Die Veraenderungen des Immunsystems mit zunehmendem Alter, Immunseneszenz genannt, resultieren in erhoehter Infektanfaelligkeit. Infolge dessen leiden aeltere Menschen unter haeufigeren und schwerer verlaufenden Infekten, Autoimmunerkrankungen und vermindertem Impfschutz. Immunseneszenz entsteht nicht nur aufgrund chronologischen Alterns, sondern wird auch durch andere teils ungeklaerte Faktoren verursacht. Cytomegalievirus (CMV) ist ein Herpesvirus, dass in immunkompetenten Menschen lebenslang persistiert. Infolge chronischer Immunaktivierung traegt CMV zur beschleunigten Immunseneszenz bei. In dieser Arbeit wurden die Alters- und CMV-induzierten Veraenderungen in humanen T-Zellen analysiert. Dafuer wurden PBMC von 50 gesunden Spendern in drei verschiedenen Altersgruppen antigenspezifisch (CMV-Proteine pp65 und IE1 und protein purified derivate von Mycobacterium tuberculosis (PPD)) und polyklonal (OKT3) in vitro stimuliert. Ein 11-Farben-Panel wurde etabliert, um die simultane Expression der Differenzierungsmarker CD45RA und CD27 und der Aktivierungsmarker CD40L, IFNg, IL2 und TNF auf T-Zellen durchflusszytometrisch zu untersuchen. Die Ergebnisse dieser Arbeit zeigten, dass die antigenabhaengige Differenzierung der Gedaechtnis-T-Zellen mit zunehmendem Alter und CMV-Infektion zunahm. Die Differenzierung der Gedaechtnis-T-Zellen in CMV-positiven Spendern war ausserdem mit der Antwortgroesse gegen pp65 korreliert. Die pp65-spezifische, aber nicht die IE1-spezifische polyfunktionale T-Zell-Antwort nahm mit zunehmendem Alter der Probanden zu. Zusammenfassend kann gesagt werden, dass CMV einen grossen Einfluss auf das Immunsystem gesunder Individuen hat. Die Differenzierung der Gedaechtnis-T-Zellen ist nicht nur abhaengig von der Infektion mit CMV, sondern auch davon, wie das Immunsytem auf CMV reagiert. Polyfunktionale CMV-spezifische T-Zellen, die wahrscheinlich CMV kontrollieren, sind vorhanden und werden nicht von dysfunktionalen T-Zellen im Alter verdraengt. / Ageing of the immune system, also called immunosenescence, is a phenomenon that leads to increased susceptibility to infections in elderly people. Persistent cytomegalovirus (CMV) infection induces strong T cell responses in humans and is thought to be one of the driving forces of immunosenescence. In this work CMV-induced alterations in the T cell compartment of human individuals were analysed in terms of frequencies and absolute numbers per ml blood. Peripheral blood mononuclear cells (PBMC) from 50 donors in three different age groups were stimulated in vitro and examined for the phenotypic markers CD45RA and CD27 and the effector molecules CD40L, IFNg, IL2 and TNF by polychromatic flow cytometry. The frequency of responding polyfunctional T cells to stimulation with OKT3 or CMV peptide pools phosphoprotein 65 (pp65) or immediate-early protein1 (IE1) increased with the age of the donor. The memory subset distribution differed between CMV-seronegative and CMV-seropositive donors and was correlated with the response size in the CMV-seropositive group. Polyfunctional T cells expressed quantitatively and qualitatively more activation marker on a per cell level than monofunctional cells and therefore appeared to be more effective. Furthermore, polyfunctional T cells expressed reduced amounts of surface CD3, CD4 and CD8 compared to monofunctional or non-responding T cells. In summary, the results indicate that CMV has a significant influence on the immune system of healthy people. The memory subset composition of the entire T cell compartment depends on how the immune system responds to CMV (large or small responses) rather than the presence or absence of infection. Irrespectively of response size or age a robust population of polyfunctional CMV-specific T cells is present and may be in control of CMV.

Introdução: Cistite Hemorrágica (CH) é uma grave complicação do Transplante de Células-Tronco Hematopoiéticas (TCTH) Alogênico. Sua incidência varia de 12 a 25,5%. A forma precoce desenvolve-se devido aos efeitos tóxicos de certos quimioterápicos usados no regime de condicionamento, especialmente Ciclofosfamida. Já a CH tardia ocorre a partir do terceiro dia após o TCTH e sua etiologia é multifatorial. Vários fatores de risco para o desenvolvimento de CH tardia foram descritos, incluindo Doença do Enxerto Contra o Hospedeiro (DECH) aguda, doador não relacionado, infecções por vírus urotrópicos, sexo masculino e condicionamento mieloablativo. Materiais e Métodos: O presente estudo tem como objetivos descrever a incidência de CH em pacientes adultos e pediátricos submetidos a TCTH alogênico e identificar fatores de risco associados ao desenvolvimento de CH nesta população. Foram analisados dados de prontuário de 347 pacientes submetidos a TCTH Alogênico no Hospital de Clínicas de Porto Alegre no período de Janeiro de 2001 a Dezembro de 2014. Resultados: CH ocorreu em 42 pacientes (12,1%, IC: 8,9 - 16%), em uma média de 53.4 dias após o procedimento (desvio padrão: 28.1 dias). Apenas 1 paciente (2,4%) desenvolveu CH precoce, com início dos sintomas no D+1. Entre os 41 pacientes que desenvolveram CH tardia, BK vírus foi o principal agente viral identificado. CH ocorreu em 12.8% dos pacientes que receberam condicionamento mieloablativo e em 10.5% dos restantes (P = 0,704). Dos 197 pacientes que apresentaram DECH aguda, 35 (17,8%) desenvolveram CH e somente 7 (4,9%) apresentaram CH na ausência de DECH aguda (P < 0,001). CH foi mais frequente também em pacientes do sexo masculino (P = 0,027). Conclusão: A incidência de CH em nossa amostra foi semelhante à encontrada em outros trabalhos. DECH aguda e sexo masculino estiveram associados a um maior risco de desenvolvimento de CH. / Introduction: Hemorrhagic cystitis (HC) is a serious complication of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) afecting 12 to 25.5% of the patients. The early-onset form of HC develops during or until 72 hours after the conditioning regimen containing high doses of certain chemotherapy drugs such as Busulfan and especially Cyclophosphamide. Late-onset HC occurs from the third day on after HSCT and its etiology is multifactorial. Several risk factors for the late-onset form have been reported including graft-versus-host disease (GVHD), unrelated donor, urotropic infections, male gender and myeloblative conditioning regimen. Methods: This study aims to evaluate the incidence of HC in adult and pediatric patients undergoing Allogeneic HSCT and to identify risk factors associated with the development of HC in this population. Medical records of 347 patients who underwent Allogeneic HSCT at Hospital de Clínicas, Porto Alegre, Brazil, from January 2001 to December 2014 were analyzed. Results: HC occurred in 42 patients (12.1% CI: 8.9 - 16%) at an average of 53.4 days after the procedure (standard deviation: 28.1 days). Only one of them developed early-onset HC, with onset of symptoms on D+1. Among the 41 patients who developed late-onset HC, BKV was the main identified viral agent. HC developed in 12.8% of the patients treated with myeloablative conditioning and in 10.5% of the remaining patients (P = 0.704). Of the 197 patients with acute GVHD, 35 (17.8%) developed HC and only 7 (4.9%) showed HC in the absence of GVHD (P<0.001). HC was also more frequent in males than females (P = 0.027). Conclusion: The incidence of HC in our sample was similar to that found in other studies. In our cohort of patients being male and having acute GVHD increased the risk of developing HC.

Orientador: Luís Gustavo Modelli de Andrade / Resumo: Introdução: O Brasil é o maior serviço público de transplantes do mundo sendo a quase totalidade do seu financiamento custeado pelo sistema único de saúde. Os regimes de imunossupressão também são inteiramente financiados pelo Estado através do Componente Especializado de Assistência Farmacêutica, mas são pouco estudados sob a perspectiva econômica. O objetivo primário foi realizar uma análise de custo efetividade do regime de imunossupressão de tacrolimo com micofenolato comparado a tacrolimo associado a imTOR pelo período de um ano após o transplante renal. O objetivo secundário foi avaliar a não inferioridade destes dois regimes. Materiais e Métodos: Tratou-se de um estudo retrospectivo de transplantados renais realizados no Hospital das Clínicas de Botucatu, comparando protocolos de imunossupressão em 12 meses após o transplante. O primeiro esquema de imunossupressão foi o utilizado até outubro de 2015 e o segundo esquema adotado após mudança do protocolo do serviço. Foram feitas comparações entre os dois regimes de imunossupressão para avaliar a não inferioridade sendo definida como: o somatório dos eventos de rejeição aguda, descontinuidade, perda do enxerto e óbito ao fim de um ano. Para as análises de custo foi construído um modelo de análise de decisão no software TreeAge Pro 2009. As probabilidades de ocorrência dos eventos foram calculadas em cada grupo na amostra histórica. Foram conduzidas análises de sensibilidade visando obter uma simulação realista dos custos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Brazil is the largest public transplant service in the world, with almost all of its costs funded by the public health system. The immunosuppressive regimens are also fully funded by the state. These regimes were studied in terms of effectiveness, but they were poorly studied from an economic perspective. The primary objective was to conduct a cost-effectiveness analysis of the immunosuppressive regimen of tacrolimus with mycophenolate compared to tacrolimus associated with imTOR for a period of one year after kidney transplantation. The secondary objective was to assess the non-inferiority of these two regimens. Materials and Methods: This was a retrospective study of kidney transplants carried out at UNESP, Univ Estadual Paulista, comparing immunosuppressive protocols at 12 months after transplantation. The first immunosuppressive regimen was the one used until October 2015 and the second regimen adopted after changing the service protocol. Comparisons were made between the two immunosuppressive regimens to evaluate non-inferiority, defined as a composite end-point of biopsy proven acute rejection, graft loss, death, and adverse events leading to drug discontinuation. A decision analysis model was constructed with TreeAge Pro 2009 software to perform the cost analyzes. The probability of occurrence of clinical events was calculated for each group in the historical sample. Sensitivity analyzes were conducted in order to obtain a realistic simulation of the fina... (Complete abstract click electronic access below) / Mestre

The oxysterol, 25-hydroxycholesterol (25-HC), is important for sterol metabolism and emerging evidence suggests that 25-HC plays a more critical role in immunity and infection. However, the precise antiviral mechanism and the target of 25- HC remains unclear. Here efforts were made to investigate the link between viral infection and the triggering of the 25-HC associated interferon (IFN) response, and how this dynamically alters the endogenous level of 3-hydroxy- 3-methylglutaryl-CoA reductase (HMGCR), a key enzyme that catalyses the production of the precursor of cholesterol and oxysterols. In this thesis I have sought to specifically explore the temporal changes and role of HMGCR in DNA virus (cytomegalovirus) and RNA (Influenza) virus infections. I hypothesise that HMGCR is a target for 25-HC associated IFN-mediated host defence against viral infection. To characterise HMGCR and test this hypothesis, the following objectives were defined: (1). To establish an experimental system to quantitatively study the endogenous HMGCR protein level; (2). To investigate the mechanism of the down-regulation of HMGCR involved in the IFN-mediated innate immune response; (3). To study the behaviour of HMGCR in the influenza virus induced 25-HC associated IFN-mediated innate immune response; (4). To study the behaviour of HMGCR in the cytomegalovirus induced 25-HC associated IFN-mediated innate immune response. Chapter 3, describes establishing an experimental system for the quantification of endogenous HMGCR levels. Different protein detection methods, including a modified western blot protocol and immunostaining, were tested. The results of RNA interference of HMGCR demonstrate that under lipid-deficient condition with the supplementation of mevastatin (an HMGCR inhibitor) the modified western blot protocol specifically detects endogenous HMGCR. This chapter lays the foundational work for the temporal analysis and testing the role of HMGCR in infection. In Chapter 4, the mechanism of the degradation of HMGCR following 25-HC and IFN treatments, in wild-type and Ch25h−/− mouse bone marrow derived macrophages (BMDMs), was investigated. Similar to 25-HC, IFN-γ treatment results in the drop of both the transcript and protein abundance of HMGCR in wild-type BMDMs. Differential temporal analysis of RNA and protein alterations and the use of proteasome inhibitors reveals that both 25-HC and IFN-γ lead to a marked reduction of HMGCR protein via a proteasomal degradation mechanism within early times of treatments. Further, the immediate reduction of HMGCR levels induced by IFN-γ was completely abrogated in Ch25h−/− BMDMs. Hence, the reduction of HMGCR following IFN-γ treatment is due to the de novo synthesis in macrophages of 25-HC. However, the decrease of Hmgcr gene expression was observed in not only wild-type but also Ch25h−/− BMDMs, suggesting additional mechanisms for regulating Hmgcr RNA levels. These results demonstrate the mechanism of the down-regulation of HMGCR resulted from the induction of IFN response during viral infection, is only partially due the de novo synthesis of 25-HC. In chapter 5, influenza A virus was used to investigate the role of HMGCR in the IFN-mediated innate immune response. The inhibition of HMGCR by RNA interference inhibited viral growth, suggesting the requirement of HMGCR for optimal intracellular viral growth. Viral infection in wild-type murine BMDMs reduced the endogenous HMGCR levels. However, the reduction of HMGCR at early times was prevented in Ch25h−/− BMDMs. Intriguingly, the decrease of HMGCR at late time points was still observed in Ch25h−/− BMDMs. These results indicate that the down-regulation of HMGCR with influenza virus infection in BMDMs at early times is completely due to the de novo synthesis of 25-HC; whereas at late times alternative pathways or mechanisms exist. Additionally, human epithelial A549 cells and A549/PIV5-V cells that are deficient in STAT1 were used to study the role of IFN pathway in the down-regulation of HMGCR at late times during viral infection. Results from these studies show that at late times the reduction of HMGCR is due to IFN-independent mechanisms. Chapter 6, extends these investigations to the herpes virus murine cytomegalovirus and infection of BMDMs. HMGCR is known to be essential for cytomegaloviral infections and 25-HC, statin and RNAi inhibition of HMGCR restrict viral growth. 25-HC is shown to reduce HMGCR at immediate early times of infection. However, most notably, the down-regulation of HMGCR was also observed in Ch25h−/− BMDMs at late times with murine cytomegalovirus infected BMDMs. These results confirm that alternative pathways or mechanisms exist, playing roles in the crosstalk between cholesterol metabolism and innate immune response. Collectively, this study characterises the role of HMGCR in the 25-HC associated IFN-mediated host defence against viral infection. Results indicate that, in addition to the IFN-mediated host response, alternative pathways or other mechanisms also result in the down-regulation of HMGCR during viral infection. HMGCR is at the crossroad of different pathways or mechanisms, and is therefore not only targeted by 25-HC. Hence, further questions can be addressed from these results: (1). What are the alternative pathways or mechanisms for the down-regulation of HMGCR? (2). How do these pathways or mechanisms work in hosts’ immune system? Answering these questions can contribute to refining the pathway map of innate immunity and understanding the precise role of HMGCR, or even the sterol biosynthesis pathway, in hosts’ immune response against pathogens.

Viruses are obligate intracellular pathogens. Therefore, their successful replication, at every stage from attachment to assembly and egress, is dependent on host cell functions. The host cell in turn engages mechanisms to counteract virus replication. As a result, viruses have evolved mechanisms to evade these counteracting measures as well as ways to reshape the cellular environment into one that’s favourable for successful replication. Systematic studies offer a platform for unravelling virus-cell interactions and in particular can address three important aspects 1) increase our understanding of basic biology of the virus, 2) identify and characterise novel cellular functions 3) provide important leads for novel targets for antiviral therapy. In this study, I investigated two aspects of virus host interaction; the role of microRNAs (miRNAs) in virus infection and the role of interferon inducible genes in virus infection. Human cytomegalovirus (HCMV) is a β herpes virus that infects humans. HCMV maintains a persistent lifelong infection in the host involving a cycle of latency and reactivation. Infection of healthy individuals with HCMV results in relatively minor symptoms. In contrast, infection of individuals with a compromised immune system, as in the case of organ transplant recipients and AIDS patients, can cause significant morbidity and mortality. In common with other herpes viruses, HCMV expresses multiple small regulatory RNAs called miRNAs. HCMV encodes at least 14 miRNAs. Identifying the targets of these miRNAs will help us understand their functional importance during infection. Recently, a biochemical technique called Cross-Linking, Ligation and Sequencing of Hybrids (CLASH), was developed by Tollervey and colleagues, representing the most advanced systematic technique for the identification of miRNA targets. We adapted this approach to identify high confidence miRNA targets during HCMV infection. However, the protocol was sub-optimal and presented us with technical challenges. Although high quality data sets were not generated, the work was crucial for the establishment of the system which is now generating promising data. Virus-cell interactions can also be elucidated by probing for host factors that are important for virus replication. Type I interferon is a highly effective inhibitor of HCMV replication. Treatment of cells with interferon results in up regulation of multiple effectors known as interferon stimulated genes (ISGs). How these genes block HCMV replication is poorly understood. A library of more than 380 ISG expressing lentiviruses was screened to determine the effects of individual ISGs on HCMV replication. The screen was performed in primary human fibroblast cells and a glioblastoma cell line called U373s. Multiple inhibitory ISGs were identified including well characterised ISGs such as cGAS, STAT2, NOD2, DDX60 and HPSE as well as novel candidates TXNIP, ELF1, FAM46C, MT1H and CHMP5. Five ISGs were identified as HCMV replication enhancers including previously published ISGs BST2 and IFITM1 and novel enhancers ODC1, BCL3 and IL28RA. siRNA screens against top hits demonstrated that STAT2, CPT1A and cGAS are dominant inhibitory factors during HCMV infection and knockdown of these genes can partially rescue HCMV replication following interferon treatment. Finally, using a corresponding rhesus ISG library we show that rhesus SAMHD1 effectively inhibits HCMV replication while human SAMHD1 has no effect, suggesting that HCMV expresses a species-specific inhibitor of SAMHD1. This study defines interferon stimulated pathways important for HCMV replication and identifies multiple novel host factors that both restrict and enhance HCMV replication. These studies demonstrate the effectiveness of using systematic approaches for the identification of novel host virus interactions.

Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants. In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values. Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation. Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.

Cytomegalovirus (CMV) is a β-herpesvirus that, after primary infection, establishes a life-long persistence in the human host. Up to 90% of humans are infected with CMV, that is kept under control by CMV-specific CD8+ and CD4+ T cells. In patients with an impaired cellular immunity, however, CMV infections can be life-threatening. Thus, it is vital to identify risk factors and target high-risk patients. In this thesis we have evaluated low-dose valacyclovir prophylaxis in renal transplant patients and studied CMV-specific T cell immunity in healthy and renal transplant patients. In renal transplant patients, the CMV serostatus of both the recipient (R) and the donor (D) has a major impact on the risk of developing CMV disease. In the high-risk D+/R- population, &gt;50% are likely to develop CMV disease in the absence of prophylaxis and/or pre-emptive therapy. We have used low-dose valacyclovir prophylaxis for high-risk renal transplant patients, and graft and patient survival up to 5 years after transplantation was comparable to data reported for other prophylactic protocols. The incidence of CMV disease and graft rejection during the first year after transplantation was also comparable to that achieved with other protocols, and without the adverse effects reported for other therapies. In the D+/R+ population, with a 15-35% risk of developing CMV disease, it is important to identify those individuals that are subject to a higher risk because of risk factors other than CMV serostatus. We therefore measured several immunologic parameters in renal transplant patients and in immunocompetent individuals with latent and primary CMV infection. In patients with a primary symptomatic CMV infection, CMV-specific CD8+ T cells peaked within a month after onset of symptoms but declined rapidly. In renal transplant patients, we found that the reduction in IFNγ-producing CMV-specific CD4+ T cells at 2 months post-transplantation may predict high-grade CMV DNAemia.

The quality of immune responses develops from birth into adulthood and in the context of the host microbial environment. The aim of this work was to study immune maturation during childhood, and how this process can be affected by the common herpesviruses; Epstein-Barr virus (EBV) and cytomegalovirus (CMV). In paper I we studied monocytes, an important cell type for immunity in the newborn. We showed that the neonatal monocyte subsets exist in similar frequencies as adult subsets, and have a potent capacity for pro-inflammatory cytokine production. In paper II, III and IV we studied the effects of EBV and CMV infections on immune cell function in children. In paper II we found that monocyte-induced NK-cell production of IFN-γ, and plasma IFN-γ levels, were decreased in 2-year old EBV- and/or CMV-seropositive children and mostly so in co-infected children. In paper III we found that in 5-year old children, EBV and CMV co-infection was associated with the highest levels of differentiated NKG2C+ NK cells. CMV+ children had higher plasma IFN-γ and IL-15 levels and higher NK-cell cytotoxic capacity. In vitro PBMC systems showed elevated frequencies of NKG2C+ NK cells in the presence of EBV-infected cells. In paper IV we showed that a child’s age and subsequent capacity for anti-viral cytokine production affects in vitro EBV infection in terms of B-cell proliferation and B-cell acquisition of memory phenotype. PBMC from CMV+ children had lower EBV-induced accumulation of switched memory B cells, which was connected to high prevalence of CD57+CD8+ T cells and IFN-γ production. Taken together, this thesis work shows that monocyte subsets at birth can give potent functional responses and that latency with EBV and CMV has a significant effect on the differentiation process and functional capacity of anti-viral effector cells during childhood. This in turn could affect responses to related or unrelated infections or even to non-invasive antigens such as allergens. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 17, 2009). Includes bibliographical references.