Download June 6, 2014 Montefiore ~ Cherkasky Auditorium Bronx, New York

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
Jose Gabriel Mantilla Arango
Matias Jaureguiberry
Brett Baskovich
Sangeeta Jayakar
Miriam Ben-Dayan
Lizandra Jimenez
Yuri Chaves Martins
Anne Kessler
Hong Cheng
Adriana Maria Knopfelmacher
Sonia Elhadad
Deqiong Ma
Yanan Fang
Mark McCarron
Ignacio Guerrero
Bezawit Megra
Laleh Hakima
Xia Qian
10:30 PM – 12:00 PM
Elizabeth Richards
Henry Shikani
Daniel Shin
Karin Skalina
Tadakimi Tomita
Mike Veenstra
Dionna Williams
Rama Yakubu
Jiahong Yao
Valerio Zolla
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
3:00 PM – 5:00 PM
Break – Afternoon Refreshments
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+/24CSCs 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 activationinduced 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-1treated 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 vesselconstriction 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 ET ARB 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. ET ARB
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 autophagosomelysosome 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 1.5fold 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 actinrich, 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 qRTPCR 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 miR375 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 comorbidities, 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, estradiol17B 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 nonsyndromic 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 thyroxineproducing 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 nonpathogenic 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 PrP c
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 PrP c 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-TOMESENCHYMAL 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 Tregmediated 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 anergyassociated 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 monocytogenesbased 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 bradyzoitespecific 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.