June 6, 2014

Montefiore ~ Cherkasky Auditorium Bronx, New York

Montefiore ~ Cherkasky Auditorium Friday, June 6, 2014

9:30 AM – 10:15 AM Poster Set-up (Grand Hall)

Posters will displayed for the entire session Nicole Anayannis Evan Himchak Elizabeth Richards Jose Gabriel Mantilla Arango Matias Jaureguiberry Henry Shikani Brett Baskovich Sangeeta Jayakar Daniel Shin Miriam Ben-Dayan Lizandra Jimenez Karin Skalina Yuri Chaves Martins Anne Kessler Tadakimi Tomita Hong Cheng Adriana Maria Knopfelmacher Mike Veenstra Sonia Elhadad Deqiong Ma Dionna Williams Yanan Fang Mark McCarron Rama Yakubu Ignacio Guerrero Bezawit Megra Jiahong Yao Laleh Hakima Xia Qian Valerio Zolla

10:30 PM – 12:00 PM Oral Presentations (Cherkasky Auditorium)

Brandi Freeman, Predoctoral Fellow Mentor: Dr. Mahalia Desruisseaux

Esther Adler, Fellow Montefiore Medical Center

Loreto Carvallo Torres, Research Fellow Mentor: Dr. Joan W. Berman

Rachel Ames, Predoctoral Fellow Mentor: Dr. Fernando Macian

12:00 PM – 1:30 PM Buffet Luncheon (Grand Hall)

1:30 PM – 1:45 PM Pathology Department Photo (Gardens)

1:45 PM – 3:00 PM Guest Speaker (Cherkasky Auditorium)

Alexander Tarakhovsky, MD, PhD Irene Diamond Professor of Immunology and

Director of Immune Cell Epigenetics and Signaling The Rockefeller University

“Control of gene expression by histone mimics”

3:00 PM Break – Afternoon Refreshments 3:00 PM – 5:00 PM Viewing & Judging of Posters (Grand Hall)

MOLECULAR DIAGNOSTICS FROM STAINED CYTOLOGY SMEARS

Esther Adler, Laleh Hakima, Evan Pieri, Eli Grunblatt, Andrew Seymour, Samer Khader, Antonio Cajigas, Mark Suhrland, Maja Oktay, Sumanta Goswami

Mutational analyses are increasingly important for guiding treatment decisions of lung non-small cell carcinomas (NSCC) and for surgical decisions of thyroid nodules with indeterminate cytological diagnoses. Both lung and thyroid lesions are frequently diagnosed using only fine need aspiration (FNA) biopsy obtained material. Molecular diagnostic tests of cytological samples are most commonly done using cell blocks. However, insufficient cellularity of cell blocks often represents an obstacle to the performance of these tests. Here we used PNA clamp technology to we assess if we can detect commonly encountered mutations in non-small lung and thyroid carcinomas using cytological direct smears and thus improve patient service.

We collected cytology smears from 31 cases of NSCLC and 22 cases of papillary carcinoma of thyroid. Eligible cases had either a cell block or corresponding paraffin embedded tissue and sufficient diagnostic material available to sacrifice one or two slides. Source material included FNA, bronchial wash and bronchial brush cytology specimens and biopsy or surgical resections surgical pathology specimens. We have tested our approach on KRAS, EGFR and BRAF. The detection system utilizes DNA and RNA extracted from cytology pap and Diff Quik stained smears containing abnormal cells. Mutation and deletion detection is performed by using a specific qPCR that only amplifies the mutant DNA. Translocation detection is done by a qRT-PCR process utilizing primers at both the 3’ and 5’ ends of the fusion protein. This technology can be used on 50 calls and can detect 2% mutation positive cells.

Our results obtained from cytology smears were compared to the results obtained by a NY State approved reference lab which used cell blocks from the corresponding cases. We tested EGFR mutations and deletions, KRAS mutations and ALK translocations. Our data from 24 lung cancer cases were 100% in agreement with the reference lab for EGRR testing; 6 EGFR mutation positive (2 exon 21, 3 exon 20 and 1 exon 19) and 18 EGFR negative. For KRAS testing we were 100% in agreement with 8 KRAS mutation positive (6 codon 12 and 2 codon 13) cases. Among the 8 negative cases, we detected 1 case as positive which was reported as negative by the reference lab (codon 12). We tested 2 ALK translocation positive samples; one from a tissue slide and one from a PAP smear along with three negative samples and found 100% agreement with the previously reported results. However, we detected 1 sample which was positive for KRAS codon 12 mutation to be also positive for ALK translocation. That sample had not been tested for ALK translocation before. In our thyroid cancer cohort we had only 1 BRAFV600 positive sample which identified positive as well and 8 samples not tested by the reference lab, none of which were positive for BRAFV600 mutation. In conclusion, using our approach we can detect commonly encountered mutations in non-small lung and thyroid carcinomas using cytological direct smears and thus improve patient service.

HIGH DENSITY OF METASTATIC INTRAVASATION SITES (TMEM) CORRELATE WITH STEM CELL RICH TUMOR MICROENVIRONMENTS

Sumanta Goswami, Esther Adler, Eli Grunblatt, Maja Oktay

Background: Chemotherapy targets proliferative tumor cells but misses two low proliferative populations; highly motile cells and cancer stem cells (CSCs). Patients with high proportions of these cells are poor treatment candidates and are at risk for developing metastases. To differentiate chemotherapy responders from nonresponders, a method of identifying tumors with high proportions of treatment resistant cells is needed. Using immunohistochemistry (IHC) we previously identified the microanatomical sites of breast cancer cell intravasation, named TMEM. TMEM density positively correlates with distant metastases and the proportion of migratory cells, which are CSC enriched. We now questioned if TMEM rich microenvironments are also enriched for CSCs.

Design: Cells were collected from 47 human breast invasive ductal carcinomas by fine needle aspiration (FNA) of surgical resections and the percentage of CD44+/24- CSCs were identified using flow cytometry. TMEM density in histologic sections of the corresponding resections were evaluated with IHC using a triple stain for Mena expressing cancer cells, macrophages and vascular endothelial cells.

Results: TMEM density positively correlated with the percentage of CSCs across all clinical subtypes (r= .92, p<.001). There was a significant difference in tumor size between cases with high percentage of CSC and high TMEM density (P<.001). The percentage of CSC and TMEM density did not correlate with the degree of differentiation, mitotic index or lymph node status.

Conclusion: Microenvironments associated with metastasis in human breast cancer are enriched for CSCs. The proportion of CSCs can be successfully assessed using flow cytometry on FNA collected breast cancer cells. Additional studies are under way to determine if this approach is useful in predicting chemotherapy response.

USING PRIMARY TUMOR CELLS FROM PATIENTS TO INVESTIGATE THE

EARLY STEPS OF METASTATIC DESSEMINATION

Jeanine Pignatelli, Sumanta Goswami, Xiaoming Chen, Esther Adler, Joan Jones, John Condeelis, Maja H Oktay

Background: Cell lines are widely used as in vitro models to study cancer cell

biology. Despite numerous advantages there is growing evidence that cell lines do not faithfully represent tumor cells in vivo due to intra- and inter-laboratory cell line heterogeneity and drift away from the phenotype of the original tumor due to immortalization and time in culture. This is a serious concern especially if cell lines are assumed to be valid models for evaluating the pathobiology of breast cancer and/ or the likely response to novel targeted therapies. We have developed a novel approach to study the initial step of metastatic dissemination, transendothelial migration (TEM), in vitro using primary breast cancer cells from patients.

Design: We obtained breast cancer cells from 25 surgical resections of invasive ductal carcinomas of the breast by fine needle aspiration (FNA) using 25 gauge needle and developed conditions suitable for short term in vitro survival and intravital labeling of these cells. We assessed their TEM activity using subluminal-to-luminal transwell assay engineered with human umbilical vein endothelial cells (HUVEC) and visualized TEM by confocal microscopy. Actin regulatory protein Mena isoform expression pattern known to be associated with disseminating tumor cells in vivo (MenaINV high Mena11a low) was used to confirm the presence of TEM-competent cells on the luminal side of the assay.

Results: From all 25 cases, using FNA we obtained a 90-95% pure cancer cell population containing viable cells suitable for labeling with intravital tracker dyes. Cancer cells from three major clinical subtypes; ERPR+/Her2-, triple negative and Her2+ were capable of TEM in vitro. In all cases but one, TEM competent cells showed in vivo Mena isoform expression pattern, MenaINV high Mena11a low. By comparison, cancer cell lines such as MDA-MB 231 did not consistently demonstrate either TEM or the in vivo Mena splice variant expression pattern.

Conclusion: Cancer cells obtained from patient breast cancers by FNA can be successfully used in vitro to study TEM and potentially to assess the effect of novel drugs that target cancer cell TEM and thereby intravasation.

NFAT1 REGULATES CD4+ T CELL EXHAUSTION

Rachel Ames, Li-Min Ting, Kami Kim, Fernando Macian

Chronic infection of a host results in reduced responsiveness of both CD4+ and

CD8+ T cells. This phenotype, known as T cell exhaustion, has been shown occur in

many chronic viral infections and more recently in non-viral infections such as

Plasmodium and other parasites. Unlike the functional memory cells that typically form

after successful clearance of a pathogen, exhausted T cells fail to fully activate upon

further interaction with their cognate antigen, resulting in reduced proliferation and

secretion of effector cytokines. The expression of cell-surface inhibitory molecules such

as PD-1 and LAG-3 is upregulated in exhausted T cells and blockade of these signals

restores T cell function. However, the mechanisms that underlie the induction of this

phenotype have not been fully characterized. In T cells, members of the NFAT family of

transcription factors are not only responsible for the expression of many activation-

induced genes but are also crucial for the induction of transcriptional programs that

inhibit T cell activation and maintain tolerance. Our results show that the expression of

exhaustion-associated molecules, such as LAG-3 and PD-1, is calcium/calcineurin

dependent. We also show that NFAT1-deficient CD4+ T cells are more resistant to

exhaustion induced by repetitive exposure to antigen in vitro and fail to induce the

expression of LAG-3. Furthermore, our data show that unlike wild-type T cells that

become exhausted in mice infected with Plasmodium yoelii, NFAT1-deficient CD4+ T

cells maintain expression of effector cytokines after Plasmodium infection. We propose

that NFAT1 is a key regulator of several programs of T cell inactivation that include T

cell exhaustion, in which the activity of this transcription factor is required for the

expression of the inhibitory factors that negatively regulate T cell function.

DISRUPTION OF THE VIRAL E2 GENE IN HPV-ASSOCIATED HEAD AND NECK

SQUAMOUS CELL CARCINOMA

N. Anayannis, N.F. Schlecht, M. Ben-Dayan, R.V. Smith, Y. Wang, T.J. Belbin, R.D. Burk, S.M. Leonard, C.B. Woodman, J.L. Parish, M.B. Prystowsky

Human papillomavirus (HPV)-16 is the predominant genotype in HPV+ head and

neck squamous cell carcinoma (HNSCC). Integration of the HPV genome into human

DNA often disruptes the HPV E2 gene. E2 regulates transcription of the E6 and E7 HPV

oncoproteins and is associated with malignant progression and decreased

radiosensitivity when disrupted in cervical cancer. In HPV+ HNSCC, disruption of E2

and its impact on expression of HPV oncoproteins is not well studied. We hypothesized

that disruption of the E2 oncogene would result in deregulated transcription of E6 & E7.

We also hypothesized that E2 would be intact in the majority of HPV positive

oropharyngeal cancer (OPSCC) specimens because HPV+ OPSCC appears to be more

radiosensitive then HPV negative OPSCC. Expression of HPV-16 E6, E7 & E2 in 35

primary HNSCC specimens was assessed by qRT-PCR. Disruption of E2 in HNSCC

specimens was determined using a PCR-based method. E2 disruption data was

combined with HPV expression data and various clinical and molecular data previously

gathered on these samples. This includes data such as tumor origin and staging, host

gene expression and methylation. In the oropharynx, 66% HPV+ tumors appear to have

an intact E2 oncogene while 34% have disrupted E2. Additionally, disruption of the E2

gene appeared to correlate with decreased E6 & E7 expression. Though HPV-16

infection is more commonly seen in OPSCC than in HNSCC from other primary sites,

our data suggest that E2 disruption, a surrogate for viral integration into the host

genome, is less prevalent in OPSCC.

DESMOID-TYPE FIBROMATOSIS: RADIOLOGIC-PATHOLOGIC CORRELATION

AND CLINICAL PREDICTORS OF RECURRENCE IN AN 18-YEAR

RETROSPECTIVE COHORT.

Jose G. Mantilla Arango, Elina Shustef, Esperanza Villanueva-Siles

Background: Desmoid-type fibromatoses are rare, locally aggressive, fibrous neoplasms with an extensive differential diagnosis. These tumors are estimated to have a 20-45% risk of recurrence. Higher risk of recurrence has been associated with age, tumor size, and mutation of the CTNNB1 gene. Given this elevated risk, finding specific criteria to predict in which patients recurrences will be more likely might allow for more effective treatment. We aimed to establish specific criteria to predict the risk of recurrence and help guide individualized surveillance and treatment.

Design: We conducted a retrospective cohort study of 51 patients diagnosed with desmoid-type fibromatosis between 1995 and 2008. The subset of patients and clinical information was extracted from our clinical information system using Clinical Looking Glass; (Emerging Health Information Technology, Yonkers, NY). The initial imaging modalities, diagnoses, and rates of recurrence 5-13 years post-diagnosis were reviewed. Risk of recurrence was determined based on age, sex, anatomic site, and surgical margin status.

Results: Of the 51 patients, 18 developed post-operative recurrences during the follow up period (34.6%). The average time to recurrence was 18.3 months. Females were more frequently affected (68.6%), and also showed a non-significant higher recurrence rate (42.9%), compared to that of their male counterparts (18.8%) The median age at diagnosis was 37.5 years (range 5-81), with 72% of the patients below the age of 50.

The most common anatomic site was the abdominal wall (30.8%), followed by the mesentery (13.5%) and upper extremities (13.5%). Patients with tumors located in the upper extremities showed an increased risk of recurrence compared with other sites (RR 2.47, P<0.05, CI95% 1.3-4.8). There was a non-significant increase in recurrence for patients with positive and unknown surgical margins status.

32 patients had undergone imaging studies prior to surgery. The majority of patients received a CT scan as the initial imaging modality (68.5%), followed by MRI (21.8%). The most common radiologic diagnosis was “unspecified mass/neoplasm” in 31.3%, followed by sarcoma (28.1%), hematoma (21.8%) and desmoid-type fibromatosis (21.8%)

Conclusion: Overall, tumors located in the upper extremities showed an increased risk of recurrence. Age, sex, race, and margin status did not significantly impact this risk. In addition, radiologic diagnosis is commonly non-specific. Thus, a definitive diagnosis is best made on histology by an experienced surgical pathologist. Further studies are needed for determining immunohistochemical or molecular predictors of recurrence.

A PIPELINE FOR CURATION OF GENETIC VARIANTS THROUGH OMIM

Brett Baskovich, Kinnari Upadhyay, Philip Meyer, Carole Oddoux, Harry Ostrer

Interpreting genetic variants remains a difficulty of high-throughput genetic

testing. The Online Mendelian Inheritance in Man (OMIM) website provides a curated

database of over 12,000 genes and their variants with interpretations of pathogenicity,

explanations and literature sources. Algorithms including SIFT, PolyPhen2, and

Mutation Taster consider amino acid change, location in the protein, and conservation.

One method of dealing with the uncertainty in pathogenicity is to provide a

confidence level; the International Agency for Research on Cancer (IARC) has

developed a scoring system on a scale of 1 to 5, from almost certainly nonpathogenic to

almost certainly pathogenic (3 being indeterminate).

Axiom microarrays were performed on 196 members of each of the Bronx's 3

majority ethnic groups: African Americans, Puerto Ricans, and Dominicans. Pooled

whole-exome sequencing was also done on the Dominicans.

A pipeline was written in BioPerl to parse the results and query OMIM and

ClinVar based on the variants' nucleotide position. Useful parameters were extracted

from these and other databases including pathogenicity, 1000Genomes results and

ESP6500 results. For all results called pathogenic, literature sources were manually

reviewed and an IARC score was assigned. Scores were also obtained from SIFT,

Polyphen2, and MutationTaster.

The Axiom software called 305,513 variants. The pipeline resulted in 1440 known

variants of which 338 were reported as pathogenic. Of the more common variants, the

majority were given an IARC score of 3 (35%) or 4 (42%). However, 10% were

considered likely nonpathogenic (1 or 2) after reviewing the literature. Several variants

had allele frequencies too high to be pathogenic given the disease prevalence.

Whole-exome sequencing identified 62 additional pathogenic variants per OMIM

in the Dominicans, 19 of which we scored a 4 or 5.

There was no correlation in IARC scoring results with the algorithms' results. The

results among the three programs also varied widely (55% of the variants had one

prediction opposite the others).

A large number of variants identified as pathogenic by OMIM and ClinVar were

determined to be inconclusive or polymorphic upon review. Most of those called 1 or 2

were actually identified as polymorphic by the papers cited in OMIM. Most calls of 3

were made due to weak evidence such as one patient who was a compound

heterozygote.

The variation with the scoring algorithms is not surprising, as our scoring was

based on the literature (linkage studies and functional studies), whereas the algorithms

consider the amino acid change.

HPV-POSITIVE HEAD AND NECK SQUAMOUS CELL CARCINOMA IS ASSOCIATED WITH THE OVEREXPRESSION OF P14ARF

Miriam M Ben-Dayan, Nicole VJ Anayannis, Nicole Kawachi, Robert D Burk, Nicolas F Schecht, Thomas J Belbin, and Michael B Prystowsky

Head and Neck Squamous Cell Carcinoma (HNSCC) is an invasive cancer caused by various factors including Human Papillomavirus (HPV) infection. Two tumor suppressors, p53 and Rb are inactivated by the HPV-16 E6 and E7 proteins, respectively. The CDKN2A locus encodes two tumor suppressors, p16ink4a (INK4a) and p14ARF (ARF), and is frequently silenced in cancer cells. INK4a, a member of the Rb pathway, is overexpressed in HPV-positive (HPV+) HNSCC and is currently used as a marker for HPV infection. ARF is known for its role in the p53 pathway, and is transcribed by E2F-1 (a binding partner of Rb). While ARF is overexpressed in cervical cancer, expression of this splice variant has not been studied in HPV+ HNSCC. We tested the hypothesis that ARF expression is increased in HPV+ HNSCC.

Experiments were done on paired HNSCC patient tumor and adjacent normal samples. mRNA expression of the CDKN2A splice variants in HPV+ and HPV- samples were assessed by qRT-PCR. Both DNA and RNA tests were used for HPV typing of the tumor samples. Wilcoxon rank-sum and signed rank tests were performed on the resulting CDKN2A splice variant expression levels.

Comparing the tumor-normal ratios between HPV+ and HPV- samples, there was a significant increase in ARF expression in the HPV+ tumors (p=0.02). There was no significant difference in INK4a expression due to elevated transcript levels in both tumor and normal samples. These results show that ARF is overexpressed in HPV+ HNSCC primary tumors. Future studies will focus on identifying a mechanism for ARF expression in infected cells to explain the difference between INK4a and ARF in HPV+ tissue.

ROLE OF HIV TAT PROTEIN IN THE REGULATION OF GENE EXPRESSION IN MACROPHAGES

Loreto Carvallo, J. Eduardo Fajardo, Matias Jaureguiberry,

Peter Gaskill, Joan W. Berman

Despite the success of cART, 40-70% of HIV infected people develop cognitive and motor deficits termed HIV-associated neurocognitive disorders (HAND). Macrophages are the major cell type infected in the CNS. They produce cytokines, chemokines, and low levels of virus or viral proteins that promote neuroinflammation that ultimately result in neuronal damage, dysfunction and cell death, playing a key role in the progression of HAND. HIV-infected macrophages are long-lived cells that produce low levels of virus, as well as one of the early viral proteins, tat. Tat is the transactivator of the virus and is still produced despite cART. Tat also interacts with host genes to alter their expression. We and others demonstrated that tat induces cytokine secretion in macrophages, including CCL2, that mediates neuroinflammation and is highly expressed in the CNS of HIV infected people with HAND. In this study we are examining the role of the tat, a viral protein essential for replication and transcriptional regulation of HIV, in the regulation of gene expression in human macrophages. Using THP-1 cells, a human monocyte/macrophage cell line, we generated stable cells that express Tat-Flag by infection with lentivirus. We performed ChIP-seq analysis of these cells and found 67 DNA regions in promoters or genes associated with binding of complexes containing tat. Among these genes were neurocan, C5, CRFL2, APBA1, and Col6A6. We are confirming the association of tat with these sequences by ChIP assay and we are examining the expression of these genes in our THP-1 cell lines as well in HIV infected primary human macrophages as compared to uninfected macrophages by RT-qPCR. Our data indicate new mechanisms by which tat alters host gene expression and the contribution of these dysregulated genes to the pathogenesis of HAND.

ENDOTHELIN-1 TREATMENT INDUCES EXPERIMENTAL CEREBRAL MALARIA IN

Plasmodium berghei NK65 INFECTED MICE

Yuri Chaves Martins, Herbert B Tanowitz, Louis M Weiss, Mahalia S Desruisseaux

Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice causes severe CNS disease and is widely used as a model of experimental cerebral malaria (ECM). By contrast, the non-neurotropic rodent parasite, P. berghei NK65 (PbN), causes severe malarial disease in C57BL/6 mice, but not ECM. Preliminary data indicate that blockade of the endothelin receptor A prevents the development of ECM, suggesting that Endothelin-1 (ET-1) contributes to the pathogenesis of the disease. We hypothesized that treatment of PbN-infected mice with exogenous ET-1 would trigger the development of ECM. Mice were infected with 106 PbN-parasitized red blood cells and treated with either ET-1 or saline from 3 to 8 days post infection (dpi). PbA-infected mice served as positive control. Saline-treated PbN-infected mice did not display ECM, surviving until 12 dpi; whereas PbN-infected mice treated with ET-1 exhibited neurological signs and behavioral alterations characteristic of ECM, dying 4-8 dpi. ET-1 did not affect parasitemia; however, it significantly worsened hypothermia and weight loss during the course of infection. ET-1-treated uninfected mice had a modest reduction in rectal temperature, but no alterations in body weight or behavior. ET-1-treated PbN-infected mice demonstrated leukocyte adhesion to the cerebrovascular endothelia and petechial hemorrhages throughout the brain parenchyma which were not evident in saline-treated PbN-infected mice or ET-1 treated uninfected mice at 6 dpi. Intravital microscopy of the brain microcirculation showed significant arteriolar vessel-constriction in ET1-treated, PbN-infected mice and in PbA-infected mice. ET-1 treated uninfected mice and saline-treated PbN-infected mice displayed significantly lower levels of brain arteriolar constriction.

NEW MULTIANTIBODY STRATEGY OF EIGHT COLOR FLOW CYTOMETRY FOR PAUCICELLULAR SPECIMENS

Hong Cheng, John Pizzolo and Howard Ratech

Flow cytometry immunophenotyping has become a popular and useful diagnostic

tool in the hematopathology laboratory. It not only provides a high degree of efficiency and sensitivity, but also is more reproducible than microscopy. However, one of the most important clinical challenges is to analyze paucicellular specimens. Fine needle aspiration of lymph node or mass, as well as body fluids, especially cerebrospinal fluid, usually contain limited amount of cells. It is critical to characterize their immunophenotype and direct proper patient care. In order to provide clinicians with high quality and cost efficient service, we have designed a new screening panel using eight color channels and ten antibodies for these paucicellular specimens.

The ten antibodies include an immature blast marker (CD34), three B-cell lineage markers (CD19, Kappa and Lambda), three T-cell markers (CD3, CD4, and CD8), two differential markers (CD5 and CD10), and one gating marker (CD45). CD3 and CD5 share a same color channel to highlight the T-cell population. CD8 and CD10 share a same color channel since they are usually not co-expressed in the same cell population. For 43 specimens, we have run the new panel along with routine panels. Then we compared the results from both panels and plotted the data in a single variable linear regression chart. The correlation coefficient (R) is 0.98. Up to now, we have performed the new panel flow cytometry on 127 specimens. The flow cytometry results and final surgical pathologic results are collected and compared. This new screening panel revealed a compatible result with final surgical reports in all tested specimens.

Our data suggest that this new flow cytometry panel provides a reliable, high quality and cost-efficient screening tool for paucicellular specimens.

THE BONE MARROW STROMAL CELLS PLAY A MAJOR ROLE IN CONTROLLING

MULTIPLE MYELOMA CELLS MIGRATION THROUGH PRODUCTION OF

CHEMOKINES

Sonia Elhadad and David Fooksman

Multiple myeloma (MM) is a disease resulting from the transformation of plasma

cells. Myeloma develops in 1-4 per 100,000 people per year. The survival is 5-7 years or longer with advanced treatments. One main obstacle to MM treatment is the localization of myeloma cells in the bone marrow due to their adhesion to stromal cells and multiple environmental signals, thus making them resistant to treatment. Factors that play a role in inhibiting multiple myeloma cells egress from the bone marrow are not clearly defined, neither are the roles of the immune stromal cells in this function. Therefore, it is important to define these factors and the role of the immune stromal microenvironment in normal and pathological setting. This knowledge will help define new therapy for the multiple myeloma condition, and other types of cancer.

In preliminary in vitro data, we identified two chemokines, Sphingosine 1phosphate, S1P, and the neuroimmune guidance cue, netrin-1, as good candidates in myeloma cells migration and adhesion respectively, in the bone marrow. Using a combination of in vivo and in vitro studies, we will assess how these factors regulates normal and pathologic plasma cells transformation and egress from the bone marrow.

HIGHT FLUORSECENCE LYMPHOCYTE CELL COUNTS ARE NOT AN EARLY

MARKER FOR SEPSIS

Yanan Fang, Evan Himchak, Elizabeth Richards, and Jacob Rand

In a recent study, high fluorescence lymphocyte cell (HFLC) counts were proposed as an early sepsis marker. The objective of our study is to determine the clinical significance of increased HFLC and its potential use as an early sepsis marker in a large tertiary medical center.

We retrospectively reviewed the HFLC percentages in 1000 consecutive patients at our hospital. We then studied a cohort of 33 consecutive patients with positive blood cultures and reviewed the serial laboratory data and clinical history. The percentage of HFLC was collected with a Sysmex XE-5000 automatic hematological analyzer.

Out of 1000 patients, 43 had elevated HFLC% (defined as >1%). Among the 43 patients, 3 had sepsis with positive blood culture, 2 had influenza, 8 had neutropenic fever (1 had CMV, 1 had influenza B, 1 had pulmonary aspergillosis), 6 had neutropenia, 6 had upper respiratory infection, 4 had asthma exacerbation, 1 had pneumonia, 3 had pulmonary embolus, 3 had fever after vaccine, 2 had urinary infection, 5 had other causes.

In addition, among the 33 blood culture positive patients studied sequentially, 9 developed increased HFLC%. In all of the patients, HFLC% was not increased at the time of positive blood culture. Two of the patients were found to have contaminated blood cultures with a viral illness, but increase in HFLC% was also not initially seen. Interestingly, HFLC% increased later with decreasing white blood cell count and worsening symptoms.

HFLC counts are not a marker for early sepsis; however, it might still be a useful indicator of late sepsis where its significance deserves further investigation.

THE ROLE OF ENDOTHELIN-1 IN THE VASCULAR PATHOBIOLOGY OF CEREBRAL MALARIA

Brandi D. Freeman, Yuri Chaves Martins, and Mahalia Desruisseaux

The vasoactive peptide, endothelin-1 (ET-1), has been shown to mediate blood

brain barrier (BBB) permeability, inflammation, and vascular tone, and may be important in cerebral malaria (CM) pathogenesis. We previously reported that ET-1 was important in regulating CBF, brain microvascular hemorrhage and mortality in our experimental CM (ECM) model. These actions were mediated by ET-1 activation of the ETA receptor. To test the hypothesis that ET-1 is involved in the pathological process of ECM, we investigated ETA receptor mediated signaling in mice infected with Plasmodium berghei ANKA (PbA). ETA receptor blockers (ETARB) significantly improved survival in ECM mice. In addition, BBB permeability and protein levels of angiopoietin-2 and VCAM-1 were significantly lower in ECM mice treated with ETARB than mice treated with saline. ETARB prevented the ECM-induced decrease in angiopoietin-1 in PbA-infected mice. CM is associated with astrogliosis in both human disease and in experimental models. Our preliminary data indicate that astrogliosis is associated with abnormal protein levels of connexin 43 (Cx43), a gap junction protein critical in gliosis and BBB integrity. ETARB prevented the PbA-induced dysregulation of Cx43. We hypothesize that ET-1 mediates vascular dysfunction in ECM potentially by regulating neuroinflammation and Cx43 expression. JNK, a downstream substrate of ET-1, regulates Cx43 expression and function, and is important in CM. Our data indicate that ET-1 may mediate the vascular pathology and neuroinflammation in ECM via regulation of JNK signaling and subsequent Cx43 dysregulation. The ET-1 pathway may thus be a potential therapeutic target as an adjunct in the treatment of human CM.

REGULATION OF CD4+ T CELL ACTIVATION BY DIETARY LIPIDS AND THEIR EFFECTS ON MACROAUTOPHAGY

Ignacio Guerrero and Fernando Macian

Previous studies have reported a dysregulation of the immune responses in mice in response to lipid challenges. However, the cellular processes that are affected by dietary lipid load are still not completely understood. Autophagy is an essential catabolic process through which cellular components are degraded by the lysosomal machinery. Whereas, upregulation of autophagy in response to fatty acids has been demonstrated in a variety of cell types, such as neurons, epithelial or muscle cells, an increased number of recent reports have shown inhibition of macroautophagy in response to high concentrations of lipids, most likely due to a defect in autophagosome-lysosome fusion. In our lab we have demonstrated that macroautophagy is an actively regulated process in T cells that is necessary in order to provide the bio-energetic requirements for T cell activation. Therefore, the dysregulation of macroautophagy under high dietary lipid load in mice may have an effect on the activity of T cells, and consequently on the adaptive immune response. Our preliminary data shows defective cell proliferation and cytokine secretion upon stimulation of the T cell receptor in CD4+ T cells isolated from mice that have been chronically fed with a high fat diet for 4 months when compared to mice fed with a control diet. Interestingly, those cells show also a defect in the activation-induced macroautophagy, detected as a decreased turnover of LC3-II, a protein present in the autophagosome membrane. Following these results, we have also performed invitro assays to assess CD4+ T cell responses when challenged with increasing concentrations of saturated or unsaturated free fatty acids. These experiments show a decrease in TCR activation-induced cell proliferation as well as cytokine secretion in a dose-dependent manner when the CD4+ T cells were challenged with either palmitic or oleic acid. Interestingly, this effect was more pronounced in aged mice (22 months) when compared to young mice (3 months). Our results indicate, thus, that increased lipid stress can dysregulate macroautophagy and cause defective T cell responses; and also support that a decreased ability of T cells to respond to lipotoxic stress with age may underlie some of the characteristic immunosenescence-associated defects in the T cell compartment.

EFFECTS OF BUPRENORPHINE ON MONOCYTE MIGRATION ACROSS THE BLOOD BRAIN BARRIER IN THE CONTEXT OF HIV-1 INFECTION

Matias Jaureguiberry, Loreto Carvallo, Dionna Williams, and Joan. W. Berman

HIV-1 enters the CNS early after peripheral infection and results in chronic neuroinflammation that leads to cognitive and motor deficits termed HIV associated neurocognitive disorders (HAND) in more than 50% of infected people. HIV enters the CNS by transmigration of infected monocytes across the blood brain barrier. Drug abuse is a major risk factor for HIV-1 infection and opioids have been shown to alter the severity of HAND. Buprenorphine is a therapeutic for opioid dependency. It is a partial agonist of μ, and a full antagonist of κ, opioid receptors, but its effects on monocyte migration relevant to the development of neuroinflammation have not been studied. We showed that treatment of human monocytes with buprenorphine and CCL2, a chemokine highly elevated in the CNS of people with HAND, reduces CCL2-induced migration, suggesting that it may limit monocyte migration into the CNS in response to inflammatory chemokines. In this project we will examine the effects of buprenorphine on the mechanisms of CCL2induced CD14+CD16+ monocyte migration, a mature subpopulation critical for the development of HAND.

A ROLE FOR APOLIPOPROTEIN E IN INVASION IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

Sangeeta K. Jayakar, Olivier D. Loudig, Margaret Brandwein-Gensler, Ryung S.

Kim, Michael B. Prystowsky, Geoffrey Childs, Jeffrey E. Segall, Thomas J. Belbin Head and neck squamous cell carcinomas (HNSCC) have a poor patient prognosis, which is attributed to their invasive nature. Our goal was to identify novel genes that are important in HNSCC invasion. We utilized genome-wide expression data generated from HNSCC patient tumor samples that each exhibited a different “pattern of invasion”- a histological parameter, which in a previous study, correlated significantly with the appearance of local recurrence and decreased overall survival of the patients after surgery.

RNA from flash frozen tumor samples was analyzed using the Illumina® HumanHT-12 v4 Expression BeadChip. From FFPE tumor-cell enriched cores from the same patient tumors, RNA was analyzed using the Illumina® WG-DASL array. We selected genes from the two microarray platforms that were overexpressed at least 1.5-fold in the more invasive tumors. Ingenuity Pathway Analysis was used to prioritize which genes that were overexpressed in the microarrays that should be assayed for their effects on invasion in vitro. Candidate genes were transiently knocked down with siRNA in the cell line UMSCC1. Ability to invade was determined by using in vitro transwell invasion assays.

Analysis of two microarray experiments showed 104 genes that were overexpressed in the more invasive tumors compared to the less invasive tumors. Ingenuity pathway analysis identified 51 genes out of the 104 that fell into the top five functional categories of cell death, cell to cell signaling and interaction, cellular assembly and organization, cellular movement, and cell morphology. Invasion ability of UMSCC1 cells was impaired significantly by knockdown of 16 out of the 51 genes. The gene with the most significant effect on invasion from this screen was APOE (apolipoprotein E). This initial screen of global gene expression data in combination with pattern of invasion has revealed APOE as a novel gene that may play a critical role in HNSCC invasion. Downstream signaling of APOE receptors and how this may interact with invasion are currently being assessed.

MICRORNA-375 SUPPRESSES EXTRACELLULAR MATRIX DEGRADATION AND INVADOPODIAL ACTIVITY IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

Lizandra Jimenez, Ved P. Sharma, John Condeelis, Thomas Harris,

Michael B. Prystowsky, Geoffrey Childs and Jeffrey E. Segall

Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer, with a five-year survival rate of around 50%. Our research group previously identified microRNA-375 (miR-375) as the most consistently down-regulated miRNA in tumor samples when compared to paired normal samples. In that study, patients in the lowest quartile of miR-375 expression had significantly decreased disease-specific survival, increase incidence of loco-regional recurrence and distant metastasis. We have previously observed increased miR-375 expression in HNSCC cells resulted in diminished cell invasion in vitro. One major determining feature of the ability of cells to invade is their capability to degrade extracellular matrix barriers. Invadopodia are actin-rich, proteolytic structures, which can mediate degradation of extracellular matrix barriers. We have set out to evaluate the impact of miR-375 expression on extracellular matrix (ECM) degradation and invadopodial activity. For the detection of invadopodial ECM degradation, the fluorescent gelatin matrix degradation assay was conducted in combination with immunostaining of cortactin and Tks5 (invadopodial markers). Invadopodium precursors were defined as puncta of Tks5 and cortactin staining colocalized not associated with gelatin degradation; mature invadopodia were defined as Tks5 and cortactin colocalized puncta that were associated with gelatin degradation holes. Cellular protein levels of cortactin and Tks5 were assessed by western blot analyses. Immunoprecipitation experiments were conducted to evaluate levels of phosphocortactin (pY421). The gene expression assessments of invadopodia-associated proteins were performed with Taqman qRT-PCR assays. Secreted protease levels were assessed by Proteome Profiler™ Human Protease Array and ELISA analyses. We identified that miR-375 over-expression in HNSCC cells results in significantly reduced ECM degradation and number of mature invadopodia compared to their empty vector control cells, while the numbers of invadopodium precursors was not significantly altered. MiR-375 over-expressing HNSCC cells do not show reduced cellular levels of cortactin and Tks5 or levels of phosphocortactin. The gene expression of another invadopodia-associated protein, Tks4, was significantly suppressed in UMSCC1 miR-375 over-expressing cells. Many proteases with diminished secretion levels in UMSCC1 miR-375 over-expressing cells, including Kallikrein-5, -6, -10, -13 and MMP-9, were identified. The gene expression of these kallikreins and MMP-9 were significantly reduced in UMSCC1 miR-375 over-expressing cells as well. The gene expression and secreted protein levels of MMP-9 were diminished in UMSCC47 miR-375 over-expressing cells compared to its control line. We are currently testing candidate target proteins of miR-375 for possible involvement in the diminished matrix degradation and invadopodia maturation of HNSCC cells. In conclusion, increased miR-375 expression may suppress the invasive properties of HNSCC through diminished invadopodia activity. Improvements in HNSCC patient outcome may be obtained by clearly understanding the mechanism by which miR-375 expression diminishes HNSCC invasion.

HUMAN IMMUNOPHENOTYPING: A SMALL VOLUME, WHOLE BLOOD APPROACH FOR FLOW CYTOMETRY

Anne Kessler and Kami Kim

Standardized immunophenotyping techniques, namely flow cytometry, are important for the characterization and comparison of human immune responses in healthy and diseased states. Easily administered, whole blood panels requiring small volumes are ideal in most settings; however, these assays have particular relevance in low-resource settings, pediatric cohorts, and studies involving patients with multiple co-morbidities, as the methodology is simple, time-efficient, and requires minimal blood (~100 ul) for each assay. Using preliminary samples from Jacobi Medical Center (JMC), we are developing and optimizing small volume, whole blood immunophenotyping panels for use in our pediatric cohort studies. In our protocol, 100 ul of whole blood is stained with the appropriate antibodies and subsequently subjected to a red blood cell lysis step (no wash). Fluorescence-minus-one controls are used to ensure staining specificity for the accurate division of immune cell populations and subsets, and TruCOUNT technology is used for absolute cell quantification. A blood draw to data acquisition timeline is in place to ensure immune cell viability (as determined by DAPI staining), general accuracy, and consistency across specimens, as we do not freeze our samples. Currently, our panel includes basic T lymphocyte and monocyte subset markers; however, we have validated the agreement of our methods relative to those used at JMC, so we are expanding the current cocktail to include additional T lymphocyte and monocyte markers, as well as B cell lymphocyte markers, which allows for more comprehensive immune profiling of our study population. These panels are not only imperative to our current work but also have wide application in the field of human immunophenotyping, owing to their minimal time and sample requirements.

FRASIER SYNDROME CONFIRMED BY WT1 MUTATION ANALYSIS AFTER CHROMOSOME AND FISH ANALYSES IN A GIRL WITH SHORT STATURE,

DELAYED PUBERTY AND RENAL DISEASE

Adriana Knopfelmacher, KH Ramesh, Harry Ostrer, Linda Cannizzaro, Paul Levy, Marcela Del Rio, Ping Zhou.

Frasier syndrome is a disorder characterized by 46 XY karyotype, gonadal

dysgenesis and progressive glomerulopathy with a mutation in the WT1 gene at intron 8 or 9. Most cases have normal female external genitalia, streak gonads with a risk to develop gonadoblastoma. Patients present with nephrotic syndrome due to non-specific focal segmental glomerulosclerosis (FSGS) leading to end-stage renal disease in the 2nd or 3rd decade of life and/or with delayed puberty. We present a case of a 46 XY phenotypic female who was diagnosed with Frasier Syndrome after intensive work up for primary amenorrhea.

The patient is a 13 year old female with a past medical history remarkable for nephrotic syndrome due to FSGS since age 4, ureteropelvic junction repair, and kidney transplant at age 8 and growth retardation with delayed onset of puberty. The physical exam indicated <3rd percentile for height and weight and no secondary sexual characters. She has no other developmental delays. Imaging revealed prepubertal figures of uterus and ovaries (both ovaries < 1 cc). Blood chemistry results show hypergonadotrophic hypogonadism with LH of 27.4 UI/ml, FSH of 184 mUI/ml, estradiol-17B 5 pg/ml and progesterone of <8 ng/dl. ACTH and cortisol levels were within normal range. Karyotype revealed a 46 XY complement and FISH confirmed presence of a Y chromosome with an intact SRY. Based on clinical and cytogenetic findings a mutation analysis of the WT1 gene was suggested and sequencing revealed a mutation in intron 9, IVS9+5G>A, establishing the diagnosis of Frasier syndrome. The patient was started on estrogen supplementation. Repeat pelvic sonogram confirmed presence of ovaries in the pelvic area. The family received genetic counseling and the patient was given hormonal therapy to induce puberty. She is also scheduled for a bilateral gonadectomy due to the risk of gonadoblastoma and other tumors.

In our patient the diagnosis was made during a work up of short stature and delayed puberty. The importance of trying to make the diagnosis as early as possible in disorders of sex development with a 46 XY karyotype lies in the risk of the gonads to develop tumors most frequently but not limited to gonadoblastoma and institute the appropriate hormonal and surgical management.

Our findings support the idea of including cytogenetic and WT1 gene mutation analyses as part of the work up in young girls who present with steroid resistant nephrotic syndrome. Further research to institute such guidelines in young phenotypic females or males with ambiguous genitalia with steroid resistant nephrotic syndrome will be needed.

COMPREHENSIVE EVALUATION OF GENOMIC VARIANTS IN A MULTIPLEX AUTISTIC FAMILY

Deqiong Ma, Rizwan Naeem, Carole Oddoux, Brett Abrahams

Objective: The genetics of autism spectrum disorders is complex and remains

largely unclear. Recent whole exome sequencing studies have advanced our understanding of its genetic heterogeneity and led to the discovery of rare de novo mutations implicating novel biochemical pathways in disease pathogenesis. An integrated model of de novo and inherited genetic variants have yielded greater power indicating the importance of inherited rare variants in the etiology of ASD (He, Sanders et al. 2013).

Methods: A multiplex family with 6 non-syndromic autistic members was recruited. Affymetrix CytoScan® HD SNP Array and whole exome sequencing (WES) using NimbleGen’s capture array and Illumina Hiseq 2000 are employed for a comprehensive evaluation of both de novo and inherited variants in the family.

Results: 3 affecteds were sequenced so far. The initial screen on 574 SFARI autism candidate genes identified a novel non-synonymous mutation at SCFD2 (rs79025139, p.G283V). Its heterozygous deletion was previously reported in two non-syndromic autistic patients (Pinto, et al, 2010). SCFD2 is a secretion pathway (GO: 0046903) gene, which has shown a significant enrichment in autistic cases. Interestingly, three affecteds shared another heterozygous non-synonymous mutation at BLZF1, which falls to the same secretion pathway. In addition, a novel deleterious heterozygous mutation within ATP13A4 [c.T1916G:p.V639G; SIFT score =0] was detected. This gene has been recently linked to autism (Vallipuram J, 2010; Lesca, 2012).

Conclusion: These findings provide further evidence for the involvement of secretion pathway. Additional investigation including confirmation and family segregation study will be followed up.

A DE NOVO 10.79 Mb INTERSTITIAL DELETION AT 2q13-14.2 INVOLVING PAX8 CAUSING HYPOTHYROIDISM AND MULLERIAN AGENESIS: A NOVEL CASE

REPORT AND LITERATURE REVIEW

Deqiong Ma, Netra Prasad Punjabi, Elaine Pereira, Joy Samanich, Chhavi Agarwal, Jianli Li, Chih-Kang Huang, K.H. Ramesh, Linda Cannizzaro,

Robert Marion, Rizwan Naeem

Proximal long arm interstitial deletions at 2q12q14 are rarely reported. Literature review revealed three cases carrying both de novo and inherited deletion at 2q12q14.1 suggesting variable expressivity and possible incomplete penetrance. A recurrent 1.71 Mb genomic imbalance at 2q13 has been linked to increased risk of developmental delay and dysmorphism. Herein we report a case of 12 years-old girl with global developmental delay, hypotonia, short stature, clinodactyly, hallus valgus and pes planus. She was recently diagnosed with non-autoimmune and possible congenital hypothyroidism (CH) through laboratory testing. Ultrasound indicated a normal size and position of thyroid. Soon after the initial genetics visit, mullerian agenesis was observed as part of workup for acute abdomen. Genetic investigation revealed a de novo 10.79 Mb deletion at 2q13q14.2 (111,548,932-122,336,492) by array comparative genomic hybridization (aCGH), which was consistent with a retrospective chromosomal analysis. It involves more than 88 UCSC genes, 38 of which are OMIM genes, 7 of which are disease-causing genes and 3 of which show a dominant inheritance mode including GLI2, IL1B and PAX8. Interestingly, PAX8 is a member of the paired-box gene family and expressed in embryogenesis of the thyroid, Mullerian and renal/upper urinary tracts and carcinomas from each of these sites. It is essential for the formation of thyroxine-producing follicular cells. Loss-of-function effect has been reported for the majority of its point mutations and small indels suggesting possible haploinsufficiency effect. Although the recognized cause of congenital hypothyroidism is mainly due to thyroid dysgenesis or hypoplasia, some CH patients with certain point mutations present normal thyroid size. In addition, a novel mutation in PAX8 causing a severe form of hypothyroidism has been associated with abnormalities in the urogenital tract. Taken altogether, the unique clinical manifestation of the patient with possible congenital hypothyroidism and mullerian agenesis could be attributed to the complete one-copy deletion of PAX8 gene. A prospective investigation is merited to fully evaluate the pathogenic effect of the interstitial deletion of 2q13q14.2.

THE ROLE OF SYNDECAN-1 ON PLASMA CELL DIFFERENTATION AND FUNCTION

Mark McCarron and David Fooksman

The production of high affinity antibodies by plasma cells forms the mechanistic

basis of long-term immune protection and all currently successful vaccines. Understanding the factors that control the differentiation of plasma cells from activated B-lymphocytes will be key to the development of future vaccines and therapies directed against HIV and other pathogens that currently elude vaccination efforts.

Plasma cells are routinely identified and characterized by surface expression of syndecan-1 (also known as CD138). However, the role of syndecan-1 on plasma cell differentiation and function is currently unknown. Here we use a syndecan-1 knockout (SDC1-/-) mouse to characterize the function of syndecan-1.

The production of antigen-specific antibodies was significantly reduced in SDC1-/- mice compared to wild type mice following immunization with a model antigen in adjuvant. Consistent with that observation, cell transfer studies demonstrated that B cells lacking syndecan-1 expression were intrinsically deficient in forming plasma cells. Furthermore, plasma cells lacking syndecan-1 expression were less mature than their wild-type counterparts. Interestingly, transfer of SDC1-/- B cells into an IL6-deficient host rescued plasma cell formation suggesting that IL6 cooperates with syndecan-1 to drive plasma cell differentiation.

Future studies will aim to address the cellular source of IL6 and whether IL-6 can directly bind to syndecan-1. In total, these data suggest an essential role for syndecan-1 in plasma cell generation.

PRPc : FRIEND OR FOE IN HIV CNS PATHOGENSIS?

Bezawit Megra, Dionna Williams, Mike Veenstra, Toni Roberts, Joan W. Berman

HIV-1 enters the CNS soon after peripheral infection and causes chronic neuroinflammation and CNS damage that leads to cognitive impairment in greater than 50% of HIV infected people. PrPc (protease resistant prion protein) is the non-pathogenic cellular isoform of the human prion protein that is constitutively expressed in the CNS and is involved in several physiological processes that are disrupted during HIV neuropathogenesis. Using flow cytometry, we showed that monocyte surface PrPc

expression is increased with HIV infection. In addition, HIV infection as well as treatment of CNS cells with CCL2, a chemokine that is highly elevated in the brain of HIV infected people, causes increased PrPc shedding. This shed PrPc induces the production of inflammatory mediators from CNS cells suggesting that sPrPc participates in mechanisms that mediate NeuroAIDS. PrPc is expressed on monocytes and brain microvascular endothelial cells and is essential for the transmigration of monocytes across the blood brain barrier (BBB). To determine the effect of shed PrPc on the transmigration of monocyte across the BBB, we used our in vitro BBB model which consists of astrocytes and endothelial cells co-cultured on opposite sides of 0.3um pore insert. Soluble PrPc treatment blocked the transmigration of monocytes across the BBB, suggesting that it could be an initial mechanism of protection against the influx of monocytes across the barrier during early stages of HIV infection that results in viral seeding and neuroinflammation. Thus, PrPc may be both protective and neuroinflammatory depending upon its temporal and spatial expression during HIV neuropathogenesis.

N-CADHERIN/FGFR PROMOTES METASTASIS THROUGH EPITHELIAL-TO-

MESENCHYMAL TRANSITION AND STEM/PROGENITOR CELL-LIKE PROPERTIES

Xia Qian and Rachel Hazan

N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis. N-cadherin caused fibroblast growth factor receptor (FGFR) upmodulation, resulting in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties, involving Snail and Slug upregulation, mammosphere formation and aldehyde dehydrogenase activity. N-cadherin potentiation of the FGFR stimulated extracellular signal regulated kinase (ERK) and protein kinase B (AKT) phosphorylation resulting in differential effects on metastasis. Although ERK inhibition suppressed cyclin D1 expression, cell proliferation and stem/progenitor cell properties, it did not affect invasion or EMT. Conversely, AKT inhibition suppressed invasion through Akt 2 attenuation, and EMT through Snail inhibition, but had no effect on cyclin D1 expression, cell proliferation or mammosphere formation. These findings suggest N-cadherin/FGFR has a pivotal role in promoting metastasis through differential regulation of ERK and AKT, and underscore the potential for targeting the FGFR in advanced ErbB2-amplified breast tumors.

TAU ABNORMALITIES, INFLAMMATION AND AXONAL DAMAGE IN MURINE CEREBRAL MALARIA

Henry Shikani and Mahalia Desruisseaux

Cerebral malaria (CM) is a potentially fatal complication of disease associated

with Plasmodium falciparum infection. Despite successful anti-malarial treatment, approximately 20% of CM survivors develop long-term neurological deficits; however, the mechanisms that mediate this are not well understood. Neuronal injury has been linked to neurocognitive impairment in neurodegenerative disease and may contribute to the deficits seen in CM. In this regard, damage to neuronal axons has been observed in both human and murine experimental CM (ECM). Furthermore, improper regulation of tau, an axonal protein important for microtubule stability and cytoskeletal organization, has been demonstrated in mouse and human disease. We hypothesized that the neuronal injury observed in ECM results, in part, from abnormalities in tau. Improper regulation of tau results in an increase in its phosphorylated levels. We quantified protein levels of three forms of phosphorylated tau known to be pathological in Alzheimer’s disease (Ser396/404; Ser202; Ser202/Thr205) in several brain regions of mice with ECM and compared our findings with uninfected mice and mice infected with a less neurotropic malarial strain. In the same regions, we also quantified the level of SMI 32, a marker of axonal damage. Phosphorylated tau and SMI 32 were elevated throughout the brains of mice with neurological disease. Treatment of ECM mice with the immunotherapeutic PHF-1 antibody, which clears phosphorylated tau in mouse models of Alzheimer's disease, prevented axonal damage in certain brain regions, suggesting that this protein is contributing to the neuronal injury in ECM. Abnormal tau regulation has previously been linked to dysregulated inflammation, a common feature of CM. We hypothesized that the aberrant tau phosphorylation in ECM is associated with the hyper-inflammation which typically occurs. mRNA levels of several inflammatory cytokines in the brains of our experimental mice were found to be consistently elevated during neurological disease. The increases in these cytokines may contribute to the atypical tau regulation in ECM. Our goal is to further establish abnormal tau as a hallmark of CM. This protein may prove to be a viable target to ameliorate both the neuronal damage and neurocognitive impairment which occur during disease.

NFAT-DEPENDENT TRANSCRIPTIONAL MECHANISMS REGULATE THE SUPPRESSION OF EFFECTOR T CELL ACTIVATION BY REGULATORY T CELLS

Daniel S Shin, Ayana Jordan, Samik Basu, Rajan M Thomas,

Andrew D Wells, and Fernando Macian

Suppression by regulatory T cells (Tregs) is an essential mechanism of peripheral tolerance that controls autoreactive T cells by inhibiting activation-induced proliferation and cytokine expression. Despite intense research focused on the characterization of the development and function of Tregs, the mechanisms responsible for the inactivation of effector T cells by Tregs remain yet to be fully elucidated. Previous studies have shown that in response to anergizing stimuli, members of the Nuclear Factor of Activated T cells (NFAT) direct the expression of a specific set of genes, whose products are responsible for the inhibition of T helper (Th) cell effector function. Here, we show that effector T cells from Nfat1-/- mice are also more resistant to Treg-mediated suppression in vitro and in vivo. Interestingly, effector Th1 cells stimulated in the presence of Tregs behave similarly to T cells responding to an anergizing stimulus, translocating NFAT1 into the nucleus while failing to adequately activate AP-1. As a consequence, these cells up-regulate the expression of NFAT-dependent anergy-associated genes, which are required for efficient Treg-mediated suppression. Our results describe, thus, the existence of an overarching mechanism of T cell inactivation and indicate that, as described in anergic cells, Treg-mediated suppression of effector T cells results from the activation of NFAT-regulated gene expression.

THE USE OF LOW-INTENSITY FOCUSED ULTRASOUND WITH A LISTERIA-BASED VACCINE FOR THE TREATMENT OF PROSTATE

CANCER

Karin Skalina, Lisa Scandiuzzi, Huagang Zhang, Indranil Basu, Laibin Liu and Chandan Guha

Focused ultrasound (FUS) is a noninvasive, nontoxic targeted therapy that

generates ultrasonic waves delivering thermal and mechanical energies to the target region. Low intensity focused ultrasound (LOFU) is an alternative form of FUS that does not produce a temperature increase significant enough to cause ablation, thus resulting in sublethal damage of the cells. We assessed whether a prostate tumor treated with LOFU could enhance the immunogenecity of a Listeria monocytogenes-based vaccine conjugated to human prostate serum antigen (PSA). Our results demonstrated that the Listeria-based PSA (LM-PSA) vaccine was effective as a monotherapy in reducing prostate cancer growth in wild-type mice. However, when mice were treated with LOFU in conjunction with LM-PSA vaccine we observed an enhanced effect on reducing tumor burden when compared to each treatment individually. In addition, immunological analyses showed a lower infiltration of CD11b+Gr1+ cells in mice receiving LOFU and LM-PSA combined therapy as compared to the single treated mice. Overall our results demonstrated that LOFU therapy enhances the immunogenecity of a tumor-antigen specific vaccine. These findings provide the basis for effective targeted therapy that induces an anti-cancer response with minimal toxicity.

THE TOXOPLASMA GONDII CYST WALL PROTEIN CST1 IS CRITICAL FOR THE STRUCTURAL INTEGRITY AND CHRONIC PERSISTENCE

Tadakimi Tomita, David J. Bzik, Yan Fen Ma, Barbara A. Fox, Kami Kim,

Louis M. Weiss The obligate intracellular protozoan parasite Toxoplasma gondii infects 30% of humans. As infection proceeds it differentiates into a latent form (bradyzoites) within a modified parasitophorous vacuole termed the tissue cyst. Through the generation and screening of cyst specific monoclonal antibody library, we identified a 250 kDa cyst wall glycoprotein (CST1) that contains multiple SRS domains and a large mucin-like domain. Deletion of CST1 (Δcst1) resulted in the formation of fragile brain cysts and reduced the number of cysts in the CNS of infected mice, suggesting a role for CST1 in the persistence of cysts in central nervous system. TEM of brain cysts revealed that the cyst wall of Δcst1 parasites was much thinner and disorganized compared with the wild type cyst wall. Transcriptomic analysis of in vitro cysts demonstrated that Δcst1 parasites had impaired bradyzoite specific gene upregulations. Complementation of Δcst1 parasite with full length CST1 restored cyst sturdiness, brain cyst burden, and bradyzoite gene upregulation whereas complementation with CST1 lacking the mucine domain failed to restore these phenotypes. Taken together, we have shown that the CST1 and its mucin domain are required for the cyst wall structure, parasite differentiation, and chronic persistence in mice. CST1 is the first cyst wall protein identified with the function of cyst wall integrity for cyst architecture. Currently there is no treatment that can eliminate quiescent tissue cysts preventing latent infection and reactivation disease. This study provides important insights into the disruption of cyst wall and can led to improved treatments for chronic toxoplasmosis.

REVERSE TRANSCRIPTASE INHIBITORS INCREASE MONOCYTE ADHESION TO THE BBB ENDOTHELIUM: IMPLICATIONS FOR NEUROAIDS

Mike Veenstra*, Dionna W. Williams* and Joan W. Berman

Combined antiretroviral therapy (cART) has decreased the mortality associated

with HIV. However, cART has been less successful in treating or preventing the establishment of a spectrum of cognitive disorders that occur in 40-70% of HIV infected individuals, termed HAND. The blood brain barrier (BBB) is vulnerable during HAND, due to both viral and host factors, as the diapedesis of HIV infected monocytes may compromise its integrity. It is unclear whether cART can mitigate these processes, or whether the antiviral drugs comprising cART regimens may adversely affect the specialized endothelial cells of the BBB. We developed a technique that enables us to study the effects of the reverse transcriptase inhibitors (RTI’s), the backbone of cART regimens, on the human BBB using an in vitro model. We determined that junctional proteins critical for maintaining BBB function were upregulated following RTI treatment, which increased monocyte adhesion to the endothelium. This suggests that this may render the barrier more permeable and facilitate the transmigration of monocytes across the BBB, which could exacerbate the neuroinflammation that mediates HAND.

THE DISCOVERY OF NOVEL TOXOPLASMA GONDII CYST WALL PROTEINS

Rama Yakubu, Tadakimi Tomita, Yan Fen Ma, Louis Weiss

Our laboratory has identified the T. gondii gene (TGME49_064660) encoding CST1, using a tissue cyst specific mAb, Salmon E, which was cloned from a hybridoma library of about 1000 mAbs. We have observed that in the CST-1 knockout (KO) putative cyst wall proteins appear to be diffusing from the cyst into the host cell cytoplasm. We refer to this as the “leaky cyst” phenotype, and think it is a direct result of the absence of CST1 and its glycoepitopes that we believe form a matrix scaffold for cyst wall formation. In support of this model, when a CST-1 KO is complemented with a CST-1 gene lacking its mucin domain the CST1-KO phenotype of a fragile cyst wall and disrupted cyst wall layer are not rescued. We propose that additional bradyzoite-specific proteins exist in the T. gondii cyst wall which contribute to its structural and functional properties and that these proteins interact with CST1 and its glycoepitopes to form a cyst wall protein complex.

Screening of the hybridoma library resulted in the identification of mAbs that reacted to CST1 and which were instrumental in the identification of the CST1 gene. Having identified CST1 as a major component of the cyst wall and having created a CST-1 KO, we are now able to examine the remaining mAbs in this library to identify those mAbs that react with other components of the cyst wall using both immunofluorescence (IFA) and immunoblotting techniques. Using this technique I have already identified 5 mAbs in the library that are cyst wall reactive in both the WT and CST1-KO and, therefore, recognize different protein(s) than CST1.

T. gondii is one of the most successful protozoan parasites partly because it forms persistent latent cysts that last for the life of its hosts, and the cyst wall is the critical biological structure for this persistence. Understanding the formation of the cyst wall should enable us to design therapeutic approaches to disrupt formation of latent brain cysts, preventing chronic T. gondii infection.

SLUG PROMOTES SURVIVAL DURING METASTASIS THROUGH SUPPRESSION OF PUMA-MEDIATED APOPTOSIS

Seaho Kim, Jiahong Yao, Kimita Suyama, Xia Qian, Bin-Zhi Qian, Sanmay Bandyopadhyay, Olivier Loudig, Carlos De Leon-Rodriguez, Zhenni Zhou,

Jeffrey Segall, Fernando Macian, Larry Norton, and Rachel. B. Hazan

Tumor cells must overcome apoptosis to survive throughout metastatic

dissemination and distal organ colonization. Here we show in the Polyoma Middle T mammary tumor model that N-cadherin expression causes Slug upregulation, which in turn promotes carcinoma cell survival. Slug was dramatically upregulated in metastases relative to primary tumors. Consistent with a role in metastasis, Slug knockdown in carcinoma cells suppressed lung colonization by decreasing cell survival at metastatic sites, but had no effect on tumor cell invasion or extravasation. In support of this idea, Slug inhibition by shRNA, sensitized tumor cells to apoptosis by DNA damage, resulting in caspase-3 and PARP cleavage. The pro-survival effect of Slug was found to be caused by direct repression of the pro-apoptotic gene, Puma, by Slug. Consistent with a pivotal role for a Slug-Puma axis in metastasis, inhibition of Puma by RNA interference in Slug-knockdown cells rescued lung colonization, whereas Puma overexpression in control tumor cells suppressed lung metastasis. The survival function of the Slug-Puma axis was confirmed in human breast cancer cells, where Slug knockdown increased Puma expression and inhibited lung colonization. This study demonstrates a pivotal role for Slug in carcinoma cell survival, implying that disruption of the Slug-Puma axis may impinge on the survival of metastatic cells.

ULTRASTRUCTURAL BIOCHEMICAL AND FUNCTIONAL CHANGES IN AGING LYMPHATIC VESSELS

Valerio Zolla, Tsoy Nizamutdinova, Daisuke Maejima, Brian Scharf, Cristina C.

Clement, Sabriya Stukes, Arturo Casadevall, David Fooksman, Anatoliy Gashev, Laura Santambrogio

The role of lymphatic vessels is to transport fluid, soluble molecules, and immune

cells to the draining lymph node. Herein we analyze how the aging process affects the dynamic and structural elements of lymph flow. Ultrastructural, biochemical and proteomic analysis indicated loss of matrix protein in aged lymph vessels, as well as an increased in protein oxidative modifications. This resulted in loss of lymphatic contractions amplitude and frequency with reduction of total productivity of lymph pump, as measured in vivo on lymph collectors. Functionally, this impairment translated into a reduce ability to in vivo bacterial transport to the draining lymph nodes, as determined by time-lapse microscopy. Ultrastructural analysis also indicated a decrease in the thickness of the endothelial cell glycocalix and redox proteomic determined increased in glycation and carbonylation of endothelial cell glycocalix structural proteins. The glycocalix changes in structure resulted in increased vessel permeability. Altogether, our data provide a mechanistic analysis of how the anatomical and biochemical changes, as occurring in aged lymphatic vessels, compromise lymphatic transport.