INTRODUÇÃO: A síndrome de Sjögren primária (SSp) é uma doença autoimune inflamatória crônica que afeta principalmente as glândulas exócrinas, levando aos sintomas de síndrome sicca. O olho seco é uma das características mais importantes dessa síndrome e um estudo recente relatou redução da atividade da DNase I em lágrimas de pacientes com olho seco de várias etiologias. Portanto, postulamos que pacientes com SSp possam ter anticorpos direcionados à DNase I. MÉTODOS: Avaliamos em um estudo de corte transversal 85 pacientes com SSp (conforme os critérios de classificação do American European Consensus Group Criteria, 2002), 50 pacientes com artrite reumatoide (AR) (American College of Rheumatology Criteria/ 1987) sem sintomas de síndrome sicca e 88 voluntários saudáveis. A reatividade IgG anti-DNase I foi detectada por ELISA utilizando a enzima de pâncreas bovino como antígeno e confirmada por Imunoblotting. RESULTADOS: A idade e sexo foram comparáveis nos três grupos (p > 0,05). A anti-DNase I foi detectada em 43,5% dos pacientes com SSp, conforme determinado por ELISA. Em contraste, essa reatividade estava ausente em todos os pacientes com AR (p= 0,0001). Comparações adicionais dos pacientes com SSp com (n= 37) e sem (n= 48) anti-DNase I revelaram que o primeiro grupo tinha níveis séricos de IgG mais altos (2293,2 ± 666,2 vs. 1483,9 ± 384,6 mg/dL, p= 0,0001) e uma frequência maior de leucopenia não induzida por drogas (43% vs. 19%, p= 0,02). A análise de regressão logística multivariada mostrou que apenas os níveis de IgG foram independentemente associados com o anti-DNase I. CONCLUSÃO: Descrevemos uma alta frequência de anticorpos anti-DNase I em pacientes com SSp associados a níveis séricos de IgG mais elevados. A falta dessa reatividade em pacientes com AR sem sintomas de sicca sugere que esse anticorpo pode ser útil no diagnóstico diferencial dessas doenças / INTRODUCTION: Primary Sjögren\'s syndrome (pSS) is a chronic inflammatory autoimmune disease that mainly affects exocrine glands. Dry eye is one of the most important features of this syndrome and a recent study reported reduced DNase I activity in tears of patients with dry eye of various etiologies. We therefore postulated that patients with pSS may have antibodies targeting DNase I. METHODS: We have evaluated in a cross-sectional study 85 pSS patients (American European Consensus Group Criteria/ 2002), 50 rheumatoid arthritis (RA) patients (American College of Rheumatology Criteria/ 1987) without sicca symptoms and 88 healthy volunteers. The IgG anti-DNase I reactivity was detected by ELISA using bovine pancreas enzyme as antigen and confirmed by Immunoblotting. RESULTS: Age/ gender were comparable in the three groups (p > 0.05). Anti-DNase I was detected in 43.5% of the pSS patients as determined by ELISA. In contrast, this reactivity was absent in all RA patients (p= 0.0001). Further comparison of pSS patients with (n= 37) and without (n= 48) anti-DNase I revealed that the former group had higher IgG serum levels (2293.2 ± 666.2 vs. 1483.9 ± 384.6 mg/dL, p= 0.0001) and a higher frequency of non-drug induced leukopenia (43% vs. 19%, p= 0.02). A multivariate logistic regression analysis identified that only IgG levels were independently associated with anti-DNase I. CONCLUSION: We describe a high frequency of anti-DNase I antibodies in pSS patients associated with higher serum IgG levels. The lack of this reactivity in RA patients without sicca symptoms suggests that this antibody may be helpful in the differential diagnosis of these diseases

Autoimmune diseases occur when the immune system attacks and destroys healthy body tissue. Autoimmunity is known to cause a wide range of disorders, and is suspected to be responsible for many more. Most autoimmune disorders are chronic and cause severe morbidity for the patients, and are also costly for society. A majority of these disorders are today considered as complex diseases with incompletely known etiology. Hence, model systems for studying the pathogenesis of autoimmunity are important to unravel its causes. Autoimmune Polyendocrine Syndrome Type 1 (APS-1), (OMIM 240300), is a rare autoimmune disorder. Patients with APS-1 progressively develop multiple organ-specific autoimmune lesions involving both endocrine and non endocrine tissues. Typical autoimmune disease components in APS-1 are hypoparathyroidism, Addison’s disease, vitiligo, alopecia and type 1 diabetes. The gene preventing APS-1 has been identified and designated Autoimmune Regulator (AIRE). It has been shown that mutations of AIRE cause loss of tolerance to self-structures, resulting in organ-specific autoimmunity. Although APS-1 is a rare syndrome occurring mainly in genetically isolated populations, the disease components of APS-1 are, in isolated forms, not unusual in the general population and affect many patients. Hence, APS-1 is an attractive model disease for studies of molecular mechanisms underlying organ-specific autoimmunity. This thesis concerns investigations in which two novel autoantigens are identified in APS-1 and used in serological diagnosis of the disease. NALP5, is identified as a parathyroid autoantigen - an important finding since autoimmune hypoparathyroidism is one of the cardinal symptoms of APS-1. Additionally, KCNRG is identified as a bronchial autoantigen in APS-1 patients with respiratory symptoms. Finally, studies that compare the immune response in APS-1 patients and the mouse model for APS-1 are presented.

Type 1 diabetes (T1D) is a chronic disease caused by destruction of the insulin producing beta-cells in the pancreas. The incidence of T1D has increased rapidly, especially in the Western world and among young children. The pathogenesis of T1D is not fully understood, but the beta-cells are believed to be destroyed by an autoimmune process initiated years before the onset of T1D. During this pre-clinical period, autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA) and the tyrosine phosphatase-like protein IA-2 (IA-2A) can be detected and are used to identify individuals at risk of T1D. The major genetic determinant for T1D is the HLA class II genes, but also polymorphism in the insulin gene and CTLA-4 gene are associated with T1D. The risk genes cannot explain the rapid increase in incidence of T1D, therefore a role for different environmental factors has been suggested. The aim was to study the prevalence of beta-cell autoantibodies in children from the general population in relation to known genetic and environmental risk factors, and in young patients with T1D in high and low incidence areas. Short duration of breast-feeding was associated with an increased risk of developing beta-cell autoantibodies in children from the general population at 5-6 years of age. We found an association between positivity for GADA and/or IAA at the age of 5-6 years and a short duration of total breastfeeding, and also between positivity for GADA, IA-2A and/or IAA and a short duration of exclusive breast-feeding. Our findings suggest that breast-feeding has a long term protective effect on the risk of beta-cell autoimmunity in children from the general population. The T1D related risk genes were not associated with beta-cell autoantibodies other than GADA in children from the general population at 5-6 years of age. Children with the DR4-DQ8 haplotype were more often positive for GADA than children without this haplotype. We found no association of GADA with DR3-DQ2 haplotype or between these two haplotypes and any of the other autoantibodies. Our results suggest that beta-cell autoimmunity in children from the general population is not strongly associated with any risk genes of T1D other than DR4-DQ8. In the non-diabetic children with allergic heredity GADA was detectable in almost all children, IA-2A in about half and IAA in 10% of the children. The levels low of these autoantibodies fluctuated with age and different patterns of fluctuations were seen for GADA and IA-2A, which may reflect differences in the immune response to the autoantigens. In patients with newly diagnosed T1D, we found some differences between patients from a high incidence country (Sweden) and a country with a lower incidence (Lithuania). Among the Swedish patients, the prevalence of IAA and GADA or multiple autoantibodies was higher than in Lithuanian patients. The risk genes DR4-DQ8 and the heterozygous high risk combination DR4-DQ8/DR3-DQ2 was more common among the Swedish patients than Lithuanian patients. Patients with low levels of IAA had higher levels of HbA1c and ketones, indicating that patients without IAA or with low levels of IAA have a more severe onset of T1D. Our findings indicate that beta-cell autoimmunity is more pronounced in a high incidence area compared to an area with a lower incidence. In conclusion, short duration of breast-feeding is a risk factor for beta-cell autoantibodies in children from the general population, and the beta-cell autoantibodies in these children are not associated with specific risk genes. Children with newly diagnosed T1D in a high incidence area carry risk genes and have autoantibodies more often than newly diagnosed children from an area with a lower incidence, perhaps indicating different disease phenotypes.

Das La-Protein wurde als Autoantigen bei Autoimmunpatienten, die an SLE oder Sjögren-Syndrom erkrankt sind, entdeckt. Es kommt in phosphorylierter Form im Zellkern aller Eukaryonten vor und nimmt Aufgaben bei der Faltung, Prozessierung und nukleären Retention von RNA-Polymerase III-Transkripten wahr. Unter normalen zellulären Bedingungen ist das La-Protein außerdem in der Lage, zwischen Zellkern und Zytoplasma zu pendeln. Bei Zellstress, der nach UV-Exposition oder während einer viralen Infektion entsteht, wird das Protein verstärkt im Zytoplasma beobachtet, wo es an der Cap-unabhängigen Translation zellulärer und ggf. viraler Proteine beteiligt ist. Wird in der Zelle daraufhin Apoptose induziert, so ist das La-Protein auf der Zellmembran bzw. in apoptotischen Körperchen nachweisbar. Ein wesentlicher Bestandteil dieser Arbeit war die Untersuchung verschiedener monoklonaler anti-La-Antikörper. Einige wenige konnten durch wiederholte Immunisierung von Mäusen mit rhLa-Protein generiert werden. Im Gegensatz dazu resultierte die einmalige Übertragung von gegen das hLa-Protein aktivierten CD4+ T-Zellen auf eine hLaTg-Maus in der Gewinnung mehrerer La-spezifischer Antikörper. Die Sequenzanalyse der Gene, die für die variablen Antikörperdomänen codieren, bestätigte, dass es sich um individuell rekombinierte und hypermutierte Immunglobuline handelt. Die Antikörper zeichneten sich außerdem durch unterschiedliche Eigenschaften bei der Bindung von humanem und murinem La-Protein in der Immunfluoreszenz, im Immunoblot oder während der Immunpräzipitation aus. Für die IgG-Antikörper konnten die Epitopbereiche innerhalb des La-Proteins eingegrenzt werden. Auffällig waren die kurzen linearen Peptidepitope, die von den auf konventionelle Art erzeugten Antikörpern gebunden wurden. Hingegen erkannten alle Antikörper, die aus dem adoptiven T-Zell-Transfer hervorgegangen waren, Konformationsepitope. Darüber hinaus wurde gezeigt, dass einige mAks aber auch anti-La-Patientenseren die reduzierte von der oxidierten Form des La-Proteins unterscheiden können. Unerwartet ist die Erkenntnis, dass sich offensichtlich zahlreiche B-Zellen mit anti-La-Spezifität von wenigen variablen Ketten ableiten und dass diese bei einer herkömmlichen Immunisierung entweder nicht aktiviert werden (und deshalb nicht in der Milz zu finden sind) oder sogar eliminiert werden. Der Import des La-Proteins in den Zellkern wird durch die klassischen Transportmoleküle Karyopherin-α und Karyopherin–β vermittelt. Für den Shuttlingprozess muss das Protein auch wieder aus dem Kern exportiert werden. Da es kontroverse Daten bezüglich eines Crm1-abhängigen Kernexports gab, wurde das Shuttlingverhalten von GFP-La-Fusionsproteinen in dieser Arbeit genauer analysiert. Mit Hilfe von Heterokaryonexperimenten konnte bestätigt werden, dass sowohl das hLa- als auch das mLa-Protein zwischen humanen und murinen Zellkernen pendeln kann und dass der Export unabhängig von Crm1 stattfindet. Aufgrund der kurzen Verweildauer im Zytoplasma schienen die Proteine quantitativ im Zellkern vorzuliegen, doch ein Teil konnte stets in den im Heterokaryon enthaltenen Nachbarzellkernen detektiert werden. Die Verwendung von N-terminal deletierten La-Fragmenten, die alle über das C-terminale NLS verfügten, gab Aufschluss über die Regulation des Shuttlings. Es zeigte sich, dass die Menge des exportierten Proteins von einem nukleären Retentionspartner festgelegt wird, der das La-Protein bindet und dadurch im Zellkern festhält. Wird diese Assoziation aufgehoben, gelangt das La-Protein in das Zytoplasma. Dort ist es allerdings nicht detektierbar, da das NLS einen umgehenden Import zurück in den Zellkern hervorruft. Zusätzlich wurde die Auswirkung von zellulärem Stress (z. B. durch ROS) auf die intrazelluläre Lokalisation des Proteins untersucht. Unter oxidativen zellulären Bedingungen wird einerseits die Wechselwirkung mit dem nukleären Retentionspartner aufgehoben und andererseits findet kein Kernimport über Karyopherin-α mehr statt. Aus diesem Grund reichert sich das La-Protein nun verstärkt im Zytoplasma an. Darüber hinaus wurde nachgewiesen, dass das La-Protein von apoptotischen Zellen freigesetzt wird und daraufhin auf die Membran von Nachbarzellen binden kann. Die Bindungs-eigenschaften wurden mit rhLa-Protein genauer untersucht. Das La-Protein war auf Endothel- und Epithelzellen nachweisbar und die Bindung fand sowohl bei Inkubation auf Eis als auch bei 37 °C statt. Da das La-Protein auch über DNA-Bindungseigenschaften verfügt, war es in der Lage, DNA auf der Zelloberfläche zu immobilisieren. Innerhalb von PBMCs wurde es selektiv auf Antigen-präsentierenden Zellen nachgewiesen. Diese Eigenschaften lassen eine Beteiligung des Proteins bei der Induktion von anti-dsDNA-Antikörpern in Autoimmunpatienten vermuten. Es ist bekannt, dass die Bedingungen (Virusinfektion, UV-Exposition), die zur Translokation des La-Proteins auf die Zelloberfläche führen, bei SLE-Patienten Krankheitsschübe auslösen können. Bisher wurden anti-La-Autoantikörper aber eher nicht als pathophysiologisch erachtet, da sie bei der Bindung an bereits apoptotische Zellen keine weiteren Schäden verursachen können. Jedoch wurde in dieser Arbeit gezeigt, dass das La-Protein apoptotischer Zellen auf der Oberfläche von lebenden Zellen in der Umgebung nachgewiesen werden kann. Daran könnten anti-La-Autoantikörper binden und eine Komplement- oder NK-Zell-vermittelte Zerstörung der Nachbarzellen hervorrufen. Dadurch entstehen zusätzliche Gewebeschäden. Im Chromfreisetzungstest waren NK-Zellen tatsächlich in der Lage, La-dekorierte Zielzellen Antikörper-abhängig zu lysieren, sofern zusätzliche in vitro Stimuli präsent waren, die z. B. eine virale Infektion simulierten. Die Immuntherapie von Tumoren ist auf bestimmte Zielstrukturen auf den Tumorzellen angewiesen, über welche die Wirkstoffe spezifisch zu den maligne transformierten Zellen gebracht werden. Die Therapeutika, die sich oft von mAks gegen diese Zielstrukturen ableiten, müssen für verschiedene Tumorarten individuell entwickelt werden. Da das La-Protein von apoptotischen Zellen freigesetzt wird und auf die Membran benachbarter (bestrahlungsresistenter) Zellen binden kann, ist es in Kombination mit einer vorangegangenen Bestrahlung als universelle Zielstruktur für die Immuntherapie nutzbar. Aus diesem Grund wurde unter Verwendung eines ausführlich in dieser Arbeit charakterisierten anti-La-Antikörpers ein rekombinantes bispezifisches Antikörperderivat entwickelt. Es ist in der Lage, das La-Protein auf der Oberfläche von Tumorzellen zu binden und auf diesen zytotoxische T-Lymphozyten zu immobilisieren. Durch die Quervernetzung werden die T-Lymphozyten aktiviert und induzieren in den Zielzellen Apoptose. Das neue Antikörperderivat verspricht eine vielseitige Anwendung in Kombination mit Strahlentherapie oder auch mit rekombinanten Antikörpermolekülen, die gegen spezifische Zielstrukturen auf den Tumorzellen gerichtet sind.

The studies presented in this thesis focus on human and canine models for autoimmune disease, with the main aim to gain new knowledge about disease mechanisms and to further evaluate the dog as a model for autoimmune disease. Autoimmune Polyendocrine Syndrome type 1 (APS-1) is a hereditary human multiorgan disease caused by mutations in the autoimmune regulator (AIRE) gene. Hallmarks of APS-1 are chronic mucocutaneous candidiasis caused by Candida albicans, together with the autoimmune endocrine disorders hypoparathyroidism and adrenocortical failure. Many human diseases have an equivalent disease in dogs. Because humans share environment, and in part life style with the dogs they provide an interesting model for further genetic studies. Immune responses to Candida albicans in APS-1 patients displayed an increased secretion of the proinflammatory cytokine IL-17A and similar results were also found in AIRE deficient mice. Anticytokine autoantibodies to IL-17A, IL-17F and IL-22 were detected in APS-1 patients, and a radioligand binding assay for measuring these autoantibodies was developed and evaluated. In the canine studies we investigated whether canine diabetes mellitus could serve as a model for human autoimmune diabetes mellitus. Furthermore, we investigated type I IFN responses in Nova Scotia duck tolling retriever dogs with a systemic autoimmune disease resembling human SLE. Four assays were used in search for signs of humoral autoimmunity in diabetic dogs. However, no evidence for a type 1 diabetes-like phenotype in dogs was found. Sera from Nova Scotia duck tolling retrievers suffering from steroid-responsive meningitis arteritis elicited an increased expression of IFN-inducible genes in the canine MDCK cell line. This suggests that these dogs have an IFN signature, as seen in human SLE.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Thesis (Ph.D. in Biostatistics) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 53-56). Online version available via ProQuest Digital Dissertations.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in platelets from patients with increasing cholesterol levels. A possible association between cholesterol levels and inflammatory markers, such as oxidized low density lipoprotein (oxLDL), highly sensitive C-reactive protein (hsCRP) and oxLDL autoantibodies was also investigated. The following groups were studied: group I (< 150 mg/dl), group II (151 to 200 mg/dl); group III: (201 to 250 mg/dl); group IV (> 251 mg/dl) of cholesterol. The results demonstrated that both ATP and ADP hydrolysis were enhanced as a function of cholesterol levels. The LDL levels increased concomitantly with total cholesterol levels. The triglyceride levels were increased in the group with total cholesterol above 251 mg/dl. oxLDL levels were elevated in groups II, III and IV. hsCRP was elevated in the group with cholesterol higher than 251 mg/dl. oxLDL autoantibodies were elevated in groups III and IV. TBARS content was enhanced as a function of cholesterol levels. In summary, hypercholesterolemia is associated with an enhanced of inflammatory response and ATP and ADP hydrolysis. The increase in NTPDase activity is possibly related to a compensatory response to the inflammatory and pro-oxidative state associated with hypercholesterolemia. / A atividade da NTPDase (EC 3.6.1.5, apyrase, CD39) foi verificada em plaquetas de pacientes com diferentes níveis de colesterol. Uma possível associação entre os níveis de colesterol e os marcadores inflamatórios como LDL oxidado (oxLDL), proteína C reativa ultrasensível (hsCRP) e anticopos anti-LDL oxidado (Anti-oxLDL) foi investigado. Os seguintes grupos foram estudados: grupo I (< 150 mg/dl), grupo II (151 a 200 mg/dl); grupo III: (201 a 250 mg/dl); grupo IV (> 251 mg/dl) de colesterol. Os resultados demonstraram que a hidrólise dos nucleotídeos (ATP e ADP) aumentou em função dos níveis de colesterol. Os níveis de LDL aumentaram concomitantemente com os níveis de colesterol total. Os níveis de triglicerídeos foram elevados no grupo com colesterol total acima de 251 mg/dl. Os níveis de oxLDL foram elevados nos grupos II, III and IV. A hsCRP foi elevada no grupo com cholesterol maior que 251 mg/dl. Os Anti-oxLDL foram elevados nos grupos III e IV. O conteúdo de TBARS foi aumentando em função dos níveis de colesterol. Em resumo, a hipercolesterolemia está associada com o aumento da resposta inflamatória e hidrólise de ATP e ADP. O aumento da atividade da NTPDase está possivelmente relacionado com uma resposta compensatória ao estado inflamatório e pró-oxidativo associado com a hipercolesterolemia.

Background: In recent years mechanical circulatory assist devices became an established option in bridging patients with refractory heart failure to heart transplantation. One of the alleged limitations of mechanical devices is a high degree of antibody production with possible deleterious effect on subsequent heart transplantation outcome. Aim: The main goal of this study is to assess the role of antibodies on the outcome of surgical treatment of patients with end- stage heart failure. Method: Firstly, we present a literature review on the current state of knowledge of possible immunologic mechanisms involved in antibody production in left ventricular assist device (LVAD) recipients, new methods of antibody detection, desensitization strategies and overview of published evidence assessing the impact of sensitization on post-transplantation outcome. In the experimental part of our study we prospectively evaluated the presence of anti-Angiotensin II Type 1 Receptor (AT1R) antibodies in 83 Heart Mate II (HMII) recipients who were implanted at our institution between 2008 and 2012 and survived the first 60 days. On-device survival and device malfunction, major infection, major bleeding and neurologic dysfunction were compared between antibody positive and antibody negative recipients. Out of a total...