[Poster title] - WEB . WHRSD . ORG - Whitman-Hanson

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Feb 22, 2013 (4 years and 3 months ago)

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CURRICULUM OVERVIEW

This high school biology unit is designed to present students in a typical high school
setting with the following aspects of Cell Biology and Immunology:



Students will be able to list and describe the two main defensive strategies of the
immune system: innate immunity and acquired immunity.


Students will be able to distinguish between antibody
-
mediated immunity and cell
-
mediated immunity.


Students will be able to compare and contrast the cell components of the immune
system.


Students will be able to distinguish between antigens and antibodies.


Students will be able to list and draw the different classes of immunoglobulins.


Students will be able to describe the basics of an ELISA assay and practical uses of
this type of lab assay.


Students will research and describe the disease tuberculosis and how it interacts with
the human immune system.


Students will perform a bacterial transformation.


Students will relate the bacterial transformation lab to evolution in general and to the
evolution of bacterial resistance in particular.


Students will communicate their knowledge of the immune system with two
narrative essays.

This unit may be incorporated into several units of study (biochemistry, disease and
immunity, evolution, microbiology, biotechnology) of a typical biology, advanced
biology or anatomy and physiology class. It is meant to complement and extend existing
curricula rather than exist as a stand
-
alone unit.


Hiding in Plain Site: The Immune System, Tuberculosis, and
Antibiotic Resistance

ABSTRACT # 818999



This past summer I worked in the Cruikshank lab at the Boston University School
of Medicine. The Cruikshank research team determined that mannose
lipoarabinomannan, (ManLAM), a cell wall component of
M. tuberculosis

can
decrease S1P sensitivity of activated T cells as well as to alter production of selected
cytokines. These alterations likely affect the normal immune response by changing T
cell homing patterns and decreasing production of response cytokines thereby
increasing the mTB’s chances of survival and optimal infection. This research along
with the history of tuberculosis inspired me to create a high school biology unit. This
unit is designed to link the study of the immune system with evolution through a closer
examination of
mTB
, particularly, its two main forms: latent TB infection and active
TB disease. The unit includes teacher presentations and demonstrations, an internet
activity entitled TB in the Media, and laboratory activities. Students are first
introduced to the basic functions of the immune system through its defense against
Mycobacterium tuberculosis infection. Then, students work through a simulated
ELISA (Enzyme
-
linked
immunosorbent

assay) to see its importance in diagnosing
disease. Finally, we turn to the emergence of multi
-
drug resistant
mTB

as students
carry out a laboratory exercise on bacterial transformation. Students present a
summary of their learning through the creation of a pamphlet/poster where they
investigate a specific disease and describe its symptoms, treatments, suggested causes
and immune system interactions
.

Mark Stephansky,
1
; Jillian Richmond,
2

and William Cruikshank,
2

1
Whitman
-
Hanson Regional High School, Whitman, MA 02382,
2
Pulmonary
Center, Boston University School of Medicine, Boston, MA 02118

MATERIALS & METHODS

A Sampling of Laboratory Experiences Encountered During Summer Research
Program in Immunology

Human
Jurkat

T
-
lymphocyte cell line and Human Primary T
-
lymphocytes

Human
Jurkat

T
-
lymphocyte cells lines were obtained from ATCC and were cultured in
RPMI complete medium supplemented with 25mM HEPES, 10% fetal bovine serum,
and 1% Penicillin/Streptomycin. Primary T lymphocytes were isolated from whole
blood via density gradient centrifugation and were cultured in RPMI complete
medium. Both primary cells and cell lines were incubated at 37ºC, 5% CO2. Cell lines
were kept in a logarithmic growth phase for the entirety of experiments.



CURRICULUM TIME REQUIREMENT


Total time for this unit is approximately 7 class periods (7 hours


although the
computer exercises may be completed by students at home or at the library). The
immune system web tutorial is 1 class period, the antibody investigation is 1 class
period, the virtual ELISA is 1 class period, the ELISA Classroom Simulation is 1 class
period, the bacterial transformation lab requires 3


60 minute lab periods.

BACKGROUND



Tuberculosis, second only to HIV as the worldwide cause of death from infection,
has haunted humanity for the past 500,000 years. Currently one
-
third of the world’s
population is infected with the TB
bacterium.
1

The unique characterization of the
Mycobacterium tuberculosis
(
mTB
) cell wall has been implicated in not only
protecting the pathogen but in increasing its adaptive ability.



Recent
TB research has focused on T
-
lymphocyte function and migration.
Lymphocytes are the key cells of acquired immunity. T
-
lymphocytes migrate to the
thymus to mature, while B
-
lymphocytes mature in the bone marrow. Naïve T cells
constantly circulate between blood and lymph nodes in search of pathogen
-
derived
antigen. The migration into lymph nodes is controlled by the action of adhesion
receptors and chemokines, particularly CCL19 and
CCL21,
2

both of which are
produced in the high endothelial venules of lymph nodes, while
egress is controlled by
sphingosine 1
-
phosphate (S1P)
gradients.
3
This
is significant in that in order for T
cells to carry out their immune functions in peripheral tissue they must
first experience
physical contact with antigen presenting cells, in particular, dendritic cells within a
lymph node. (When dendritic cells bind foreign antigen they become activated and
migrate to lymph nodes where antigen sampling by Naïve T cells takes place.) The
activated T cells must then egress
from lymph
nodes to carry out their response.



The TB cell wall is unique among prokaryotes and a major contributor to the
pathogen’s virulence and survival. Two of the principle components of the cell wall
are
mannose lipoarabinomannan
(ManLAM) and mycolyl arabinogalactans (AG).
During TB infection
ManLAM portions are thought break off and directly insert
into lipid raft microdomains (cholesterol and phosphoinositol rich regions).
When
primary human T cells were treated with
ManLAM,
migration to S1P was
completely
blocked.
4

Another cell wall component, Arabinogalactan,
is part of the
mycolyl arabinogalactan
-
peptidoglycan (mAGP) complex, a linking of the free lipids
and cell wall
proteins
5

(Fig
1.) that surround the cell membrane. AG is thought to play
a role in the regulation of specific cytokines responsible for protecting the TB
granuloma that surrounds the bacterium
.

This past summer the Cruikshank lab
determined that
ManLAM can
decrease S1P sensitivity of activated T cells as well as
to alter production of selected
cytokines.
4
These
alterations likely affect the normal
immune response by changing T cell homing patterns and decreasing production of
response cytokines thereby increasing the mTB’s chances of survival and optimal
infection.


Fig 1: Mycobacterium Cell Wall

1.
outer lipids

2.

mycolic

acid

3.
Polysaccharides


(arabinogalactan)

4.

peptidoglycan

5.

plasma membrane

6.

lipoarabinomannan
(LAM)

7.

phosphatidylinositol

mannoside

8.

cell wall skeleton


REFERENCES

1.
K.
Todar
, Mycobacterium tuberculosis and Tuberculosis (2009). Available at
http://www.textbookofbacteriology.net/tuberculosis.html (18 February 2010).

2.
Drennan
, M.,
Elewaut
, D.,
Hogquist
, K. 2008.
Thymic

emigration: Sphingosine
-
1
-
phosphate receptor
-
1
-
dependent models and beyond.
Eur

J. of Immun.39:4:925
-
930.

3.
Allende
, M.L., Dreier, J.L.,
Mandala
, S., and
Proia
, R.L. 2004. Expression of the sphingosine
-
1 phosphate
receptor, S1P1, on T
-
cells controls
thymic

emigration. J. Biol. Chem. 279, 15396

15401.

4.
J. Richmond & W. Cruikshank, BUMC dept. of Pulmonary, Allergy, and Critical Care Medicine;
Unpublished Data

5.
Birch HL, et al. (2008) Biosynthesis of
mycobacterial

arabinogalactan: Identification of
a novel α(1

>3)
arabinofuranosyltransferase. Mol Microbiol 69:1191

1206.

ACKNOWLEDGEMENTS


I would like to sincerely thank the American Association of Immunologists for this
opportunity, Dr. William Cruikshank, Jillian Richmond and the entire lab at Boston
University Medical School Pulmonary Center for their wisdom, patience, and support of
this profession
-
changing experience.

Figure 3.

IVIS
in vitro
imaging was performed on mice subjected to ManLAM I.T.
following I.V. injection of
Qdot

labeled NWNATS. Compared to the PBS control
(left) the ManLAM treated mouse (right) showed a twofold increase in T cell
concentration within lymphoid tissue. Concentrations are highest in the lymph nodes
which drain the lungs in mice. The noninvasive fluorescent IVIS imaging system
allowed for live analysis of T cell migration while mice were under sedation.

IVIS Imaging of
Qdot

labeled NWNATS in Mice Subjected to ManLAM I.T.
4

Isolation of mouse peripheral T
-
lymphocytes

T
-
lymphocytes were obtained from 6 week old C57BL/6J mice by removing the
axillary
,
cervical, inguinal, and
mediastinal

nodes and suspending in RPMI complete media.
Cells were dissociated from lymph node
stroma

and filtered to remove excess tissue. In
addition, the spleen was
perfused

with media and the supernatant collected. A red
blood cell
lysis

was performed, then cells were washed, and
resuspended

in media.
Isolated T
-
lymphocytes were
resuspended

in RPMI. The Boston University
Institutional Animal Care and Use Committee Approved all experiments.


Jurkat

cell stimulation and Arabinogalactan pretreatment

2.5x105
Jurkat

cells were isolated and allowed to incubate at 37ºC, 5% CO2 for 24 hours
prior to PHA stimulation. Following incubation cells were pretreated with 10ng/
mL

Arabinogalactan (AG provided by Colorado State University TBVRTM Contract) for
2 hours. Cell stimulation was performed using
Phaseolus

vulgaris

agglutinin (PHA,
Sigma
-
Aldrich) at 100μg/
mL

and
Ionomycin

calcium salt (Sigma
-
Aldrich) at 2μM.
Stimulated cell suspensions were allowed to incubate for 72 hours prior to supernatant
collection and cytokine analysis by ELISA.


Figure 2.
All cells pretreated with arabinogalactan had no detectable TNF
-
α or IL
-
4
concentrations, this may be attributed to concentrations outside the standard range (R2
= 0.98 & 0.934, respectively). IL
-
4 concentration for the untreated cells was 1.159
ng
/
mL

indicating that AG may inhibit IL
-
4 production.

Figure 4.

Schematic representation of “sandwich” ELISA (Enzyme
-
linked
immunosorbent

assay) screening set. ELISA is a biochemical technique used mainly in
immunology to detect the presence of an antibody or an antigen in a sample.

BACTERIAL TRANSFORMATION

A major objective of this curriculum unit is for students to forge a link between the
inability of a compromised immune system to completely eradicate a pathogen borne
threat with the ability of bacteria to evolve resistance to antibiotic drugs designed to aid
the immune system. Students research tuberculosis and the stages of disease. They learn
about bacterial transformation and how bacteria receive resistance genes to antibiotics,
Fig 5. Students then conduct their own bacterial transformation lab Fig 6.

Figure 5.

Representation (left) of how bacteria may receive antibiotic resistance genes
from their environment. Scanning electron micrograph (right) showing Multidrug
-
Resistant
Mycobacterium tuberculosis
and possible effective treatments.
Mag

15549X.
CDC.

Figure 6.

Schematic representation of bacterial transformation procedure.

CONCLUSIONS


This has certainly been an enlightening experience. One of my main reasons for applying
to the program was to become better informed and more confident in my knowledge of
the workings of the immune system. My summer research experience certainly delivered.
My experience in the Cruikshank lab at BUMC has bolstered my own knowledge and also
provided a number of anecdotes to share with my students not only about immune system
research, but life in the lab in general.