archives@tulane.edu / In order to develop better therapeutics and treatment strategies for tuberculosis (TB) infection, it is imperative to understand interactions that occur in the host in response to the bacilli that contribute to disease progression. Modeling of TB in simple animal models such as mice and zebrafish is often incomplete as there are evolutionary differences, as well as structural issues, that reduce the faithfulness to human TB infection. The non-human primate model of TB infection provides the added benefit of providing a long-established model of SIV infection that recapitulates HIV infection in humans and has been expanded to model TB/HIV co-infection. Here, we have sought to identify correlates of protection in TB and TB/SIV co-infection using rhesus macaques. In the first experiment, we used two strains of Mycobacterium tuberculosis (Mtb), CDC1551 and Erdman, to investigate strain-specific mechanisms of virulence. As increased virulence of Mtb Erdman was associated with excess inflammatory responses, we sought to evaluate a host-directed therapeutic in a lethal challenge model of Mtb CDC1551 infection. We found that use of a type I interferon antagonist significantly improved host survival in the absence of antibiotic treatment and survival was associated with the presence of increased levels of granzyme B producing T cells. A major producer of granzyme B, mucosal-associated invariant T (MAIT) cells was investigated in Mtb/SIV co-infection, but was found to not contribute to protection in TB or TB/SIV. In order to further expand our model of Mtb/SIV co-infection, we co-infected latent Mtb-infected rhesus macaques with a non-pathogenic strain of SIV, SIVmac239ΔGY, and administered a CD4-depleting antibody, CD4R1, in place of SIVmac239 co-infection. Using SIVΔGY, we found that virulent viral replication was necessary for TB reactivation. Using both SIVΔGY and antibody-mediated CD4+ T cell depletion, we found that immune responses are disregulated in Mtb/SIV reactivators in a divergent manner, illustrating the presence of SIV-dependent factors that contribute to TB/SIV reactivation. Overall, these results indicate that immune mechanisms, especially those of inflammation, are significant in determining host outcomes. Developing ways to better control inflammation are necessary to supplement antibiotic treatment and cure TB. / 1 / Allison Bucsan

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Thesis advisor: Kenneth Williams / Proliferating cell nuclear antigen (PCNA) is a DNA polymerase δ auxiliary protein during cell cycle. However, PCNA is also present in quiescent cells undergoing DNA repair. This study investigates the expression of PCNA in CNS macrophages of SIV infected Rhesus macaque and examines the presence of PCNA in monocytes prior to differentiation into CNS macrophages. Accumulation of macrophages in the SIV infected brain, together with the creation of multinucleated giant cells (MNGC), form SIV lesions, which lead to neurological disorders. From the twelve animals used in this study, four were analyzed by flow cytometry to detect PCNA expression in monocytes, but no expression of PCNA was detected. Peripheral blood mononuclear cells (PBMC) of five animals were also analyzed using cytospin preparations to confirm results obtained by flow cytometry. The study did not find induction of PCNA mRNA by qRT-PCR in infected animals. Animals that developed SIVE were analyzed by immunohistochemistry to identify PCNA in recently immigrated macrophages. Together results suggest that 1) PCNA is not expressed in monocytes; 2) PCNA in monocyte/macrophages is induced in SIV infected animals and it is not associated with cell cycle; 3) Recently immigrated monocytes of CNS are PCNA negative and 4) majority PCNA+ macrophages in the SIVE lesions are perivascular macrophages. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The human immunodeficiency virus-1 (HIV-1) currently infects 35 million people globally. HIV preferentially infects CD4+ T cells, a critical component of the host immune system, causing their rapid depletion, which has a broad negative impact on host immunity. Chronic HIV infection also results in increased expression of inhibitory immune regulatory proteins, which is associated with impaired functionality of a wide range of immune cells. This clinical phenomenon, referred to as "immune exhaustion," precludes the slow and eventual failure of host immunity against co-infecting pathogens and is the hallmark of AIDS-related disease. In Aim 1 of our studies, we used whole blood from Indian-origin rhesus monkeys to distinguish 12 discrete immune cell subsets utilizing antibody staining and flow cytometric cell sorting. The segregated "naïve" (uninfected) cell subsets will be characterized by RNASeq gene expression analysis, and will be used as the baseline population for a bioinformatics "deconvolution" method comparing the same cell subsets from simian immunodeficiency virus (SIV)-infected cell populations. We successfully developed an antibody panel that distinguishes activated and resting CD4+ T cells, activated and resting CD8+ T cells, activated and resting B cells, activated and resting NK cells, plasmacytoid dendritic cells, myeloid dendritic cells, and monocytes. We also developed a separate isolation protocol for neutrophils using different densities of Percoll. Finally, we optimized cell sorting of CD4+ and CD8+ T cells in order to obtain sufficient amounts of high quality RNA for future RNASeq gene expression analysis. As an adjunct to our above work, in Aim 2 of our experiments we sought to quantify the expression of immune inhibitory proteins that are increased upon the various cell subsets during SIV infection. We set out to optimize an antibody panel targeting three proteins of interest, PD-L1, LAG-3, and TIM-3, for the study of whole blood from SIV-infected rhesus monkeys. This data will be used to compliment our RNASeq dataset developed in Aim 1. By utilizing a biotinylated antibody specific for PD-L1 along with fluorescently conjugated streptavidin, we were able to detect PD-L1-positive cells and allow for amplification of positive fluorescence. We also performed multiple evaluations of monoclonal antibodies specific to either LAG-3 or TIM-3 and determined the concentrations at which detection was best for LAG-3+ and TIM-3+ cells.

Thesis advisor: Welkin Johnson / HIV associated cardiovascular disease is likely due to multiple factors ranging from accelerated aging, the direct effects of HIV proteins, and increased inflammation and immune activation. Monocytes/macrophages play roles in the development and progression of HIV and cardiovascular disease. Increased monocyte/macrophage inflammation and immune activation associated with HIV infection likely contributes to the increased risk of cardiovascular disease development associated with HIV infection. To further understand the role of monocytes/macrophages in the development of HIV-associated cardiovascular disease we: 1) assessed monocyte activation longitudinally to determine if they correlate with and can be predictive of cardiac fibrosis and inflammation; 2) we examined cardiac tissues from the SIV-infected CD8+ T-lymphocyte depleted animals to determine the effects of monocyte/macrophage inflammation on cardiac fibrosis; 3) in parallel we examined cardiovascular tissues from HIV+ individuals on durable cART to determine if aortic and cardiac inflammation persists with infection and if soluble factors (sCD163) correlated with intima-media thickness and fibrosis; 4) we next examined the effects of blocking leukocytes trafficking to the heart on SIV-associated cardiac inflammation and fibrosis; 5) and finally we examined if targeting monocyte/macrophage activation (as opposed to traffic) directly using MGBG decreases SIV-associated cardiovascular pathology, inflammation and fibrosis. We found that early increased monocyte activation was predictive of animals that developed cardiac fibrosis and SIV encephalitis (SIVE). Animals with both cardiac fibrosis and SIVE had increased macrophage inflammation in the heart, suggesting that there is a link between cardiac and CNS inflammation seen with HIV infection (Chapter 2). We found in a SIV-infected CD8+ T-lymphocyte depletion model of rapid AIDS increased prevalence of cardiac disease compared to nondepleted animals, and increased cardiac inflammation that correlated with cardiac fibrosis. Monocyte/macrophage traffic to the heart occurred later with SIV infection, possibly with the development of AIDS (Chapter 3). In post-mortem human tissues studies we found that inflammation in aorta and heart correlated with increased soluble CD163, and correlated with aortic intima-media thickness and cardiac fibrosis with HIV infection (Chapter 4). Blocking leukocyte traffic to the heart using an anti-α4 antibody decreased macrophage inflammation in the heart that correlated with decreased cardiac fibrosis (Chapter 5). Using MGBG, a polyamine biosynthesis inhibitor that directly targets monocyte/macrophage activation, we found decreased inflammation in the carotid artery and heart correlated with decreased carotid artery intima-media thickness and cardiac fibrosis (Chapter 6). Overall these studies provide evidence for ongoing monocyte/macrophage cardiovascular inflammation with HIV and SIV infection. Macrophage inflammation correlates with markers of cardiovascular disease (fibrosis and intima-media thickness, cardiomyocyte damage). Directly targeting monocyte/macrophage traffic (anti-α4 antibody) and activation (MGBG) decreased cardiovascular pathology, inflammation, fibrosis, and intima-media thickness. Taken together, the data in this thesis indicate that targeting monocytes/macrophages in conjunction with combination anti-retroviral therapy could alleviate cardiovascular disease in HIV-infected individuals. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

No description available.

archives@tulane.edu / Background: Individuals with human immunodeficiency virus (HIV) and undergoing antiretroviral therapy (ART) exhibit high levels of circulating inflammatory cytokines and proteins, which are strongly correlated with shortened time to death and disease. To target damaging inflammation at the source, the drivers of inflammation must be identified. Adipose tissue is a massive organ that contains adipocytes and immune cells capable of producing pro-inflammatory mediators. Dysregulated adipose tissue is implicated in the pathogenesis of obesity and related diseases, such as type 2 diabetes, that are likewise reported in persons with chronic HIV infection. Adipose tissue was therefore explored as a contributor to circulating inflammation in patients with HIV using the rhesus macaque model. Simian immunodeficiency virus (SIV) closely models HIV regarding pathogenesis, including CD4+ T cell depletion, induction of a viral reservoir, and development of opportunistic infections before succumbing to Acquired Immunodeficiency Syndrome (AIDS) and death. Methods: Subcutaneous adipose tissue (SQAT) from SIV-infected rhesus macaques was characterized using confocal microscopy to describe the major immune cell subsets. Adipose tissue homogenates and plasma were analyzed for expression of genes and proteins related to inflammatory processes using antibody and RNA-based fluorescent multiplex bead technology for protein and gene quantitation, respectively. The functions of adipose tissue immune cells during SIV infection were measured with stimulation and phagocytosis assays. / 1 / Marissa Fahlberg

No description available.

Thesis advisor: Kenneth Williams / Approximately 15% of individuals infected with Human Immunodeficiency Virus (HIV) develop a neurological condition that consists of motor dysfunction and cognitive deterioration in late stage disease that is known as the AIDS dementia complex (ADC). This condition is mirrored in rhesus macaques infected with Simian Immunodeficiency Virus (SIV), which can be more easily studied. This project analyzed different macrophage populations in rhesus macaques infected and uninfected with SIV at early and terminal stage disease. Single and double immunohistochemistry stains were performed for the known macrophage and microglial markers CD163, CD16, CD68, Mac387, HAM56, and Iba-1, as well as for the SIV-p28 viral protein. Photographs and observations of the tissue stainings demonstrated that early after infection with SIV, there is an increase in perivascular macrophages and monocytes surrounding vessels and tissue edges, and the SIV-p28 protein is already present. There is also an observed change in the morphology of the microglia to an active, ramified state. After the development of AIDS and SIVE, the increase in all of the macrophage markers and the accumulation of activated microglia are clearly visible, especially surrounding and within lesions. Furthermore, these markers can be used to categorize the encephalitic lesions as “new” or “old” based on the presence or absence of Mac387 within the cells. All lesions contained CD68+ and HAM56+ macrophages, but “new” lesions presented with a relatively high count of Mac387+ macrophages that were newly imported from the periphery, whereas “old” lesions lacked Mac387+ cells. / Thesis (BS) — Boston College, 2009. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

Thesis advisor: Kenneth C. Williams / Thesis advisor: Welkin Johnson / Neuropathogenesis of HIV-associated neurocognitive disorders (HAND) is likely instigated by chronic immune activation in response to residual infection in the central nervous system (CNS), where combined antiretroviral therapy (cART) has limited access. Monocyte/macrophages (Mo/Mϕ) constitute the predominant population of infected cells in the CNS and play a major role in HIV-induced neuropathogenesis. Emergence of compartmentalized HIV subpopulations in the brain corresponds with accumulation of HIV-infected Mo/Mϕ and is consistent with acquired immune deficiency syndrome (AIDS)-related neuropathology. We used a rhesus macaque model of neuroAIDS to elucidate the role of Mo/Mϕ in establishing CNS infection and the emergence of compartmentalized virus in the brain. To do this, we: 1) performed phylogenetic analysis of viral sequences from peripheral and CNS compartments and determined the incidence of Mo/Mϕ infection in CNS tissues to identify sources of CNS viral subpopulations that emerge with AIDS-related neuropathology; 2) optimized a method for obtaining single genome viral sequences from Mo/Mϕ populations extracted from tissues and 3) performed phylogenetic analysis of viral sequences from bone marrow (BM) and CNS Mo/Mϕ and determined the incidence of Mo/Mϕ infection in the BM to assess whether BM Mo/Mϕ are sources of infected Mo/Mϕ that accumulate in the CNS with AIDS-related neuropathology. We found that animals with AIDS-related neuropathology had a higher incidence of Mo/Mϕ infection and compartmentalized SIV subpopulations in CNS tissues compared to animals without neuropathology. Additionally, CSF virus, which is used to assess the presence of CNS virus compartmentalization in living patients, was not compartmentalized even with significant compartmentalization in the brain and severe AIDS-related neuropathology (Chapter 2). Relative to animals without CNS pathology, animals with AIDS-related neuropathology had a higher incidence of Mo/Mϕ infection in the BM and viral sequences from BM and CNS perivascular Mo/Mϕ clustered with sequences from trafficking monocytes and CNS tissues (Chapter 4). The results suggest that infected Mo/MΦ in CNS tissues are sources of compartmentalized virus and that infected Mo/Mϕ in the BM are sources of infected Mo/Mϕ that accumulate in the CNS with AIDS-related neuropathology. In summary, the data in this dissertation suggest that targeting Mo/Mϕ may prevent CNS infection and inflammation associated with HIV-induced neuropathogenesis. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.