Browsing by Subject "Tumor Necrosis Factor-alpha"
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Item Attenuation of The Host Innate Cytokine Response By The Human Cytomegalovirus Immediate-Early 2 Protein Ie86(2006-12-20) Taylor, Roger Travis; Bresnahan, Wade A.Human cytomegalovirus infects a majority of the human population and is a significant cause of life-long morbidity and mortality in neonates and patients with an impaired immune system. Human cytomegalovirus infection has a profound effect on host cell, and expression of new viral proteins interferes with the ability of the host response to effectively limit virus persistence and the initation of a latent infection. This aim of this dissertation was to identify how human cytomegalovirus attenuates the host innate immune response early during infection. Specifically, I have employed genetic and biochemical approaches to identify the HCMV immediate-early 2 protein, IE86, as an interferon beta antagonist. IE86 expression also blocks the expression of a number of proinflammatory chemokines, including RANTES, MIG and IL-8 during human cytomegalovirus infection. I have further demonstrated that IE86 mediates the attenuation of cytokine and chemokine expression by targeting the nuclear factor kappa B pathway early during infection. Using gel shift analysis I have demonstrated that IE86 blocks nuclear factor kappa B DNA binding to target promoters, including the interferon beta promoter. Since IE86 is one of the first viral proteins to be expressed during human cytomegalovirus infection, it can rapidly block cytokine and chemokine expression thereby suppressing the antiviral response and limiting the recruitment of effecter cells. The attenuation of the innate immune response by IE86 likely enhances virus replication and contributes to persistence within the host. This work addresses a number of unanswered questions about human cytomegalovirus's interactions with the host, and has identified a previously unrecognized mechanism employed by human cytomegalovirus to evade the host immune response. A better understanding of IE86's ability to attenuate cytokine expression may be key to designing novel antiviral therapy or development of an effective vaccine to prevent human cytomegalovirus infection and disease.Item Characterization of a Small Molecule Smac Mimetic's Role in Inducing Apoptosis in Human Cancer Cells(2008-09-19) Yalcin-Chin, Asligul; Wang, XiaodongInhibitor of apoptosis proteins (IAPs) regulates apoptosis by inhibiting caspases. This inhibition mechanism is an escape from death used by some human cancers. Second mitochondria-derived activator of caspases (Smac), a mitochondria-released protein during apoptosis, binds to IAPs BIR domains with four amino acid residues (AVPI) and releases the inhibition caused on caspases by IAPs. With the idea of designing a Smac mimicking drug, that will induce apoptosis in cancer cells, we synthesized a small molecule Smac mimetic compound. I tested the ability of the Smac mimetic compound to induce apoptosis on several human cancer cells in combination with chemotherapeutic agents. Unexpectedly, in 25% of the cancer cells we tested, Smac mimetic treatment alone caused apoptosis. Of the cancer cells that were sensitive to Smac mimetic, MDA-MB231 human breast cancer cells and HCC44, HCC461, H2126 lung cancer cells had the highest sensitivity. In addition, a majority of the lung cancer cell lines I tested were sensitive to TNF and/or TRAIL in combination with Smac mimetic. We identified the target of Smac mimetic to be XIAP, cIAP1, and cIAP2 in both Smac mimetic induced and TNF/Smac mimetic induced apoptosis. Moreover, we were able to mimic the Smac mimetic effect by triple knockdown experiments of IAPs in TNF induced cell death. Furthermore, we identified the target of Smac mimetic to be XIAP in the TRAIL pathway. This work identifies the targets and mechanism of Smac mimetic induced cell death in cancer cells.Item Identification and Characterization of a New E1 That Activates Ubiquitin and FAT10(2008-05-13) Chiu, Yu-Hsin; Chen, Zhijian J.Ubiquitination is one of many post-translational modifications in eukaryotes. Three enzymes (E1, E2, and E3) are involved in conjugating ubiquitin to protein substrates. I identified a novel E1-like protein, E1-L2, which is homologous to the ubiquitin E1 and another E1 involved in the activation of the ubiquitin-like protein ISG-15 (E1-L1). E1-L2 activates both ubiquitin and FAT10, a ubiquitin-like protein. Interestingly, E1-L2 can transfer ubiquitin to only a subset of E2 enzymes, Ubc5 and Ubc13, but not Ubc3 and E2-25K, suggesting that E1-L2 may function in certain ubiquitination reactions. E1-L2, but not E1 or E1-L1, forms a thioester with FAT10 in vitro. The formation of the thioester bond requires the active site cysteine residue of E1-L2 and the C-terminal diglycine motif of FAT10. In addition, endogenous FAT10 forms a thioester with E1-L2 in cells stimulated with tumor necrosis factor-alpha and interferon-gamma, which induce FAT10 expression. Silencing of E1-L2 expression by RNAi impaired the formation of FAT10 conjugates in cells. Furthermore, E1-L2 deficient embryos died before embryonic day 13.5, suggesting that E1-L2 is essential for early embryonic development. Since the FAT10-deficient mice develop normally, it is likely that specific ubiquitination reactions catalyzed by E1-L2 play an important role in animal development.Item Mechanisms of Sensitization to Chemotherapy in Non-Small Cell Lung Cancer(2012-07-20) Greer, Rachel Marie; Minna, John D.Lung cancer is the leading cause of cancer-related deaths world-wide for men and women, due in part to late detection of disease and inherent resistance to treatment. This body of work focuses on resistance to treatment, specifically chemotherapy and understanding ways to sensitize non-small cell lung cancers to existing chemotherapies. Using a large panel of non-small cell lung cancers, response to common platinum-based doublet chemotherapy was tested, and compared in a statistical fashion to each chemotherapy as a single agent, to understand the breadth of responses, and have a baseline of response to improve upon. The rest of this work endeavors to make chemotherapies more effective at tumor kill by targeting tumor specific alterations. One such targeted approach focused on miR337, and its ability to influence paclitaxel sensitivity through introduction of miR337 mimics and antagomiRs was evaluated using MTS based assays; increasing miR337 levels in moderately paclitaxel-sensitive or completely paclitaxel resistant cells sensitized the cells at least ten-fold to paclitaxel. Non-small cell lung cancers have frequent mutations in p53 (>80%). Targeting of the p53 promoter region with agRNAs, in p53-mutant containing cell lines induces cytotoxicity that is reminiscent of wild type p53 activity and is associated with large increases of the non-coding RNA lincRNAp21, and can cause large sensitizations to p53-dependent chemotherapies such as doxorubicin, indicating that these agRNAp53s could be of therapeutic importance in p53-mutant lung cancers. Re-engagement of the apoptotic pathway by a small molecule (JP1201) sensitizes non-small cell lung cancers to a variety of chemotherapies. Anti-mitotic chemotherapies have the most frequent sensitization across a large panel of non-small cell lung cancers, and the largest degree of sensitization by combination with JP1201, and this sensitization is dependent on activation of the ER stress pathway. Xenograft models using cell lines recapitulate the ability of JP1201 to sensitize non-small cell lung cancers to chemotherapy, indicating that combinations with JP1201 might be effective in patients.Item The Role of TNF Signaling in Regulating Beta-Amyloid Burden in the 3xTgAD Mouse Model of Alzheimer's Disease(2008-05-13) McAlpine, Fiona E.; Tansey, Malú G.Microglial activation and overproduction of inflammatory mediators in the CNS have been implicated in Alzheimer's Disease (AD), but the precise nature of the key molecular mediators of neurotoxicity that directly contribute to neurodegeneration or loss of specific neuronal populations is less clear. The pro-inflammatory cytokine Tumor Necrosis Factor (TNF) has been implicated in AD by its elevated presence in serum and post-mortem brains of patients with AD. To test the hypothesis that TNF-dependent neuroinflammation and neurotoxicity contributes to the increased microglial burden and exacerbated pathology observed in the hippocampus and cortex of 3xTgAD mice (transgenic mouse expressing human familial AD mutations in APP, PS1, and a familial frontotemporal dementia mutation in tau) after chronic systemic LPS exposure, we inhibited TNF signaling with novel engineered dominant negative TNF inhibitors (DN-TNF) selective for soluble TNF (solTNF) or lentiviral-derived DN-TNF to achieve long-term inhibition of TNF activity and halt or delay the early stages of amyloid-associated neuropathology. In vitro, cells infected with lenti-pro-DN-TNF-IRES-GFP produce sufficient levels of DN-TNF protein to block nuclear translocation of p65 in response to stimulation by TNF. Infection with lenti-DN-TNF also blocks solTNF-induced activation of primary microglia. Results from in vivo studies indicate that short-term pharmacological inhibition as well as long-term lentiviral-driven inhibition of soluble TNF signaling decreases the accumulation of intraneuronal full length APP in hippocampus and cortex induced by chronic systemic inflammation. To our knowledge, this is the first study that selectively inhibits soluble TNF signaling in an acute manner using a pharmacologic agent, thereby directly linking endogenous TNF activity in vivo to accumulation of APP in a model of Alzheimer's disease. Targeted inhibition of soluble TNF in the central nervous system may represent a new therapeutic approach to slow the appearance of amyloid pathology, cognitive deficits, and possibly the progressive loss of neurons in AD.Item The Role of Tumor Necrosis Factor (TNF) in Microglial Activation and Progressive Degeneration of Dopaminergic Neurons(2010-05-14) Harms, Ashley Nicole Simpson; Tansey, Malú G.Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). A number of studies have implicated chronic inflammation in the pathophysiology of PD; however, it is unclear which inflammatory mechanisms directly contribute to neuronal loss in PD. A number of cytokines, including TNF, are elevated in post-mortem brain and cerebrospinal fluid of patients with PD. Previous studies from our group have shown that blocking solTNF signaling at the time of a unilateral 6-OHDA striatal lesion attenuated behavioral deficits and the acute loss of dopaminergic neuron loss by 50%. However, a critical question of clinical relevance is whether delayed solTNF signaling inhibition can prevent the progressive loss of DA neurons that occurs after a CNS insult. I report here that a single intranigral injection of a lentivirus encoding a dominant negative TNF inhibitor delivered 2 weeks after an intrastriatal 6-OHDA lesion attenuated microgliosis in SNpc and halted the progressive loss of nigral DA neurons and the associated locomotor deficits. Given the potential contribution of microglial activation to PD and the suggestion that anti-TNF therapies in the CNS may exert neuroprotective effects on vulnerable dopaminergic neuron populations, I also investigated the role of TNF in regulating microglia effector functions to gauge the potential detrimental effects of anti-TNF therapies on the microglia functional response. I found that microglia from TNF-null mice produced reduced protein levels of cytokines and chemokines in response to LPS stimulation and they displayed no cytotoxic effects on dopaminergic neuroblastoma MN9D cells when activated in vitro. I also demonstrated that microglia isolated from TNF-null mice failed to display the expected morphological changes in response to LPS stimulation, including enhanced perinuclear expression of the activation marker CD45. My results suggest that TNF plays a critical role in microglia activation, regulation of several microglia effector functions and is the primary microglia-derived inflammatory factor that compromises survival of dopaminergic neurons. Furthermore, these studies suggest that TNF-dependent neuroinflammation directly contributes to the delayed and progressive degeneration of nigral DA neurons after neurotoxic injury and further validates solTNF as a potential therapeutic target in PD.Item Safety of Tumor Necrosis Factor Inhibitor Use in Patients with Concomitant Malignancy(2018-01-23) Phan, Hiep S.; Weideman, Rick; Feagins, Linda A.BACKGROUND: Tumor necrosis factor (TNF) inhibitors are considered contraindicated in patients with a history of malignancy. However, data to support this notion is limited. We hypothesize TNF inhibitors can be used safely in patients with chronic inflammatory diseases, like IBD, who have concomitant malignancy or develop malignancy while on these agents. METHODS: Retrospective chart review performed 1996-2015 at our local VA. Cases and controls (matched 2:1 for cancer type) were identified using pharmacy and pathology databases and then charts manually reviewed. Cases were patients with inflammatory disease including inflammatory bowel disease (IBD), rheumatoid arthritis (RA), psoriatic arthritis (PsA), psoriasis or spondyloarthropathy (SpA), concomitant malignancy, and TNF inhibitor use while controls were patients with inflammatory disease, concomitant malignancy but no TNF inhibitor use. Data was collected for cases and controls including survival at 1-yr, 2-yrs, 5-yrs after malignancy diagnosis and end of study time points. RESULTS: 36 cases (3 IBD, 22 RA, 5 PsA, 2 SpA, 1 IBD+SpA, 3 psoriasis) and 70 controls (6 IBD, 44 RA, 12 psoriasis, 6 PsA, 2 SpA) were identified. Age, cancer stage at diagnosis, and Charlson comorbidity index was not significantly different between cases and controls.Treatments with other immunosuppressives at diagnosis were not significantly different between the cases and controls and cancer specific therapies were similar between cases and controls. For cases, survival at 1-yr, 2-yrs, 5-yrs and at end of study follow-ups were 32 (89%), 31(86%), 29 (81%) and 24 (64%), respectively compared to 63 (90%), 61 (87%), 51 (73%) and 45 (64%) for the control group (p=NS for all time points). For cases, recurrence rates at 1-yr, 2-yrs, 5-yrs and at end of study follow-ups were 3 (8%), 5 (14%), 6 (17%), and 8 (22%), respectively compared to 2 (3%), 5 (7%), 7 (10%), 9 (13%) for the control group (p=NS for all time points). CONCLUSION: Survival rates and cancer recurrence after a malignancy is diagnosed in patients with inflammatory diseases treated with TNF inhibitors are not different from similar patients not treated with TNF inhibitors. This preliminary data suggests that TNF inhibitors should not be withheld for fear of worsening survival or tumor recurrence after diagnosis with a malignancy if the agent is needed for adequate inflammatory disease control.Item Tumor Necrosis Factor Dependent Mechanisms and Neuroprotective Strategies in Models of Parkinson's Disease(2008-05-13) McCoy, Melissa Kay; Tansey, Malú G.Parkinson's disease is a chronic, progressive, neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra that innervate the striatum. Although the nigral cell loss that causes motor dysfunction in Parkinson's disease has been identified for some time, the mechanisms that lead to this dopaminergic neuron loss are unclear. Elevated levels of the cytokine tumor necrosis factor in cerebrospinal fluid and postmortem brains of Parkinson's patients and in animal models of the disease implicate tumor necrosis factor in contributing to disease pathology; but a specific role for this cytokine in mediating loss of dopaminergic neurons in Parkinson's disease has not been clearly established. Here I demonstrate that neutralization of soluble tumor necrosis factor in vivo with a recombinant dominant-negative tumor necrosis factor inhibitor reduced 6-hydroxydopamine-induced nigral degeneration by 50% and attenuated amphetamine-induced rotational behavior, indicative of striatal dopamine preservation in a rodent model of Parkinson's disease. Similar protective effects were observed with in vivo chronic co-infusion of dominant-negative tumor necrosis factor inhibitor with a proinflammatory initiator, low-dose lipopolysaccharide, into the substantia nigra of rodents, confirming a role for soluble tumor necrosis factor inhibitor-dependent neuroinflammation in contributing to nigral degeneration. In rat embryonic midbrain neuron/glia mixed cell cultures exposed to lipopolysaccharide, delayed administration of dominant-negative tumor necrosis factor inhibitor prevented degeneration of dopaminergic neurons despite sustained microglia activation. Addition of a dominant-negative tumor necrosis factor inhibitor also attenuated 6-hydroxydopamine-induced dopaminergic neuron toxicity in vitro. In this dissertation the ability of lentiviral-encoded dominant-negative tumor necrosis factor inhibitor to provide neuroprotection was also investigated. Intranigral delivery of lentiviral dominant-negative tumor necrosis factor inhibitor in a hemiparkinsonian rat 6-hydroxydopamine model attenuated nigral dopaminergic neuron loss and reduced behavior deficits when compared to control lentiviral-infected animals. Collectively, these data identify tumor necrosis factor signaling in contributing to dopaminergic neuron loss in vitro and in vivo in two chronic rat models of Parkinson's disease, and provide evidence that delaying the progressive degeneration of the nigrostriatal pathway in the early stages of Parkinson's disease in humans may be therapeutically feasible with agents that block soluble tumor necrosis factor signaling.Item [UT Southwestern Medical Center News](2006-09-12) McKenzie, Aline