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The aim of the thesis was to investigate the role of the immune system in fibromyalgia (FM), as part of a dynamic co-regulation between different bodily systems. FM is a chronic musculoskeletal disorder characterized by widespread pain and specific tender points, combined with other symptoms including fatigue, sleep disturbances, morning stiffness and anxiety. The main goal of the work was to identify possible dysregulation of peripheral immune and endocrine parameters in patients with FM compared to matched healthy controls. Moreover, the possible relation between symptom complaints and the specific parameters measured was also evaluated. A first approach was to investigate possible differences between FM patients and controls in the expression of cytokines, as they have been implicated in the occurrence of several of the symptoms associated with FM. Furthermore, adhesion molecules which are involved in cell-to-cell communication and immune cell trafficking were also studied. The latter are known to be regulated by both cytokines and glucocorticoids (GCs) and their expression is often found altered in patients with immune dysregulation. It was expected that subjects with FM would have an increased production of proinflammatory cytokines and/or a reduced antiinflammatory cytokine production and that certain cytokines and/or adhesion molecules would be differently regulated by dexamethasone (DEX). Unstimulated blood was used in the analysis of adhesion molecule expression by flow cytometry while stimulated whole blood cell cultures were used in cytokine flow cytometry assays. Peripheral blood mononuclear cells (PBMCs) were also cultured and the supernatants collected to determine the concentration of cytokines by biochip protein array. In addition, serum samples were used in enzyme-linked immunosorbent assays (ELISA) for quantification of soluble adhesion molecules. L-selectin was found elevated on monocytes and neutrophils of FM patients. A bias toward lower IL-4 levels was observed in FM patients. Based on studies showing differences in glucocorticoid receptor (GR) affinity and disturbances associated with loss of hypothalamic-pituitary-adrenal (HPA) axis resiliency in FM, it was hypothesized whether FM would be associated with abnormalities in glucocorticoid sensitivity. Total plasma cortisol and salivary free cortisol were quantified by ELISA and time-resolved fluorescence immunoassay, respectively. GR sensitivity through DEX inhibition of IL-6, in stimulated whole blood, was evaluated after cytokine quantification by ELISA. The corticosteroid receptors, GR alpha and mineralocorticoid receptor (MR), as well as the glucocorticoid-induced leucine zipper (GILZ) and the FK506 binding protein 5 mRNA expression were assessed in PBMCs by real-time reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, sequencing of RT-PCR products and/or genomic DNA was used for mutational analysis of the corticosteroid receptors. We observed lower basal plasma cortisol levels (borderline statistical significance) and a lower expression of corticosteroid receptors and GILZ in FM patients when compared to healthy controls. The minor allele of the MR single nucleotide polymorphism (SNP) rs5522 was found more often in FM patients than in controls. In addition, female carriers of this SNP seemed to have reduced salivary cortisol responses to a strong psychological stressor (Trier Social Stress Test) compared to non-carriers. FM patient carriers of an MR intronic SNP (rs17484245), before exon 3, were associated with significantly higher scores of depression symptoms compared to patient non-carriers. The thesis includes also a comprehensive analysis of the complexity of GR regulation and the role of alternative mRNA splicing. It focuses on the differential expression of the untranslated GR first exons, their high sequence homology among different species and how genetic determinants, without apparent relevance, may have implications in health and disease. In FM patients, GR exon 1-C expression was found lower and a significant difference was observed when comparing GR 1-C expression between antidepressant-free and patients who had taken antidepressants until two weeks before sample collection. In summary, the study shows a slight disturbance of some components of the innate immune system of FM patients and suggests an enhanced adhesion and possible recruitment of leukocytes to inflammatory sites. The reduced expression of corticosteroid receptors and possibly the reduced MR function may be associated with an impaired function of the HPA axis in these patients. A hyporesponsiveness of the HPA axis under stress or disturbances of the stress response could make these patients more vulnerable to cytokines and inflammation which, compounded by lower antiinflammatory mediators, may sustain some of the symptoms that contribute to the clinical picture of FM.
Early life adversity (ELA) poses a high risk for developing major health problems in adulthood including cardiovascular and infectious diseases and mental illness. However, the fact that ELA-associated disorders first become manifest many years after exposure raises questions about the mechanisms underlying their etiology. This thesis focuses on the impact of ELA on startle reflexivity, physiological stress reactivity and immunology in adulthood.
The first experiment investigated the impact of parental divorce on affective processing. A special block design of the affective startle modulation paradigm revealed blunted startle responsiveness during presentation of aversive stimuli in participants with experience of parental divorce. Nurture context potentiated startle in these participants suggesting that visual cues of childhood-related content activates protective behavioral responses. The findings provide evidence for the view that parental divorce leads to altered processing of affective context information in early adulthood.
A second investigation was conducted to examine the link between aging of the immune system and long-term consequences of ELA. In a cohort of healthy young adults, who were institutionalized early in life and subsequently adopted, higher levels of T cell senescence were observed compared to parent-reared controls. Furthermore, the results suggest that ELA increases the risk of cytomegalovirus infection in early childhood, thereby mediating the effect of ELA on T cell-specific immunosenescence.
The third study addresses the effect of ELA on stress reactivity. An extended version of the Cold Pressor Test combined with a cognitive challenging task revealed blunted endocrine response in adults with a history of adoption while cardiovascular stress reactivity was similar to control participants. This pattern of response separation may best be explained by selective enhancement of central feedback-sensitivity to glucocorticoids resulting from ELA, in spite of preserved cardiovascular/autonomic stress reactivity.
With the advent of highthroughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. The dissection of IG repertoires promises to transform our understanding of the adaptive immune system dynamics. Advances in sequencing technology now also allow the use of the Ion Torrent Personal Genome Machine (PGM) to cover the full length of IG mRNA transcripts. The applications of this benchtop scale HTS platform range from identification of new therapeutic antibodies to the deconvolution of malignant B cell tumors. In the context of this thesis, the usability of the PGM is assessed to investigate the IG heavy chain (IGH) repertoires of animal models. First, an innovate bioinformatics approach is presented to identify antigendriven IGH sequences from bulk sequenced bone marrow samples of transgenic humanized rats, expressing a human IG repertoire (OmniRatTM). We show, that these rats mount a convergent IGH CDR3 response towards measles virus hemagglutinin protein and tetanus toxoid, with high similarity to human counterparts. In the future, databases could contain all IGH CDR3 sequences with known specificity to mine IG repertoire datasets for past antigen exposures, ultimately reconstructing the immunological history of an individual. Second, a unique molecular identifier (UID) based HTS approach and network property analysis is used to characterize the CLLlike CD5+ B cell expansion of A20BKO mice overexpressing a natural short splice variant of the CYLD gene (A20BKOsCYLDBOE). We could determine, that in these mice, overexpression of sCYLD leads to unmutated subvariant of CLL (UCLL). Furthermore, we found that this short splice variant is also seen in human CLL patients highlighting it as important target for future investigations. Third, the UID based HTS approach is improved by adapting it to the PGM sequencing technology and applying a custommade data processing pipeline including the ImMunoGeneTics (IMGT) database error detection. Like this, we were able to obtain correct IGH sequences with over 99.5% confidence and correct CDR3 sequences with over 99.9% confidence. Taken together, the results, protocols and sample processing strategies described in this thesis will improve the usability of animal models and the Ion Torrent PGM HTS platform in the field if IG repertoire research.