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Thesis advisor: Tricia H. Burdo / Human immunodeficiency virus-associated peripheral neuropathy (HIVPN) continues to be a prevalent comorbidity of HIV infection, despite virologic control due to effective antiretroviral therapy (ART). Symptoms include bilateral tingling, numbness, and pain in distal extremities. Severity of symptoms is associated with a loss of intraepidermal nerve fiber density (IENFD) in the feet. Damage to the dorsal root ganglia (DRG) has also been observed in postmortem tissue analysis from patients with HIV-PN. Treatment options are limited due to a lack of understanding of the disease pathogenesis. Chronic monocyte activation and accumulation of macrophages in peripheral nervous system (PNS) tissues has been reported but few studies have directly demonstrated the role of monocyte/macrophage activation and traffic in the pathogenesis of HIV-PN. The central hypothesis of this thesis is that monocyte activation and traffic mediates PNS neuronal damage. We addressed this hypothesis in several ways. In chapter 2, we describe pathology seen in a rapid disease progression animal model of HIV-PN. We found that an early loss of IENFD preceded a loss of small diameter DRG neurons. In chapter 3, we associated DRG pathology with an accumulation of inflammatory macrophages surrounding DRG neurons. Increased monocyte traffic to the DRG was associated with severity of DRG pathology and with a loss of IENFD. In chapter 4, we directly tested the impact of monocyte traffic on DRG pathology by blocking leukocyte traffic with an anti-VLA-4 antibody, natalizumab. Blocking cell traffic reduced accumulation of macrophages in the DRG and improved pathology. Next we treated animals with methylglyoxal-bisguanylhydrazone (MGBG) to specifically target myeloid cells and reduce their activation. MGBG treatment improved DRG pathology and reduced accumulation of macrophages in tissues. Having demonstrated the role of monocyte traffic and activation, we aimed to identify signaling proteins and inflammatory proteins associated with PNS pathology. We found elevated monocyte chemoattractants in DRG tissue and elevated markers of monocyte activation in plasma that were associated with a loss of IENFD. Together, these studies demonstrate that systemic monocyte activation, macrophage accumulation in DRG tissue, and monocyte traffic plays a major role in SIV-PN pathogenesis. These studies provide novel insight into immune mechanisms that impact neuronal loss during SIV infection. Thus, modulating macrophage activation and reducing monocyte traffic may have therapeutic benefits to patients suffering from or at risk of developing HIV-PN. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

<p>In the present studies, aspects of some disease activity measures in rheumatoid arthritis (RA) have been investigated, including the effect of glucocorticoids on this activity. In RA, serum interleukin(IL)-6 levels were elevated and were shown to have a circadian rhythm, with peak levels in the morning, declining towards low or normal levels in the afternoon and evening. In contrast, serum levels of tumour necrosis factor(TNF) alpha were low and stable. In other connective tissue diseases, serum TNF alpha levels were elevated but without circadian variation, while IL-6 levels were low and stable. Nocturnal administration (at 2:00 a.m.) of low-dose prednisolone a few hours before the early morning peak of IL-6 was shown to be significantly more effective in reducing clinical symptoms of disease activity and serum IL-6 levels than the traditional morning administration (at 7:30 a.m.) of the same dose of prednisolone. Circulating monocytes are activated in RA, expressing receptors related to adhesion and phagocytosis. Treatment with glucocorticoids suppressed the expression of these receptors on monocytes, and this may be one mechanism of the beneficial effect of glucocorticoids in RA. Endogenous levels of cortisol seem to play a minor role in expression of monocyte receptors. The different acute phase markers used to assess disease activity in RA showed good corrrelations with each other and with serum IL-6 levels. There were especially strong corrrelations between C-reactive protein (CRP) and Serum amyloid protein A (SAA), and between fibrinogen and erythrocyte sedimentation rate (ESR). Fibrinogen and CRP showed stronger correlation than ESR with the Modified Health Assessment Questionnaire (MHAQ) score and with the neutrophil count. Four simple objective function tests were each compared with the HAQ score a with a radiological joint damage score (Larsen score). The objective function tests correlated with the MHAQ score, and each of these two methods of assessing physical disability correlated with pain, CRP and ESR. In addition, most of the objective function tests correlated significantly with radiological joint damage, while the MHAQ score did not.</p>

In the present studies, aspects of some disease activity measures in rheumatoid arthritis (RA) have been investigated, including the effect of glucocorticoids on this activity. In RA, serum interleukin(IL)-6 levels were elevated and were shown to have a circadian rhythm, with peak levels in the morning, declining towards low or normal levels in the afternoon and evening. In contrast, serum levels of tumour necrosis factor(TNF) alpha were low and stable. In other connective tissue diseases, serum TNF alpha levels were elevated but without circadian variation, while IL-6 levels were low and stable. Nocturnal administration (at 2:00 a.m.) of low-dose prednisolone a few hours before the early morning peak of IL-6 was shown to be significantly more effective in reducing clinical symptoms of disease activity and serum IL-6 levels than the traditional morning administration (at 7:30 a.m.) of the same dose of prednisolone. Circulating monocytes are activated in RA, expressing receptors related to adhesion and phagocytosis. Treatment with glucocorticoids suppressed the expression of these receptors on monocytes, and this may be one mechanism of the beneficial effect of glucocorticoids in RA. Endogenous levels of cortisol seem to play a minor role in expression of monocyte receptors. The different acute phase markers used to assess disease activity in RA showed good corrrelations with each other and with serum IL-6 levels. There were especially strong corrrelations between C-reactive protein (CRP) and Serum amyloid protein A (SAA), and between fibrinogen and erythrocyte sedimentation rate (ESR). Fibrinogen and CRP showed stronger correlation than ESR with the Modified Health Assessment Questionnaire (MHAQ) score and with the neutrophil count. Four simple objective function tests were each compared with the HAQ score a with a radiological joint damage score (Larsen score). The objective function tests correlated with the MHAQ score, and each of these two methods of assessing physical disability correlated with pain, CRP and ESR. In addition, most of the objective function tests correlated significantly with radiological joint damage, while the MHAQ score did not.

Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2014-08-01T12:58:03Z No. of bitstreams: 1 Dissertação Izabela.pdf: 1302143 bytes, checksum: ab457123bfc94643d499a3062e8b2cc2 (MD5) / Made available in DSpace on 2014-08-01T12:58:03Z (GMT). No. of bitstreams: 1 Dissertação Izabela.pdf: 1302143 bytes, checksum: ab457123bfc94643d499a3062e8b2cc2 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / O teste de detecção de pirogênio é preconizado nas farmacopeias como teste de segurança imprescindível para a avaliação da qualidade de produtos injetáveis. Os métodos alternativos ao teste de pirogênio em coelhos são o Teste de Lisado de Amebócitos de Limulus (LAL) e o Teste de Ativação de Monócitos (MAT). Esses métodos ainda não podem substituir o teste em coelhos por completo, pois no caso do LAL os resultados podem não ser confiáveis quando a análise é realizada na presença de algumas substâncias interferentes com alto teor de lipídios e proteínas (encontrados nos medicamentos biológicos) e glucanas, além disso, o teste só detecta endotoxinas. Em relação ao MAT, já que o teste é sensível para todos os tipos de pirogênios e tem o mesmo mecanismo biológico responsável pela reação de febre em humanos, o Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recomendou sua utilização desde que fique demonstrada a equivalência de seus resultados ao teste em coelhos, em conformidade com a regulamentação aplicável. Verifica-se assim, que a literatura carece de dados que envolvam a comparação entre a dose limite que causa febre em coelhos e a correspondência para o MAT relacionado ao ácido lipoteicóico (ALT), sendo assim, esse estudo tem como objetivo principal avaliar a utilização do MAT na detecção da contaminação de ALT de S. aureus em Cloreto de Sódio 0,9 % apirogênico artificialmente contaminado, através do estabelecimento de curva dose-resposta de ALT em coelhos; curva concentração-resposta de ALT para o MAT e avaliação em paralelo dos resultados obtidos utilizando ALT no teste em coelhos, LAL cromogênico e MAT. A resposta de febre foi observada a partir de 75.000 ng de ALT/Kg nos coelhos e no MAT para sangue criopreservado/IL-1β foi estabelecida em 50.000 ng/mL de ALT, ou 5,41 UEE/mL. O teste de LAL apresentou resultado falso-reativo a partir de 10.000 ng/mL de ALT. Os resultados apresentados neste estudo fornecem informações importantes sobre a comparação entre o teste de pirogênio em coelhos, MAT e LAL, contribuindo com dados para a validação do MAT, envolvendo outros pirogênios que não a endotoxina, e também para a aceitação deste teste pelos órgãos regulatórios no Brasil visando uma possível substituição do uso de animais, garantindo, assim, a segurança da saúde da população. / The pyrogenic test is preconized in the Pharmacopeias as a safety test indispensable for the quality of evaluation of injectable products. The alternative methods to the rabbit pyrogen test are the Limulus Amebocite Lysate (LAL) Test and the Monocyte Activation Test (MAT). These methods cannot replace the rabbit test completely yet, for in the case of LAL the results may not be reliable when the analysis is carried out in the presence of some interfering substances with high content of lipids and proteins (found in biological medicines) and glucans, moreover, the test only detects endotoxins. Concerning the MAT, since the test is sensitive to all types of pyrogens and has the same biological mechanism responsible for the fever reaction in humans, the Interagency Coordinating Committee on the Validation of Alternative Methods (Comitê de Coordenação Interagências sobre Validação de Métodos Alternativos - ICCVAM) have recommended its utilization provided that the equivalence of its results from the rabbit test, in conformity with the applicable regulation. It can be verified, then, that the literature lacks the data which involves the comparison between the limit dose that causes fever in rabbits and the consequence for the MAT related to the lipoteichoic acid (LTA), that being so, the aim of this study is mainly the utilization of MAT in the detection of the contamination of LTA of S. aureus in apyrogenic 0.9 % Sodium Chloride artificially contaminated, through the establishment of the dose-response curve of LTA in rabbits; concentration-response curve of LTA for the MAT and parallel evaluation of the results obtained by utilizing LTA in the test in rabbits, chromogenic LAL and MAT. The fever response was observed from 75.000 ng of LTA/Kg in rabbits and in MAT and for cryopreserved blood/IL-1β was established in 50.000 ng/mL of LTA, or 5,41 UEE/mL. The LAL test presented a false-positive result from 10.000 ng/mL of ALT. The results presented in this study provide important information on the comparison between the rabbit pyrogen test, MAT and LAL, contributing with data to the evaluation of MAT, involving pyrogens other than endotoxin, and also to the acceptance of this test by the regulatory organs in Brazil aiming at a possible animal substitution, and guaranteeing, then, the health security of the population.