Analysing gene expression of IFN-ɑ stimulated genes in patients with SLE using direct and indirect methods
(2021) MOBY01 20202Degree Projects in Molecular Biology
- Abstract
- Systemic Lupus Erythematosus (SLE) is an autoimmune disease where the immune system is attacking the body and its tissues. This leads to a chronic inflammation that can affect different parts of the body and therefore present itself in a vast variety of symptoms. Depending on the activity of the disease it can go up in flares where the patients experience symptoms and then go down into recession. Both the adaptive and innate immune system is involved as well as both genetic and environmental factors. Among other things, it is the formation of autoantibodies, immune complexes and a decreased ability to clear cell debris from apoptotic cells that starts the cascade of events that lead to the chronic inflammation. One important factor in all... (More)
- Systemic Lupus Erythematosus (SLE) is an autoimmune disease where the immune system is attacking the body and its tissues. This leads to a chronic inflammation that can affect different parts of the body and therefore present itself in a vast variety of symptoms. Depending on the activity of the disease it can go up in flares where the patients experience symptoms and then go down into recession. Both the adaptive and innate immune system is involved as well as both genetic and environmental factors. Among other things, it is the formation of autoantibodies, immune complexes and a decreased ability to clear cell debris from apoptotic cells that starts the cascade of events that lead to the chronic inflammation. One important factor in all this is the cytokine interferon alpha (IFN-ɑ) which is mainly produced by plasmacytoid dendritic cells (pDCs) as a response to the cell debris and immune complexes. IFN-ɑ then lower the threshold of B cell receptor (BCR) activation and promote B cell differentiation into autoreactive B cells, as well as promote T-cell activation. This is known as the type I IFN signature that has been observed in patients with SLE. By measuring this signature it would be possible to prevent flares of the disease and to treat the patients with drugs aimed to inhibit the type I IFN system. There is therefore a clinical need for a measuring method that can be used routinely.
This study evaluates and compares two different methods to measure the type I IFN signature, one already established a direct method where peripheral blood mononuclear cells (PBMC) are isolated from patient blood samples and the expression of seven different IFN induced genes are analysed. The other method is an indirect method where patient sera is incubated on human epithelial tumor cells (WISH cells) and the same genes are analysed and compared to the PBMC samples. The results were also compared to clinical data and the effect on freezing and thawing RNA and sera was also evaluated.
Although there were some coherent samples between the methods the overall result suggests that there is a need for improvement on the indirect method. In regard to freezing and thawing samples, RNA is stable enough for freezing and thawing once but sera is not. (Less) - Popular Abstract (Swedish)
- Små molekyler med stor inverkan - interferon alfas roll i Systemisk Lupus Erythematosus (SLE)
Systemisk Lupus Erythematosus är en autoimmun sjukdom där immunförsvaret attackerar kroppens egna vävnader. Detta leder till en kronisk inflammation som kan yttra sig i en rad olika manifestationer beroende på var inflammationen sker. Sjukdomsaktiviteten går i perioder, dvs att den går upp i skov där patienten upplever symptom för att sedan gå ner i recession. Det som kännetecknar sjukdomen är en så kallad typ I interferon (IFN) signatur där celler från immunförsvaret producerar interferon, främst IFN-ɑ. Interferon är signalmolekyler som instruerar andra celler i immunförsvaret att bli aktiva. Detta skapar då en kaskad av händelser som leder... (More) - Små molekyler med stor inverkan - interferon alfas roll i Systemisk Lupus Erythematosus (SLE)
Systemisk Lupus Erythematosus är en autoimmun sjukdom där immunförsvaret attackerar kroppens egna vävnader. Detta leder till en kronisk inflammation som kan yttra sig i en rad olika manifestationer beroende på var inflammationen sker. Sjukdomsaktiviteten går i perioder, dvs att den går upp i skov där patienten upplever symptom för att sedan gå ner i recession. Det som kännetecknar sjukdomen är en så kallad typ I interferon (IFN) signatur där celler från immunförsvaret producerar interferon, främst IFN-ɑ. Interferon är signalmolekyler som instruerar andra celler i immunförsvaret att bli aktiva. Detta skapar då en kaskad av händelser som leder till den kroniska inflammationen. Den här typ I IFN signaturen är av intresse för den kliniska världen då man kan, genom att mäta den, få möjlighet att förutspå sjukdomsaktivitet och behandla patienten med korrekt medicin för att förhindra eventuell organskada.
Idag finns det metoder för att mäta typ I IFN signaturen men av olika anledningar så är det svårt att använda dessa kliniskt på ett rutinmässigt sätt. Vilket är varför vi har utvärderat och jämfört två metoder för att kunna mäta typ I IFN signaturen. Den första metoden är en etablerad metod som kräver nytagna prover från patienten. Det är en direkt metod där celler från blodet isoleras och RNA renas fram för att sedan analysera uttrycket av sju olika gener som stimuleras av IFN-ɑ. Ett förhöjt uttryck ger en indikation på en pågående typ I IFN signatur i patienten. Den andra metoden är en indirekt metod där serum från blodet inkuberas med en typ av tumörcell kallad WISH cell, därefter isoleras RNA fram och uttrycket av samma gener analyseras, som i den direkta metoden. Fördelen med denna metod är att serum kan användas, vilket rutinmässigt på kliniken tas på alla patienter och sparas i biobank.
Resultatet visade att den indirekta metoden behöver omarbetas och optimeras då patienter som var positiva för en typ I IFN signatur för den direkta metoden blev negativa i den indirekta. Vidare behöver ett större material analyseras och jämföras med kliniska data.
Handledare: Anders Bengtsson & Birgitta Gullstrand
Examensarbete 30 hp i Molekylärbiologi 2021
Biologiska institutionen, Lunds universitet (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9042370
- author
- Israelsson, Linn
- supervisor
- organization
- course
- MOBY01 20202
- year
- 2021
- type
- M2 - Bachelor Degree
- subject
- language
- English
- id
- 9042370
- date added to LUP
- 2021-03-24 11:59:09
- date last changed
- 2021-03-24 11:59:09
@misc{9042370,
abstract = {{Systemic Lupus Erythematosus (SLE) is an autoimmune disease where the immune system is attacking the body and its tissues. This leads to a chronic inflammation that can affect different parts of the body and therefore present itself in a vast variety of symptoms. Depending on the activity of the disease it can go up in flares where the patients experience symptoms and then go down into recession. Both the adaptive and innate immune system is involved as well as both genetic and environmental factors. Among other things, it is the formation of autoantibodies, immune complexes and a decreased ability to clear cell debris from apoptotic cells that starts the cascade of events that lead to the chronic inflammation. One important factor in all this is the cytokine interferon alpha (IFN-ɑ) which is mainly produced by plasmacytoid dendritic cells (pDCs) as a response to the cell debris and immune complexes. IFN-ɑ then lower the threshold of B cell receptor (BCR) activation and promote B cell differentiation into autoreactive B cells, as well as promote T-cell activation. This is known as the type I IFN signature that has been observed in patients with SLE. By measuring this signature it would be possible to prevent flares of the disease and to treat the patients with drugs aimed to inhibit the type I IFN system. There is therefore a clinical need for a measuring method that can be used routinely.
This study evaluates and compares two different methods to measure the type I IFN signature, one already established a direct method where peripheral blood mononuclear cells (PBMC) are isolated from patient blood samples and the expression of seven different IFN induced genes are analysed. The other method is an indirect method where patient sera is incubated on human epithelial tumor cells (WISH cells) and the same genes are analysed and compared to the PBMC samples. The results were also compared to clinical data and the effect on freezing and thawing RNA and sera was also evaluated.
Although there were some coherent samples between the methods the overall result suggests that there is a need for improvement on the indirect method. In regard to freezing and thawing samples, RNA is stable enough for freezing and thawing once but sera is not.}},
author = {{Israelsson, Linn}},
language = {{eng}},
note = {{Student Paper}},
title = {{Analysing gene expression of IFN-ɑ stimulated genes in patients with SLE using direct and indirect methods}},
year = {{2021}},
}