm Phase 2 Trial of Elective Nodal Dose Reduction for Patients With Locoregionally Advanced Squamous Cell Carcinoma of the Head and Neck.

PURPOSE: To evaluate a novel chemoradiation therapy (CRT) regimen for patients with squamous cell carcinoma of the head and neck (SCCHN) incorporating a lower intensity modulated radiation therapy dose to electively treated neck lymph nodes than is currently standard. METHODS AND MATERIALS: Eligible patients had locally advanced SCCHN of the oral cavity, oropharynx, larynx, or hypopharynx. The 7-week CRT course consisted of weekly cisplatin at 35 mg/m2 concurrently with sequential-boost intensity modulated radiation therapy: 36 Gy to high- and low-risk planning target volumes followed by a sequential boost to the high-risk planning target volume to 70 Gy. The primary endpoint was elective nodal failure. Secondary endpoints were survival, toxicity, feeding tube duration, and quality of life evaluated by the FACT-HN and QOL-RTI surveys. RESULTS: Between 2011 and 2014, 54 patients were enrolled, 31 (57%) of whom had human papillomavirus (HPV)-positive disease. Of the patients, 35 (65%) had stage IVa disease. The median follow-up period for survivors was 36 months (range, 12-66 months). Elective nodal failure did not develop in any patient. The actuarial 3-year survival rate for the entire cohort was 91% (95% confidence interval [CI] 0.79-0.96); for the HPV-negative group, 85% (95% CI 0.61-0.95); and for the HPV-positive group, 96% (95% CI 0.77-0.99). Common grade 3 toxicities were dysphagia (79%), mucositis and/or stomatitis (41%), nausea (20%), xerostomia (13%), vomiting (11%), and neutropenia (10%). The median feeding tube duration was 142 days. Patient FACT-HN scores were higher at 3, 6, and 12 months versus at the end of treatment (P < .0001). Total FACT-HN scores returned to pretreatment baseline by 6 months. Overall QOL-RTI scores were lower from pretreatment to the end of treatment through 12 months (P = .0001). CONCLUSIONS: This CRT regimen for patients with advanced SCCHN demonstrated the potential feasibility of reducing the elective dose to the neck, a topic that requires additional study in future clinical trials.

School-Aged Outcomes following Prenatal Methamphetamine Exposure: 7.5-Year Follow-Up from the Infant Development, Environment, and Lifestyle Study.

OBJECTIVE: To assess the relationship between prenatal methamphetamine exposure (PME) and behavior problems at age 7.5 years and the extent to which early adversity mediated this relationship. STUDY DESIGN: The multicenter, longitudinal Infant Development, Environment, and Lifestyle study enrolled 412 mother-infant pairs at 4 sites. Methamphetamine-exposed participants (n = 204) were identified by self-report and/or gas chromatography/mass spectrometry confirmation of amphetamine and metabolites in infant meconium. Matched participants (n = 208) denied methamphetamine use and had a negative meconium screen. At the 7.5-year follow-up, 290 children with complete Child Behavior Checklist data and an early adversity index score were available for analysis (n = 146 exposed). RESULTS: PME was significantly associated with an increased early adversity index score (P < .001) and with increased externalizing, rule-breaking behavior, and aggressive behavior (P < .05). Early adversity was also associated with higher externalizing behavior scores. Early adversity significantly mediated the relationship between PME and behavioral problems. After adjusting the mediation model for sex, prenatal tobacco, alcohol, and marijuana exposures, and study site, the association of PME with early adversity remained significant. CONCLUSIONS: Though PME is associated with behavioral problems, early adversity may be a strong determinant of behavioral outcome for children exposed to methamphetamine in utero. Early adversity significantly mediated the relationship between PME and behavioral problems.

Prenatal methamphetamine exposure and neonatal and infant neurobehavioral outcome: results from the IDEAL study.

BACKGROUND: Methamphetamine (MA) use among pregnant women is an increasing problem in the United States. How MA use during pregnancy affects neonatal and infant neurobehavior is unknown. METHODS: The Infant Development, Environment, and Lifestyle (IDEAL) study screened 34,833 subjects at 4 clinical centers. Of the subjects, 17,961 were eligible and 3705 were consented, among which 412 were enrolled for longitudinal follow-up. Exposed subjects were identified by self-report and/or gas chromatography/mass spectroscopy (GC/MS) confirmation of amphetamine and metabolites in meconium. Comparison subjects were matched (race, birth weight, maternal education, insurance), denied amphetamine use, and had a negative meconium screen. Both groups included prenatal alcohol, tobacco, and marijuana use, but excluded use of opiates, lysergic acid diethylamide, or phencyclidine. The Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scale (NNNS) was administered within the first 5 days of life and again at 1 month to 380 enrollees (185 exposed, 195 comparison). Analysis of variance (ANOVA) tested exposure effects on NNNS summary scores at birth and 1 month. General linear model (GLM) repeated-measures analysis assessed the effect of MA exposure over time on the NNNS scores with and without covariates. RESULTS: By 1 month of age, both groups demonstrated higher quality of movement (P = .029), less lethargy (P = .001), and fewer asymmetric reflexes (P = .012), with no significant differences in NNNS scores between the exposed and comparison groups. Over the first month of life, arousal increased in exposed infants but decreased in comparison infants (P = .031) and total stress was decreased in exposed infants, with no change in comparison infants (P = .026). CONCLUSIONS: Improvement in total stress and arousal were observed in MA-exposed newborns by 1 month of age relative to the newborn period.

Risk of neurobehavioral disinhibition in prenatal methamphetamine-exposed young children with positive hair toxicology results.

BACKGROUND: The objective was to evaluate the effects of prenatal methamphetamine exposure (PME) and postnatal drug exposures identified by child hair analysis on neurobehavioral disinhibition at 6.5 years of age. METHODS: Mother-infant pairs were enrolled in the Infant Development, Environment, and Lifestyle (IDEAL) Study in Los Angeles, Honolulu, Tulsa, and Des Moines. PME was determined by maternal self-report and/or positive meconium results. At the 6.5-year follow-up visit, hair was collected and analyzed for methamphetamine, tobacco, cocaine, and cannabinoid markers. Child behavioral and executive function test scores were aggregated to evaluate child neurobehavioral disinhibition. Hierarchical linear regression models assessed the impact of PME, postnatal substances, and combined PME with postnatal drug exposures on the child's neurobehavioral disinhibition aggregate score. Past year caregiver substance use was compared with child hair results. RESULTS: A total of 264 children were evaluated. Significantly more PME children (n = 133) had hair positive for methamphetamine/amphetamine (27.1% versus 8.4%) and nicotine/cotinine (38.3% versus 25.2%) than children without PME (n = 131). Overall, no significant differences in analyte hair concentrations were noted between groups. Significant differences in behavioral and executive function were observed between children with and without PME. No independent effects of postnatal methamphetamine or tobacco exposure, identified by positive hair test, were noted and no additional neurobehavioral disinhibition was observed in PME children with postnatal drug exposures, as compared with PME children without postnatal exposure. CONCLUSIONS: Child hair testing offered a noninvasive means to evaluate postnatal environmental drug exposure, although no effects from postnatal drug exposure alone were seen. PME, alone and in combination with postnatal drug exposures, was associated with behavioral and executive function deficits at 6.5 years.

G protein-linked signaling pathways in bipolar and major depressive disorders.

The G-protein linked signaling system (GPLS) comprises a large number of G-proteins, G protein-coupled receptors (GPCRs), GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP), phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD) and bipolar disorder (BPD). This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC) and anterior cingulate (ACC) were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, "activated" cAMP signaling activity in BPD and "blunted" cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects.

Prenatal methamphetamine exposure and inhibitory control among young school-age children.

OBJECTIVE: To examine the association between prenatal methamphetamine exposure and inhibitory control in 66-month-old children followed since birth in the multicenter, longitudinal Infant Development, Environment, and Lifestyle study. STUDY DESIGN: The sample included 137 children with prenatal methamphetamine exposure and 130 comparison children matched for race, birth weight, maternal education, and type of insurance. Inhibitory control, an executive function related to emotional and cognitive control, was assessed using a computerized Stroop-like task developed for young children. Hierarchical linear modeling tested the relationship between the extent of prenatal methamphetamine exposure (heavy, some, or none) and accuracy and reaction time outcomes, adjusting for prenatal exposure to alcohol, tobacco, and marijuana; age; sex; socioeconomic status; caregiver IQ and psychological symptoms; Child Protective Services report of physical or sexual abuse; and site. RESULTS: In adjusted analyses, heavy prenatal methamphetamine exposure was related to reduced accuracy in both the incongruent and mixed conditions on the Stroop-like task. Caregiver psychological symptoms and Child Protective Services report of physical or sexual abuse were associated with reduced accuracy in the incongruent and mixed consitions and in the incongruent conditions, respectively. CONCLUSION: Heavy prenatal methamphetamine exposure, along with caregiver psychological distress and child maltreatment, are related to subtle deficits in inhibitory control during the early school-age years.

Prenatal methamphetamine exposure and childhood behavior problems at 3 and 5 years of age.

OBJECTIVE: We evaluated behavior problems in children who were prenatally exposed to methamphetamine (MA) at ages 3 and 5 years. METHODS: The Infant Development, Environment, and Lifestyle study, a prospective, longitudinal study of prenatal MA exposure and child outcome, enrolled subjects postpartum in Los Angeles, California; Honolulu, Hawaii; Des Moines, Iowa; and Tulsa, Oklahoma. Prenatal exposure was determined by maternal self-report and/or meconium results. Exposed and comparison groups were matched on race, birth weight, public health insurance, and education. Mothers in the comparison group denied use and had a negative meconium screen for amphetamines. Prenatal exposures to tobacco, alcohol, or marijuana occurred in both groups. At ages 3 and 5 years, 330 children (166 exposed and 164 comparison) were assessed for behavior problems by using the caregiver report on the Child Behavior Checklist. General linear mixed models were used to determine the effects of prenatal MA exposure, including heavy exposure (≥3 days per week), age, and the interaction of exposure and age on behavior problems with adjustment for other drugs of abuse and environmental risk factors. RESULTS: MA exposure was associated with increased emotional reactivity and anxious/depressed problems at both ages and externalizing and attention-deficit/hyperactivity disorder problems by age 5 years. Heavy exposure was related to attention problems and withdrawn behavior at both ages. There were no effects of MA on the internalizing or total behavior problems scales. CONCLUSIONS: This first report of behavior problems in patients as young as 3 years associated with MA exposure identifies an important public health problem. Continued follow-up can inform the development of preventive intervention programs.

Phase II trial of hyperfractionated intensity-modulated radiation therapy and concurrent weekly cisplatin for Stage III and IVa head-and-neck cancer.

PURPOSE: To investigate a novel chemoradiation regimen designed to maximize locoregional control (LRC) and minimize toxicity for patients with advanced head-and-neck squamous cell carcinoma (HNSCC). METHODS AND MATERIALS: Patients received hyperfractionated intensity modulated radiation therapy (HIMRT) in 1.25-Gy fractions b.i.d. to 70 Gy to high-risk planning target volume (PTV). Intermediate and low-risk PTVs received 60 Gy and 50 Gy, at 1.07, and 0.89 Gy per fraction, respectively. Concurrent cisplatin 33 mg/m(2)/week was started Week 1. Patients completed the Quality of Life Radiation Therapy Instrument pretreatment (PRE), at end of treatment (EOT), and at 1, 3, 6, 9, and 12 months. Overall survival (OS), progression-free (PFS), LRC, and toxicities were assessed. RESULTS: Of 39 patients, 30 (77%) were alive without disease at median follow-up of 37.5 months. Actuarial 3-year OS, PFS, and LRC were 80%, 82%, and 87%, respectively. No failures occurred in the electively irradiated neck and there were no isolated neck failures. Head and neck QOL was significantly worse in 18 of 35 patients (51%): mean 7.8 PRE vs. 3.9 EOT. By month 1, H&N QOL returned near baseline (mean 6.2, SD = 1.7). The most common acute Grade 3+ toxicities were mucositis (38%), fatigue (28%), dysphagia (28%), and leukopenia (26%). CONCLUSIONS: Hyperfractionated IMRT with low-dose weekly cisplatin resulted in good LRC with acceptable toxicity and QOL. Lack of elective nodal failures despite very low dose per fraction has led to an attempt to further minimize toxicity by reducing elective nodal doses in our subsequent protocol.

Evolutionary sequence modeling for discovery of peptide hormones.

There are currently a large number of "orphan" G-protein-coupled receptors (GPCRs) whose endogenous ligands (peptide hormones) are unknown. Identification of these peptide hormones is a difficult and important problem. We describe a computational framework that models spatial structure along the genomic sequence simultaneously with the temporal evolutionary path structure across species and show how such models can be used to discover new functional molecules, in particular peptide hormones, via cross-genomic sequence comparisons. The computational framework incorporates a priori high-level knowledge of structural and evolutionary constraints into a hierarchical grammar of evolutionary probabilistic models. This computational method was used for identifying novel prohormones and the processed peptide sites by producing sequence alignments across many species at the functional-element level. Experimental results with an initial implementation of the algorithm were used to identify potential prohormones by comparing the human and non-human proteins in the Swiss-Prot database of known annotated proteins. In this proof of concept, we identified 45 out of 54 prohormones with only 44 false positives. The comparison of known and hypothetical human and mouse proteins resulted in the identification of a novel putative prohormone with at least four potential neuropeptides. Finally, in order to validate the computational methodology, we present the basic molecular biological characterization of the novel putative peptide hormone, including its identification and regional localization in the brain. This species comparison, HMM-based computational approach succeeded in identifying a previously undiscovered neuropeptide from whole genome protein sequences. This novel putative peptide hormone is found in discreet brain regions as well as other organs. The success of this approach will have a great impact on our understanding of GPCRs and associated pathways and help to identify new targets for drug development.

Perinatal iron deficiency results in altered developmental expression of genes mediating energy metabolism and neuronal morphogenesis in hippocampus.

The human and rat hippocampus is highly susceptible to iron deficiency (ID) during the late fetal, early neonatal time period which is a peak time of regulated brain iron uptake and utilization. ID during this period alters cognitive development and is characterized by distinctive, long-term changes in hippocampal cellular growth and function. The fundamental processes underlying these changes are not entirely understood. In this study, ID-induced changes in expression of 25 genes implicated in iron metabolism, including cell growth and energy metabolism, dendrite morphogenesis, and synaptic connectivity were assessed from postnatal day (P) 7 to P65 in hippocampus. All 25 genes showed altered expression during the period of ID (P7, 15, and 30); 10 had changes on P65 after iron repletion. ID caused long-term diminished protein levels of four factors critical for hippocampal neuron differentiation and plasticity, including CamKII alpha, Fkbp1a (Fkbp12), Dlgh4 (PSD-95), and Vamp1 (Synaptobrevin-1). ID altered gene expression in the mammalian target of rapamycin (mTOR) pathway and in a gene network implicated in Alzheimer disease etiology. ID during late fetal and early postnatal life alters the levels and timing of expression of critical genes involved in hippocampal development and function. The study provides targets for future studies in elucidating molecular mechanisms underpinning iron's role in cognitive development and function.

A bridge between academic and community radiation oncology treatment planning.

PURPOSE: To evaluate Telesynergy (TS) as a method of interactive treatment planning between academic and community radiation oncology departments. METHODS: Through a grant from the National Cancer Institute to improve cancer outcomes for underserved populations, community radiation oncologists at New Hanover Regional Medical Center (NHRMC) in Wilmington, North Carolina, partnered with those at the University of North Carolina (UNC) in Chapel Hill, North Carolina. TS suites were installed at both sites to facilitate teleconferencing and review of treatment planning for intensity-modulated radiation therapy (IMRT). Patients with locally advanced head and neck cancer at NHRMC who were enrolled on a clinical trial of chemoirradiation underwent IMRT planning utilizing commercial software. NHRMC physicians contoured tumor targets and adjacent healthy organs. Physics staff at NHRMC generated an initial IMRT plan for each patient. Radiation oncologists at UNC then reviewed individual IMRT plans via TS conferences. RESULTS AND CONCLUSION: Between August 2004 and August 2005, seven IMRT plans were reviewed in eight TS conferences. Physician contours of tumor targets and healthy organs, dose volume histograms, IMRT beams, and isodose distributions were shared during each TS conference successfully. Median time for each session was 35 minutes (range, 30 to 75). Physician satisfaction with the interactive planning process was high at both NHRMC and UNC. A cycle would likely evolve of initial intensive use of TS conferences, to gradual use for ongoing quality control, then greater use as the treatment planning technology undergoes its next change. Complex IMRT treatment planning review was feasible between an academic and community hospital via TS with a high level of physician participant satisfaction.

Periareolar injection for localization of sentinel nodes in breast cancer patients.

The goal of this study was to evaluate the periareolar injection of technetium 99m sulfur colloid to identify axillary sentinel nodes and compare the number of sentinel lymph nodes identified with preoperative lymphoscintigraphy to intraoperative biopsy using a handheld gamma probe. A total of 104 consecutive patients diagnosed with invasive breast cancer participated in this prospective study, with 81 patients receiving an intradermal periareolar injection and 23 patients receiving an intradermal peritumoral injection of filtered technetium 99m sulfur colloid. Preoperative lymphoscintigraphy was performed for sentinel node mapping and localization. In addition to selective sentinel node biopsy, axillary dissection was performed on all patients to determine false-negative rates. Routine histologic staining was performed on all identified nodes, along with immunohistochemical staining of sentinel nodes negative on initial routine staining. With an intradermal periareolar injection, the sentinel node identification rate was 91.4% (74/81), axillary metastatic rate 35.1% (26/74), sentinel node positive only 61.5% (16/26), and false negative 3.8% (1/26). With an intradermal peritumoral injection, the sentinel node identification rate was 91.3% (21/23), axillary metastatic rate 42.9% (9/21), sentinel node positive only 88.9% (8/9), and false negative 0% (0/9). A total of 241 sentinel nodes were identified with biplanar lymphoscintigraphy and 173 sentinel nodes were harvested during surgery, yielding a 28.2% increase in sentinel nodes identified with lymphoscintigraphy. This study demonstrates that intradermal periareolar injection of filtered technetium 99m sulfur colloid is successful in identifying axillary sentinel nodes with a low false-negative rate. Preoperative lymphoscintigraphy aids in the identification and surgical planning of sentinel node biopsy and provides an objective measure of surgical performance.

99m Tc anti-CD 15 monoclonal antibody (LeuTech) imaging improves diagnostic accuracy and clinical management in patients with equivocal presentation of appendicitis.

BACKGROUND: Appendicitis frequently presents in an atypical fashion leading to misdiagnosis or a delay in diagnosis. This is particularly true in early cases where the patient may be erroneously discharged from an emergency department and will invariably return with perforated appendicitis. The standard of care is hospital admission for observation or early operation. Adjunctive imaging tests have been used with mixed results in this equivocal patient population. The authors studied a promising new monoclonal antibody, 99mTc-labeled anti-CD 15 (LeuTech; Palatin Technologies, Inc., Princeton, NJ), which specifically targets neutrophils and may be used for imaging appendicitis. This prospective, multicenter, open-label study evaluated the diagnostic efficacy and clinical impact of LeuTech scintigraphy for detecting appendicitis in patients with an equivocal presentation. METHODS: A total of 200 patients (121 females, 79 males; age range 5-86 years; mean age 30.5 +/- 16.5 years) completed the study. Management plan was formulated before and reassessed following LeuTech imaging to determine impact on management. Following intravenous injection of LeuTech, the abdomen was imaged with a standard gamma camera for 30 to 90 minutes. RESULTS: Fifty-nine patients had a histopathologic diagnosis of acute appendicitis. LeuTech identified 53 of 59 patients with appendicitis (90% sensitivity) and was negative in 122 of 141 patients without appendicitis (87% specificity). Accuracy, positive predictive value, and negative predictive value were 88%, 74%, and 95%, respectively. Diagnostic efficacy was unchanged in a subgroup of 48 pediatric patients (5-17 years). Diagnostic images for appendicitis were achieved within 8 minutes postinjection in 50% of patients and within 47 minutes in 90% of patients. Significant shifts in patient management decisions were evident following LeuTech results. LeuTech was well tolerated with no serious adverse events reported. CONCLUSION: LeuTech is a convenient, safe, rapid, and sensitive imaging test for diagnosis of appendicitis and favorably impacts patient management in adult and pediatric patients with equivocal signs and symptoms.

(99m)Tc sestamibi breast imaging for the examination of patients with dense and fatty breasts: multicenter study.

PURPOSE: To evaluate the accuracy of scintimammography as an adjunct to physical examination and mammography in the detection of breast cancer in women with dense and fatty breasts. MATERIALS AND METHODS: A total of 558 women were prospectively enrolled from 42 centers in North America. Images were interpreted by readers blinded to the subjects' clinical history, mammographic findings, and other test results. The Breast Imaging Reporting and Data System classification was used to describe breast density. Parenchymal patterns of "heterogeneously dense" and "extremely dense" were used to classify breasts as dense, whereas "almost entirely fat" and "numerous vague densities" defined fatty breasts. Between-group differences were evaluated with the 2 test for categorical variables and Student t test for continuous variables. Accuracy of scintimammography was assessed against the core laboratory histopathologic evaluation, the standard. The 95% CIs around point estimates of sensitivity, specificity, and positive and negative predictive values were calculated with the normal approximation to the binomial distribution. RESULTS: The analyses were based on 580 breasts with an abnormality; 276 (48%) breasts were dense and 228 had a malignant lesion. Diagnostic properties for scintimammography of fatty versus dense breasts were, respectively, sensitivity, 72% versus 70%; specificity, 80% versus 78%; positive predictive value, 72% versus 67%; negative predictive value, 81% versus 81%; and accuracy, 77% versus 75% (all not significant). Scintimammography led to similar and significant changes in the posttest likelihood of cancer for both dense and fatty breasts. CONCLUSION: The diagnostic accuracy of scintimammography is not affected by breast density.