Elevations in Tumor Necrosis Factor Alpha and Interleukin 6 From Neuronal-Derived Extracellular Vesicles in Repeated Low-Level Blast Exposed Personnel.

Objective: The purpose of this pilot study was to determine if military service members with histories of hundreds to thousands of low-level blast exposures (i. e., experienced breachers) had different levels of serum and neuronal-derived extracellular vesicle (EV) concentrations of interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNFα), compared to matched controls, and if these biomarkers related to neurobehavioral symptoms. Methods: Participants were experienced breachers (n = 20) and matched controls without blast exposures (n = 14). Neuronal-derived EVs were isolated from serum and identified with mouse anti-human CD171. Serum and neuronal-derived EVs were analyzed for IL-6, IL-10, and TNFα using an ultra-sensitive assay. Results: Serum TNFα concentrations were decreased in breachers when compared to control concentrations (p < 0.01). There were no differences in serum concentrations of IL-6, IL-10, or the IL-6/IL-10 ratio between breachers and controls (p's > 0.01). In neuronal-derived EVs, TNFα and IL-6 levels were increased in breachers compared to controls (p's < 0.01), and IL-10 levels were decreased in the breacher group compared to controls (p < 0.01). In breachers the IL-6/IL-10 ratio in neuronal-derived EVs was higher compared to controls, which correlated with higher total Rivermead Post-concussion Questionnaire (RPQ) scores (p's < 0.05). Conclusions: These findings suggest that exposure of personnel to high numbers of low-level blast over a career may result in enduring central inflammation that is associated with chronic neurological symptoms. The data also suggest that peripheral markers of inflammation are not necessarily adequate surrogates for central neuroinflammation.

Remote blast-related mild traumatic brain injury is associated with differential expression of exosomal microRNAs identified in neurodegenerative and immunological processes.

BACKGROUND: Blast traumatic brain injury (TBI) and subconcussive blast exposure have been associated, pathologically, with chronic traumatic encephalopathy (CTE) and, clinically, with cognitive and affective symptoms, but the underlying pathomechanisms of these associations are not well understood. We hypothesized that exosomal microRNA (miRNA) expression, and their relation to neurobehavioral outcomes among Veterans with blunt or blast mild TBI (mTBI) may provide insight into possible mechanisms for these associations and therapeutic targets. METHODS: This is a subanalysis of a larger Chronic Effects of Neurotrauma Consortium Biomarker Discovery Project. Participants (n = 152) were divided into three groups: Controls (n = 35); Blunt mTBI only (n = 54); and Blast/blast+blunt mTBI (n = 63). Postconcussive and post-traumatic stress symptoms were evaluated using the NSI and PCL-5, respectively. Exosomal levels of 798 miRNA expression were measured. RESULTS: In the blast mTBI group, 23 differentially regulated miRNAs were observed compared to the blunt mTBI group and 23 compared to controls. From the pathway analysis, significantly dysregulated miRNAs in the blast exposure group correlated with inflammatory, neurodegenerative, and androgen receptor pathways. DISCUSSION: Our findings suggest that chronic neurobehavioral symptoms after blast TBI may pathomechanistically relate to dysregulated cellular pathways involved with neurodegeneration, inflammation, and central hormonal regulation.

Neuronally-derived tau is increased in experienced breachers and is associated with neurobehavioral symptoms.

Military and law enforcement breachers are exposed to many low-level blasts during their training and occupational experiences in which they detonate explosives to force entry into secured structures. There is a concern that exposure to these repetitive blast events in career breachers could result in cumulative neurological effects. This study aimed to determine concentrations of neurofilament light (NF-L), tau, and amyloid-beta 42 (Aß42) in serum and in neuronal-derived extracellular vesicles (EVs) in an experienced breacher population, and to examine biomarker associations with neurobehavioral symptoms. Thirty-four participants enrolled in the study: 20 experienced breachers and 14 matched military or civilian law enforcement controls. EV tau concentrations were significantly elevated in experienced breachers (0.3301 ± 0.5225) compared to controls (-0.4279 ± 0.7557; F = 10.43, p = 0.003). No statistically significant changes were observed in EV levels of NF-L or Aß42 or in serum levels of NF-L, tau, or Aß42 (p's > 0.05). Elevated EV tau concentrations correlated with increased Neurobehavioral Symptom Inventory (NSI) score in experienced breachers (r = 0.596, p = 0.015) and predicted higher NSI score (F(1,14) = 7.702, p = 0.015, R2 = 0.355). These findings show that neuronal-derived EV concentrations of tau are significantly elevated and associated with neurobehavioral symptoms in this sample of experienced breachers who have a history of many low-level blast exposures.

Extracellular Vesicle Proteins and MicroRNAs Are Linked to Chronic Post-Traumatic Stress Disorder Symptoms in Service Members and Veterans With Mild Traumatic Brain Injury.

Symptoms of post-traumatic stress disorder (PTSD) are common in military populations, and frequently associated with a history of combat-related mild traumatic brain injury (mTBI). In this study, we examined relationships between severity of PTSD symptoms and levels of extracellular vesicle (EV) proteins and miRNAs measured in the peripheral blood in a cohort of military service members and Veterans (SMs/Vs) with chronic mTBI(s). Participants (n = 144) were divided into groups according to mTBI history and severity of PTSD symptoms on the PTSD Checklist for DSM-5 (PCL-5). We analyzed EV levels of 798 miRNAs (miRNAs) as well as EV and plasma levels of neurofilament light chain (NfL), Tau, Amyloid beta (Aß) 42, Aß40, interleukin (IL)-10, IL-6, tumor necrosis factor-alpha (TNFα), and vascular endothelial growth factor (VEGF). We observed that EV levels of neurofilament light chain (NfL) were elevated in participants with more severe PTSD symptoms (PCL-5 ≥ 38) and positive mTBI history, when compared to TBI negative controls (p = 0.024) and mTBI participants with less severe PTSD symptoms (p = 0.006). Levels of EV NfL, plasma NfL, and hsa-miR-139-5p were linked to PCL-5 scores in regression models. Our results suggest that levels of NfL, a marker of axonal damage, are associated with PTSD symptom severity in participants with remote mTBI. Specific miRNAs previously linked to neurodegenerative and inflammatory processes, and glucocorticoid receptor signaling pathways, among others, were also associated with the severity of PTSD symptoms. Our findings provide insights into possible signaling pathways linked to the development of persistent PTSD symptoms after TBI and biological mechanisms underlying susceptibility to PTSD.

Extracellular vesicle concentrations of glial fibrillary acidic protein and neurofilament light measured 1 year after traumatic brain injury.

Traumatic brain injury (TBI) is linked to long-term symptoms in a sub-set of patients who sustain an injury, but this risk is not universal, leading us and others to question the nature of individual variability in recovery trajectories. Extracellular vesicles (EVs) are a promising, novel avenue to identify blood-based biomarkers for TBI. Here, our aim was to determine if glial fibrillary acidic protein (GFAP) and neurofilament light (NfL) measured 1-year postinjury in EVs could distinguish patients from controls, and whether these biomarkers relate to TBI severity or recovery outcomes. EV GFAP and EV NfL were measured using an ultrasensitive assay in 72 TBI patients and 20 controls. EV GFAP concentrations were elevated in moderate and severe TBI compared to controls (p's < 0.001) and could distinguish controls from moderate (AUC = 0.86) or severe TBI (AUC = 0.88). Increased EV GFAP and EV NfL levels were associated with lower 1-year Glasgow Outcome Scale-Extended (GOS-E) score (p's < 0.05). These findings suggest that blood-derived EV concentrations of GFAP and NfL drawn even 1 year after injury are higher in TBI patients compared to controls, and are related to injury severity and poor recovery outcomes, suggesting that TBIs alter the activity of these biomarkers, likely contributing to individual variability in recovery.

Poor Sleep Quality is Linked to Elevated Extracellular Vesicle-Associated Inflammatory Cytokines in Warfighters With Chronic Mild Traumatic Brain Injuries.

Background: Elevations of inflammatory cytokine levels occur immediately after mild traumatic brain injury (mTBI) and can persist for years. These elevations have been associated with neuropsychological outcomes, including depression and PTSD symptoms. Sleep disorders, another common sequelae of mTBI, are independently associated with inflammation in otherwise healthy individuals. However, whether sleep and inflammation are linked in chronic mTBI has not been reported. Methods: A retrospective cross-sectional cohort of warfighters was used to investigate the hypothesis that inflammation may be linked to sleep quality in chronic mTBI. Clinical history, peripheral blood samples, and sleep quality scores were collected from 182 warfighters (n = 138 mTBI; n = 44 controls) during enrollment in the Chronic Effects of Neurotrauma Consortium study. Biomarkers of inflammation (IL-6, IL-10, TNFα cytokines) from plasma and plasma-derived extracellular vesicles (EVs) were quantified using single molecule array. Relationships between sleep quality and cytokine levels were assessed, controlling for age, sex, and BMI. Using clinical cutoff scores for sleep quality, mTBI patients were then divided into "good" and "poor" sleepers and cytokine levels compared between groups. Results: In mTBI participants, sleep quality was significantly associated with EV levels of IL-10 [ß (SE) = 0.11 (0.04), p = 0.01] and TNFα [ß (SE) = 0.07 (0.03), p < 0.01]. When divided according to "good" versus "poor" sleepers, those reporting poor sleep had significantly elevated EV IL-10 compared to those reporting good sleep [ß (SE) = 0.12 (0.04), p < 0.01]. Plasma-derived associations were not significant. No associations were found between sleep quality and cytokine levels in controls. Conclusion: These results suggest a significant relationship between sleep quality and chronic inflammation in mTBI patients. Clinically, mTBI patients with a high likelihood of sleep disorders demonstrate elevated levels of inflammatory cytokines. Signal from EVs, though smaller in magnitude, may have stronger clinical associations than from plasma. Sleep-focused interventions may also serve to regulate chronic inflammatory processes in these patients. Larger prospective studies are needed to investigate the mechanisms and therapeutic implications of the likely bi-directional relationship between sleep and inflammation following mTBI.

Exosomal neurofilament light: A prognostic biomarker for remote symptoms after mild traumatic brain injury?

OBJECTIVE: To measure exosomal and plasma levels of candidate blood biomarkers in veterans with history of mild traumatic brain injury (mTBI) and test their relationship with chronic symptoms. METHODS: Exosomal and plasma levels of neurofilament light (NfL) chain, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and vascular endothelial growth factor (VEGF) were measured using an ultrasensitive assay in a cohort of 195 veterans, enrolled in the Chronic Effects of Neurotrauma Consortium Longitudinal Study. We examined relationships between candidate biomarkers and symptoms of postconcussive syndrome (PCS), posttraumatic stress disorder (PTSD), and depression. Biomarker levels were compared among those with no traumatic brain injury (TBI) (controls), 1-2 mTBIs, and repetitive (3 or more) mTBIs. RESULTS: Elevated exosomal and plasma levels of NfL were associated with repetitive mTBIs and with chronic PCS, PTSD, and depression symptoms. Plasma TNF-α levels correlated with PCS and PTSD symptoms. The total number of mTBIs correlated with exosomal and plasma NfL levels and plasma IL-6. Increased number of years since the most recent TBI correlated with higher exosomal NfL and lower plasma IL-6 levels, while increased number of years since first TBI correlated with higher levels of exosomal and plasma NfL, as well as plasma TNF-α and VEGF. CONCLUSION: Repetitive mTBIs are associated with elevated exosomal and plasma levels of NfL, even years following these injuries, with the greatest elevations in those with chronic PCS, PTSD, and depression symptoms. Our results suggest a possible neuroinflammatory and axonal disruptive basis for symptoms that persist years after mTBI, especially repetitive.

Exosomal MicroRNAs in Military Personnel with Mild Traumatic Brain Injury: Preliminary Results from the Chronic Effects of Neurotrauma Consortium Biomarker Discovery Project.

Chronic symptoms after mild traumatic brain injury (mTBI) are common among veterans and service members, and represent a significant source of morbidity, with those who sustain multiple mTBIs at greatest risk. Exosomal micro-RNAs (miRNAs), mediators of intercellular communication, may be involved in chronic TBI symptom persistence. Exosomal miRNA (exomiR) was extracted from 153 participants enrolled in the Chronic Effect of Neurotrauma Consortium (CENC) longitudinal study (no TBI, n = 35; ≥ 3 mTBIs (rTBI), n = 45; 1-2 mTBIs, n = 73). Analyses were performed with nCounter® Human miRNA Expression Panels and Ingenuity Pathway Analysis (IPA) for identification of gene networks associated with TBI. Generalized linear models were used to analyze the predictive value of exomiR dysregulation and remote neurobehavioral symptoms. Compared with controls, there were 17 dysregulated exomiRs in the entire mTBI group and 32 dysregulated exomiRs in the rTBI group. Two miRNAs, hsa-miR-139-5p and hsa-miR-18a-5p, were significantly differentially expressed in the rTBI and 1-2 mTBI groups. IPA analyses showed that these dysregulated exomiRs correlated with pathways of inflammatory regulation, neurological disease, and cell development. Within the rTBI group, exomiRs correlated with gene activity for hub-genes of tumor protein TP53, insulin-like growth factor 1 receptor, and transforming growth factor beta. TBI history and neurobehavioral symptom survey scores negatively and significantly correlated with hsa-miR-103a-3p expression. Participants with remote mTBI have distinct exomiR profiles, which are significantly linked to inflammatory and neuronal repair pathways. These profiles suggest that analysis of exosomal miRNA expression may provide novel insights into the underlying pathobiology of chronic TBI symptom persistence.

Changes in Plasma von Willebrand Factor and Cellular Fibronectin in MRI-Defined Traumatic Microvascular Injury.

The neuropathology of traumatic brain injury (TB) is diverse, including primary injury to neurons, axons, glial cells, vascular structures, and secondary processes, such as edema and inflammation that vary between individual patients. Traumatic microvascular injury is an important endophenotype of TBI-related injury. We studied patients who sustained a TBI requiring ER evaluation and had an MRI performed within 48 h of injury. We classified patients into 3 groups based on their MRI findings: (1) those that had evidence of traumatic microvascular injury on susceptibility or diffusion weighted MRI sequences without frank hemorrhage [Traumatic Vascular Injury (TVI) group; 20 subjects]. (2) those who had evidence of intraparenchymal, subdural, epidural, or subarachnoid hemorrhage [Traumatic Hemorrhage (TH) group; 26 subjects], and (3) those who had no traumatic injuries detected by MRI [MRI-negative group; 30 subjects]. We then measured plasma protein biomarkers of vascular injury [von Willebrand Factor (vWF) or cellular fibronectin (cFn)] and axonal injury (phosphorylated neurofilament heavy chain; pNF-H). We found that the TVI group was characterized by decreased expression of plasma vWF (p < 0.05 compared to MRI-negative group; p < 0.00001 compared to TH group) ≤48 h after injury. cFN was no different between groups ≤48 h after injury, but was increased in the TVI group compared to the MRI-negative (p < 0.00001) and TH (p < 0.00001) groups when measured >48 h from injury. pNF-H was increased in both the TH and TVI groups compared to the MRI-negative group ≤48 h from injury. When we used the MRI grouping and molecular biomarkers in a model to predict Glasgow Outcome Scale-Extended (GOS-E) score at 30-90 days, we found that inclusion of the imaging data and biomarkers substantially improved the ability to predict a good outcome over clinical information alone. These data indicate that there is a distinct, vascular-predominant endophenotype in a subset of patients who sustain a TBI and that these injuries are characterized by a specific biomarker profile. Further work to will be needed to determine whether these biomarkers can be useful as predictive and pharmacodynamic biomarkers for vascular-directed therapies after TBI.

Higher exosomal phosphorylated tau and total tau among veterans with combat-related repetitive chronic mild traumatic brain injury.

OBJECTIVE: The objective of the study is to measure plasma and exosomal levels of tau, phosphorylated tau (p-tau), and amyloid beta (Aß) in Veterans with historical mild traumatic brain injury (mTBI) and chronic neuropsychological symptoms. METHODS: Tau, p-tau, Aß40, and Aß42 were measured by ultrasensitive immunoassay in plasma and exosomes from 195 Veterans enrolled in the Chronic Effects of Neurotrauma Consortium Multicenter Observational Study. Protein biomarkers were compared among groups with and without mTBI with loss of consciousness (LOC) or post-traumatic amnesia (PTA), and also in those with and without repetitive (≥3) mTBI (rTBI) compared to those with 0 (TBI-neg) and 1-2 mTBI. RESULTS: There were no differences in measures of plasma and exosomal protein levels among mTBI with LOC or PTA, mTBI with alteration of consciousness only or TBI-neg. Exosomal tau and exosomal p-tau were elevated in rTBI compared to those with 2 or fewer mTBIs and TBI-neg (p < 0.05). Elevations of exosomal tau and p-tau significantly correlated with post-traumatic and post-concussive symptoms, with exosomal tau also relating specifically to cognitive, affective, and somatic post-concussive symptoms (p < 0.05). CONCLUSION: rTBI is associated with elevations of exosomal p-tau and exosomal tau, suggesting that blood-based exosomes may provide a peripheral source of informative, centrally derived biomarkers in remote mTBI and that rTBI may contribute to chronic neuropsychological symptoms.

Phosphodiesterase-5 inhibition potentiates cerebrovascular reactivity in chronic traumatic brain injury.

BACKGROUND: Traumatic cerebrovascular injury (TCVI), a common consequence of traumatic brain injury (TBI), presents an attractive therapeutic target. Because phosphodiesterase-5 (PDE5) inhibitors potentiate the action of nitric oxide (NO) produced by endothelial cells, they are candidate therapies for TCVI. This study aims to: (1) measure cerebral blood flow (CBF), cerebrovascular reactivity (CVR), and change in CVR after a single dose of sildenafil (ΔCVR) in chronic TBI compared to uninjured controls; (2) examine the safety and tolerability of 8-week sildenafil administration in chronic symptomatic moderate/severe TBI patients; and as an exploratory aim, (3) assess the effect of an 8-week course of sildenafil on chronic TBI symptoms. METHODS: Forty-six subjects (31 chronic TBI, 15 matched healthy volunteers) were enrolled. Baseline CBF and CVR before and after administration of sildenafil were measured. Symptomatic TBI subjects then completed an 8-week double-blind, placebo-controlled, crossover trial of sildenafil. A neuropsychological battery and neurobehavioral symptom questionnaires were administered at each study visit. RESULTS: After a single dose of sildenafil, TBI subjects showed a significant increase in global CVR compared to healthy controls (P < 0.001, d = 0.9). Post-sildenafil CVR maps showed near-normalization of CVR in many regions where baseline CVR was low, predominantly within areas without structural abnormalities. Sildenafil was well tolerated. Clinical Global Impression (CGI) scale showed a trend toward clinical improvement while on sildenafil treatment. FINDINGS: Single-dose sildenafil improves regional CVR deficits in chronic TBI patients. CVR and ΔCVR are potential predictive and pharmacodynamic biomarkers of PDE5 inhibitor therapy for TCVI. Sildenafil is a potential therapy for TCVI.

Imaging of Cerebrovascular Function in Chronic Traumatic Brain Injury.

Traumatic cerebrovascular injury (TCVI) is a common pathologic mechanism of traumatic brain injury (TBI) and presents an attractive target for intervention. The aims of this study were to assess cerebral blood flow (CBF) and cerebrovascular reactivity (CVR) using magnetic resonance imaging (MRI) to assess their value as biomarkers of TCVI in chronic TBI, characterize the spatial distribution of TCVI, and assess the relationships between each biomarker and neuropsychological and clinical assessments. Forty-two subjects (27 chronic TBI, 15 age- and gender-matched healthy volunteers) were studied cross-sectionally. CBF was measured by arterial spin labeling and CVR by assessing the MRI-blood oxygen level-dependent signal with hypercapnia challenge. A focused neuropsychological battery adapted from the TBI Common Data Elements and neurobehavioral symptom questionnaires were administered at the time of the imaging session. Chronic TBI subjects showed a significant reduction in mean global, gray matter (GM), and white matter (WM) CVR, compared with healthy volunteers (p < 0.001). Mean GM CVR had the greatest effect size (Cohen's d = 0.9). CVR maps in chronic TBI subjects showed patchy, multifocal CVR deficits. CBF discriminated poorly between TBI subjects and healthy volunteers and did not correlate with CVR. Mean global CVR correlated best with chronic neurobehavioral symptoms among TBI subjects. Global, GM, and WM CVR are reliable and potentially useful biomarkers of TCVI in the chronic stage after moderate-to-severe TBI. CBF is less useful as biomarker of TCVI. CVR correlates best with chronic TBI symptoms. CVR has potential as a predictive and pharmacodynamic biomarker for interventions targeting TCVI.

Localized cortical chronic traumatic encephalopathy pathology after single, severe axonal injury in human brain.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with repetitive mild impact traumatic brain injury from contact sports. Recently, a consensus panel defined the pathognomonic lesion for CTE as accumulations of abnormally hyperphosphorylated tau (p-tau) in neurons (neurofibrillary tangles), astrocytes and cell processes distributed around small blood vessels at sulcal depths in irregular patterns within the cortex. The pathophysiological mechanism for this lesion is unknown. Moreover, a subset of CTE cases harbors cortical ß-amyloid plaques. In this study, we analyzed postmortem brain tissues from five institutionalized patients with schizophrenia and history of surgical leucotomy with subsequent survival of at least another 40 years. Because leucotomy involves severing axons bilaterally in prefrontal cortex, this surgical procedure represents a human model of single traumatic brain injury with severe axonal damage and no external impact. We examined cortical tissues at the leucotomy site and at both prefrontal cortex rostral and frontal cortex caudal to the leucotomy site. For comparison, we analyzed brain tissues at equivalent neuroanatomical sites from non-leucotomized patients with schizophrenia, matched in age and gender. All five leucotomy cases revealed severe white matter damage with dense astrogliosis at the axotomy site and also neurofibrillary tangles and p-tau immunoreactive neurites in the overlying gray matter. Four cases displayed p-tau immunoreactivity in neurons, astrocytes and cell processes encompassing blood vessels at cortical sulcal depths in irregular patterns, similar to CTE. The three cases with apolipoprotein E ε4 haplotype showed scattered ß-amyloid plaques in the overlying gray matter, but not the two cases with apolipoprotein E ε3/3 genotype. Brain tissue samples from prefrontal cortex rostral and frontal cortex caudal to the leucotomy site, and all cortical samples from the non-leucotomized patients, showed minimal p-tau and ß-amyloid pathology. These findings suggest that chronic axonal damage contributes to the unique pathology of CTE over time.

Beta-amyloid auto-antibodies are reduced in Alzheimer's disease.

Accumulation and cytotoxicity of amyloid beta (Aß) are understood as the major cause of Alzheimer's disease (AD). There is evidence that naturally occurring antibodies against amyloid beta (Aß) protein play a role in Aß-clearance, and such a mechanism appears to be impaired in AD. In the present study, the anti-Aß antibodies in the serum from individuals with and without late onset AD were measured using ELISA and dot-blot methods. Aß auto-antibodies in serum were mainly targeted to Aß1-15 epitope and its titer was significantly lower in AD patients than elderly non-AD controls (NC). The dot-blot analysis further demonstrated that auto-antibodies against fibrillar Aß42, Aß1-15 and Aß16-30 epitopes were all in a lower level in AD than in NC. The isotypes of the auto-antibodies were mainly non-inflammatory IgG2 type. We also analyzed the relationship of auto-Aß antibody levels with the genotypes of apolipoprotein E (ApoE) and ANKK1/DRD2 gene.

A peptide prime-DNA boost immunization protocol provides significant benefits as a new generation Aß42 DNA vaccine for Alzheimer disease.

Immunotherapy has the potential to provide a possible treatment therapy to prevent or delay Alzheimer disease. In a clinical trial (AN1792) in which patients received this immunotherapy and received active Aß1-42 peptide immunizations, treatment was stopped when 6% of patients showed signs of meningoencephalitis. Follow up on these patients led to the conclusion that the antibody response was beneficial in removing Aß1-42 from brain but an accompanying inflammatory Th1 T cell response was harmful. As a safe alternative treatment targeting the same self protein, Aß1-42, in brain, we and others are working on a DNA Aß1-42 immunization protocol as the immune response to DNA immunizations differs in many aspects from immunizations with peptide antigens. Because the immune response to DNA vaccination has different kinetics and has a significantly lower antibody production, we evaluated two different prime boost regimens, Aß1-42 DNA prime/Aß1-42 peptide boost and Aß1-42 peptide prime/Aß1-42 DNA boost for their effectiveness in antibody production and possible side effects due to inflammatory T cell responses. While both boost regimes significantly enhanced the specific antibody production with comparable antibody concentrations, the absence of the Aß1-42 T cell response (no proliferation and no cytokine production) is consistent with our previous findings using this DNA Aß1-42 trimer immunization and greatly enhances the safety aspect for possible clinical use.

cPLA2α knockout mice exhibit abnormalities in the architecture and synapses of cortical neurons.

Cytosolic phospholipase A2α (cPLA2α) affects membrane fluidity and permeability by catalyzing the hydrolysis of membrane phospholipids. We hypothesize that cPLA2α deficiency induces rigidity and architectural changes in cell membranes, especially in large cortical neurons. These membrane changes are discernible using light and electron microscopy. Through careful comparison with wild-type counterparts, we observed significant morphological changes in cortical neurons of cPLA2α knockout mice. These changes included the following: (1) increased numbers of nucleoli and enlarged nuclei, (2) narrower spaces between the inner and outer nuclear membranes, (3) reduced numbers of nuclear pores and altered nuclear pore structure, and (4) morphological changes in synaptic clefts. These results further suggest that cPLA2α and its cleaved arachidonic acids play important roles in cortical neuronal maturation and in normal neurochemical processes.

DNA immunization against amyloid beta 42 has high potential as safe therapy for Alzheimer's disease as it diminishes antigen-specific Th1 and Th17 cell proliferation.

The pathogenesis of Alzheimer's disease (AD) has been strongly associated with the accumulation of amyloid beta (Aß) peptides in brain, and immunotherapy targeting Aß provides potential for AD prevention. A clinical trial in which AD patients were immunized with Aß42 peptide was stopped when 6% of participants showed meningoencephalitis, apparently due to an inflammatory Th1 immune response. Previously, we and other have shown that Aß42 DNA vaccination via gene gun generates a Th2 cellular immune response, which was shown by analyses of the respective antibody isotype profiles. We also determined that in vitro T cell proliferation in response to Aß42 peptide re-stimulation was absent in DNA Aß42 trimer-immunized mice when compared to Aß42 peptide-immunized mice. To further characterize this observation prospectively and longitudinally, we analyzed the immune response in wild-type mice after vaccination with Aß42 trimer DNA and Aß42 peptide with Quil A adjuvant. Wild-type mice were immunized with short-term (1-3× vaccinations) or long-term (6× vacinations) immunization strategies. Antibody titers and isotype profiles of the Aß42 specific antibodies, as well as cytokine profiles and cell proliferation studies from this longitudinal study were determined. Sufficient antibody titers to effectively reduce Aß42, but an absent T cell proliferative response and no IFNγ or IL-17 secretion after Aß42 DNA trimer immunization minimizes the risk of inflammatory activities of the immune system towards the self antigen Aß42 in brain. Therefore, Aß42 DNA trimer immunization has a high probability to be effective and safe to treat patients with early AD.

The relationship of cardiovascular risk factors to Alzheimer disease in Choctaw Indians.

OBJECTIVES: To test the hypothesis that cardiovascular risk factors (CRFs) influence predisposition to and the clinical course of Alzheimer disease (AD), the authors compared Choctaw Indians, a group with known high CRF with white persons with AD. In addition to CRF history, the authors investigated the frequency of apolipoprotein E4 (apoE4) genotype andplasma homocysteine (HC) levels. METHOD: The authors compared 39 Choctaw Indians with AD and 39 Choctaw Indians without AD to 39 white persons with AD with all groups similar in age. CRF history included diabetes, hypertension, high cholesterol or hypolipidemic agent use, or myocardial infarction. The authors also compared plasma HC concentration and apoE4 allele frequency. RESULTS: Choctaw persons with AD differed significantly from white persons with AD in history of hypertension, diabetes, and in HC values but not from Indians without AD. There was a significantly lower apoE4 allele frequency in Choctaw Indian AD than white persons with AD, and both AD groups had an affected first degree relative significantly more often than Indian controls. There was no relationship between the number of CRF and age at onset among Indians or whites, whereas HC concentration was associated with significantly earlier age of onset for Choctaw Indians but not for whites. CONCLUSIONS: This small study suggests that in Choctaw Indians modifiable risk factors may play more of a role in disease pathogenesis than in whites and that nonmodifiable risk factors such as apoE4 may play less of a role.

Analysis of three plasmid systems for use in DNA A beta 42 immunization as therapy for Alzheimer's disease.

In an effort to optimize DNA immunization-elicited antibody production responses against A beta 1-42 (A beta 42) as a therapy for Alzheimer's disease (AD), comparisons were made between three distinct plasmid systems using gene gun delivery. Plasmids encoding A beta 42 monomer and a novel A beta 42 trimeric fusion protein were evaluated in conjunction with CMV or Gal4/UAS promoter elements. It was found that vaccination A beta 42 trimer under the Gal4/UAS promoter elicited high levels of anti-A beta 42 antibody production. Serum antibody levels from Gal4/UAS-A beta 42 trimer immunized mice were found to be 16.6+/-5.5 microg/ml compared to 6.5+/-2.5 microg/ml with Gal4/UAS-A beta 42 monomer or even less with CMV-A beta 42 trimer. As compared to monomeric A beta 42 or A beta 42 trimer expressed under the CMV promoter, injection of the Gal4/UAS-A beta 42 trimer induced high levels of A beta 42 antigen expression in tissue suggesting a mechanism for the increase in anti-A beta 42 antibody. Antibodies were found to be primarily IgG1 suggesting a predominant Th2 response (IgG1/IgG2a ratio of 9). Serum from A beta 42 trimer-vaccinated mice was also found to identify amyloid plaques in the brains of APP/PS1 transgenic mice. These results demonstrate the potential therapeutic use of Gal4/UAS DNA A beta 42 trimer immunization in preventing Alzheimer's disease.

DNA beta-amyloid(1-42) trimer immunization for Alzheimer disease in a wild-type mouse model.

CONTEXT: DNA beta-amyloid(1-42) (Abeta42) trimer immunization was developed to produce specific T helper 2 cell (T(H)2)-type antibodies to provide an effective and safe therapy for Alzheimer disease (AD) by reducing elevated levels of Abeta42 peptide that occur in the brain of patients with AD. OBJECTIVE: To compare the immune response in wild-type mice after immunization with DNA Abeta42 trimer and Abeta42 peptide. DESIGN AND INTERVENTION: Wild-type mice received either 4 microg of DNA Abeta42 trimer immunization administered with gene gun (n = 8) or intraperitoneal injection of 100 microg of human Abeta42 peptide with the adjuvant Quil A (n = 8). Titers, epitope mapping, and isotypes of the Abeta42-specific antibodies were analyzed. MAIN OUTCOME MEASURES: Antibody titers, mapping of binding sites (epitopes), isotype profiles of the Abeta42-specific antibodies, and T-cell activation. RESULTS: DNA Abeta42 trimer immunization resulted in antibody titers with a mean of 15 microg per milliliter of plasma. The isotype profile of the antibodies differed markedly. A predominant IgG1 antibody response was found in the DNA-immunized mice, indicating a T(H)2 type of immune response (IgG1/IgG2a ratio of 10). The peptide-immunized mice showed a mixed T(H)1/T(H)2 immune response (IgG1/IgG2a ratio of 1) (P < .001). No increased T-cell proliferation was observed in the DNA-immunized mice (P = .03). CONCLUSION: In this preliminary study in a wild-type mouse model, DNA Abeta42 trimer immunization protocol produced a T(H)2 immune response and appeared to have low potential to cause an inflammatory T-cell response.