Chronic hepatitis C virus (HCV) infection is associated with cirrhosis, autoimmunity and lymphoproliferative disorders. We have previously reported a differential regulation of T and B lymphocytes by HCV core protein in vitro. In this report, we employed a translational approach to characterize the activation status of peripheral B cells from individuals with chronic HCV infection and to explore potential mechanisms for B-cell dysregulation in the setting of HCV infection. In contrast to the T-cell suppression observed in HCV-infected individuals, B cells exhibit a non-specific polyclonal activation phenotype, characterized by significantly higher levels of (1) the early activation marker, CD69, (2) the costimulatory molecule, CD86, and (3) the CCR5 chemokine receptor, CD195, when compared with B cells from healthy donors in response to phytohaemagglutinin (PHA) stimulation. Importantly, tumour necrosis factor- and Apo-L-related leucocyte-expressed ligand-1 (TALL-1), also known as B-lymphocyte stimulator (BLYS), was found to be up-regulated on the surface of B cells from HCV patients in response to PHA as well as HCV core antigen stimulation. This up-regulation of TALL-1 was associated with vigorous memory B-cell responses to viral antigenic stimulation. Additionally, suppressor of cytokine signalling-1 (SOCS-1), a negative feedback immunoregulator that is inhibited in B lymphocytes by HCV core in vitro, was also inhibited in B cells from HCV patients when compared with healthy donors. These findings suggest that TALL-1 over-expression and SOCS-1 suppression are associated with aberrant B-cell activation, providing a plausible basis for the B-cell clonal expansion underlying the lymphoproliferative disorders and autoimmune phenomena observed during chronic HCV infection.

Inflammaging is a hallmark of human aging. Defined as low-grade, chronic inflammation, it is characterized by heightened proinflammatory cytokines in the blood and tissues and predicts morbidity and mortality. Despite this, the etiology of inflammaging and its role in infection have remained elusive, an issue this thesis addressed. First, we provided a comprehensive overview of an intranasal Streptococcus pneumoniae colonization model (Chapter 2). We described in detail the colonization technique, and demonstrated how to isolate and phenotype recruited cells, quantify bacterial load and measure production of immune mediators in the nasopharynx. Since both myeloid cell recruitment and tumour necrosis factor (TNF) production were increased following S. pneumoniae colonization with age, we investigated whether TNF directly augmented monocyte frequency (Chapter 3). TNF increased CCR2 expression on monocytes in old mice, leading to their enhanced egress from the bone marrow, resulting in enrichment of this population in the circulation. Monocyte numbers directly influenced plasma IL-6 levels, and this negatively impacted anti-bacterial responses, as monocyte blockade improved pneumococcal clearance in old mice. Lastly, to better understand the fundamental source of inflammaging, we studied the impact of the host microbiome on its development. This work was rooted in Elie Metchnikoff’s early predictions that leakage of intestinal bacterial products could dysregulate macrophage function, resulting in inflammation that would progress aging (Chapter 4). We showed that old mice had increased intestinal permeability, aberrant expression of cellular junction genes and increased microbial translocation from the gut to the blood. Germ-free mice lived longer than their conventionally colonized counterparts, and were protected from the development of inflammaging and defective macrophage function. Together, these studies resolve a major disparity in the field by demonstrating that systemic TNF production is initiated by increased levels of circulating bacterial products, driving functional defects in myeloid cells, which ultimately impairs anti-bacterial immunity. / Thesis / Candidate in Philosophy

The aim of the study was to examine the immune response and different immunoprotective strategies in experimental type 1 diabetes mellitus. The autoimmune destruction of the insulin-producing pancreatic β-cells that leads to type 1 diabetes is complex and incompletely understood. Activated immune cells infiltrate the pancreatic islets at an early stage of the disease, and they produce and release cytokines, which may contribute to β-cell dysfunction and death. Several immunomodulatory agents with different mechanisms have recently been developed in order to suppress cytokine function such as MDL 201, 449A, a novel transcriptional inhibitor of TNF-α. At least in rodent β-cells, many of the toxic actions of cytokines depend on the synthesis of nitric oxide (NO). Aminoguanidine (AG), an inhibitor of NO formation, might therefore be an interesting compound for prevention of type 1 diabetes. Another substance that could influence the course of events leading to this disease is the pituitary hormone prolactin (PRL), since it has the ability to activate different immune cells. We have studied the effects of AG, PRL and MDL 201, 449A on the development of hyperglycaemia and pancreatic insulitis in multiple low dose streptozotocin induced autoimmune diabetes in mice. The natural course after syngeneic islet transplantation of pancreatic islets in NOD mice, a model of type 1 diabetes mellitus was also investigated. AG and PRL were also studied in vitro on cultured isolated rodent pancreatic islets. We suggest that the insulin-producing cells are specifically targeted by the inflammatory response after syngeneic islet transplantation in type 1 diabetic mice. Our data do not exclude a role for NO in type 1 diabetes, but it raises concerns about the use of AG as a therapeutic agent since an increased mortality and no decline in diabetes frequency was observed. AG did not seem to be directly harmful to β-cell function, but it could affect pancreatic and islet blood flows. PRL and MDL 201, 449A could both counteract hyperglycaemia and insulitis in the early phase of autoimmune diabetes.

Background: Uterine fibriod (UF) or leiomyoma is the most frequent benign tumour upon lower genital tract and represents the most frequent indication for hysterectomy. The aetiology remains still unknown. The genetic factors contributing for the development of UF are being intensively investigated. The aim of our study was to look for possible genetic markers which could be used as prognostic tools for evaluation of an increased risk for development of UF. Methods: The study group enrolled 102 patients diagnosed with UF and 145 healthy controls. Ultrasonographic examination of the pelvis was performed and a single blood sample was taken in all women. Histological verification followed the surgery in the patient group. The principal of the cytokine gene polymorphisms detection is based on PCR reaction with sequence-specific primers. Results: A large spectrum of Th1/Th2 cytokine gene polymorphisms in patients with uterine fibroid was compared with control group. The frequencies of the majority of tested cytokine gene SNP in the patient cohort were not statistically different from the cytokine SNP in the control group. However, an intriguing association between polymorphisms of the IL-4 gene promotor at positions -590 C/T and -33 C/T, and the risk of leiomyoma was observed. The CC genotype of IL-4 at position...

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-07T12:49:00Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Mayara final [08-09-2016].pdf: 6000027 bytes, checksum: 8aba9ff907d1176a530d19ec2bb6fad1 (MD5) / Made available in DSpace on 2017-04-07T12:49:00Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Mayara final [08-09-2016].pdf: 6000027 bytes, checksum: 8aba9ff907d1176a530d19ec2bb6fad1 (MD5) Previous issue date: 2015-09-02 / CAPES / Doença inflamatória intestinal descreve um grupo heterogêneo de doenças inflamatórias crônicas do trato gastrointestinal. Os dois principais tipos de DII são retocolite ulcerativa idiopática e doença de Crohn. A patogênese dessas doenças é caracterizada pela inflamação persistente no intestino, envolvendo uma interação entre fatores genéticos, ambientais e imunológicos. Foram investigados aspectos clínico-epidemiológicos e analisados os polimorfismos dos genes da reposta imune em pacientes brasileiros com doença inflamatória intestinal em diferentes formas anátomo-clínicas. Um total de 101 pacientes foram analisados (43 - retocolite ulcerativa idiopática e 58 - doença de Crohn) para os polimorfismos dos genes do fator de necrose tumoral alfa (TNF-α -308 G/A; rs1800629), interleucina-10 (IL-10 -1082 G/A; rs1800896), domínio do recrutamento e ativação da caspase 15/receptor tipo NOD2 (CARD15/NOD2; rs2066844 e rs2066845), receptor tipo NOD contendo domínio pirina – NLRP1 (rs12150220), NLRP3 (rs35829419) e interleucina -1beta (IL-1β -511T/C; rs16944). A forma anatómica-clínica de DC predominante foi a fistulizante (29,31%), seguida por inflamatória (27,58%) e estenosante (27,58%). O grupo controle foi composto por 91 indivíduos saudáveis. Os genes do receptor tipo NOD contendo domínio pirina 1 e 3 e do domínio do recrutamento e ativação da caspase 15/receptor tipo NOD2 variantes R702W e G908R não foram associados à susceptibilidade a doença inflamatória intestinal. Em relação ao polimorfismo da interleucina 10, nenhuma diferença estatística foi encontrada entre os genótipos e alelos para a doença inflamatória intestinal comparado aos controles. Fator de necrose tumoral alfa mostrou uma associação estatisticamente significativa entre pacientes e controles de retocolite ulcerativa idiopática que sugere que a presença do alelo A predispõe o aparecimento de retocolite ulcerativa idiopática, mas não doença de Crohn. Verificou-se ainda que o genótipo AG da interleucina 1 foi associado com o desenvolvimento de retocolite ulcerativa idiopática. Os resultados sugerem que os polimorfismos de única base do fator de necrose tumoral alfa e da interleucina 1 estão envolvidos com a retocolite ulcerativa idiopática e podem contribuir para a patogênese na população brasileira estudada. / Inflammatory bowel disease describes a heterogeneous group of chronic inflammatory diseases of the gastrointestinal tract. The two main types of inflammatory bowel disease are ulcerative colitis and Crohn disease. The pathogenesis of the disease is characterized by unpredictable attacks of inflammation of the intestine, besides involving an interaction between genetic, environmental and immunological factors. Clinical and epidemiological aspects were investigated and the polymorphisms of genes of the immune response in Brazilian patients with inflammatory bowel disease in different anatomic-clinical forms were analyzed. A total of 101 patients were analyzed (43 - ulcerative colitis and 58 - Crohn disease) for the tumour necrosis factor alpha (TNF-α -308 G/A; rs1800629), interleukin-10 (IL-10 -1082 G/A; rs1800896), caspase activation and recruitment domains 15/ NOD like receptor 2 (CARD15/NOD2; rs2066844 and rs2066845), NOD like receptor pyrin domain containing – NLRP1 (rs12150220), NLRP3 (rs35829419) and interleukin-1beta (IL-1β 511T/C; rs16944) genes polymorphisms. The anatomic-clinical form of Crohn disease predominant was the fistulizing (29.31%), followed by inflammatory (27.58%) and stricturing (27.58%). A control group was composed by 91 healthy subjects group. NOD like receptor pyrin domain containing 1 and 3 and caspase activation and recruitment domains 15/ NOD like receptor 2 genes R702W and G908R variants were not associated to inflammatory bowel disease susceptibility. With respect to the polymorphism of interleukin-10, no statistical difference was found between the genotypes and alleles for inflammatory bowel disease compared to controls. Tumour necrosis factor alpha showed a statistically significant association between ulcerative colitis patients and controls which suggests that the presence of A allele predisposes the onset of ulcerative colitis but not Crohn disease. It was found yet that AG genotype of interleukin-1beta was associated with the development of ulcerative colitis. The results suggest that the tumour necrosis factor alpha and interleukin-1beta single nucleotide polymorphisms are involved with ulcerative colitis and may be contributing to pathogenesis in Brazilian population.

Background: Uterine fibriod (UF) or leiomyoma is the most frequent benign tumour upon lower genital tract and represents the most frequent indication for hysterectomy. The aetiology remains still unknown. The genetic factors contributing for the development of UF are being intensively investigated. The aim of our study was to look for possible genetic markers which could be used as prognostic tools for evaluation of an increased risk for development of UF. Methods: The study group enrolled 102 patients diagnosed with UF and 145 healthy controls. Ultrasonographic examination of the pelvis was performed and a single blood sample was taken in all women. Histological verification followed the surgery in the patient group. The principal of the cytokine gene polymorphisms detection is based on PCR reaction with sequence-specific primers. Results: A large spectrum of Th1/Th2 cytokine gene polymorphisms in patients with uterine fibroid was compared with control group. The frequencies of the majority of tested cytokine gene SNP in the patient cohort were not statistically different from the cytokine SNP in the control group. However, an intriguing association between polymorphisms of the IL-4 gene promotor at positions -590 C/T and -33 C/T, and the risk of leiomyoma was observed. The CC genotype of IL-4 at position...

Cardiovascular diseases are a broad group of diseases, such as heart attack and heart failureaffecting the cardiovascular system. The primary cause of cardiovascular diseases isatherosclerosis, and its progression is brought about by oxidative stress and a complex chronicinflammation reaction cascade. Of central importance are proinflammatory cytokines, regulatedby multiple factors, including interleukin (IL) 17A. This project aims to investigate the effectof IL-17A on the inflammatory response of human vascular endothelial cells by quantifyingchemokine C-X-C motif ligand-1 (CXCL1) release when exposed or not to otherproinflammatory mediators such as TNF-𝛼, IL-6 and IL-1β. To investigate this, humanumbilical cord endothelial cells were cultured and then stimulated with IL-17A alone or incombination with other cytokines, namely IL-6/sIL6R, IL-1β, or TNF-𝛼. After an appropriateincubation time following the stimulations, the supernatants of the cells were collected, and theamount of CXCL1 was analysed with ELISA or qPCR, respectively. At a lower concentration(10ng/ml), IL-17A failed to induce a significant level of CXCL1 release from endothelial cells.However, IL-17A + TNF-𝛼 (5ng/ml) greatly enhanced, higher than inductions from individualtreatments combined, level of CXCL1 release from endothelial cells. Furthermore, combiningIL-17A with IL-1β or IL-6 induced non-abundant and abundant upregulation in CXCL1 release,respectively. On transcription level, the amount of CXCL1 mRNA induced by IL-17A alonewas non-significant, but stimulation with TNF-𝛼 and IL-17A + TNF-𝛼 induced significantlyupregulated expression of CXCL1. In conclusion, we found that IL-17A induced synergeticrelease of CXCL1 in human vascular endothelial cells with TNF-𝛼. In addition, the synergisticimpact of IL-17A and TNF-𝛼 in terms of CXCL1 induction in vascular endothelial cells wasevident on a transcriptional level. Our data imply that combined blockage of IL-17A and TNF-𝛼 could have an enhanced therapeutic effect on vascular inflammation.

Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/T_H17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.