Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 5 uppsatser.

Thesis (M.Sc.)--Victoria University of Wellington, 2009. / "Malaghan Institute of Medical Research." Includes bibliographical references.

Thèse (M.Sc.)--Université Laval, 2009. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

The clock-controlled nuclear receptor REV-ERBα has emerged as a critical regulator of multiple pathways involved in metabolism, development and immunity. Recent evidence has highlighted a major role for the clock in epithelial cells regulating lung inflammation, mediated by control of neutrophil chemokine expression. In this thesis, I examined the role of REV-ERBα in pulmonary immunity, using in-vivo gene targeting and nebulised lipopolysaccharide (LPS), a model for gram-negative bacterial infection, ex-vivo cell biology approaches and in vitro cell models. Initial studies of Rev-Erbα knock-out mice revealed an increase in pulmonary neutrophilia and inflammation upon aerosolised LPS challenge. Moreover, by selectively deleting the REV-ERBα DNA binding domain (DBD) in the mouse bronchial epithelium, I observed exaggerated inflammatory responses to LPS and augmented CXCL5 secretion. Interestingly, a dual deletion of REV-ERBα DBD and REV-ERBβ in mouse bronchial epithelium had a more dramatic effect on neutrophil recruitment and chemokine secretion than deletion of just the REV-ERBα DBD; in both basal and bacterial challenged conditions. Ex-vivo analysis revealed bronchial epithelial cells and macrophages both responded to novel REV-ERBα synthetic ligand GSK1362 but displayed divergent inflammatory responses in presence of this compound. Finally, I observed a striking loss of REV-ERBα protein upon pro-inflammatory challenge. Further analysis revealed this degradation was dependent on the 26S proteasome and driven by sumoylation and ubiquitination of REV-ERBα. However, by using novel REV-ERB ligand GSK1362, these post-translational modifications were blocked and the protein protected from degradation. Collectively, my results propose a new model for a central role for REV-ERBα in conferring clock control to lung neutrophilic inflammation. I have also identified a feed-forward circuit activated by inflammatory stimuli, leading to suppression of the endogenous anti-inflammatory REV-ERBα protein. Finally, I have discovered a novel mechanism for small-molecule regulation of REV-ERBα, operating via suppression of endogenous protein ubiquitination process. These observations implicate REV-ERBα as a novel therapeutic target in human inflammatory disease.

Objectives – Cognitive ageing is an inevitable part of human life. Research from disciplines such as epidemiology, medicine and neuroscience implicate a wide range of determinants in the pathophysiological processes that lead to clinical symptoms of neurodegeneration. Markers of systemic inflammation are postulated to play an important role in mechanisms underlying a neuro-pathological cascade, either directly, through neuro-inflammatory processes, or through the mediating effect of diseases that are associated with cognitive deficits, such as cardiovascular disease and variation and disruption to cerebral blood flow. This may be particularly important in people with type 2 diabetes, where the increased prevalence of vascular events and glycaemic upset along with elevated levels of various circulating biomarkers, have been implicated in accelerated cognitive decline. Increasingly, evidence suggests a contribution of vascular disease state in the development of Alzheimer’s disease in which inflammation could be a significant factor. Determining the direction of association between individual markers of inflammation and altered cognitive performance is important in order to understand the possible role of inflammation in the development of cognitive decline and to inform the development of preventive clinical interventions. Therefore investigating these risk factors in relation to the trajectory of age related cognitive decline is crucial; in this respect, longitudinal evidence, detecting change in cognitive performance over a defined period of time, is most appropriate. To date, the majority of evidence is inconclusive, predominantly due to methodological obstacles embedded in the prospective design of cognitive ageing studies and in the investigation of a complex disease state, such as insufficient follow up period and restricted cognitive assessment. Since associations reported from modelling late life cognitive change in epidemiological studies may be the result of confounding variables, such as gender, vascular risk factors/disease, education attainment and social status, investigating the causal nature of inflammatory mediators in cognitive decline, has proved more problematic. Additionally, even a casual association may be due to ‘reverse causation’. One method of unravelling such associations is through the use of genetic association, where the exposure variable of interest (such as genetic variants affecting plasma biomarker levels) is modelled against the outcome, thereby overcoming some of the problems of confounding and reverse causation inherent in non-genetic epidemiological studies. Aim – The primary aim of this thesis was to test for associations of baseline measures of acute-phase proteins (fibrinogen and C-reactive protein) and central pro-inflammatory cytokines (interleukin – 6 and tumour necrosis –α) with four-year change and estimated life-time change in cognitive ability in older people with type 2 diabetes. The second aim was to explore the association between fibrinogen-related SNPs (SNPs shown previously to be associated with altered plasma fibrinogen levels) and cognitive ability in the general population. Methods –Data from the Edinburgh Type 2 Diabetes Study (the ET2DS), a prospective epidemiological study of older people with type 2 diabetes were available, including that collected at a baseline clinic (2006-07) on 1066 participants, mean age 67.9 years (SD 4.2). For the present study, follow up cognitive assessment was carried out after four-years (2010-11) at which cognitive data was collected on 828 survivors. Cognitive ability at both time points was assessed using the same, comprehensive, seven neuropsychological tests battery, including measures of fluid as well as crystallised intelligence (vocabulary test). Principal component analysis was conducted to derive a general cognitive factor ‘g’ and a general inflammatory factor, derived from individual cognitive scores and from baseline measures of four inflammatory markers (fibrinogen and C-reactive protein, interleukin – 6 and tumour necrosis –α), respectively. Genotype and cognitive data were collected from seven, well-established population-based cohorts with clearly defined sampling frames and data collection procedures. Five cohorts comprised of community-dwelling elderly people, living in central Scotland (AAA Trial, n = 2061, EAS, n = 534; ET2DS, n = 1045; LBC 21, n = 517; LBC 36, n=1005) and two large were cohorts based in England (ELSA; n = 5458; and Whitehall II; n = 3400). In total, genotype and cognitive data were available for 14033 participants, age range between 60 to 80 years. In all studies cognition was assessed on three cognitive domains: memory, executive functioning and information processing. Compatibility of cognitive data allowed for calculation of a general cognitive factor ‘g’ that was comparable between all cohorts. The instrument variables consisted of 61 fibrinogen-related polymorphisms within 13 different loci. These were identified through a detailed literature search as well as through search of relevant, genetic databases. Results – in the ET2DS, the age and sex-adjusted analyses revealed statistically significant associations between raised plasma inflammatory markers and poorer ‘g’ at follow-up; this was observed for all biomarkers, with the strongest associations detected for IL-6 and the general inflammation factor (p values <0.001). These findings persisted in linear regression models of baseline biomarker levels with four-year cognitive change as well as estimated life-time change – here the general inflammatory factor and plasma IL-6 levels were the strongest predictors. Adjustment for conventional vascular risk factors and cardiovascular disease attenuated the associations of cognitive decline with fibrinogen, CRP and TNF-α; associations were largely attenuated in analyses assessing IL-6 and the general inflammation factor and tended to remain statistically significant. Meta-analysis was conducted in order to explore associations between pre-selected fibrinogen-related SNPs and impairment in general cognitive ability as indexed by ‘g’. The analysis identified five plasma fibrinogen-related SNPs that were significantly associated with impaired ‘g’ at the nominal threshold level of p < 0.05. These were: rs2070016 (FGB gene); rs2070016 (FGA gene); rs1800497 (ANKK1 gene); rs4251961 (IL1RN gene) and rs1130864 (CRP gene). Discussion – the results of the ET2DS indicate that in an elderly diabetic population, there is a significant relationship between baseline levels of circulating inflammatory markers and four-year cognitive change as well as estimated life-time cognitive decline. These associations were generally independent of common cardiovascular risk factors and events, suggesting a possible pathway where cytokine-induced activation of glial cells may be responsible for the consequent neuro-inflammatory processes resulting in declined cognitive ability. The lack of some associations may be due to a relatively short follow up period. The main strength of the ET2DS was the availability of prospective cognitive data, the large sample size and the use of a comprehensive cognitive battery, including a vocabulary test for crystallised intelligence and thus calculation of estimated life-time cognitive change. Genetic association analysis indicated a significant association between five preselected SNPs each located within different genes (in general, genes associated with inflammation), and impaired general cognitive ability. This provides some support for a causal role of inflammation in age-related general cognitive impairment. One of the major strength was the use of a large dataset and the applied methodological approach. Meta-analysis was conducted on raw, prospectively generated data, allowing determination of the cognitive phenotype variable. / The main outcomes of this thesis suggest that systemic inflammation may indeed be involved in aetiology of age-related cognitive decline, possibly via neuro-inflammation. Further epidemiological investigation should involve a measurement of biomarkers trajectories in modelling cognitive change. Use of a stronger genetic instrument for inflammatory biomarkers, modeled against cognitive decline rather than cognitive ability as in the current study could further advance knowledge of the bio-pathological mechanisms underlying age-related cognitive decline. Results could ultimately inform subsequent investigations in the form of a randomised control trial, testing an evidence-based anti-inflammatory clinical intervention in diabetic populations as well as the general populations.

Chronic inflammation is associated with a dysregulation of the immune response. Inflammation is also associated with decreased reproductive capacity in women, however an exact mechanism has yet to be identified. Physiological states such as obesity and disease states such as endometriosis are both associated with chronic inflammation, an insufficient immune response, and infertility in women; therefore these two conditions serve as excellent models to study the effects of inflammation on reproductive function. Early indications of inflammation may aid in early detection of immune dysregulation associated with different physiological and pathological conditions. One way to measure immune balance between pro- and anti-inflammatory states in the female urogenital tract is by observing changes in the bacterial species that populate the mucosal surface. Commensal bacteria that make up the microbiome play a critical role in the development and maturation of the immune system in humans. Because these microbes and the host’s immune system are constantly influencing each other, several immunological conditions and disease states have been shown to have an altered microbial profile than that of healthy individuals. The goal for this study was to examine how triggers of inflammation alter the peripheral immune response, urogenital microbial communities, and reproductive function. Specifically, our aims were to 1) use an animal model of obesity to determine whether this physiological model of inflammation decreases immune protection of the urogenital microbiome and alters ovarian function; and 2) use endometriosis as a disease model of inflammation to assess whether the presence of endometriotic lesions alters urogenital microbial dynamics, and also whether surgical intervention restores commensal bacterial profiles to that of a non-disease state. The results of this study revealed that the urogenital microbial community dynamics were altered in both our obese and disease models of inflammation compared to their respective controls. In the obesity study, we also found that our obese model had decreased markers of inflammation, which may be due to dietary composition. In the endometriosis study, we observed that patients with disease had a unique urogenital microbiome profile and that surgery had an effect in shifting the urogenital microbial profiles of several patients. Overall, our long-term goal is to determine whether the urogenital microbiome is a good indicator of immune stress and if alternative therapies can alter microbial community dynamics, eliminate immune stress associated with disease. Ultimately we are looking at the microbiome as an indicator of overall immune health and implementing alternative diagnostic and treatment strategies to immune diseases that affect reproductive function in women.

Background: Previous studies have shown that patients with obsessive-compulsive disorder (OCD) have elevated interleukin and chemokine levels in plasma. The purpose of this study was to investigate and validate whether a group of cytokines and chemokines are elevated in a cohort of young adult OCD patients. Methods: A total of 43 patients (11 male/32 female) and 45 controls (15 male/30 female) with OCD were included in the study. The subjects were assessed with the Structured Clinical Interview for DSM-IV Axis I Disorders- Clinical Version or Mini-International Neuropsychiatric Interview. The control group was screened with the Alcohol Use Disorders Identification Test. Proximity extension assay (PEA) was used to measure plasma levels of IL-6, IL-8, MIP-1α, and IL-10. Results: A factor analysis for the cytokines was performed and logistic regression analysis revealed that the cytokines as a group have a significant association for OCD (P=0.031, OR: 2.2) and IL-8 was the cytokine with the highest significance (P=0.007) for the patient group. Conclusion: These findings suggest that this group of cytokines are associated with OCD diagnosis and strengthens previous findings of immune activity in the etiology of OCD. Therefore cytokines and chemokines could have an active role in the etiology of OCD and PEA could be useful in the search for biomarkers.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O Acetato de lupeol (FAL), isolado do lÃtex extraÃdo do caule de Himatanthus drasticus (APOCYNCEAE), à quimicamente classificado como sendo um triterpeno pentacÃclico pertencente à classe do lupano, foi avaliado em modelos de nocicepÃÃo e inflamaÃÃo. No teste das contorÃÃes abdominais induzidas por Ãcido acÃtico (10 ml/kg, i.p.) em camundongos, a FAL (50 e 100 mg/kg, i.p.) reduziu de forma significativa o nÃmero de contorÃÃes abdominais em 56 e 61%, respectivamente, e a indometacina (10 mg/kg, i.p.) reduziu em 66%. No teste da formalina, a FAL (25 e 50 mg/kg, i.p.) reduziu de forma significativa o tempo gasto pelo animal lambendo a pata, tanto na fase inicial (21 e 46,5%, respectivamente) quanto na fase tardia (57,6 e 61,3%, respectivamente) e a morfina (7,5 mg/kg, i.p.) reduziu em 62 e 91%, respectivamente. O prÃ-tratamento com Naloxona (2 mg/kg, i.p) reverteu de modo significativo, os efeitos da FAL e da morfina tanto na fase inicial quanto na tardia do teste da formalina. No edema de pata induzido por carragenina, a FAL (10, 25 e 50 mg/kg,i.p.) reduziu de modo significativo, o volume do edema na 1Â, 2 e 3 hora apÃs a aplicaÃÃo da carragenina (1%, 50&#956;l, s.p.). AnÃlise histopatolÃgica do tecido de pata de camundongo submetido à carragenina, demonstrou reduÃÃes significativas no edema e do infiltrado celular. Na marcaÃÃo imunohistoquÃmica, em tecido de pata de camundongo submetida ao estÃmulo da carragenina, a FAL (50mg/kg, i.p.) promoveu uma discreta reduÃÃo na expressÃo de TNF- &#945;, porÃm causou uma significante reduÃÃo dos nÃveis de iNOS teciduais. No edema de pata induzido por dextrano, a FAL (12,5 e 25 mg/kg, i.p.) reduziu de modo significativo, o volume do edema na 2 e 3 hora apÃs a aplicaÃÃo de dextrano (12%, 50&#956;l, s.p.). Na peritonite induzida por carragenina, a FAL (1, 10 e 20 mg/kg, i.p.) diminuiu de forma significativa, o nÃmero de leucÃcitos em 56, 80 e 92%, respectivamente. A Pentoxifilina (1 e 25mg/kg, i.p.) inibiu em 39 e 68%, respectivamente o nÃmero de leucÃcitos. No teste da inibiÃÃo da atividade da enzima mieloperoxidase (MPO), a FAL (10, 25, 50 e 100 &#956;g/ml) reduziu a atividade da MPO em 36, 80, 79 e 74 %, respectivamente. A FAL nÃo demonstrou atividade antioxidante no teste do DPPH. Em conjunto, esses dados revelam que a FAL apresenta atividade antinociceptiva, que pode ser explicada pela habilidade deste composto em mimetizar efeitos de opiÃides endÃgenos, e antiinflamatÃria, explicada pela diminuiÃÃo da expressÃo de TNF-&#945; e iNOS, bem como pela diminuiÃÃo da atividades da mieloperoxidase, resultando na inibiÃÃo da migraÃÃo de leucocitÃria para o foco da inflamaÃÃo. / The lupeol acetate (FAL), isolated from the latex extracted of the stem of Himatanthus drasticus (APOCYNCEAE) is chemically classified as a pentacyclic triterpene belonging to the lupane class, was evaluated in nociception and inflammation models. In the writhing test induced by acetic acid (10 ml/kg, i.p.) in mice, FAL (50 and 100 mg/kg, i.p.) significantly reduced the number of writhing in 56 and 61%, respectively, and indomethacin (10 mg/kg, i.p.) reduced by 66%. In the formalin test, FAL (25 and 50 mg/kg, i.p.) significantly reduced the time spent by the animal licking the paw, both in the initial phase (21 and 46.5%, respectively) and in the late phase (57 , 6, and 61.3%, respectively) and morphine (7.5 mg/kg, i.p.) reduced by 62 and 91%, respectively. Pretreatment with naloxone (2 mg/kg, i.p.) significantly reversed the effects of FAL and morphine in both the early and in late phase in formalin test. In the carrageenan induced paw oedema, FAL (10, 25 and 50 mg/kg, i.p.) reduced in significant way the oedema volume in the 1st, 2nd and 3rd hour after carrageenan application (1%, 50&#956;l, s.p.). Histopathologic analysis of mice paw tissue subjected to carrageenan, showed significant reductions in oedema and cellular infiltration. In the immunohistochemical staining in mice paw tissue subjected to carrageenan stimulus, the FAL (50mg/kg, i.p.) induced a slight reduction in the expression of TNF-&#945;, but caused a significant reduction of tissue iNOS levels. In the dextran induced paw oedema, FAL (12.5 and 25 mg/kg, i.p.) significantly reduced the oedema volume in the 2nd and 3rd hour after dextran application (12%, 50&#956;l, s.p.). In carrageenan-induced peritonitis, FAL (1, 10 and 20 mg/kg, i.p.) significantly reduced the number of leukocytes at 56, 80 and 92%, respectively. Pentoxifylline (1 and 25mg/kg, i.p.) inhibited by 39 and 68%, respectively, the number of leukocytes. In the myeloperoxidase (MPO) enzyme inhibition test, FAL (10, 25, 50 and 100 &#956;g/ml) reduced the activity of MPO at 36, 80, 79 and 74%, respectively. The FAL showed no antioxidant activity in DPPH test. Together, these data reveal that the FAL has antinociceptive activity, which can be explained by its ability in mimicking the endogenous opioids effects, and antiinflammatory, explained by TNF-&#945; and iNOS expression decreased, as well as by the myeloperoxidase activity decreasing, resulting in inhibition of leukocyte migration to the focus of inflammation.

Abstract This work engaged Finnish females affected with osteoarthritis (OA) of the hand to define the role of common sequence variations within the genes of the important structural protein of cartilage, aggrecan (AGC1), and the genes of inflammatory mediators, the interleukin 1 gene cluster and interleukin 6 (IL6), as possible risk factors for the disease. Also, a genome-wide linkage analysis was performed in a sample consisting of Finnish families with multiple individuals affected with hip and knee OA in order to reveal new chromosomal areas that are likely to contain disease associated variations. OA is a chronic disease that leads to the degeneration of articular cartilage in synovial joints. The etiology of the disease is for the most part unknown. Joints of the hand, hip and knee are most commonly affected, and obesity, trauma and excess mechanical stress are known risk factors for the disease. OA also has a significant genetic component. AGC1 carries a variable number of tandem repeats (VNTR) polymorphism, which may be significant for the biomechanical properties of cartilage. It was shown that the most common allele with 27 tandem repeats is protective against hand OA (HOA) (odds ratio 0.46, 95% confidence interval 0.27–0.78). Also, carrying two copies of any of the shorter or longer alleles increased the risk of the disease. Inflammation seems to play a role in the etiology of OA and certain polymorphisms within the interleukin 1 gene cluster and IL6 have been previously shown to increase the transcription of these molecules and to associate with OA. In this study it was shown that the G alleles in three common IL6 promoter single nucleotide polymorphism (SNP) sites are associated with the risk of more severe forms of HOA (p =  0.001 for GGG haplotype). A SNP in IL1B associated with the bilateral form of the disease (p =  0.006) and two IL1B-IL1RN extended haplotype alleles were associated with the same phenotype. Genome-wide and fine mapping linkage analyses recognized chromosomal locus 2q21 with a multipoint LOD score of 3.96. Despite the association analyses of several candidate genes within the locus, no disease-associating sequence variants were identified.

CXCR4 is a chemokine receptor that has been linked to several disease related pathways including: HIV-1 proliferation, autoimmune disorders, inflammatory disease and cancer metastasis. The interaction of the C-X-C chemokine receptor type 4 (CXCR4) with C-X-C chemokine ligand 12 (CXCL12) plays a key role in triggering these disease related pathways. Various antagonists for these receptors have been synthesized and tested, but many are not useful clinically either because of toxicity or poor pharmacokinetics. Some of the most extensive CXCR4 antagonist libraries stem from a class of compounds, p-xylyl-enediamines, which all feature a benzene ring as the core of the compound. This work focuses on the design and synthesis of a new class of compounds that show potential as CXCR4 antagonists by using heterocyclic aromatic rings (2,6-pyridine, 2,5-furan, 2,5-pyrazine and 3,4-thiophene) as the core of the scaffold. After synthesis, these analogues were probed through a variety of assays and techniques by our collaborators in the Shim lab at Emory University including: preliminary binding assays, Matrigel invasion assays, carrageenan mouse paw edema tests, and in silico analysis. In silico analysis also probed 2,5-thiophene-based analogues previously synthesized. This work has produced the beginnings of a new library of CXCR4 antagonists and has identified fifteen hit compounds that are promising leads for further testing and modification.