Browsing by Subject "Spinal cord injury"

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Open Access
Immunotherapeutic action of extracellular vesicles and effects of TLR signaling to immune dysfunction of SCI patients
(2017-03) Güçlüler, Gözde
The primary aim of this thesis is to extend the breadth of in vivo application of externally loaded exosomes as prophylactic or therapeutic carriers against disease treatment. Exosomes are secreted from all cells and could be purified from all bodily fluids; however, engineering of exosomes to carry specific ligands post-purification is a daunting task. Herein, we show that lyophilization of exosomes together with the biological cargo alone or in combination of CpG ODN motifs, model protein antigen ovalbumin or lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution successfully internalizes these cargos within exosomes. Furthermore, the bioactivity of the loaded ligand(s) surpasses the unloaded free ligand activities. When tested in vivo, exosome incorporated ligand(s) proved to be significantly effective against model tumors such as E.G7 thymoma or established melanoma models. The mechanism behind this elevated immune activity is the ability of exosomes to be delivered to target cells and boost immune antigen dependent immune activation. Our in vitro findings revealed that encapsulation of CpG ODN into exosomes enhances immunostimulatory activity of CpG ODN than free form as evidenced by superior levels of cytokines like IL6, IL12 and Type-I and II interferons. This magnified immune activity might be partly due the increased APC activation observed as elevated CD86/MHCII surface marker expression. Immunization of C57/Bl6 mice with exosomal CpG ODN plus OVA induced strong Th1-biased anti-OVA response. Following thymoma induction in naive and OVAimmunized animals, >85% of exosomal vaccine treated mice cleared tumors whereas almost all naive animals were positive for tumor. This data suggests that CpG ODN encapsulation into exosomes improve immunostimulatory activity, provide better anti-OVA immunity thereby contribute effective tumor clearance in mice. A second aim of this thesis was to establish that it is feasible to load exosomes with more than two ligands. Next, invariant natural killer T (iNKT) cell ligand αGC was included within exosomes as the third element next to OVA and CpG ODN. Initial in vivo studies revealed that exosomes containing αGC were significantly more potent in inducing antigen dependent immune responses in comparison to free form of CpG ODN, OVA and αGC. In therapeutic tumor vaccine model, two exosome injections (@d: 9 and d: 15) were done to B16-OVA tumor bearing animals and tumor regression was followed. Mice that had triple exosomal ligands significantly reduced tumors compared to mice treated with non-exosomal ligands. This study confirmed that exosomes with triple ligands could be effectively control established tumor development. In this thesis, the elucidation of the involvement of extracellular vesicles (EVs) on the pathogenesis of autoimmune/autoinflammatory diseases was studied. The underlying mechanism in BD pathogenesis is still unclear. We found that one of the human cathelicidin group members, antimicrobial peptide LL37 along with EVs were elevated in active BD patients` plasmas. Strikingly, majority of plasma LL37 was associated with circulating EVs. We found that there was a strong correlation between i) LL37 level, ii) EV #/ml plasma and iii) cytokine production. In the last part of this thesis, one of the possible mechanisms of immune dysfunction contributing to severe neurological deterioration of chronic spinal cord injured (SCI) patients was unearthed. We aimed to investigate whether there is a correlation between susceptibility to infections of chronic SCI patients within the context of impaired innate recognition of pathogen associated molecular patterns (PAMPs). Our data implicated that although there was no dysfunction of B cell, or CD4+ Treg activity, but sensing TLR7 and TLR9 ligands by monocytes and pDCs were ablated in patients with SCI, leading to lower IFNγ and IP10 production along with costimulatory molecule expression, that could explain the immunological dysfunction in patient with SCI contributing to persistent complications.
Open Access
Impaired toll like receptor-7 and 9 induced immune activation in chronic spinal cord injured patients contributes to immune dysfunction
(Public Library of Science, 2017) Gucluler, G.; Adiguzel, E.; Gungor, B.; Kahraman, T.; Gursel, M.; Yilmaz, B.; Gursel, I.
Reduced immune activation or immunosuppression is seen in patients withneurological diseases. Urinary and respiratory infections mainly manifested as septicemia and pneumonia are the most frequent complications following spinal cord injuries and they account for the majority of deaths. The underlying reason of these losses is believed to arise due to impaired immune responses to pathogens. Here, we hypothesized that susceptibility to infections of chronic spinal cord injured (SCI) patients might be due to impairment in recognition of pathogen associated molecular patterns and subsequently declining innate and adaptive immune responses that lead to immune dysfunction. We tested our hypothesis on healthy and chronic SCI patients with a level of injury above T-6. Donor PBMCs were isolated and stimulated with different toll like receptor ligands and T-cell inducers aiming to investigate whether chronic SCI patients display differential immune activation to multiple innate and adaptive immune cell stimulants. We demonstrate that SCI patients' B-cell and plasmacytoid dendritic cells retain their functionality in response to TLR7 and TLR9 ligand stimulation as they secreted similar levels of IL6 and IFNα. The immune dysfunction is not probably due to impaired T-cell function, since neither CD4+ T-cell dependent IFNγ producing cell number nor IL10 producing regulatory T-cells resulted different outcomes in response to PMA-Ionomycin and PHA-LPS stimulation, respectively. We showed that TLR7 dependent IFNγ and IP10 levels and TLR9 mediated APC function reduced substantially in SCI patients compared to healthy subjects. More importantly, IP10 producing monocytes were significantly fewer compared to healthy subjects in response to TLR7 and TLR9 stimulation of SCI PBMCs. When taken together this work implicated that these defects could contribute to persistent complications due to increased susceptibility to infections of chronic SCI patients. © 2017 Gucluler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Open Access
Intestinal microbiota in patients with spinal cord injury
(Public Library of Science, 2016) Gungor, B.; Adiguzel, E.; Gursel, I.; Yilmaz, B.; Gursel, M.
Human intestinal flora comprises thousands of bacterial species. Growth and composition of intestinal microbiota is dependent on various parameters, including immune mechanisms, dietary factors and intestinal motility. Patients with spinal cord injury (SCI) frequently display neurogenic bowel dysfunction due to the absence of central nervous system control over the gastrointestinal system. Considering the bowel dysfunction and altered colonic transit time in patients with SCI, we hypothesized the presence of a significant change in the composition of their gut microbiome. The objective of this study was to characterize the gut microbiota in adult SCI patients with different types of bowel dysfunction. We tested our hypothesis on 30 SCI patients (15 upper motor neuron [UMN] bowel syndrome, 15 lower motor neuron [LMN] bowel syndrome) and 10 healthy controls using the 16S rRNA sequencing. Gut microbial patterns were sampled from feces. Independent of study groups, gut microbiota of the participants were dominated by Blautia, Bifidobacterium, Faecalibacterium and Ruminococcus. When we compared all study groups, Roseburia, Pseudobutyrivibrio, Dialister, Marvinbryantia and Megamonas appeared as the genera that were statistically different between groups. In comparison to the healthy group, total bacterial counts of Pseudobutyrivibrio, Dialister and Megamonas genera were significantly lower in UMN bowel dysfunction group. The total bacterial count of Marvinbryantia genus was significantly lower in UMN bowel dysfunction group when compared to the LMN group. Total bacterial counts of Roseburia, Pseudobutyrivibrio and Megamonas genera were significantly lower in LMN bowel dysfunction group when compared to healthy groups. Our results demonstrate for the first time that butyrate-producing members are specifically reduced in SCI patients when compared to healthy subjects. The results of this study would be of interest since to our knowledge, microbiome-associated studies targeting SCI patients are non-existent and the results might help explain possible implications of gut microbiome in SCI. Copyright © 2016 Gungor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Open Access
Investigation of immune impairment status in primary immune deficiency and spinal cord injury
(2021-02) Evcili, İrem
Primary 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.