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.
Viability of cd4+T cells and Heligmosomoides Babbileo
By Gabriel PadillaCSUSM Biology Major
My name is Gabriel Padilla, and I am a junior Biology majorat California State University San Marcos. While attendingCSUSM, I have been lucky enough to get involved inundergraduate research. I was given the opportunity toresearch, design, conduct, and present my own scientificexperiment. It has been a wonderful and rewardingopportunity, and I am excited to share it with you!
Helminths, nematode parasites, are a substantial causeof morbidity and disease. It is estimated that over one billion people in the worldare infected by these worms. Visit the CDCs website to learn more about helminths
T Helper Cell responseo CD4+ T cells recruit other cells and damage the parasite bysecreting 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
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 treatinflammatory and/or autoimmune diseases.NematodeparasiteExcretory/SecretoryProduct
H. bakeri is a nematode and a mouse parasite. It is a good model for human hookworm infection H. bakeri produces excretory/secretory products.
1. I wanted to assess if the presence of live H. bakeriworms affects CD4+ T cell viability.2. I wanted to assess if the presence of CD4+ T cellsaffects female H. bakeri motility and reproduction.
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. 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. bakeri worms from mouse small intestine;placed in same 48-well plate 24h incubation at 37°C in 3%O2:5% CO2:92% N gasmixture
After 24 hour incubation, the following dependent variables weremeasured:CD4+ T cell viability• Trypan Blue exclusionIn-vitro H. bakeri egg output• I counted the number of eggs present with a dissectionmicroscopeH. bakeri worm motility• I looked at the worms under a microscope and scored themwith 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
CD4+ T cell viability will not be affected by the presenceof H. bakeri.o H. bakeri excretory/secretory products should suppress T cellfunction, but not kill T cells.• This is because the parasites want to exploit their hosts for living. Ifthey kill their hosts, then the parasites will die too.
Live: t=1.2, p= 0.24, DF = 54;Dead: t=0.1, p=0.92, DF=54H. bakeri withCD4+T cellsCD4+T cells ONLYLive Cells Dead CellsAverage#ofCD4+TcellsTreatment Group
H. bakeri will have decreased motility andproduce fewer eggs in vitro.o This is because mouse CD4+ T cells secretecytokines which can damage the worms.
t= 1.12, p=0.27, DF=56H. bakeri H. bakeri andCD4+ T cellsTreatment
H. bakeri H. bakeri andCD4+ T cellsTreatmentt= 0.65, p= 0.51, DF= 56
Maybe ↑ Worm Excretory/Secretory products= T cell inhibition. Maybe Cytokines produced by CD4+T cells areineffective at damaging parasitesMaybe the CD4+T cells need the presence of other immune cells todamage 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 oneparasite in the wells.
1.) By learning more about the molecular and cellularinteractions between host immune cells and parasitesNew medications can be developed to expel theparasites. 2.)Helminth excretory/secretory products haveimmunoregulatory 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 excretorysecretory products to use them to treat autoimmune diseasessuch as Inflammatory Bowel Disease, Asthma, Arthritis, andeven Type 1 Diabetes!
Dr. Deborah Kristan Samantha Lang The RISE Program Students in Dr. Kristan’s lab
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