published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Philosophy

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and subsequent destruction of synovial joints. Although its causes remain largely unknown, a substantial genetic contribution is known to exist. During the last decades the benefits of early aggressive treatment have become evident, and more potent therapeutic options have become available. These advances have increased the demands for rapid accurate diagnosis and prognostic markers of disease course and therapy response. The ‘rheumatoid factor’ (RF) has long been used as a diagnostic and prognostic marker of RA. In this thesis, the utility of measuring antibodies to cyclic citrullinated peptides (CCP) was investigated. In a population-based arthritis incidence study, 69 very early arthritis patients (symptom duration < 3 months) were identified. The anti-CCP test, performed at baseline and related to diagnosis at the 2-year follow-up, had a diagnostic specificity for RA of 96% and a sensitivity of 44%, both of which were superior to RF. In a prospective cohort of 242 incident cases of RA (symptom duration < 1 year), 64% of the patients tested positive for anti-CCP at baseline (equal to RF). Despite receiving more active anti-rheumatic therapy, the anti-CCP-positive patients had a more aggressive disease course during 3 years as compared to those testing negative. The 158VV genotype of Fcγ Receptor type IIIA (FcγRIIIA), which binds IgG with higher affinity than 158FF, was associated with an increased susceptibility to RA in men, but not in women. Previous studies report conflicting results, and none stratified according to gender. The 158V/F polymorphism of FcγRIIIA was not found to influence outcome of anti-tumour necrosis factor therapy in 282 RA patients, contradicting hints from previous studies. Genetic variation in proteins of the inflammasome, an interleukin-1 (IL-1) regulating intracellular protein complex, is associated with rare autoinflammatory conditions and possibly with Crohn’s disease. In this first study on genetic variation of the inflammasome in RA, we describe a compound polymorphism of the genes CIAS1 and TUCAN that associates both with susceptibility to RA and to the severity of the disease. Hypothetically, these genes may identify a subgroup of RA patients that would benefit from anti-IL-1 therapy. This thesis work emphasizes the benefits of testing for anti-CCP in the diagnosis and outcome prediction in early arthritis. FcγRIIIA genotype is likely to affect RA susceptibility and further work should apply a gender perspective. Inflammasome genetics may influence the risk of developing RA. Additional studies are warranted to settle whether it also identifies a subgroup of RA patients benefiting from IL-1 targeted therapy.

Cancer is a disease that is caused by mutations in somatic cells. Metastasis is the major cause of death from cancer and often complicates treatment. Malignant tumours secrete degradative enzymes such as cathepsin L which degrade the extracellular matrix to facilitate tumour invasion and metastasis. The immune system does not normally recognize and eradicate tumours because they arise from self tissues to which the immune system is tolerant. Self antigens are poorly immunogenic because they lack T cell help. In this study, a foreign glucosidase was conjugated to self rabbit cathepsin L using glutaraldehyde to specifically provide T helper cell epitopes. The conjugate was used to immunise two male rabbits. A second pair of rabbits (male and female), was primed with sheep cathepsin L (to induce T helper cell activation) and received rabbit cathepsin L boosters. A third pair of rabbits which served as a control was immunised with sheep cathepsin L. The two pairs of test rabbits made high avidity antibodies against rabbit cathepsin L, showing a similar response to control rabbits when antibodies were tested in an ELISA. Western blot analysis showed that these anti-cathepsin L autoantibodies were specific for rabbit cathepsin L. Rabbits which were immunised with the conjugate were · inoculated with sheep cathepsin L nine weeks after the final inoculation with the conjugate. Analysis of antibodies in an ELISA showed that antibody responses against rabbit cathepsin L were augmented in a manner that is characteristic of memory responses. Low titre antibodies against sheep cathepsin L were also produced. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Rheumatoid arthritis (RA) is a chronic inflammatory disease with an unknown aetiology characterized by joint destruction. Both genetic and environmental factors contribute to the disease development with HLA-DRB1* alleles and smoking identified as most important. The disease is characterized by the presence of autoantibodies, originally by rheumatoid factor (RF) and more recently by anti citrullinated protein/peptide antibodies (ACPA) and antibodies against carbamylated peptides (CarP). These autoantibodies are present, not only after the onset of disease, but also prior to the onset of symptoms. The development of RA is a gradual process lasting several years before the onset of any joint symptom, but when and if there is a temporal difference in the development both between and within the different antibody systems is currently unknown. B-cells produce the antibodies, and a subset of B-cells, i.e., B-regulatory (Breg) cells, produces interleukin-10, and thus have the ability to down-regulate pro-inflammatory cytokines. Whether the Breg cells are involved in the pathogenesis of RA is, as yet, unknown. The aim of this thesis was to increase knowledge of the pathophysiological processes in the development of RA through identification of factors involved. The analyses involved detection of autoantibodies to post-translationally modified peptides/proteins in addition to RF isotypes, cell surface markers on immune cells in asymptomatic individuals, who have an increased risk of developing RA. In a co-analysis of the registers of patients with RA attending the Department of Rheumatology, with the registers from population based screening programmes within the Biobank of Northern Sweden, blood samples collected from individuals prior to the onset of symptoms were identified, as were those from population control subjects. A cohort of pre-symptomatic individuals also donated samples at the time of receiving a diagnosis of RA. First-degree relatives (FDR) of patients with RA were also identified and included for analyses. The levels of ten different ACPAs, i.e., (fibrinogen (Fib) α563-583(573), Fibα580-600(591), Fibβ62-81a(72), Fibβ62-81b(74), Fibβ36-52, a-enolase (CEP-1), triple helical collagen type II (citC1III), filaggrin (Fil307-324), vimentin (Vim) 2-17, and Vim60-75) were measured using the ImmunoCAP ISAC system (Phadia/ThermoFischer, Uppsala, Sweden) in blood samples from individuals before the onset of symptoms and when diagnosed with RA in comparison with those in population based controls. In a subset of samples, the levels of anti-CarP antibodies were measured using ELISA coated with anti-CarP-FCS, as well as analysis of RF of IgM, IgG and IgA isotype using the EliA assay (Phadia, Uppsala, Sweden). Breg cells were analysed both with and without stimulation ex vivo along with other cell types using flow cytometry in samples from patients with RA, their first degree relatives (FDR) and healthy controls. In paper I it was shown that levels of ACPA were initially restricted to a few antibodies but disseminated over time to involve additional different antibodies. The levels of antibodies to CEP-1, Fibß36-52, and filaggrin were significantly increased. In paper II, anti-CarP antibodies were positive in 5-13% of the individuals negative for the various ACPA studied. The presence of anti-CarP antibodies was significantly related to radiological destruction of joints at baseline, at follow-up after 24 months and to the radiological progress between baseline and 24months. In paper III, the relationships between the frequencies of RF isotypes, the ten different ACPA, anti-CCP2 and anti-CarP antibodies before the onset of any symptoms and the presence of certain combinations of antibodies were associated with a very high risk of developing RA. In paper IV Breg cells from patients with RA are functionally impaired and FDR showed a similar pattern by responding less to stimulation ex vivo than cells from healthy controls. In conclusion, individuals who subsequently develop RA have an increased number and amount of ACPAs, anti-CarP antibodies and RF of IgM, IgG and IgA isotype, several years before symptom onset. Most of the different antibodies analysed remain associated with disease development after adjustments for each separate antibody. In FDRs, Breg cells were functionally altered in that they produce less IL-10 and consequently contribute to a more inflammation-prone status, as in their relatives with RA. These findings contribute to information about the development of RA as well as a given individual’s risk(s) of developing RA and its progression.

ĮVADAS: Po kraujo perpylimo į organizmą patenka eritrocitų membranoje esantys antigenai, kurie gali sukelti specifinių aloantikūnų gamybą. Pacientams, kuriems taikoma ilgalaikė hemotransfuzinė terapija, antikūnai prieš kliniškai svarbius kraujo grupių antigenus atsiranda 1- 38 % atvejų. Po kurio laiko aloantikūnai prieš eritrocitų antigenus recipientų kraujuje jau nebeaptinkami, todėl po pakartotinio kontakto su tuo pačiu antigenu, po hemotransfuzijos, aloantikūnai sukels hemolizinę potransfuzinę reakciją. TYRIMO MEDŽIAGA IR METODAI: Buvo ištirtas 674 recipientų kraujas, sergančių hematologinėmis ir onkohematologinėmis ligomis. Antikūnų paieška ir identifikavimas buvo atliekami naudojant Kumbso reakcijas (tiesioginę ir netiesioginę). REZULTATAI: Aloantikūnų atsiradimas po hemotransfuzijų ir antikūnų pasiskirstymas buvo analizuojami 2 metų laikotarpyje (2004- 2006). • Aloantikūnai nustatyti 36,36 %, alo- ir autoantikūnai– 52,27 %, autoantikūnai– 11,36 % tiriamųjų. Aloantikūnų prieš kraujo grupių sistemų antigenus paplitimas: Rh (58,75 %), Kell (11,25 %), MNS (7,5 %), Duffy (6,25 %), Lutheran (6,25 %), Lewis (5,00 %), Kidd (5,00 %) • Aloantikūnai po alogeninių eritrocitų masės transfuzijų atsirado 14,77 % pacientų prieš Rh (68,42 %), Lutheran (15,79 %), Kell (10,53 %), Lewis (5,26 %) ir Kidd (5.26 %) kraujo grupių sistemų antigenus. Aloantikūnų išsilaikymas recipientų kraujyje buvo analizuojamas 5 metų laikotarpyje (2000- 2006). • Po 1 mėnesio buvo nebeaptinkami 30,16 % visų... [toliau žr. visą tekstą] / BACKGROUND: Individuals exposed to red blood cell alloantigens through transfusion may produce antibodies. Clinically significant red blood cells alloantibodies develop in 1- 38 % of patient, who receive multiple transfusion. Red cell antibodies, which can become undetectable over time, can cause delayed hemolytic transfusion reactions after incompatible blood transfusions. STUDY DESIGN, METHODS: Blood samples of 674 patients with hematologic and hematooncologic diseases, who undergo transfusion were studied. Antibodies screening and identification were performed by direct and indirect Coombs test. RESULTS: We examined the alloimmmunization risk and antibodies incidence over the period of 2 years (2004-2006). • Alloantibodies were found in 36,36 %, allo- and autoantibodies in– 52,27 %, autoantibodies in– 11,36 % patients. Alloantibodies against blood group system antigens incidence: Rh (58,75 %), Kell (11,25 %), MNS (7,5 %), Duffy (6,25 %), Lutheran (6,25 %), Lewis (5,00 %), Kidd (5,00 %) • We found that overall immunization rate in patients was 14, 77 % against Rh (68,42 %), Lutheran (15,79 %), Kell (10,53 %), Lewis (5,26 %) ir Kidd (5.26 %) blood group systems antigens. We examine the persistence of clinically significant red blood cell alloantibodies over a period of 5 years (2000-2006). • After 1 months 30,16 % antibodies become undetectable, after 3 months- 60,32 %, after 5 years– 88,89 %. Red blood cell alloantibodies persistence (in months): anti-E (9,0± 9,61), anti-C... [to full text]

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.