Objetivo: determinar a expressão da elafina, um inibidor da elastase, sobre o endométrio de mulheres com e sem hidrossalpinge. Design: estudo caso-controle. Local: Laboratório universitário de pesquisa Pacientes: Dez mulheres normais em idade fértil (controles) e nove mulheres com hidrossalpinge (casos) diagnosticados por histerossalpingografia ou por laparoscopia. Intervenções: análise de imuno-histoquímica da elafina em biópsias endometriais obtidas durante a menstruação, usando o software ImageJ do NIH. Principal medida do(s) desfecho(s): Intensidade da coloração do 3,3 '-diaminobenzidina (DAB) nos neutrófilos e na porção luminal, glandular, do estroma do endométrio dos controles e casos. A intensidade do pixel variava de 0(escuro) a 255 (branco). A análise estatística para a intensidade do DAB foi feito por meio do teste equações de estimação generalizadas (GEE). Resultados: Não houve diferença estatística entre os grupos quando a intensidade do DAB foi medida em glândulas estroma e neutrófilos. A mediana do número de neutrófilos foi significativamente maior no grupo de hidrossalpinge, comparadas com controles [7 (0-255) vs 28 (1-228) e mediana (intervalo), P <0,0001 - teste de Mann-Whitney). A expressão da Elafina está reduzida no lúmen do endométrio em casos de hidrossalpinge [3,9 (3,5-4,2)], quando comparadas com controles [2,7 (2,2-3,2)] [média (IC 95%), P = 0,0001 - GEE, níveis mais baixos = maior intensidade do DAB]. Conclusões: O endométrio de mulheres com hidrossalpinge tem um aumento do número de neutrófilos e menor expressão da elafina. Estes achados sugerem que a atividade da elastase esteja aumentada no endométrio de mulheres com hidrossalpinge. / Objective: To determine the expression of elafin, an elastase inhibitor, on the endometrium of women with and without hydrosalpinx. Design: Case-control study. Setting: University laboratory research Patients: Ten normal fertile women (controls) and nine women with hydrosalpinx (cases) diagnosed either by hysterosalpingography or by laparoscopy. Interventions: Immunohistochemical analysis of elafin on endometrial biopsies obtained during menses, using NIH ImageJ software. Main outcome measure(s): Intensity of 3,3'-Diaminobenzidine (DAB) staining on luminal, glandular, stromal and neutrophil endometrium of controls and cases. Pixel intensity varies from 0(dark) to 255(white). Statistical analysis for DAB intensity was done using generalized estimating equations (GEE). Results: There were no statistical differences between groups when DAB intensity was measured in glands, stroma and neutrophils. The median number of neutrophils was significantly higher in the hydrosalpinx group, compared to controls [7(0-255) vs. 28(1- 228); median (range), P<0.0001 – Mann-Whitney test). Elafin expression is reduced in the lumen of endometrium from hydrosalpinx cases [3.9(3.5-4.2)], compared to controls [2.7(2.2-3.2)] [mean (95% CI), P=0.0001 – GEE, lower levels=higher DAB intensity]. Conclusions: The endometrium of women with hydrosalpinx has an increased number of neutrophils and lower expression of elafin. These findings suggest that elastase activity is increased in endometrium of women with hydrosalpinx.

Objetivo: determinar a expressão da elafina, um inibidor da elastase, sobre o endométrio de mulheres com e sem hidrossalpinge. Design: estudo caso-controle. Local: Laboratório universitário de pesquisa Pacientes: Dez mulheres normais em idade fértil (controles) e nove mulheres com hidrossalpinge (casos) diagnosticados por histerossalpingografia ou por laparoscopia. Intervenções: análise de imuno-histoquímica da elafina em biópsias endometriais obtidas durante a menstruação, usando o software ImageJ do NIH. Principal medida do(s) desfecho(s): Intensidade da coloração do 3,3 '-diaminobenzidina (DAB) nos neutrófilos e na porção luminal, glandular, do estroma do endométrio dos controles e casos. A intensidade do pixel variava de 0(escuro) a 255 (branco). A análise estatística para a intensidade do DAB foi feito por meio do teste equações de estimação generalizadas (GEE). Resultados: Não houve diferença estatística entre os grupos quando a intensidade do DAB foi medida em glândulas estroma e neutrófilos. A mediana do número de neutrófilos foi significativamente maior no grupo de hidrossalpinge, comparadas com controles [7 (0-255) vs 28 (1-228) e mediana (intervalo), P <0,0001 - teste de Mann-Whitney). A expressão da Elafina está reduzida no lúmen do endométrio em casos de hidrossalpinge [3,9 (3,5-4,2)], quando comparadas com controles [2,7 (2,2-3,2)] [média (IC 95%), P = 0,0001 - GEE, níveis mais baixos = maior intensidade do DAB]. Conclusões: O endométrio de mulheres com hidrossalpinge tem um aumento do número de neutrófilos e menor expressão da elafina. Estes achados sugerem que a atividade da elastase esteja aumentada no endométrio de mulheres com hidrossalpinge. / Objective: To determine the expression of elafin, an elastase inhibitor, on the endometrium of women with and without hydrosalpinx. Design: Case-control study. Setting: University laboratory research Patients: Ten normal fertile women (controls) and nine women with hydrosalpinx (cases) diagnosed either by hysterosalpingography or by laparoscopy. Interventions: Immunohistochemical analysis of elafin on endometrial biopsies obtained during menses, using NIH ImageJ software. Main outcome measure(s): Intensity of 3,3'-Diaminobenzidine (DAB) staining on luminal, glandular, stromal and neutrophil endometrium of controls and cases. Pixel intensity varies from 0(dark) to 255(white). Statistical analysis for DAB intensity was done using generalized estimating equations (GEE). Results: There were no statistical differences between groups when DAB intensity was measured in glands, stroma and neutrophils. The median number of neutrophils was significantly higher in the hydrosalpinx group, compared to controls [7(0-255) vs. 28(1- 228); median (range), P<0.0001 – Mann-Whitney test). Elafin expression is reduced in the lumen of endometrium from hydrosalpinx cases [3.9(3.5-4.2)], compared to controls [2.7(2.2-3.2)] [mean (95% CI), P=0.0001 – GEE, lower levels=higher DAB intensity]. Conclusions: The endometrium of women with hydrosalpinx has an increased number of neutrophils and lower expression of elafin. These findings suggest that elastase activity is increased in endometrium of women with hydrosalpinx.

Objetivo: determinar a expressão da elafina, um inibidor da elastase, sobre o endométrio de mulheres com e sem hidrossalpinge. Design: estudo caso-controle. Local: Laboratório universitário de pesquisa Pacientes: Dez mulheres normais em idade fértil (controles) e nove mulheres com hidrossalpinge (casos) diagnosticados por histerossalpingografia ou por laparoscopia. Intervenções: análise de imuno-histoquímica da elafina em biópsias endometriais obtidas durante a menstruação, usando o software ImageJ do NIH. Principal medida do(s) desfecho(s): Intensidade da coloração do 3,3 '-diaminobenzidina (DAB) nos neutrófilos e na porção luminal, glandular, do estroma do endométrio dos controles e casos. A intensidade do pixel variava de 0(escuro) a 255 (branco). A análise estatística para a intensidade do DAB foi feito por meio do teste equações de estimação generalizadas (GEE). Resultados: Não houve diferença estatística entre os grupos quando a intensidade do DAB foi medida em glândulas estroma e neutrófilos. A mediana do número de neutrófilos foi significativamente maior no grupo de hidrossalpinge, comparadas com controles [7 (0-255) vs 28 (1-228) e mediana (intervalo), P <0,0001 - teste de Mann-Whitney). A expressão da Elafina está reduzida no lúmen do endométrio em casos de hidrossalpinge [3,9 (3,5-4,2)], quando comparadas com controles [2,7 (2,2-3,2)] [média (IC 95%), P = 0,0001 - GEE, níveis mais baixos = maior intensidade do DAB]. Conclusões: O endométrio de mulheres com hidrossalpinge tem um aumento do número de neutrófilos e menor expressão da elafina. Estes achados sugerem que a atividade da elastase esteja aumentada no endométrio de mulheres com hidrossalpinge. / Objective: To determine the expression of elafin, an elastase inhibitor, on the endometrium of women with and without hydrosalpinx. Design: Case-control study. Setting: University laboratory research Patients: Ten normal fertile women (controls) and nine women with hydrosalpinx (cases) diagnosed either by hysterosalpingography or by laparoscopy. Interventions: Immunohistochemical analysis of elafin on endometrial biopsies obtained during menses, using NIH ImageJ software. Main outcome measure(s): Intensity of 3,3'-Diaminobenzidine (DAB) staining on luminal, glandular, stromal and neutrophil endometrium of controls and cases. Pixel intensity varies from 0(dark) to 255(white). Statistical analysis for DAB intensity was done using generalized estimating equations (GEE). Results: There were no statistical differences between groups when DAB intensity was measured in glands, stroma and neutrophils. The median number of neutrophils was significantly higher in the hydrosalpinx group, compared to controls [7(0-255) vs. 28(1- 228); median (range), P<0.0001 – Mann-Whitney test). Elafin expression is reduced in the lumen of endometrium from hydrosalpinx cases [3.9(3.5-4.2)], compared to controls [2.7(2.2-3.2)] [mean (95% CI), P=0.0001 – GEE, lower levels=higher DAB intensity]. Conclusions: The endometrium of women with hydrosalpinx has an increased number of neutrophils and lower expression of elafin. These findings suggest that elastase activity is increased in endometrium of women with hydrosalpinx.

Coronary atherosclerosis and plaque rupture leads to acute coronary thrombosis and myocardial infarction. Current treatment involves re-establishing vessel patency, but no treatments have been developed to target post-infarction inflammatory pathways. Such treatments may reduce cardiomyocyte injury, attenuate adverse remodelling and improve clinical outcome. Inflammation within the infarcted myocardium is associated with chemotaxis of neutrophils and monocytes to the site of injury. Early reperfusion therapy amplifies this inflammatory cell influx. Neutrophil release a variety of pro-inflammatory factors, including human neutrophil elastase (HNE). HNE has a wide range of substrates. Preclinical studies have demonstrated that neutrophil depletion or inhibition of neutrophil elastase attenuates post-ischemic inflammatory reperfusion injury within the myocardium. Recruitment of monocytes into the infarcted myocardium is followed by maturation and differentiation into macrophages. Macrophages play a key role in orchestrating inflammation and repair. Therapeutic manipulation of this healing process will only come from understanding mechanisms and targeting reparative pathways. “Ultrasmall superparamagnetic iron oxide particles” (USPIOs) extravasate through capillaries and are phagocytosed by tissue inflammatory cells. These cells are predominately macrophages, but neutrophils have also been shown to take up USPIOs. USPIO-enhanced MRI can identify areas of inflammation in models inflammation in various tissues. Therefore we hypothesised that USPIO enhanced MRI could identify and assess cellular inflammation of the myocardium. During coronary artery bypass graft surgery (CABG), the myocardium receives an immediate ischaemic insult that is exacerbated by post-ischaemic reperfusion inflammatory responses leading to increased myocardial injury. CABG surgery can therefore be used as a clinical model of myocardial infarction and inflammation. We investigated this with blood markers of inflammation, MRI scanning and USPIO. Elafin inhibits the destructive and inflammatory HNE enzyme. Beyond this elafin inhibits inflammatory cytokines and modulates the innate and adaptive immune systems. In preclinical studies elafin treatment is associated with reduced myocardial injury. As such, elafin has a marked potential for the treatment of cardiovascular disease involving inflammation. Therefore, we hypothesised that elafin will reduce perioperative ischaemic myocardial injury and inflammation in patients undergoing elective coronary artery bypass graft surgery. We demonstrated for the first time that USPIOs are taken up by the infarct tissue in patients with recent myocardial infarction and by the peri-infarct myocardium to a lesser degree. This represents a novel non-invasive method to further study cardiac inflammation and therapeutic interventions. All patients undergoing CABG surgery demonstrated >10-fold elevation above the 99th centile of cardiac troponin by high sensitivity assay (hs-cTnI) indicating the current universal definition of type 5 myocardial infarction lacks specificity. A peak hs-cTnI at 6 hours following CABG surgery appears to be related to the surgical process and non-specific myocardial injury whilst a continuing increase at 24 hours suggests myocardial infarction. We would suggest hs-cTnI sampling at 6 and 24 hours post CABG surgery together with ECG assessment for the routine detection and diagnosis of type 5 MI. Differing levels of humoral makers inflammation post CABG surgery occurred, and did not correlate directly with the length of cardiopulmonary bypass time or hs-cTnI release. For the first time we identified differing levels of inflammatory cell infiltrate into the myocardium post CABG. This varied from none to levels similar to infarcted myocardial tissues. Elafin did not attenuate myocardial ischemia-reperfusion injury and inflammation. Post-hoc analysis identified reduced cTnI concentrations at 6 hours in Elafin treated patients and it is possible that a bigger dose would have conferred protection out to 48 hours. Elafin did not attenuate the cellular infiltration into the myocardium post CABG surgery, but did appear to reduce inflammation in renal tissue. USPIO enhanced CMR holds major promise in the non-invasive assessment of myocardial inflammation post surgery.

Natural antimicrobials are peptides that are essential components of the innate immune system, providing broad-spectrum protection against bacteria, yeasts and some viruses. In addition to their innate immune activity, they exhibit properties suggesting they interact with the adaptive immune system. These functions imply they may be of particular importance in pregnancy. Intrauterine infection is responsible for approximately one third of cases of preterm labour, and normal labour is considered an inflammatory process, associated with leukocyte invasion of the uterine tissues and increased cytokine production. Little is known, however, about natural antimicrobial expression in pregnant reproductive tract. The aim of this thesis was thus to characterize natural antimicrobial production in pregnancy. The study focused on two main areas - the lower genital tract, comprised of the vagina and cervix; and the innermost fetal membrane, the amnion. In the lower genital tract, levels of natural antimicrobials were determined in samples of cervicovaginal secretions collected from pregnant women, using enzyme linked immunosorbance assay (ELISA). In addition Taqman quantitative PCR and ELISAs were used to investigate natural antimicrobial production by cell lines derived from endocervical, ectocervical and vaginal epithelium. It was found that elafin and secretory leucocyte protease inhibitor (SLPI) were found at high concentrations in cervicovaginal secretions, but levels were diminished in women with the common vaginal infection bacterial vaginosis (p<0.05). Cells derived from the vaginal epithelium expressed greater amounts of elafin than cervically derived cells. However, elafin and SLPI production could be stimulated in endocervical cells by the bacterial product lipopolysaccharide, a response that was not seen in the vaginal cell line. Natural antimicrobial production in the amnion was examined in tissue explants and primary cultured amnion cells, using a combination of Taqman PCR and ELISAs. In addition, cDNA microarray was carried out to investigate factors controlling amniotic antimicrobial production, and the involvement of signalling pathways was studied using specific pathway inhibitors. It was shown that the amnion expressed five antimicrobials: human beta defensins (HBD) 1, 2 and 3, SLPI and elafin. Expression of HBD2 was significantly upregulated following normal labour (p<0.05), with production in primary amnion epithelial cells dramatically increased by IL-1ß. The pattern of HBD2 expression in response to IL-1ß was biphasic, which suggested involvement of a secondary gene product. Several putative influential factors were identified by cDNA micorarray, including the NF-kappaB cofactor NFkappaBinhibitorζ. Its relationship to HBD2 was explored. The involvement of both NF-kappaB and mitogen activated protein (MAP) kinase p38 signalling appeared crucial in the response. This work has shown that natural antimicrobials are expressed by both the lower genital tract, where infections that are associated with preterm labour originate, and in the amnion, which is the fetus last line of defence to infection. They may have an important role in the prevention of infection associated preterm labour. Further characterization of these responses may increase understanding of the physiology, and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

Dendritic cells (DC) are sentinels of the immune system that display an extraordinary capacity to present antigen to naïve T cells and initiate immune responses. DCs are distributed throughout the lungs in the conducting airways of the tracheobronchial tree and in the parenchymal lung, and play a pivotal role in controlling the immune response to inhaled antigens. The respiratory surface is continually exposed to potentially injurious particulates and pathogenic organisms, to which tightly regulated innate and adaptive immunological responses are made. The airways are usually sterile in healthy individuals. However, patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) have increased susceptibility to microbial infections and increased neutrophil elastase (NE) in lung secretions. This thesis was designed to test the hypotheses that; (i) excess NE may result in a dysregulation of lung DCs function in pulmonary chronic diseases, and (ii) the natural NE inhibitors in the respiratory system are able to rescue the NE-mediated dysregulation of DCs and potentially enhance their antigen presenting activity. The data in this thesis demonstrate that purified human NE down-regulated murine bone marrow (BM)-derived DC co-stimulatory molecules (CSM; CD40, CD80 and CD86), which was due to its proteolytic activity. NE-treated LPS-matured DCs were less efficient at presenting ovalbumin (OVA) peptide to naïve OVAspecific transgenic (D011.10) T cells. In addition, immature DCs (iDC) simultaneously treated with LPS and NE failed to mature fully and produced significantly less IL-12 and TNF-α than DCs matured in the presence of LPS alone. Similarly, treatment of mature DC (mDC) with pooled and individual COPD and CF sputum samples caused a reduction in CD80 and CD86 levels (but not CD40) which positively correlated with the NE concentration present in the samples. The demonstration that NE could adversely affect DC phenotype and function suggested that augmentation of NE inhibitors could reverse this process and preserve DC function in inflammatory microenvironments. Over-expression of an NE specific inhibitor (elafin) in the lungs of mice (using either replication-deficient adenovirus [Ad] or elafin transgenic [eTg] mice) increased the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung DCs in in vivo models. Replication-deficient Ad vectors encoding NE inhibitors, namely elafin, secretory leukocyte protease inhibitor (SLPI) and α1-protease inhibitor (α1-PI), were also used to infect DCs in vitro, to further study the effect of these NE-inhibitors on DCs in isolation. These findings suggest that purified NE and NE-containing lung inflammatory secretions are powerful down-regulators of DC maturation, resulting in reduced capacity of these potent APCs to efficiently present antigens; whereas, NE inhibitors could boost immunity by increasing the activation state and/or number of DCs.

<p>Abstract Redacted.</p> / Master of Science (MSc)

<p>Sexually transmitted infections (STIs), especially HIV/AIDS and HSV-2, continue to be a devastating burden on societies around the world. The close link between HSV-2 and HIV-1, the role of inflammation in driving these infections, and the limited success and availability of prophylactic and therapeutic measures underscore the need for continued search of alternative means of protection. Characterization of endogenous antimicrobials, especially those local to the female genital tract and actively regulating inflammatory and antiviral responses, could be beneficial for microbicidal trials. Although regulators of mucosal immunity, such as serine antiproteases, trappin-2 and elafin (Tr/E), have been associated with resistance to HIV-1, their antiviral activity remains poorly understood. Thus, the research presented in this thesis centers on characterization of antiviral properties of Tr and E individually and their potential mechanisms in defense against HSV-2 and HIV-1 in the female genital mucosa. Chapter 2 examines Tr/E contribution to antiviral host defense responses elicited by a synthetic mimic of viral dsRNA, polyI:C. Chapter 3 documents the presence and characteristics, including potential mechanisms, of antiviral activity of Tr/E against in vitro and in vivo HSV-2 infection. Chapters 4 and 5 determine the contribution of Tr/E to the natural anti-HIV-1 protection of CVL and structural characteristics, mode(s) of action, and cellular distribution/localization of antiviral Tr/E proteins. Therein, we present novel properties of each Tr/E by demonstrating their inhibitory and multiple effects against both HSV-2 and HIV-1. These effects appear to be mediated either through virus or cells and be associated with altered viral attachment/entry, transcytosis and infection, innate viral recognition, modulated inflammation and increased antiviral protection of cells. Reported antiviral activity of Tr/E was also contextual and exerted, at least in HEC-1A cells, via autocrine/paracrine mode and depended on elafin’s nuclear localization and its unmodified N-terminus. Tr/E may represent viable candidates for further studies in the field of STIs.</p> / Doctor of Philosophy (PhD)