Faculty Bibliography

PURPOSE: We establish a mechanical injury model for articular cartilage to assess the sensitivity of diffusion tensor imaging (DTI) in detecting cartilage damage early in time. Mechanical injury provides a more realistic model of cartilage degradation compared with commonly used enzymatic degradation. METHODS: Nine cartilage-on-bone samples were obtained from patients undergoing knee replacement. The 3 Tesla DTI (0.18 x 0.18 x 1 mm3 ) was performed before, 1 week, and 2 weeks after (control zero, mild, and severe) injury, with a clinical radial spin-echo DTI (RAISED) sequence used in our hospital. We performed stress-relaxation tests and used a quasilinear-viscoelastic (QLV) model to characterize cartilage mechanical properties. Serial histology sections were dyed with Safranin-O and given an OARSI grade. We then correlated the changes in DTI parameters with the changes in QLV-parameters and OARSI grades. RESULTS: After severe injury the mean diffusivity increased after 1 and 2 weeks, whereas the fractional anisotropy decreased after 2 weeks (P < 0.05). The QLV-parameters and OARSI grades of the severe injury group differed from the baseline with statistical significance. The changes in mean diffusivity across all the samples correlated with the changes in the OARSI grade (r = 0.72) and QLV-parameters (r = -0.75). CONCLUSION: DTI is sensitive in tracking early changes after mechanical injury, and its changes correlate with changes in biomechanics and histology. Magn Reson Med, 2016. (c) 2016 Wiley Periodicals, Inc.

Vitiligo, characterized by progressive melanocyte death, can be initiated by exposure to vitiligo-inducing phenols (VIPs). VIPs generate oxidative stress in melanocytes and activate the master antioxidant regulator NRF2. While NRF2-regulated antioxidants are reported to protect melanocytes from oxidative stress, the role of NRF2 in the melanocyte response to monobenzone, a clinically relevant VIP, has not been characterized. We hypothesized that activation of NRF2 may protect melanocytes from monobenzone-induced toxicity. We observed that knockdown of NRF2 or NRF2-regulated antioxidants NQO1 and PRDX6 reduced melanocyte viability, but not viability of keratinocytes and fibroblasts, suggesting that melanocytes were preferentially dependent upon NRF2 activity for growth compared to other cutaneous cells. Furthermore, melanocytes activated the NRF2 response following monobenzone exposure and constitutive NRF2 activation reduced monobenzone toxicity, supporting NRF2's role in the melanocyte stress response. In contrast, melanocytes from individuals with vitiligo (vitiligo melanocytes) did not activate the NRF2 response as efficiently. Dimethyl fumarate-mediated NRF2 activation protected normal and vitiligo melanocytes against monobenzone-induced toxicity. Given the contribution of oxidant-antioxidant imbalance in vitiligo, modulation of this pathway may be of therapeutic interest

Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and beta-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote beta-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies beta-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-beta, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

A workshop entitled "Radiation-Induced Fibrosis: Mechanisms and Opportunities to Mitigate" (held in Rockville, MD, September 19, 2016) was organized by the Radiation Research Program and Radiation Oncology Branch of the Center for Cancer Research (CCR) of the National Cancer Institute (NCI), to identify critical research areas and directions that will advance the understanding of radiation-induced fibrosis (RIF) and accelerate the development of strategies to mitigate or treat it. Experts in radiation biology, radiation oncology and related fields met to identify and prioritize the key areas for future research and clinical translation. The consensus was that several known and newly identified targets can prevent or mitigate RIF in pre-clinical models. Further, basic and translational research and focused clinical trials are needed to identify optimal agents and strategies for therapeutic use. It was felt that optimally designed preclinical models are needed to better study biomarkers that predict for development of RIF, as well as to understand when effective therapies need to be initiated in relationship to manifestation of injury. Integrating appropriate endpoints and defining efficacy in clinical trials testing treatment of RIF were felt to be critical to demonstrating efficacy. The objective of this meeting report is to (a) highlight the significance of RIF in a global context, (b) summarize recent advances in our understanding of mechanisms of RIF,

Reperfusion injury following acute myocardial infarction is associated with significant morbidity. Activation of neuronal or non-neuronal cholinergic pathways in the heart has been shown to reduce ischemic injury and this effect has been attributed primarily to muscarinic acetylcholine receptors. In contrast, the role of nicotinic receptors, specifically alpha-7 subtype (α7nAChR) in the myocardium remains unknown which offers an opportunity to potentially repurpose several agonists/modulators that are currently under development for neurologic indications. Treatment of ex vivo and in vivo rat models of cardiac ischemia/reperfusion (I/R) with a selective α7nAChR agonist (GTS21) showed significant increases in left ventricular developing pressure, and rates of pressure development without effects on heart rate. These positive functional effects were blocked by co-administration with methyllycaconatine (MLA), a selective antagonist of α7nAChRs. In vivo, delivery of GTS21 at the initiation of reperfusion, reduced infarct size by 42% (p<0.01) and decreased tissue reactive oxygen species (ROS) by 62% (p<0.01). Flow cytometry of MitoTracker Red stained mitochondria showed that mitochondrial membrane potential was normalized in mitochondria isolated from GTS21 treated compared to untreated I/R hearts. Intracellular ATP concentration in cultured cardiomyocytes exposed to hypoxia/reoxygenation was reduced (p<0.001), but significantly increased to normoxic levels with GTS21 treatment, and this was abrogated by MLA pretreatment. Activation of stress-activated kinases, JNK and p38MAPK, were significantly reduced by GTS21 in I/R. We conclude that targeting myocardial 17nAChRs in I/R may provide therapeutic benefit by improving cardiac contractile function through a mechanism that preserves mitochondrial membrane potential, maintains intracellular ATP and reduces ROS generation, thus limiting infarct size.

Study question: What happens to telomere length during human meiotic maturation and pre-implantation development? What is the impact of DNA damage and telomere attrition on human development? Summary answer: DNA damage and telomere attrition limit oocyte maturation in vitro. Telomeres are short in oocytes, increase markedly, and develop increased heterogeneity during pre-implantation development. What is known already: Telomere length reflects aging in many cell types. Sperm, which emerge from spermatogonia throughout the life of the man, have long telomeres. Oocytes from mouse and women maintain short telomeres. Telomerase, the enzyme that lengthens telomeres, is minimally active in mouse and human oocytes and pre-implantation embryos until blastocyst stage. Lacking appreciable telomerase activity, early mouse embryos elongate telomeres via Alternative Lengthening of Telomeres (ALT), which provides robust telomere elongation, but increased genomic instability. We do not know whether ALT occurs during early human development, and if so, when it takes place. Study design, size, duration: 60 immature germinal vesicle (GV) and metaphase I (M1) human oocytes donated to research from women ages 18-45 were collected and in vitro matured (IVM) for up to 48 hours following the subjects' retrieval. Additionally, 28 cryopreserved human embryos donated to research from 7 couples (age 27-42 years old), at the New York University Langone Fertility Center. Participants/materials, setting, methods: Immature oocytes were in vitro matured to metaphase II (M2). Frozen embryos between the 2 pronuclear (2PN) and day 3 (8-10-cell) stage were thawed and dissociated into single blastomere, intact blastocysts were processed whole. Telomere length was evaluated using Single Cell Amplification of Telomere Repeats PCR (SCATRPCR), expressed as a telomere to reference gene ratio (T/R ratio). DNA damage was assessed by immunoflorescent staining. Statistical analysis was performed using one-way ANOVA or T-test where appropriate. Main results and the role of chance: During oocyte maturation, immunostaining revealed that oocytes which arrested at the GV stage and failed to mature, contained robust and abundant DNA damage signaling on their chromosomes compared with successfully matured M2s (mean total florescent units = 35073.4 +/- 10051.8 vs. 843.7 +/- 74.9). Telomere length however did not differ significantly between arrested GV and M2 oocytes (mean T/R ratio = 0.074 +/- 0.040 vs 0.105 +/- 0.067). Telomere length increased significantly (p < 0.05) between M2 oocytes and both 2PN embryos (mean T/R ratio = 0.837 +/- 0.546) and blastocysts (mean T/R ratio = 0.634 +/- 0.260). The most significant elongation (p < 0.0002) occurred by day 2 (2-4 cell) (mean T/ R ratio = 0.957 +/- 767). Between day 2 and day 3 (mean T/R ratio = 0.385 +/- 0.418) telomere length decreased. These data suggest early activation of a telomere lengthening mechanism, prior to zygotic genome activation. Additionally, Intra-embryo telomere length increased until its peak day 3 (Coefficient of Variation = 108.51%) and was at its lowest at the blastocyst stage (Coefficient of Variation = 40.97%) when telomerase becomes active. Together these data suggest that an Alternative Lengthening of Telomeres (ALT) mechanism may be responsible for both increases in telomere length and increased heterogeneity among blastomeres within early embryos. Limitations, reasons for caution: The limited sample size for some of the earliest embryonic stages in addition to the freezing method for the embryos may have affected quality, and an inability to infer developmental ability as the embryos were not cultured to blastocyst. Wider implications of the findings: Our study is the first to perform a molecular characterization of telomere dynamics and the role of DNA damage in human oocytes and embryos at the single cell level and provides the first evidence that ALT is part of normal embryonic development in humans

Pancreatic cancer is a devastating disease where the 5-year survival is less that 10%. Recent studies have shown that ?? T cells are a dominant lymphocyte subset in pancreatic cancer and they promote the progression of the disease. With the emerging use of T cell- based therapy for cancer, ?? T cells are an attractive target for novel immunotherapy in pancreatic cancer.

BACKGROUND/OBJECTIVES: Atypical and severe clinical manifestations of primary and recurrent herpes simplex virus (HSV) infections may present to a pediatric dermatologist for evaluation. The purpose of this study was to characterize the clinical features of the population diagnosed with HSV referred to a pediatric dermatology office. METHODS: This retrospective case series examined patients diagnosed with HSV in a pediatric dermatology practice at an academic medical center from 2005 to 2015. Characteristics of the population were collected and analyzed. RESULTS: In this study of 48 children diagnosed with HSV, 33% presented at age 2 years or younger, with approximately half having exhibited initial symptoms before 2 years of age; 39.6% of the population had six or more outbreaks per year. The outbreaks were equally divided between unifocal and multifocal presentations, with 60% of children without any labial or mucosal involvement. Suppressive treatment was initiated in 33% of patients; the average age at initiation was 6 years. CONCLUSION: Our data characterize a subset of immunocompetent young children who present to pediatric dermatologists with frequent HSV outbreaks that are often multifocal and involve cutaneous sites, with or without mucosal involvement.

Recent data suggest that intraneuronal accumulation of metabolites of the amyloid-beta-precursor protein (APP) is neurotoxic. We observed that transgenic mice overexpressing in neurons a human APP gene harboring the APPE693Q (Dutch) mutation have intraneuronal lysosomal accumulation of APP carboxylterminal fragments (APP-CTFs) and oligomeric amyloid beta (oAbeta) but no histological evidence of amyloid deposition. Morphometric quantification using the lysosomal marker protein 2 (LAMP-2) immunolabeling showed higher neuronal lysosomal counts in brain of 12-months-old APPE693Q as compared with age-matched non-transgenic littermates, and western blots showed increased lysosomal proteins including LAMP-2, cathepsin D and LC3. At 24 months of age, these mice also exhibited an accumulation of alpha-synuclein in the brain, along with increased conversion of LC3-I to LC3-II, an autophagosomal/autolysosomal marker. In addition to lysosomal changes at 12 months of age, these mice developed cholinergic neuronal loss in the basal forebrain, GABAergic neuronal loss in the cortex, hippocampus and basal forebrain and gliosis and microgliosis in the hippocampus. These findings suggest a role for the intraneuronal accumulation of oAbeta and APP-CTFs and resultant lysosomal pathology at early stages of Alzheimer's disease-related pathology.Molecular Psychiatry advance online publication, 25 October 2016; doi:10.1038/mp.2016.189.

BACKGROUND:Dalbavancin is a novel lipoglycopeptide antibiotic that has potent in vitro activity against Gram-positive microorganisms. METHODS:We performed a phase 1, open-label, multicenter study to investigate the pharmacokinetics (PK) and safety of a single dose of intravenous dalbavancin in hospitalized pediatric subjects 3 months to 11 years of age. We combined these data with previously collected adolescent PK data and performed a population PK analysis. RESULTS:Model development was performed using 311 dalbavancin plasma concentrations from 43 subjects. The median age was 5.9 years (range: 0.3-16.9). A 3-compartment, linear PK model was developed. Based on simulations, the following age-dependent dosing regimen was found to achieve similar dalbavancin exposure to that in adults administered a 2-dose regimen: children 6 to <18 years of age, 12 mg/kg (1000 mg maximum) on day 1 and 6 mg/kg (500 mg maximum) on day 8 and children 3 months to <6 years of age, 15 mg/kg (1000 mg maximum) on day 1 and 7.5 mg/kg (500 mg maximum) on day 8. Similarly, the following age-dependent regimen was found to match adult exposure after a single-dose (1500 mg): 6 to <18 years of age, 18 mg/kg (1500 mg maximum) on day 1 and 3 months to <6 years of age, 22.5 mg/kg (1500 mg maximum) on day 1. Nineteen subjects experienced 36 treatment-emergent adverse events. Five of 36 adverse events were assessed as possibly or probably related to treatment. CONCLUSIONS:Dalbavancin pediatric dosing that matched adult exposure was identified. Overall, dalbavancin was well tolerated in our study population.