Familial influences on the full range of variability in attention and activity levels during adolescence: A longitudinal twin study.

To investigate familial influences on the full range of variability in attention and activity across adolescence, we collected maternal ratings of 339 twin pairs at ages 12, 14, and 16, and estimated the transmitted and new familial influences on attention and activity as measured by the Strengths and Weaknesses of Attention-Deficit/Hyperactivity Disorder Symptoms and Normal Behavior Scale. Familial influences were substantial for both traits across adolescence: genetic influences accounted for 54%-73% (attention) and 31%-73% (activity) of the total variance, and shared environmental influences accounted for 0%-22% of the attention variance and 13%-57% of the activity variance. The longitudinal stability of individual differences in attention and activity was largely accounted for by familial influences transmitted from previous ages. Innovations over adolescence were also partially attributable to familial influences. Studying the full range of variability in attention and activity may facilitate our understanding of attention-deficit/hyperactivity disorder's etiology and intervention.

The genetics of impulsivity: evidence for the heritability of delay discounting.

BACKGROUND: Delay discounting (DD), a decline in the subjective value of reward with increasing delay until its receipt, is an established behavioral model of impulsive choice, a key component of a broader impulsivity construct. Greater DD, i.e., a tendency to choose smaller immediate over larger delayed rewards, has been implicated as a potential intermediate phenotype (endophenotype) for addictive disorders and comorbid externalizing psychopathology, particularly in adolescence. However, genetic and environmental origins of DD remain unclear. Accordingly, the goal of the present study was to assess heritability of DD, an important aspect of its utility as an endophenotype. METHODS: A commonly used computerized procedure involving choice between varying amounts of money available immediately and a standard amount of $100 presented at variable delays was administered to a population-based sample of twins aged 16 and 18 (n = 560, including 134 monozygotic and 142 dizygotic pairs). DD was quantified using area under the discounting curve and the k coefficient estimated by fitting a hyperbolic model to individual data. Heritability was assessed using linear structural equation modeling of twin data. RESULTS: The genetic analysis revealed significant heritability of both DD measures (area under the discounting curve: 46% and 62%; k: 35% and 55% at age 16 and 18, respectively). CONCLUSIONS: The present study provides evidence for heritability of both model-based and model-free DD measures and suggests that DD is a promising intermediate phenotype for genetic dissection of impulsivity and externalizing spectrum disorders.

Long-term test-retest reliability of delayed reward discounting in adolescents.

Delay discounting (DD), a decline in subjective value of a reward with increasing temporal delay in receipt of that reward, is an established behavioral indicator of impulsivity. Preference for smaller-immediate over larger-delayed rewards has been implicated in the basic neurobehavioral mechanisms of risk for addictive disorders and related externalizing psychopathology. Establishing long-term stability of DD in adolescence is a necessary step towards its validation as an intermediate phenotype, or marker of risk, in neurobiological and genetic studies. Previous studies have demonstrated moderate to high test-retest reliability of DD, however, these studies utilized adult samples and examined relatively short retest intervals. Due to continuing development of brain and behavior, stability of temporal discounting behavior in adolescence may differ from that in adulthood. Here, two cohorts of adolescents aged 16 (n=126) and 18 (n=111) were administered a computerized test of DD and re-tested two years later. DD rate showed a modest but significant decrease with age, suggesting a reduction in overall impulsivity from middle to late adolescence. Significant test-retest correlations were observed in both cohorts (.67 and .76, respectively, p<.001) indicating longitudinal stability of individual differences in decision-making behavior during middle and late adolescence. This article is part of a Special Issue entitled: insert SI title.

Neural correlates of inhibitory control and functional genetic variation in the dopamine D4 receptor gene.

BACKGROUND: The dopamine D4 receptor gene (DRD4) has been implicated in psychiatric disorders in which deficits of self-regulation are a prominent feature (e.g., attention-deficit hyperactivity disorder and substance use disorders) and in dopamine D4 receptor insensitivity within prefrontal regions of the brain. Our hypothesis was that carriers of 7-repeats in the Variable Number of Tandem Repeats (VNTR) of DRD4 (7R+) would recruit prefrontal brain regions involved in successful inhibitory control to a lesser degree than non-carriers (7R-) and demonstrate less inhibitory control as confirmed by observation of locally reduced blood oxygenation level dependent (BOLD) % signal change and lower accuracy while performing "No-Go" trials of a Go/No-Go task. METHODS: Participants (age=18, n=62, 33 females) were recruited from the general population of the St. Louis, Missouri region. Participants provided a blood or saliva sample for genotyping, completed drug and alcohol-related questionnaires and IQ testing, and performed a Go/No-Go task inside of a 3T fMRI scanner. RESULTS: Go/No-Go task performance did not significantly differ between 7R+ and 7R- groups. Contrast of brain activity during correct "No-Go" trials with a non-target letter baseline revealed significant BOLD activation in a network of brain regions previously implicated in inhibitory control including bilateral dorsolateral prefrontal, inferior frontal, middle frontal, medial prefrontal, subcortical, parietal/temporal, and occipital/cerebellar brain regions. Mean BOLD % signal change during "No-Go" trials was significantly modulated by DRD4 genotype, with 7R+ showing a lower hemodynamic response than 7R- in right anterior prefrontal cortex/inferior frontal gyrus, left premotor cortex, and right occipital/cerebellar areas. Follow-up analyses suggested that 7-repeat status accounted for approximately 5-6% of the variance in the BOLD response during "No-Go" trials. DISCUSSION: The DRD4 7-repeat allele may alter dopaminergic function in brain regions involved in inhibitory control. When individuals must inhibit a prepotent motor response, presence of this allele may account for 5-6% of the variance in BOLD signal in brain regions critically associated with inhibitory control, but its influence may be associated with a greater effect on brain than on behavior in 18-year-olds from the general population.

Effects of nicotine deprivation and replacement on BOLD-fMRI response to smoking cues as a function of DRD4 VNTR genotype.

INTRODUCTION: Reactivity to smoking cues is an important factor in the motivation to smoke and has been associated with the dopamine receptor 4 variable number tandem repeat (DRD4 exon III VNTR) polymorphism. However, little is known about the associated neural mechanisms. METHODS: Non-treatment-seeking Caucasian smokers completed overnight abstinence and viewed smoking and neutral cues during 2 separate functional magnetic resonance imaging scans while wearing either a nicotine or placebo patch (order randomized) and were genotyped for the DRD4 VNTR. We conducted mixed-effects repeated-measures analyses of variance (within-subject factor: nicotine or placebo patch; between-subject factor: DRD4 long [L: ≥ 1 copy of ≥ 7 repeats] or short [S: 2 copies ≤ 6 repeats] genotype) of 6 a priori regions of interest. RESULTS: Relative to neutral cues, smoking cues elicited greater activity in bilateral ventral striatum and left amygdala during nicotine replacement and deactivation in these regions during nicotine deprivation. A patch × DRD4 interaction was observed in the left amygdala, an area associated with appetitive reinforcement and relapse risk, such that S allele carriers demonstrated greater activation on active patch than on placebo patch. CONCLUSIONS: Brain systems associated with reward salience may become primed and overreactive at nicotine replacement doses intended for the first step of smoking cessation and may become inhibited during nicotine withdrawal in DRD4 S but not in DRD4 L carriers. These findings are consistent with the role of these regions in drug reinforcement and suggest a differential influence of nicotine replacement on amygdala activation in the association of incentive salience with smoking stimuli across DRD4 genotypes.

Attention-Deficit/Hyperactivity Disorder, Autistic Traits, and Substance Use Among Missouri Adolescents.

BACKGROUND: Although existing literature demonstrates the association of attention-deficit/hyperactivity disorder (ADHD) with both substance use (SU) and autism spectrum disorder (ASD), few studies have examined rates of SU among adolescents with elevated ASD symptoms, with or without comorbid ADHD. Clinic-based studies suggest a possible protective effect of ASD against SU, but this has not been confirmed in population-based studies. OBJECTIVE: We examined alcohol, tobacco, and drug use in adolescents with either ADHD, elevated autistic traits, or both as compared with controls. METHODS: Subjects (N = 2937) who were 13 to 17 years old from a Missouri population-based large sibship sample were assessed for ADHD, autistic traits, and SU with the use of parent-report questionnaires. The Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition ADHD symptom criterion (Criterion A) was applied to the Strengths and Weaknesses of ADHD-symptoms and Normal-behavior (SWAN) questionnaire item responses to determine ADHD diagnosis. The presence of elevated autistic traits was defined as a raw Social Responsiveness Scale (SRS) score of 62 (95th percentile for this sample) or higher. SU was determined with the use of three items from the Child Behavior Checklist (CBCL). Statistical methods used included logistic and fractional polynomial regression. RESULTS: As compared with controls, adolescents with ADHD were at increased risk for alcohol, tobacco, and drug use whether or not they had elevated autistic traits. Adolescents with elevated autistic traits were at significantly increased risk for drug use other than alcohol and tobacco, even if they did not have ADHD. Among those with raw SRS scores in the range of about 20 (normal) to 80 (consistent with mild to moderate ASD), adolescents with ADHD had higher levels of SU than control individuals with similar levels of autistic traits. However, strong conclusions cannot be drawn regarding individuals with very low or very high SRS scores as a result of sparse data. CONCLUSIONS: This study confirms previous research showing an increased risk of SU among adolescents with ADHD. It also provides new information indicating that adolescents with high levels of autistic traits are at elevated risk for alcohol and tobacco use if they have comorbid ADHD; in addition, they may be at high risk for other drug use, even if they do not have comorbid ADHD. Therefore, it should not be assumed that adolescents with mild to moderate ASD have a low risk of SU, especially if ADHD is also present.

Autologous CLL cell vaccination early after transplant induces leukemia-specific T cells.

BACKGROUND: Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. METHODS: We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. RESULTS: At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8+ T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. CONCLUSION: Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. TRIAL REGISTRATION: Clinicaltrials.gov NCT00442130. FUNDING: NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Translational Research Program.

Detecting T-cell reactivity to whole cell vaccines: Proof of concept analysis of T-cell response to K562 cell antigens in CML patients.

BCR-ABL(+) K562 cells hold clinical promise as a component of cancer vaccines, either as bystander cells genetically modified to express immunostimulatory molecules, or as a source of leukemia antigens. To develop a method for detecting T-cell reactivity against K562 cell-derived antigens in patients, we exploited the dendritic cell (DC)-mediated cross-presentation of proteins generated from apoptotic cells. We used UVB irradiation to consistently induce apoptosis of K562 cells, which were then fed to autologous DCs. These DCs were used to both stimulate and detect antigen-specific CD8(+) T-cell reactivity. As proof-of-concept, we used cross-presented apoptotic influenza matrix protein-expressing K562 cells to elicit reactivity from matrix protein-reactive T cells. Likewise, we used this assay to detect increased anti-CML antigen T-cell reactivity in CML patients that attained long-lasting clinical remissions following immunotherapy (donor lymphocyte infusion), as well as in 2 of 3 CML patients vaccinated with lethally irradiated K562 cells that were modified to secrete high levels of granulocyte macrophage colony-stimulating factor (GM-CSF). This methodology can be readily adapted to examine the effects of other whole tumor cell-based vaccines, a scenario in which the precise tumor antigens that stimulate immune responses are unknown.

Effects of HIV and early life stress on amygdala morphometry and neurocognitive function.

Both HIV infection and high levels of early life stress (ELS) have been related to abnormalities in frontal-subcortical structures, yet the combined effects of HIV and ELS on brain structure and function have not been previously investigated. In this study we assessed 49 non-demented HIV-seropositive (HIV+) and 47 age-matched HIV-seronegative healthy control (HC) adults. Levels of ELS exposure were quantified and used to define four HIV-ELS groups: HC Low-ELS (N = 20); HC High-ELS (N = 27); HIV+ Low-ELS (N = 24); HIV+ High-ELS (N = 25). An automated segmentation tool measured volumes of brain structures known to show HIV-related or ELS-related effects; a brief neurocognitive battery was administered. A significant HIV-ELS interaction was observed for amygdala volumes, which was driven by enlargements in HIV+ High-ELS participants. The HIV+ High-ELS group also demonstrated significant reductions in psychomotor/processing speed compared with HC Low-ELS. Regression analyses in the HIV+ group revealed that amygdala enlargements were associated with higher ELS, lower nadir CD4 counts, and reduced psychomotor/processing speed. Our results suggest that HIV infection and high ELS interact to increase amygdala volume, which is associated with neurocognitive dysfunction in HIV+ patients. These findings highlight the lasting neuropathological influence of ELS and suggest that high ELS may be a significant risk factor for neurocognitive impairment in HIV-infected individuals.

A phase I trial of adeno-associated virus serotype 1-γ-sarcoglycan gene therapy for limb girdle muscular dystrophy type 2C.

γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16-38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10(9) viral genomes in 100 µl, the second group received a single injection of 1.5 × 10(10) viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10(10) viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7-10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.

Neural correlates of inhibitory control in adult attention deficit/hyperactivity disorder: evidence from the Milwaukee longitudinal sample.

Only a few studies have investigated the neural substrate of response inhibition in adult attention deficit hyperactivity disorder (ADHD) using Stop-Signal and Go/No-Go tasks. Inconsistencies and methodological limitations in the existing literature have resulted in limited conclusions regarding underlying pathophysiology. We examined the neural basis of response inhibition in a group of adults diagnosed with ADHD in childhood and who continue to meet criteria for ADHD. Adults with ADHD (n=12) and controls (n=12) were recruited from an ongoing longitudinal study and were matched for age, IQ, and education. Individuals with comorbid conditions were excluded. Functional magnetic resonance imaging (fMRI) was used to identify and compare the brain activation patterns during correct trials of a response-inhibition task (Go/No-Go). Our results showed that the control group recruited a more extensive network of brain regions than the ADHD group during correct inhibition trials. Adults with ADHD showed reduced brain activation in the right frontal eye field, pre-supplementary motor area, left precentral gyrus, and the inferior parietal lobe bilaterally. During successful inhibition of an inappropriate response, adults with ADHD display reduced activation in fronto-parietal networks previously implicated in working memory, goal-oriented attention, and response selection. This profile of brain activation may be specifically associated with ADHD in adulthood.

Recombinant adeno-associated virus type 2 pseudotypes: comparing safety, specificity, and transduction efficiency in the primate striatum. Laboratory investigation.

OBJECT: Although several clinical trials utilizing the adeno-associated virus (AAV) type 2 serotype 2 (2/2) are now underway, it is unclear whether this particular serotype offers any advantage over others in terms of safety or efficiency when delivered directly to the CNS. METHODS: Recombinant AAV2-green fluorescent protein (GFP) serotypes 2/1, 2/2, 2/5, and 2/8 were generated following standard triple transfection protocols (final yield 5.4 × 10(12) particles/ml). A total of 180 µl of each solution was stereotactically infused, covering the entire rostrocaudal extent of the caudoputamen in 4 rhesus monkeys (Macaca mulatta) (3.0 ± 0.5 kg). After 6 weeks' survival, the brain was formalin fixed, cut at 40 µm, and stained with standard immunohistochemistry for anti-GFP, anticaspase-2, and cell-specific markers (anti-microtubule-associated protein-2 for neurons and anti-glial fibrillary acidic protein for glia). Unbiased stereological counting methods were used to determine cell number and striatal volume. RESULTS: The entire striatum of each animal contained GFP-positive cells with significant labeling extending beyond the borders of the basal ganglia. No ischemic/necrotic, hemorrhagic, or neoplastic change was observed in any brain. Total infusate volumes were similar across the 4 serotypes. However, GFP-labeled cell density was markedly different. Adeno-associated virus 2/1, 2/2, and 2/5 each labeled < 8000 cells/mm(3), whereas serotype 8 labeled > 21,000 cells, a 3- to 4-fold higher transduction efficiency. On the other hand, serotype 8 also labeled neurons and glia with equal affinity compared with neuronal specificities > 89% for the other serotypes. Moderate caspase-2 colabeling was noted in neurons immediately around the AAV2/1 injection tracts, but was not seen above the background anywhere in the brain following injections with serotypes 2, 5, or 8. CONCLUSIONS: Intrastriatal delivery of AAV2 yields the highest cell transduction efficiencies but lowest neuronal specificity for serotype 8 when compared with serotypes 1, 2, and 5. Only AAV2/1 revealed significant caspase-2 activation. Careful consideration of serotype-specific differences in AAV2 neurotropism, transduction efficiency, and potential toxicity may affect future human trials.

Effects of nicotine withdrawal on verbal working memory and associated brain response.

Previous literature has reported effects of nicotine withdrawal on brain function during cognitive tasks such as verbal working memory (VWM). Mechanisms of these withdrawal effects have not been clearly identified. Functional neuroimaging offers an objective method to examine brain mechanisms associated with observable behavior and subjective reports. To investigate these mechanisms, 12 smokers were administered a 2-Back VWM challenge during two functional magnetic resonance imaging sessions. Participants abstained from smoking prior to both sessions; however, they applied a nicotine patch before one session and a placebo patch prior to the other. Among regions that exhibited a significant response to the 2-Back during either session, withdrawal was associated with significantly greater deactivation in left and right temporal poles and left medial frontal gyrus. The magnitude of task-related activation showed a significant inverse relationship to craving in the majority of regions during placebo administration. Also, individual brain responses varied more during placebo, suggesting inefficient neural processing. Results suggest that differences in brain response to a VWM challenge during abstinence may be attributed to increased craving. Further deactivation of regions associated with the default network (medial frontal and anterior temporal clusters) during the placebo condition suggests further suspension of default activity, possibly to compensate for inefficient neural processing.

Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages.

Directed gene transfer into specific cell lineages in vivo is an attractive approach for both modulating gene expression and correcting inherited mutations such as emphysema caused by human alpha1 antitrypsin (hAAT) deficiency. However, somatic tissues are mainly comprised of heterogeneous, differentiated cell lineages that can be short lived and difficult to specifically transfect. Here, we describe an intratracheally instilled lentiviral system able to deliver genes selectively to as many as 70% of alveolar macrophages (AMs) in the mouse lung. Following a single in vivo lentiviral transduction, genetically tagged AMs persisted in lung alveoli and expressed transferred genes for the lifetime of the adult mouse. A prolonged macrophage lifespan, rather than precursor cell proliferation, accounted for the surprisingly sustained presence of transduced AMs. We utilized this long-lived population to achieve localized secretion of therapeutic levels of hAAT protein in lung epithelial lining fluid. In an established mouse model of emphysema, lentivirally delivered hAAT ameliorated the progression of emphysema, as evidenced by attenuation of increased lung compliance and alveolar size. After 24 weeks of sustained gene expression, no humoral or cellular immune responses to hAAT protein were detected. Our results challenge the dogma that AMs are short lived and suggest that these differentiated cells may be a possible target cell population for in vivo gene therapy applications, including the sustained correction of hAAT deficiency.

A mammalian cell-based assay for screening inhibitors of RNA cleavage.

RNA cleavage is a catalytic reaction which defines many types of RNA processing events, including those of metabolite-sensing riboswitch, self-splicing introns, mRNA splicing, tRNA processing, polyA-cleavage, and various small ribozymes such as hairpin and hammerhead ribozyme. In this chapter, we describe a general methodology for developing a mammalian cell-based high-throughput screening assay useful for identifying small molecules capable of inhibiting RNA cleavage in mammalian cells. In the specific assay described, a plasmid DNA vector in which the expression of a luciferase reporter gene is controlled by hammerhead ribozyme cleavage was stably introduced into the human 293 cell line. Such a cell line enabled the rapid screening of chemical compound libraries and the identification of cell membrane-permeable inhibitory molecules capable of blocking ribozyme cleavage. The general strategy described later could in principle be adapted to identify small molecule inhibitors of many types of RNA cleavage reactions.

Genetically engineered T cells to target EGFRvIII expressing glioblastoma.

Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.

A fMRI Study of Verbal Working Memory, Cardiac Output, and Ejection Fraction in Elderly Patients with Cardiovascular Disease.

Cardiovascular disease (CVD) is associated with cognitive deficits even in the absence of stroke. We examined the relationship between cardiac performance, as measured by cardiac output (CO) and ejection fraction (EF), and brain activity during a verbal working memory (VWM) task in elderly CVD patients who tend to be at increased risk for vascular cognitive impairments. Seventeen patients were recruited from a cohort participating in an ongoing prospective study examining the effects of CVD on cognitive function in the elderly. Participants were diagnosed with CVD (age 68±8) and completed a 2-back VWM task in a 1.5T fMRI paradigm. CO and EF were calculated from echocardiogram measures. Task-related activation was averaged in a priori regions of interest. The relationship between CO, EF, and 2-back-related activity was modeled using partial correlations (two-tailed p<.05) controlling for age and 2-back accuracy. All participants were globally cognitively intact as indicated by Mini-Mental Status Exam and Dementia Rating Scale scores. Mean accuracy on the 2-back was 78±9% while reaction time averaged 1,027±192 ms. Mean CO and EF values showed a large range (CO: 3.55 to 6.31; EF: 0.36 to 0.76) but average values were within the normal range. After controlling for age and 2-back accuracy, lower EF was related to decrease in left insula activity (r=0.61, p=0.03). There were trends for EF to be related to accuracy (r=0.47, p=0.09) and reaction time (r=-0.48, p=0.09). CO was also related to insula activity (r=0.60, p=0.04) and activity in the supplementary motor area activity (r=0.66, p=0.01). Cardiac performance was related to decreased efficiency in task related brain areas and tended to be related to performance on a VWM task in elderly patients with CVD. Results have implications for a line of investigation indicating that cardiac and systemic vascular indices could be used as proxy measures to examine mechanisms of cerebrovascular dysfunction in the elderly.

Soluble receptor-mediated selective inhibition of VEGFR and PDGFRbeta signaling during physiologic and tumor angiogenesis.

The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFbeta receptor (PDGFRbeta) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRbeta signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRbeta (Ad sPDGFRbeta) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRbeta was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRbeta and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRbeta effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRbeta inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.

Successful inhibition of intracranial human glioblastoma multiforme xenograft growth via systemic adenoviral delivery of soluble endostatin and soluble vascular endothelial growth factor receptor-2: laboratory investigation.

OBJECT: Glioblastoma multiforme (GBM) is characterized by neovascularization, raising the question of whether angiogenic blockade may be a useful therapeutic strategy for this disease. It has been suggested, however, that, to be useful, angiogenic blockade must be persistent and at levels sufficient to overcome proangiogenic signals from tumor cells. In this report, the authors tested the hypothesis that sustained high concentrations of 2 different antiangiogenic proteins, delivered using a systemic gene therapy strategy, could inhibit the growth of established intracranial U87 human GBM xenografts in nude mice. METHODS: Mice harboring established U87 intracranial tumors received intravenous injections of adenoviral vectors encoding either the extracellular domain of vascular endothelial growth factor receptor-2-Fc fusion protein (Ad-VEGFR2-Fc) alone, soluble endostatin (Ad-ES) alone, a combination of Ad-VEGFR2-Fc and Ad-ES, or immunoglobulin 1-Fc (Ad-Fc) as a control. RESULTS: Three weeks after treatment, magnetic resonance imaging-based determination of tumor volume showed that treatment with Ad-VEGFR2-Fc, Ad-ES, or Ad-VEGFR2-Fc in combination with Ad-ES, produced 69, 59, and 74% growth inhibition, respectively. Bioluminescent monitoring of tumor growth revealed growth inhibition in the same treatment groups to be 62, 74, and 72%, respectively. Staining with proliferating cell nuclear antigen and with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling showed reduced tumor cell proliferation and increased apoptosis in all antiangiogenic treatment groups. CONCLUSIONS: These results suggest that systemic delivery and sustained production of endostatin and soluble VEGFR2 can slow intracranial glial tumor growth by both reducing cell proliferation and increasing tumor apoptosis. This work adds further support to the concept of using antiangiogenesis therapy for intracranial GBM.