Investigation of immune impairment status in primary immune deficiency and spinal cord injury

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buir.advisorGürsel, İhsan
dc.contributor.authorEvcili, İrem
dc.date.accessioned2021-02-10T08:27:46Z
dc.date.available2021-02-10T08:27:46Z
dc.date.copyright2021-02
dc.date.issued2021-02
dc.date.submitted2021-02-08
dc.descriptionCataloged from PDF version of article.en_US
dc.descriptionThesis (M.S.): Bilkent University, Department of Molecular Biology and Genetics, İhsan Doğramacı Bilkent University, 2021.en_US
dc.descriptionIncludes bibliographical references (leaves 114-124).en_US
dc.description.abstractPrimary Immunodeficiencies (PIDs) are raised by immune impairment causing disorders. In the patients carrying deficiency in any part of immune system, various defects and symptoms are observed. Generally, abnormality against microbial agents are seen in patients and different therapies are applied depending on defect. That’s why it is crucial to characterize how immune system works in PIDs for better understating of disease and treatment options. In this study, we investigated two different mutations (CD27 and STAT1 GOF) and their effects on innate and adaptive arm of immune system. A trans-membrane receptor, CD27, is a one of the leukocyte differentiation markers for T, B and NK cells. It is involved in the activation of these cells by binding to the co-stimulatory molecule CD70 and mediates cell survival. In CD27 deficiency, loss of T cell dependent B cell responses and antibody production along with T cell dysfunction are mainly seen. Herein, immunological phenotypes of two patients with genetic CD27 deficiency were described. CD27 expression of the PBMCs, CD3+ T and CD19+ B cells obtained from the patients were absent in patients. Frequency of CD19+/CD24hi/CD38hi transitional B cells were higher compared to healthy controls. Reversed CD4+/CD8+ T cell ratio was observed in one patient. After IFN-β stimulation, the phosphorylation level of STAT1 in CD4+ T cells was increased in CD27-/- patients as compared to healthy group. There was no difference in phospho-STAT3 levels between CD27-/- and healthy donor CD4+ T cells upon IL-6 stimulation. However, decreased STAT5 phosphorylation levels were observed upon IL-2 stimulation of CD4+ T cells of CD27-/-. When PBMCs from healthy and CD27 deficient patients were stimulated with PMA/Ionomycin and analyzed for cytokine secretions, increased IFN-, IL-4 and IL-17a secretion were investigated. Through activation of different TLR and inflammasome pathways, normal IFN- and IL-1β responses were observed while increased TNF-α and IL-10 was present in patients. Results suggest that patients’ both humoral and cellular immune responses could be impaired in CD27 deficiency. STAT1 is a member of the STAT protein family whose mutations can cause gain-of-function (GOF) or loss of function (LOF). Recurrent widespread infections are common in both mutations. In this study, the responses of innate and adaptive immune systems of 6 patients from different hospitals known to have STAT1 GOF were examined. To determine the STAT1 phosphorylation and dephosphorylation levels, cells were treated with IFN-β and CD4+ T cell phosphorylation levels were analyzed by flow cytometry. Th1 and Th17 responses were examined since imbalanced Th1 and Th17 response is known in STAT1 GOF patients. To identify defects in innate immunity, cytokine ELISA was performed through different PRR ligands. For neutrophil activities, cells were examined as untreated and treated form by microscopically and fluorometrically. As a result, it was found that p-STAT1 levels and p-STAT1 positive CD4+ T cells of all patients were higher than healthy controls which approves STAT1 GOF mutation in patients. Although the STAT1 phosphorylation level was high, it was determined that IFN- released due to Th1 response was low in patients. Th17 response were low in patients which is supported by current infection of Chronic Mucocutaneous Candidiasis. Type II interferon responses of the patients to the ligands of the endosomal and inflammatory pathways were lower than healthy controls while normal IL-12 secretion was observed. Some patients had increased NETotic activities at the basal levels supported by spontaneous NET formation, while some patients were not able to induce NETosis through PMA stimulation. Impaired immune status was observed in STAT1 GOF patients as well. Spinal cord injury (SCI) is damage occurring in the spinal cord and nerves within the spinal canal that causes completely or partially loss of sensation around injury site. Since uncontrolled immune response occurs at the post-traumatic injury site in SCI patients and this situation increases the damage caused by the lesion, it is important to examine the immune system responses in these patients. In this study, we tried to determine how innate and adaptive immune system responses of SCI patients with and without infection differ. Percentages of T, B, NK cells and pDCs were determined from whole blood. Reversed CD4/CD8 ratio was observed in two patients. Increased Treg and pDCs cell percentage was seen in patients while B and NK cell percentages are different from patient to patient. Released IFN- , IL-17 and IL-10 levels were determined by flow cytometry upon PMA/Ionomycin. Normal Th17, increased Th1 and Treg was investigated in SCI patients. NETotic activities were examined microscopically and fluorometrically and via ROS production. Although NETotic activities of neutrophils of all patients were similar in healthy subjects, fluorometric results showed that some patients had more NETotic activities compared to healthy controls. It was also found that patients produced lower amounts of ROS as a result of incubation with PMA. These results suggest that SCI patients become more susceptible to infection due to the low response to the pathogen despite increased neutrophil activity and the immunosuppressive effect of the amount of IL-10 caused by excess Treg activity.en_US
dc.description.provenanceSubmitted by Betül Özen (ozen@bilkent.edu.tr) on 2021-02-10T08:27:46Z No. of bitstreams: 1 İrem Evcili MSc Thesis imzalı.pdf: 12466353 bytes, checksum: 5ca66a8a9cd63b34dac851ece20f392c (MD5)en
dc.description.provenanceMade available in DSpace on 2021-02-10T08:27:46Z (GMT). No. of bitstreams: 1 İrem Evcili MSc Thesis imzalı.pdf: 12466353 bytes, checksum: 5ca66a8a9cd63b34dac851ece20f392c (MD5) Previous issue date: 2021-02en
dc.description.statementofresponsibilityby İrem Evcilien_US
dc.embargo.release2021-08-08
dc.format.extentxix, 127 leaves : color illustrations, charts, graphics ; 30 cm.en_US
dc.identifier.itemidB150684
dc.identifier.urihttp://hdl.handle.net/11693/55035
dc.language.isoEnglishen_US
dc.rightsinfo:eu-repo/semantics/openAccessen_US
dc.subjectCD27en_US
dc.subjectSTAT1 GOFen_US
dc.subjectSpinal cord injuryen_US
dc.subjectPrimary immune deficiencyen_US
dc.subjectInnate and adaptive immunityen_US
dc.titleInvestigation of immune impairment status in primary immune deficiency and spinal cord injuryen_US
dc.title.alternativeBirincil bağışıklık yetersizliği ve omurilik yaralanmasında bağışıklık bozukluğu durumunun araştırılmasıen_US
dc.typeThesisen_US
thesis.degree.disciplineMolecular Biology and Genetics
thesis.degree.grantorBilkent University
thesis.degree.levelMaster's
thesis.degree.nameMS (Master of Science)

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