Viability of CD4+ T cells and Heligmosomoides bakeri female worms during co-

incubation

By Gabriel PadillaCSUSM Biology Major

Hello! 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!

Background 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

Human response to Parasites

T Helper Cell responseo 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

Parasite Excretory/Secretory Products

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

Heligmosomoides bakeri

H. bakeri is a nematode and a mouse parasite.

It is a good model for human hookworm infection

H. bakeri produces excretory/secretory products.

My Research Goals

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. bakeri motility and reproduction.

Methods: Day 1 Experimental Groups:

o CD4+ T cells only (n = 4 wells)o H. bakeri worms only (n = 4 wells)o H. bakeri + CD4+ T cells (n = 4 wells)o The T cells by themselves and the H. bakeri by themselves are

“control groups” so I can compare them to the H. bakeri combined 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. bakeri worms from mouse small intestine; placed in same 48-well plate

24h incubation at 37°C in 3%O2:5% CO2:92% N gas mixture

Experimental Layout

Methods: Day 2 After 24 hour incubation, the following dependent variables were

measured:CD4+ T cell viability

• Trypan Blue exclusion

In-vitro H. bakeri egg output• I counted the number of eggs present with a dissection

microscope

H. bakeri worm 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

Hypothesis 1 CD4+ T cell viability will not be affected by the

presence of H. bakeri. o H. bakeri excretory/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.

T cell viability was unaffected by H. bakeri

Live: t=1.2, p= 0.24, DF = 54;Dead: t=0.1, p=0.92, DF=54

H. bakeri with CD4+T cells

CD4+T cells ONLY

Live Cells Dead Cells

Avera

ge #

of

CD

4+ T

cells

Treatment Group

Hypothesis 2

H. bakeri will have decreased motility and produce fewer eggs in vitro.

o This is because mouse CD4+ T cells secrete cytokines which can damage the worms.

Worm motility did not change in the presence of CD4+ T cells

t= 1.12, p=0.27, DF=56

H. bakeri H. bakeri and CD4+ T cellsTreatment

H. bakeri egg output did not change in the presence of CD4+ T

cells

H. bakeri H. bakeri and CD4+ T cellsTreatment

t= 0.65, p= 0.51, DF= 56

Why was Hypothesis 2 not supported?

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 cultureMaybe I needed to add more CD4+T cells in the wells, or more than one

parasite in the wells.

This research is important!

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!

Special Thanks Dr. Deborah Kristan Samantha Lang The RISE Program Students in Dr. Kristan’s lab

Literature Cited Behnke, J.M., Menge, D.M. and Noyes, H. (2009). Heligmosomoides bakeri: 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 Heligmosomoides polygyrus 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/