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Cannabidiol (CBD), a non-psychotropic phytocannabinoid with structural similarity to Δ 9 -tetrahydrocannabinol (THC), is currently being investigated as a therapeutic for its immunosuppressive effects. One disease for which CBD is extensively researched is multiple sclerosis (MS), a demyelinating, autoimmune disorder, and its murine model counterpart, experimental autoimmune encephalomyelitis (EAE). The focus of this dissertation aimed to analyze the transcriptomic brain pathways in EAE and its comparison to MS in addition to CBD’s immunosuppressive mechanisms in the innate and adaptive immune systems. Evidence presented here showed that transcriptomic signaling pathways in the EAE brain of mice with clinical symptoms were similar to the transcriptome of active lesions from MS patients. The transcriptomic analysis also presented two differentially expressed genes that were increased in CBD-treated, asymptomatic EAE mouse brains: oxytocin and vasopressin. Expression of these genes was also increased in naïve, CBD-treated mouse brains, which may indicate potential as efficacy biomarkers. Subsequently, as disease progression requires input from the innate and adaptive immune systems, the mechanisms of CBD were analyzed under naïve and stimulatory conditions in macrophages and splenocytes. In macrophages, CBD exerted an anti-inflammatory effect by dampening the M1 polarization phenotype, decreasing pro-inflammatory cytokines and chemokines, reducing TNF-α through intracellular TACE retention, and diminishing the translocation of RelA to the nucleus. Notably, similar impact of CBD on TACE was evident in naïve macrophages, suggesting that CBD exerted an effect under naïve conditions. In splenocytes, CBD exhibited a long-term effect on the percentage of various immune populations during naïve and splenic T cell activation (with anti-CD3/anti-CD28) conditions but only provided temporary relief and short-term from TNF-α and IFN-γ cytokine secretion. CBD also increased early mRNA expression of Tnfa in CBD in stimulated splenocytes. In naïve splenocytes, CBD impacted key immune mediators discovered from a transcriptomic re-analysis of human neuroblastoma cells, including decreased early expression of Noxo1 but increased expression of Ctsb. In summary, this dissertation presented evidence that CBD impacts the immune system from the transcriptional level in the brain, the innate and adaptive immune systems at the cellular level, and the overall EAE disease phenotype.

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Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome is a complex disease with unknown cause. It affects about 0.4 percent of the population. It is a chronic disease that manifests itself in an impairment fatigue that is impaired by physical exertion. Purpose: The purpose of the study was to illustrate how people diagnosed with ME/CFS experience the primary care treatment. Method: An empirical questionnaire with mixed method was used. Most questionnaires were quantitative, and one question was qualitative. Result: ME patients may wait several years and meet several doctors before they get their diagnosis. The result showed that most respondents felt that they did not receive good treatment in primary care. Conclusion: People with ME/CFS want to be treated with respect and want primary care to listen and take them seriously. In a partnership between the primary care and healthcare personnel, they could meet more equally, and the ME sufferers would not have to wait several years to meet several doctors before receiving the assistance and support they need in their illness. Clinical significance: New research on how healthcare professionals can help people with ME/CFS is needed. Research would also be needed on how the role of nurse could be extended to be a key part of primary care for people with ME/CFS.

Bakgrund: Myalgic Encephalomyelitis eller Chronic Fatigue Syndrome (ME/CFS) omfattar ett spektrum av olika symptom som bland annat påverkar de autonoma och neurologiska systemen, kognitiv funktion och ger immunologiska störningar med mera. De karakteristiska symptomen är oförklarlig kronisk trötthet, ansträngningsutlöst fysisk och mental uttröttbarhet Post Exertional Malaise (PEM). Trots forskning inom ett flertal områden är den underliggande molekylära orsaken bakom ME/CFS inte fastställd. Flertalet hypoteser om sjukdomsorsaken finns, varav en är att ME/CFS är en autoimmun sjukdom. Syfte: Syftet med litteraturstudien är att undersöka huruvida det finns autoimmuna aspekter i ME/CFS. Metod: Systematisk litteraturstudie utifrån vetenskapliga artiklar, publicerade 2010—2020 i databasen PubMed. Resultat: Studieresultaten är inte helt entydiga när det kommer till att påvisa autoimmuna aspekter i ME/CFS. Antikroppsstudier riktade mot neuronalt protein hos ME/CFS-patienter och behandlingar riktade mot antikroppar, immunoadsorption och rituximab, gav negativa resultat. Däremot observeras HSP60 (heat shock protein 60) antikroppar för specifika korsreaktiva epitoper i en undergrupp av ME/CFS-patienter, vilket stämmer överens med infektionsutlöst autoimmunitet. Även i de två genstudierna, HLA-association (human leucocyte antigen) och SNP (single nucleotide polymorphism) genotypning i immungener, observeras karakteristiska riskgener för autoimmun sjukdom, tydligast resultat observerades hos de patienter som har en infektionsutlöst ME/CFS. Slutsats: Trots delvis negativa resultat ges visst stöd för hypotesen då dessa indikerar autoimmuna aspekter i en undergrupp av infektionsutlöst ME/CFS. / Background: Myalgic Encephalomyelitis or Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease which is characterized by unexplained and persistent post exertional fatigue (PEM) and a myriad of symptoms related to neurological disturbance, immunological, cognitive and autonomous dysfunction. Despite biomedical research from a disparate field of expertise the pathogenesis and etiology of ME/CFS is not well-understood. Several hypotheses regarding the pathogenesis have been proposed one of which is that ME/CFS is an autoimmune disease. Aim: The purpose of the literature study is to investigate whether there are autoimmune aspects in ME/CFS. Method: A systematic literature study based on scientific articles, published 2010-2020 in the PubMed database. Results: The study results are not entirely consistent when it comes to detecting autoimmune aspects in ME/CFS. Antibody studies targeting neuronal proteins in ME/CFS-patients and antibody treatments, immunoadsorption, and rituximab yielded negative results. In contrast, HSP60 (heat shock protein 60) antibodies for specific cross-reactive epitopes are observed in a subset of ME/CFS patients, which corresponds to infection-triggered autoimmunity. The two gene studies, HLA (human leucocyte antigen) association and genotyping of SNP (single nucleotide polymorphism) in immune genes, observed characteristic risk genes for autoimmune disease. Significant results were observed in ME/CFS- patients with an infection-triggered onset. Conclusion: Despite partially negative results, some support exists for the hypothesis as results indicate autoimmune aspects in ME/CFS with an infection-triggered onset.

Die Multiple Sklerose (MS) ist die häufigste chronisch-entzündliche Erkrankung des zentralen Nervensystems und die häufigste Ursache erworbener Behinderung im jungen Erwachsenenalter. Histopathologisches Kennzeichen der MS sind fokale Läsionen, die durch Demyelinisierung, Entzündungsinfiltrat, gliale Narbenbildung, Astrozytose, Oligodendrozytenschädigung und unterschiedlich stark ausgeprägte Axondegeneration gekennzeichnet sind. Die Läsionen können im gesamten ZNS auftreten. Bei der Pathogenese der MS geht man von einem multifaktoriellen Geschehen aus. Nach dem derzeitigen Wissenstand ist die MS eine T-Zell-vermittelte Autoimmunerkrankung gegen bestimmte Bestandteile des zentralen Nervensystems, bei der zusätzlich genetische, epidemiologische und Umweltfaktoren eine Rolle spielen. MicroRNAs (miRNAs) haben wichtige Funktionen in der Genregulation und sind in viele physiologische Zellprozesse involviert. Man geht daher davon aus, dass miRNAs auch eine wichtige Rolle in der Pathogenese der MS spielen. Sowohl an MS-Patienten als auch im Tiermodell werden diesbezügliche Untersuchungen durchgeführt. Das wichtigste Tiermodell der MS ist die experimentelle autoimmune Enzephalomyelitis (EAE). In der vorliegenden Arbeit wurde die miRNA-Regulation ausgewählter miRNAs in EAE-Läsionen von C57/BL6 Mäusen (MOG35-55-induziert) und Weißbüschelaffen (MOG1-125-induziert) im Gegensatz zu Kontrollgewebe ermittelt und mit der bekannten miRNA-Regulation in aktiven humanen MS-Läsionen verglichen. Aus den EAE-Rückenmarksläsionen der Mäuse und den makrosezierten EAE-Hirnläsionen der Weißbüschelaffen wurde zunächst die RNA extrahiert. Im Anschluss erfolgte die Umschreibung in eine cDNA und dann eine miRNA-Detektion und -Quantifizierung mit Hilfe einer qPCR. Es konnte gezeigt werden, dass die miRNA-Regulation in den EAE-Läsionen von Mäusen und Weißbüschelaffen mit der miRNA-Hoch- und -Herabregulation in den aktiven humanen MS-Läsionen vergleichbar ist. Die im Menschen konservierten und regulierten miRNAs miR-155, miR-142-3p, miRNA-146a, miR-146b und miR-21 waren auch in Maus und Weißbüschelaffe in ähnlicher Weise hochreguliert.

Glycine receptor antibodies have been identified in a few patients with progressive encephalomyelitis with rigidity and myoclonus (PERM), a highly disabling disorder characterised by rigidity, spasm and brainstem symptomatology. The clinical characteristics of patients with glycine receptor antibodies have not yet been fully described and it is not clear whether GlyR-Abs are pathogenic or just an epiphenomenon. This study examined the clinical features and immunotherapy responses of 45 patients; characterised the GlyR-Ab pathogenicity, subunit specificity and binding to different brain region in vitro, and examined mice injected with GlyR-Abs to model the disease in vivo. Most of the patients were classified as PERM but some patients had symptomatology beyond the classical motor manifestations and there were four patients with tumours (thymomas and lymphomas). GlyR-Ab titres were varied in serum and CSF, but there was intrathecal synthesis in the six patients with suitable samples. Most patients were very disabled but almost all showed excellent responses to immunotherapies. The antibodies were mainly IgG1 and IgG3 subclasses, activated complement on glycine receptor-transfected HEK cells at room temperature, and caused internalisation and lysosomal degradation of the glycine receptors at 37°C. GlyR-Abs bound to rodent spinal cord and brainstem co-localising with monoclonal antibodies to GlyRα1 on the surface of neurons. GlyR-IgG injected intra-peritoneally led to impairment in forced walking ability, sensorimotor function and coordination. Analysis of the brain showed that animals injected with patients' IgG, but not control IgG, had antibodies bound to the brainstem, spinal cord, cerebellum and caudate, co-localising with GlyRα1 monoclonal antibody. Intra-cerebroventricular injection of GlyR-IgG caused an anxiety-like behaviour in mice but no evident motor disturbances. These results provide the first evidence of in vitro and in vivo pathogenicity of the GlyR-Abs, supporting the use of long term immunosuppression in these patients to provide them with a good prognosis.

Resultados obtidos pelo nosso grupo demonstraram, in vitro, que a PGE2 é capaz de modular a sobrevivência de linfócitos TCD4+ protegendo essas células da morte. Dentro do modelo de EAE, nossa hipótese é que a PGE2 liberada pelas APCs, durante a fase de indução, module a sobrevivência de linfócitos autorreativos específicos induzindo a doença. Realizamos o tratamento de camundongos submetidos à EAE com indometacina durante 5 dias e notamos que houve redução da EAE associada à redução de linfócitos produtores de IFN-γ, IL-17 e GM-CSF, e macrófagos infiltrantes e microglias ativadas, no SNC. O tratamento alterou a freqüência de células em proliferação e a frequência de células produtoras de IFN-γ e IL-17 na periferia e a concentração dessas citocinas. Esses resultados sugerem que a indometacina reduz o desenvolvimento da EAE e sua resposta antígeno-específica demonstrando a sua importância na modulação das respostas de linfócitos T na autoimunidade. / Results obtained by our group demonstrated in vitro that PGE2 is able to modulate CD4+ T cells survival protecting these cells from death. Within the EAE model, we hypothesized that PGE2 released by APCs during the induction phase, modulate survival of autoreactive specific lymphocytes by induction the disease. We carried out the treatment of EAE in mice subjected to indomethacin for 5 days and noticed that there is reduction of EAE associated with decreased IFN-γ, IL-17 and GM-CSF producing T cells, and infiltrating macrophages and activated microglia in the CNS. The results suggest that indomethacin reduces EAE and its antigen-specif response demonstrating their importance in the modulation of T lymphocyte responses in autoimmunity.

O estudo dos efeitos do estresse em modelos animais contribui para investigar os mecanismos de sinalização envolvidos nas enfermidades humanas. O projeto foi dividido em duas partes. Primeiramente investigou-se o efeito do estresse crônico imprevisível (ECI) na evolução clínica da encefalomielite autoimune experimental (EAE) e as concentrações circulantes de corticosterona (CORT). O ECI agravou os sinais clínicos da EAE em ratos, mas as concentrações plasmáticas de CORT durante o desenvolvimento da doença pareceram depender unicamente do desafio imunológico. Posteriormente investigaram-se os mediadores inflamatórios e o estresse oxidativo em diversas áreas do SNC de ratos submetidos à isquemia e reperfusão mesentérica. Foram observadas alterações distintas, conforme o tecido analisado, na expressão gênica de NOS e COX, atividade da NOS Ca2+-dep e da arginase e no conteúdo de TBARS. Conclui-se que, nas primeiras horas de reperfusão intestinal, as estruturas analisadas reagem diferentemente ao estresse oxidativo. / The study of stress effects in different animal models help to clarify signalization mechanisms involved in human diseases. The present project was divided in two parts. Firstly, we investigated the effects of chronic unpredictable stress (CUS) on the course of experimental autoimmune encephalomyelitis (EAE) and circulating corticosterone (CORT) concentrations. CUS aggravated the clinical signs of the disease in Lewis rats. However, plasma CORT during the development of clinical signs seemed to be solely dependent on the immunological challenge. Secondly, we investigated the oxidative and defense mechanisms in several CNS regions of rats submitted to an intestinal ischemia-reperfusion model. There were distinct results, as tissue analysis, in genic expression of NOS and COX, calcium-dependent nitric oxide synthase and arginase activities and TBARs content. Based on these results, we concluded that, at least during the first two hours of intestinal reperfusion, CNS lesions may be efficiently prevented by defense mechanisms able to modulate the oxidative injury.