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Antibody Testing for use in Immunohistochemistry and the Investigation of Precociously Induced Maturation of the Gastrointestinal Tract in Young Nude Rats

Gidlund, Catherine (2016) MOBT01 20161
Degree Projects in Molecular Biology
Abstract
Background: Rat pups are born with an immature intestine adapted for the effective uptake of milk nutrients and with high permeability for bioactive molecules from the maternal milk until weaning. During weaning the immune system is activated after stimulation with e.g. dietary antigens, leading to an inflammatory response in the gut. This response is believed to be important for progress of gut maturation which involves changes in the small intestine from a foetal- to adult-like state. Recent studies indicate that immune cells, especially T cells, could play a key role in this and that treatment with immunosuppressive drugs delay gut maturation. The aim of this project was to further investigate the role of the immune system in the... (More)
Background: Rat pups are born with an immature intestine adapted for the effective uptake of milk nutrients and with high permeability for bioactive molecules from the maternal milk until weaning. During weaning the immune system is activated after stimulation with e.g. dietary antigens, leading to an inflammatory response in the gut. This response is believed to be important for progress of gut maturation which involves changes in the small intestine from a foetal- to adult-like state. Recent studies indicate that immune cells, especially T cells, could play a key role in this and that treatment with immunosuppressive drugs delay gut maturation. The aim of this project was to further investigate the role of the immune system in the maturation of the gastrointestinal (GI) tract using immunodeficient nude young rats as the model.

Methods: At first different antibodies were tested with immunohistochemistry (IHC) to see if they could be used in the investigation of GI maturation. Thymus deficient (NIH-Foxn1rnu, nude) suckling rats were treated with a dietary antigen (PHA) or a protease (trypsin) known to induce precocious GI maturation. Whereafter, the maturity of the intestines was investigated, i.e., organ growth and the proportion of adult-like epithelium was studied by morphometric methods and the presence of CD3+ cells was studied using IHC techniques.

Results: The α-CD3 antibody was found to be useful for the further IHC studies of GI maturation. My combined results revealed that the treated nude rats showed intestinal maturation, similar to that seen at weaning in euthymic suckling rats. This included increased length of the proximal villi, tendency of increased crypt depth and change from foetal-like vacuolated cells to adult-like non-vacuolated cells in the distal epithelium. Surprisingly, CD3+ cells could be observed in the small intestine of the nude athymic young rats, however, in a lower amount than in the euthymic young rats.

Conclusion: This study showed that the intestinal maturation process could be induced in nude young rats. However, since CD3+ cells could be detected in the nude rats this indicate that thymus-independent T cells might be involved in maturation of the gut after birth. These discoveries might lead to a better understanding of maturation of the GI tract and hopefully help in the search for treatment of Necrotizing Enterocolitis (NEC) in preterm humans born too early with an immature gut. (Less)
Popular Abstract (Swedish)
Sambandet mellan mognaden av immunsystemet och mag- tarmkanalen

Mognaden av mag-tarmkanalen tros vara sammankopplad med mognaden av immunförsvaret efter födseln. Nyligen gjorda studier indikerar att immunceller, framförallt T-celler, kan ha en roll i detta eftersom behandling med immunhämmande mediciner kan fördröja mognadsprocessen hos unga råttor.
Råttor föds med en omogen tarm anpassad för upptag av näringsämnen och med hög permeabilitet för bioaktiva molekyler från modersmjölken. Under de första veckorna av en råttunges liv är den beroende av det skydd som mjölkmolekyler så som antikroppar och tillväxtfaktorer ger, på grund av deras omogna immunsystem. Under avvänjningen, från modersmjölk till fast föda, kommer immunsystemet att... (More)
Sambandet mellan mognaden av immunsystemet och mag- tarmkanalen

Mognaden av mag-tarmkanalen tros vara sammankopplad med mognaden av immunförsvaret efter födseln. Nyligen gjorda studier indikerar att immunceller, framförallt T-celler, kan ha en roll i detta eftersom behandling med immunhämmande mediciner kan fördröja mognadsprocessen hos unga råttor.
Råttor föds med en omogen tarm anpassad för upptag av näringsämnen och med hög permeabilitet för bioaktiva molekyler från modersmjölken. Under de första veckorna av en råttunges liv är den beroende av det skydd som mjölkmolekyler så som antikroppar och tillväxtfaktorer ger, på grund av deras omogna immunsystem. Under avvänjningen, från modersmjölk till fast föda, kommer immunsystemet att aktiveras efter stimulering av olika födoantigener. Detta leder till en inflammatorisk respons i tarmarna som tros vara viktig för att stimulera mognadsprocessen.
Idag föds många barn förtidigt med bl.a. en omogen mag-tarmkanal vilket medför en ökad risk att utveckla tarmsjukdomar, så som NEC (nekrotiserande enterokolit). En omogen tarm med en oönskad passage av främmande antigen över tarmväggen kan medverka i att orsaka autoimmuna sjukdomar och allergier. En sådan ”öppen” tarm kan liknas den hos diande råttor. Förhoppningarna är att kunna behandla barn genom att inducera mognad av mag-tarmkanalen. Det har tidigare visats att det är möjligt att inducera mognad av tarmen i diande råttor genom behandling med födoämnesantigen eller enzym.
Syftet med detta projekt var att undersöka immunsystemets roll under mognaden av mag- tarmkanalen genom att försöka inducera mognad hos unga råttor med en immunbrist (nude) som en modell.
Metod och Resultat
Diande nude-råttor, med avsaknad av thymus-beroende T-celler, behandlades med ett antigen (PHA) eller ett enzym (trypsin) för att inducera mognad, varefter förändringar i tarmen undersöktes såsom tillväxt, morfologiska förändringar och närvaro av T-celler.
De behandlade nude- råttorna visade tarmmognad, som liknar den som ses vid avvänjning av normala råttungar, inkluderat ändringen av tarmen från ett foster-lik till ett mer vuxen-lik stadium. Intressant nog hittades T-celler (CD3+) även i tarmarna från nude-råttorna, dock i mindre mängd än i de normala råttorna. Dessa T-celler kan eventuellt vara orsaken till de mognadseffekter som även observerades i nude-råttorna.
Denna studie visar att mognad av mag-tarmkanalen kan induceras även i råttor med immunbrist och avsaknad av thymus-beroende T-celler. Dessa resultat leder till en bättre förståelse av sambandet mellan immunsystemet och mognaden av mag-tarmkanalen och kan förhoppningsvis få oss ett steg närmare en förbättrad behandling av förtidigt-föda barn med en omogen tarm.

Handledare: Ester Arévalo Sureda and Björn Weström
Examensarbete – masterexamen 60 hp i Medicinska Biologi/Molekylärbiologi 2015/2016
Biologiska institutionen, Lunds universitet (Less)
Please use this url to cite or link to this publication:
author
Gidlund, Catherine
supervisor
organization
course
MOBT01 20161
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8893029
date added to LUP
2016-10-05 13:47:02
date last changed
2016-10-07 09:38:23
@misc{8893029,
  abstract     = {Background: Rat pups are born with an immature intestine adapted for the effective uptake of milk nutrients and with high permeability for bioactive molecules from the maternal milk until weaning. During weaning the immune system is activated after stimulation with e.g. dietary antigens, leading to an inflammatory response in the gut. This response is believed to be important for progress of gut maturation which involves changes in the small intestine from a foetal- to adult-like state. Recent studies indicate that immune cells, especially T cells, could play a key role in this and that treatment with immunosuppressive drugs delay gut maturation. The aim of this project was to further investigate the role of the immune system in the maturation of the gastrointestinal (GI) tract using immunodeficient nude young rats as the model. 

Methods: At first different antibodies were tested with immunohistochemistry (IHC) to see if they could be used in the investigation of GI maturation. Thymus deficient (NIH-Foxn1rnu, nude) suckling rats were treated with a dietary antigen (PHA) or a protease (trypsin) known to induce precocious GI maturation. Whereafter, the maturity of the intestines was investigated, i.e., organ growth and the proportion of adult-like epithelium was studied by morphometric methods and the presence of CD3+ cells was studied using IHC techniques. 

Results: The α-CD3 antibody was found to be useful for the further IHC studies of GI maturation. My combined results revealed that the treated nude rats showed intestinal maturation, similar to that seen at weaning in euthymic suckling rats. This included increased length of the proximal villi, tendency of increased crypt depth and change from foetal-like vacuolated cells to adult-like non-vacuolated cells in the distal epithelium. Surprisingly, CD3+ cells could be observed in the small intestine of the nude athymic young rats, however, in a lower amount than in the euthymic young rats. 

Conclusion: This study showed that the intestinal maturation process could be induced in nude young rats. However, since CD3+ cells could be detected in the nude rats this indicate that thymus-independent T cells might be involved in maturation of the gut after birth. These discoveries might lead to a better understanding of maturation of the GI tract and hopefully help in the search for treatment of Necrotizing Enterocolitis (NEC) in preterm humans born too early with an immature gut.},
  author       = {Gidlund, Catherine},
  language     = {eng},
  note         = {Student Paper},
  title        = {Antibody Testing for use in Immunohistochemistry and the Investigation of Precociously Induced Maturation of the Gastrointestinal Tract in Young Nude Rats},
  year         = {2016},
}