Faculty Bibliography

INTRODUCTION: According to a recent study from the Center of Disease Control and Prevention, 1 in every 10 Americans aged 12 and older reported chronic use of antidepressants. Chronic use of serotonin re-uptake inhibitors (SSRI) has been linked to impaired bone mineral accrual during skeletal development and osteoporosis [1,2]. We investigated the effect of fluoxetine, the most commonly prescribed SSRI in the U.S., on the complex program of bone regeneration in two disparate models of fracture repair in mice, followed by a thorough assessment of the in vitro mineralization capacity of primary osteoprogenitor cells (OPCs). We hypothesized that fluoxetine exerts a negative effect on osteoblast proliferation and differentiation during the process of fracture repair, resulting in a less mineralized and weaker bony callus. METHODS: Twelve week-old C57BL/6J mice were used following the IACUC guidelines at our institution. Fluoxetine was delivered in the drinking water at 10 mg/kg/day dose during the 3 weeks before surgeries to simulate chronic SSRI use [3]. Bone fracture repair through endochondral ossification was analyzed using a well-established femur fracture model stabilized with an intramedullary rod. Fracture callus was examined at 14 and 28 days. Intramembranous ossification was analyzed using a 1-mm monocortical tibial defect model. Here, injuries were allowed to heal for 7 or 14 days. Samples were subjected to microCT analysis, histomorphometry, TRAP and ALP histochemistry and immunolabeling for osteocalcin and runx2. A set of fractured femurs at d28 was subjected to 4-point biomechanical bending tests. All mice were continuously treated with fluoxetine during the repair period, except for a group of mice in which we aimed at understanding how discontinuation of the SSRI at the time of fracture would affect fracture healing (Fig. 1A). For the in vitro studies, bone marrow stromal cells were cultured in growth media alone or in presence of 5, 10 or 20 microM fluoxetine, with and without serotonin. Cell proliferation was measured using a BrdU colorimetric assay and apoptotic cells were detected by TUNEL labeling. bMSCs were also cultured in osteogenic differentiation media alone or with the aforementioned fluoxetine concentrations. Mineralization activity was analyzed by alizarin red staining and ALP activity and the expression of osteogenic markers was evaluated by qRT-PCR. An additional set of in vitro experiments was carried out with serotonin supplementation at 50mM in growth media or osteogenic media. Cell proliferation and osteogenic differentiation were examined. Student's t test with Holm-Sidak correction were used to quantify differences described in this study. Error bars represent standard deviation. An asterisk symbol (*) denotes a p value of less than 0.05. RESULTS: Fluoxetine-treated mice developed a normal cartilaginous callus at 14 days after fracture. At 28 days, the fluoxetine-treated animals demonstrated a significantly smaller and biomechanically weaker bony callus (Fig. 1B). In order to further dissect the mechanism that resulted in a smaller osseous regenerate, we studied the healing process of monocortical tibial defects as an intramembranous model of bone healing, which confirmed a direct effect of fluoxetine on osteoblast differentiation and mineralization. In vitro studies established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. Interestingly, histologic and microCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus (Fig. 1). DISCUSSION: In summary, our current study shows that chronic fluoxetine treatment negatively affects bone healing by inhibiting proliferation, osteoblast differentiation and mineralization. Data from this study and others provide strong evidence that chronic SSRI use leads to osteoporosis, which is associated with an increased fracture risk. In a translation arm of our study, we aimed at studying the effect of fluoxetine cessation at the time of fracture. In this group, we surprisingly encountered the consistent formation of non-unions with persistent fibrous tissue interposition. Further studies are now focusing at understanding this intriguing finding. (Figure Presented)

Introduction With an ever-increasing number of bone fractures, physicians are constantly faced with patients with delayed unions or non-unions. Aminocaproic acid has been approved and used in joint replacement and spinal fusion surgeries as an anti-fibrinolytic to limit perioperative blood loss (1-3). The efficacy and safety of aminocaproic acid has been studied extensively, and it has been shown to have a very safe and favorable risk profile (2,3). Cuellar et al. showed that aminocaproic acid significantly enhanced spine fusion in an animal model (4). We investigated aminocaproic acid as a pro-osteogenic agent and test whether it can be used as an adjunct to enhance bone regeneration after long-bone fracture in contrast to axial bone. Methods In order to investigate the osteoinductive capacity of aminocaproic acid, we performed an in vitro analysis with mouse marrow mesenchymal progenitor cells cultured with 150, 200, 250 ug/ml aminocaproic acid. The gene expression of osteogenic markers was assessed by quantitative RT-PCR and cell proliferation was measured by BrdU assay. For the in vivo study, we employed two well-established mouse models using 12 week-old male C57BL/6J mice to evaluate intramembranous healing (1 mm monocortical tibia defect) and endochondral healing (femur fracture). Immediately after euthanasia, the tibiae and femurs were dissected, fixed in paraformaldehyde then scanned at high resolution with a Bruker SkyScan microCT. Ten mum resolution images were used to assess bone volume, bone volume/total volume, trabecular thickness, cortical thickness and overall regenerate volume. Histology was completed using Movat's pentachrome and aniline blue to detect cartilaginous and osseous tissues. Histomorphometric analyses were completed as follows: the stained slides were photographed using a Leica digital imaging system (5X objective). The digital images were imported into Adobe Photoshop CC 2015 and the region of interest was set to 106 x 106 pixels. The number of aniline blue/alcian blue stained pixels was determined using the magic wand tool (tolerance setting; 60, histogram pixel setting; cache level 1) by a single blinded investigator, and confirmed by a second independent investigator. Two-tailed Student's t-tests were used to determine significant differences between data sets that are normally distributed. For non-normally distributed data sets, Mann-Whitney U test was used. Significance was attained at p < 0.05 and all statistical analyses were performed with Graphpad Prism software (GraphPad Software, San Diego, California). Results In vitro data showed no significant difference in cell proliferation of mouse bone marrow cells treated at varying concentrations of aminocaproic acid. Osteogenic gene expression, specifically alkaline phosphatase, osterix and osteocalcin, were all significantly decreased at all concentrations of aminocaproic acid treatment while collagen I gene expression was significantly decreased only at higher concentrations (Figure 1). When looking at the femur fracture model (endochondral healing) a significant difference in cartilage volume was noted both on day 14 (p=0.007) and day 21 (p=0.045) (Figure 2). Contrasting the cartilage findings, bone volume on histomorphometric analysis in the femur fracture model was significantly increased in the treatment group compared to the control group on both day 14, p= 0.059 and day 21, p=0.032 (Figure 3). No difference was detected in bone volume in micro CT analysis at either day 14 or day 21 for the femur fracture model. A significant difference in early fracture healing was observed in the tibial defect (intramembranous) model at day 7 in the control group compared to the treatment group in both histomorphometric analyses (p=0.038) and microCT data, where bone volume was reduced in the treated group at day 7 compared to controls (p<0.05). Discussion: Aminocaproic acid is an important hemostasis pharmacologic used to decrease blood loss in joint replacements and spinal fusions but its effects on bone remodeling are not completely understood. We sought to analyze the differences in the effect of aminocaproic acid on the appendicular skeleton versus its effects on the axial skeleton. Our results show a decrease in early bone formation and osteogenic gene expression but no difference in cellular proliferation, which suggests that aminocaproic acid interferes with osteogenic differentiation. Additionally the decrease in cartilage volume in the femur fracture model could indicate that aminocaproic acid interferes with chondrogenesis, which is essential to endochondral ossification and should be further investigated. (Figure Presented)

Pancreatic cancer is the third leading cause of cancer mortality in both men and women in the United States, with poor response to current standard of care, short progression-free and overall survival. Immunotherapies that target cytotoxic T lymphocyte antigen-4, programmed cell death protein-1, and programmed death-ligand 1 checkpoints have shown remarkable activities in several cancers such as melanoma, renal cell carcinoma, and non-small cell lung cancer due to high numbers of somatic mutations, combined with cytotoxic T-cell responses. However, single checkpoint blockade was ineffective in pancreatic cancer, highlighting the challenges including the poor antigenicity, a dense desmoplastic stroma, and a largely immunosuppressive microenvironment. In this review, we will summarize available clinical results and ongoing efforts of combining immune checkpoint therapies with other treatment modalities such as chemotherapy, radiotherapy, and targeted therapy. These combination therapies hold promise in unleashing the potential of immunotherapy in pancreatic cancer to achieve better and more durable clinical responses by enhancing cytotoxic T-cell responses.

Purpose: GPR143 regulates melanosome biogenesis and organelle size in pigment cells. The mechanisms underlying receptor function remain unclear. G protein-coupled receptors (GPCRs) are excellent pharmacologic targets; thus, we developed and applied a screening approach to identify potential GPR143 ligands and chemical modulators. Methods: GPR143 interacts with beta-arrestin; we therefore established a beta-arrestin recruitment assay to screen for compounds that modulate activity. Because GPR143 is localized intracellularly, screening with the wild-type receptor would be restricted to agents absorbed by the cell. For the screen we used a mutant receptor, which shows similar basal activity as the wild type but traffics to the plasma membrane. We tested two compound libraries and investigated validated hits for their effects on melanocyte pigmentation. Results: GPR143, which showed high constitutive activity in the beta-arrestin assay, was inhibited by several compounds. The three validated inhibitors (pimozide, niclosamide, and ethacridine lactate) were assessed for impact on melanocytes. Pigmentation and expression of tyrosinase, a key melanogenic enzyme, were reduced by all compounds. Because GPR143 appears to be constitutively active, these compounds may turn off its activity. Conclusions: X-linked ocular albinism type I, characterized by developmental eye defects, results from GPR143 mutations. Identifying pharmacologic agents that modulate GPR143 activity will contribute significantly to our understanding of its function and provide novel tools with which to study GPCRs in melanocytes and retinal pigment epithelium. Pimozide, one of three GPR143 inhibitors identified in this study, maybe be a good lead structure for development of more potent compounds and provide a platform for design of novel therapeutic agents.

Identification of biomarkers for early detection of lung cancer (LC) is important, in turn leading to more effective treatment and reduction of mortality. Serological proteome analysis (SERPA) was used to identify proteins around 34 kD as ECH1 and HNRNPA2B1, which had been recognized by serum autoantibody from 25 LC patients. In the validation study, including 90 sera from LC patients and 89 sera from normal individuals, autoantibody to ECH1 achieved an area under the curve (AUC) of 0.799 with sensitivity of 62.2% and specificity of 95.5% in discriminating LC from normal individuals, and showed negative correlation with tumor size (rs = -0.256, p = 0.023). Autoantibody to HNRNPA2B1 performed an AUC of 0.874 with sensitivity of 72.2% and specificity of 95.5%, and showed negative correlation with lymph node metastasis (rs = -0.279, p = 0.012). By using longitudinal preclinical samples, autoantibody to ECH1 showed an AUC of 0.763 with sensitivity of 60.0% and specificity of 89.3% in distinguishing early stage LC from matched normal controls, and elevated autoantibody levels could be detected greater than 2 y before LC diagnosis. ECH1 and HNRNPA2B1 are autoantigens that elicit autoimmune responses in LC and their autoantibody can be the potential biomarkers for the early detection of LC.

Cellular potassium import systems play a fundamental role in osmoregulation, pH homeostasis and membrane potential in all domains of life. In bacteria, the kdp operon encodes a four-subunit potassium pump that maintains intracellular homeostasis, cell shape and turgor under conditions in which potassium is limiting. This membrane complex, called KdpFABC, has one channel-like subunit (KdpA) belonging to the superfamily of potassium transporters and another pump-like subunit (KdpB) belonging to the superfamily of P-type ATPases. Although there is considerable structural and functional information about members of both superfamilies, the mechanism by which uphill potassium transport through KdpA is coupled with ATP hydrolysis by KdpB remains poorly understood. Here we report the 2.9 A X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA and a water molecule at a canonical cation site in the transmembrane domain of KdpB. The structure also reveals two structural elements that appear to mediate the coupling between these two subunits. Specifically, a protein-embedded tunnel runs between these potassium and water sites and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. On the basis of these observations, we propose a mechanism that repurposes protein channel architecture for active transport across biomembranes.

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.

Optic gliomas are brain tumors characterized by slow growth, progressive loss of vision, and limited therapeutic options. Optic gliomas contain various amounts of myxoid matrix, which can represent most of the tumor mass. We sought to investigate biological function and protein structure of the myxoid matrix in optic gliomas to identify novel therapeutic targets. We reviewed histological features and clinical imaging properties, analyzed vasculature by immunohistochemistry and electron microscopy, and performed liquid chromatography-mass spectrometry on optic gliomas, which varied in the amount of myxoid matrix. We found that although subtypes of optic gliomas are indistinguishable on imaging, the microvascular network of pilomyxoid astrocytoma, a subtype of optic glioma with abundant myxoid matrix, is characterized by the presence of endothelium-free channels in the myxoid matrix. These tumors show normal perfusion by clinical imaging and lack histological evidence of hemorrhage organization or thrombosis. The myxoid matrix is composed predominantly of the proteoglycan versican and its linking protein, a vertebrate hyaluronan and proteoglycan link protein 1. We propose that pediatric optic gliomas can maintain blood supply without endothelial cells by using invertebrate-like channels, which we termed primitive myxoid vascularization. Enzymatic targeting of the proteoglycan versican/hyaluronan and proteoglycan link protein 1 rich myxoid matrix, which is in direct contact with circulating blood, can provide novel therapeutic avenues for optic gliomas of childhood.

The COP9 signalosome removes Nedd8 modifications from the Cullin subunits of ubiquitin ligase complexes, reducing their activity. Here we show that mutations in the Drosophila COP9 signalosome subunit 1b (CSN1b) gene increase the activity of ubiquitin ligases that contain Cullin 1. Analysis of CSN1b mutant phenotypes revealed a requirement for the COP9 signalosome to prevent ectopic expression of Epidermal growth factor receptor (EGFR) target genes. It does so by protecting Capicua, a transcriptional repressor of EGFR target genes, from EGFR pathway-dependent ubiquitination by a Cullin 1/SKP1-related A/Archipelago E3 ligase and subsequent proteasomal degradation. The CSN1b subunit also maintains basal Capicua levels by protecting it from a separate mechanism of degradation that is independent of EGFR signaling. As a suppressor of tumor growth and metastasis, Capicua may be an important target of the COP9 signalosome in cancer.