Gabriel Padilla, a junior biology major at CSUSM, conducted an undergraduate research experiment comparing CD4+ T cell viability and H. bakeri worm motility and reproduction alone and in combination. The experiment found no significant differences between the combined and separate conditions. Possible explanations for the negative results include not enough T cells or parasites in the culture or the need for other immune cells. The research aims to help develop new drugs to expel parasites and use helminth secretions to treat autoimmune diseases.
2. My name is Gabriel Padilla, and I am a junior Biology major
at California State University San Marcos. While attending
CSUSM, I have been lucky enough to get involved in
undergraduate research. I was given the opportunity to
research, design, conduct, and present my own scientific
experiment. It has been a wonderful and rewarding
opportunity, and I am excited to share it with you!
3. Helminths, nematode parasites, are a substantial cause
of morbidity and disease.
It is estimated that over one billion people in the world
are infected by these worms.
Visit the CDC's website to learn more about helminths
4. T Helper Cell response
o CD4+ T cells recruit other cells and damage the parasite by
secreting cytokines such as:
• Interleukin-4, Interleukin-5, Interleukin-9, Interleukin-
13, Transforming Growth Factor-β
• Click here to learn more about the human immune system
5. May damage and/or inhibit T cell function
Can regulate and even suppress the host immune system
An exciting area of new research, and can treat
inflammatory and/or autoimmune diseases.
Nematode
parasite
Excretory/Secretory
Product
6. H. bakeriis a nematode and a mouse parasite.
It is a good model for human hookworm infection
H. bakeriproduces excretory/secretory products.
7. 1. I wanted to assess if the presence of live H. bakeri
worms affects CD4+ T cell viability.
2. I wanted to assess if the presence of CD4+ T cells
affects female H. bakerimotility and reproduction.
8. Experimental Groups:
o CD4+ T cells only (n = 4 wells)
o H. bakeriworms only (n = 4 wells)
o H. bakeri+ CD4+ T cells (n = 4 wells)
o The T cells by themselves and the H. bakeriby themselves are
“control groups” so I can compare them to the H.
bakericombined with T cells to look for differences.
I Isolated mouse CD4+ T cells using sterile techniques;
placed in cell culture media in a 48-well plate
I Removed H. bakeriworms from mouse small intestine;
placed in same 48-well plate
24h incubation at 37°C in 3%O2:5% CO2:92% N gas
mixture
9.
10. After 24 hour incubation, the following dependent variables were
measured:
CD4+ T cell viability
• Trypan Blue exclusion
In-vitro H. bakeriegg output
• I counted the number of eggs present with a dissection
microscope
H. bakeriworm motility
• I looked at the worms under a microscope and scored them
with my 0-4 motility scoring scale
• 4- spontaneous full body movement
• 3- spontaneous head and tail movement
• 2- full body movement after probing
• 1- head and tail movement after probing
• 0- No movement after probing
11. CD4+ T cell viability will not be affected by the presence
of H. bakeri.
o H. bakeriexcretory/secretory products should suppress T cell
function, but not kill T cells.
• This is because the parasites want to exploit their hosts for living. If
they kill their hosts, then the parasites will die too.
12. Live: t=1.2, p= 0.24, DF = 54;
Dead: t=0.1, p=0.92, DF=54
H. bakeriwith
CD4+T cells
CD4+T cells ONLY
Live Cells Dead Cells
Average#ofCD4+Tcells
Treatment Group
13. H. bakeriwill have decreased motility and
produce fewer eggs in vitro.
o This is because mouse CD4+ T cells secrete
cytokines which can damage the worms.
14. t= 1.12, p=0.27, DF=56
H. bakeri H. bakeriand
CD4+ T cellsTreatment
15. H. bakeri H. bakeriand
CD4+ T cells
Treatment
t= 0.65, p= 0.51, DF= 56
16. Maybe ↑ Worm Excretory/Secretory products
= T cell inhibition.
Maybe Cytokines produced by CD4+T cells are
ineffective at damaging parasites
Maybe the CD4+T cells need the presence of other immune cells to
damage the parasites.
Not enough CD4+ T cells or parasites in culture
Maybe I needed to add more CD4+T cells in the wells, or more than one
parasite in the wells.
17. 1.) By learning more about the molecular and cellular
interactions between host immune cells and parasites
New medications can be developed to expel the
parasites.
2.)Helminth excretory/secretory products have
immunoregulatory capabilities.
o Current clinical trials use whole worms.
o Excretory/secretory products may have fewer side effects.
o Hopefully we will soon be able to extract these excretory
secretory products to use them to treat autoimmune diseases
such as Inflammatory Bowel Disease, Asthma, Arthritis, and
even Type 1 Diabetes!
18. Dr. Deborah Kristan
Samantha Lang
The RISE Program
Students in Dr. Kristan’s lab
19. Behnke, J.M., Menge, D.M. and Noyes, H. (2009). Heligmosomoidesbakeri: a model for
exploring the biology and genetics of resistance to chronic gastrointestinal nematode
infections. Parasitology 136, 1565-1580.
Brooker, S. (2010). Estimating the global distribution and disease burden of intestinal
nematode infections: Adding up the numbers – A review. Int. J. Parasitol. 10, 1137-1144.
Hotez, P., Brindley, P.J., Bethony, J.M., King, C.H., Pearce EJ, Jacobson J (2008). Helminth
infections: the great neglected tropical diseases. J Clin Invest 118, 1311–1321.
Maizels, R.M., Hewitson, J.P., Murray, J., Harcus, Y.M., Dayer, B., Filbey, K.J., Grainger, J.R.,
McSorley, H.J., Reynolds, L.A., Smith, K.A. (2012). Immune modulation and modulators in
Heligmosomoidespolygyrus infection. Exp. Parasitol. 132, 76-89
McSorley, H.J., Hewitson, J.P., Maizels, R.M. (2013). Immunomodulation by helminth
parasites: Defining mechanisms and mediators. Int. J. Parasitol.
Navarro, S., Ferreira, I., Loukas, A. (2013). The hookworm pharmacopoeia for inflammatory
diseases. Int J. Parasitol. 43, 225-231.
http://babayan.bio.ed.ac.uk/index.html
http://www.nobelprize.org/educational/medicine/immunity//immune-detail.html
http://www.noh-sign.net/serological_plates.htm
http://www.cdc.gov/parasites/sth/
Notes de l'éditeur
Trypan Blue works by entering dead cells, but live cells are impermeable to it. Live cells will look light colored under the microscope, and dead cells will look dark blue under the microscope.
There are no significant differences in live cells or dead cells between treatment groups, so my hypothesis was supported.
There was no significant difference in average worm motility between groups, so my second hypothesis was not supported.
There was no significant difference in average number of eggs visible in a well, so my 2nd hypothesis was not supported.
