Mike Keller

Long-Term Cytomegalovirus Infection Leads to Significant Changes in the Composition of the CD8+ T-Cell Repertoire, Which May Be the Basis for an Imbalance in the Cytokine Production Profile in Elderly Persons

ABSTRACT In spite of the present belief that latent cytomegalovirus (CMV) infection drives CD8+ T-cell differentiation and induces premature immune senescence, no systematic studies have so far been performed to compare phenotypical and functional changes in the CD8+ T-cell repertoire in CMV-infected and noninfected persons of different age groups. In the present study, number, cytokine production, and growth potential of naïve (CD45RA+ CD28+), memory (CD45RA− CD28+), and effector (CD45RA+ CD28− or CD45RA− CD28−) CD8+ T cells were analyzed in young, middle-aged, and elderly clinically healthy persons with a positive or negative CMV antibody serology. Numbers and functional properties of CMVpp65495-503-specific CD8+ T cells were also studied. We demonstrate that aging as well as CMV infection lead to a decrease in the size of the naïve CD8+ T-cell pool but to an increase in the number of CD8+ effector T cells, which produce gamma interferon but lack substantial growth potential. The size of the CD8+ memory T-cell population, which grows well and produces interleukin-2 (IL-2) and IL-4, also increases with aging, but this increase is missing in CMV carriers. Life-long latent CMV infection seems thus to diminish the size of the naïve and the early memory T-cell pool and to drive a Th1 polarization within the immune system. This can lead to a reduced diversity of CD8 responses and to chronic inflammatory processes which may be the basis of severe health problems in elderly persons.

Evidence of premature immune aging in patients thymectomized during early childhood.

While the thymus is known to be essential for the initial production of T cells during early life, its contribution to immune development remains a matter of debate. In fact, during cardiac surgery in newborns, the thymus is completely resected to enable better access to the heart to correct congenital heart defects, suggesting that it may be dispensable during childhood and adulthood. Here, we show that young adults thymectomized during early childhood exhibit an altered T cell compartment. Specifically, absolute CD4+ and CD8+ T cell counts were decreased, and these T cell populations showed substantial loss of naive cells and accumulation of oligoclonal memory cells. A subgroup of these young patients (22 years old) exhibited a particularly altered T cell profile that is usually seen in elderly individuals (more than 75 years old). This condition was directly related to CMV infection and the induction of strong CMV-specific T cell responses, which may exhaust the naive T cell pool in the absence of adequate T cell renewal from the thymus. Together, these marked immunological alterations are reminiscent of the immune risk phenotype, which is defined by a cluster of immune markers predictive of increased mortality in the elderly. Overall, our data highlight the importance of the thymus in maintaining the integrity of T cell immunity during adult life.

T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor repertoire

Atherosclerosis is a multifactorial, chronic-inflammatory disease for which the underlying cause remains unknown. It is also well documented that T-cells are among the first cells to migrate into the arterial intimal vessel layer, but their function there is still unexplained. Clinical and experimental data have provided evidence that atherosclerosis starts as an autoimmune reaction based on humoral and cellular immunity against a phylogenetically highly conserved stress protein, heat shock protein 60 (HSP60). In the present study, we phenotypically characterized T-cells from endarterectomized specimens of the carotid artery, and tested their reactivity to human HSP60. In addition, the T-cell receptor repertoire of the T-cell lines was defined by immunoscope analysis. We found a mixed population of CD4(+) and CD8(+) intralesional T-cells, with a slight predominance of CD8(+) cells. IFN-gamma production prevailed over IL-4 production. The T-cell reaction against human HSP60 was significantly increased in intralesional cells compared to peripheral T-cells. The lesion-derived T-cells showed an oligoclonally-restricted repertoire, in contrast to the polyclonal pattern of PBMC. These results clearly show that HSP60 is a major antigenic candidate, and that an oligoclonal T-cell expansion takes place in advanced human atherosclerotic lesions.

Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

ABSTRACT We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

Reversal of Human Cytomegalovirus Major Immediate-Early Enhancer/Promoter Silencing in Quiescently Infected Cells via the Cyclic AMP Signaling Pathway

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (MIE) enhancer contains five functional cyclic AMP (cAMP) response elements (CRE). Because the CRE in their native context do not contribute appreciably to MIE enhancer/promoter activity in lytically infected human fibroblasts and NTera2 (NT2)-derived neurons, we postulated that they might have a role in MIE enhancer/promoter reactivation in quiescently infected cells. Here, we show that stimulation of the cAMP signaling pathway by treatment with forskolin (FSK), an adenylyl cyclase activator, greatly alleviates MIE enhancer/promoter silencing in quiescently infected NT2 neuronal precursors. The effect is immediate, independent of de novo protein synthesis, associated with the phosphorylation of ATF-1 serine 63 and CREB serine 133, dependent on protein kinase A (PKA) and the enhancers CRE, and linked to viral-lytic-cycle advancement. Coupling of FSK treatment with the inhibition of either histone deacetylases or protein synthesis synergistically activates MIE gene expression in a manner suggesting that MIE enhancer/promoter silencing is optimally relieved by an interplay of multiple regulatory mechanisms. In contrast, MIE enhancer/promoter silence is not overcome by stimulation of the gamma interferon (IFN-γ) signaling pathway, despite the enhancer having two IFN-γ-activated-site-like elements. We conclude that stimulation of the cAMP/PKA signaling pathway drives CRE-dependent MIE enhancer/promoter activation in quiescently infected cells, thus exposing a potential mode of regulation in HCMV reactivation.

Breaking Human Cytomegalovirus Major Immediate-Early Gene Silence by Vasoactive Intestinal Peptide Stimulation of the Protein Kinase A-CREB-TORC2 Signaling Cascade in Human Pluripotent Embryonal NTera2 Cells

ABSTRACT The triggering mechanisms underlying reactivation of human cytomegalovirus (HCMV) in latently infected persons are unclear. During latency, HCMV major immediate-early (MIE) gene expression breaks silence to initiate viral reactivation. Using quiescently HCMV-infected human pluripotent embryonal NTera2 cells (NT2) to model HCMV reactivation, we show that vasoactive intestinal peptide (VIP), an immunomodulatory neuropeptide, immediately and dose-dependently (1 to 500 nM) activates HCMV MIE gene expression. This response requires the MIE enhancer cyclic AMP response elements (CRE). VIP quickly elevates CREB Ser133 and ATF-1 Ser63 phosphorylation levels, although the CREB Ser133 phosphorylation level is substantial at baseline. VIP does not change the level of HCMV genomes in nuclei, Oct4 (pluripotent cell marker), or hDaxx (cellular repressor of HCMV gene expression). VIP-activated MIE gene expression is mediated by cellular protein kinase A (PKA), CREB, and TORC2. VIP induces PKA-dependent TORC2 Ser171 dephosphorylation and nuclear entry, which likely enables MIE gene activation, as TORC2 S171A (devoid of Ser171 phosphorylation) exhibits enhanced nuclear entry and desilences the MIE genes in the absence of VIP stimulation. In conclusion, VIP stimulation of the PKA-CREB-TORC2 signaling cascade activates HCMV CRE-dependent MIE gene expression in quiescently infected NT2 cells. We speculate that neurohormonal stimulation via this signaling cascade is a possible means for reversing HCMV silence in vivo.

Quality of Training Effectiveness Assessment (QTEA); A neurophysiologically based method to enhance flight training

Today, flight trainers use objective measures of task performance and additional estimated, subjective data to assess the cognitive workload and situation awareness of trainees. This data is very useful in training assessment but trainees can succeed at performing a task purely by accident (referred to as ldquomiserable successrdquo). Additionally the trainee can be in a less than optimal for learning cognitive state when the instructor operator applies brute force training tasks and methods with little regard to the learning curve which can result in the training being too easy or more often too difficult thereby inducing negative learning. In order to provide the instructor with additional quantitative data on student performance, we have designed the quality of training effectiveness assessment (QTEA) concept. QTEA is conceived as a system that allows the trainer to assess a student in real-time using sensors that can quantify the cognitive and physiological workload. Patterns of cognitive and physiological behavior can be established for experts to provide a learning benchmark towards which new trainees can be trained. Using QTEA, the trainer can quantify the studentpsilas workload level in real-time so that the scenarios can be adjusted to an optimal intensity. The cognitive and physiological measures also serve as a quantitative manifestation of a studentpsilas learning curve and it will be possible for the trainer to detect plateaus in learning. QTEA is under development but once fielded as an operational system, the trainer will be able to assess the needs for further training in a student. The basic idea of QTEA is to give the trainer a real-time picture of the performance of a trainee based on human physiological and cognitive data, flight technical, and mission specific data. In this paper, we are presenting the QTEA framework in the context of an ongoing project that studies trainee performance in a simulated Urban Close-Air Support Task.

Neurophysiological workload assessment in flight

Over the past few years, we developed a neurocognitive assessment system called the cognitive avionics tool set (CATS). This system has been used in fixed and rotary wing aircraft and in automobiles to assess operator workload using physiological and neurocognitive markers. Sensors that we used in the past include dense array electroencephalogram (EEG), electrocardiogram (ECG), galvanic skin response (GSR), pulse oximetry (sPO2), respiration (amplitude and frequency), and non-contact measures such as facial feature point location, facial temperature differences, and eye tracking. We recently integrated a new sensor called the PhotrodeTM manufactured by SRICO. The Photrode is an optical device that detects electrical fields. The device operates by sending a beam of light through a Mach-Zehnder Interferometer (MZI). An electric field modulates the light in a way that can be detected by an optical receiver. Because bio-electrical fields travel through clothing, the Photrode can detect ECG activity remotely, eliminating the need for wires being directly attached to the individual. The Photrode is capable of detecting EEG activity remotely through clothing. This paper provides an overview of CATS.

Multi-Sensory Methods to Aid Pilot Spatial Orientation and Upset Recovery in Real Flight

The goal of the spatial orientation enhancement system (SOES) research program was to develop, integrate, evaluate, and refine a multi-sensory system that provides spatial orientation cues to pilots to avoid loss of control due to spatial disorientation and loss of situation awareness. The spatial orientation enhancement (SOES) program combined the existing concept of synthetic vision and enhanced the inherent spatial orientation capabilities of that system with tactile and audio cues to convey aircraft attitude. This paper describes part III of a three part study. This part tested general aviation (GA) maneuvers using a GA pilot population. Part I was a simulation experiment in the dynamic environment simulator (DES), testing military maneuvers using an F-22 flight model. Part II was a flight test of military maneuvers onboard the Harvard Mark IV operated by the National Research Council of Canada (NRC). Funding was provided by the NASA Langley Research Center under the aviation safety program (AvSP), the US Air Force Research laboratory (AFRL), and Rockwell Collins

Workload Assessment in Flight using Dense Array Eeg

As cockpit technologies advance and increase information-rich content is provided to aircrews, and it is possible that we are reaching the cognitive limits of the human operator. With additional layers of automation, crew alertness becomes equally important as high levels of workload. Foundational tools, methods, and technology components to quantitatively assess cognitive limits and to characterize operator state are needed to improve aircraft safety and enable full realization of the next generation air transport system (NGATS). Over the last two years, we have built up a neural imaging capability onboard our computerized airborne research platform (CARP) research aircraft, a Beech Bonanza. A similar system shall be deployed on the National Research Council (NRC, Canada) Bell 412 Advanced Systems Research Aircraft (ASRA). Flight trials on the ASRA are slated for November, 2006. We have collected preliminary physiological data using the CARP in flight to demonstrate that minute EEG signals can in fact be collected in the ecologically valid context of real flight. A secondary goal of our work was to develop data synchronization and artifact removal methods. In future research, we hope to automate these methods and collect physiological data to develop sophisticated operator state classification and feedback models. This research program is a collaborative effort between the National Research Council Canada and the Operator Performance Laboratory (OPL)