T lymphocytes are an essential component of the adaptive immune response and are highly versatile in function. Each T cell has a unique T cell receptor that can recognize an antigenic peptide in the context of the major his to compatibility complex (MHC) encoded molecules, thus offering a high degree of specificity to the immune response. T cells play a central role in the development of an effective host immune response and the quantitative and qualitative regulation of the T cell response is critical. T cells develop in the thymus, an important primary immune organ, where immature thymocytes undergo differentiation and maturation. Through the process of thymic differentiation, immature cluster of differentiation (CD)4-CD8- thymocytes progress to a CD4+CD8+ stage and are subjected to positive and negative selection to give rise to MHC restricted, single positive CD4+ or CD8+ naive T cells that emigrate from the thymus and populate the peripheral lymphocyte pool. Thymic atrophy is well known to occur naturally during the process of aging with thymocyte depletion and reduced thymic output. Along with age associated changes leading to atrophy, the thymus is exquisitely sensitive to starvation and several stresses. In addition, thymic atrophy is a characteristic feature during several viral, bacterial and parasitic infections. Egress of immature thymocytes, loss of thymic populations due to sensitivity to glucocorticoids and cytokine modulation, etc. have been variously proposed to be involved in this process. However there is limited understanding on the numerous mechanisms involved and the crosstalk between these diverse pathways. In this study, a model for thymic atrophy during acute Salmonella enterica serovar Typhimurium (S. typhimurium) infection was developed. S. typhimurium is a Gram negative bacterium that resides and grows in intracellular compartments within host cells. It causes gastroenteritis in humans but leads to typhoid like disease in mice, similar to that caused by S. typhi in humans. Initially, it was established that acute infection of C57BL/6 mice with 108 CFU S. typhimurium, via the oral, i.e. the physiological, route of infection leads to extensive depletion (8-10 fold) of thymocytes in an infection-dependent manner. Infected mice had higher CFU burden in the Peyer’s patches, spleen, liver, and mesenteric lymph node (MLN) as compared to the thymus. The thymic atrophy was dependent upon the infection caused by live S. typhimurium since oral feeding of mice even with higher doses (1010 CFU) of heat-killed bacteria did not lead to thymic atrophy. The susceptible populations in the thymus were identified by staining for expression of CD4 and CD8 on cell surface using specific monoclonal antibodies tagged to fluorophores, e.g. Fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively. The double labelled samples were analyzed by flow cytometry. Interestingly, significant death of CD4+CD8+, the major population of thymocytes, but not single positive thymocytes or peripheral lymphocytes (MLN and spleen cells), was observed at later stages during infection. To gain greater understanding of the processes involved, the mechanisms leading to thymic atrophy were investigated. To this purpose, small molecule inhibitors and mice lacking key molecules important for the immune response were utilized. Also, various assays to assess death of thymocytes, including analysis of death markers such as Annexin V based detection of membrane flipping and caspase activation were performed. I. The extrinsic death pathway involving Fas/FasL interactions is a major death pathway. Therefore, the expression and functional role of the components of the pathway in this model of thymocyte death was investigated. It was observed that thymocytes from infected mice expressed more Fas and Fas ligand (FasL) on their surface than cells from uninfected mice. To address the role of the death receptor, Fas, infection studies were performed with lpr mice that lack functional Fas expression. The depletion of CD4+CD8+ thymocytes in lpr mice was comparable to that in C57BL/6 mice indicating that it was independent of the Fas pathway. However, extensive loss of mitochondrial membrane potential was observed upon analysis with mitochondrial potential specific dyes MitoTracker Red and DiOC6. Most likely, the intrinsic death pathway involving mitochondrial depolarization is involved in this model of thymic atrophy. II. Since thymocytes are known to be sensitive to glucocorticoids both in vitro and in vivo, the involvement of the same in this model of thymic atrophy was assessed. The amounts of cortisol, a glucocorticoid, as detected by ELISA, were elevated during infection. To investigate the functional implication of the increase in cortisol, studies were performed using RU486, a glucocorticoid receptor antagonist. RU486 did not modulate cortisol amounts and treatment of mice with RU486 did not affect CFU burden or survival of mice. However there was a moderate rescue in the number of viable CD4+CD8+ thymocytes, with only a 3-4 fold drop as compared to the 8-10 fold drop in vehicle treated infected mice. III. As glucocorticoids appeared to play a partial role in this model, it was reasonable to assume that other pathways were also involved in the thymic atrophy. The quantitative and qualitative modulation of the cytokine milieu has a profound effect upon the thymus. In fact, inflammatory cytokines, Tnfα and Ifnγ, increased upon infection. In order to study the role of Ifnγ mediated inflammatory responses in this model, infection studies with Ifnγ-/- mice were performed. Ifnγ-/- mice had higher CFU and lower survival; however the drop in thymocyte numbers was 3-4 fold as compared to the 8-10 fold drop in the infected C57BL/6 mice, again indicating a partial involvement of the Ifnγ mediated pathways. In order to study the interactions, if any, between the two pathways mentioned above, corticosteroid signaling was blocked in the Ifnγ-/- mice with RU486. Upon infection, the number of CD4+CD8+ thymocytes was significantly higher in Ifnγ-/- mice treated with RU486 (~1.5 fold drop in viable thymocyte numbers) along with lower caspase 3 activity and mitochondrial damage. Importantly, cortisol amounts in infected Ifnγ-/- mice were comparable to those in infected C57BL/6 mice and the administration of RU486 did not modulate Tnfα and Ifnγ cytokine amounts in sera. Thus, the glucocorticoid and Ifnγ mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S. typhimurium nfection. IV. Although thymic atrophy is known to occur, a detailed characterization of cell surface changes in thymocyte populations has not been performed. To investigate this aspect, thymocytes and MLN cells from uninfected and infected animals were stained for cell surface expression of CD3, CD4, CD5, CD8, CD24, CD25, CD44, CD69, MHC I and MHC II. This analysis was initially performed by studying the changes in expression of these molecules within the total thymocyte and MLN populations. Although there was no change in the expression of CD25 and MHC II in the total thymocyte population upon infection, CD24 expression reduced, whereas, the expression of CD3, CD5, CD44, CD69 and MHC I increased. Notably, changes in the frequency of expression of CD3, CD69 and MHC I were observed before the development of extensive thymic atrophy. The depletion of majority of the CD4+CD8+ thymocytes enriches the mature CD4+ or CD8+ thymocyte population This was corroborated with the observation that, upon in vitro stimulation with PMA and Ionomycin (pharmacological agents used to activate T cells) the residual thymocytes from infected mice produced more IL2 compared to thymocytes from uninfected mice. Subsequently, cells were stained with anti-CD4-FITC, anti-CD8-PE and a third biotinylated antibody, which was detected by a streptavidin-APC conjugate, against one of the remaining six markers. This three colour analysis made it possible to determine the changes in the expression of the third marker in each of the CD4-CD8-, CD4+CD8+, CD4+ and CD8+ populations upon infection. Distinct differences were observed in the phenotypes of uninfected and infected CD4+CD8+ thymocytes and the latter were CD3high, CD5high, CD24low, CD69high and MHC Ihigh indicating that the surviving population had a possibly more mature phenotype. Also, the changes in the phenotypes of the thymocyte populations were dependent upon the extent of thymic atrophy as indicated by time course and CFU studies with C57BL/6 and BALB/c mice respectively. Finally, the roles of glucocorticoids, Ifnγ and Nos2 in modulation of expression of these markers during infection were addressed. Interestingly, the expression of CD3, CD24 and MHC class I significantly correlated with increase in the number of surviving thymocytes upon inhibition of glucocorticoids signaling and in Ifnγ-/- mice. The implications of these changes in the thymocyte surface phenotype during thymic atrophy are discussed. V. Finally, the roles of downstream signalling molecules in S. typhimurium induced thymic atrophy were studied. Although the MAP kinase family members, Erk, Jnk and p38 have been implicated to play a role in the positive and/or negative selection of thymocytes during development, their role in infection induced thymocyte depletion has not been studied. Interestingly, the amounts of Jnk and pJnk, but not p38, increased in thymocytes upon infection. Importantly, pJnk amounts increased predominantly in CD3-/low thymocytes during infection. Furthermore, inhibition of Jnk signalling, using a specific inhibitor SP600125, lead to an increase in survival of CD4+CD8+ thymocytes during infection due to multiple reasons: lowering of cortisol, Tnfα and Ifnγ amounts, and better maintenance of thymic architecture. Thus, inhibition of Jnk mediated signaling protected CD4+CD8+ and CD3-/low thymocytes from death during S. typhimurium infection. Overall, the main conclusions of this study are as follows: First, extensive analysis of the surface phenotype of cells during thymic atrophy throws light on the sensitive and resistant thymocyte populations, thus offering a potential predictive marker profile. Second, glucocorticoids, Ifnγ and, importantly, Jnk mediated signaling play functional roles in the death of immature CD4+CD8+ thymocytes during S. typhimurium infection. The mechanistic details uncovered in this study may be important in designing effective strategies for reducing thymic atrophy during other infections. In fact, enhancement of thymic output may lead to greater numbers and diversity of thymic T cell emigrants in the periphery which is likely to enhance host responses during infections.

The thesis deals with the role of a family in particular life phases of a person suffering from the rare disease, spinal muscular atrophy. There are three main parts. The first one defines the term family, namely its system, features and functions, and explores the particularities of a family living with a handicapped member. The second part focuses on the spinal muscular atrophy, primarily on the development objectives of individual life phases of the people suffering from the disease. Thirdly, there is a section aiming at describing the role of the people taking care of the people challenged by spinal muscular atrophy from the early childhood to the middle adulthood. Furthermore, the diploma thesis points out the effort of these people to become independent and live their lives to the fullest with the help of care assistants, partners as well as start their families. For a better comprehension of the situation, there have been made semi-structured interviews with twelve families with a member of various age suffering from spinal muscular atrophy, inquiring not only the challenged persons, but also their parents and siblings.

Critically ill patients admitted to the intensive care unit (ICU) commonly develop severe muscle wasting and weakness and consequently impaired muscle function. This not only delays respirator weaning and ICU discharge, but has deleterious effects on morbidity, mortality, financial costs, and quality of life of survivors. Acute Quadriplegic Myopathy (AQM) is one of the most common neuromuscular disorders underlying ICU muscle wasting and paralysis, and is a consequence of modern intensive care interventions, although the exact causes remain unclear. Muscle gene/protein expression, intracellular signalling, post-translational modifications, muscle membrane excitability, and contractile properties at the single muscle fibre level were explored in order to unravel the mechanisms underlying the muscle wasting and weakness associated with AQM and how this can be counteracted by specific intervention strategies. A unique experimental rat ICU model was used to address the mechanistic and therapeutic aspects of this condition, allowing time-resolved studies for a period of two weeks. Subsequently, the findings obtained from this model were translated into a clinical study. The obtained results showed that the mechanical silencing of skeletal muscle, i.e., absence of external strain (weight bearing) and internal strain (myosin-actin activation) due to the pharmacological paralysis or sedation associated with the ICU intervention, is likely to be the primary mechanism triggering the preferential myosin loss and muscle wasting, features specifically characteristic of AQM. Moreover, mechanical silencing induces a specific gene expression pattern as well as post-translational modifications in the motor domain of myosin that may be critical for both function and for triggering proteolysis. The higher nNOS expression found in the ICU patients and its cytoplasmic dislocation are indicated as a probable mechanism underlying these highly specific modifications. This work also demonstrated that passive mechanical loading is able to attenuate the oxidative stress associated with the mechanical silencing and induces positive effects on muscle function, i.e., alleviates the loss of force-generating capacity that underlie the ICU intervention, supporting the importance of early physical therapy in immobilized, sedated, and mechanically ventilated ICU patients.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

No description available.

Background Multiple sclerosis (MS) is an autoimmune disease characterised by inflammatory damage to the central nervous system (CNS). Accumulated CNS injury can be quantified as brain atrophy, definable as a reduction in brain parenchymal fraction (BPF). BPF correlate with disability in MS and is used routinely as an endpoint in clinical trials. In 2009/2010, a new MS clinical care program, that includes follow-up of BPF, was introduced at Umeå University Hospital (NUS). Levels of neurofilament light polypetide (NFL) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) are markers of axonal and astrocytic injury, respectively, and also potential surrogate biomarkers for BPF decline. The goals of this thesis were to establish age-adjusted values of BPF in healthy individuals and to relate these to the BPF values from individuals with MS as well as to the levels of NFL and GFAP in CSF. Another goal was to investigate if expanded disability status scale (EDSS)-worsening could be predicted in a clinical MS cohort and if BPF measurements could contribute to such predictions. Methods A group of 111 healthy individuals volunteered to participate in the studies. A total of 106 of these underwent MRI with BPF measurements, 53 underwent lumbar puncture (LP) with measurement of NFL and GFAP and 48 underwent both MRI and LP. Three different automatic and one manual method were utilised to determine BPF. A literature search on BPF in healthy individuals was performed for the purpose of a systematic review. For studying disability progression in MS, all individuals with MS followed at NUS and included in the Swedish MS registry were included if they had matched data on BPF, EDSS and lesion load as part of clinical follow-up (n=278). Results BPF as well as NFL and GFAP levels in CSF were all associated with age. NFL was associated with BPF and GFAP, but only the association with GFAP was retained when adjusting for age. Significant differences were found between different methods for BPF determination. In the MS population, BPF was associated with EDSS. Only progressive disease course could predict EDSS worsening. Conclusion The data on BPF and levels of NFL and GFAP in CSF of healthy individuals can aid in the interpretation of these variables in the setting of MS. Knowledge on differences in BPF data from different methods for BPF determination can be useful in comparing data across studies, but also highlights the need for a commonly accepted gold standard. The correlation between GFAP and NFL levels in CSF may indicate an association between glial and axonal turnover that is independent of the aging effect on the brain. However, the low number of volunteers for LP precluded clear conclusions. An association between BPF and EDSS was seen in the MS group. The ability to predict EDSS worsening in the clinical MS cohort was limited.

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the commonest motor neuron diseases of adult- and childhood onset. Alterations of the RNA binding protein TDP-43 are associated with most cases of ALS, while SMA is caused by deletion of the Survival Motor Neuron (SMN1) gene. SMN has been well characterised in its role in the assembly of the cellular machinery that carries out splicing of pre-mRNA, but is thought to have other functions in RNA metabolism unrelated to pre-mRNA splicing. It is conceivable that specific aspects of RNA handling are disrupted in both SMA and ALS. A variety of genetic, molecular and neuropathological approaches were applied to investigate a potential common pathway in these diseases. The spectrum of genetic mutations underlying motor neuron disorders were explored by screening patient DNA. Cell culture and mouse models were used to test the hypothesis that altered pre-mRNA splicing causes motor neuron death. Human neuropathological specimens were examined for changes in proteins involved in RNA metabolism. The results indicate that altered pre-mRNA splicing is a late occurrence in disease and more likely to be a consequence rather than the cause of motor neuron degeneration. However, the notion that RNA metabolism is highly relevant to motor neuron diseases was strengthened by the discovery of mutations in another RNA binding protein, FUS, in cases of ALS without TDP-43 pathology. Overall the findings highlight the need to consider disruption of mRNA transport and regulation of mRNA translation in future motor neuron disease research.

Objetivo: estudiar la frecuencia de positividad de las pruebas serológicas en pacientes con biopsias compatible con enfermedad celiaca. Material y métodos: estudio transversal. Se incluyeron pacientes con biopsia duodenal histológicamente compatible con enfermedad celiaca y determinación de anticuerpos antigliadina, antiendomisio y antitransglutaminasa IgA. Definimos como caso de enfermedad celiaca a quienes tuvieran biopsia positiva y anticuerpos antiendomisio y/o antitransglutaminasa positivos. Resultados: 31 pacientes fueron incluidos de los cuales 6 fueron antiendomisio positivo, 5 fueron antitransglutaminasa positivo y antigliadina fue positivo en 14. Por lo tanto de 31 pacientes con cambios histológicos compatibles con enfermedad celiaca sólo 10 tuvieron serología diagnóstica. Sólo uno de los pacientes tuvo positividad tanto para antitransglutaminasa como para antiendomisio. Conclusiones: a) encontramos que la mayoría de biopsias de duodeno con un cuadro histológico sugerente de enfermedad celiaca no se corresponden con serología diagnóstica de esta enfermedad; b) encontramos baja coincidencia en la positividad serológica entre antiendomisio y antitransglutaminasa. / Objective: to study the frequency of positive serology for celiac disease (CD) in patients with duodenal biopsies suggestive of this disease. Material and methods: cross sectional study. We included patients with duodenal biopsies histologically compatible with CD and antigliadin, antiendomysial and IgA antitransglutaminase antibodies. We defined a “case” of CD if there was a positive biopsy and either antiendomisial or antitransglutaminase positive antibodies. Results: thirty one patients were included in our study. Six were antiendomysial positive and 5 antitransglutaminase positive while the antigliadin was positive in 14 cases. Therefore, out of 31 patients only 10 had a serology compatible with CD and only one had positive both antibodies, antiendomysial and antitransglutaminase. Conclusions: a) we have found that most of the duodenal biopsies compatible with CD are not diagnosed with positive serology; and b) we found a low correlation between serological diagnostic tests.

Cerebral small-vessel damage manifests as white matter hyperintensities and cerebral atrophy on brain MRI and is associated with aging, cognitive decline and dementia. We sought to examine the interrelationship of these imaging biomarkers and the influence of hypertension in older individuals. We used a multivariate spatial covariance neuroimaging technique to localize the effects of white matter lesion load on regional gray matter volume and assessed the role of blood pressure control, age and education on this relationship. Using a case-control design matching for age, gender, and educational attainment we selected 64 participants with normal blood pressure, controlled hypertension or uncontrolled hypertension from the Northern Manhattan Study cohort. We applied gray matter voxel-based morphometry with the scaled subprofile model to (1) identify regional covariance patterns of gray matter volume differences associated with white matter lesion load, (2) compare this relationship across blood pressure groups, and (3) relate it to cognitive performance. In this group of participants aged 60-86 years, we identified a pattern of reduced gray matter volume associated with white matter lesion load in bilateral temporal-parietal regions with relative preservation of volume in the basal forebrain, thalami and cingulate cortex. This pattern was expressed most in the uncontrolled hypertension group and least in the normotensives, but was also more evident in older and more educated individuals. Expression of this pattern was associated with worse performance in executive function and memory. In summary, white matter lesions from small-vessel disease are associated with a regional pattern of gray matter atrophy that is mitigated by blood pressure control, exacerbated by aging, and associated with cognitive performance.

A perda muscular ou atrofia é uma condição associada a importantes doenças sistêmicas humanas, incluindo diabetes, câncer e insuficiência renal. Há diversos estudos de perfis transcricionais mostrando que um conjunto comum de genes, denominados atrogenes, é modulado nos músculos atrofiados. No entanto, as alterações transcricionais que desencadeiam a reversão ou atenuação da atrofia muscular ainda não foram caracterizadas a nível molecular até o momento. Para identificar os principais genes envolvidos na recuperação da massa muscular esquelética, utilizamos a técnica de microarray e RT-PCR para investigar genes diferencialmente expressos durante a reversão da atrofia do músculo Elevador do ânus (EA) sensível a andrógeno, no modelo de castração e reposição de testosterona. Como esperado, a maioria dos genes expressos de maneira diferencial comportam-se como atrogenes e respondem à atrofia induzida pela castração. Porém, observou-se pela primeira vez um grupo de sete genes (APLN, DUSP5, IGF1, PIK3IP1, KLHL38, PI15 e MKL1) que não responderam à castração, mas exclusivamente à reposição de testosterona. Considerando que quase todas as proteínas codificadas por esses genes estão associadas à reversão da atrofia e podem funcionar como reguladores da proliferação/crescimento celular, nossos resultados abrem novas perspectivas sobre a existência de anti-atrogenes / Muscle wasting or atrophy is a condition associated with major human systemic diseases including diabetes, cancer, and kidney failure. There is accumulating evidence from transcriptional profiles showing that a common set of genes, termed atrogenes, is modulated in atrophying muscles. However, transcriptional changes that trigger reversion or attenuation of muscle atrophy have not been characterized at the molecular level until now. To identify key genes involved in the recovery of skeletal muscle mass, we have used cDNA microarrays and RT-PCR to investigate genes differentially expressed during the atrophy reversion of the androgen-sensitive Levator ani muscle (LA), in the well-established model of castration and testosterone replacement. As expected, most of the differential expressed genes behave as atrogenes and responded to castration-induced atrophy. Strikingly, seven genes (APLN, DUSP5, IGF1, PIK3IP1, KLHL38, PI15, and MKL1) did not respond to castration but exclusively to the testosterone replacement. Considering that almost all proteins encoded by these genes are associated to reversion of atrophy and may function as regulators of cell proliferation/growth, our results open new perspectives on the existence of a new group of genes, determined anti-atrogenes