Introduction: Bronchiectasis is a common chronic debilitating respiratory condition. Patients suffer daily cough, excess sputum production and recurrent chest infections because of inflamed and permanently damaged airways. The pathogenesis of bronchiectasis is poorly understood. Pulmonary pathology shows excess neutrophilic airways inflammation, but despite this over two thirds of patients are chronically infected with potential pathogenic microorganisms. The acute inflammatory response is a protective mechanism that is evolved to eliminate invading organisms and should ideally be self-limiting and lead to complete resolution. The driver for persistent neutrophilic airway inflammation in bronchiectasis is unknown, but infection is considered to play a major role. AIMS The main aims of this thesis were to: (i) Characterize neutrophils in the serum and airways in bronchiectasis in the stable state and during exacerbations; (ii) Cohort study to establish if LXA4 deficiency correlates with disease severity (iii) Characterize lipids in bronchiectasis airways and peripheral blood to establish the correlation of LXA4 to disease severity; (iv) To investigate a potential mechanism for low levels of LXA4 in bronchiectasis, lipoxin biosynthetic genes expression will be measured; (v) Assess the anti-inflammatory and pro resolution effect of LXA4 on neutrophils and monocyte derived macrophages from healthy volunteers; (vi) Assess the anti-inflammatory and pro resolution effect of LXA4 on neutrophils during exacerbations in bronchiectasis and community acquired pneumonia. Methods (I) To establish the serum neutrophil subtype in stable state and following antibiotic treatment in patients with bronchiectasis, the following studies were done. Inclusion criteria: Patients aged 18-80 were recruited. All had an established radiological diagnosis of bronchiectasis (CT of the chest). Patients had clinically significant bronchiectasis, aetiology being either idiopathic or post infection. Exclusion Criteria: current smokers or ex-smokers of less than 1 year; >20 pack year history; cystic fibrosis; active allergic bronchopulmonary aspergillosis; active tuberculosis; poorly controlled asthma; severe COPD requiring nebulised bronchodilators or long term oxygen therapy; patients on aspirin or leukotriene inhibitors, pregnancy or breast feeding, active malignancy. A. 6 patients with mild bronchiectasis, 6 patients with severe bronchiectasis and 6 healthy volunteers were recruited. Serum and airways neutrophils were subsequently isolated. Neutrophil apoptosis, CD11b and CD62L expression, myeloperoxidase release, superoxide generation, phagocytosis and killing of GFP labeled bacteria were assessed. B. To compare serum with airways neutrophils function, bacterial phagocytosis and killing of GFP labeled bacteria was done, with both serum and airways neutrophils. Samples were obtained from the above group of patients. C. To establish neutrophil function following antibiotic treatment, 6 bronchiectasis patients at the beginning (day1) and the end (day14) of intravenous antibiotic therapy for an exacerbation were studied. As a control group, 6 community acquired pneumonia patients at the beginning (day1) and the end (day 5) of intravenous antibiotic therapy for infection were studied. Induced sputum and peripheral blood was taken at day1 and 5, where able. Phagocytosis and killing of GFP labeled bacteria was assessed and the two groups compared. (II) To address if lipoxin A4 deficiency correlates with disease severity, a cohort study was done in bronchiectasis patients. 169 patients were recruited and followed up for 1 year. Assessments done were Bronchiectasis severity index, systemic inflammatory markers (white cell count, ESR and c-reactive protein), Forced Expired Volume in 1sec, Forced Vital Capacity and its ratio, antibiotic courses in 1 year, hospital admissions in 1 year, sputum microbiology, quality of life assessments by Leicester Cough Questionnaire and St. Georges Respiratory Questionnaire, interleukin 8, myeloperoxidase, neutrophil elastase and leukotriene B4 (from sputum). (III) To assess effect of lipoxin on disease severity, 6 healthy volunteers, 10 patients with mild disease, 15 with moderate and 9 with severe disease were recruited. Disease severity was calculated as per the bronchiectasis severity index. All participants had 60mls of blood taken and underwent a bronchoscopy. Two segments of the lungs were washed out from bronchiectasis patients, an area affected by bronchiectasis and an area unaffected by bronchiectasis. This led to patients acting as their own internal control. Serum and airways neutrophils (from both segments) were subsequently isolated. Assessments done were systemic inflammatory markers (white cell count, ESR and c-reactive protein), serum lipoxin A4 and the cathelicidin LL-37, Forced Expired Volume in 1sec, Forced Vital Capacity and its ratio, transfer factor for carbon monoxide, antibiotic courses in 1 year, hospital admissions in 1 year and sputum microbiology. Phagocytosis and bacterial killing were assessed by both serum and airways neutrophils. From bronchoalveolar lavage fluid (BALF), I measured myeloperoxidase and neutrophil elastase. For both serum and BALF, lipidomics were obtained. (IV) To address the impact of anatomic compartment, gene expression was measured in from endobronchial brushings from the same cohort of bronchiectasis patients and controls as above, where samples were available. qPCR was performed for the following eicosanoid biosynthetic genes- 5 Lipoxygenase (LOX), 15 LO-A, 15LO-B and leukotriene (LT) A4 hydrolase. (V) To assess the anti inflammatory and pro resolution effect of LXA4 on neutrophils and monocyte derived macrophages from healthy volunteers, freshly isolated PMN will be treated with LXA4 or vehicle control. Spontaneous apoptosis was measured. fMLF and cytochalasin B was added and the inflammatory response assessed measuring myeloperoxidase (MPO), free neutrophil elastase (NE), CD11b, CD18 and CD62L. Human monocytes and PMNs were isolated from bronchiectasis patients. Following differentiation, LXA4 treated or control adherent, washed MDMs will be incubated with apoptotic stained PMNs. Efferocytosis was analyzed by flow cytometry. (VI) To establish the effect of Lipoxin A4 on neutrophil function following antibiotic treatment, the same study group used to evaluate aim 1 was taken. As a control group, 6 community acquired pneumonia patients at the beginning (day1) and the end (day 5) of oral or intravenous antibiotic therapy for infection were studied. Induced sputum and peripheral blood was taken at day1 and 5, where able. Phagocytosis and killing of GFP labeled bacteria and the effect of Lipoxin A4 was assessed and the two groups compared. Serum and sputum lipidomics were obtained in bronchiectasis exacerbations on day 1 and day 14. Serum lipidomics was obtained in pneumonia on day 1 and day 5. RESULTS (I) Neutrophil sub type study (Studied on healthy volunteers/ mild/ severe bronchiectasis) Peripheral blood neutrophils from bronchiectasis patients showed that there was significantly more viable neutrophils in mild and severe bronchiectasis compared to healthy volunteers, p=0.002 and p=0.005 respectively. In addition, there was significantly less apoptotic neutrophils in mild and severe bronchiectasis compared to healthy volunteers, p=0.0003 and p < 0.0001 respectively. There was a significantly higher level of CD11b in the mild (p=0.01) and severe bronchiectasis (p=0.01) compared to healthy volunteers. There was more CD62L shedding (p=0.02) and myeloperoxidase release (p=0.04) in bronchiectasis compared to healthy volunteers. There was lesser phagocytosis in mild (p=0.04) and severe (p=0.03) bronchiectasis compared to healthy volunteers. This led to lesser bacterial killing in mild (p=0.04) and severe (p=0.0004) bronchiectasis compared to healthy volunteers.

No description available.

Introducción: Los derrames pleurales (DP) son una patología común, aunque su fisiopatología no está del todo aclarada. El sistema de la angiogénesis se ha visto implicado en procesos inflamatorios y neplásicos, pero ha sido poco estudiado en el espacio pleural. Objetivos: 1) Analizar los niveles pleurales y plasmáticos de factores activadores e inhibidores de la angiogénesis en diferentes categorías de DP. 2) Estimar su asociación con los marcadores bioquímicos habituales, el recuento celular, los marcadores inflamatorios, el sistema de la fibrinolisis y el de las metaloproteinasas. 3) Estudiar su asociación con la presencia de complicaciones pleurales en los DP infecciosos. Pacientes y método: 21 pacientes de cada uno de los siguientes DP: empiema o paraneumónico complicado, paraneumónico no complicado, tuberculoso, neoplásico y trasudado. Se determinaron en sangre y pleura los marcadores bioquímicos habituales (glucosa, proteínas, ADA, LDH, pH), celularidad (leucocitos y recuento diferencial), marcadores inflamatorios (elastasa, IL-8, TNF-alfa), sistema de la fibrinolisis (activadores del plasminógeno u-PA, t-PA e inhibidores PAI-1, PAI-2), sistema de las metaloproteinasas (MMP-2, MMP-9 y sus inhibidores TIMP-1, TIMP-2) y sistema de la angiogénesis (activadores factor de crecimiento endotelial vascular (VEGF total y libre), factor de crecimiento fibroblástico básico (b-FGF) e inhibidores endostatina y trombospondina-1 (TSP-1). Paquete estadístico: SPSS.Resultados: Los niveles de VEGF y b-FGF fueron más altos en los exudados que en los trasudados (p < 0.001) y en los empiemas y paraneumónicos complicados que en los no complicados (p < 0.001). La endostatina no mostró diferencias significativas en los diferentes grupos de DP excepto dentro de los paraneumónicos, donde los niveles fueron superiores en los no complicados que en los empiemas y paraneumónicos complicados (p = 0.002). La TSP-1 mostró niveles superiores en los exudados que en los trasudados y en los empiemas y paraneumónicos complicados que en los paraneumónicos no complicados (p < 0.001). En los exudados pleurales observamos una correlación positiva entre VEGF, b-FGF y TSP-1 y los niveles bajos de glucosa y de pH y los niveles altos de LDH, la IL-8, la elastasa y el TNF-alfa. En los exudados pleurales observamos una asociación positiva entre VEGF, b-FGF y TSP-1 y los PAIs y la MMP-9 y una asociación negativa con el t-PA y la MMP-2. Excepto para la TSP-1, los factores implicados en la angiogénesis presentaron niveles superiores en pleura que en plasma. Excepto para la endostatina en los DP paraneumónicos, no se evidenció correlación entre los niveles pleurales y plasmáticos de los factores implicados en la angiogénesis. Los DP paraneumónicos con cifras más altas de VEGF, b-FGF y TSP-1 presentaron mayor incidencia de paquipleuritis.Conclusiones: 1) Los DP exudados muestran valores más elevados de VEGF, b-FGF y TSP-1 que los trasudados, siendo el grupo de los empiemas y paraneumónicos complicados los que presentan los niveles más altos. Asimismo no existe correlación entre los niveles pleurales y plasmáticos, sugiriendo una respuesta compartimentalizada a nivel pleural. 2) En los exudados pleurales existe una correlación positiva entre VEGF, b-FGF y TSP-1 y los niveles bajos de glucosa y de pH, los niveles altos de LDH, los marcadores de inflamación, los PAIs y la MMP-9 y una correlación negativa con el t-PA y la MMP-2. 3) Los DP bacterianos paraneumónicos con cifras más altas de VEGF, b-FGF y de TSP-1 presentan mayor incidencia de paquipleuritis y podrían ser predictores de la presencia de complicaciones pleurales tardías. 4) Con este trabajo se aportan datos novedosos del papel de la angiogénesis en la fisiopatología de los DP exudados y de su asociación con la inflamación, el sistema de la fibrinolisis y la matriz extracelular. / Introduction: Pleural effusion is a common clinical entity, though its pathophysiology is still uncertain. The angiogenesis system has been implicated in inflammatory and neoplastic processes; nevertheless, it has been little studied in relation to the pleural space. Aims: 1) To analyze pleural and plasma levels of the activator and inhibitor factors of angiogenesis in the various types of pleural effusion; 2) To estimate the association of these factors with related biochemical markers, leukocyte count, inflammatory markers, fibrinolysis system parameters and metalloproteinases; and 3) To study the association between the angiogenesis factors and the presence of complications in infectious pleural effusions. Patients and method: Samples from 21 patients with each of the following etiological types of pleural effusion were studied: empyema or complicated parapneumonic, non-complicated parapneumonic, tuberculous, neoplastic and transudative effusions. Plasma and pleural fluid related biochemical markers (glucose, proteins, ADA, LDH, pH), cellularity (leukocyte count and differential count), inflammatory markers (elastase, IL-8, TNF-alpha), fibrinolysis system (plasminogen activators-PA, t-PA-and inhibitors-PAI-1, PAI-2), metalloproteinase system (MMP-2, MMP-9 and their inhibitors, TIMP-1, TIMP-2) and angiogenesis system (activators-total and free vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (b-FGF)-and inhibitors-endostatin and thrombospondin-1 (TSP-1). Statistical package: SPSS.Results: VEGF and b-FGF were higher in exudates than in transudates (p<0.001) and in complicated parapneumonic patients and empyema than in non-complicated parapneumonic patients (p<0.001). Endostatin showed no significant differences in the various effusion groups, except the parapneumonic, where levels were higher in the non-complicated than in the empyema and complicated parapneumonic patients (p=0.002). TSP-1 showed higher levels in the exudates than in the transudates and in complicated parapneumonic effusions and empyema than in non-complicated parapneumonic effusions (p<0.001). In pleural exudates there was a positive correlation of VEGF, b-FGF and TSP-1 with low glucose and pH and high LDH, IL-8, elastin and TNF-alfa. Furthermore, VEGF, b-FGF and TSP-1 showed a positive association with PAIs and MMP-9 and a negative association with t-PA and MMP-2 in pleural exudates. With the exception of TSP-1, the factors implicated in angiogenesis presented higher levels in pleural fluid than in plasma. Except for endostatin in parapneumonic PE, there was no correlation between pleural and plasma levels of the angiogenesis factors. Parapneumonic PE, which showed highest values of VEGF, b-FGF and TSP-1 presented a higher incidence of pachypleuritis.Conclusions: 1) Exudative pleural effusions showed higher VEGF, b-FGF and TSP-1 values than transudative effusions, with the empyema and complicated parapneumonic groups displaying the highest values. There was no correlation between pleural and plasma concentrations, suggesting a compartmentalized response at the pleural level. 2) Exudates showed a positive correlation of VEGF, b-FGF and TSP-1 with low glucose and pH, and high LDH, inflammation markers, PAIs and MMP-9, and a negative correlation with t-PA and MMP-2. 3) Bacterial parapneumonic effusions with higher levels of VEGF, b-FGF and TSP-1 presented a higher incidence of pachypleuritis and could be predictors of late-onset pleural complications. 4) This study provides new data on the role of angiogenesis in the pathophysiology of exudative pleural effusions and on the association between angiogenesis and inflammation, the fibrinolysis system and the extracellular matrix.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse manifestations affecting multiple organs. Current understanding of its pathogenesis remains largely inadequate despite recent progress in SLE research on the characterization of immune system dysfunction and its link with heritable and environmental factors. Dendritic cells (DCs) are immune cells that express pattern recognition receptors (PRRs) for the recognition of pathogen associated molecular patterns (PAMPs). Aberrant functions of DCs have been reported in SLE including recognition of self-nucleic acids, presentation of self-antigens and strong induction of interferon response, depending on their expression of PRRs. Dectin-1 is a non-toll like receptor PRR that is highly expressed in DCs for the recognition of pathogenic carbohydrates found mostly in fungi. Recognition of fungal carbohydrates by dectin-1 promotes DC maturation and production of pro-inflammatory cytokines that preferentially skew towards a T helper-17 (Th17) response which has been found to be engaged in the pathogenesis of SLE and other autoimmune diseases. Therefore, the current investigation aimed to study the expression of dectin-1 and the effect of dectin-1 activation in DCs from SLE patients. Dectin-1 expression on CD14+ monocytes from peripheral blood of SLE patients and healthy controls was measured by flow cytometry. SLE patients (mean+/-SD = 92.05+/-3.471%) were found to have higher dectin-1 expression on CD14+ monocytes compared to controls (mean+/-SD = 83.7+/-14.64%) (p=0.02). Monocyte derived DCs (MoDCs) were then derived from CD14+ monocytes in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. Pre-treated moDCs with curdlan, a dectin-1 specific ligand, showed increased expression of costimulatory molecules including CD83 and CD86 and enhanced production of IL-1β compared with healthy controls (all p<0.05). Curdlan treated moDCs were further co-cultured with CD4+ na?ve T cells and polarization into Th17 cells was subsequently evaluated by measuring the percentage of Th17 per moDCs-na?ve T cells and expression of intracellular IL-17. Curdlan treated moDCs from SLE patients were found to have enhanced Th17 polarization capacity (mean+/-SEM = 28.33+/-5.64%) compared with controls (mean+/-SEM = 12.77+/-2.56%) (p<0.05). To address the mechanism of Th17 polarization, expression of caspase-1 which promotes the production of IL-1β was measured. Curdlan treated moDCs in SLE patients expressed higher levels of caspase-1 detected in the cell lysate as measured using Western Blot compared with healthy controls (p<0.05). In conclusion, compared with healthy individuals, SLE moDCs were more matured and activated in response to β-glucan as shown by their higher expression of co-stimulatory molecules, enhanced production of IL-1β and stronger Th17 polarizing effect. These findings suggest functional dysregulation of dectin-1 expressing DCs in patients with SLE which may be involved in the pathogenesis of this condition. / published_or_final_version / Medicine / Master / Master of Philosophy

Persistence in human macrophages is central to the virulence of Mycobacterium tuberculosis, which is the causative agent of tuberculosis. Although the intracellular parasitism is apparent, molecular determinants of mycobacterial virulence are not well understood. The current investigation identified virulent genes of M. tuberculosis by measuring survivability of Mycobacterium smegmatis recombinants inside a human monocytic cell line THP-1 after acquiring various virulent gene candidates of M. tuberculosis. These gene candidates included nine virulent gene candidates suggested by other studies, five genomic polymorphisms identified in hypervirulent strains of M. tuberculosis using microarray-based comparative genomic hybridization, and ten single nucleotide polymorphisms identified in the hypervirulent strains using full genome sequencing. Interestingly, only recombinants harboring a truncated Rv2820c and a known virulent gene mce1A survived significantly better than vector control after six hours of ex vivo infection. As nucleotide sequencing indicated that the truncated Rv2820c loses around 60% of gene at 3’ end, ex vivo survivability of M. smegmatis recombinants harboring the last 60% of Rv2820c as well as the intact Rv2820c was measured, but was similar to that of vector control. The 3’ truncated portion itself did not alter mycobacterial survivability ex vivo, but its presence did compromise the survival advantage gained due to the truncated Rv2820c. To determine whether the truncated and the intact Rv2820c could enhance mycobacterial virulence in vivo, these two alleles were transformed into Mycobacterium marinum and their recombinants were used to infect zebrafish. In vivo infection showed that zebrafish infected with the recombinant harboring truncated Rv2820c died significantly faster than vector control, whereas the recombinant harboring intact Rv2820c behaved similarly to vector control. Results indicated that the truncated Rv2820c, but not the intact Rv2820c, could enhance mycobacterial virulence both ex vivo and in vivo. Additional nucleotide sequencing revealed that the 3’ truncation in Rv2820c is caused by a Beijing/W-defining deletion RD207 and is commonly found in Beijing/W strains of M. tuberculosis. Non-Beijing/W strains possess the intact Rv2820c conversely. Since Beijing/W strains have proven to be more virulent than non-Beijing/W strains both ex vivo and in vivo, the truncated Rv2820c may be one of the Beijing/W-specific virulence determinants. To confirm that Rv2820c of Beijing/W strains really enhances M. tuberculosis survival in human macrophages, the truncated Rv2820c was transformed into non-Beijing/W M. tuberculosis strains and their recombinants were used to infect THP-1 cells. Ex vivo infection confirmed that the truncated Rv2820c could enhance M. tuberculosis survival inside human macrophages, but is unlikely to induce a different profile of cytokine secretion from infected macrophages. In conclusion, the current study demonstrated that the truncated Rv2820c of Beijing/W strains could enhance mycobacterial virulence both ex vivo and in vivo. Enhanced phenotypic virulence, however, was not observed for the intact Rv2820c of non-Beijing/W strains. The truncated Rv2820c may be one of the Beijing/W-specific virulence determinants and collaboratively contribute to the high phenotypic virulence of this family. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies worldwide with very poor prognosis. It is generally believed that accumulation of irreversible alterations in critical oncogenes and tumor suppressor genes during the long-term inflammation finally leads to the hepatocellular pathogenesis. Although under intensive investigation, the molecular pathogenesis of HCC still remains to be further elucidated. In this study, we aimed to identify novel genetic alterations critical to the pathogenesis of HCC, especially in hot regions with recurrent chromosomal instability. Amplification of broad regions of 8q is one of the most frequent genetic alterations in HCC, suggesting the existence of oncogenes in addition to MYC at 8q24. By screening the publicly available microarray database and clinical samples, we found frequent amplification and overexpression of Serum and Glucocorticoid Kinase 3 (SGK3) in clinical HCC specimens, and SGK3 genomic activation was significantly associated with poor outcome of HCC patients. Functional assays revealed that SGK3 could increase G1/S cell cycle progression, cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. We provided evidences that SGK3 could promote HCC growth and survival through inactivating GSK3-β and BAD respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1, has more significance than overexpression of AKT in predicting poor outcome of HCC patients. Our findings suggested the existence of an AKT-independent SGK3 pathway, which may function in parallel with AKT pathway in the pathogenesis of HCC. In addition to large chromosomal alterations, small changes in nucleotides may also make substantial contributions to carcinogenesis. Recent advances in high-throughput deep sequencing technology have provided a powerful tool to understand the whole cancer transcriptome and identify novel genetic alterations related to cancer progression. In this study, we identified a high proportion of allele imbalance in genes related to cellular stress response by sequencing the whole transcriptome of 3 paired HCC tissues. A novel nucleotide variation which resulted in a R438H amino acid change was identified in the coding region of the gene Oxidative Stress Induced Growth Inhibitor 1 (OSGIN1), and the variant 438H form of OSGIN1 was found to be specifically retained in the tumor tissues in a cohort of HCC patients. OSGIN1 was found to be closely associated with chemotherapeutic reagents and exhibited strong tumor suppressive function in HCC by directly inducing cell apoptosis. The wild type OSGIN1 was found to have stronger tumor suppressive function than the variant allele, and this might be due to their different ability to localize to mitochondria. The significantly decreased basal apoptotic index in HCC patients carrying OSGIN1 variant allele and their poor prognosis further suggested that the specific retention of 438H OSGIN1 might be important in HCC progression. In summary, we found a frequently amplified oncogenic SGK3 signaling pathway, as well as the allele-specific imbalance of tumor suppressive OSGIN1 in the pathogenesis of HCC. Further characterization of their mechanisms in hepatocarcinogenesis may help provide novel prognostic biomarkers and therapeutic targets in HCC treatment. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Lupus nephritis is characterized by the production of anti-dsDNA antibodies, deposition of immune complexes within the kidney parenchyma, proliferation of resident renal cells and induction of inflammatory and fibrotic processes. Approximately 70% of patients with lupus nephritis show immune aggregates along the tubular basement membrane, which is accompanied by an influx of infiltrating cells and increased intra-renal expression of IL-6. Much attention has focused on the inflammatory processes in the kidney during pathogenesis of lupus nephritis whereas mechanisms of fibrogenesis are less well characterized. Tubulo-interstitial injury is a key indicator of poor prognosis of renal function. Given that the tubulo-interstitium occupies over 80% of the kidney volume, injury to this compartment will have a major impact on renal function. There is evidence to show that proximal tubular epithelial cells (PTEC) undergo epithelial-to-mesenchymal transition (EMT) during pathological disorders and adopt a fibroblastic morphology with increased fibrogenic potential. We have previously demonstrated that anti-dsDNA antibodies bound directly to the surface of PTEC through cross-reactive proteins, which were subsequently internalized and translocated to the nucleus where they induced functional changes. Using a proteomic approach, this study identified the cross-reactive antigens that mediated anti-dsDNA antibody binding and intracellular localization in PTEC and the functional consequences thereafter, focusing on EMT and fibrogenic events. Human polyclonal anti-dsDNA antibodies isolated from patients with lupus nephritis bound to Ku70 in plasma membrane extracts isolated from PTEC, and to Ku70, Ku80 and major vault protein in cytosolic and nuclear fractions. Anti-dsDNA antibodies increased synthesis of Ku70, Ku80 and major vault protein in PTEC in a time-dependent manner. Expression of these proteins was localized to proximal tubules especially those undergoing atrophy, and staining was more prominent in renal biopsies from patients with lupus nephritis compared to non-lupus renal disease or control specimens. Binding of anti-dsDNA antibodies to PTEC increased phosphorylation of MAPK and PKC signaling pathways that was accompanied by a concomitant increase in IL-6, IL-8 and TGF-1 secretion and synthesis of β-catenin, fibroblast specific protein-1, fibronectin and laminin. Inhibition of MAPK and PKC signaling pathways with specific inhibitors revealed differential regulation of inflammatory and fibrotic processes by these signaling pathways. In this respect, increased ERK, p38 MAPK, JNK and PKC phosphorylation in PTEC following anti-dsDNA antibody stimulation enhanced IL-6, IL-8 and fibronectin synthesis, whereas increased ERK and JNK phosphorylation upregulated TGF-β1 secretion. Increased β-catenin synthesis was mediated through JNK and PKC phosphorylation. Taken together, our data suggest that PTEC contribute to the pathogenesis of renal inflammation and fibrosis in lupus nephritis. We hypothesize that anti-dsDNA antibodies bind to Ku70 on the plasma membrane of PTEC to mediate inflammation, cell activation and increased fibrogenesis. Although synthesis of EMT markers was increased in PTEC after anti-dsDNA antibody stimulation, transition to a fibroblastic morphology was not observed under our experimental setting suggesting that induction of the EMT cascade is an early event before phenotypic alterations. / published_or_final_version / Medicine / Master / Master of Philosophy