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Johansson, Siri, 2021. Uttrycksnivåer för interleukin-2 (IL-2)-mRNA och -protein hos Nova Scotia duck tolling retriever. Second cycle, A2E. Uppsala: SLU, Dept. of Animal Breeding and Genetics

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Abstract

Immunmedierad reumatisk sjukdom (IMRD) är en autoimmun sjukdom med ökad predisponering
hos rasen Nova Scotia duck tolling retriever, också känd som tollare. IMRD, som är en del i den så
kallade tollarsjukan, karakteriseras främst av smärta och stelhet i leder samt oftast positivt test avseende autoantikroppar (ANA). IMRD är en genetiskt komplex sjukdom, med åtskilliga identifierade riskgener av vilka flertalet är involverade i olika delar av immunsystemet. Beroende på vilken
typ av ANA-mönster drabbade hundar uppvisar, kan i tillägg en skillnad i riskgener observeras.
Patogenesen bakom IMRD är emellertid inte helt kartlagd.
Målet i studien var att undersöka basuttrycksnivåer för cytokinet interleukin-2 (IL-2), på mRNAnivå och proteinnivå, hos friska tollare jämfört med friska beaglar. Med avstamp i detta kan IL-2-
nivåer i framtiden jämföras mellan friska tollare och tollare insjuknade i IMRD. IL-2 utövar viktiga
funktioner för att stimulera immunförsvaret, men också essentiella funktioner för att upprätthålla
den immunologiska toleransen. Hos både människor och djur har främst minskade IL-2-nivåer observerats vid flera olika autoimmuna sjukdomar. I studien togs blodprov från friska tollare och beaglar. Studien utfördes genom enzyme-linked immunosorbent assay (ELISA), där 12 tollare och 14
beaglar deltog. Av dessa hundar deltog även 9 tollare och 3 beaglar i real-time reverse-transcription
polymerase chain reaction (RT2
-PCR).
Variationsvidden för koncentrationer IL-2-protein hos tollarna (n=12) uppmättes genom ELISA till
2,5-6,7 ng/l, med en median på 3,3 ng/l. Hos beaglarna (n=11) låg variationsvidden på 2,6-7,9 ng/l,
med en median på 5,8 ng/l. En trend observerades där tollarna generellt tycktes ligga lägre än beaglarna i uppmätta IL-2-nivåer. Ingen signifikant skillnad kunde emellertid detekteras mellan de båda
raserna. Ett fynd i studien var en signifikant åldersrelaterad minskning av IL-2-nivåer hos äldre
individer jämfört med yngre individer inom tollargruppen. Uttrycket av IL-2-mRNA hos tollarna
(n=8) bestämdes genom RT2
-PCR till ett ΔCt-spann mellan 11,1–13,54. Samma spann hos beaglarna
(n=3) låg mellan 12,2–13,25. Ingen skillnad i mRNA-uttrycket för IL-2 kunde uppvisas mellan tollarna jämfört med beaglarna. I studien uppvisades emellertid signifikanta skillnader mellan de båda
raserna avseende uttrycket av 4 andra gener; CRP, IL-10, IL-1R1 samt TYK2. Ingen korrelation
avseende IL-2-mRNA och IL-2-protein kunde detekteras i studien.
Det kan inte uteslutas att tollare generellt uttrycker lägre nivåer IL-2 än andra hundraser, något som
i teorin skulle kunna utgöra en initierande faktor för IMRD-utveckling tillsammans med andra riskfaktorer. I framtiden krävs emellertid en större grupp hundar för att kunna säkerställa eventuell signifikans. IL-2 är en komplex cytokin, vilket illustreras av dualiteten avseende dess stimulerande
respektive hämmande reglering på immunförsvaret. Om IL-2 visar sig vara involverad i IMRD, kan
det därför också spekuleras i huruvida IL-2-nivåer kan ses fluktuera beroende på var i sjukdomsprocessen djuret befinner sig i. Mycket forskning kvarstår emellertid innan slutsatser kan dras kring
eventuell koppling mellan IL-2 och IMRD.

,

Immune-mediated rheumatic disease (IMRD) is an autoimmune disease that shows an increased
predisposition in the Nova Scotia duck tolling retriever (NSDTR) breed. In Sweden, IMRD is a part
of a disease complex that is often referred to as ”toller disease”. IMRD is characterized by pain and
stiffness in joints, usually with an additional positive test for autoantibodies (ANA). The disease is
genetically complex but several genetic risk factors have been identified, most of which are involved
in the immune system. Earlier research have found that depending on what type of ANA-patterns
the IMRD-positive dogs present, they can be associated with different genetic risk factors. The pathogenesis of IMRD has not been fully characterized.
The aim of this study was to determine basal expression levels of the cytokine interleukin-2 (IL-2),
in mRNA and in protein form, in healthy NSDTRs compared to healthy beagles. For future prospects, this study may contribute to assess IL-2 levels in healthy NSDTRs compared to IMRD-positive NSDTRs. IL-2 has been shown to participate in several ways to stimulate the immune system,
but IL-2 also performs essential functions in maintaining the immunological tolerance. In both humans and animals, decreased levels of IL-2 has often been associated with several autoimmune diseases. In this study, blood was collected from healthy NSDTRs and beagles. The study was performed by enzyme-linked immunosorbent assay (ELISA), where 12 NSDTRs and 14 beagles participated, Out of these dogs 9 NSDTRs and 3 beagles also participated in real-time reverse-transcription polymerase chain reaction (RT2
-PCR).
The range of IL-2 concentrations in the participating NSDTRs (n=12) was with ELISA measured at
2,5-6,7 ng/l, with a median of 3,3 ng/l. The range of the beagles (n=11) was measured at 2,6-7,9
ng/l, with a median of 5,8 ng/l. An observation was made regarding a possible trend in which the
NSDTRs appeared to present lower concentrations than the beagles in measured IL-2 levels. However, no significant difference could be detected between the two groups. One finding in the study
was a significant age related decrease of IL-2 protein levels in older individuals compared to younger
individuals in the NSDTR group. The expression of IL-2 mRNA in the NSDTRs (n=8) was measured with RT2
-PCR at a ΔCt range of 11,1–13,54. The same range of the beagles was measured at
12,2-13,25. No significant difference in the IL-2 mRNA expression could be detected between NSDTRs and beagles. However, significant differences between the groups were detected regarding 4
other genes; CRP, IL-10, IL1R1 and TYK2. No correlation concerning IL-2 mRNA and IL-2 protein
could be distinguished in this study.
The possibility that NSDTRs in general express lower IL-2 levels than other breeds can not be ruled
out. This could in theory present an initiating factor in IMRD-development, along with other risk
factors. In the future, however, a larger group of dogs is required to be able to ensure any significance. IL-2 is a complex cytokine, which is illustrated by the duality concerning IL-2’s stimulating
and inhibitory regulation of the immune system. If IL-2 is found to be involved in IMRD, it can
therefore also be speculated as to whether IL-2 levels can be seen to fluctuate depending on where
in the process of the disease the animal is in. However, a lot of research remains before any conclusions can be made about the association between IMRD and IL-2.

Main title:Uttrycksnivåer för interleukin-2 (IL-2)-mRNA och -protein hos Nova Scotia duck tolling retriever
Authors:Johansson, Siri
Supervisor:Andersson, Göran and Hamlin, Helene
Examiner:Wernersson, Sara
Series:UNSPECIFIED
Volume/Sequential designation:UNSPECIFIED
Year of Publication:2021
Level and depth descriptor:Second cycle, A2E
Student's programme affiliation:VY002 Veterinary Medicine Programme 330 HEC
Supervising department:(VH) > Dept. of Animal Breeding and Genetics
Keywords:IMRD, cytokin, immunmedierad reumatisk sjukdom, NSDTR, tollare, tollarsjuka, auto-immunitet, hund, ELISA, RT2-PCR
URN:NBN:urn:nbn:se:slu:epsilon-s-16786
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-s-16786
Subjects:Animal diseases
Language:Swedish
Deposited On:16 Jun 2021 10:21
Metadata Last Modified:17 Jun 2021 01:01

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