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Characterization of circulating T cell-specific exosomes as a possible biomarker in type 1 diabetes

Mitrovic, Christina (2015) MOBM01 20142
Degree Projects in Molecular Biology
Abstract
Type 1 diabetes, also known as juvenile diabetes or insulin-dependent diabetes, is a growing and serious health problem worldwide with highest incidence in developed countries and among children. It is a T cell-mediated autoimmune disease, characterized by destruction of β-cells of the pancreatic islets of Langerhans, resulting in deficient insulin secretion and hyperglycemia. The cause of the disease is still unknown, however it has been suggested that environmental, genetic as well as immunological factors play important roles in the development of the disease. Exosomes are spherical fragments of membrane, released from the endosomal compartment or shed from the membranes of most cell types. They may influence the behaviour of target... (More)
Type 1 diabetes, also known as juvenile diabetes or insulin-dependent diabetes, is a growing and serious health problem worldwide with highest incidence in developed countries and among children. It is a T cell-mediated autoimmune disease, characterized by destruction of β-cells of the pancreatic islets of Langerhans, resulting in deficient insulin secretion and hyperglycemia. The cause of the disease is still unknown, however it has been suggested that environmental, genetic as well as immunological factors play important roles in the development of the disease. Exosomes are spherical fragments of membrane, released from the endosomal compartment or shed from the membranes of most cell types. They may influence the behaviour of target cells in multiple ways i.e. by direct stimulation of target cells after binding to stimulating receptors or by delivery of proteins or miRNAs between cells. In this study, we aimed at investigating whether T-EXOs could be detected in the blood circulation and whether they could be used as surrogate biomarkers of T cell activity in T1D. Exosomes were isolated by ultracentrifugation and characterized by flow cytometry, based on the expression of TcR, which is selectively expressed only by T cells, and the exosome specific marker CD63. In the first set of experiments, we have identified T-EXOs in the supernatant of in vitro activated T cells to validate our assay. Thereafter, we investigated circulating T-EXOs in serum samples from newly diagnosed T1D patients and autoantibodies positive children. We demonstrated that in vitro activated T cells secrete exosomes that express TcR. While T-EXOs were not detected in any of the autoantibodies positive children, 60% of T1D patients had detectable T-EXOs in the serum. These results demonstrate for the first time the presence of circulating T cell-specific exosomes that might represent a valuable T cell biomarker in T1D that could be used to follow disease progression. (Less)
Popular Abstract (Swedish)
T cell-exosomer som biomarkör vid Typ 1-Diabetes

Typ 1-diabetes (T1D) är en autoimmun sjukdom, där kroppens T-celler (immunceller) angriper bukspottskörtelns insulinproducerande betaceller. Insulin är ett viktigt hormon, som reglerar blodsockerhalten i kroppen. Destruktion av betacellerna leder till nedsatt insulinproduktion och därmed höga blodsockerhalter, som följaktligen leder till andra komplikationer. Orsaken till sjukdomens utveckling är ännu oklar, men interaktioner mellan genetiska anlag och miljöfaktorer, i form av exempelvis virusinfektioner, tros bidra till T-cellernas autoreaktivitet (självförstörelse). T1D utgör en väldigt liten del av alla diabetesformer och utvecklas främst hos ungdomar. Idag behandlas sjukdomen med... (More)
T cell-exosomer som biomarkör vid Typ 1-Diabetes

Typ 1-diabetes (T1D) är en autoimmun sjukdom, där kroppens T-celler (immunceller) angriper bukspottskörtelns insulinproducerande betaceller. Insulin är ett viktigt hormon, som reglerar blodsockerhalten i kroppen. Destruktion av betacellerna leder till nedsatt insulinproduktion och därmed höga blodsockerhalter, som följaktligen leder till andra komplikationer. Orsaken till sjukdomens utveckling är ännu oklar, men interaktioner mellan genetiska anlag och miljöfaktorer, i form av exempelvis virusinfektioner, tros bidra till T-cellernas autoreaktivitet (självförstörelse). T1D utgör en väldigt liten del av alla diabetesformer och utvecklas främst hos ungdomar. Idag behandlas sjukdomen med insulininjektioner. T1D är ett allvarligt hälsoproblem, som ständigt ökar världen över. Som en konsekvens av detta uppmärksammas forskning om nya strategier för att förutsäga, förebygga och bota sjukdomen.

Syftet med min studie var att detektera och karaktärisera T cell-exosomer i humant blodserum hos nydiagnostiserade T1D barn, i syfte att upptäcka en möjlig biomarkör för dem cirkulerande
autoreaktiva T-celler.

Exosomer är små vesiklar (50-100 nm) med varierande funktioner, vilka inkluderar antigen presentation, facilitering av immunresponser samt möjliggöra cell till cell kommunikation. De utsöndras av de flesta celler och består av ett membran av specifika fetter, RNA och en unik sammansättning av proteiner. Trots att exosomer från olika ursprung har samma uppbyggnad, kan det genetiska materialet skiljas åt, något som är av stort intresse vad gäller studier inom klinisk diagnostik samt för nya behandlingsstrategier för olika sjukdomar. Humant blodserum anses vara en lättillgänglig kroppsvätska, där olyckligtvis T-celler själva inte är närvarande och kan inte studeras. Däremot, lyckades vi i den här studien att isolera exosomer som har utsöndrats av dessa autoreaktiva T-celler. Detta resultat baserades på karaktärisering av exosomerna, där T-cell receptorer påvisades på cellytan. Denna identifiering av cirkulerande T-cell biomarkörer är av avgörande betydelse för att kunna identifiera individer i riskzonen för att utveckla T1D men också för att övervaka sjukdomsframfarten samt immunterapiforskning. Tillgängliga data om exosomer tyder starkt på att dessa vesiklar kan representera framtidens biomarkörer inom medicin.

Handledare: Corrado Cilio och Luis Sarmiento
Examensarbete 30 hp i medicinsk biologi 14/15
Clinical Research Center, Lunds universitet (Less)
Please use this url to cite or link to this publication:
author
Mitrovic, Christina
supervisor
organization
course
MOBM01 20142
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
7765424
date added to LUP
2015-08-20 15:56:24
date last changed
2015-08-20 15:56:24
@misc{7765424,
  abstract     = {Type 1 diabetes, also known as juvenile diabetes or insulin-dependent diabetes, is a growing and serious health problem worldwide with highest incidence in developed countries and among children. It is a T cell-mediated autoimmune disease, characterized by destruction of β-cells of the pancreatic islets of Langerhans, resulting in deficient insulin secretion and hyperglycemia. The cause of the disease is still unknown, however it has been suggested that environmental, genetic as well as immunological factors play important roles in the development of the disease. Exosomes are spherical fragments of membrane, released from the endosomal compartment or shed from the membranes of most cell types. They may influence the behaviour of target cells in multiple ways i.e. by direct stimulation of target cells after binding to stimulating receptors or by delivery of proteins or miRNAs between cells. In this study, we aimed at investigating whether T-EXOs could be detected in the blood circulation and whether they could be used as surrogate biomarkers of T cell activity in T1D. Exosomes were isolated by ultracentrifugation and characterized by flow cytometry, based on the expression of TcR, which is selectively expressed only by T cells, and the exosome specific marker CD63. In the first set of experiments, we have identified T-EXOs in the supernatant of in vitro activated T cells to validate our assay. Thereafter, we investigated circulating T-EXOs in serum samples from newly diagnosed T1D patients and autoantibodies positive children. We demonstrated that in vitro activated T cells secrete exosomes that express TcR. While T-EXOs were not detected in any of the autoantibodies positive children, 60% of T1D patients had detectable T-EXOs in the serum. These results demonstrate for the first time the presence of circulating T cell-specific exosomes that might represent a valuable T cell biomarker in T1D that could be used to follow disease progression.},
  author       = {Mitrovic, Christina},
  language     = {eng},
  note         = {Student Paper},
  title        = {Characterization of circulating T cell-specific exosomes as a possible biomarker in type 1 diabetes},
  year         = {2015},
}