Translational Biomarker Research for Militarily Relevant Populations in Neurocognitive Diseases

In recent decades more soldiers are being mobilized to conflict areas, such as the over 2 million service members, who have been deployed to Iraq and Afghanistan since October 2001, which includes but is not limited to Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF); or the 700,000 service veterans deployed to the Persian Gulf War in 1990-91 in the US. The UK mobilized over 46,000 military personnel to Iraq, 9,500 British troops to Afghanistan and 50,000 troops to the Gulf War. Soldiers are being exposed to traumatic events such as physical and psychological trauma, as well as chemical exposure and therefore service members are at risk of postdeployment health-related issues, associated commonly with post-traumatic stress disorder (PTSD) and traumatic brain injury (TBI) among OEF/OIF veterans, as well as Gulf War Illness (GWI) among the Persian Gulf War Veteran population. Although progress has been made in identifying underlying pathology for TBI and PTSD and acute as well as sub-acute biomarkers have been identified, with commercially available tests on the horizon, the work presented here addresses a critical but underinvestigated issue, the need for chronic biomarkers for these conditions, as they can go undetected for an extended period of time. Additionally, more evidence has surfaced that discusses how symptoms related to mild TBI (mTBI) can last for years after the insult, emphasizing the importance of investigatjng biomarkers at a late timepoint after injury as, owing to the mild nature of the injury, the condition was often undiagnosed at the time. PTSD itself still lacks an objective measure that can capture its complexity, whereas co-morbidity of PTSD with TBI further complicates the issue. The other mentioned militarily relevant condition, termed GWI, faces similar issues. Veterans deployed to the Persian Gulf War in 1991 suffer from a disease that has shown to exhibit persistent multisymptom complexity. No biomarker has been identified for this particular population thus making objective diagnosis difficult. Besides the identification of clinical biomarkers, much research has been done in preclinical models, yet there is still a need to verify and validate such animal models in order to demonstrate their utility. Once the validity of a preclinical model has been confirmed, investigation of pathogenic mechanisms in those models has the potential to reveal therapeutic targets of relevance to the human condition. Chapter 1 will discuss epidemiology, current clinical diagnosis and pathophysiology of TBI, PTSD and GWI as well as the status of biomarker research in each of these three areas. The thesis then focuses on the identification of plasma biomarkers in human patient populations, specifically in military populations suffering from TBI, PTSD or both at chronic time points post traumatic exposure (Chapters 2 & 3). In Chapter 4, we then explore whether or not such changes are present in our established animal model of TBI. In Chapter 5 we investigate peripheral biomarkers in plasma samples from Gulf War veterans and in two animal models of GWI. Given the complexity of TBI, PTSD and GWI clinical presentation and pathogenesis and their heterogeneity in human populations, it is anticipated that a valid biomarker for broad application will in fact require assessment of many markers to create a panel that can support diagnosis. The lipidomic and proteomic analyses I employed in this work are approaches with the required breadth and lack of bias to be successful in such an undertaking, and I hope that the work described in this thesis provides a foundation for future development of such biomarker panels

Translational potential of long-term decreases in mitochondrial lipids in a mouse model of Gulf War Illness

Gulf War Illness (GWI) affects 25% of veterans from the 1990–1991 Gulf War (GW) and is accompanied by damage to the brain regions involved in memory processing. After twenty-five years, the chronic pathobiology of GWI is still unexplained. To address this problem, we examined the long-term consequences of GW exposures in an established GWI mouse model to identify biological processes that are relevant to the chronic symptoms of GWI. Three-month old male C57BL6 mice were exposed for 10 days to GW agents (pyridostigmine bromide and permethrin). Barnes Maze testing conducted at 15- and 16-months post-exposure revealed learning and memory impairment. Immunohistochemical analyses showed astroglia and microglia activation in the hippocampi of exposed mice. Proteomic studies identified perturbation of mitochondria function and metabolomics data showed decreases in the Krebs cycle compounds, lactate, β-hydroxybutyrate and glycerol-3 phosphate in the brains of exposed mice. Lipidomics data showed decreases in fatty acids, acylcarnitines and phospholipids, including cardiolipins in the brains of exposed mice. Pilot biomarker studies showed that plasma from exposed mice and veterans with GWI had increases in odd-chain, and decreases in long-chain, acylcarnitines compared to their respective controls. Very long-chain acylcarnitines were decreased in veterans with GWI compared to controls. These studies suggest that mitochondrial lipid disturbances might be associated with GWI and that further investigation is required to determine its role in the pathophysiology of this illness. Targeting mitochondrial function may provide effective therapies for GWI, and that lipid abnormalities could serve as biomarkers of GWI

Author Correction: Oleoylethanolamide treatment reduces neurobehavioral deficits and brain pathology in a mouse model of Gulf War Illness

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Relative resistance of HIV-1 founder viruses to control by interferon-alpha

Background: Following mucosal human immunodeficiency virus type 1 (HIV-1) transmission, type 1 interferons (IFNs) are rapidly induced at sites of initial virus replication in the mucosa and draining lymph nodes. However, the role played by IFN-stimulated antiviral activity in restricting HIV-1 replication during the initial stages of infection is not clear. We hypothesized that if type 1 IFNs exert selective pressure on HIV-1 replication in the earliest stages of infection, the founder viruses that succeed in establishing systemic infection would be more IFN-resistant than viruses replicating during chronic infection, when type 1 IFNs are produced at much lower levels. To address this hypothesis, the relative resistance of virus isolates derived from HIV-1-infected individuals during acute and chronic infection to control by type 1 IFNs was analysed. Results: The replication of plasma virus isolates generated from subjects acutely infected with HIV-1 and molecularly cloned founder HIV-1 strains could be reduced but not fully suppressed by type 1 IFNs in vitro. The mean IC50 value for IFNα2 (22 U/ml) was lower than that for IFNβ (346 U/ml), although at maximally-inhibitory concentrations both IFN subtypes inhibited virus replication to similar extents. Individual virus isolates exhibited differential susceptibility to inhibition by IFNα2 and IFNβ, likely reflecting variation in resistance to differentially up-regulated IFN-stimulated genes. Virus isolates from subjects acutely infected with HIV-1 were significantly more resistant to in vitro control by IFNα than virus isolates generated from the same individuals during chronic, asymptomatic infection. Viral IFN resistance declined rapidly after the acute phase of infection: in five subjects, viruses derived from six-month consensus molecular clones were significantly more sensitive to the antiviral effects of IFNs than the corresponding founder viruses. Conclusions: The establishment of systemic HIV-1 infection by relatively IFNα-resistant founder viruses lends strong support to the hypothesis that IFNα plays an important role in the control of HIV-1 replication during the earliest stages of infection, prior to systemic viral spread. These findings suggest that it may be possible to harness the antiviral activity of type 1 IFNs in prophylactic and potentially also therapeutic strategies to combat HIV-1 infection

Brain‐derived neurotropic factor polymorphisms, traumatic stress, mild traumatic brain injury, and combat exposure contribute to postdeployment traumatic stress

Abstract Background: In addition to experiencing traumatic events while deployed in a combat environment, there are other factors that contribute to the development of posttraumatic stress disorder (PTSD) in military service members. This study explored the contribution of genetics, childhood environment, prior trauma, psychological, cognitive, and deployment factors to the development of traumatic stress following deployment. Methods: Both pre‐ and postdeployment data on 231 of 458 soldiers were analyzed. Postdeployment assessments occurred within 30 days from returning stateside and included a battery of psychological health, medical history, and demographic questionnaires; neurocognitive tests; and blood serum for the D2 dopamine receptor (DRD2), apolipoprotein E (APOE), and brain‐derived neurotropic factor (BDNF) genes. Results: Soldiers who screened positive for traumatic stress at postdeployment had significantly higher scores in depression (d = 1.91), anxiety (d = 1.61), poor sleep quality (d = 0.92), postconcussion symptoms (d = 2.21), alcohol use (d = 0.63), traumatic life events (d = 0.42), and combat exposure (d = 0.91). BDNF Val66 Met genotype was significantly associated with risk for sustaining a mild traumatic brain injury (mTBI) and screening positive for traumatic stress. Predeployment traumatic stress, greater combat exposure and sustaining an mTBI while deployed, and the BDNF Met/Met genotype accounted for 22% of the variance of postdeployment PTSD scores (R 2 = 0.22, P < 0.001). However, predeployment traumatic stress, alone, accounted for 17% of the postdeployment PTSD scores. Conclusion: These findings suggest predeployment traumatic stress, genetic, and environmental factors have unique contributions to the development of combat‐related traumatic stress in military service members

Ensuring smoothly navigable approximation sets by Bézier curve parameterizations in evolutionary bi-objective optimization

The aim of bi-objective optimization is to obtain an approximation set of (near) Pareto optimal solutions. A decision maker then navigates this set to select a final desired solution, often using a visualization of the approximation front. The front provides a navigational ordering of solutions to traverse, but this ordering does not necessarily map to a smooth trajectory through decision space. This forces the decision maker to inspect the decision variables of each solution individually, potentially making navigation of the approximation set unintuitive. In this work, we aim to improve approximation set navigability by enforcing a form of smoothness or continuity between solutions in terms of their decision variables. Imposing smoothness as a restriction upon common domination-based multi-objective evolutionary algorithms is not straightforward. Therefore, we use the recently introduced uncrowded hypervolume (UHV) to reformulate the multi-objective optimization problem as a single-objective problem in which parameterized approximation sets are directly optimized. We study here the case of parameterizing approximation sets as smooth Bézier curves in decision space. We approach the resulting single-objective problem with the gene-pool optimal mixing evolutionary algorithm (GOMEA), and we call the resulting algorithm BezEA. We analyze the behavior of BezEA and compare it to optimization of the UHV with GOMEA as well as the domination-based multi-objective GOMEA. We show that high-quality approximation sets can be obtained with BezEA, sometimes even outperforming the domination- and UHV-based algorithms, while smoothness of the navigation trajectory through decision space is guaranteed

Robust evolutionary bi-objective optimization for prostate cancer treatment with high-dose-rate brachytherapy

We address the real-world problem of automating the design of high-quality prostate cancer treatment plans in case of high-dose-rate brachytherapy, a form of internal radiotherapy. For this, recently a bi-objective real-valued problem formulation was introduced. With a GPU parallelization of the Multi-Objective Real-Valued Gene-pool Optimal Mixing Evolutionary Algorithm (MO-RV-GOMEA), good treatment plans were found in clinically acceptable running times. However, optimizing a treatment plan and delivering it to the patient in practice is a two-stage decision process and involves a number of uncertainties. Firstly, there is uncertainty in the identified organ boundaries due to the limited resolution of the medical images. Secondly, the treatment involves placing catheters inside the patient, which always end up (slightly) different from what was optimized. An important factor is therefore the robustness of the final treatment plan to these uncertainties. In this work, we show how we can extend the evolutionary optimization approach to find robust plans using multiple scenarios without linearly increasing the amount of required computation effort, as well as how to deal with these uncertainties efficiently when taking into account the sequential decision-making moments. The performance is tested on three real-world patient cases. We find that MO-RV-GOMEA is equally well capable of solving the more complex robust problem formulation, resulting in a more realistic reflection of the treatment plan qualities

Selective induction of apoptosis in leukemic B-lymphoid cells by a CD19-specific TRAIL fusion protein

Although the treatment outcome of lymphoid malignancies has improved in recent years by the introduction of transplantation and antibody-based therapeutics, relapse remains a major problem. Therefore, new therapeutic options are urgently needed. One promising approach is the selective activation of apoptosis in tumor cells by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study investigated the pro-apoptotic potential of a novel TRAIL fusion protein designated scFvCD19:sTRAIL, consisting of a CD19-specific single-chain Fv antibody fragment (scFv) fused to the soluble extracellular domain of TRAIL (sTRAIL). Potent apoptosis was induced by scFvCD19:sTRAIL in several CD19-positive tumor cell lines, whereas normal blood cells remained unaffected. In mixed culture experiments, selective binding of scFvCD19:sTRAIL to CD19-positive cells resulted in strong induction of apoptosis in CD19-negative bystander tumor cells. Simultaneous treatment of CD19-positive cell lines with scFvCD19:sTRAIL and valproic acid (VPA) or Cyclosporin A induced strongly synergistic apoptosis. Treatment of patient-derived acute B-lymphoblastic leukemia (B-ALL) and chronic B-lymphocytic leukemia (B-CLL) cells resulted in strong tumoricidal activity that was further enhanced by combination with VPA. In addition, scFvCD19:sTRAIL prevented engraftment of human Nalm-6 cells in xenotransplanted NOD/Scid mice. The pre-clinical data presented here warrant further investigation of scFvCD19:sTRAIL as a potential new therapeutic agent for CD19-positive B-lineage malignancies