Aicardi-Goutières syndrome (AGS) is a rare inflammatory encephalopathy mimicking in utero acquired viral infection. Cardinal findings comprise leukodystrophy, basal ganglia calcifications and cerebral atrophy along with cerebrospinal fluid lymphocytosis and elevated interferon-α. In the majority of cases AGS is inherited as an autosomal recessive trait and caused by mutations in six genes including RNASEH2A, RNASEH2B, RNASEH2C, TREX1, SAMHD1 and ADAR1, all of which encode enzymes acting on nucleic acid species. Most patients present with first neurological signs in early infancy and experience severe global developmental delay. Here, we report on the unusual divergent phenotype of two siblings who both carry the most frequent AGS causing p.A177T (c.529G > A) RNASEH2B mutation in the homozygous state. While one sibling showed a typical AGS presentation with early onset and severe statomotor and mental impairment, the older sibling was intellectually completely normal. She was only diagnosed because of mild spasticity of the legs and serological signs of autoimmunity. These findings highlight the phenotypic variability of AGS and suggest that AGS may be underdiagnosed among children with mild cerebral palsy.

Background: Multiple sclerosis (MS) is an autoimmune disease which attacks the central nervous systems (CNS) white substance. The damages cause MS-plaques which lead to disability in the CNS. There are currently around 20,000 people in Sweden who live with the diagnosis and every year another 1,000 people develop MS. It is twice as common for women to suffer from MS in comparison with men. Objective: The purpose of the study was to investigate the efficacy and safety of the alemtuzumab and daclizumab infusion drugs. Methods: This work is a literature study based on 6 scientific articles. Results: Based on at least one of the studied parameters, which are improvement in Expanded Disability Status Scale (EDSS), annual relapse rate and sustained accumulation of disability (IFS), showed that all four studies on alemtuzumab provided a higher therapeutic effect than IFN-β-1a. The two studies on daclizumab showed in one article in comparison with placebo a better treatment effect. The article comparing daclizumab with IFN-β-1a did not show major differences between the preparations but a reduced annual relapse rate was observed. Conclusion: The studies showed that both alemtuzumab and daclizumab yield better effect than IFN-β-1a and placebo. Treatment with alemtuzumab gave slightly better results in persistent worsening of the disease (IFS) compared with daclizumab, IFN-β- 1a and placebo. Patients who were treated with alemtuzumab had worse adverse events in comparison to the group of patients who were treated with IFN-β-1a. This shows that the safety of treatment with alemtuzumab was lower in comparison to treatment with IFN-β-1a. Most fatal outcomes were found during treatment with alemtuzumab. These aspects makes one consider whether alemtuzumab may or may not be the best treatment option. Also daclizumab had some serious side effects, but those did not occur as much as with alemtuzumab treatment. IFN-β-1a has been a safer drug to use as compared to alemtuzumab and dalclizumab. On the other hand, the mechanism of action differs between the three drugs, which means that these can be tailored more individually to MS-affected patients and a more individual-based drug treatment may be possible.

O estabelecimento de uma resposta TH1 com a produção de IL-12, IFN-gama e de óxido nítrico é crucial no controle do Trypanosoma cruzi, o qual pode colonizar o SNC de crianças e pacientes imunossuprimidos. A inflamação exacerbada em decorrência da persistência de um estímulo antigênico gera o acúmulo de substâncias potencialmente citotóxicas, como mediadores pró-inflamatórios e radicais livres. A partir da infecção de camundongos imunodeficientes (IL-12p40KO) com T. cruzi Sylvio X10/4, avaliamos os danos causados à medula espinal com enfoque na inflamação e neurodegeneração. Além da desmielinização, alta reatividade glial e morte de neurônios no ponto mais tardio da doença, constatamos uma baixa produção de mediadores inflamatórios nas primeiras semanas após a infecção, acompanhada pela proliferação ascendente do parasita no tecido nervoso. Acreditamos que um atraso na produção de IFN-gama seja responsável pela ativação tardia ou ineficiente dos fagócitos da medula espinal, favorecendo a disseminação descontrolada do protozoário e subsequentes danos teciduais. / The establishment of a TH1 response with IL-12, IFN-gamma and nitric oxide production is crucial for controlling the proliferation of Trypanosoma cruzi, which may colonize the CNS of children and immunosuppressed hosts. The exacerbated inflammation due to the persistence of an antigenic stimulus results on the accumulation of potentially cytotoxic substances, such as pro-inflammatory mediators and free radicals. By the infection of immunodeficient mice (IL-12p40KO) with T. cruzi Sylvio X10/4 parasites we evaluated the spinal cord damages, focusing on the inflammation and neurodegeneration. Besides demyelization, high glial reactivity and neuron death at the latest stage of the disease, we noticed low production of inflammatory mediators during the first weeks of the infection, accompanied by an ascendant parasite proliferation in the nervous tissue. We believe that a delay on IFN-gamma production is responsible for the late or inefficient phagocyte activation in the spinal cord, contributing to the uncontrolled protozoan proliferation and subsequent tissue injury.

O câncer colorretal é o terceiro em mortalidade, e apesar das classificações moleculares, entre 30% e 40% dos pacientes apresentam recidiva após quimioterapia, o que aponta a necessidade de novas estratégias terapêuticas. É crescente o estudo de vetores virais para a terapia gênica do câncer, e dados do nosso laboratório mostraram que a entrega combinada dos genes Arf (supressor tumoral e parceiro funcional de p53) e interferon-beta (IFNbeta, citocina imunomodulatória), via vetores adenovirais sob o controle do promotor responsivo à p53 causou indução de morte celular massiva e um efeito imunomodulatório importante. Entretanto, esses resultados foram observados em modelos murinos. O presente trabalho avalia como a combinação da modulação das vias de p53 e IFNbeta impacta células de carcinoma colorretal humano em ensaios in vitro. Para isto, utilizamos nosso adenovírus para transferir p53 (p53) e IFN? como uma estratégia combinada para induzir a morte celular na presença de um modulador imunológico. As linhas celulares HCT116, HCT116p53-/- toleraram uma MOI (multiplicidade de infecção) de 25, enquanto uma MOI de 100 foi suportada por HT29 (mutante p53 R273H). O promotor PG, dependente de p53, como esperado, proporcionou níveis de expressão de GFP reduzidos em HCT116 p53 - / - e HT29, mas a expressão foi aumentada em todas as linhas celulares quando co-transduzida com o vetor codificando p53, mas não com IFNbeta. Em geral, HCT116 e HCT116p53-/- foram mais sensíveis à transferência de p53, enquanto o HT29 foi particularmente afetado pela combinação de p53 + IFNbeta. Esta tendência reflectiu-se na viabilidade celular (curva de crescimento, MTT), formação de colónias e acumulação de células hipodiplóides. Os níveis de morte celular correlacionaram-se com a atividade da caspase 3/7 e coloração com o Anexina V. Os marcadores de morte imunogênico ATP e calreticulina também foram aumentados, especialmente para o HT29 tratado com INFbeta sozinho ou em combinação com p53. Além disso, observamos aumento da expressão de TP63, TP73, bem como alvos transcricionais de p53, como p21 e NOXA, enquanto SESTRIN foi reduzido em ambas as linhagens HCT, mas aumentou em HT29. Adicionalmente, testamos a combinação p53 + IFNbeta em associação com quimioterápicos, revelando cooperação entre a transferência gênica e a doxorrubicina ou 5-FU, mesmo nas menores doses testadas. Ensaios em andamento incluem a avaliacao da ativacao de celulas dentriticas humanas expostas para as celulas tumorais tratadas com os vetores portadores de p53 e/ou IFNbeta. Com este projeto, nossa abordagem de transferência gênica revelará a resposta aos transgenes em modelo humano e também abrirá o caminho para estudar o envolvimento do sistema imune humano / Colorectal cancer is the third leading cause of cancer death and despite new molecular classifications, 30% to 40% of all patients will relapse after chemotherapy, pointing the need for new and innovative therapeutic strategies. The number of studies about viral vectors for gene therapy is growing, and previous data from our laboratory indicates that the combined delivery of Arf (a tumor suppressor gene and functional partner of p53) and interferon beta (IFNbeta, an immunomodulator cytokine), under a p53 responsive promoter, induced massive cell death and an important immunomodulatory effect. However, these results were only observed in murine models. Here we present an RGD-modified adenovirus whose transgene is under the control of a p53 responsive promoter (PG), used to transfer p53 (p53) and IFNbeta as a combined strategy to induce cell death in the presence of an immune modulator. Cell lines HCT116, HCT116 p53 -/- tolerated a MOI (multiplicity of infection) of 25, while a MOI of 100 was supported by HT29 (mutant p53 R273H). The p53-dependent PG promoter, as expected, provided reduced GFP expression levels in HCT116 p53 -/- and HT29, but the expression was increased in all cell lines when co-transduced with the p53 vector, but not with IFNbeta . In general, HCT116 and HCT116p53-/- were more sensitive to p53 gene transfer while HT29 was particularly affected by the combination of p53 + IFNtbeta. This trend was reflected in cell viability (growth curve, MTT), colony formation and accumulation of hypodiploid cells. Levels of cell death correlated with caspase 3/7 activity and staining with Annexin V. Immunogenic markers were also increased, especially for HT29 treated with INFbeta and p53/IFNbeta, as we detected exposure of calreticulin and release of ATP. Also, we observed increased expression of TP63, TP73 as well as p53 transcriptional targets, such as p21 and NOXA; SESTRIN was reduced in both HCTs but increased in HT29. Additionally, we tested the p53+IFNb combination in association with chemotherapeutics, revealing cooperation between gene transfer and doxorubicin or 5-FU even at the lowest doses tested. Ongoing studies include the evaluation of human dendritic cells exposed to tumor cells treated with the vectors for p53/IFNbeta. In conclusion, our combined gene transfer approach has potentiated the killing of colorectal cancer cell lines and may provide an immunomodulatory effect

As complicações hepáticas secundárias à infecção crônica pelo vírus da hepatite C (HCV) são uma importante causa de morte em portadores da infecção pelo vírus da imunodeficiência humana (HIV). Pacientes com coinfecção HIV/HCV apresentam progressão acelerada da fibrose hepática, na qual há participação da resposta inflamatória do sistema imunológico, e requerem maior atenção no tratamento da hepatite C e de suas reações adversas. Assim, os objetivos principais do estudo foram tipificar e comparar os polimorfismos -607 e -137 da interleucina-18 (IL-18), +874 do interferon gama (IFN-?? e -308 e -238 do fator de necrose tumoral alfa (TNF- ?? em quatro grupos (coinfecção HIV/HCV, monoinfecção pelo HIV, monoinfecção pelo HCV e controles saudáveis); investigar a associação dos alelos e genótipos desses polimorfismos com a resposta ao tratamento da hepatite C (respondedor e não respondedor), graus de atividade necroinflamatória (METAVIR A0A1 vs. A2A3) e de fibrose hepática (METAVIR F0-F2 vs. F3F4) em portadores do HCV e identificar os sinais e sintomas relacionados às reações adversas do tratamento da hepatite C. Os dados foram coletados nos prontuários médicos e no sistema informatizado do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto e os polimorfismos tipificados pela técnica de reação em cadeia da polimerase com iniciadores de sequência específica. Participaram do estudo 400 indivíduos, distribuídos em quatro grupos de 100, predominantemente constituídos por homens com idade média entre 33 e 50 anos. Na avaliação geral, os genótipos -238 G/G (TNF-?? e +874 A/A (IFN-?? foram mais frequentes no grupo coinfecção HIV/HCV em relação ao monoinfecção pelo HCV. O genótipo -308 G/A e o alelo -308 A (TNF-?? foram associados com a susceptibilidade à coinfecção HIV/HCV e o genótipo -308 G/G e o alelo -308 G (TNF-?), com proteção. No grupo coinfecção HIV/HCV, a frequência do genótipo - 137 G/C (IL-18) foi maior nos sujeitos com atividade necroinflamatória A0A1 que nos com A2A3. Nos pacientes com fibrose F3F4, o genótipo -238 G/G (TNF-?? foi mais frequente no grupo coinfecção HIV/HCV que no monoinfecção pelo HCV e naqueles com F0-F2, o genótipo +874 A/A (IFN-?? também foi mais frequente no grupo coinfecção HIV/HCV. A frequência do genótipo +874 T/T (IFN-??, dentre os pacientes do grupo coinfecção HIV/HCV, foi maior naqueles com fibrose F3F4 que nos com F0-F2. Não foram encontradas associações estatisticamente significantes entre as frequências alélicas e genotípicas e os tipos de resposta ao tratamento da hepatite C nos pacientes do grupo coinfecção HIV/HCV; nos do monoinfecção pelo HCV, houve diferenças nas frequências alélicas e genotípicas (posição -238 do TNF-?) entre pacientes respondedores e não respondedores. Os principais sinais e sintomas relacionados às reações adversas do tratamento da hepatite C foram mialgia, febre, fraqueza, cefaleia e hiporexia. Anemia, hiporexia e vômito foram mais frequentes no grupo coinfecção HIV/HCV. Conclui-se que há relação dos alelos e genótipos de citocinas com a gravidade da doença hepática e resposta ao tratamento da hepatite C. Adicionalmente, algumas reações adversas ao tratamento foram mais pronunciadas em coinfectados HIV/HCV / Hepatic complications secondary to chronic infection by hepatitis C virus (HCV) are a major cause of death in people infected by the human immunodeficiency virus (HIV). Patients with HIV/HCV coinfection present rapid progression of liver fibrosis, with involvement of the immune system\'s inflammatory response, and require more attention in hepatitis C treatment and its adverse reactions. The main goals of this study were to typify and compare the polymorphisms -607 and -137 of the interleukin-18 (IL-18), +874 of the interferon gamma (IFN-?? and -308 and -238 of the tumor necrosis factor-alpha (TNF-?? in four groups (HIV/HCV coinfection, HIV monoinfection, HCV monoinfection and healthy controls), to investigate the association of the alleles and genotypes of these polymorphisms with response to hepatitis C treatment (responder and non-responder), degrees of necroinflammatory activity (METAVIR A0A1 vs. A2A3) and of liver fibrosis (METAVIR F0-F2 vs. F3F4) in HCV patients and to identify the signs and symptoms related to adverse reactions of hepatitis C treatment. Data were collected on medical records and on the computerized system of the Hospital das Clínicas of the University of São Paulo Ribeirão Preto Medical School and the polymorphisms were typified using the polymerase chain reaction technique with sequence specific primers. The study included 400 individuals, distributed in four groups of 100, predominantly consisting of men with an average age between 33 and 50 years. In the overall evaluation, genotypes -238 G/G (TNF-?? and +874 A/A (IFN-?? were more frequent in the HIV/HCV coinfection group compared to HCV monoinfection. The genotype -308 G/A and allele -308 A (TNF-?? were associated with susceptibility to HIV/HCV coinfection and the genotype -308 G/G and allele -308 G (TNF-?), with protection. In the HIV/HCV coinfection group, the frequency of genotype -137 G/C (IL-18) was greater in subjects with necroinflammatory activity A0A1 than in the ones with A2A3. In patients with fibrosis F3F4, genotype -238 G/G (TNF-?? was more frequent in the HIV/HCV coinfection than in the HCV monoinfection group and in those with fibrosis F0-F2, genotype +874 A/A (IFN-?? was also more frequent in the HIV/HCV coinfection group. The frequency of genotype +874 T/T (IFN-??, among patients of the HIV/HCV coinfection group, was higher in those with fibrosis F3F4 compared to the ones with F0-F2. No statistically significant associations were found between the allele and genotype frequencies and the types of answer to hepatitis C treatment in patients of the HIV/HCV coinfection group. On the ones of the HCV monoinfection group, there were differences on the allele and genotype frequencies (position -238 of TNF-?? among responder and non-responder patients. The main signs and symptoms related to adverse reactions to hepatitis C treatment were myalgia, fever, weakness, headache and loss of appetite. Anemia, loss of appetite and vomiting were more frequent in the HIV/HCV coinfection group. It is concluded that there is relationship of the alleles and genotypes of cytokines with the severity of liver disease and response to hepatitis C treatment. Additionally, some adverse reactions to treatment were more frequent in HIV/HCV coinfected patients

A tuberculose permanece como um dos maiores problemas de saúde pública mundial. O diagnóstico precoce e o tratamento rápido e eficiente dos indivíduos com tuberculose pulmonar ativa são medidas essenciais para a redução da morbidade, mortalidade e da incidência da tuberculose no mundo. As limitações encontradas nos métodos microbiológicos tradicionais, fizeram com que metodologias alternativas fossem desenvolvidas para melhorar o diagnóstico e o prognóstico da tuberculose humana. Neste trabalho verificamos o desempenho diagnóstico do Western blotting para pesquisa de anticorpos IgG anti - Mycobacterium tuberculosis, a utilização do teste QuantiFERON® - TB Gold e a detecção de moléculas de adesão celular (ICAM-1 e selectinas) como marcadores de prognóstico. Foram acompanhados até o final do tratamento 31 pacientes com tuberculose pulmonar diagnosticados por critérios clínicos e laboratoriais. Como controles, selecionamos população de indivíduos sadios, doadores de banco de sangue e indivíduos com outras pneumopatias. Os resultados mostraram que o Western blotting apresentou sensibilidade de 94% e especificidade de 96% no diagnóstico da tuberculose pulmonar, atendendo os requisitos da OMS para testes sorológicos. O QuantiFERON® - TB Gold apresentou sensibilidade de 83% e especificidade de 100%, após ajuste do limiar de reatividade. Os resultados das moléculas de adesão celular sugerem potencial para serem utilizadas como marcadores de prognóstico da doença. Ao associarmos os resultados do Western blotting ou do QuantiFERON® - TB Gold com a baciloscopia obtivemos sensibilidade superior a 95%; e quando associados à cultura a sensibilidade encontrada foi de 100%. O Western blotting mostrou ser uma ferramenta útil como auxiliar no diagnóstico da tuberculose pulmonar mesmo em pacientes com baciloscopia negativa. / Tuberculosis remains a major public-health problem. Rapid diagnosis and prompt treatment is the cornerstone to reduce morbidity, mortality and incidence of tuberculosis in the world. Alternative methods have been developed to overcome the limitations presented by conventional microbiological methods and to improve the diagnosis and prognosis of tuberculosis. In this study we verified the diagnostic performance of Western blotting for IgG anti-M.tuberculosis antibodies detection, QuantiFERON® - TB Gold and circulating adhesion molecules (ICAM-1 and Selectins) as prognosis markers. Thirty-one patients were followed-up during the treatment. Active pulmonary tuberculosis was diagnosed by clinical and laboratorial criteria. As group control healthy individuals, blood donors and patients with other lung diseases were included. Western blotting results showed a high performance with sensitivity of 94% and specificity of 96% for the diagnosis of pulmonary tuberculosis, attending WHO requirements for serological tests. After adjusting the threshold, QuantiFERON® - TB Gold showed sensitivity of 83% and specificity of 100%. The results of adhesion molecules suggested potential to use the test as prognosis markers. Combining Western blotting or QuantiFERON® - TB Gold with acid-fast bacilli (AFB) smear results, the overall sensitivity increase to more than 95%, and when combined with culture the overall sensitivity was 100%. Together, these findings, suggest that Western blotting could be a very useful supplementary tool for pulmonary tuberculosis, especially in patients with AFB smear negative.

Das 26S Proteasom ist ein großer Proteinase-Komplex, der aus 32 unterschiedlichen Untereinheiten aufgebaut ist. Das 26S Proteasom ist involviert in die ATP-abhängige De-gradation von ubiquitinierten Proteinen, die eine Vielfalt an zellulären Prozessen wie Signaltransduktion, Stressantwort, transkriptionelle Regulation, Chromosomen-Segregation, DNA-Reparatur, Zellzyklus-Steuerung und die Prozessierung von Peptiden für die MHC I Antigen Präsentation regulieren. Die Prozessierung von Peptiden wird ver-stärkt durch eine Interferon ? stimulierbare Variante des Proteasoms übernommen, dem so genannten Immunoproteasom. Die Biogenese dieses großen Komplexes ist ein komplizierter Mechanismus, welcher Expression und Assemblierung der proteasomalen Untereinheiten beinhaltet. In Eukaryonten sind für die Assemblierung und Maturierungsprozesse Helferproteine notwendig. In Mammalia übernimmt diese Funktion das Proteasom maturation Protein POMP. POMP ist wahrscheinlich auch bei der Biogenese des Immunoproteasoms von Bedeutung, da die mRNA von POMP durch Interferon ? induziert wird. Um die Regulation dieser Induktion zu untersuchen wurde der Promotor von POMP für die erste Fragestel-lung dieser Arbeit charakterisiert und seine Induzierbarkeit durch Interferon ? untersucht. Es konnte nachgewiesen werden, dass die erhöhte mRNA-Menge durch Interferon ?-Stimulation nicht auf eine Promotor-Induktion, sondern auf post-transkriptionelle Ereig-nisse zurückzuführen ist. In der zweiten Fragestellung dieser Arbeit sollte die Genregulation des Proteasoms unter Stressbedingungen untersucht werden. Der Stress wurde durch Inhibition der proteolyti-schen Aktivität des Proteasoms ausgelöst. Wie seit längerem bekannt ist, werden in Bakterien und Hefe die ATP-abhängigen Pro-teasekomplexe über ein kompliziertes regulatorisches Netzwerk gesteuert. Über die transkriptionelle Regulation des Mammalia Proteasoms war bisher wenig bekannt. Im Rahmen der hier vorliegenden Dissertation konnte gezeigt werden, dass die Reduktion der proteolytischen Aktivität des Proteasoms durch Behandlung von Mammalia-Zellen mit Proteasom-Inhibitoren durch eine gesteigerte Genexpression der proteasomalen Unter-einheiten kompensiert wird. Alle proteasomalen Untereinheiten werden konzertiert hoch-reguliert. Exemplarisch an der proteasomalen Untereinheit Rpt1(S7) und an dem Matu-rierungsfaktor POMP konnte eine posttranskriptionelle Regulation unter Proteasom-Inhibitor Einfluss ausgeschlossen werden. Die vom Inhibitor induzierte Genaktivierung resultiert in einer de novo Protein-Synthese und führt daher zu einer gesteigerten de no-vo Biogenese des Proteasoms. Dieses Phänomen ist begleitet durch eine vermehrte Ex-pression vom POMP. Damit konnte erstmals gezeigt werden, dass die Menge an Protea-som in Mammalia auf transkriptioneller Ebene reguliert wird und dass vermutlich ein au-toregulatorischer feedback-Mechanismus eine verminderte proteolytische Aktivität kom-pensieren kann. Diese Daten werden durch Ergebnisse der CAT-Reportergen-Assays des ?1(?)-Promotors gestützt. Exemplarisch konnte gezeigt werden, dass die Aktivität dieses Promotors in Anwesenheit von Proteasom-Inhibitoren ansteigt. Die induzierbare Promotorregion konnte bis auf 130 bp eingegrenzt werden. Innerhalb dieser Promotorse-quenz konnte die Bindung eines Transkriptionsfaktors (Nrf2) durch EMSA-Technik nach-gewiesen werden. / The 26 S proteasome is a high molecular mass proteinase complex that is built by of least 32 different protein subunits. The 26S proteasome is involved in the ATP-dependent degradation of ubiquitinated proteins that regulate a variety of cellular proc-esses including signal transduction, stress response, transcriptional control, chromosome segregation, DNA repair, cell cycle progression and processing of antigenic Peptides for the MHC I pathway. Biogenesis of this large complex is a complicated process compris-ing expression, assembly and maturation of all subunits. This crucial step is supported by POMP (proteasome maturation protein). POMP mRNA is induced by Interferon gamma (IFN ?). We investigated this phenomenon via Reportergen assays with the Promoter re-gion of POMP. POMP mRNA seems not to be regulated on a trancriptional level, but on posttranscriptional events. ATP-dependent protease complexes in bacteria and yeast are systems that undergo a highly sophisticated network of gene expression regulation. However, regulation of mammalian proteasome gene expression has been neglected so far as a possible control mechanism for the amount of proteasomes in the cell. We showed that treatment of cells with proteasome inhibitors and the concomitant impairment of proteasomal enzyme activ-ity induce a transient and concerted up-regulation of all mammalian 26S proteasome subunit mRNAs. Proteasome inhibition in combination with inhibition of transcription re-vealed that the observed up-regulation is mediated by coordinated transcriptional activa-tion of the proteasome genes and not by post-transcriptional events. Our experiments also demonstrate that inhibitor-induced proteasome gene activation results in enhanced de novo protein synthesis of all subunits and in increased de novo formation of the pro-teasome. This phenomenon is accompanied by enhanced expression of the proteasome maturation factor POMP. Thus, our experiments present first evidence that the amount of proteasomes in mammalia is regulated at the transcriptional level and that an auto regu-latory feedback mechanism exists that allows the compensation of reduced proteasome activity. These data are also supported by CAT reportergene assays with the protea-somal subunit ?1(?)-promoter. Exemplary we show the increase of CAT activities in re-sponse to proteasome inhibition. We can restrict the region of the promoter to 130 bp and identify Nrf2 as a possible candidate for a transcription factor via EMSA.

Infektionen lösen eine Immunantwort aus, welche u.a. durch Induktion von Interferonen (IFN) für die Expression von antibakteriell oder antiviral wirkenden Proteinen sorgt. Eines der am stärksten induzierten Gene nach Typ I IFN Stimulation ist das Interferon stimulated gene 15 (ISG15). ISG15 gehört zu den Ubiquitin-ähnlichen Proteinen und wird über eine Enzymkaskade kovalent an zu modifizierende Proteine konjugiert. Sowohl freiem ISG15, als auch ISG15-Konjugaten wurden antivirale Funktionen nachgewiesen. In der vorliegenden Arbeit wurde die Regulation von ISG15, der ISGylierung und der an der ISGylierung beteiligten Enzyme vergleichend nach Typ I und Typ II IFN Stimulation untersucht. In HeLa Zellen zeigte sich, dass ISG15 und ISG15-Konjugate sowohl nach Typ I, als auch nach Typ II IFN Stimulation gebildet werden. Die Unterschiede in der Menge der ISGylierten Proteine konnten in Zusammenhang mit der unterschiedlich starken Expression der an der ISGylierung beteiligten Enzyme gebracht werden. Durch Inhibition der Expression der einzelnen Enzyme wurde weiterhin nachgewiesen, dass nach Typ I und Typ II IFN Stimulation das gleiche E2-Enzym (UBE2L6), aber unterschiedliche E3-Ligasen an der ISGylierung beteiligt sind. Während die ISGylierung nach Typ I IFN Stimulation größtenteils von der E3-Ligase Herc5 abhängt, zeigte sich, dass nach Typ II IFN Stimulation weitere noch nicht identifizierte ISG15 E3-Ligasen an dem Prozess beteiligt sind. Microarry- und siRNA-Analysen führten zur Identifizierung der potentiellen ISG15 E3-Ligasen UBE3A und ARIH1. Dabei zeigte sich, dass UBE3A nach Typ I und ARIH1 nach Typ II IFN Stimulation einen Einfluss auf die ISGylierung von Proteinen haben. Diese Ergebnisse machen deutlich, dass in HeLa Zellen Typ I und Typ II Interferone die ISGylierung induzieren, wobei sich die Enzymkaskade zwischen den Interferonen auf Ebene der E3-Ligasen unterscheidet. Diese Ergebnisse geben neue Einblicke in die Regulation der IFN vermittelten ISGylierung. / Infections initiate the induction of an immune response that lead to induction of interferons followed by expression of antibacterial and antiviral proteins. One of the most upregulated genes after type I interferon (IFN) stimulation is the Interferon stimulated gene 15 (ISG15). ISG15 belongs to the family of ubiquitin-like modifiers and is covalently attached to target proteins via an enzyme cascade. It was shown that free ISG15 as well as ISG15-conjugates exhibit antiviral functions. In this thesis the regulation of ISG15, ISGylation and the involvement of enzymes after type I and type II IFN treatment were analysed. It could be demonstrated that ISG15 and ISG15-conjugates are generated after type I as well as type II IFN treatment in HeLa cells. Differences in the amount of ISGylated proteins were shown to correlate with the expression of proteins of the enzyme cascade. Inhibition of these enzymes demonstrated that the E2-enzyme UBE2L6 is involved in the ISGylation process after both types of IFN stimulation, whereas different E3-ligases are implicated in ISGylation of proteins after type I and type II IFN stimulation. It could be shown that the E3-ligase Herc5 is involved in type I IFN mediated ISGylation, whereas other not yet identified ISG15 E3-ligases are involved in type II IFN mediated ISGylation. Microarry- and siRNA-assays led to the identification of the putative ISG15 E3-ligases UBE3A and ARIH1, whereas UBE3A had an influence on type I IFN mediated ISGylation and ARIH1 affected the type II IFN mediated ISGylation. In this thesis it could be demonstrated that both types of interferons are able to induce ISGylation in HeLa cells, but different E3 ligases are involved in conjugation of ISG15 to its target proteins after type I and type II IFN induction. These observations give new insights into the regulation of IFN mediated ISGylation.

Psoriasis ist eine komplexe entzündliche Erkrankung der Haut. Charakteristisch ist eine dichte Infiltration von T-Lymphozyten und eine Hyperproliferation der Epithelschicht. Heutzutage belegen die Ergebnisse vieler experimteller Studien, daß Psoriasis eine T-Zell-induzierte Erkrankung ist. Die Spezifität der T-Zell-stimulierenden Antigene ist noch unbekannt. Voraussetzung für die Charakterisierung psoriatischer T-Zellen ist die Isolation von T-Zell-Klonen aus psoriatischen Plaques, deren Restimulation in vitro qualitativ und quantitativ erfaßbar ist. Hierfür haben wir einen hochsensiblen gamma-Interferon Elispot-Assay etabiert, der die Aktivität der T-Zellen aus Hautplaques erfaßt. Zudem zeigen wir, daß man aktivierte T-Zell-Klone mittels CD25-Markierung isolieren und anschließend klonieren kann. Unsere Ergebnisse können als eine Grundlage für weitere Versuche dienen, die die Spezifität von T-Zell-Klonen aus psoriatischen Plaques charakterisieren sollen. / Psoriasis is a complex inflammatory disease of the skin characterized by a dense infiltration of T-lymphocytes and a hyperproliferation of the epithelial layer. A host of experimental and clinical data suggest that psoriasis is a T cell mediated disorder. The nature of T-cell-stimulating antigens is still unknown. One way to identify putative antigen(s) is the definition of T-cell-receptor specifities using randomized combinatorial peptide libraries. This requires the isolation and expansion of T cell clones from psoriatic plaques in vitro. Therefore we established a gamma-interferon Elispot-assay which allows quantification of the frequency of activated plaque-derived T cells in vitro. In addition, we show that activated T cell clones can be sorted via CD25 and cloned. The expanded clones can also be restimulated by autologous cells. Our results should be useful in the design of experiments aiming at a systematic analysis of the specifity of T cell clones present in psoriatic plaques.

Mit durchflusszytometrischen und molekularbiologischen Verfahren wurden verschiedene Subpopulationen peripherer T-Lymphozyten bei gesunden Spendern und Nierentransplantatempfängern phänotypisch und funktionell untersucht. Wir fanden, dass eine T-Zell-Population, welche unter anderem die Oberflächenmarker LFA-1 und CD57 exprimierte, nicht aber CD28, terminale Effektorzellen enthielt. Wegen der bekannten Assoziation der Expansion CD57-positiver CD8-positiver T-Zellen mit einer Infektion mit dem Zytomegalievirus (CMV), wollten wir untersuchen, ob auch CMV-spezifische Effektorzellen darin enthalten waren. Um diesen möglichen Zusammenhang zwischen Phänotyp und Spezifität zu untersuchen, wurde ein bekanntes durchflusszytometrisches Verfahren zur Erfassung antigen-spezifischer CD4-T-Zellen so modifiziert, dass damit auch antigen-spezifische CD8-T-Zellen erfasst werden konnten. Dazu wurden frisch isolierte periphere mononukleäre Zellen in vitro mit löslichen Peptiden oder Peptidgemischen inkubiert (anstelle von Erregerlysaten oder Proteinantigenen wie im bekannten Verfahren), so dass durch direkte externe Beladung von MHC-I- und MHC-II- Molekülen nicht nur CD4- sondern auch CD8-T-Zellen stimuliert wurden. Anschließend konnten CD4 - und/oder CD8-positive T-Zellen nachgewiesen werden, in welchen es zur Synthese von Interferon-gamma gekommen war (intrazelluläre Färbung), wodurch diese Zellen als antigen-spezifisch identifiziert wurden. Diese Zellen konnten dann weiter phänotypisch analysiert werden. Unter Verwendung bekannter CD8-T-Zellen-stimulierender CMV-Peptide konnte der Phänotyp CMV-spezifischer CD8-T-Zellen untersucht werden, wobei sich zeigte, dass das CD57-positive Subset tatsächlich den gößeren Teil der CMV-spezifischen CD8 T-Zellen enthielt. Andererseits konnte das neue Verfahren verwendet werden, um weitere Peptide zu identifizieren, welche eine T-Zellstimulation bewirkten (Epitopkartierung). In zwei von uns untersuchten Proteinen des CMV (pp65 und IE-1) wurden so mehrere neue CD4- und CD8-T-Zellepitope beschrieben. Die Vewendung komplexer Peptidgemische erlaubte darüber hinaus die Untersuchung der T-Zellantwort gegen ganze Proteine (repräsentiert durch die Gesamtheit aller denkbaren Epitope), was insbesondere für die Untersuchung von CD8-T-Zellen eine große Bereicherung darstellte und vom MHC-Typ unabhängig war. Wir verwendeten dieses neue Verfahren bisher zur Analyse und zum Monitoring der T-Zellantwort gegen bestimmte Erregerproteine oder -peptide (z.B. aus CMV oder HIV). Es eignet sich darüber hinaus auch zur Untersuchung der Immunantwort gegen Impfstoffe, welche zur Induktion von T-Zellen führen sollen. / Using flow-cytometric and molecular-biology methods subpopulations of peripheral blood T-lymphocytes were examined in healthy donors and renal transplant recipients with respect to phenotype and function. We found that a T-cell population that expressed the surface markers LFA-1 and CD57 (among others), but not CD28, contained terminal effector cells. Because of the known association of an expansion of CD57-positive CD8-positive T-cells with an infection with Cytomegalovirus (CMV), we wanted to examine if CMV-specific effector cells were also contained in this subset. In order to investigate this association between phenotype and specificity, we modified a known flow-cytometric method for the detection of antigen-specific CD4 T-cells in such a way that antigen-specific CD8 T-cells could also be detected. For this purpose freshly isolated mononuclear cells were incubated in vitro with soluble peptides or peptide mixes (instead of pathogen lysates or protein antigens as used in the original method) so that following direct external loading of MHC-I and MHC-II molecules not only CD4 T-cells but also CD8-T-cells were stimulated. Subsequently, CD4 and/or CD8 T-cells that had synthesized Interferon-gamma (intracellular staining), which identified them as being antigen-specific, could be detected. These cells could then be analyzed with regard to phenotype. Using known CD8-T-cell stimulating CMV-peptides, the phenotype of CMV-specific CD8 T-cells could be analyzed. Thus it was demonstrated that the majority of CMV-specific CD8 T-cells was indeed contained in the CD57-positive subset. On the other hand, this new approach allowed the identification of additional peptides that stimulated T-cells (epitope mapping). In two CMV-proteins that we examined (pp65 and IE-1) several new CD4 and CD8-T-cell epitopes were described. The use of complex peptide mixes in this approach allowed the analysis of T-cell responses to complete proteins (represented by the entirety of all possible epitopes), which was a great benefit to the analysis of CD8 T-cells and independent of MHC-type. Until now, we have used this new method for the analysis and the monitoring of the T-cell response to specific pathogen proteins or peptides (e.g. from CMV or HIV). It is suitable, moreover, for the analysis of the immune response to vaccinations that aim at the induction of T-cells.