Neutrophils and macrophages accumulate at sites of inflammation and cause chronic inflammation leading to various diseases. Therefore, to better understand chronic disease pathways it is important to investigate the properties of macrophage accumulation in inflamed tissues. The I-domain of the macrophage receptor integrin aDb2 plays a vital role in macrophage retention by binding to CEP (carboxyethyl pyrrole), a ligand available at inflammatory sites. This thesis mainly focuses on evaluating the binding site within integrin aDb2 that binds carboxyethyl pyrrole (CEP)-modified proteins. So, a recombinant plasmid construct containing the integrin I-domain was developed. Seven non-conserved amino acids were mutated by PCR-site-directed mutagenesis to create a mutant construct. After expressing in E. coli, the binding affinities of wild-type and mutant I-domains to CEP were analyzed using biolayer interferometry. It was found that a patch of seven positively charged amino acids contributes to the strong binding of the I domain to CEP.

With the introduction of antiretroviral therapy (ART), human immunodeficiency virus (HIV) has progressed to a chronic inflammatory disease with accelerated, subclinical end-organ damage, specifically cardiovascular disease (CVD). People with HIV (PWH) have higher incidence, risk, and mortality from CVD, such as atherosclerosis, diastolic dysfunction, and heart failure. Several recent clinical reports have shown that PWH have a predisposition to developing heart failure with preserved ejection fraction (HFpEF), presenting with pathological concentric hypertrophy, diffuse fibrosis, and diastolic dysfunction. As such, an investigation into immunological and molecular mechanisms promoting pathological changes in the heart is necessary. Recent clinical reports show that people living with HFpEF have elevated plasma osteopontin (Opn), and plasma Opn is a powerful predictor of HFpEF severity, HFpEF-related hospitalizations, and mortality. Second, several animal models of HFpEF phenotypes have suggested Opn is involved in driving or perpetuating diastolic dysfunction and cardiac fibrosis. Therefore, we investigated changes in Opn in a cohort of PWH and two translationally relevant models of HIV infection (non-human primates and humanized mice) to identify the pathological role of Opn in cardiac fibrosis and detail the adjunctive potential of Opn for PWH presenting with HFpEF. In Chapter 2, twenty asymptomatic, antiretroviral-treated women with HIV (WHIV) and fourteen women without HIV (HIV-women) matched on age and body mass index underwent cardiac magnetic resonance imaging (MRI) and immune phenotyping. First, we compared a number of immunological parameters to the extensive cardiac MRI parameters in WHIV. Secondly, we analyzed relationships between plasma Opn with cardiac structure and function and markers of immune activation among WHIV, HIV- women, and the whole cohort. Multivariable modeling among the whole group was performed using myocardial fibrosis and myocardial steatosis, respectively, as the dependent variable and HIV status, atherosclerotic cardiovascular disease (ASCVD) risk score, and plasma Opn as independent variables. Among WHIV, multi-variable modeling was performed using plasma Opn as the dependent variable and CD4+ T cell count, HIV viral load, and the respective immune parameter, relating to plasma OPN in bivariate analyses, as an independent variable. In Chapter 3, we investigated bulk transcriptomic changes in the left ventricle of the heart in a model of HIV infection. We utilize the highly translatable simian immunodeficiency virus (SIV)-infected rhesus macaque model to identify changes in the myocardium with and without ART. Animals were selected by viral load, with SIV-infected animals having a high titer of plasma viral load and the SIV-infected animals with ART having a reduction in viral load by several logs. We performed total RNA-Seq on left ventricle tissue from uninfected animals, SIV-infected animals, and SIV-infected animals receiving a clinically relevant ART regimen. SIV infection led to high plasma viral load, but little to no SIV RNA was detectable in the left ventricle, shown by minimal of SIV RNA+ cells in the heart and no SIV sequences identified from RNA-Seq. SIV infection produced a highly inflammatory reaction in the heart, predominated by interferon and pathogen response. Additionally, interferon gamma (IFNg) and lipopolysaccharide (LPS) were both identified as potential upstream drivers of transcriptomic changes in the heart from SIV infection. Reduction of viral load by ART reduced the interferon and cytokine response in the heart; however, SIV-infected animals receiving ART exhibited decreased expression of integral genes directly involved in fatty acid (FA) metabolism, carnitine shuttling, and beta-oxidation. In Chapter 4, we use both in vitro and in vivo modeling to identify molecular mechanisms involved in the development of cardiac fibrosis. We utilized mouse embryonic fibroblasts (MEFs) modeled cardiac fibroblasts and were stimulated with IFNg, LPS, and TGF-b for phenotypic changes in contraction and cytokine production. The interplay of Opn and cardiac fibrosis was investigated in SIV-infected macaques with/without ART and HIVinfected humanized mice with/without an Opn-inhibiting RNA aptamer. LPS-stimulated MEFs retained myofibroblast-like contractility from TGF-b stimulation, secreted inflammatory cytokines/chemokines, and produced Opn (Spp1 transcripts). SIV-infected animals had elevated plasma Opn at necropsy, accumulation of full-length Opn in the ventricle, and ventricular interstitial fibrosis. Multivariate regression identified growth differentiation factor (GDF)-15, inflammatory CD14+CD16+ monocytes, and CD163 expression on CD14+ CD16+ monocytes as independent predictors of plasma Opn during SIV infection. HIV-infected humanized mice showed increased interstitial fibrosis compared to uninfected/untreated animals, and systemic inhibition of Opn by RNA aptamer reduced left ventricle fibrosis in HIV-infected humanized mice. These studies combined a clinical cohort of WHIV, SIV-infected rhesus macaques, and HIV-infected humanized mice to determine the role of Opn in the pathophysiology of cardiac deficits from HIV infection. Our primary goals were to begin to unravel the role of Opn in the development of HFpEF phenotypes seen in PWH, detail Opn as a converging biomarker of cardiac stress and remodeling and immune dysfunction in HIV infection, and investigate the therapeutic potential of Opn to reduce cardiac fibrosis from HIV infection. While Opn has a broad spectrum of physiological and pathological functions, we aimed to frame Opn as an important protein of interest in future studies into HFpEF in PWH. / Biomedical Sciences

Psoriasis is a chronic inflammatory disease of the skin and joints associated with several comorbidities such as arthritis, diabetes mellitus and metabolic syndrome, including obesity, hypertension and dyslipidaemia, Crohn’s disease, uveitis and psychiatric and psychological diseases. Psoriasis has been described as an independent risk factor for cardiovascular diseases and thus patients with psoriasis should be monitored for the development of cardiovascular disease or metabolic syndrome. However, there is mounting evidence that psoriasis also affects the development of osteoporosis, an important metabolic disease with enormous clinical and socioeconomic impact. At present, there are still controversial opinions about the role of psoriasis in osteoporosis. A more in depth analysis of this phenomenon is of great importance for affected patients since, until now, bone metabolism is not routinely examined in psoriatic patients, which might have important long-term consequences for patients and the health system. In the present review, we summarize current knowledge on the impact of psoriatic inflammation on bone metabolism and osteoporosis.

The heme protein myeloperoxidase (MPO) is a major constituent of neutrophils. As a key mediator of the innate immune system, neutrophils are rapidly recruited to inflammatory sites, where they recognize, phagocytose, and inactivate foreign microorganisms. In the newly formed phagosomes, MPO is involved in the creation and maintenance of an alkaline milieu, which is optimal in combatting microbes. Myeloperoxidase is also a key component in neutrophil extracellular traps. These helpful properties are contrasted by the release of MPO and other neutrophil constituents from necrotic cells or as a result of frustrated phagocytosis. Although MPO is inactivated by the plasma protein ceruloplasmin, it can interact with negatively charged components of serum and the extracellular matrix. In cardiovascular diseases and many other disease scenarios, active MPO and MPO-modified targets are present in atherosclerotic lesions and other disease-specific locations. This implies an involvement of neutrophils, MPO, and other neutrophil products in pathogenesis mechanisms. This review critically reflects on the beneficial and harmful functions of MPO against the background of immune response.

No description available.

Introduction: Cancer is the leading cause of death worldwide, and the risk increases as age increases. Additionally, chronic inflammation is highly prevalent in older adults, and is associated with cancer. In this respect, physical activity (PA) seems to act as a preventive tool of both cancer, and chronic inflammation, by exerting anti-inflammatory properties. However, current knowledge on links between physical activity and inflammatory biomarkers in older adults remains limited. Therefore, the present study aims to investigate the association between objectively assessed time in different physical activity intensities and pro-, and anti-inflammatory intermediated biomarkers of cancer risk in older European adults. Material and Methods: Men and women older adults (aged 65-79 years; N = 888) were recruited from four European centers. Accelerometer-based assessment of daily time spent sedentary (SED), in light (LPA), and in moderate-to-vigorous (MVPA) PA was conducted. The inflammatory markers C-reactive protein (CRP), tumor necrosis factor-α, interleukin-6, tumor growth factor-β1, leptin, interleukin-10 and adiponectin were assessed in blood samples using enzyme-linked immunosorbent assay (ELISA), and standardized procedures were used to define indicators of the metabolic syndrome. Linear regression analysis based on isotemporal substitution modelling with 30-minutes periods of different physical activity intensities was used and the analysis was stratified by biological sex. Results: Replacing 30 minutes of either SED or LPA with a corresponding time in MVPA was associated to reduced CRP levels in older men and women (P < 0.05). In older women, reduced leptin levels were associated with reallocation of time spent in SED with both LPA and MVPA, and with reallocation of time spent in LPA with MVPA (P < 0.05). In older men, replacing time in SED with either LPA or MVPA (P < 0.01) resulted in significantly reduced leptin levels. Finally, reallocation of 30 minutes in either SED or LPA with MVPA was associated with elevated adiponectin levels in older women only (P < 0.05). Conclusion: This study suggests that different important cancer-related biomarkers of chronic inflammation in older adults are affected by different intensity thresholds of physical activity and that the impact of physical activity is independent of several important confounding factors, including smoking, disease risk and medication.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24549号 / 薬科博第166号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Chronic obstructive pulmonary disease (COPD) including emphysema is a devastating condition, increasing in prevalence in the US and worldwide. There remains no cure for COPD, rather only symptomatic treatments. Due to unique challenges of the lung, translation of therapies for acute lung injury to target chronic lung diseases like COPD has not been successful. We have been investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. During the course of this work we have developed and characterized a lug derived ECM hydrogel that exhibits “injectability,” allowing cells or dugs to be delivered in a liquid and encapsulated at body temperature. The hydrogel self assembles in <5 minutes and achieves mechanical stiffness similar to other soft tissue ECM hydrogels. The hydrogel can support 3D cell growth and encapsulated cell viability. Encapsulated hMSCs can also still be activated by simulated inflammatory environments. Naïve mouse macrophages exposed to the fully formed gel were not significantly induced to express markers for pro or anti-inflammatory polarized phenotypes, but increased expression for several secreted inflammatory mediators was observed. We also investigated a novel approach for preparing and solubilizing the isolated ECM proteins, using digestion time as a variable for controlling hydrogel density (interconnectivity), mechanical stiffness, component protein size distribution, and cell behavior on fully formed gels. The potential future impact for the presented research includes optimization for future animal studies, expansion to additional applications, and the development of new derivative materials.

Evidências sugerem o envolvimento de um estado de inflamação crônica de baixo grau na fisiopatologia do transtorno bipolar (TB). Os estudos apresentados nesta tese tiveram como objetivo explorar o papel da inflamação crônica nos mecanismos fisiopatológicos do TB através da avaliação dos níveis séricos de proteína C-reativa (PCR). A PCR é um marcador de inflamação sistêmica comumente utilizado na prática clínica, sendo considerado fator de risco para várias patologias, incluindo câncer e doença cardiovascular. O primeiro artigo, através de um estudo de meta-análise, teve como objetivo avaliar o tamanho de efeito da associação entre níveis de PCR em pacientes bipolares nas diferentes fases de humor (n=730) comparado a indivíduos controles (n=888). Pacientes bipolares apresentaram níveis de PCR significativamente elevados em comparação ao grupo controle, com moderado tamanho de efeito (effect size, ES = 0.39; 95% IC, 0.24 – 0.55; P < 0.0001). Níveis de PCR foram significativamente maiores em pacientes maníacos (ES = 0.73; 95% IC, 0.44 – 1.02; P < 0.001) e em eutímicos (ES = 0.26; 95% IC, 0.01 – 0.51; P = 0.04). O segundo artigo se propôs a revisar dados da literatura relacionados a biomarcadores periféricos potencialmente implicados na progressão do TB. Pacientes em diferentes estágios do TB apresentaram níveis alterados de marcadores de estresse oxidativo, neurotrofinas e de inflamação, incluindo a PCR, o que reforça a hipótese da inflamação crônica exercer um papel importante na fisiopatologia do TB. Em seguida, considerando a abordagem multidimensional no TB, o terceiro artigo avaliou a reatividade emocional como uma dimensão relevante para caracterizar pacientes bipolares apresentando sintomas subclínicos de humor durante a fase de remissão (N=613). Apesar de todos pacientes estarem em remissão, a maioria deles (68%) apresentou reatividade emocional anormal (hipo ou hiper-reatividade emocional). Esse estudo avaliou, também, o funcionamento psicossocial nesses pacientes e os níveis de PCR ultra-sensível como um possível marcador objetivo de hiper-reatividade emocional no TB. Os pacientes com hiper-reatividade emocional, em comparação aos pacientes com hipo- ou normal reatividade emocional, apresentaram prejuízo cognitivo e níveis de PCR significativamente mais elevados (P < 0.001). Esses resultados provêm de um estudo transversal e, portanto, conclusões sobre causalidade dessas associações não podem ser inferidas, já que outros fatores, além dos níveis de PCR, podem também contribuir para o estado inflamatório crônico observado nesses pacientes. Em suma, os resultados desta tese sugerem que a inflamação crônica de baixo grau, evidenciada pelas alterações nos níveis de PCR, parece estar implicada na fisiopatologia e na progressão do TB. Novas intervenções terapêuticas com alvo em mecanismos inflamatórios e na modulação dos níveis de PCR devem ser priorizados em estudos futuros. / Evidence suggests that chronic low-grade inflammation appears to be involved in the pathophysiology of bipolar disorder (BD). The studies presented in this thesis aimed at exploring the role of chronic inflammation in the BD pathophysiological mechanisms by assessing serum levels of C-reactive protein (CRP). CRP is a marker of low-grade inflammation widely used in clinical practice, and a risk factor for cardiovascular and malignant diseases. The first article, a meta-analysis, aimed at evaluating the effect size of the association between CRP levels in bipolar patients (n=730) compared to healthy subjects (n=888). Overall, CRP levels were significantly elevated in patients with BD versus controls (effect size, ES = 0.39; 95% CI, 0.24 to 0.55; P < .0001). CRP levels were significantly higher in manic (ES = 0.73; 95% CI, 0.44 to 1.02; P < 0.001) and euthymic (ES = 0.26; 95% CI, 0.01 to 0.51; P = 0.04). The second paper aimed at reviewing the scientific literature regarding peripheral biomarkers potentially implicated in the progression of BD. Bipolar patients within different disease’s stages presented altered levels of oxidative stress, neurotrophins and inflammatory markers, including PCR. These findings reinforce the hypothesis of the potential role of the chronic inflammation in BD pathophysiology. Regarding the multidimensional approach in BD, the third article assessed emotional reactivity as a major dimension for better characterizing remitted bipolar patients with subthreshold mood symptoms (N=613). Although all patients were in remission, most of them (68%) showed abnormal emotional reactivity (hipo- or hyper-reactivity). In addition, this study assessed the psychosocial functioning in these patients as well as the levels of high-sensitivty PCR (hsCRP) as an objective marker of emotional hyper-reactivity in BD. Patients with emotional hyper-reactivity had higher levels of PCR and cognitive impairment compared to patients with emotional hypo or normal emotional reactivity (P < 0.001). This was a crosssectional study of emotional reactivity, hsCRP levels and functional status in remitted bipolar patients, and no conclusions regarding the causality of these associations can be substantiated. Others factors could also be contributing to the chronic inflammatory state in these patients. In conclusion, the results of this thesis suggest that low-grade chronic inflammation, as evidenced by alteration in CRP levels, may be implicated in the pathophysiology as well as in the BD progression. Novel therapeutic interventions targeting inflammatory mechanisms and the modulation of CRP levels should be prioritized in future studies.

Orientador: Aparecido Antonio Camacho / Resumo: O uso de betabloqueadores tem mostrado implicações significativas na terapia de neoplasias através do bloqueio de adrenoreceptores em tecidos tumorais. O sistema nervoso autônomo simpático apresenta um perfil pró-inflamatório e por estímulo das catecolaminas ocorre à ativação de macrófagos teciduais com liberação de citocinas inflamatórias. Estudos recentes sugerem que estímulos inflamatórios crônicos podem acelerar a progressão do câncer, fato este relacionado à ativação do sistema beta adrenérgico. Assim, o objetivo do presente estudo foi avaliar a influência do emprego do propranolol sobre a evolução macroscópica dos nódulos mamários assim como a monitoração da função cardíaca. O estudo foi prospectivo, randomizado e longitudinal. Para tanto, 06 cadelas portadoras de tumores de mama (G1) receberam cloridrato de propranolol (0.2 mg/kg/BID, VO, 30 dias) e, outras 08 cadelas também com neoplasia mamária (G0) receberam apenas medicação placebo (BID, VO, 30 dias). Foram realizados exames ecocardiográfico, eletrocardiográficos convencional e Holter, paquimetria tumoral e exame histopatológico. A análise estatística foi baseada em um estudo experimental, cujos resultados foram submetidos à análise de variância (ANOVA) com medidas repetidas no tempo e em seguida Teste de Tukey. O uso do fármaco mostrou segurança sobre os parâmetros cardíacos avaliados e controle sobre o crescimento tumoral quando comparado com o grupo placebo. / Abstract: The use of beta-blockers has shown significant implications in the therapy of neoplasias through the blockade of adrenoreceptors in tumor tissues. The autonomic sympathetic nervous system presents a pro-inflammatory profile and by stimulating the catecholamines occurs to the activation of tissue macrophages with the release of inflammatory cytokines. Recent studies suggest that chronic inflammatory stimuli may accelerate the progression of cancer, a fact related to activation of the beta adrenergic system. Thus, the objective of the present study was to evaluate the influence of the use of propranolol on the macroscopic evolution of the mammary nodes as well as the monitoring of the cardiac function. The study was prospective, randomized, and longitudinal. For this, 06 bitches bearing breast tumors (G1) received propranolol hydrochloride (0.2 mg / kg / BID, VO, 30 days) and another 08 female mammary glanders (G0) received only placebo medication (BID, VO , 30 days). Echocardiographic, conventional electrocardiographic and Holter tests, tumor pachymetry and histopathological examination were performed. The statistical analysis was based on an experimental study, whose results were submitted to analysis of variance (ANOVA) with measures repeated in time and then Tukey's test. The use of the drug showed safety over the evaluated cardiac parameters and control over tumor growth when compared to the placebo group. / Mestre