Orientador: João Martins Pizauro / Coorientador: Samir Issa Samara / Coorientador: Marco Antonio de Andrade Belo / Banca: Eduardo Maffud Cilli / Banca: Monica Valdyrce dos Anjos Lopes Ferreira / Banca: Fabiana Garcia / Banca: Luiz Flávio José dos Santos / Resumo: A Aeromonas hydrophila é um patógeno oportunista com capacidade de infectar peixes, mamíferos e humanos. É cosmopolita e responsável por infecções hospitalares em humanos e causadora da aeromonose em peixes. O uso de antibióticos na água, como forma terapêutica na aquicultura, apesar de controlar a infecção, favorece o desenvolvimento de resistência microbiana através do efeito residual destes fármacos no peixe e consequentemente no consumidor final humano, tornando-se um agravo para a saúde pública. Pensando nisso, foi produzido uma imunoglobulina Y (IgY), extraída da gema do ovo de galinhas poedeiras, específica para as proteínas de membrana e citoplasmática de A. hydrophila. No início, a IgY foi validada quanto a capacidade de detectar o patógeno no tecido de tilápias-do-nilo (Oreochromis niloticus) infectadas com a bactéria homóloga. Após a validação da IgY anti- A. hydrophila, a mesma foi testada in vitro na imunoterapia, e seus efeitos na atividade fagolisossômica de macrófagos (MΦs) de tilápias-do-nilo, infectados com A. hydrophila viva opsonizada/ neutralizada ou não. Os resultados provaram a eficácia e a aplicação da IgY para o ensaio de imunofluorescência direta (IMF) e imuno-histoquímica (IHC) validando a IgY para o diagnóstico e estudo fisiopatológico da infeção por A. hydrophila em tilápias-do-nilo. Na imunoterapia in vitro a IgY anti-A. hydrophila teve efeito positivo no controle da infecção, postulando ainda, o uso dos MΦs da tilápia-do-nilo como um importante ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Aeromonas hydrophila is an opportunistic pathogen with the capacity to infect fish, mammals and humans. It is cosmopolitan and is responsible for hospital infections among humans and for aeromonosis in fish. Although using antibiotics in water for therapeutic purposes in fish-farming has the effect of controlling infection, it also favors development of microbial resistance through the residual effect of these drugs on fish and consequently on the final human consumers. Thus, their use has become a public health hazard. With the aim of investigating alternative ways of combating A. hydrophila, an immunoglobulin Y (IgY) extracted from the yolk of the eggs of laying hens that was specific for the proteins of the membrane and cytoplasm of A. hydrophila was produced. This IgY was firstly validated regarding its capacity to detect A. hydrophila in the tissues of Nile tilapias (Oreochromis niloticus) that were infected with the homologous bacterium. The anti-A. hydrophila IgY was then tested in vitro for immunotherapy to determine its effects on the phagolysosomal activity of macrophages (MΦs) in Nile tilapias that were infected with live A. hydrophila that was either opsonized/neutralized or not. The results demonstrated that application of IgY was effective. Direct immunofluorescence (IMF) tests and immunohistochemical (IHC) analysis validated IgY for diagnosing the presence of A. hydrophila and for conducting physiopathological studies on A. hydrophila in Nile tilapias. In in vi... (Complete abstract click electronic access below) / Doutor

Today, it is estimated that 35 million people are living with human immunodeficiency virus (HIV). Since its initial discovery in 1981, researchers and medical providers have worked endless hours to understand the pathology, transmission, and medical management of HIV. In the early days of HIV, life expectancy after diagnosis was 10 years. However, after the development of zidovudine (AZT) in 1987, life expectancy of HIV patients began to slowly increase, albeit still lower than that of the general population. The development of AZT opened the door for more antiretroviral drugs and more drug classes. Now, patients undergo a triple drug regimen to manage HIV. These patients are able to maintain viral suppression and are no longer experiencing opportunistic infection or other AIDS-related conditions. While HIV is medically managed, this is a chronic condition and to-date, not cured. As opposed to opportunistic infections and other AIDS-related conditions, patients are succumbing to non-AIDs related conditions such as renal, neurological, bone disorders, and liver complications. The leading non-AIDs related condition is cardiovascular disease (CVD). Even with viral suppression, HIV infection itself contributes to the pathology and development of atherosclerosis and CVD. It is clear that chronic immune activation, HIV proteins, and dyslipidemia appear to be key factors in CVD development. Since the life expectancy of HIV patients has increased, physicians are now seeing an older generation of HIV patients. Medical providers are shifting focus toward understanding the long-term effects of not just HIV, but antiretroviral therapy (ART) as well. It appears that drug interactions and long-term toxicity augment CVD development. Protease inhibitors (PIs), compared to other ART drug classes, appear to increase the risk of atherosclerosis, especially through dyslipidemia. Due to management of HIV being life-long, compliance is difficult because of high pill burden, drug-drug interactions, and drug side effects. This can result in drug failure leading HIV patients to switch to second-line ART regimens. PIs are a common component of second-line ART regimens. Compared to other ART drugs, PIs have a high genetic barrier to resistance. However, PIs have a low bioavailability requiring high dosage and/or boosting with ritonavir (RTV). Lopinavir (LPV) boosted with RTV (LPV/r) is a favorable PI as it is used in a combination pill and is the most cost effective. However, multiple studies have shown LPV/r correlates more to CVD compared to other PIs. Patients on LPV/r exhibit an increased intima-medial thickening, a hallmark characteristic of atherosclerosis and an increased risk for myocardial infarction. Unfortunately, researchers are greatly conflicted as to why this is and in general why PIs increase the risk of CVD. Future medical treatment for HIV is complex and requires long-term medical management. In recent years, integrase inhibitors (IIs) have exhibited promise to provide better lipid profiles while maintaining viral suppression. However, as this drug class is relatively new and expensive, the financial burden on HIV patients is high. The next step toward addressing the global health issue of HIV is understanding the exact mechanism of how PIs contribute to CVD. This will not only increase the life expectancy of HIV patients, but reduce drug toxicity, non-AIDS related conditions, and increase adherence and viral suppression. It is clear that future research must be focused on understanding the role PIs have in CVD development. Physicians are seeing an older generation of HIV patients, and a vast majority are on second-line regimens. By understanding this relationship, researchers could design alternative drugs to manage CVD risk, by modifying current PIs or designing entirely new drugs.

Cancer survivors typically adapt well to life following remission; however, some patients still experience lingering negative moods that may develop into depression. There are multiple factors that can contribute to depressive symptoms from somatic symptoms, to emotion and social concerns. When analyzing these contributing factors, an underlying theory of a connection between the symptoms of depression and inflammation was revealed. Although this was not shown to be a causal association, it set forward some theories on the pathogenesis of depression in cancer survivors who may have persistent inflammation following treatment. As cancer patients are a growing group due to improvements in treatment and an aging general population, early identification of high-risk survivors and research into new treatment methods is essential. Depression in survivors can lead to an inability to follow through on medical care, decreased quality of life and poorer patient health outcomes. Currently, depressive symptoms are treated primarily on an individual basis, determining if there is a common underlying biological mechanism that could help scientists develop new guidelines for the treatment of survivors. Research studies demonstrated interdependence between conditions such as chronic pain, fatigue, sleep disturbance, cognitive impairment and depression. Current published literature on each of these conditions was explored and while each of these conditions have demonstrated connections to inflammatory pathways and specific cytokines, there does not appear to be one common underlying mechanism. Preliminary research has presented some options for future treatments that can mitigate the severity of the inflammation caused during traditional cancer therapies. These preventative measures address certain cytokine pathways that have been associated with negative side effects. As emotional and social concerns can add to a survivor’s stress causing stimulation of inflammatory pathways, evidence-based methods for reducing stress have been discussed and include exercise, psychosocial and occupational therapy interventions as well as legislative advocacy for better insurance coverage. There are some limitations in the current literature on the topic as much of the survivor research centers on patients who were diagnosed with breast cancer. There are several diverse subgroups of cancer survivors including childhood, adolescent and young adult, and adult cancer survivors, and comprehensive research should be conducted across these cohorts to ascertain which groups are at a higher risk for certain symptoms and stresses. Furthermore, research into novel treatment options has focused mainly on pharmacological solutions to the negative impacts of chemotherapy. While several studies have theorized about possible, persistent biological mechanisms underlying radiotherapy, few drugs have been suggested or developed to combat the late effects of inflammation including fibrosis. Many of the suggested treatments can be given as a co-treatment alongside of chemotherapy, radiotherapy or surgery; in the future, research should be conducted on the effects of reducing inflammatory levels in patients already in remission.

Das postoperativ neu aufgetretene Vorhofflimmern (POAF) ist die häufigste Herzrhythmusstörung nach einer Herzoperation und mit einer erhöhten Morbidität und Mortalität verbunden. Das Ziel der Studie war fibrotische und inflammatorische Biomarker sowie die kalziuminduzierte Kontraktionskraft im linken und rechten Vorhof als Prädiktoren für POAF zu bewerten. Von den 229 eingeschlossenen Patienten im Sinusrhythmus, die sich einer elektiven Herzbypass-Operation unterzogen haben entwickelten 38 Patienten ein POAF. Klinische und laborchemische Daten sowie echokardiographische Befunde wurden erhoben und kalziuminduzierte Kraftmessungen von geskinnten linken und rechten atrialen Muskelfasern durchgeführt. Patienten mit POAF waren älter, hatten signifikant größere LA-Fläche und RA-Fläche, eine höhere Prävalenz der arteriellen Hypertonie und signifikant geringere TAPSE-Werte. Von den getesteten Biomarkern für Fibrose und Entzündung waren MMP-9 und Pentraxin-3 signifikant verringert und NT-Pro-BNP und GDF-15 signifikant erhöht. Patienten mit POAF hatten zusätzlich signifikant niedrigere LA-Kraftwerte (pCa 5,5 bis 4,52; p <0,04) und RA-Kraftwerte (pCa 5,0 bis pCa 4,52; p <0,04). Die univariate Analyse ergab den LA-Kraftwert bei pCa 5,5 (p = 0,033), das fortgeschrittene Alter (p = 0,033), die LA-Fläche (p = 0,013), die RA-Fläche (p = 0,081) und das TAPSE (p = 0,01) als unabhängige Prädiktoren für POAF. Die Berücksichtigung dieser Parameter könnte die Identifizierung und Risikostratifizierung von Patienten mit POAF-Risiko unterstützen. / Objective: Postoperative new-onset atrial fibrillation (POAF) is common after cardiac surgery. Much less has been reported about the relationship between fibrosis, inflammation and calcium-induced left atrial (LA) and right atrial (RA) contractile forces and POAF. We sought to identify predictors of POAF: Methods: From August 2016 through February 2018, we evaluated 229 patients who had preoperative sinus rhythm (SR) before elective primary coronary artery bypass grafting (CABG). Of 229 patients, 191 maintained SR postoperatively, whereas 38 patients developed AF. Preoperative tissue inhibitor of metalloproteinases-1 (TIMP-1), pentraxin-3, matrix metallopeptidase-9 (MMP-9), galectin-3, high-sensitive C-Reactive Protein (hs-CRP), growth differentiation factor 15 (GDF 15) and transforming growth factor –ß (TGF-ß) were measured. Clinical and echocardiographic findings (Tricuspid annular plane systolic excursion [TAPSE]for right heart function) and calcium-induced force measurements (pCa) from LA and RA-derived skinned myocardial fibers were recorded. Results: Patients with AF were older (p=0.001), had enlarged LA (p=0.0001) and RA areas (p=0.0001) and decreased TAPSE (p=0.001). Levels of MMP-9 andpentraxin-3 were decreased (p<0.05), while GDF-15 was increased (p=0.001). We detected lower LA at pCa 5.5(p<0.05), pCa 5.4 (p<0.01) and pCa 5.3 to 4.52 (p=0.0001)and RA force values (pCa 5.0 to4.52; p<0,05) in POAF patients. Multivariable analysis identified advanced age (p=0.033), decreased LA force values at pCa 5.5 (p=0.033), enlarged LA (p=0.013) and RA (p=0.081) areas and reduced TAPSE (p=0.010) independently predicted POAF. Conclusions: Advanced age, decreased LA-force value at pCa 5.5, enlarged LA and RA areas and reduced TAPSE were identified as independent predictors for POAF.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is the main cause of dementia in the elderly population. AD is pathologically characterized by the accumulation of amyloid plaques and neurofibrillary tangles that results in neurodegeneration and loss of memory function. However, diagnosis of AD and characterization of biological mechanisms that lead to pathology and modulate risk for disease has proven to be extremely difficult. Cerebrospinal fluid (CSF) contains critical biomarkers for AD such as levels of amyloid beta (Aβ) phosphorylated-tau (p-tau), total-tau (t-tau), and neurofilament light chain (NfL). The CSF levels of these biomarkers are useful in determining AD status in a patient, but data collection can be time consuming, technically difficult, and expensive. While still subject to the limitations of obtaining CSF, cell free single stranded DNA (cfssDNA) is much cheaper and more reliably measured than these biomarkers. We investigated cfssDNA as a biomarker for AD status. We observed an association between low levels of concentration isolated from CSF as a potential biomarker for diagnosis of AD. Inflammation is a vital process in the immune system. Acute inflammation plays an essential role in the normal response to tissue injury. This inflammatory response initiates a cascade of cellular activation signals in innate immune cells resulting in increased production of proinflammatory cytokines and chemokines. These chemokines are essential to the recruitment and activation of other cells in the innate and adaptive immune system. Deviations from the normal production of these chemokines can result in disease status. Recently published work has identified genetic variants that show strong associations with AD-related chemokine levels in CSF and plasma. We attempted to characterize the biological mechanisms that underlie the reported associations between the ACKR2-V41A variant and CCL2 levels and the CCRL2-V180M variant and CCL4 levels. Our data demonstrate that the ACKR2-V41A receptor has a lower CCL2 binding affinity, scavenging efficiency, and receptor upregulation compared to ACKR2-WT. For CCRL2-V180M our data demonstrate higher binding affinity with chemerin and CCL19 than CCRL2-WT. Our data also show that while CCRL2-V180M and CCRL2-WT do not directly bind with CCL4, interactions between CCRL2-V180M and CCL19 alter the secretion of CCL4 from leukocytes. These findings provide evidence for a novel biomarker for AD diagnosis, mechanistic insights into the functional impact of common genetic variants on chemokine levels, and highlight a potential role of atypical chemokines in altering the risk for AD.

C1q/TNF-related protein 3 (CTRP3) is a secreted hormone that modulates hepatic glucose and lipid metabolism. Its circulating levels are reduced in human and rodent models of obesity, a metabolic state accompanied by chronic low-grade inflammation. Recent studies have demonstrated an anti-inflammatory role for recombinant CTRP3 in attenuating LPS-induced systemic inflammation, and its deficiency markedly exacerbates inflammation in a mouse model of rheumatoid arthritis. We used genetic mouse models to explore the immunomodulatory function of CTRP3 in response to acute (LPS challenge) and chronic (high-fat diet) inflammatory stimuli. In a sublethal dose of LPS challenge, neither CTRP3 deficiency nor its overexpression in transgenic mice had an impact on IL-1β, IL-6, TNF-α, or MIP-2 induction at the serum protein or mRNA levels, contrary to previous findings based on recombinant CTRP3 administration. In a metabolic context, we measured 71 serum cytokine levels in wild-type and CTRP3 transgenic mice fed a high-fat diet or a matched control low-fat diet. On a low-fat diet, CTRP3 transgenic mice had elevated circulating levels of multiple chemokines (CCL11, CXCL9, CXCL10, CCL17, CX3CL1, CCL22 and sCD30). However, when obesity was induced with a high-fat diet, CTRP3 transgenic mice had lower circulating levels of IL-5, TNF-α, sVEGF2, and sVEGFR3, and a higher level of soluble gp130. Contingent upon the metabolic state, CTRP3 overexpression altered chemokine levels in lean mice, and attenuated systemic inflammation in the setting of obesity and insulin resistance. These results highlight a context-dependent immunomodulatory role for CTRP3.

Intervertebral disc (IVD) degeneration (DD) affects over 40% of adults, is a leading cause of disability and costs over $100 billion in economic burden annually. DD is a multifactorial process ultimately leading to tissue breakdown and loss of functionality. DD is associated with increased levels of pro-inflammatory cytokines within the disc and the catabolic effect of inflammatory stimulation on disc cell biology has been well studied. As part of its physiological functioning the disc experiences mechanical, hydrostatic, and osmotic stimuli. Cells within the disc are mechanosensitive to these signals, where hyper physiological and damaging physical signals can perpetuate degenerative effects in the disc. Despite the known contributions of inflammatory stimulation and biomechanics to DD individually, the interaction of inflammation and biomechanics in the IVD is still not well understood. The objective of this thesis is to examine the role of inflammatory stimulation on cellular biophysical properties in the disc, subsequent implications at the tissue level, and its contributions to DD. Here the cell cytoskeleton and actomyosin contractility are identified as key regulators of the response of cellular properties to inflammation. Actomyosin contractility is further identified as a regulator of well-known biological responses to inflammatory stimulation within the disc including ECM catabolism and altered disc tissue mechanics. Altered cellular biophysical properties observed in clinical human DD samples indicate the inflammatory milieu present in DD drive changes in cellular mechanics. Increasing actomyosin contractility is shown to be effective in mitigating the effects of inflammation on cellular biophysical properties and subsequent degenerative effects highlighting its potential as a therapeutic for the treatment of DD.

High molecular weight mouse nerve growth factor(H M W-NGF), in addition to its effects on certain neural elements, is also chemotactic for human polymorphonuclear leukocytes. One of the subunits of H M W-NGF is a protease of the serine family and its active site contains a serine residue and a closely-neighboring histidine residue that are both essential for proteolysis. Elimination of enzyme activity by irreversibly blocking the single serine has no effect on leukotaxis, but blocking the histidine abolishes leukotaxis. These results suggest the possibility that part of the proteolytic active site of this enzyme may have evolved to perform more than one, completely different, biologic function - proteolysis as well as nonproteolytically mediated chemotaxis.

Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-α. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (Tm(A)) and resting (Tm(R)) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of Tm(A) (but not Tm(R)) was capable of blocking monocyte apoptosis and the ability of Tm(A) to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both Tm(A) and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.

Endothelial dysfunction and/or injury is pivotal to the development of cardiovascular and inflammatory pathology. Endothelial dysfunction and/or injury has been described in Human Immunodeficiency Virus (HIV) infection. Elaboration of circulating markers of endothelial activation, such as soluble adhesion molecules and procoagulant proteins, occurs in HIV infection. Certain endothelial cells, such as those lining liver sinusoids, human umbilical vein endothelial cells, bone marrow stromal endothelial cells or brain microvascular endothelial cells, have been shown to be variably permissive for HIV infection. Entry of virus into endothelial cells may occur via CD4 antigen or galactosyl-ceramide receptors. Other mechanisms of entry including chemokine receptors have been proposed. Nevertheless, endothelial activation may also occur in HIV infection either by cytokines secreted in response to mononuclear or adventitial cell activation by virus or else by the effects of the secreted HIV-associated proteins, gp 120 (envelope glycoprotein) and Tat (transactivator of viral replication) on endothelium. Enhanced adhesiveness of endothelial cells, endothelial cell proliferation and apoptosis as well as activation of cytokine secretion have all been demonstrated. Synergy between select inflammatory cytokines and viral proteins in inducing endothelial injury has been shown. In HIV infection, dysfunctional or injured endothelial cells potentiate tissue injury, inflammation and remodeling, and accelerate the development of cardiovascular disease.