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no / Combinatorial antibody libraries were constructed from the spleen of a patient with concomitant systemic lupus erythematosus and idiopathic thrombocytopenia. Following selection of the libraries with DNA, a panel of 15 anti-DNA Fabs was isolated. Sequence analysis of these antibodies coupled with measurements of their affinities for ss- and dsDNA were used to investigate the role of somatic mutation in affinity maturation of the anti-DNA response. Examination of the germline genes used by these Fabs supports previous studies that suggest there is no restriction of the gene usage in the anti-DNA response. However, data are presented indicating that VH3 genes and the A27 V¿ paired with the J¿1 may be over-expressed in the anti-DNA repertoire. Analysis of the role of somatic mutation in increasing affinity for DNA indicates that affinity maturation has occurred and suggests that the CDR1 and CDR2 of the heavy chain are of importance in this process.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease requiring a genetic predisposition coupled with an environmental trigger in order for initiation of disease. While the exact pathoaetiology has yet to be determined, both B and T cell dysregulation are thought to contribute to disease. Histone deacetylases (HDACs) are a class of enzymes that hydrolyze the lysine bound acetyl group in both histone and non-histone proteins thereby altering protein structure and function. While the use of pan-HDAC inhibitors has proven to be effective for the treatment of a number of acute diseases, they may not be viable as therapeutics for chronic disease due to cytotoxicity and adverse side effects following long term treatment. We sought to determine whether treatment with a class I and II HDAC inhibitor (HDACi) or a specific HDAC6i would be able to ameliorate disease in lupus-prone NZB/W mice. We found that both the class I and II HDACi (ITF2357) and the HDAC6i (ACY-738) were able to decrease SLE markers of disease including splenomegaly, proteinuria, and anti-dsDNA and IgG production in the sera. Treatment with ITF2357 resulted in an increase in the number of immunosuppressive regulatory T (Treg) cells and a decrease in the pro-inflammatory Th17 phenotype. Furthermore, ITF2357 was found to increase Foxp3 acetylation leading to increased Foxp3 stability allowing for differentiation into the Treg phenotype. ACY-738 treatment was able to correct aberrant bone marrow B cell differentiation while also increasing the number of splenic Treg cells in NZB/W mice. These results suggest that HDAC inhibition is able to ameliorate SLE in NZB/W mice by altering aberrant T and B cell differentiation. Additional studies were performed to further examine the expression and function of different HDAC isoforms in immune cells. Due to the ability of HDAC inhibition to decrease markers of SLE disease as well as alter B and T cell development and differentiation, we sought to determine if specific HDAC isoforms are altered in lupus vs non lupus mice in early and late disease states. We determined the level of class IIb HDAC (HDACs 6, 9, and 10) expression in bone marrow B cells, splenic B and T cells, and glomerular cells from early- and late-disease MRL/lpr lupus-prone mice compared to healthy, age-matched C57BL/6 control mice. Expression of HDAC6 and HDAC9 were significantly increased in all of the tissues tested from MRL/lpr mice. Furthermore, both cytoplasmic and nuclear HDAC activity was increased in diseased MRL/lpr mice, and HDAC activity and expression continued to increase as disease progressed. In vitro treatment with ACY-738, a selective HDAC6i, was able to decrease cytoplasmic HDAC activity and inhibit iNOS production. Furthermore, ACY-738 was able to alter apoptosis through increased Bax expression in B cells. Treatment with ACY-738 was also able to inhibit Hsp90 expression and decrease NF-κB nuclear translocation, which are both upregulated during active SLE. Our studies indicate that HDAC activity contributes to SLE pathogenesis and that the use of isoform-selective HDAC inhibitors may be a viable treatment for SLE. / Ph. D.

No / Our objectives were to obtain monoclonal anti-endothelial cell antibodies (AECA) from systemic lupus erythematosus (SLE) patients, to characterize their antigen specificity, and their capability to induce a pro-inflammatory and pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generated by hybridoma formation with human SLE B cells. Antigen specificity was characterized by immunoblotting with enriched cell membrane fractions, by cytofluorimetry and by cell solid-phase ELISA. Endothelial activation was evaluated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechanisms of endothelial activation were investigated by assessment of NF-B by measuring the loss of its inhibitor I-B. mAb E-3 bound live EC and recognized a 42 kDa EC membrane protein, it enhanced U937 adhesiveness, E-selectin and ICAM-1 expression and IL-6 production, and caused the loss of I-B. We conclude this is the first in vitro demonstration that a human monoclonal AECA from a SLE patient reacts with a constitutive endothelial membrane antigen and induces a pro-inflammatory endothelial phenotype through NF-B activation.

Introdução: A idade ao diagnóstico do lúpus eritematoso sistêmico juvenil (LESJ) pode influenciar a expressão da doença em termos de apresentação clínica inicial, padrão de envolvimento de órgãos e achados sorológicos. Objetivo: Avaliar dados demográficos, características clínicas e alterações laboratoriais no momento do diagnóstico da doença em três grupos etários diferentes de pacientes com LESJ: grupo A com início precoce (< 6 anos), grupo B com início em idade escolar ( >= 6 e < 12 anos) e grupo C com início em adolescentes ( >= 12 e < 18 anos). Métodos: Estudo multicêntrico brasileiro de coorte retrospectiva em 10 centros de reumatologia, incluindo 847 pacientes com o diagnóstico de LESJ. Resultados: Os pacientes foram divididos em três grupos: A com 39 (4%), B com 395 (47%) e C com 413 (49%). Dos 39 pacientes com LESJ do grupo A, 3 (8%) tinham < 2 anos, 4 (10%) >= 2 e < 3 anos e 32 (82%) >= 3 e < 6 anos. Setenta e quatro pacientes com LESJ foram analisados para os níveis séricos de C1q e a deficiência completa de C1q foi observada em 3/74 (4%), todos estes pertencentes ao grupo A. Os grupos foram semelhantes quanto às altas frequências de sexo feminino, nefrite, envolvimento neuropsiquiátrico, SLEDAI-2K ( >= 8), perfil de autoanticorpos, proteínas de fase aguda elevada e baixos níveis de complemento (p > 0,05). No entanto, as frequências de febre (78% vs. 61% vs. 47%, p < 0,0001), hepatomegalia (42% vs. 29% vs. 14%, p < 0,0001), esplenomegalia (28% vs. 12% vs. 4%, p < 0,0001) e lúpus discoide (13% vs. 4% vs. 4%, p=0,020) foram significantemente maiores no grupo A em comparação com os grupos B e C. As frequências de perda de peso > 2kg (19% vs. 28% vs. 36%, p < 0,017), fotossensibilidade (34% vs. 41% vs. 51% p < 0,006), leucopenia < 4.000/mm3 (14% vs. 25% vs. 30%, p=0,048) e linfopenia < 1.500/mm3 (22% vs. 41% vs. 47%, p=0,011) foram significantemente menores no grupo A. Conclusão: O presente estudo multicêntrico identificou que a apresentação inicial de LESJ foi caracterizada por alta frequência de envolvimento de órgãos internos nos três grupos estudados e algumas características clínicas e laboratoriais distintas nos grupos de início precoce e adolescentes / Introduction: Age at diagnosis of childhood-onset systemic lupus erythematosus (cSLE) may influence disease expression in terms of initial clinical presentation, pattern of organ involvement and serological findings. Objective: To evaluate demographic data, clinical and laboratory features at disease diagnosis in three different age groups of childhood systemic lupus erythematosus (cSLE): group A early-onset ( < 6 years), group B school age ( >= 6 and < 12 years) and group C adolescent ( >= 12 and < 18 years). Methods: Brazilian multicenter cohort retrospective study in 10 Pediatric Rheumatology centers, including 847 cSLE patients. Results: Patients were divided in three groups: A with 39 (4%), B 395 (47%) and C 413 (49%). Of 39 cSLE patients of group A, 3 (8%) were < 2 years, 4 (10%) >= 2 to < 3 years and 32 (82%) >= 3 and < 6 years. Seventy-four cSLE patients were analyzed for C1q levels and complete C1q deficiency was observed in 3/74 (4%), all of them of group A. Groups were similar regarding high frequencies of female gender, nephritis, neuropsychiatric involvement, SLEDAI-2K ( >= 8), autoantibody profile, elevated acute phase proteins and low complement levels (p > 0.05). However, the frequency of fever (78% vs. 61% vs. 47%, p < 0.0001), hepatomegaly (42% vs. 29% vs. 14%, p < 0.0001), splenomegaly (28% vs. 12% vs. 4%, p < 0.0001) and discoid lupus (13% vs. 4% vs. 4%, p=0.020) was significantly higher in the group A compared to groups B and C. The frequency of weight loss > 2kg (19% vs. 28% vs. 36%, p=0.017), photosensitivity (34% vs. 41% vs. 51%, p=0.006), leukopenia < 4,000/mm3 (14% vs. 25% vs. 30%, p=0.048) and lymphopenia < 1,500/mm3 (22% vs. 41% vs. 47%, p=0.011) was significantly lower in the group A. Conclusions: Our large multicenter study identified that the initial presentation of cSLE is characterized by comparable high frequency of internal organ involvement and some distinct clinical and laboratory features in early-onset and adolescent groups

Results of the above studies thus suggested that immune dysregulation in SLE result in derangement of a spectrum of inflammatory mediators leading to possible multiple organs auto-inflammatory damages. However, the exact etio-pathogenic mechanism could not simply be explained by these phenomena. Infection has been invoked as an underlying etiology or trigger for the induction of autoimmune disease. Epstein-Barr virus (EBV) possesses multiple features that characterise its involvement in initiating or perpetuating SLE disease. Several research groups demonstrated that the peripheral blood EBV DNA load is significantly higher in SLE patients, yet cell-free viral DNA was also reported in other EBV-associated diseases such as nasopharyngeal carcinoma (NPC) and certain lymphomas, suggesting that relatively little is known about its biology and dynamic distribution in the blood circulation. In the second part of our study, we examined the cell-free and cell-associated distribution profile of EBV DNA load in SLE. Our data showed that the distribution of EBV DNA in the cell-free and cell-associated compartments exhibited a heterogeneous pattern in SLE patients. Contrary to the exclusive presence of circulating cell-free EBV DNA in NPC patients, both cell-free and cell-associated EBV DNA were detected in some SLE patients, while in others, no EBV DNA was measurable in either blood compartments. The level of cell-associated EBV viral load was significantly higher in SLE patients with active disease than those who presented with milder disease activity. This phenomenon indicated a possible association of EBV viral infection with the level of immune competence in SLE patients. It has been reported that EBV encodes proteins which shares significantly homology sequence with human IL-6, IL-8, IL-10, IL-12 and colony-stimulating factor (CSF)-1. This proposition brought our attention to the immune perturbation by EBV on the cytokine balance, possibly constitute in part, to the immune dysregulation and Th1 and Th2 dichotomy in SLE exacerbation. (Abstract shortened by UMI.) / The first section of this research study aimed to explore the messengers that influence Th1/Th2 cells differentiation, development, effector functions and hence their plausible contribution in SLE immunopathogenesis. We focused on studying the expression of cytokine and chemokine milieu that directs the traffic of T lymphocytes; co-stimulatory molecules in the activation of T lymphocytes; transcription factors T-bet and GATA-3 in regulating the differentiation of Th1 and Th2 cell lineage. We also investigated the involvement of the lymphocyte subpopulation, Th17 in the auto-inflammatory axis of SLE exacerbation. / Lit, Choi Wan. / Advisers: Christopher W.K. Lam; Y.M. Dennis Lo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3358. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 203-235). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Introdução: A idade ao diagnóstico do lúpus eritematoso sistêmico juvenil (LESJ) pode influenciar a expressão da doença em termos de apresentação clínica inicial, padrão de envolvimento de órgãos e achados sorológicos. Objetivo: Avaliar dados demográficos, características clínicas e alterações laboratoriais no momento do diagnóstico da doença em três grupos etários diferentes de pacientes com LESJ: grupo A com início precoce (< 6 anos), grupo B com início em idade escolar ( >= 6 e < 12 anos) e grupo C com início em adolescentes ( >= 12 e < 18 anos). Métodos: Estudo multicêntrico brasileiro de coorte retrospectiva em 10 centros de reumatologia, incluindo 847 pacientes com o diagnóstico de LESJ. Resultados: Os pacientes foram divididos em três grupos: A com 39 (4%), B com 395 (47%) e C com 413 (49%). Dos 39 pacientes com LESJ do grupo A, 3 (8%) tinham < 2 anos, 4 (10%) >= 2 e < 3 anos e 32 (82%) >= 3 e < 6 anos. Setenta e quatro pacientes com LESJ foram analisados para os níveis séricos de C1q e a deficiência completa de C1q foi observada em 3/74 (4%), todos estes pertencentes ao grupo A. Os grupos foram semelhantes quanto às altas frequências de sexo feminino, nefrite, envolvimento neuropsiquiátrico, SLEDAI-2K ( >= 8), perfil de autoanticorpos, proteínas de fase aguda elevada e baixos níveis de complemento (p > 0,05). No entanto, as frequências de febre (78% vs. 61% vs. 47%, p < 0,0001), hepatomegalia (42% vs. 29% vs. 14%, p < 0,0001), esplenomegalia (28% vs. 12% vs. 4%, p < 0,0001) e lúpus discoide (13% vs. 4% vs. 4%, p=0,020) foram significantemente maiores no grupo A em comparação com os grupos B e C. As frequências de perda de peso > 2kg (19% vs. 28% vs. 36%, p < 0,017), fotossensibilidade (34% vs. 41% vs. 51% p < 0,006), leucopenia < 4.000/mm3 (14% vs. 25% vs. 30%, p=0,048) e linfopenia < 1.500/mm3 (22% vs. 41% vs. 47%, p=0,011) foram significantemente menores no grupo A. Conclusão: O presente estudo multicêntrico identificou que a apresentação inicial de LESJ foi caracterizada por alta frequência de envolvimento de órgãos internos nos três grupos estudados e algumas características clínicas e laboratoriais distintas nos grupos de início precoce e adolescentes / Introduction: Age at diagnosis of childhood-onset systemic lupus erythematosus (cSLE) may influence disease expression in terms of initial clinical presentation, pattern of organ involvement and serological findings. Objective: To evaluate demographic data, clinical and laboratory features at disease diagnosis in three different age groups of childhood systemic lupus erythematosus (cSLE): group A early-onset ( < 6 years), group B school age ( >= 6 and < 12 years) and group C adolescent ( >= 12 and < 18 years). Methods: Brazilian multicenter cohort retrospective study in 10 Pediatric Rheumatology centers, including 847 cSLE patients. Results: Patients were divided in three groups: A with 39 (4%), B 395 (47%) and C 413 (49%). Of 39 cSLE patients of group A, 3 (8%) were < 2 years, 4 (10%) >= 2 to < 3 years and 32 (82%) >= 3 and < 6 years. Seventy-four cSLE patients were analyzed for C1q levels and complete C1q deficiency was observed in 3/74 (4%), all of them of group A. Groups were similar regarding high frequencies of female gender, nephritis, neuropsychiatric involvement, SLEDAI-2K ( >= 8), autoantibody profile, elevated acute phase proteins and low complement levels (p > 0.05). However, the frequency of fever (78% vs. 61% vs. 47%, p < 0.0001), hepatomegaly (42% vs. 29% vs. 14%, p < 0.0001), splenomegaly (28% vs. 12% vs. 4%, p < 0.0001) and discoid lupus (13% vs. 4% vs. 4%, p=0.020) was significantly higher in the group A compared to groups B and C. The frequency of weight loss > 2kg (19% vs. 28% vs. 36%, p=0.017), photosensitivity (34% vs. 41% vs. 51%, p=0.006), leukopenia < 4,000/mm3 (14% vs. 25% vs. 30%, p=0.048) and lymphopenia < 1,500/mm3 (22% vs. 41% vs. 47%, p=0.011) was significantly lower in the group A. Conclusions: Our large multicenter study identified that the initial presentation of cSLE is characterized by comparable high frequency of internal organ involvement and some distinct clinical and laboratory features in early-onset and adolescent groups

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