Michelle L. Dossett

Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

Interleukin-15 rescues tolerant CD8 + T cells for use in adoptive immunotherapy of established tumors

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell–mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor α chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.

Adoptive immunotherapy: Engineering T cell responses as biologic weapons for tumor mass destruction

Adoptive T cell immunotherapy is an evolving technology with the potential of providing a means to safely and effectively target tumor cells for destruction.

Structural features of T cell receptor variable regions that enhance domain stability and enable expression as single-chain VαVβ fragments

The variable (V) domains of antibodies and T cell receptors (TCRs) share sequence homology and striking structural similarity. Single-chain antibody V domain constructs (scFv) are routinely expressed in a variety of heterologous systems, both for production of soluble protein as well as for in vitro engineering. In contrast, single-chain T cell receptor V domain constructs (scTCR) are prone to aggregation and misfolding and are refractory to display on phage or yeast in their wild-type form. However, through random mutagenesis and yeast display engineering, it has been possible to isolate scTCR mutants that are properly folded and displayed on the yeast surface. These displayed mutants can serve not only as a scaffold for further engineering but also as scTCR variants that exhibit favorable biophysical properties in Escherichia coli expression. Thus, a more comprehensive understanding of the V domain mutations that allowed display would be beneficial. Our goal here was to identify generalizable patterns of important mutations that can be applied to different TCRs. We compared five different scTCRs, four from mice and one from a human, for yeast surface display. Analysis of a collection of mutants revealed four distinct regions of TCR V domains that were most important for enabling surface expression: the Valpha-Vbeta interface, the HV4 of Vbeta, and the region of the Valpha and Vbeta domains normally apposed against the constant (C) domains. Consistent with the role of the V-C interface in surface display, reconstitution of this interface, by including the constant domains of each chain, allowed V domain display and alphabeta chain association on the yeast surface, thus providing an alternative TCR scaffold. However, the surface levels of TCR achieved with engineered scTCR mutants were superior to that of the ValphaCalpha/VbetaCbeta constructs. Therefore, we describe further optimization of the current strategy for surface display of the single-chain format in order to facilitate yeast display engineering of a broader range of scTCRs.

Peripheral CD8+ T cell tolerance to self-proteins is regulated proximally at the T cell receptor.

CD8(+) T cell tolerance, although essential for preventing autoimmunity, poses substantial obstacles to eliciting immune responses to tumor antigens, which are generally overexpressed normal proteins. Development of effective strategies to overcome tolerance for clinical applications would benefit from elucidation of the immunologic mechanism(s) regulating T cell tolerance to self. To examine how tolerance is maintained in vivo, we engineered dual-T cell receptor (TCR) transgenic mice in which CD8(+) T cells recognize two distinct antigens: a foreign viral-protein and a tolerizing self-tumor protein. Encounter with peripheral self-antigen rendered dual-TCR T cells tolerant to self, but these cells responded normally through the virus-specific TCR. Moreover, proliferation induced by virus rescued function of tolerized self-tumor-reactive TCR, restoring anti-tumor activity. These studies demonstrate that peripheral CD8(+) T cell tolerance to self-proteins can be regulated at the level of the self-reactive TCR complex rather than by central cellular inactivation and suggest an alternate strategy to enhance adoptive T cell immunotherapy.

Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection

Many of the most promising tumor antigens for T-cell-based cancer immunotherapies are unmodified self-antigens. Unfortunately, the avidity of T cells specific for these antigens is limited by central tolerance during T-cell development in the thymus, resulting in decreased anti-tumor efficacy of these T cells. One approach to overcoming this obstacle is to mutate T-cell receptor (TCR) genes from naturally occurring T cells to enhance the affinity for the target antigen. These enhanced-affinity TCRs can then be developed for use in TCR gene therapy. Although TCRs with significantly enhanced affinity have been generated using this approach, it is not clear whether these TCRs, which bypass the affinity limits imposed by negative selection, remain unresponsive to the low levels of self-antigen generally expressed by some normal tissues. Here we show that 2 variants of a high-affinity WT1-specific TCR with enhanced affinity for WT1 are safe and do not mediate autoimmune tissue infiltration or damage when transduced into peripheral CD8 T cells and transferred in vivo. However, if expressed in developing T cells and subjected to thymic selection, the same enhanced-affinity TCRs signal tolerance mechanisms in the thymus, resulting in T cells with attenuated antigen sensitivity in the periphery.

Complementary and alternative medicine use by US adults with gastrointestinal conditions: Results from the 2012 National Health Interview Survey.

OBJECTIVES:Use of complementary and alternative medicine (CAM) has increased over the past two decades, and a growing body of evidence suggests that some CAM modalities may be useful in addressing gastrointestinal (GI) conditions. However, the overall pattern of CAM use for GI conditions remains unknown. We sought to elucidate the prevalence and patterns of CAM use among US adults with GI conditions.METHODS:We used the 2012 National Health Interview Survey (n=34,525), a nationally representative survey of the civilian, noninstitutionalized US population, to estimate the prevalence of CAM use among adults with GI conditions (abdominal pain, acid reflux/heartburn, digestive allergy, liver condition, nausea and/or vomiting, stomach or intestinal illness, and ulcer). We also examined the reasons for CAM use, perceived helpfulness, and disclosure of use to health-care providers among individuals who specifically used CAM to address a GI condition. Prevalence estimates were weighted to reflect the complex sampling design of the survey.RESULTS:Of the 13,505 respondents with a GI condition in the past year, 42% (n=5629) used CAM in the past year and 3% (n=407) used at least one CAM modality to address a GI condition. The top three modalities among those using CAM to address GI conditions were herbs and supplements, mind body therapies, and manipulative therapies. Of those using CAM to address a GI condition, 47% used three or more CAM therapies, and over 80% felt that it was helpful in addressing a GI condition and was important in maintaining health and well-being. Respondents told their health-care providers about use of these therapies 70% of the time.CONCLUSIONS:CAM was used by 42% of respondents with a GI condition in the past year. A small proportion use CAM specifically to address their GI condition, but the majority who do find it helpful. The most commonly used modalities in this group are herbs and supplements, and mind body and manipulative therapies.

Adoptive Immunotherapy of Disseminated Leukemia With TCR-transduced, CD8+ T Cells Expressing a Known Endogenous TCR

Adoptive T-cell immunotherapy has shown promise in the treatment of human malignancies, but the challenge of isolating T cells with high avidity for tumor antigens in each patient has limited application of this approach. The transfer into T cells of T-cell receptor (TCR) genes encoding high-affinity TCRs recognizing defined tumor-associated antigens can potentially circumvent this obstacle. Using a well-characterized murine model of adoptive T-cell immunotherapy for widely disseminated leukemia, we demonstrate that TCR gene-modified T cells can cure mice of disseminated tumor. One goal of such adoptive therapy is to establish a persistent memory response to prevent recurrence; however, long-term function of transferred TCR-transduced T cells is limited due to reduced expression of the introduced TCR in vivo in quiescent resting T cells. However, by introducing the TCR into a cell with a known endogenous specificity, activation of these T cells by stimulation through the endogenous TCR can be used to increase expression of the introduced TCR, potentially providing a strategy to increase the total number of tumor-reactive T cells in the host and restore more potent antitumor activity.

Meditation for Adults with Mild Cognitive Impairment: A Pilot Randomized Trial

To the Editor High levels of chronic stress are associated with an increased incidence of Mild Cognitive Impairment (MCI) and Alzheimers disease (AD) (1, 2) and negatively impact the hippocampus, a key AD brain region. Mindfulness Based Stress Reduction (MBSR), a standardized mindfulness meditation/yoga intervention, may decrease stress/cortisol, improve well-being, and increase hippocampal gray matter density in healthy adults. (3, 4) Studies also demonstrate that meditation selectively activates the hippocampus,(5) and experienced meditators have larger hippocampal volumes and gray matter compared to controls. (6) Within this context, our objectives were to: 1) test the safety/feasibility of MBSR in adults with MCI; 2) explore the effects of MBSR on cognition and well-being through standardized instruments, neuropsychological evaluations, and interviews.

Relaxation Response and Resiliency Training and Its Effect on Healthcare Resource Utilization.

Background Poor psychological and physical resilience in response to stress drives a great deal of health care utilization. Mind-body interventions can reduce stress and build resiliency. The rationale for this study is therefore to estimate the effect of mind-body interventions on healthcare utilization. Objective Estimate the effect of mind body training, specifically, the Relaxation Response Resiliency Program (3RP) on healthcare utilization. Design Retrospective controlled cohort observational study. Setting: Major US Academic Health Network. Sample: All patients receiving 3RP at the MGH Benson-Henry Institute from 1/12/2006 to 7/1/2014 (n = 4452), controls (n = 13149) followed for a median of 4.2 years (.85–8.4 yrs). Measurements: Utilization as measured by billable encounters/year (be/yr) stratified by encounter type: clinical, imaging, laboratory and procedural, by class of chief complaint: e.g., Cardiovascular, and by site of care delivery, e.g., Emergency Department. Subgroup analysis by propensity score matched pre-intervention utilization rate. Results At one year, total utilization for the intervention group decreased by 43% [53.5 to 30.5 be/yr] (p <0.0001). Clinical encounters decreased by 41.9% [40 to 23.2 be/yr], imaging by 50.3% [11.5 to 5.7 be/yr], lab encounters by 43.5% [9.8 to 5.6], and procedures by 21.4% [2.2 to 1.7 be/yr], all p < 0.01. The intervention group’s Emergency department (ED) visits decreased from 3.6 to 1.7/year (p<0.0001) and Hospital and Urgent care visits converged with the controls. Subgroup analysis (identically matched initial utilization rates—Intervention group: high utilizing controls) showed the intervention group significantly reduced utilization relative to the control group by: 18.3% across all functional categories, 24.7% across all site categories and 25.3% across all clinical categories. Conclusion Mind body interventions such as 3RP have the potential to substantially reduce healthcare utilization at relatively low cost and thus can serve as key components in any population health and health care delivery system.