5BBBI203 In course Assessment
Leishmaniasis is a parasitic infection caused by a protozoan from the
Trypanosomatidae family, genus Leishmania. A well established mouse model of
visceral leismaniasis disease exists using a strain (BALB/C) of mice susceptible to
the disease. An experiment was performed in order to examine the effects of an anti-
IL-10 immunomodulatory agent on cytokine production by T cells in a mouse model
of Leismaniasis. L. major promastigotes were injected into the ears of BALB/c mice.
After 4 weeks T cells were isolated from the lymph nodes and cultured for 48 hours
in the presence of 5 µg/mL of soluble Leishmania antigen (SLA). Supernatants from
these cultures were harvested and ELISA experiments performed to investigate IFNg
and IL-4 production by the T cells. Following infection, a group of mice were treated
with an anti-IL-10 immunomodulatory agent prior to isolation and culture of T cells.
For comparison, two groups of control mice were used: one group was uninfected
and untreated (no immunomodulatory agent) and the other was uninfected but
treated with the anti-IL-10 agent. Details of the samples can be found below.
Following infection, L.major infected mice developed skin lesions and showed a high
worm burden at the site of infection. The anti-IL-10 treated mice showed reduced
symptoms and reduced worm burden. Both groups of control mice were healthy.
Sample A = Untreated mouse.
Sample B = L.major infected mouse.
Sample C = L.major infected mouse treated with anti-IL-10.
Sample D = Uninfected mice treated with anti-IL-10 alone.
Samples were added to ELISA plates in the following layout – and the raw absorbance data is
indicated for each cytokine.
The deadline for submission of the coursework is 4pm Thursday 24th November. The
coursework with accompanying coversheet must be handed into the academic centre,
1st floor Henriette House.
Plate Layout
STD
4000n
g/ml
STD
2000
ng/ml
STD
1000
ng/ml
STD
500
ng/ml
STD
250
ng/ml
STD
125
ng/ml
STD
62.5
ng/ml
STD
31.2
ng/ml
STD
15.5
ng/ml
STD
7.75
ng/ml
STD
3.7
ng/ml
blank
STD
4000n
g/ml
STD
2000
ng/ml
STD
1000
ng/ml
STD
500
ng/ml
STD
250
ng/ml
STD
125
ng/ml
STD
62.5
ng/ml
STD
31.2
ng/ml
STD
15.5
ng/ml
STD
7.75
ng/ml
STD
3.7
ng/ml
blank
Sample
A
Sample
A
Sample
A
Sample
A
Sample
B
Sample
B
Sample
B
Sample
B
Sample
C
Sample
C
Sample
C
Sample
C
Sample
D
Sample
D
Sample
D
Sample
D
Plate A: O.D. values for IFNg ELISA
2.893 2.772 2.401 1.772 0.995 0.613 0.332 0.188 0.056 0.02 0.01 0.005
2.761 2.514 3.335 1.665 0.992 0.624 0.317 0.132 0.032 0.01 0.01 0.004
0.006 0.001
0.004 0.003
0.186 0.096
0.124
0.176
2.389 2.446 2.229 2.678
0.025 0.018 0.019 0.022
Plate B: O.D. values for IL-4 ELISA
1.422 0.669 0.336 0.159 0.079 0.043 0.032 0.011 0.004 0.003 0.001 0.001
1.589 0.599 0.322 0.214 0.092 0.054 0.027 0.016 0.002 0.004 0.001 0.001
0.001
0.005 0.002 0.003
0.998
1.234
1.116
1.222
0.031
0.028 0.033 0.021
0.012
0.016 0.019 0.012
Task:
You should first analyse the data you are provided with to determine the amount of IFNg and IL-4
produced by each of the four experimental samples (A-D).
a) You should plot the standard curve of the two ELISA experiments on a graph using an
appropriate analysis package (eg. Sigmaplot, etc) – ensure graphs are presented and labelled
appropriately.
b) You should present the results of your analysis of the experimental samples in a separate
graph.
c) You should describe your results (500 words maximum). Remember to include a
description of what your sample and treatment groups did in comparison to controls. You should
comment on what the sensitivity of the two ELISA experiments was and how significant, reliable and
reproducible your results are.
d) You should then give a brief discussion and conclusion for your findings (500 words
maximum). Why were these two cytokines measured? What can they tell us about how the body
fights the infection? Why might treatment with the anti-IL-10 agent effect the production of IFNg
and IL-4 by T cells and why might this effect the health of the mice. Finally, can you find any research
literature that supports these conclusions in human Leismaniasis infection?
