Red Blood Cells of Patients with Celiac Disease

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ERIC CZINN ’10
Mentor: Alessio Fasano, MD
Institution: The University of Maryland Center for Celiac Disease Research
Project: DNA Mutations In The CXCR3 Gene Of Patients With Celiac Disease.
Abstract:
Celiac disease is an autoimmune disease that affects the digestive tract and affects 1/250 Americans
and can cause intermittent diarrhea, abdominal pain, bloating and failure to thrive. Celiac disease
patients cannot tolerate gluten, a protein found primarily in wheat, barley, and rye. When gluten is
metabolized, the protein gliadin is released, causing intestinal damage. A gene, CXCR3, has been
linked to Celiac disease. The CXCR3 receptor is located along the intestinal lining, and gliadin has
been shown to bind with the CXCR3 receptor, causing the release of the protein zonulin, which in
turn increases intestinal cell permeability. Gliadin can then pass through these permeable cells and
activate the patient’s immune cells to attack the gliadin molecule, resulting in inflammation that
damages the intestinal barrier.
Specific Aims: To identify mutations (SNPs) in the DNA of celiacs in CXCR3 and zonulin in an
effort to identify genetic polymorphisms in the CXCR3 gene.
Methods: DNA was extracted from the red blood cells of patients with and without Celiac disease by
using a QIAamp blood Midi Kit (Spin Protocol). The red blood cells were lysed using protease, then
incubated in a 70o Celsius hot water bath for 10 minutes. Ethanol and cleansing solutions were then
added and centrifuged for 15 minutes to extract the DNA. The DNA was then placed in a
thermocycler for 35 cycles where the DNA was replicated in a polymerase chain reaction (PCR).
Finally the DNA was placed on an ethidium bromide gel for gel electrophoresis to confirm the
presence of DNA. The PCR reaction product was then purified, sequenced, and analyzed to find
common mutations on the gene.
Conclusions: The DNA from healthy individuals and patients with Celiac disease is now being
sequenced and analyzed for polymorphisms in CXCR3 as well as other genes linked to Celiac disease
in an effort to identify new diagnostic and/or therapeutic modalities.
MICHAEL DAVIS ‘10
Project: Cationic Contrast Agents for Cartilage CT Scans
Mentor: Dr. Mark Grinstaff, Ph.D.
Institution: Boston University Department of Biomedical Engineering
Abstract:
This summer, I participated in the Boston University Summer Research Internship Program, working
for 6 weeks in the biomedical engineering department with Dr. Mark Grinstaff. I partook in a project
to produce a new imaging agent for non-invasive scans such as CT and MRI. For instance, CT scans
of joint cartilage can detect the amount of glycosaminoglycans (GAGs), naturally occurring
substances that maintain cartilage health. Lower GAG levels often signify osteoarthritis (OA), a
disease that wears away at cartilage and causes mechanical instability.
Traditionally, a negatively charged dye is administered intravenously to the patient, providing a better
visualization of the cartilage. However, anionic agent concentration is inversely related to actual
GAG levels, risking an inaccurate scan. Positive cationic contrast agents, on the other hand, directly
correlate to GAGs due to electrostatic attraction, thereby resulting in more accurate measurements
and higher resolution images.
Prior to this summer, Dr. Grinstaff designed and tested several types of cationic agents, comparing
their effects against those of a commercially available anionic agent. As part of my research, Dr.
Grinstaff taught me to produce a particular form of cationic agent through a series of chemical
reactions. After synthesizing the agent, I helped optimize the process so that the agent can eventually
be mass produced.
MICAH LEHMANN ‘10
Project: Vascular Dysfunction in Septic Shock
Mentors: Dr. Dan Berkowitz and Dr. Gautam Sikka
Institution: The John Hopkins University School of Medicine, Department of Anesthesiology and
Critical Care Medicine
Abstract:
Dr. Dan Berkowitz and Dr. Gautam Sikka are working to determine the causes of vascular
dysfunction in septic shock. Septic shock is a condition in which the blood vessels dilate during
overwhelming bacterial infection in the bloodstream. As a consequence of the expansion of blood
vessels, blood flow decreases and prohibits adequate oxygenation of body organs. Researchers have
now established that carbon monoxide, nitric oxide, and hydrogen sulfide, gases that are generally
known to be toxic air pollutants, actually have a crucial role in controlling the relaxation and
expansion of blood vessels. Produced by enzymes within our arteries, these gases help regulate the
blood pressure and arterial function. Drs. Berkowitz and Sikka hypothesized that abnormal
production of these gases in cases of septic shock causes the vascular dysfunction that leads to multiorgan failure.
The aim of this research was to clarify the process underlying vascular dysfunction in septic shock
using genetically altered mice whose blood vessel responses mimicked those seen in septic shock.
The purpose of this internship was to refine techniques to measure the contraction and relaxation of
the mouse aorta in response to different agents.
Each experiment consisted of first anesthetizing the mouse using isofluorane. The chest cavity was
opened and a heparin solution was quickly injected into the heart to prevent blood clotting in the
aorta. The aorta was removed and was placed on a Petri dish in a buffer solution to be cleaned
carefully with the aid of a microscope. Finally, the aorta was mounted onto a myograph, which is an
instrument that amplifies and records relaxation and contraction of muscles. Contraction and
relaxation measurements and observations in the presence of a variety of drugs in buffer solution
were conducted.
In conclusion, Drs. Berkowitz and Sikka have since utilized this refined technique to determine that
increased production of hydrogen sulfide by the enzyme cystathionine –γ-lyase in sepsis and septic
shock leads to vasodilation and resultant decrease in blood pressure. Thus, the vessels are unable to
oxygenate peripheral tissues.
JOANNA RAPOPORT ‘10
Project: Studies on the Role of Mir-27a as a Regulator of the BCL3 Oncogene
Mentors: Dr. Curt Civin, Dr. Kara Schiebner, Mary Claire Hauer, and Brie Teaboldt
Institution: University of Maryland, School of Medicine
Abstract:
A major area of blood cancer research is identifying the key genes and gene regulators that are
involved in the development and growth of blood cancer cells. One such gene (called an
“oncogene”) is BCL3, which when mutated or over-expressed, can turn a normal cell into a cancer
cell and cause certain types of lymphoma. A possible regulator of BCL3 is the microRNA mir-27a.
MicroRNAs are single strands of RNA about 22 base pairs long which bind to the 3’ untranslated
region of a gene’s mRNA transcript that comes after the coding sequence for the gene. Aiming to see
if mir-27a indeed targets the BCL3 gene and causes down regulation, a basic “plasmid-insert” gene
cloning technique was performed to clone the region of the BCL3 gene where the mir-27a binds into
a luciferase plasmid.
The next stage of this process was to transfect the plasmid with the insert along with mir-27a into
human tissue cell line HEK293T and conduct a Reporter Assay. The result of the assay would be
expected to show that the mir-27a would reduce the luciferase signal, indicating that the BCL3 is a
mir-27a target as hypothesized. If such an interaction exists, confirmation could lead to potential
blood cancer treatments.
GIDEON WOLF, ‘10
Project: Crab Mortality and Lifecycle Changes
Mentors: Dr. Odi Zmora and Dr. Sook Chung
Institution: University of Maryland, Center of Marine Biotechnology
Abstract:
In an era of fractured food webs and a decline in global fisheries, this research focused on tolerance
levels of Chesapeake Bay Blue Crab larvae to various experimental treatments.
Experiment 1: An important piece of information that affects the growth rate and mortality rate of
the larval Blue Crab is the salinity level of the water. Because labs that harvest the Blue Crab are
constantly looking for the optimum salinity level for the crabs, it is crucial to observe the crabs’
response to various treatment levels. The different salinity treatment levels used were 15, 20 and 25
ppt and the control was 30 ppt. Reactions to the various treatments were quantified using growth,
molting and mortality rates. The procedure used well plates filled with 2 mL of the respective salinity
solution. Subsequently, one Z8 (end of larval stage) crab was placed in each cell and fed 10-15 fresh
nauplii per day. Results were checked daily for about one month. The 20 ppt water yielded the
highest survivorship. However, a second trial was instituted using a different batch of Z8 crabs with
the 25 ppt treatment yielding the best results and a decrease in mortality. The second trial replicatecrabs were sent to a testing facility at Piny Point, and were deemed genetically weaker compared to
other received batches. Thus salinity levels and genetics play a role in an interpretation of the results.
Experiment 2: A recent problem in modern society has been the overuse of anti bacterial/microbial
soaps. Stemming from public fears and marketing ploys, the overuse of such soaps has led to
deleterious effects to the environment. One of the ingredients commonly found in these soaps is
Triclosan, or Irgasan, (C12H7Cl3O2). In addition to skin irritations and allergic reactions, this
compound has been shown to damage important enzymes and hormone receptors. Since these
chemicals are spread throughout the environment, it is critical to understand their effect on the
ecosystem. Different dilutions of the chemical were added to well plates with Z8 larva and 30 ppt
water. The pH was checked to ensure a neutral/slightly basic level of 8.00. The experiment yielded a
100% mortality rate until a diluted solution of Irgasan was used. Even smaller dilutions were tested,
with some crabs surviving to reach megalopae stage or C2 stage. The results supported the overall
hypothesis predicting severe damage that Irgasan imposes on Blue Crab growth.
The results from both experiments are testaments to the detrimental effects of exposing organisms
to environmental stressors and confirm the sensitivity of Blue Crabs to changes in salinity levels.
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