Office of Postdoctoral Education,

School of Medicine                                                                                   




1643 Pierce Drive, Room 307

Atlanta, GA 30322

Phone: (404)-727-3302



2nd Annual Postdoctoral Fellow Research Symposium

Session I

9:00 - Agnieska Bialkowska

Haploinsufficiency of Krüppel-like Factor 5 Rescues the Tumor-initiating Effect of the ApcMin Mutation in the Intestine

Department: Medicine: Digestive Disease, Mentor: Vincent Wang, PhD


9:15 – Michael McCabe

Repressive Epigenetic Signatures Predispose CpG Islands to Hypermethylation in Non-Small Cell Lung Cancer

Department: Radiation Oncology, Mentor: Paula Vertino, PhD


9:30 – M. Melissa Gilbert

Slaughterhouse-five, the Drosophila Ortholog of Mammalian HD-PTP, is Allelic to Myopic, and Acts a Conditional Growth Suppressor Gene

Department: Cell Biology, Mentor: Kenneth Moberg, PhD


9:45 – Gang Chen

Trim3, the Human Homolog of Drosophila Brat, Regulates Neural Differentiation, Myc Expression and May Control Glioma Stem Cells Properties

Department: Pathology, Mentor: Daniel J. Brat, MD/PhD


10:00 – Divya Gupta

Receptors for Advanced Glycation Endproducts Cause Decreased Collateral Vessel Formation in the Face of Uncontrolled Diabetes

Department: Medicine: Cardiology, Mentor: W. Robert Taylor, MD/PhD


10: 15 – Brian Crawford

PPARg and Oxidative Stress Injury in the Developing Heart

Department: Pediatrics, Mentor: Mary Wagner, PhD


10:30 – Kellie Jo White

Regulation of Actin Filament Dynamics in C. elegans

Department: Dermatology, Mentor: Robert Swerlick, MD




Session II

1:15 – Liya Wang

Alterations in Cortical Thickness and White Integrity in MCI Measured by Thickness Mapping and DTI

Department: Radiology Mentor: Hui Mao, PhD


1:30 – Kristy Welshhans

Role of Zipcode Binding Protein 1 and its Phosphorylation by Src Kinase in Local Protein Synthesis and Growth Cone Guidance

Department: Cell Biology, Mentor: Gary Bassell, /PhD


1:45 – Simon Lacey

View-Independence of Visuo-Haptic Object Representations

Department: Neurology, Mentor: Krish Sathian, MD/PhD


2:00 - Vijayakuman Velu

Enhancing SIV-specific immunity in vivo by PD-1 Blockade

Department: Microbiology & Immunology, Mentor: Rama Rao Amara, PhD


2:15 – Ying Lu

Healthcare Utilization by the Elderly and Impact on Diagnosis of Melanoma

Department: Hematology & Medical Oncology, Mentor: Edmund Waller, PhD


2:30 – Richard Dunham

Hepatic Stellate Cells Preferentially Prime Regulatory T Cells

Department: Microbiology and Immunology, Yerkes National Primate Research Center, Mentor: Arash Grakoui, PhD




Session III

3:00 – Matthew Wagner

The  Ligand, Rosiglitazone, Attenuates Lung Barrier DysfunctiongPPAR- Following Chronic Alcohol Ingestion

Department: Medicine/Pulmonary Diseases Mentor: C. Michael Hart, MD


3:15 – Rami Yacoub

Streptavidin Conjugated Quantum Dots for Multiplex Detection of Cancer-Related Biomarker

Department: Hematology &Medical Oncology, Mentor: Ruth O’Regan, MD


3:30 – Bindu Chandrasekharan

Neuropeptide-epithelial Interactions Modulate Inflammatory Signaling and Epithelial Permeability in Inflammatory Bowel Disease

Department: Medicine: Digestive Diseases, Mentor: Shanthi Srinivasan, MD


3:45 – Quinghuan Xiao

Regulation of hBest1 Chloride Channel by Phophorylation: Effects of Protein Kinace C, Protein phosphatase 2A, and hypertonic stress

Department: Cell Biology, Mentor: H. Criss Hartzell, PhD


4:00 – Alyssa Long

Understanding Craniofacial Development: Lessons from a Mutant Mouse

Department: Human Genetics, Mentor: Tamara Caspary, PhD


4:15 –Nguyen Phuong

Effects of Zinc Supplementation on Growth of Children Under 5 years of age: A Meta-Analysis of Randomized Controlled Trials

Department: Global Health, Mentor: Reynaldo Martorell, PhD






Haploinsufficiency of Krüppel-like Factor 5 Rescues the Tumor-initiating Effect of the ApcMin Mutation in the Intestine


Agnieszka B. Bialkowska, Beth B. McConnell, Mandayam O. Nandan, Amr M. Ghaleb, Frank J. Gordon, and Vincent W. Yang


Mentor: Vincent W. Yang, MM, PhD


Department: Medicine/Digestive Diseases


Abstract: Inactivation of the tumor suppressor, adenomatous polyposis coli (APC), with the resultant activation of b-catenin, is the initiating event in the development of a majority of colorectal cancer. Krüppel-like factor 5 (KLF5), a pro-proliferative transcription factor, is highly expressed in the proliferating intestinal crypt epithelial cells. To determine whether KLF5 contributes to intestinal adenoma formation, we examined tumor burdens in ApcMin/+ mice and ApcMin/+/Klf5+/- mice. Compared to ApcMin/+ mice, ApcMin/+/Klf5+/- mice had a 96% reduction in the number of intestinal adenomas. Reduced tumorigenicity in the ApcMin/+/Klf5+/- mice correlated with reduced levels and nuclear localization of b-catenin as well as reduced expression of two b-catenin targets, cyclin D1 and c-Myc. In vitro studies revealed a physical interaction between KLF5 and b-catenin that enhanced b-catenin’s nuclear localization and transcriptional activity. Thus, Klf5 is necessary for the tumor-initiating activity of b-catenin during intestinal adenoma formation in ApcMin/+ mice, and reduced Klf5 expression offsets the tumor-initiating activity of the ApcMin mutation by reducing the nuclear localization and activity of b-catenin.


Source of Funding for Research: NIH grant



Repressive Epigenetic Signatures Predispose CpG Islands to Hypermethylation in Non-Small Cell Lung Cancer


Michael T. McCabe1 and Paula M. Vertino1,2

1Department of Radiation Oncology and 1,2Winship Cancer Institute, Emory University, Atlanta, GA.


Mentor:  Paula M. Vertino, PhD


Department:  Radiation Oncology


DNA methylation is responsible for the transcriptional silencing of many tumor suppressor genes in human cancers.  While most CpG islands remain unmethylated in both normal and cancer cells, a subset acquire aberrant de novo methylation during tumorigenesis via unknown mechanisms.  Recent studies have suggested that features of the underlying chromatin may regulate the susceptibility of different genes to hypermethylation.  In order to further investigate this relationship, we first established a dataset of CpG islands hypermethylated in cancer.  Utilizing the Illumina Infinium DNA methylation platform, we determined the methylation status of greater than 10,000 CpG islands in a cohort of paired non-small cell lung adenocarcinomas and adjacent normal tissues.  While the majority (~75%) of CpG islands remained unmethylated in all tumors, ~3% of CpG islands exhibited recurrent (³25% of tumors) hypermethylation.  Functional analysis revealed that these CpG islands were highly enriched in genes encoding developmental proteins, transcription factors, and homeodomain-containing proteins.  In order to understand the chromatin features that might predispose CpG islands to hypermethylation, we performed detailed analyses of ChIP-Seq experiments from a normal adult cell type.  Computational examination of 40 individual histone modifications and DNA binding proteins revealed depletion of activating histone modifications accompanied by enrichment of multiple repressive marks in the vicinity of methylation-prone CpG islands.  Combinatorial analysis of all 40 variables revealed unique patterns of histone modifications among methylation-prone CpG islands when compared to methylation-resistant CpG islands implying the potential for coordinated function of several histone modifications in the establishment of a methylation-prone chromatin.  These studies suggest that hypermethylation-prone CpG islands exhibit unique chromatin signatures in normal cells which may play a role in the targeting of aberrant methylation to these loci during tumorigenesis.


Source of Funding for Research: American Cancer Society grant PF-07-130-01-MGO and Frederick Gardner Cottrell Postdoctoral Fellowship to MTM, Georgia Cancer Coalition funds to PMV, and National Cancer Institute grants CA077337 and CA116676 to PMV.


Slaughterhouse-five, the Drosophila Ortholog of Mammalian HD-PTP, is Allelic to Myopic, and Acts a Conditional Growth Suppressor Gene


M. Melissa Gilbert and Kenneth H. Moberg


Mentor:  Kenneth H. Moberg, Ph.D.


Department:  Cell Biology


In mammalian systems, there exists a well-defined link between aberrant cellular proliferation and cell death.  Although linear signaling systems exist in Drosophila that simultaneously regulate both cellular proliferation and cell death, the existence of survival-dependent conditional tumor suppressors has largely been unexplored. We have conducted a mosaic screen in the Drosophila eye for genes that restrict growth only in a background in which cell death is blocked. We have isolated alleles of a gene we named slaughterhouse-five (sfv) that is required to restrict organ size in multiple epithelial tissues.  sfv encodes the Drosophila ortholog of mammalian His-domain protein tyrosine phosphatase (HD-PTP) and is allelic to Drosophila myopic (mop), a gene previously shown to potentiate EGFR signal transduction.  Our sfv alleles exhibit overgrowth phenotypes that are strikingly similar to ones in the Hippo/Salvador/Warts (H/S/W) pathway. We present evidence that sfv/mop is a component of the HSW pathway, and that loss of the gene drives growth by impairing HSW signaling.


Source of funding for Research:  N.I.H. grant to KHM, N.C.I. grant to KHM and NRSA

to MMG.



Trim3, the Human Homolog of Drosophila brat, Regulates Neural Differentiation, Myc Expression and may Control Glioma Stem Cells Properties


Gang Chen, X Fan, F Rahman, Y Rong, C Tucker-Burden, S Dougan, C Hadjipanayis, E G Van Meir, D J Brat      


Mentor: Daniel J Brat, MD, PhD


Department: Pathology


Glioblastoma (GBM) is the most common and most malignant primary brain tumor, yet the molecular mechanisms responsible for its development have not been fully elucidated.  Mutations in Drosophila Brain tumor (brat) result in a massively enlarged fly brain that consists of neuroblast cell population with neoplastic growth properties. Brat has been shown to direct neural differentiation at least partially through its translational suppression of Myc. We investigated the role of brat homologs in zebrafish neural differentiation and in human brain tumors. In zebrafish, we used a morpholino oligo directed at the brat homolog Trim3b and found a central nervous system phenotype characterized by an enlarged midbrain, expansion of the ventricular system and an enrichment of neuroblastic cells showing differentiation immediately adjacent to the ventricles. In situ hybridization for Notch1a and nestin showed reduced expression compared to age-matched control siblings, consistent with a lower cell number, but also showed an even greater reduction in markers of cellular differentiation, including GFAP and Hu, suggesting impaired differentiation capacity. Trim3 is highly expressed in human brain but the function of Trim3 is largely unknown. A substantial subset of GBMs (25-30%) shows loss of heterozygosity (LOH) of chromosome 11p15.5, the site of Trim3. We found the mRNA expression of Trim3 was markedly reduced in human GBM cell lines compared to human astrocytes and also found that Trim3 was reduced in human GBM specimens compared to normal brain, consistent with results from mining existing gene expression data sets (oncomine).  Since Drosophila brat is critical to neural differentiation and regulates Myc, we explored whether human Trim3 might regulate glioma stem cell properties by its regulation of Myc. We found that the forced expression of Trim3 in human GBM cell lines led to reduced Myc protein expression. This inverse relationship between Trim3 and Myc also held true in GBM samples.


Source of Funding for Research: NIH 5R01NS053727-04



Receptors for Advanced Glycation Endproducts Cause Decreased Collateral Vessel Formation in the Face of Uncontrolled Diabetes


Divya Gupta, Daiana Weiss, Catherine Norton, Robert E Guldberg and W R Taylor


Mentor: W Robert Taylor, MD, PhD


Department: Medicine/Cardiology


Introduction:  Diabetes is associated with multiple vascular complications and is associated with the development of poor collateral vasculature.  The precise cellular and molecular processes that are involved in impaired collateral development in diabetes are not well defined.  Chronic diabetes leads to the formation of advanced glycation endproducts (AGEs) which can alter molecular functions through the Receptor for Advanced Glycation Endproducts (RAGE).  Thus, the major goal of this study was to explore the contribution of RAGE to impaired collateral growth in the setting of diabetes mellitus (DM).

Hypothesis:  Decreased RAGE expression will lead to improved collateral vessel formation in the setting of hyperglycemia.

Methods:  Four groups of mice on a C57/Bl6 background were created: Wild type (WT), WT+DM, RAGE Knockout (RAGE KO), RAGE KO+DM. Streptozocin was given intraperitoneally to induce diabetes. After 4-5 weeks of DM, the left femoral artery was ligated for a hind limb ischemia model.  Laser Doppler Perfusion Imaging (LDPI) was used for quantitive evaluation of collateral vessel development over 21 days.  Alpha-actin smooth muscle staining was used for quantitative anatomic evaluation of neovascularizatoin. Spontaneous running duration was employed as a functional endpoint.

Results: WT+DM mice had the lowest level of blood flow recovery  over the 21 day period of all three groups,  with a significant difference when compared to RAGE KO+DM (34 +/- (use special symbols) 6.3, 80.8 +/- 12; p< 0.001).(does this meanthis was the only sig difference ??? maybe add table with all data?? Also add units)  These significant differences between WT+DM and RAGE KO+DM mice were also present when assessing data obtained from the running wheels (463 +/- 40, 9383 +/- 342 (in meters); p<0.001) and histology when assessing the  number of new vessels (-9.6 +/- 38.2, 409.1 +/- 86.9; p< 0.05).

Conclusions:  RAGE appears to play a significant role in the hindrance of collateral vessel development in the face of hyperglycemia.




PPARg and Oxidative Stress Injury in the Developing Heart


Brian H. Crawford, Ming Shen, Ronald W. Joyner, Mary B. Wagner, and Guoliang Ding


Mentor: Mary B. Wagner, PhD


Department: Pediatrics


The peroxisome proliferator-activated receptor-g (PPARg) is a nuclear receptor that is expressed in many tissues, including myocardium. Recent studies have shown that PPARg directly binds to the promoters of two anti-oxidative enzymes, manganese-superoxide dismutase (SOD2) and catalase (CAT), and regulates their expression in heart and other tissue. Numerous studies have shown that the newborn (NB) heart is more sensitive to reperfusion injury than the adult (AD) heart; however, the mechanism remains unclear. In this study, we investigated the acute effects of oxidative stress, by application of hydrogen peroxide (H2O2), in NB (1-4 days) and AD (3-4 mos) rabbit heart. Isolated ventricular cardiomyocytes were paced by field stimulation (cycle length 1000 ms) while sarcomere shortening and calcium transients were recorded before and after 9 minutes exposure to a 200 mM H2O2 solution at room temperature. The H2O2 treatment significantly decreased the sarcomere shortening (61%) in NB with no change in AD cardiomyocytes. However, there was no significant change of calcium transient in either NB or AD cardiomyocytes.  mRNA assays indicated that the expression of PPARg and SOD2 were significantly higher (51% and 121% , respectively) in AD compared to NB cardiomyocytes. Perfusion with 200μM H2O2 resulted in a significant increase in the mRNA expression of PPARg (178%) and SOD2 (67%) in the NB heart. The AD heart showed no significant change after H2O2 treatment.  Additionally, the ability of PPARg to up-regulate SOD2 and CAT was evaluated by treating NB and AD rabbit heart with PPARg activator, rosiglitazone (ROSI).  Treatment with ROSI significantly increased mRNA levels of SOD2 by 115% in NB and 88% in AD, and CAT by 93% in NB and 81% in AD heart. This data collectively suggests that (a) isolated NB rabbit ventricular cells are more susceptible to oxidative stress caused by acute application of H2O2 than AD cells which may be due to lower expression of PPARg; and (b) the activation of PPARg may stimulate an anti-oxidative effect in cardiomyocytes by up-regulating the antioxidant enzymes, SOD2 and CAT.


Source of Funding for Research: EECRC Seed Grant




Role of ATP Release and Purinergic Receptors in Irritant-induced EGFR Activation


Kellie J. White1, 2, Vincent J. Maffei1, Marvin Newton-West1, 2, and Robert A. Swerlick1,2. 

1 Department of Dermatology Emory University School of Medicine, Atlanta, GA. 2 Dermatology, Atlanta VA Affairs Hospital, Atlanta, GA


Mentor:  Robert A. Swerlick, MD


Department:  Dermatology


Irritant contact dermatitis (ICD) occurs rapidly following exposure to an irritant compound and exhibits key features of inflammation.  Despite a significant body of research about ICD, information regarding early events following exposure to an irritant compound remains limited.  Previously, we reported on the role of metalloproteases and the epidermal growth factor receptor (EGFR) following stimulation with the model irritant sodium lauryl sulfate (SLS) in HaCaT cells.  Early time point treatment of HaCaT cells confirmed that unlike TGFa that resulted in phosphorylation of EGFR and ERK after 1 min of stimulation, p-ERK was not present until after 5 min of SLS stimulation, suggesting involvement of an intermediate.  Consequently, assessment of ATP release following SLS treatment showed an increase in extracellular ATP, consistent with previously published data.  We hypothesized that ATP interaction with purinergic receptors may result in EGFR activation.  Consistent with this hypothesis, ATP-g-S (300mM) resulted in time-dependent ERK activation and de novo protein synthesis of early growth factor (egr)-1 in HaCaT cells that were sensitive to the protease inhibitor marimastat and the specific EGFR inhibitor PD168393.  Moreover, IL-8, VEGF, and egr-1 mRNAs were also increased, and induction was sensitive to protease and EGFR inhibitors.  Finally, nonspecific metabotropic P2Y purinergic receptor inhibitors were used to inhibit protein and mRNA induction.  A3P5AS and PPAD had no effect on SLS-induced pro-inflammatory genes.  Reactive blue 2, however, decreased SLS- and ATP-g-S-, but not TGFa-induced, VEGF and egr-1 mRNAs.  Interestingly, there was little effect on IL-8 mRNA.  Finally, neither AMP nor adenosine was capable of inducing egr-1 or any of the pro-inflammatory genes of interest.  Our data suggest a role for irritant-mediated ATP release and epithelial cell activation via P2Y purinergic receptors in ICD.


Source of Funding for Research:  NIH Ruth L. Kirschstein National Research Service Award, Department of Dermatology, Dermatology Foundation



Role of Zipcode Binding Protein 1 and its Phosphorylation by Src Kinase in Local Protein Synthesis and Growth Cone Guidance


Kristy Welshhans, Yukio Sasaki, Gary J. Bassell


Mentor: Gary J. Bassell, PhD


Department: Cell Biology


The local protein synthesis of β-actin in axons and growth cones is an important mechanism that has been demonstrated to regulate growth cone guidance. An mRNA binding protein, zipcode binding protein 1 (ZBP1), binds to a 54 nucleotide sequence within the 3’ untranslated region (UTR) of β-actin mRNA and regulates its localization and translation. It has previously been shown in neuroblastoma cells that the binding of ZBP1 to β-actin mRNA results in translational repression, which can be relieved by Src phosphorylation of ZBP1 at Tyr396. We have extended this study in neuronal growth cones to demonstrate that BDNF stimulates the local translation of a β-actin mRNA reporter in a 3’UTR- and Src kinase-dependent manner. Furthermore, we have found that overexpression of a non-phosphorylatable mutant of ZBP1 suppresses attractive growth cone turning, but does not affect axon outgrowth. We are now extending these studies using ZBP1 knockout (ZBP1 KO) mice. Growth cones cultured from ZBP1 KO mice exhibit defects in filopodial dynamics and growth cone turning; however, axon outgrowth appears normal. These results suggest that local phosphorylation of ZBP1 by Src kinase results in local translation of β-actin mRNA, thereby regulating multiple aspects of growth cone motility, including growth cone guidance.


Source of Funding for Research: NINDS (F32NS064727) to KW and NIH (RO1 HD46368-06) to GJB.



View-Independence of Visuo-Haptic Object Representations


Simon Lacey1, Marisa Pappas1, Alexandra Kreps1, Kevin Lee1, K. Sathian1,2,3,4

1Depts. of Neurology, 2Rehabilitation Medicine, 3Psychology, Emory Univ. School of Medicine, Atlanta, GA. 4Rehabilitation R&D Center of Excellence, Atlanta VAMC, Decatur, GA.


Mentor: Krish Sathian, MD, PhD


Department: Neurology


We previously showed that cross-modal recognition of unfamiliar objects is view-independent, in contrast to view-dependence within-modally in both vision and haptics. Does the view-independent bisensory representation underlying cross-modal recognition arise from integration of unisensory view-dependent representations, or intermediate unisensory but view-independent representations? Two psychophysical experiments sought to distinguish between these alternative models. In both experiments, participants began from baseline within-modal view-dependence for object recognition in both vision and haptics. The first experiments induced within-modal view-independence by perceptual learning, which was completely and symmetrically transferred cross-modally: visual view-independence acquired through visual learning also resulted in haptic view-independence and vice versa. In the second experiment, both visual and haptic view-dependence were transformed to view-independence by either haptic-visual or visual-haptic cross-modal learning. We conclude that cross-modal view-independence fits with a model in which unisensory view-dependent representations are directly integrated into a bisensory view-independent representation, rather than being gated by unisensory view-independent representations.


Source of Funding for Research: N.I.H., N.S.F. and V.A. grants to K.S.



Enhancing SIV-specific Immunity in vivo by PD-1 Blockade


Vijayakumar Velu1, Kehmia Titanji1, Baogong Zhu2, Sajid Husain1, Annette Pladevega1, Lilin Lai1, Thomas H. Vanderford3, Lakshmi Chennareddi1, Guido Silvestri3, Gordon J. Freeman2, Rafi Ahmed1, Rama Rao Amara1

1Emory Vaccine Center, Yerkes National Primate Research Center, Emory University School of Medicine, Atlanta, GA, 2Harvard Medical School, Boston, MA, 3University of Pennsylvania, Philadelphia, PA.


Mentor:  Rama Rao Amara, PhD


Department:  Microbiology and Immunology


Chronic immunodeficiency virus infections are characterized by dysfunctional cellular and humoral antiviral immune responses. As such, immune modulatory therapies that enhance and/or restore the function of virus-specific immunity may protect from disease progression. Here, we investigate the safety and immune restoration potential of the blockade of co-inhibitory receptor programmed death-1 (PD-1) during chronic SIV infection in macaques. We demonstrate that PD-1 blockade using an antibody to PD-1 is well tolerated and results in rapid expansion of virus-specific CD8 T cells with improved functional quality. This enhanced T cell immunity was seen in the blood and also in the gut, a major reservoir of SIV infection. PD-1 blockade also resulted in proliferation of memory B cells and increases in SIV envelope-specific antibody. These improved immune responses were associated with significant reductions in plasma viral load and also prolonged the survival of SIV-infected macaques. Impressively, blockade was effective during the early (wk10) as well as late (~wk90) phases of chronic infection even under conditions of severe lymphopenia. These results demonstrate enhancement of both cellular and humoral immune responses during a pathogenic immunodeficiency virus infection by blocking a single inhibitory pathway and identify a novel therapeutic approach for HIV/AIDS.


Source of Funding for Research: NIH R01AI074417, FNIH P51RR00165




Paradoxical Effects of Interferon-γ in Allogeneic Bone Marrow Transplant: Donor Dendritic Cells Regulate Graft-Versus-Leukemia and Graft-Versus-Host Disease Activities


Ying Lu, Jian-Ming Li, Wayne Harris and Edmund Waller


Mentor:  Edmund K. Waller, MD, PhD


Department:  Hematology and Medical Oncology


Both host and donor dendritic cells (DCs) have been shown to play a critical role in regulating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after MHC-mismatched bone marrow transplantation (BMT). In contrast to host DCs, much less is known about the mechanisms donor DCs may use to modulate donor T-cell activation. A clinical report has suggested an association between donor plasmacytoid DC in the graft and leukemia relapses after allogeneic BMT. Using allogeneic MHC-mismatched BMT (C57BL/6→B10.BR) in mice bearing a T lymphoblastic leukemia LBRM, we have previously reported that recipients transplanted with purified CD11b- DC in combination with purified HSC and T-cells had 45% increased long-term leukemia-free-survival, higher numbers of interferon-γ (IFN-γ) producing donor T-cells as well as higher levels of serum IFN-γ. The aim of the present work is to further define whether production of IFN-γ by donor T-cells is necessary for the augmentation of GVL effect seen with CD11b- donor DC and define the mechanism that donor CD11b- DC can augment GVL of donor T-cells without causing fatal GVHD. To evaluate the role for IFN-γ produced by donor T-cells, we used IFN-γ knockout (KO) mice as donors in our BMT model. Recipients of IFN-γ KO donor T-cells in combination with wild-type HSC and CD11b- DC died rapidly with 0% survival at day 80 compared with 65% survival among tumor-bearing recipients of donor CD11b- with wild-type HSC and T-cells and 75% survival in mice transplanted with wild-type cells in the absence of LBRM. Moreover, the addition of donor CD11b- DC to IFN-γ KO donor T-cells did not lead to further augmentation of GVHD. These data supported a role for donor T-cell-derived IFN-γ in the enhanced GVL activity seen among recipients of donor CD11b- DC, but did not explain the lack of increased GVHD. As a potent pro-inflammatory cytokine initiating immune response in GVHD, IFN-γ has also been demonstrated to show a suppressive effect during GVHD as a result of IFN-γ-inducible indoleamine-2,3-dioxygenase(IDO) gene expression. Our results showed that following IFN-γ treatment, IDO levels of freshly isolated CD11b- DCs were up-regulated. Furthermore, in vitro co-culture of CD11b- DC with syngeneic T-cells in the presence of allogeneic antigen also demonstrated increased IDO levels on the co-cultured DCs. Taken together, our data support a model in which donor CD11b- DCs initially induce Th1 polarization of activated donor T-cells that secret high levels of IFN-γ in the microenvironment. High local levels of IFN-γ subsequently induce IDO expression in DC, resulting in down-modulation of T-cell allo-reactivity and GVHD.

Source of Funding for Research: N.I.H. grant to E.K.W


Hepatic Stellate Cells Preferentially Prime Regulatory T Cells


Richard M. Dunham, Raghavan Chinnadurai, Timothy L. Denning and Arash Grakoui


Mentor: Arash Grakoui, PhD


Department: Microbiology and Immunology, Yerkes National Primate Research Center


The liver microenvironment has long been understood as immunosuppressive, though the mechanisms underlying this phenomenon are not well understood. It has recently been shown that hepatic stellate cells (HSC), a population of nonparenchymal cells in the liver, can present antigen to CD4+ T cells. However, little is known of the way in which these cells influence the differentiation of CD4+ T cells. Importantly, activation of hepatic stellate cells (HSC) is associated with production of the Treg polarizing molecules TGFβ and retinoic acid (RA). We therefore hypothesized that HSC would prime naïve T cells to differentiate into Treg. To test this hypothesis we investigated the molecular interactions between primary murine HSC and naïve CD4+ T cells from the OT-II TCR transgenic strain. We confirmed that HSC do prime proliferation and cytokine production from naïve CD4+ T cells and that HSC preferentially prime Treg in the presence of exogenous TGFβ. RA produced by metabolism of retinol in HSC by the enzymes RALDH-1 and -2 was essential for Treg conversion at lower TGFβ concentrations. Thus, HSC represent a population of liver APC that preferentially induce the generation of Treg and therefore may play a role in the tolerogenic nature of the liver.


Source of Funding for Research: EVC/CFAR Flow Cytometry Core P30 AI050409, Cancer Research Institute Investigator Award (AG), the Yerkes Research Center Base Grant RR-00165 and the Public Health Service AI070101 (AG).



The PPAR- Ligand, Rosiglitazone, Attenuates Lung Barrier Dysfunction Following Chronic Alcohol Ingestion


Matthew C. Wagner, Dean J. Kleinhenz, and C. Michael Hart


Mentor:  C. Michael Hart, MD


Department: Medicine/Pulmonary Diseases

Chronic alcohol consumption increases the incidence of Acute Respiratory Distress Syndrome in part through alcohol-induced, NADPH oxidase and eNOS-mediated increases in oxidative stress in the lung. We hypothesized that rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPAR) ligand that attenuates NADPH oxidase expression, would attenuate alcohol-induced pulmonary oxidative stress and alveolar capillary barrier dysfunction.  To explore this hypothesis, C57Bl/6 mice consumed standard chow ± ethanol (EtOH, 20% w/v) in drinking water for 12 weeks.  After 11 weeks, mice were gavaged daily with rosiglitazone (10 mg/kg) or vehicle for 1 week.  Mice were then treated ± lipopolysaccharide (LPS, 2mg/kg IP) 6 h and 3 h prior to sacrifice.  LPS treatment stimulated lung barrier dysfunction evidenced by increases in bronchoalveolar lavage (BAL) protein levels.  Chronic EtOH ingestion enhanced LPS-mediated increases in BAL protein, whereas rosiglitazone attenuated BAL protein levels in EtOH and EtOH + LPS-treated mice.  In vitro studies employing HUVEC monolayers on transwell filters confirmed that EtOH (0.1% for 72 h)-induced endothelial barrier dysfunction was attenuated by rosiglitazone. Chronic EtOH ingestion increased levels of eNOS protein expression in lung tissue, an increase that was also attenuated by rosiglitazone treatment.  These findings indicate that PPAR may represent a novel therapeutic target for reducing the barrier disruptive effects of alcohol on the lung despite continued EtOH ingestion. 

Source of Funding for Research: NIAAA and the VA Research Service.



Streptavidin Conjugated Quantum Dots for Multiplex Detection of Cancer-Related Biomarker


Rami Yacoub1,  Seema Harichand-Herdt1, Vanita Cohen2, Roberd M. Bostick1, Ruth M.O'Regan1

1Winship Cancer Institute, Emory University, Atlanta, GA; 2Department of Epidemiology, Emory University, Atlanta, GA


Mentor: Ruth M. O’Regan, MD


Department: Hematology and Medical Oncology


Introduction: Identification of biomarkers in cancers requires multiplex simultaneous profiling of cancer cells or tumor tissue, along with accurate measurements of their expressional level. Immunohistochemistry (IHC) is currently one of the most common detection methods in pathology and clinical diagnosis. However, it remains semi-quantitative, subjective with considerable inter-observer variation, and with no ability to detect multiple targets on a single sample of tissue.  Semiconductor nanocrystals, also known as quantum dots (QDs), represent a new class of fluorescent labels with unique optical properties and the potential to overcome limitations of traditional IHC techniques. We have used streptavidin-conjugated quantum dots (SAQD) in QD based-IHC assay. This method helps to address the two major deficiencies of antibody-based labeling QD: the limited availability of species-specific secondary antibodies, the size of the QD conjugate after antibody attachment, and the instability of many antibody–antigen interactions which lead in these cases to dissociation of the antibody-conjugated QD from the protein of interest. Biotin detection by streptavidin has numerous advantages; It is one of the tightest interactions known (K d = 10–13 M), with an off-rate on the order of days. In addition, streptavidin displays little nonspecific binding and is significantly smaller than an IgG antibody (60 versus 150 kDa), and a large number of streptavidin conjugates are commercially available. Methods: We evaluated the use of SAQD with distinct emission spectra to “stain” formalin-fixed, paraffin-embedded (FFPE) breast cancer, normal colon, and tonsil tissues using a panel of primary antibodies targeting known biomarkers. Standard IHC controls were used for comparison and validation of our QD-IHC protocol.  Most, but not all, antibody and quantum dot combinations worked with equal efficacy.  The stains were performed by layering each antibody and QD in a sequential fashion. Results:  Staining of two, three, and four antigens per slide was accomplished successfully on FFPE tonsil, breast tumor and normal colon tissues.  QD-IHC processed tissues were imaged and analyzed using Olympus IX71 inverted fluorescence microscope (Olympus Imaging America Inc., PA, USA) with a long-pass emission filter, and CRI Nuance spectral analyser (CRI Inc., Woburn, MA) used to capture image files at 10 nm intervals from 500 to 800 nm at 20x and 100x magnification.



Neuropeptide-epithelial Interactions Modulate Inflammatory Signaling and Epithelial Permeability in Inflammatory Bowel Disease


Bindu Chandrasekharan, Shanthi V Sitaraman & Shanthi Srinivasan


Mentor: Shanthi Srinivasan, MD


Department: Medicine/Digestive Diseases


Introduction: Enteric neurons producing NPY constitute 25% of the sub mucosal and 5% of myenteric plexi neurons of the murine enteric nervous system (ENS). We determined if NPY could modulate inflammatory signaling through TNF-α in the ENS, and modulate epithelial permeability in experimental colitis. Methods: Colitis was induced in mice by 3% DSS in drinking water for 5 days. On day 6, RNA was obtained from distal colonic enteric ganglia isolated by laser capture micro dissection (LCM), and NPY expression was assessed by Real time PCR. Enteric neuronal cultures stimulated with TNF-α for 24 h were tested for NPY expression by RT-PCR.  Enteric neurons transfected with deletion sequences of the NPY promoter (-1078, -769, -278) were stimulated with TNF-α (10 picomol) for 5 h, and NPY promoter activity was determined by luciferase assay. Caco-2 BBE epithelial cells grown on filters were treated with NPY and trans epithelial resistance (TER as Ohms/cm2) measured at 0, 30, 60 and 120 min. The claudin-2 protein in control and NPY treated cells were compared by Western blotting. Results: Real-time PCR demonstrated 3.7 fold increase in NPY expression in the enteric ganglia of DSS- treated WT mice compared to controls (P <0.001). NPY expression was up regulated in enteric neurons treated with TNF- α (P <0.001). TNF-α production was also higher in enteric neurons exposed to NPY (P <0.01).  NPY promoter activity was two-fold up regulated in enteric neurons treated with TNF-α (P <0.05).  NPY decreased the TER and increased claudin-2 protein in CaCo-2 BBE cells, thus increasing epithelial permeability (P < 0.05). Conclusions: We identified the roles of NPY in augmenting inflammatory signaling and increasing colonic epithelial permeability. Further studies on regulation of cytokine and epithelial tight junction protein expression by neuropeptides would help in understanding the development and progression of inflammation in IBD.


Source of Funding for Research:   Crohn’s and Colitis Foundation of America (CCFA)


Regulation of hBest1 Chloride Channel by Phophorylation: Effects of Protein Kinace C, Protein phosphatase 2A, and hypertonic stress


Qinghuan Xiao, Kuai Yu, Andrew Prussia, Yuan-Yuan Cui, and H. Criss Hartzell


Mentor: H. Criss Hartzell, PhD      


Department: Cell Biology 


hBest1, the founding member of the new family of ca activated chloride channel, is genetically linked to Best vitelliform macular degeneration (BVMD). hBest1 have been reported to incorporate 32P. However, it is unknown where is the potential phosphorylation site(s), and what kinase(s) can phosphorylate hBest1, and whether phosphorylation play a role in hBest1 channel regulation. Here we identified a PKC phosphorylation site (S358) located in the previously identified region responsible for channel rundown (amino acids 350-390). Channel rundown was attenuated by the PKC activator PMA, and the protein phosphatase inhibitors, okadaic acid and calyculin. The pseudo-phosphorylated mutation S358E abolished the rundown, and S358A mutation abolished the effects of PMA and okadaic acid. Furthermore, coimmunoprecipitation of hBest1 with PP2A was enhanced by PMA and the phosphorylation-mimicking S358E mutation, and was inhibited by BIM and S358A mutation, suggesting channel rundown was finely controlled by PKC and PP2A at S358. We also find that hypertonic stress inhibited hBest1 currents. The inhibition was reduced by PMA and okadaic acid, and completely blocked by S358E mutation, suggesting hypertonic stress dephosphorylates hBest1 at S358. Ceramide, a PP2A activator, mimicked the effects of hypertonic inhibition of hBest1 current, and dihydroceramide, an inactive form of ceramide, inhibited the effects of both ceramide and hypertonic solution. Furthermore, a neutral sphingomyelinase inhibitor, manumycin A significantly blocked the effect of hypertonic stress, suggesting that hypertonic stress inhibited hBest1 current through activation of neutral sphingomyelinase to release ceramide. These finding provides insights into how bestrophin are physiologically regulated by PKC and PP2A, and how bestrophins are regulated by volume change.


Source of Funding for Research: NIH (EY014852 and GM60448)



Understanding Craniofacial Development: Lessons from a Mutant Mouse


Alyssa Long and Tamara Caspary


Mentor: Tamara Caspary, PhD


Department: Human Genetics


The development of the mammalian face requires a complex and coordinated series of signaling interactions to occur in the correct time and space for the outgrowth of facial primordial. What happens if this delicate process is perturbed? We are investigating the contributions of apoptosis, proliferation, neural crest cell specification and migration, and signaling interactions to the developing mouse midface (frontonasal – maxillary) region.

Apoptotic peptidase activating factor 1 (Apaf1) is a key component of the mitochondria-mediated cell death pathway. Apaf1-deficient mice die perinatally, exhibiting central nervous system defects such as exencephaly and occasional craniofacial malformations. We have identified a mouse line that carries a chemically-induced mutation in Apaf1. We believe this mutation results in a nonfunctional protein as homozygous mutant embryos show defects in neural tube closure and craniofacial development similar to those seen in targeted Apaf1-null mouse lines. To better understand the processes that generate the mammalian face, we are examining mutant embryos at two developmental stages: early, embryonic day 8.5-10.5, and late, embryonic day 15.5-17.5. We observe differences in the expression of signaling molecules during the initial specification and outgrowth of facial primordia in addition to later defective ossification of several bones in the skull. Thus, our results continue to define the important processes in craniofacial development.




Effects of Zinc Supplementation on Growth of Children Under 5 Years of Age: A Meta-Analysis of Randomized Controlled Trials


Phuong Nguyen, Usha Ramakrishnan and Reynaldo Martorell


Mentor: Reynaldo Martorell, PhD


Department: Hubert Department of Global Health


Background: Zinc interventions have received much attention as a cost-effective and promising strategy to improve child health, but their roles in preventing growth retardation in early childhood remain unclear.

Objective: Meta-analyses of randomized controlled trials were conducted to evaluate the effect of zinc interventions on the growth of children < 5 y old.

Method: Eligible studies were identified by PubMed database search and other methods. Weighted mean effect sizes and 95% CI were calculated for changes in height, weight and weight-for- height Z-scores (WHZ) using random effect models. Tests for homogeneity and stratified analyses by predefined characteristics were conducted using meta-regression techniques. Tests for publication bias were done using funnel plots.

Results: Forty-three trials (56 datasets) had relevant information. There was no evidence of publication bias. Zinc interventions had a small positive effect on change in WHZ (effect size: 0.06; 95% CI: 0.006, 0.11) but no effect on height or weight gain (effect size: 0.07; 95% CI: -0.03, 0.17 and 0.06; 95% CI: -0.10, 0.23, respectively). The results were similar across categories of age, duration of intervention, and baseline anthropometric status.

Conclusions: Contrary to the earlier findings, we failed to detect any benefits of zinc supplementation in improving early childhood growth. Other strategies are needed to prevent stunting.


Source of Funding for Research: The Micronutrient Initiative, Ottawa, Canada