The mammalian central nervous system is not able to regenerate neurons lost upon injury. In contrast, anamniote vertebrates show a remarkable regenerative capacity and are able to replace damaged cells and restore function. Recent studies have shown that in naturally regenerating vertebrates, such as zebrafish, inflammation is a key processes required for the initiation of regeneration. These findings are in contrast to many studies in mammals, where the central nervous system has long been viewed as an immune-privileged organ with inflammation considered one of the key negative factors causing lack of neuronal regeneration. In this review, we discuss similarities and differences between naturally regenerating vertebrates, and those with very limited to non-existing regenerative capacity. We will introduce neural stem and progenitor cells in different species and explain how they differ in their reaction to acute injury of the central nervous system. Next, we illustrate how different organisms respond to injuries by activation of their immune system. Important immune cell types will be discussed in relation to their effects on neural stem cell behavior. Finally, we will give an overview on key inflammatory mediators secreted upon injury that have been linked to activation of neural stem cells and regeneration. Overall, understanding how species with regenerative potential couple inflammation and successful regeneration will help to identify potential targets to stimulate proliferation of neural stem cells and subsequent neurogenesis in mammals and may provide targets for therapeutic intervention strategies for neurodegenerative diseases.

Technological advancement allowed for the advent of single-cell technologies capable of measuring a large number of cellular features simultaneously. These technologies have been subsequently used to shed light on the heterogeneity of cellular systems previously considered homogeneous, identifying the exclusive features of individual cells within cellular niches. Today, single-cell technologies represent an essential tool for studying the underlying immunological mechanisms correlating with disease. In this context, cytometry is one of the diverse high-throughput methods capable of examining more than 50 features per cell. However, utilising cytometry at its full potential requires the development of optimized assays. Additionally, the resulting high-dimensional data represent a challenge for existing computational techniques. This thesis attempts to address these challenges. The first part of the thesis is focused on developing a non-linear embedding algorithm for rapid analysis of cytometry datasets called EmbedSOM. The comparison of EmbedSOM with other state-of-the-art algorithms suggested the superiority of EmbedSOM with faster runtime. This is critical for the analysis of large datasets with millions of cells. Furthermore, EmbedSOM has additional functionality such as landmark guided...

The role of homologous recombination deficiency (HRD) in lower grade glioma (LGG) has not been elucidated, and accurate prognostic prediction is also important for the treatment and management of LGG. The aim of this study was to construct an HRD-based risk model and to explore the immunological and molecular characteristics of this risk model. The HRD score threshold = 10 was determined from 506 LGG samples in The Cancer Genome Atlas cohort using the best cut-off value, and patients with highHRDscores had worse overall survival. A total of 251 HRD-related genes were identified by analyzing differentially expressed genes, 182 of which were associated with survival. A risk score model based on HRD-related genes was constructed using univariate Cox regression, least absolute shrinkage and selection operator regression, and stepwise regression, and patients were divided into high- and low-risk groups using the median risk score. High-risk patients had significantly worse overall survival than lowrisk patients. The risk model had excellent predictive performance for overall survival in LGG and was found to be an independent risk factor. The prognostic value of the riskmodel was validated using an independent cohort. In addition, the risk score was associated with tumor mutation burden and immune cell infiltration in LGG. High-risk patients had higher HRD scores and “hot” tumor immune microenvironment, which could benefit from poly-ADP-ribose polymerase inhibitors and immune checkpoint inhibitors. Overall, this big data study determined the threshold of HRD score in LGG, identified HRD-related genes, developed a risk model based on HRD-related genes, and determined the molecular and immunological characteristics of the risk model. This provides potential new targets for future targeted therapies and facilitates the development of individualized immunotherapy to improve prognosis.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Histopathologic examination of the tumor microenvironment demonstrates the presence of a vast repertoire of infiltrating lymphocytes and antigen presenting cells (APC’s). Recent studies establish a strong correlation between the tumor microenvironment cell composition and prognostic value in terms of cell type, location and ratio, referred to as a tumor’s immunoscore. More specifically, the relationship between T regulatory (Treg) cell to T effector (Teff) cell percentage predominates as a mechanism of tumor immune evasion. Further investigation of the factors influencing the development of Treg and Teff cells is therefore warranted. Gammainterferon‐inducible lysosomal thiol reductase (GILT) acts to influence antigenic processing and presentation by MHC class II cells, ultimately impacting lymphocyte development. Evaluation of the role of GILT expression in MHC class II+ APC’s with respect to Treg and Teff cell development in primary melanoma lesions, to our knowledge, has not been reported. Therefore our investigation focuses on elucidating a plausible relationship between GILT presence and Treg to Teff cell ratio. The aim of our study is to examine a possible association between GILT expression in APC’s and Treg:Teff cell ratio. We hypothesized GILT expression in melanoma cells would result in a decreased Treg to Teff ratio or an enhanced T cell‐mediated response. Our study included 17 de‐identified primary melanoma specimens previously stained and scored for Treg, Teff, CD8, MHC class II and GILT. Scoring was performed through identification of four areas per specimen with highest Treg and Teff cell density. These four areas were then averaged with ± standard deviation (SD). With use of landmark association, these four areas were identified and scored for MHC class II and GILT in APC’s and tumor cells with consideration to presence/absence, intensity and frequency of staining. Statistical significance was not reached relative to our hypothesized relationship of a decreased Treg to Teff cell ratio in the presence of GILT+ MHC class II. Similarly, we did not reach statistical significance when comparing individual cell types to GILT, MHC class II and GILT + MHC class. In our study, we were unable reach statistical significance relative to our proposed correlation between MHC class II and GILT presence leading to a decreased Treg to Teff cell ratio or enhanced T‐cell mediated immune response. A major limitation of our study included the small sample size leading to a probable type II error, prompting the need for further investigation of the factors influencing the Treg to Teff cell ratio within the melanoma tumor microenvironment on a larger scale.

Ein neuartiger Ansatz für die Immunphänotypisierung wird vorgestellt. Die Durchflusszytometrie (FACS) ist die übliche Methode für die Charaketrisierung des Immunsystems. Jedoch ist die Verfügbarkeit von frischem Vollblut, sowie ein schnelle Probenlogistik Vorraussetzung für die Analyse. Als potentialle Alternative werden epigentische qPCR Assays vorgestellt. Für die Quantifizierung von B- und NK-Zellen wurden epigenetische qPCR Systeme etabliert. Anhand eines erweiterten epigenetischen Markerpanels wurde die klinische Anwendung in drei Kohorten getestet: a) 41 Patienten mit primären Immundefizienzen (PID); b) 19 Neugeborene mit und ohne PID und c) 28 Patienten nach einer Stammzelltransplantation (SZT). In Kohorte a) und c) konnte die Äquivalenz der Ergebnisse mit FACS bestätigt werden. Diskrepanzen bei der regulatorischen T-Zell Quantifizierung in einzelnen PID Patienten wurde festgestellt, welche durch Mutationen verursacht wurden, die die Integrität der analysierten Proteine beeinflussen. Zudem konnte die Anwendung der epigentischen Quantifizierung in Trockenblutkarten von Neugeborenen gezeigt werden. Dies würde die Anwendung auch im Neugeborenen-Screening für die Erkennung von PIDs ermöglichen. Für die Anwendung in der SZT konnte gezeigt werden, dass das epigenetische System eine frühe Analyse der Immunrekonstitution ermöglicht, welche eine prädiktive Aussage über das Überleben der Patienten erlaubt. Patienten, welche eine Immunantwort der Lymphozyten gegen eine Virusinfektion bereits am Tag 26 nach Transplantation aufwiesen, hatten eine signifikant höhere Überlebenschance als Patienten ohne Immunantwort. Zusammengefassend zeigen die Daten, dass die epigenetische Systeme für klinische Anwendungen eine zuverlässige Methode darstellt. Die Aussagekraft der Daten ist aufgrund der Studiengröße noch limitiert, und komplizierte klinische Szenarien erschweren die Evaluierung. Deshalb sind weitere Studien erforderlich, um das gezeigte Potenzial zu validieren. / A novel approach for immunophenotyping for clinical applications is presented here. Flow cytometry is currently a common method to characterize the immune system but requiring fresh whole blood and good sample logistics which is not always available. To overcome this limitations, epigenetic qPCR assays are introduced as potential alternative. New epigenetic qPCR systems to quantiy B and NK cells have been established. Using an extended epigenetic marker panel, clinical applications were tested in three patient cohorts: a) 41 patients with different primary immunodeficiencies (PID); b) 19 newborns with and without PID and c) 28 patients after stem cell transplantation (SCT). In cohort a) and c) the equivalence of the epigenetic quantification with flow cytometry was confirmed. However, discrepancies between both methods for regulatory T-cell quantification were found in individual PID patients caused by disease-associated mutations affecting the integrity of the respective protein. Furthermore, the epigenetic quantification using dried blood spots from newborns was demonstrated. This would allow the implementation of epigenetic immunophenotyping in neonatal screening for the detection of congenital immunodeficiencies. For the application in SCT, it was shown that the epigenetic system allows an early analysis of immune reconstitution, which may allow a prediction of the patients' overall survival. Patients who showed an immune response of lymphocytes against viral infections at day 26 after transplantation had a significantly higher survival rate. In summary, the available data show that epigenetic immunophenotyping is a reliable analytical method for various clinical applications. The significance of the data is still limited due to the size of the study and complicated clinical scenarios make the evaluation of individual measurements difficult. Therefore, further extensive investigations are needed to clinically validate the demonstrated potential.

Coeliac disease (CD) is a clinically heterogeneous autoimmune disease with variable presentation and progression triggered by gluten intake. Molecular or genetic factors contribute to disease heterogeneity, but the reasons for different outcomes are poorly understood. Transcriptome studies of tissue biopsies from CD patients are scarce. Here, we present a high-resolution analysis of the transcriptomes extracted from duodenal biopsies of 24 children and adolescents with active CD and 21 individuals without CD but with intestinal afflictions as controls. The transcriptomes of CD patients divide into three groups—a mixed group presenting the control cases, and CD-low and CD-high groups referring to lower and higher levels of CD severity. Persistence of symptoms was weakly associated with subgroup, but the highest marsh stages were present in subgroup CD-high, together with the highest cell cycle rates as an indicator of virtually complete villous atrophy. Considerable variation in inflammation-level between subgroups was further deciphered into immune cell types using cell type de-convolution. Self-organizing maps portrayal was applied to provide high-resolution landscapes of the CD-transcriptome. We find asymmetric patterns of miRNA and long non-coding RNA and discuss the effect of epigenetic regulation. Expression of genes involved in interferon gamma signaling represent suitable markers to distinguish CD from non-CD cases. Multiple pathways overlay in CD biopsies in different ways, giving rise to heterogeneous transcriptional patterns, which potentially provide information about etiology and the course of the disease.

Les cellules neurales expriment des molécules dites immunes qui peuvent exercer des rôles différents de ceux exercés dans le système immunitaire. Les molécules du CMH-I classiques présentent des peptides représentatifs du contenu protéique de chaque cellule aux sentinelles du système immunitaire. Cependant, il est documenté que ces molécules ont aussi des fonctions « non immunes ». En effet, les molécules du CMH-I classiques jouent un rôle dans l'établissement et la plasticité des synapses. Sur divers types cellulaires, elles peuvent aussi interagir avec des récepteurs membranaires en cis, moduler leur stabilité à la membrane et en conséquence leur activité. RAE-1 est un membre de la famille des molécules du CMH-I, décrite initialement dans le système nerveux central embryonnaire. Pour le système immunitaire, RAE-1 est un ligand du récepteur activateur NKG2D, exprimé par les cellules NK, NKT, les lymphocytes T γδ et CD8+. RAE-1 est peu ou pas exprimé dans la plupart des tissus adultes. Son expression est induite par le stress génotoxique, la transformation tumorale ou l'infection virale ce qui permet au système immunitaire d'éliminer les cellules « malades » grâce à l'activation des cellules cytotoxiques exprimant NKG2D. Je décris l'expression de RAE-1 par les cellules neurales progénitrices et le rôle non immun de cette molécule dans la prolifération cellulaire. L'expression de RAE-1 est fortement corrélée au niveau de prolifération cellulaire et est dépendante du facteur de croissance EGF. / Neural cells express immune molecules which roles differ from those in the immune system. Classical MHC-I molecules present peptides originated from the proteic content of each cell to patrolling immune cells. However, these molecules can also have nonimmune roles. Indeed, classical MHC-I molecules participate in the establishment of synapses and synaptic plasticity. They can also interact in cis with different membrane receptors on different cell types, and modulate the receptors' membrane stability and activity. RAE-1, a member of MHC-I family, was initially described in the embryonic central nervous system. In the immune system, RAE-1 is a ligand of the activating receptor NKG2D, expressed by NK cells and by NKT, γδT and some CD8+ T lymphocytes. RAE-1 is weakly or not expressed in most adult tissues. Its expression is induced by genotoxic stress, tumoral transformation or viral infection and triggers the elimination of transformed cells by the cytotoxic immune cells which express NKG2D. I describe here the expression of RAE-1 by neural progenitor cells and its role in cell proliferation. RAE-1 expression level is highly correlated with the rate of cell proliferation and depends on the presence of epidermal growth factor (EGF). Exposition to EGF induces the colocalization of RAE-1 and phosphorylated EGF-receptor (EGFR) inside lipid rafts and endocytosed vesicles, which supports a role of RAE-1 as a partner of EGFR. RAE-1 expression is also induced in the nervous tissue in different models of CNS pathologies. In these conditions, RAE-1 could be expressed by proliferating microglia under the control of M-CSF.

A histiocitose de células de Langerhans (HCL) é uma neoplasia mieloide inflamatória comumente afetando pacientes pediátricos e apresenta frequentemente mutações ativadoras somáticas em genes da via MAPK, incluindo BRAF e MAP2K1. Vários estudos sugerem que as células lesionais da HCL podem recrutar e modular células inflamatórias e cujas citocinas parecem fornecer sinais recíprocos de sobrevivência celular. Para o presente estudo foram selecionados 15 casos de HCL (10 crianças, 5 adultos), sendo as amostras de tecido avaliadas através de imunoistoquímica utilizando marcadores para macrófagos (CD68 e CD163), células dendríticas maduras (CDm) (CD83 e CD208), linfócitos T regulatórios (LTregs) (CD4, CD25 e FOXP3) e linfócitos citotóxicos (LCs) (CD56, CD57, perforina e granzima B). Além disso, marcadores de células B (CD20), células T (CD3, CD8) e confirmatórios de HCL foram analisados. Todos os casos de HCL foram positivos para S100, CD1a, CD207 e CD4; enquanto que Bcl-2 e Ciclina D1 foram positivos em 13/15 (86,7%) casos. No microambiente imune intralesional, macrófagos M2 (CD68+/CD163+), seguidos por LTregs, foram as populações celulares mais predominantes. Em quantidade significativamente menor, foram observadas CDm, seguidas por escassos LCs. Considerando a população linfoide, linfócitos T CD3+ foram mais numerosos do que linfócitos B CD20+. Dentro dos linfócitos T, linfócitos T CD4+ foram mais numerosos do que linfócitos T CD8+ (p<0,05). Nossos resultados sugerem que a infiltração de células imunes na HCL, provavelmente através de mecanismos pró-tumorais, inflamatórios e/ou imunossupressores mediados por citocinas, pode promover o desenvolvimento e sobrevivência das células lesionais da HCL, fornecendo uma justificativa para a combinação de imunoterapia e terapia gênica (BRAF) na HCL / Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia often affecting children with constitutively somatic activating mutations in MAPK pathway genes including BRAF and MAP2K1. Several studies suggest that LCH cells can recruit and modulate inflammatory cells and whose cytokines appear provide reciprocal survival signals. For the present study, 15 cases of LCH (10 children, 5 adults) were selected, and the tissue samples were evaluated through immunohistochemistry using markers for macrophages (CD68 and CD163), mature dendritic cells (mDC) (CD83 and CD208), regulatory T-cells (Tregs) (CD4, CD25 and FOXP3) and cytotoxic lymphocytes (CLs) (CD56, CD57, perforin and granzime B). Moreover, B-cell (CD20), T-cell (CD3, CD8) and LCH markers were analyzed. All LCH cases were positive for S100, CD1a, CD207 and CD4, while Bcl-2 and Cyclin D1 were positive in 13/15 cases (86.7%). In the immune microenvironment, M2-polarized macrophages (CD68+/CD163+), followed by LTregs, were the predominant cell populations. In a significantly lower amount, mDC were observed, followed by scarce CLs. Moreover, CD3+ Tcells than CD20+ B-cells were more numerous (p>0.05), the former presenting a higher number of CD4+ than CD8+ T-cells (p<0.05). Our results suggest that immune cell infiltration in LCH, probably through cytokine-mediated pro-tumoral, inflammatory and/or immunosupressive mechanisms, can promote LCH cell development and survival, providing a rationale for combining immunotherapy and BRAF-targeted therapy in LCH

<p>Patients with peritonitis or malignant obstructive jaundice (HPB⁺) have an increased morbidity and mortality due to sepsis. An altered cell-mediated immunity in the intestinal mucosa might promote gut barrier failure, increased endotoxin and cytokine release and bacterial translocation (BT) in these conditions. A clinically relevant rat model of polymicrobial peritonitis induced sepsis by cecal ligation and puncture (CLP) was used. Septic animals demonstrated a superficial injury in the small intestinal mucosa, and a significant reduction in T lymphocytes in the villi, as well as increased number of macrophages in the villi and in the MLNs as compared to sham. CLP caused increased concentration of TNF-α and IL-6 in ascitic fluid. CLP + the immunomodulator Linomide decreased the TNF-α level, reduced mucosal damage and attenuated the changes in T lymphocytes and macrophages observed following CLP. CLP + selective cyclooxygenase (COX)-2 inhibitor (SC-236) or nonselective COX inhibitor (indometacin) decreased the amount of macrophages in the mucosa and the MLNs compared to untreated CLP. CLP + indometacin decreased T lymphocytes in the villi and MLNs. SC-236 + CLP reduced mucosal injury and cytokine release as compared to indometacin. An increased rate of apoptosis in both the mucosa and MLNs was seen following CLP; COX inhibitors enhanced this phenomenon in the MLNs.</p><p>BT occurred infrequently in patients with acute peritonitis and in HPB⁺ there was no evidence of BT. Peritonitis and HPB⁺causes significant inflammatory cellular reactions as increased endotoxin and cytokine plasma levels and an altered immune cell distribution in MLNs, in HPB⁺a high rate of apoptosis in MLNs was observed. </p><p>An altered pattern of immunocompetent cells within the mucosa and in MLNs was found in experimental and clinical peritonitis as in HPB⁺.Lymphocyte depletion may be a result of increased apoptosis, which could reduce the ability of septic or jaundice patients to eradicate infection.</p>

Patients with peritonitis or malignant obstructive jaundice (HPB+) have an increased morbidity and mortality due to sepsis. An altered cell-mediated immunity in the intestinal mucosa might promote gut barrier failure, increased endotoxin and cytokine release and bacterial translocation (BT) in these conditions. A clinically relevant rat model of polymicrobial peritonitis induced sepsis by cecal ligation and puncture (CLP) was used. Septic animals demonstrated a superficial injury in the small intestinal mucosa, and a significant reduction in T lymphocytes in the villi, as well as increased number of macrophages in the villi and in the MLNs as compared to sham. CLP caused increased concentration of TNF-α and IL-6 in ascitic fluid. CLP + the immunomodulator Linomide decreased the TNF-α level, reduced mucosal damage and attenuated the changes in T lymphocytes and macrophages observed following CLP. CLP + selective cyclooxygenase (COX)-2 inhibitor (SC-236) or nonselective COX inhibitor (indometacin) decreased the amount of macrophages in the mucosa and the MLNs compared to untreated CLP. CLP + indometacin decreased T lymphocytes in the villi and MLNs. SC-236 + CLP reduced mucosal injury and cytokine release as compared to indometacin. An increased rate of apoptosis in both the mucosa and MLNs was seen following CLP; COX inhibitors enhanced this phenomenon in the MLNs. BT occurred infrequently in patients with acute peritonitis and in HPB+ there was no evidence of BT. Peritonitis and HPB+ causes significant inflammatory cellular reactions as increased endotoxin and cytokine plasma levels and an altered immune cell distribution in MLNs, in HPB+ a high rate of apoptosis in MLNs was observed. An altered pattern of immunocompetent cells within the mucosa and in MLNs was found in experimental and clinical peritonitis as in HPB+. Lymphocyte depletion may be a result of increased apoptosis, which could reduce the ability of septic or jaundice patients to eradicate infection.