Thesis (Ph. D.)--University of St Andrews, February 2009. / Restricted until 23rd February 2012.

The MHC class I allele, HLA-B27, is strongly associated with a group of inflammatory arthritic conditions collectively known as spondyloarthropathies (SpA). Ankylosing spondylitis (AS) shows the strongest association with 90-95 % of patients being HLA-B27 positive. The relationship between HLA-B27 and SpA has been known for over 30 years, however despite ongoing research, the reason for this association has not yet been elucidated. In more recent years, research has focused on intrinsic properties of the HLA-B27 allele, in particular its propensity to misfold, forming homodimers. It has been proposed that these homodimers could be associated with the disease process through the activation of an ER stress response known as the unfolded protein response (UPR), or through aberrant recognition at the cell surface. We have investigated whether the expression of HLA-B27 is associated with the activation of the UPR. We have studied the expression of BiP, and the cleavage of XBP1 and ATF6 using stable and transiently expressing cell lines. We have also investigated the formation of non-B27 homodimers using a human cell line stably expressing HLA-B8, and finally we have studied the expression of homodimers in exosomes, small immunomodulatory vesicles released from numerous cell types. The results presented here lead us to conclude that in vitro studies of the UPR are complicated, prone to a number of technical issues, and may therefore not be appropriate for gaining information that would be of significant use when comparing to the real disease scenario. Our data suggest that non-B27 dimers may be strongly influenced by both the overexpression of MHC class I heavy chains and also the redox environment within the cell. We have isolated a novel fully folded, beta-2m-associated, MHC class I homodimer in exosomes and have detected a novel HLA-A and HLA-B mixed heavy chain dimer. Our results suggest that these dimers form through interactions between the cysteine residues in the cytoplasmic tail and that these dimers form in exosomes because they contain lower levels of the important antioxidant glutathione when compared to whole cells. Together, these results define a new MHC class I structure present on exosomes at significant levels, which could potentially influence immune recognition by both antigen-specific T cell receptors and NK family receptors. The data also poses questions about whether these novel structures, when they involve HLA-B27, could influence the pathogenesis of spondyloarthropathies.

The aim of the project was to develop a PCR method to detect HLA-B27 at the Immunology Department of St. James hospital in Dublin. The HLA-B27 gene is common among patients with ankylosing spondylitis (AS). Ninety percent of patients with AS have the HLA-B27 gene and it is therefore counted as a risk factor and could be used as part of the diagnosis. Twenty-two frozen blood samples from patients with AS or suspected AS were donated from the rheumatology department at St. James hospital. PCR is a well known and common technique, many hospital laboratories have a PCR machine and therefore PCR is a good choice for detection of the HLA-B27 gene. A multiplex PCR was developed where a PCR control, primers to the β-globin gene, was used in the same tube as the HLA-B27 primers, to secure that the PCR worked in every tube. Finally a blind test was performed to test the specificity of the PCR. The result shows that the specificity was 100%. Of all patient samples, sixteen was HLA-B27 positive and six were HLA-B27 negative. In addition, optimal conditions for the PCR and the way to extract DNA from frozen blood were successfully established. For future diagnosis, the described PCR can be used to detect the HLA-B27 gene in patients and it can be considered as a start for further development of a real-time PCR for detection of the HLA-B27.

Disertacija skirta nustatyti sergančiųjų spondiloartropatijomis (SpA) (AS, ReA, PsA, EnA) KMT pokyčių dėsningumus stuburo juosmeninėje ir šlaunikaulių proksimalinėse dalyse bei įvertinti ryšį tarp kaulų masės pokyčių ir ligos specifinių veiksnių (ligos trukmės, fizinės negalios ir judėjimo funkcijos sumažėjimo, vartojamų medikamentų, ligos aktyvumo). Darbe nustatyta, kad sergančiųjų SpA KMT nesiskiria nuo sergančiųjų reumatoidiniu artritu, ir yra reikšmingai mažesnis lyginant su sveikų asmenų KMT ir stuburo juosmeninėje, ir šlaunikaulių proksimalinėse dalyse. Panašūs KMT pokyčiai stuburo juosmeninėje ir šlaunikaulių proksimalinėse dalyse būdingi SpA pacientams, sergantiems įvairiomis SpA grupės ligomis, bei nepriklauso nuo vyraujančio sąnarių pažeidimo tipo. KMT pokyčius geriau atspindi ligos trukmė, skaičiuojant ją ne nuo klinikinės diagnozės nustatymo momento, o nuo pirmųjų ligos simptomų pasireiškimo. Ligos trukmei ilgėjant – šlaunikaulių KMT mažėja, o stubure – didėja. Vidutinis ir didelis ligos aktyvumas, kurį nustatė gydytojas reumatologas, turi įtakos stuburo juosmeninės ir šlaunikaulių proksimalinių dalių KMT sumažėjimui. KMT mažėjimas ir stuburo juosmeninėje, ir šlaunikaulių proksimalinėse dalyse susijęs su SpA sergančiojo judėjimo funkcijos sumažėjimu. Šį ryšį tiksliausiai atspindi tarpkulkšnelinio atstumo matmuo – šlaunikaulių proksimalinių dalių KMT rodmenys mažiausi, kai stuburo paslankumas įvertintas sunkiu sumažėjimo lygiu. Gliukokortikoidų kumuliacinė dozė... [toliau žr. visą tekstą] / The aim of this work was to determine consistent patterns of BMD changes at the lumbar spine and the upper part of the left and right femur in patients with SpA (AS, ReA, PsA, EnA) and to assess relation between changes of bone mass and specific factors of the disease (duration of the disease, physical disability and immobility, activity of the disease, medications in use). It was established that in patients with SpA BMD is the same as in patients with rheumatoid arthritis and is significantly lower in comparison with BMD of healthy subjects measured at the lumbar spine and upper part of the left and right femur. Similar BMD changes at the lumbar spine and upper part of the left and right femur are characteristic of SpA patients with various diseases belonging to SpA group and do not depend on the predominant type of joint lesion. The duration of the disease reflects changes in BMD better when it is calculated not from the time of the establishment of clinical diagnosis, but from the time of onset of first clinical symptoms. BMD decrease at the upper part of the left and right femur and increase at the spine with the increase of the duration of disease. Moderate and high activity of the disease established by rheumatologist contribute to BMD loss at the lumbar spine and upper parts of the both femurs. BMD reduction at the lumbar spine and the upper part of the left and right femur is associated with the decrease of mmobility of SpA patients. Intermalleolar distance is the... [to full text]

Disertacija skirta nustatyti sergančiųjų spondiloartropatijomis (SpA) (AS, ReA, PsA, EnA) KMT pokyčių dėsningumus stuburo juosmeninėje ir šlaunikaulių proksimalinėse dalyse bei įvertinti ryšį tarp kaulų masės pokyčių ir ligos specifinių veiksnių (ligos trukmės, fizinės negalios ir judėjimo funkcijos sumažėjimo, vartojamų medikamentų, ligos aktyvumo). Darbe nustatyta, kad sergančiųjų SpA KMT nesiskiria nuo sergančiųjų reumatoidiniu artritu, ir yra reikšmingai mažesnis lyginant su sveikų asmenų KMT ir stuburo juosmeninėje, ir šlaunikaulių proksimalinėse dalyse. Panašūs KMT pokyčiai stuburo juosmeninėje ir šlaunikaulių proksimalinėse dalyse būdingi SpA pacientams, sergantiems įvairiomis SpA grupės ligomis, bei nepriklauso nuo vyraujančio sąnarių pažeidimo tipo. KMT pokyčius geriau atspindi ligos trukmė, skaičiuojant ją ne nuo klinikinės diagnozės nustatymo momento, o nuo pirmųjų ligos simptomų pasireiškimo. Ligos trukmei ilgėjant – šlaunikaulių KMT mažėja, o stubure – didėja. Vidutinis ir didelis ligos aktyvumas, kurį nustatė gydytojas reumatologas, turi įtakos stuburo juosmeninės ir šlaunikaulių proksimalinių dalių KMT sumažėjimui. KMT mažėjimas ir stuburo juosmeninėje, ir šlaunikaulių proksimalinėse dalyse susijęs su SpA sergančiojo judėjimo funkcijos sumažėjimu. Šį ryšį tiksliausiai atspindi tarpkulkšnelinio atstumo matmuo – šlaunikaulių proksimalinių dalių KMT rodmenys mažiausi, kai stuburo paslankumas įvertintas sunkiu sumažėjimo lygiu. Gliukokortikoidų kumuliacinė dozė... [toliau žr. visą tekstą] / The aim of this work was to determine consistent patterns of BMD changes at the lumbar spine and the upper part of the left and right femur in patients with SpA (AS, ReA, PsA, EnA) and to assess relation between changes of bone mass and specific factors of the disease (duration of the disease, physical disability and immobility, activity of the disease, medications in use). It was established that in patients with SpA BMD is the same as in patients with rheumatoid arthritis and is significantly lower in comparison with BMD of healthy subjects measured at the lumbar spine and upper part of the left and right femur. Similar BMD changes at the lumbar spine and upper part of the left and right femur are characteristic of SpA patients with various diseases belonging to SpA group and do not depend on the predominant type of joint lesion. The duration of the disease reflects changes in BMD better when it is calculated not from the time of the establishment of clinical diagnosis, but from the time of onset of first clinical symptoms. BMD decrease at the upper part of the left and right femur and increase at the spine with the increase of the duration of disease. Moderate and high activity of the disease established by rheumatologist contribute to BMD loss at the lumbar spine and upper parts of the both femurs. BMD reduction at the lumbar spine and the upper part of the left and right femur is associated with the decrease of mmobility of SpA patients. Intermalleolar distance is the... [to full text]

The Human Leukocyte Antigen (HLA)-B27 is a Major Histocompability Complex (MHC) class I antigen that is strongly associated with development of a group of closely related arthritic diseases, collectively known as the spondyloarthropathies (SpA). However, the mechanism by which HLA-B27 confers this susceptibility is unclear. Studies have shown that HLA-B27 heavy chains can form classical heterotrimers associated with peptide and β2-microglobulin (B27HT), and also non-classical heavy chain homodimers (B27₂). B27₂ assemble intracellularly during maturation and are also expressed at the cell surface following endosomal recycling of B27HT. A pathogenic role for B27₂ has been proposed in two of the current theories of pathogenesis: the B27 homodimer theory and the B27 misfolding and UPR theory. Yet, determinations of the extent, distribution, and triggers of B27₂ expression, as well as the functional consequences of its receptor interactions in AS pathogenesis, have been hampered by the lack of a specific detection reagent. Therefore, to investigate the role of B27₂ in AS, we generated a novel antibody to B27₂ – HD6 – using phage display technology, which binds to in vitro refolded B27₂ but not B27HT complexes by ELISA. This thesis provides evidence that HD6-reactive molecules, which include B27₂, are expressed at the cell surface in both cell lines and in the context of a disease setting. Recognition is B27-specific and strongly correlated with the magnitude of B27 expression, which could account for the lack of staining in some cell subsets. Moreover, staining was comparable in cell lines expressing the disease-associated B*27:05 and the less disease-associated subtype B*27:09. In addition, I have shown cells expressing physiologic levels of B27, including EBV-transformed BCLs and AS patient PBMCs, are capable of expressing the HD6 epitope upon low pH treatment. Interestingly, these ‘acid-inducible HD6’ molecules were absent from cells lacking a functional PLC. Finally, I have shown that HD6-reactive molecules can derive from pre-existing folding B27 molecules at the cell surface, which may be inhibited by the addition of exogenous B27-binding peptides. These findings are consistent with a mechanism of pathogenesis involving the surface expression and recognition of B27₂ and/or other aberrantly folded forms of B27, as proposed in the homodimer theory. HD6 will be a powerful tool to address the potential pathogenic role of B27₂ in SpA and may additionally have therapeutic potential.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-12T17:38:37Z No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-12T17:38:59Z (GMT) No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-12T17:39:02Z (GMT). No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2004-04-20 / Introduction: Chlamydia trachomatis is the bacteria responsible for the most prevalent sexually transmitted disease worldwide. Most of the infections in men and women is asymptomatic and when undiagnosed and untreated may reach the joints causing not only arthritis, but also other acknowledged complications related to the female reproductive system. Objective: To investigate C. trachomatis DNA in the urine and synovial fluid from patients with spondyloarthropathies (SpA) and rheumatoid arthritis (RA) and evaluate serum anti-C. trachomatisIgG and IgM antibodies. Methods: The population consisted of 15 patients with spondyloarthropathies, being nine with undifferentiated spondyloarthropathy (US) and six with reactive arthritis (ReA) (group I), and 15 patients with rheumatoid arthritis (RA) (group II). The chlamydial DNA was assessed in synovial fluid and urine samples of all patients by Amplicor PCR. The anti-chlamydial IgG and IgM antibodies were quantified through indirect imunofluorescence (IIF), while 15 patients of group I were typed for HLA-B27 by the use of flow citometry. Social demographical data and all information on sexual behavior and presence of symptoms were collected through a (questionnaire in the form of) an interview. Results: C. trachomatis DNA was found in only one synovial fluid sample from patient with ReA (6,7%). In two patients with RA, chlamydial DNA was identified in the urine sample (13,3%). The anti-chlamydial IgG antibodies were present in eight patients of the population studied; being three patients from group I (20%), and five from group II (33,3%). The greatest titer of this antibody 1/256 was associated with the presence of chlamydial DNA in a patient from group II. The IgM antibody was not detected in any of the samples from both groups. Four individuals from group II (26,7%) were HLA-B27 positive and its presence was 16 related to sacroiliitis. Conclusion: The results in this study show that in patients with spondyloarthropathies and rheumatoid arthritis, presenting a picture of articular activity one might not exclude C. trachomatisas the triggering agent. / Introdução: A Chlamydia trachomatis é a bactéria responsável pela doença sexualmente transmissível mais prevalente no mundo. A maioria das infecções em homens e mulheres é assintomática e, quando não diagnosticada e tratada, pode alcançar as articulações e causar artrite, isto sem mencionar outras complicações conhecidas relacionadas ao aparelho reprodutor feminino. Objetivos: Pesquisar o DNA de C. trachomatis no líquido sinovial e urina, em pacientes com espondiloartropatias e artrite reumatóide (AR) e avaliar a presença de anticorpos séricos IgG e IgM anti-C. trachomatis nestes dois grupos de doenças. Identificar o antígeno HLA-B27 em pacientes com espondiloartropatias. Metodologia: A população do estudo consistiu de 15 pacientes com espondiloartropatias: nove com espondiloartropatia indiferenciada (EI) e seis com artrite reativa (ARe) (grupo I) e 15 pacientes com AR (grupo II). O DNA clamidial foi pesquisado em amostras de líquido sinovial e urina de todos eles empregandose a PCR (Amplicor Roche). Os anticorpos IgG e IgM anti-clamidiais foram quantificados por imunofluorescência indireta (IFI), enquanto o HLA-B27 foi tipado em 15 pacientes do grupo I por citometria de fluxo. Os dados sócio-demográficos, de comportamento sexual e presença de sintomas foram obtidos através de questionário na forma de entrevista. Resultados: O DNA da C. trachomatis foi evidenciado apenas em uma amostra de líquido sinovial do grupo I (6,7%), sendo o paciente portador de ARe. Em dois pacientes com AR, o DNA de clamidial foi identificado na urina (13,3%). Os anticorpos IgG anti-clamidiais estavam presentes em oito pacientes da população estudada, três do grupo I (20%) e cinco do grupo II (33,3%). O maior título desse anticorpo (1/256) associou-se com a presença do DNA clamidial na urina de um paciente do grupo II. O anticorpo IgM não foi detectado em nenhuma amostra 14 dos dois grupos. O antígeno HLA-B27 foi positivo em quatro indivíduos do grupo II (26,7%) e sua presença relacionou-se com sacroiliite. Conclusões: Os resultados desse estudo indicam que em portadores de espondiloartropatias e artrite reumatóide, com quadro articular em atividade, a C. trachomatisnão pode ser excluída como agente desencadeador.

This thesis comprised patients with chronic rheumatic diseases. The studies aimed to elucidate food sensitivity by measuring mucosal inflammatory reactivity and thereby a possible link between the gut and joints. In all the studies, the mucosal path technique was used to evaluate the rectal mucosal response to rectal challenge with gluten and/or cow’s milk protein (CM). In some patients with primary Sjögren’s syndrome (pSS) and the genetic susceptibility genes HLA DQ2, mucosal reactivity measured with nitric oxide (NO) was found after rectal gluten challenge without detectable serum antibodies to gluten or transglutaminase. This gluten sensitivity was not linked to coeliac disease. After rectal CM challenge, a rectal mucosal inflammatory response measured with NO and myeloperoxidase (MPO) was detected in 38% of pSS patients, all of whom fulfilled the criteria for irritable bowel syndrome. In a questionnaire study of self-experienced adverse reactions to food, 27% of patients with rheumatoid arthritis (RA) reported intolerance to various foods and CM in particular. After rectal CM challenge performed in RA patients (n=27), strong mucosal reactivity to CM was observed in a few patients and a moderate increase in 23%. After gluten challenge, a moderate increase in mucosal reactivity was found in 35% of patients. No correlation to self-perceived intolerance and mucosal reactivity measured with NO and MPO was seen. Inflammation of the gut is a prominent feature of spondyloarthropathies (SpA). After rectal challenges with CM protein and gluten, an increase in rectal NO production was seen in 26% and 19% respectively (p<0.001). An increase in the mucosal release of MPO as a sign of neutrophil activation was seen in the CM- and gluten-sensitive patients. NO production in SpA patients was more enhanced compared with RA and pSS patients and could contribute to the increased barrier permeability described in SpA patients.