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HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.

This study tested the hypothesis that older adults would have a stronger response for substance P (facilitatory) but weaker response to β-endorphin (inhibitory), in magnitude as well as time course. Eight younger and 9 older adults underwent 3 experimental sessions using well validated laboratory pain models: cold pressor task, contact heat pain, and a nonpainful control. Blood was collected through an indwelling catheter at baseline and 3, 15, 30, 45, and 60 minutes after stimuli administration. Older adults had higher baseline levels of both neuropeptides suggesting increased peripheral activity compared with younger adults. After the cold pressor task, older adults demonstrated a quick and strong release of substance P with dramatic recovery, whereas young adults maintained a constant low-grade response. Unlike substance P, β-endorphin increased between 3 and 15 minutes for both groups with the upsurge substantially higher for older adults. After heat pain, younger adults had an immediate surge in circulating substance P and β-endorphin that was more pronounced than among older adults. However, levels of substance P for younger adults slowly tapered whereas they continued to climb for the older adults through 30 minutes. β-endorphin peaked at 30 minutes for both groups and returned to baseline. No changes were observed during the nonpainful control session., Perspective: Older adults had higher baseline levels of substance P and β-endorphin suggesting increased peripheral activity compared with younger adults. After pain evocation, older adults demonstrated a more intense early response for both neuropeptides suggesting peripheral mechanisms involved in the response to pain may change with age., (Copyright © 2017 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Although it is well documented that infectious diseases can pose threats to biodiversity, the potential long-term consequences of pathogen exposure on individual fitness and its effects on population viability have rarely been studied. We tested the hypothesis that pathogen exposure causes physiological carry-over effects with a pathogen that is uniquely suited to this question because the infection period is specific and time limited. The fungus Pseudogymnoascus destructans causes white-nose syndrome (WNS) in hibernating bats, which either die due to the infection while hibernating or recover following emergence from hibernation. The fungus infects all exposed individuals in an overwintering site simultaneously, and bats that survive infection during hibernation clear the pathogen within a few weeks following emergence. We quantified chronic stress during the active season, when bats are not infected, by measuring cortisol in bat claws. Free-ranging Myotis lucifugus who survived previous exposure to P. destructans had significantly higher levels of claw cortisol than naïve individuals. Thus, cryptic physiological carry-over effects of pathogen exposure may persist in asymptomatic, recovered individuals. If these effects result in reduced survival or reproductive success, they could also affect population viability and even act as a third stream in the extinction vortex. For example, significant increases in chronic stress, such as those indicated here, are correlated with reduced reproductive success in a number of species. Future research should directly explore the link between pathogen exposure and the viability of apparently recovered populations to improve understanding of the true impacts of infectious diseases on threatened populations., (© 2016 Society for Conservation Biology.)

Early social environment can play a significant role in shaping behavioural development. For instance, in many social mammals and birds, isolation rearing results in individuals that are less exploratory, shyer, less social and more aggressive than individuals raised in groups. Moreover, dynamic aspects of social environments, such as the nature of relationships between individuals, can also impact the trajectory of development. We tested if being raised alone or socially affects behavioural development in the family-living tree skink, Egernia striolata . Juveniles were raised in two treatments: alone or in a pair. We assayed exploration, boldness, sociability and aggression repeatedly throughout each juvenile's first year of life, and also assessed social interactions between pairs to determine if juveniles formed dominant-subordinate relationships. We found that male and/or the larger skinks within social pairs were dominant. Developing within this social environment reduced skink growth, and subordinate skinks were more prone to tail loss. Thus, living with a conspecific was costly for E. striolata . The predicted negative effects of isolation failed to materialize. Nevertheless, there were significant differences in behavioural traits depending on the social environment (isolated, dominant or subordinate member of a pair). Isolated skinks were more social than subordinate skinks. Subordinate skinks also became more aggressive over time, whereas isolated and dominant skinks showed invariable aggression. Dominant skinks became bolder over time, whereas isolated and subordinate skinks were relatively stable in their boldness. In summary, our study is evidence that isolation rearing does not consistently affect behaviour across all social taxa. Our study also demonstrates that the social environment plays an important role in behavioural development of a family-living lizard., Competing Interests: We have no competing interests.

Early developmental environment can have profound effects on individual physiology, behaviour, and learning. In birds and mammals, social isolation during development is known to negatively affect learning ability; yet in other taxa, like reptiles, the effect of social isolation during development on learning ability is unknown. We investigated how social environment affects learning ability in the family-living tree skink (Egernia striolata). We hypothesized that early social environment shapes cognitive development in skinks and predicted that skinks raised in social isolation would have reduced learning ability compared to skinks raised socially. Offspring were separated at birth into two rearing treatments: (1) raised alone or (2) in a pair. After 1 year, we quantified spatial learning ability of skinks in these rearing treatments (N = 14 solitary, 14 social). We found no effect of rearing treatment on learning ability. The number of skinks to successfully learn the task, the number of trials taken to learn the task, the latency to perform the task, and the number of errors in each trial did not differ between isolated and socially reared skinks. Our results were unexpected, yet the facultative nature of this species' social system may result in a reduced effect of social isolation on behaviour when compared to species with obligate sociality. Overall, our findings do not provide evidence that social environment affects development of spatial learning ability in this family-living lizard.

Protocols of temporal summation (TS) of pain typically involve the delivery of brief repetitive noxious pulses of a constant intensity while measuring the perceived intensity of pain after each pulse. The size percept of noxious repetitive stimulation has been poorly characterized. Furthermore, no studies have investigated age differences in TS of cold pain. The current study examined TS of pain intensity and the perceived size of the painful area during repetitive noxious heat and cold pulses in healthy younger (n = 104) and older adults (n = 40). Trials of 10 brief repetitive noxious heat or cold pulses were delivered to the upper extremities. Participants rated the perceived size of the painful area or intensity of pain after each pulse. The magnitude of change for the size percept and intensity for pain were calculated for each trial. The results indicated that older adults experienced greater TS of the size percept of cold stimuli compared with younger adults. Additionally, older women experienced greater TS of the size percept of heat stimuli compared with older men and all younger participants. No overall age or sex differences were found in the TS of pain intensity for cold or heat trials. These results suggest dysfunctional modulation of the spatial percept of the painful stimuli by older adults, and in particular older women, during repetitive noxious thermal pulses.

Based on the success in animal models of type 1 diabetes (T1D), clinical trials of adoptive regulatory T cell (Treg) therapy are underway using ex vivo expanded polyclonal Tregs. However, pre-clinical data also demonstrate that islet-specific Tregs are more potent than polyclonal Tregs at reversing T1D. Translation of this approach into man will require methods to generate large populations of islet-specific Tregs which, to date, has proved to be a major hurdle. Here we demonstrate the feasibility of lentiviral-mediated T cell receptor (TCR) gene transfer to confer antigen specificity on polyclonal human Tregs. Targeting has been achieved using TCRs isolated from human islet-specific and viral-specific CD4 + T cell clones. Engineered T cells demonstrated expression of ectopically-delivered TCRs, resulting in endowment of cognate antigen-specific responses. This enabled antigen-specific suppression at increased potency compared to polyclonal Tregs. However, cells transduced with islet-specific TCRs were less responsive to cognate antigen than viral-specific TCRs, and in some cases, required additional methods to isolate functional antigen-specific Tregs. This study demonstrates the potential of TCR gene transfer to develop islet-specific Treg therapies for effective treatment of T1D, but also highlights that additional optimisation may be required to achieve its full potential., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation in vitro or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1β, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 μM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects. IMPORTANCE We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells., (Copyright © 2017 American Society for Microbiology.)

The primary aim of this study was to test the hypothesis that a patient's subjective assessments of the dentist's technical competence, quality of care, and anticipated restoration longevity during a restorative visit are predictive of restoration outcome. This prospective cohort study involved 3,326 patients who received treatment for a defective restoration in a permanent tooth, participated in a baseline patient satisfaction survey, and returned for follow-up. Of the 4,400 restorations that were examined by 150 dentists, 266 (6%) received additional treatment after baseline. Reporting satisfaction with the technical skill of the dentist or quality of the dental work at baseline was not associated with retreatment after baseline. However, patients' views at baseline that the fee was reasonable (odds ratio [OR], 1.6) was associated with retreatment after baseline, whereas satisfaction at baseline with how long the filling would last (OR, 0.6) was associated with less retreatment. These findings suggest that retreatment occurs more often for patients who at baseline are satisfied with the cost or who at baseline have less confidence in the restoration. The authors found no associations between restoration retreatment and the patients' baseline evaluations of the technical skills of their dentists or perceptions of quality care., Knowledge Transfer Statement: Dental patients' ratings of their dentist's skills were not related to a restoration needing retreatment. Patients focus on other aspects of the dental visit when making this judgment.

Background: The objectives were to: (1) quantify patient satisfaction with treatment for early dental caries overall, and according to whether or not (2a) the patient received invasive treatment; (2b) was high-risk for dental caries, and had dental insurance; and (3) encourage practitioners to begin using non-invasive approaches to early caries management., Methods: Ten practitioners recorded patient, lesion, and treatment information about non-cavitated early caries lesions. Information on 276 consecutive patients with complete data was included, who received either non-invasive (no dental restoration) or invasive (dental restoration) treatment. Patients completed a patient satisfaction questionnaire and were classified as dissatisfied if they did not "agree" or "strongly agree" with any of 14 satisfaction items., Results: Patients had a mean (± SD) age of 41.8 (±15.8) years, 64% were female and 88% were white. Twenty-five percent (n = 68) were dissatisfied in at least one of the 14 satisfaction items. Satisfaction levels did not significantly vary by patient's gender, race, caries risk category, or affected tooth surface location. Overall, 11% (28 of 276) received invasive treatment; satisfaction did not differ between patients who had invasive or non-invasive treatment. Seven patients received invasive treatment at their request even though that was not what their practitioner recommended; 5 out of 6 were satisfied with their treatment nonetheless., Conclusions: About one-fourth of patients treated for non-cavitated early caries were dissatisfied with at least some aspect of their dental care experience. Satisfaction of patients who received invasive treatment did not differ from those who received non-invasive treatment.

Effective clearance of virally infected cells requires the sequential activity of innate and adaptive immunity effectors. In human immunodeficiency virus (HIV) infection, naturally induced cell-mediated immune responses rarely eradicate infection. However, optimized immune responses could potentially be leveraged in HIV cure efforts if epitope escape and lack of sustained effector memory responses were to be addressed. Here we review leading HIV cure strategies that harness cell-mediated control against HIV in stably suppressed antiretroviral-treated subjects. We focus on strategies that may maximize target recognition and eradication by the sequential activation of a reconstituted immune system, together with delivery of optimal T-cell responses that can eliminate the reservoir and serve as means to maintain control of HIV spread in the absence of antiretroviral therapy (ART). As evidenced by the evolution of ART, we argue that a combination of immune-based strategies will be a superior path to cell-mediated HIV control and eradication. Available data from several human pilot trials already identify target strategies that may maximize antiviral pressure by joining innate and engineered T cell responses toward testing for sustained HIV remission and/or cure., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Acute pain arises from activation of myelinated (A delta) and unmyelinated (C) nociceptive afferents, leading to first (A-fiber) or second (C-fiber) pain sensations. The current study sought to investigate first and second pain within glabrous and hairy skin sites in human upper limbs. Fifty healthy adults (25 male/25 female, 18-30 years old, mean = 20.5 ± 1.4 years) participated in a psychophysical study investigating electronically rated, thermal first and second pain sensations within the glabrous skin at the palm and hairy skin of the forearm. Repeated measures analysis of variance indicated that the threshold for first pain was lower (more sensitive) than for second pain (P = .004), for glabrous as well as hairy skin, and thresholds at glabrous skin were higher than for hairy skin (P = .001). Hairy skin presented a steeper slope for testing, whereas there were no differences in slope between first and second pain. The study findings support assumptions associated with mechanistic differences between first and second pain sensations, while offering a novel method for producing first and second pain with the same thermal stimulus. Efforts to understand abnormalities among people with clinical pain and development of new therapeutic agents will benefit from specific psychophysical methods., Perspective: This article presents a novel method for directly comparing first and second pain within the same thermal stimulus. The ability to directly compare first and second pain sensations can aid in understanding pain abnormalities in clinical pain and development of therapeutic aids., (Copyright © 2016 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Background Aims: Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a "first-in-human" clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation., Methods: To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture., Results: aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced., Discussion: Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease., Competing Interests: The authors share a patent entitled “Methods to expand a T Regulatory Cell Master Cell Bank”; US Patent # 13/639,927., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: A prospective cohort study that included dentists in The National Dental Practice-Based Research Network was conducted to quantify 12-month failures of restorations that were repaired or replaced at baseline. The study tested the hypothesis that no significant differences exist in failure percentages between repaired and replaced restorations after 12 months. It also tested the hypothesis that certain dentist, patient, and restoration characteristics are significantly associated with the incidence of restoration failure., Methods: Dentists recorded data for 50 or more consecutive defective restorations. The restorations that were either - repaired or replaced were recalled after 12 months and characterized for developing defects., Results: Dentists (N = 195) recorded data on 5,889 restorations; 378 restorations required additional treatment (74 repaired, 171 replaced, 84 teeth received endodontic treatment, and 49 were extracted). Multivariable logistic regression analysis indicated that additional treatment was more likely to occur if the original restoration had been repaired (7%) compared with replaced (5%) (odds ratio [OR], 1.6; P < .001; 95% confidence interval [CI], 1.2-2.1), if a molar was restored (7%) compared with premolars or anterior teeth (5% and 6%, respectively) (OR, 1.4; P = .010; 95% CI, 1.1-1.7), and if the primary reason was a fracture (8%) compared with other reasons (6%) (OR, 1.3; P = .033; 95% CI, 1.1-1.6)., Conclusions: An additional treatment was more likely to occur within the first year if the original restoration had been repaired (7%) compared with being replaced (5%). However, repaired restorations were less likely to need an aggressive treatment (replacement, endodontic treatment, or extraction) than replaced restorations., Practical Implications: One year after repair or replacement of a defective restoration, the failure rate was low. However, repaired restorations were less likely to need an aggressive treatment than replaced restorations.

HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: JW, JJD, MCH, KLH, JD, and PDG are employees of Sangamo Biosciences Inc.

Objectives: Laryngeal research is typically conducted on animal species that have similar features to the human larynx, yet little is known of the frequency characteristics of those animals. This study examines frequency characteristics of dog, cat, pig, and sheep across emotional communication events., Study Design: Prospective study., Methods: Acoustic data were gathered in domestic environment, with fundamental frequency (f o ) computation of vocalization during happy, distress, and food request activities., Results: Dogs demonstrated reduced f o range and reduced vocalization as a factor of age (18 semitones for adult male dog, three semitones for aged male dog). When two barks were present, the second bark was typically shorter in length and had a higher f o . Male cat was observed to be more talkative and exhibited a much higher purr f o than his female housemate. Cat purr was varied by sex between the typical 25 Hz and a significantly higher f o , consistent with literature on use of cry-purr by some cats. Lambs exhibited phonation breaks, and an overall frequency range of 28 semitones in lambs compared with 49 semitones for mature sheep. Piglets exhibited f o range of 44 semitones with higher f o with distress and lowest f o for grunting., Conclusions: Aged animals followed similar phonatory patterns of humans, with change of f o for communication need. The male cat was found to purr differently from female cats. Pigs and cats may have nonlaryngeal source signal coupling. Animal phonation changes with emotion. Observed f o patterns may improve interpretation of phonation among laboratory animals in laryngeal research., (Copyright © 2016 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

In carcinogen-driven cancers, a high mutational burden results in neoepitopes that can be recognized immunologically. Such carcinogen-induced tumors may evade this immune response through "immunoediting," whereby tumors adapt to immune pressure and escape T cell-mediated killing. Many tumors lack a high neoepitope burden, and it remains unclear whether immunoediting occurs in such cases. Here, we evaluated T cell immunity in an autochthonous mouse model of pancreatic cancer and found a low mutational burden, absence of predicted neoepitopes derived from tumor mutations, and resistance to checkpoint immunotherapy. Spontaneous tumor progression was identical in the presence or absence of T cells. Moreover, tumors arising in T cell-depleted mice grew unchecked in immune-competent hosts. However, introduction of the neoantigen ovalbumin (OVA) led to tumor rejection and T cell memory, but this did not occur in OVA immune-tolerant mice. Thus, immunoediting does not occur in this mouse model - a likely consequence, not a cause, of absent neoepitopes. Because many human tumors also have a low missense mutational load and minimal neoepitope burden, our findings have clinical implications for the design of immunotherapy for patients with such tumors., Competing Interests: The authors have declared that no conflict of interest exists.

CD4(+)CD25(+)FoxP3(+) thymic-derived regulatory T cells (tTregs) are indispensable for maintaining immune system equilibrium. Adoptive transfer of tTregs is an effective means of suppressing graft-versus-host disease (GVHD) in murine models and in early human clinical trials. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an ubiquitin-conjugating enzyme that mediates nuclear factor κB (NF-κB) activation, plays an essential role in modulating regulatory T cell survival and function. MicroRNAs (miRNAs) are noncoding RNAs, which mediate RNA silencing and posttranscriptional gene repression. By performing comprehensive TaqMan Low Density Array miRNA assays, we identified 10 miRNAs differentially regulated in human tTreg compared with control T cells. One candidate, miR-146b, is preferentially and highly expressed in human naive tTregs compared with naive CD4 T cells. miRNA prediction software revealed that TRAF6 was the one of the top 10 scored mRNAs involved tTreg function with the highest probability as a potential miR-146b target. Antagomir-mediated knockdown of miRNA-146b, but not another miRNA-146 family member (miRNA-146a), enhanced TRAF6 expression. TRAF6, in turn, increases NF-κB activation, which is essential for tTreg function as well as Foxp3 protein and antiapoptotic gene expression, and downregulates proapoptotic gene expression. miR-146b knockdown increased the nuclear localization and expression of genes regulated by NF-κB, which was associated with enhanced tTreg survival, proliferation, and suppressive function measured in vitro and in vivo. TRAF6 inhibition had the opposite effects. We conclude that an miR-146b-TRAF6-NF-κB-FoxP3 signaling pathway restrains regulatory T cell survival, proliferation, and suppressor function. In vitro exposure of human tTregs to miR-146b antagomirs can be exploited to improve the clinical efficacy of human adoptive tTreg transfer in a GVHD setting., (© 2016 by The American Society of Hematology.)

Pain among individuals with knee osteoarthritis (OA) is associated with significant disability in older adults, and recent evidence demonstrates enhanced experimental pain sensitivity. Although previous research showed considerable heterogeneity in the OA clinical pain presentation, less is known regarding the variability in responses to experimental pain. The present study included individuals with knee OA (n = 292) who participated in the Understanding Pain and Limitations in Osteoarthritic Disease study and completed demographic and psychological questionnaires followed by a multimodal quantitative sensory testing (QST) session. Quantitative sensory testing measures were subjected to variable reduction procedures to derive pain sensitivity index scores, which in turn were entered into a cluster analysis. Five clusters were significantly different across all pain sensitivity index variables (P < 0.001) and were characterized by: (1) low pain sensitivity to pressure pain (N = 39); (2) average pain sensitivity across most modalities (N = 88); (3) high temporal summation of punctate pain (N = 38); (4) high cold pain sensitivity (N = 80); and (5) high sensitivity to heat pain and temporal summation of heat pain (N = 41). Clusters differed significantly by race, gender, somatic reactivity, and catastrophizing (P < 0.05). Our findings support the notion that there are distinct subgroups or phenotypes based on experimental pain sensitivity in community-dwelling older adults with knee OA, expanding previous findings of similar cluster characterizations in healthy adults. Future research is needed to further understand the pathophysiological mechanisms underlying pain within these subgroups, which may be of added value in tailoring effective treatments for people with OA.

Background: Because of its high global burden, determining biopsychosocial influences of chronic low back pain (CLBP) is a research priority. Psychological factors such as pain catastrophizing are well established. However, cognitive factors such as working memory warrant further investigation to be clinically useful., Objective: The purpose of this study was to determine how working memory and pain catastrophizing are associated with CLBP measures of daily pain intensity and movement-evoked pain intensity., Design: This study was a cross-sectional analysis of individuals with ≥3 months of CLBP (n=60) compared with pain-free controls (n=30)., Method: Participants completed measures of working memory, pain catastrophizing, and daily pain intensity. Movement-evoked pain intensity was assessed using the Back Performance Scale. Outcome measures were compared between individuals with CLBP and those who were pain-free using nonparametric testing. Associations were determined using multivariate regression analyses., Results: Participants with CLBP (mean age=47.7 years, 68% female) had lower working memory performance (P=.008) and higher pain catastrophizing (P<.001) compared with pain-free controls (mean age=47.6 years, 63% female). For individuals with CLBP, only working memory remained associated with daily pain intensity (R(2)=.07, standardized beta=-.308, P=.041) and movement-evoked pain intensity (R(2)=.14, standardized beta=-.502, P=.001) after accounting for age, sex, education, and interactions between pain catastrophizing and working memory., Limitations: The cross-sectional design prevented prospective analysis. Findings also are not indicative of overall working memory (eg, spatial) or cognitive performance., Conclusion: Working memory demonstrated the strongest association with daily pain and movement-evoked pain intensity compared with (and after accounting for) established CLBP factors. Future research will elucidate the prognostic value of working memory on prevention and recovery of CLBP., (© 2016 American Physical Therapy Association.)

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

Unlabelled: Laboratory-based studies show that acute aerobic and isometric exercise reduces sensitivity to painful stimuli in young healthy individuals, indicative of a hypoalgesic response. However, little is known regarding the effect of aging on exercise-induced hypoalgesia (EIH). The purpose of this study was to examine age differences in EIH after submaximal isometric exercise and moderate and vigorous aerobic exercise. Healthy older and younger adults completed 1 training session and 4 testing sessions consisting of a submaximal isometric handgrip exercise, vigorous or moderate intensity stationary cycling, or quiet rest (control). The following measures were taken before and after exercise/quiet rest: 1) pressure pain thresholds, 2) suprathreshold pressure pain ratings, 3) pain ratings during 30 seconds of prolonged noxious heat stimulation, and 4) temporal summation of heat pain. The results revealed age differences in EIH after isometric and aerobic exercise, with younger adults experiencing greater EIH compared with older adults. The age differences in EIH varied across pain induction techniques and exercise type. These results provide evidence for abnormal pain modulation after acute exercise in older adults., Perspective: This article enhances our understanding of the influence of a single bout of exercise on pain sensitivity and perception in healthy older compared with younger adults. This knowledge could help clinicians optimize exercise as a method of pain management., Competing Interests: There are no other conflicts of interest to report with regard to this work for any of the authors., (Copyright © 2016 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Temporal summation (TS) of pain protocols typically involve the delivery of brief repetitive noxious stimuli held at a constant intensity and measuring the consequent increase in the perceived intensity of pain sensations. To date, no studies have examined the effect of a TS protocol on the perceived spatial dimensions of the pain experience and its interaction with age. This study used a new TS protocol that examined changes in the perceived size of the painful area in 22 younger adults and 20 older adults. Four trials of ten brief heat pulses delivered at a constant intensity were administered on the volar forearm. Interpulse intervals (IPIs) were 2.5 seconds or 3.5 seconds. Subjects rated the peak pain intensity (trials 1 and 3) or the size of the painful area (trials 2 and 4) after each pulse on a 0-100 scale. The magnitude of summation was calculated for each trial. Three seconds and 6 seconds after delivering the last heat pulse, the subjects rated the intensity or the size of any remaining pain (aftersensations). The results indicated that older adults compared to younger adults exhibited significantly greater summation of size ratings for the 2.5-second and 3.5-second IPI trials and size of pain aftersensations at 3 seconds following the 2.5-second IPI TS trial. These results suggest that aging is associated with enhanced endogenous facilitation of the perceived size of pain. The potential clinical and mechanistic implications of enhanced TS of size of pain remain unknown and warrant further investigation.

Objective: Symptomatic knee osteoarthritis (OA) is a condition commonly associated with increased pain, disability, and functional limitations. Given the poor correspondence between radiographic evidence and clinical pain, central sensitization has been implicated as a potential mechanism underlying pain facilitation in knee OA. Sex may be a moderator of centrally mediated changes in knee OA pain; however, few studies have systematically assessed this. Therefore, the aim of this study was to examine differences in peripheral and central sensitization in men and women with symptomatic knee OA, as well as to determine whether these differences vary across age (middle age versus older age)., Methods: Participants (n = 288) between the ages of 45 and 85 years completed a battery of quantitative sensory pain procedures assessing sensitivity to contact heat, cold pressor, mechanical pressure, and punctate stimuli. Differences in temporal summation (TS) were examined, as well as measures of clinical pain and functional performance., Results: When compared to men, women exhibited greater sensitivity to multiple pain modalities (i.e., lower heat, cold, pressure thresholds/tolerances, greater TS of pain); however, there were no sex differences in clinical pain, with the exception of greater widespread pain observed in women. Although there were select age-related differences in pain sensitivity, sex differences in pain varied minimally across the age cohort., Conclusion: Overall, these findings provide evidence for greater overall sensitivity to experimental pain in women with symptomatic knee OA compared to men, suggesting that enhanced central sensitivity may be an important contributor to pain in this group., (© 2016, American College of Rheumatology.)

Background: Shoulder pain and surgery are common among older adults. However, the extent to which older age affects recovery after shoulder surgery is not well understood., Objective: To assess influence of older age on postoperative recovery factors 3 and 6 months after shoulder arthroscopy., Design: Prospective cohort study., Setting: University-affiliated outpatient orthopedic surgical center., Patients: A convenience sample of 139 persons between 20 and 79 years of age who experienced shoulder pain, had musculoskeletal dysfunction based on imaging and physician assessment, and were scheduled for an arthroscopic shoulder procedure., Main Outcome Measures: Postoperative outcomes were compared among younger, middle-aged, and older adults before surgery and at 3 and 6 months after surgery using analysis of variance modeling. Movement-evoked pain and an experimental laboratory correlate of pain processing were assessed at each time point. The influence of older age on 3- and 6-month pain outcomes were determined via multivariate regression analyses after accounting for preoperative, intraoperative, and postoperative prognostic factors., Results: Older adults had higher movement-evoked pain intensity (F2,108 = 5.18, P = .007) and experimental pain response (F2,111 = 7.24, P = .001) at 3 months compared with young and middle-aged adults. After controlling for key prognostic factors, older age remained a positive predictor of 3-month movement-evoked pain (R(2) = 0.05; standardized [St.] β = 0.263, P = .031) and experimental pain response (R(2) = 0.07; St. β = 0.295, P = .014). Further, older age remained a positive predictor of movement-evoked pain at 6 months (R(2) = 0.04; St. β = 0.231, P = .004), despite no age group differences in outcome. Older age was found to be the strongest predictor of 3- and 6-month movement-evoked pain., Conclusion: Older adults may experience more pain related to movement, as well as endogenous pain excitation, in the first few months after shoulder arthroscopy. Future age-related research should consider use of movement-evoked pain intensity and experimental pain response as pain outcomes, as well as the utility of such measures in clinical care., (Copyright © 2016 American Academy of Physical Medicine and Rehabilitation. Published by Elsevier Inc. All rights reserved.)

We studied the safety and clinical outcomes of patients treated with umbilical cord blood (UCB)-derived regulatory T cells (Tregs) that expanded in cultures stimulated with K562 cells modified to express the high-affinity Fc receptor (CD64) and CD86, the natural ligand of CD28 (KT64/86). Eleven patients were treated with Treg doses from 3-100 × 10(6) Treg/kg. The median proportion of CD4(+)FoxP3(+)CD127(-) in the infused product was 87% (range, 78%-95%), and we observed no dose-limiting infusional adverse events. Clinical outcomes were compared with contemporary controls (n = 22) who received the same conditioning regimen with sirolimus and mycophenolate mofetil immune suppression. The incidence of grade II-IV acute graft-versus-host disease (GVHD) at 100 days was 9% (95% confidence interval [CI], 0-25) vs 45% (95% CI, 24-67) in controls (P = .05). Chronic GVHD at 1 year was zero in Tregs and 14% in controls. Hematopoietic recovery and chimerism, cumulative density of infections, nonrelapse mortality, relapse, and disease-free survival were similar in the Treg recipients and controls. KT64/86-expanded UCB Tregs were safe and resulted in low risk of acute GVHD., (© 2016 by The American Society of Hematology.)

Objectives: Use of a rubber dam during root canal treatment is considered the standard of care because it enhances patient safety and optimises the odds of successful treatment. Nonetheless, not all dentists use a rubber dam, creating disconnect between presumed standard of care and what is actually done in clinical practice. Little is known about dentists' attitudes towards use of the rubber dam in their practices. The objectives were to: (1) quantify these attitudes and (2) test the hypothesis that specific attitudes are significantly associated with rubber dam use., Setting: National Dental Practice-Based Research Network (NationalDentalPBRN.org)., Participants: 1490 network dentists., Outcome Measures: Dentists completed a questionnaire about their attitudes towards rubber dam use during root canal treatment. Three attitude scales comprised 33 items that used a 5-point ordinal scale to measure beliefs about effectiveness, inconvenience, ease of placement, comparison to other isolation techniques and patient factors. Factor analysis, cluster analysis and multivariable logistic regression analysed the relationship between attitudes and rubber dam use., Results: All items had responses at each point on the 5-point scale, with an overall pattern of substantial variation across dentists. Five attitudinal factors (rubber dam effectiveness; inconvenient/time-consuming; ease of placement; effectiveness compared to Isolite; patient factors) and 4 clusters of practitioners were identified. Each factor and cluster was independently and strongly associated with rubber dam use., Conclusions: General dentists have substantial variation in attitudes about rubber dam use. Beliefs that rubber dam use is not effective, inconvenient, time-consuming, not easy to place or affected by patient factors, were independently and significantly associated with lower rubber dam use. These attitudes explain why there is substantial discordance between presumed standard of care and actual practice., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Older adults are at an increased risk to develop frequent and prolonged pain. Emerging evidence proposes a link between immune changes and pain, which is consistent with the inflammation theory of aging and the increased incidence of age-related diseases. This study tested the hypothesis that older adults show greater immune responses to experimental pain compared to younger individuals. Study subjects (8 younger and 9 older healthy adults) underwent 3 experimental sessions using well-validated human experimental pain models: the cold pressor task (CPT), focal heat pain (FHP), and a non-painful thermal control. Blood was collected through an indwelling catheter at baseline and 3, 15, 30, 45, 60, and 90 min post-stimuli administration. Pro-inflammatory cytokines (TNF-α IL-6 and IL-8) peaked at the same time points for both groups, with greater elevations among older subjects for TNF-α and IL-8 in both pain models and elevations in IL-6 only for CPT. Anti-inflammatory cytokines (IL-4, IL-5, and IL-10) generally peaked later for the older subjects, with increased elevations for FHP but not the CPT. These data are consistent with the assertion that age-related immune system dysregulation may account for the increased prevalence of pain in older adults., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: A prospective cohort study that included dentists in The National Dental Practice-Based Research Network was conducted to quantify 12-month failures of restorations that were repaired or replaced at baseline. The study tested the hypothesis that no significant differences exist in failure percentages between repaired and replaced restorations after 12 months. It also tested the hypothesis that certain dentist, patient, and restoration characteristics are significantly associated with the incidence of restoration failure., Methods: Dentists recorded data for 50 or more consecutive defective restorations. The restorations that were either repaired or replaced were recalled after 12 months and characterized for developing defects., Results: Dentists (N = 195) recorded data on 5,889 restorations; 378 restorations required additional treatment (74 repaired, 171 replaced, 84 teeth received endodontic treatment, and 49 were extracted). Multivariable logistic regression analysis indicated that additional treatment was more likely to occur if the original restoration had been repaired (7%) compared with replaced (5%) (odds ratio [OR], 1.6; P < .001; 95% confidence interval [CI], 1.2-2.1), if a molar was restored (7%) compared with premolars or anterior teeth (5% and 6%, respectively) (OR, 1.4; P = .010; 95% CI, 1.1-1.7), and if the primary reason was a fracture (8%) compared with other reasons (6%) (OR, 1.3; P = .033; 95% CI, 1.1-1.6)., Conclusions: An additional treatment was more likely to occur within the first year if the original restoration had been repaired (7%) compared with being replaced (5%). However, repaired restorations were less likely to need an aggressive treatment (replacement, endodontic treatment, or extraction) than replaced restorations., Practical Implications: One year after repair or replacement of a defective restoration, the failure rate was low. However, repaired restorations were less likely to need an aggressive treatment than replaced restorations., (Copyright © 2015 American Dental Association. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Chronic low back pain (CLBP) is a highly prevalent and disabling musculoskeletal pain condition among older adults. Transcutaneous electrical nerve stimulation (TENS) is commonly used to treat CLBP, however response to TENS in older adults compared with younger adults is untested. In a dose-response study stratified by age, 60 participants with axial CLBP (20 young, 20 middle-aged, 20 older) received four 20-minute sessions of high-frequency high-intensity TENS over a 2- to 3-week period in a laboratory-controlled setting. Experimental measures of pain sensitivity (mechanical pressure pain detection threshold) and central pain excitability (phasic heat temporal summation and heat aftersensations) were assessed before and after TENS. Episodic or immediate axial CLBP relief was assessed after TENS via measures of resting pain, movement-evoked-pain, and self-reported disability. Cumulative or prolonged axial CLBP relief was assessed by comparing daily pain reports across sessions. Independent of age, individuals experienced episodic increase in the pressure pain detection threshold and reduction in aftersensation after TENS application. Similarly, all groups, on average, experienced episodic axial CLBP relief via improved resting pain, movement-evoked pain, and disability report. Under this design, no cumulative effect was observed as daily pain did not improve for any age group across the 4 sessions. However, older adults received higher TENS amplitude across all sessions to achieve TENS responses similar to those in younger adults. These findings suggest that older adults experience similar episodic axial CLBP relief to that of younger individuals after high-frequency, high-intensity TENS when higher dose parameters are used., Perspective: This study examined age group differences in experimental and axial CLBP response to TENS, delivered under the current recommended parameters of strong, but tolerable amplitude. Older adults had comparable TENS response although at higher TENS amplitude than younger adults, which may have important mechanistic and clinical implications., (Copyright © 2015 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Objectives: The prevalence of knee osteoarthritis (OA) is increasing with the aging population and is exacerbated by the growing numbers of obese older adults. Low levels of vitamin D, measured by serum 25-hydroxyvitamin D (25(OH)D), in older adults and obese individuals are correlated with several negative health conditions, including chronic pain. This cross-sectional study sought to examine the interactive influence of 25(OH)D levels and obesity on knee OA pain and functional performance measures., Methods: The sample consisted of 256 (63% female) racially diverse (55% black/African Americans) middle-aged and older adults (mean age 56.8 y). Blood was collected for analysis of 25(OH)D by high-performance liquid chromatography. Participants provided self-report regarding knee OA pain and underwent a lower extremity functional performance test., Results: Results demonstrated that obesity was associated with lower levels of 25(OH)D. Participants with adequate 25(OH)D levels reported significantly less knee OA pain compared with participants with deficient or insufficient levels, regardless of obesity status. Furthermore, there was a significant interaction between obesity and 25(OH)D levels for lower extremity functional performance, such that obese individuals with adequate 25(OH)D levels demonstrated better performance than those obese participants with deficient or insufficient 25(OH)D levels., Discussion: The mechanisms by which adequate 25(OH)D levels are associated with pain severity and improved function have not been completely elucidated. It may be that the pleiotropic role of biologically active 25(OH)D influences pain and pain processing through peripheral and central mechanisms. Alternatively, higher levels of pain may lead to reduced outdoor activity, which may contribute to both obesity and decreased vitamin D. Thus, investigating vitamin D status in obese and nonobese individuals with knee OA warrants further study.

The current study evaluated age differences in conditioned pain modulation using a test stimulus that provided the opportunity to evaluate changes in heat pain sensitivity, sensitization, and desensitization within the same paradigm. During this psychophysical test, pain intensity clamping uses REsponse Dependent STIMulation (REDSTIM) methodology to automatically adjust stimulus intensity to maintain a desired pain rating set-point. Specifically, stimulus intensity increases until a pre-defined pain rating (the setpoint) is exceeded, and then decreases until pain ratings fall below the setpoint, with continued increases and decreases dictated by ratings. The subjects are blinded in terms of the setpoint and stimulus intensities. Younger and older subjects completed two test sessions of two REDSTIM trials, with presentation of conditioning cold stimulation between the trials of one session but not the other. The results indicated that conditioning cold stimulation similarly decreased the overall sensitivity of younger and older subjects, as measured by the average temperature that maintained a setpoint rating of 20 (on a scale of 0-100). The conditioning stimulus also significantly enhanced sensitization following ascending stimulus progressions and desensitization following descending stimulus progressions in older subjects relative to younger subjects. Thus, older subjects experienced greater swings in sensitivity in response to varying levels of painful stimulation. These results are discussed in terms of control over pain intensity by descending central modulatory systems. These findings potentially shed new light on the central control over descending inhibition and facilitation of pain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: After conducting a media campaign focusing on the importance of oral and pharyngeal cancer (OPC) examinations, we assessed mechanisms of behavior change among individuals receiving an OPC examination for the first time., Methods: We used data from 2 waves of telephone surveys of individuals residing in 36 rural census tracts in northern Florida (n = 806). The second survey occurred after our media intervention. We developed media messages and modes of message delivery with community members via focus groups and intercept interviews. We performed a mediation analysis to examine behavior change mechanisms., Results: Greater exposure to media messages corresponded with heightened concern about OPC. Heightened concern, in turn, predicted receipt of a first-time OPC examination, but only among men., Conclusions: We extended earlier studies by measuring an outcome behavior (receipt of an OPC examination) and demonstrating that the putative mechanism of action (concern about the disease) explained the link between a media intervention and engaging in the target behavior. Improving the quality of media campaigns by engaging community stakeholders in selecting messages and delivery methods is an effective strategy in building public health interventions aimed at changing behaviors.

Despite the ability of antiretroviral therapy to minimize human immunodeficiency virus type 1 (HIV-1) replication and increase the duration and quality of patients' lives, the health consequences and financial burden associated with the lifelong treatment regimen render a permanent cure highly attractive. Although T cells play an important role in controlling virus replication, they are themselves targets of HIV-mediated destruction. Direct genetic manipulation of T cells for adoptive cellular therapies could facilitate a functional cure by generating HIV-1-resistant cells, redirecting HIV-1-specific immune responses, or a combination of the two strategies. In contrast to a vaccine approach, which relies on the production and priming of HIV-1-specific lymphocytes within a patient's own body, adoptive T-cell therapy provides an opportunity to customize the therapeutic T cells prior to administration. However, at present, it is unclear how to best engineer T cells so that sustained control over HIV-1 replication can be achieved in the absence of antiretrovirals. This review focuses on T-cell gene-engineering and gene-editing strategies that have been performed in efforts to inhibit HIV-1 replication and highlights the requirements for a successful gene therapy-mediated functional cure.

Background: Pain intensity clamping uses the REsponse-Dependent Stimulation (REDSTIM) methodology to automatically adjust stimulus intensity to maintain a desired pain rating set-point which is continuously monitored from a subject's real-time pain ratings. REDSTIM blinds subjects regarding the pain intensity set-point, supporting its use for assessing intervention efficacy. By maintaining the pain intensity at a constant level, a potential decrease in pain sensitivity can be detected by an increase in thermode temperature (unknown to the subject) and not by pain ratings alone. Further, previously described sensitizing and desensitizing trends within REDSTIM provide a novel insight into human pain mechanisms overcoming limitations of conventional testing methods. The purpose of the present study was to assess the test-retest reliability of pain intensity clamping using REDSTIM during three separate sessions., Methods: We used a method for testing changes in pain sensitivity of human subjects (REDSTIM) where the stimulus temperature is modulated to clamp pain intensity near a desired set-point. Temperature serves as the response variable and is used to infer pain sensitivity. Several measures were analyzed for reliability including average temperature and area under the curve (AUC). Intraclass correlation coefficients were calculated for each measure at pain rating set-points of 20/100 and 35/100., Results: Sixteen healthy individuals (mean age = 21.6 ± 3.9) participated in three experiments two days apart at both pain rating set-points. Most reliability coefficients were in the moderate to substantial range (r's = 0.79 to 0.94) except for the negative AUC (r = 0.52), but only at the 20/100 pain rating set-point., Conclusions: The present study supports the test-retest reliability of pain intensity clamping using the REDSTIM methodology while providing a novel tool to examine human pain modulatory mechanisms and overcoming common shortcomings of conventional quantitative sensory testing methods.

Study Design: Single-blind randomized trial., Objectives: To compare the effects of cervical and shoulder thrust manipulation (TM) and exercise on pain sensitivity, and to explore associations with clinical outcomes in patients with shoulder pain., Background: Experimental studies indicate that spinal TM has an influence on central pain processes, supporting its application for treatment of extremity conditions. Direct comparison of spinal and peripheral TM on pain sensitivity has not been widely examined., Methods: Seventy-eight participants with shoulder pain (36 female; mean ± SD age, 39.0 ± 14.5 years) were randomized to receive 3 treatments of cervical TM (n = 26), shoulder TM (n = 27), or shoulder exercise (n = 25) over 2 weeks. Twenty-five healthy participants (13 female; mean ± SD age, 35.2 ± 11.1 years) were assessed to compare pain sensitivity with that in clinical participants at baseline. Primary outcomes were changes in local (eg, shoulder) and remote (eg, tibialis anterior) pressure pain threshold and heat pain threshold occurring over 2 weeks. Secondary outcomes were shoulder pain intensity and patient-rated function at 4, 8, and 12 weeks. Analysis-of-variance models and partial-correlation analyses were conducted to examine comparative effects and the relationship between measures., Results: At baseline, clinical participants demonstrated lower local (mean difference, - 1.63 kg; 95% confidence interval [CI]: -2.40, -0.86) and remote pressure pain threshold (mean difference, -1.96 kg; 95% CI: -3.09, -0.82) and heat pain threshold (mean difference, -1.15°C; 95% CI: -2.06, -0.24) compared to controls, suggesting enhanced pain sensitivity. Following intervention, there were no between-group differences in pain sensitivity or clinical outcome (P>.05). However, improvements were noted, regardless of intervention, for pressure pain threshold (range of mean differences, 0.22-0.32 kg; 95% CI: 0.03, 0.43), heat pain threshold (range of mean differences, 0.30-0.58; 95% CI: 0.06, 0.96), pain intensity (range of mean differences, -1.79 to -1.45; 95% CI: -2.34, -0.94), and function (range of mean differences, 3.15-3.82; 95% CI: 0.69, 6.20) at all time points. We did not find an association between pain sensitivity changes and clinical outcome (P>.05). Conclusion Clinical participants showed enhanced pain sensitivity, but did not respond differently to cervical or peripheral TM. In fact, in this sample, cervical TM, shoulder TM, and shoulder exercise had similar pain sensitivity and clinical effects. The lack of association between pain sensitivity and clinical pain and function outcomes suggests different (eg, nonspecific) pain pathways for clinical benefit following TM or exercise., Level of Evidence: Therapy, level 1b.

Objectives: (i) To quantify the importance that dentists place on caries risk factors when developing a caries treatment plan and (ii) to test the hypothesis that the ratings of importance for specific factors are significantly associated with whether or not the dentist performs caries risk assessment (CRA)., Methods: This study used a cross-sectional study design consisting of a questionnaire survey. The study queried dentists who worked in outpatient dental practices who were affiliated with the Dental Practice-Based Research Network Japan (JDPBRN), which seeks to engage dentists in investigating research questions and sharing experiences and expertise (n = 282). Participants (n = 189) were asked to rate the importance of caries risk factors when developing a caries treatment plan in both adult and pediatric patients., Results: Oral hygiene status was rated as the most important risk factor when developing a treatment plan in both adult and pediatric patients, whereas the use of fluorides was rated as the least important. Results of multiple logistic regression analysis showed that the odds ratios for the decision to perform CRA in the adult patient for past caries experience and use of fluorides were 2.61 (95% confidence interval [CI]: 1.29-5.29) and 1.85 (95% CI: 1.12-3.04), respectively, whereas that for oral hygiene was 3.84 (95% CI: 1.15-12.79) and use of fluorides 1.79 (95% CI: 1.06-3.03) in the pediatric patient., Conclusions: These results suggest that enhancing dentists' concept of the importance of current use of fluorides when developing a treatment plan may increase the percentage of dentists who conduct CRA in both adult and pediatric patients (clinicaltrials.gov registration number: NCT01680848)., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Roadways pose serious threats to animal populations. The installation of roadway mitigation measures is becoming increasingly common, yet studies that rigorously evaluate the effectiveness of these conservation tools remain rare. A highway expansion project in Ontario, Canada included exclusion fencing and ecopassages as mitigation measures designed to offset detrimental effects to one of the most imperial groups of vertebrates, reptiles. Taking a multispecies approach, we used a Before-After-Control-Impact study design to compare reptile abundance on the highway before and after mitigation at an Impact site and a Control site from 1 May to 31 August in 2012 and 2013. During this time, radio telemetry, wildlife cameras, and an automated PIT-tag reading system were used to monitor reptile movements and use of ecopassages. Additionally, a willingness to utilize experiment was conducted to quantify turtle behavioral responses to ecopassages. We found no difference in abundance of turtles on the road between the un-mitigated and mitigated highways, and an increase in the percentage of both snakes and turtles detected dead on the road post-mitigation, suggesting that the fencing was not effective. Although ecopassages were used by reptiles, the number of crossings through ecopassages was lower than road-surface crossings. Furthermore, turtle willingness to use ecopassages was lower than that reported in previous arena studies, suggesting that effectiveness of ecopassages may be compromised when alternative crossing options are available (e.g., through holes in exclusion structures). Our rigorous evaluation of reptile roadway mitigation demonstrated that when exclusion structures fail, the effectiveness of population connectivity structures is compromised. Our project emphasizes the need to design mitigation measures with the biology and behavior of the target species in mind, to implement mitigation designs in a rigorous fashion, and quantitatively evaluate road mitigation to ensure allow for adaptive management and optimization of these increasingly important conservation tools.

Objectives: Few studies have examined dentists' subjective ratings of importance of caries risk factors or tested whether dentists use this information in treatment planning. This study tested several hypotheses related to caries risk assessment (CRA) and individualized caries prevention (ICP)., Methods: Data were collected as part of a questionnaire entitled 'Assessment of Caries Diagnosis and Caries Treatment' completed by 547 practitioners who belong to The Dental Practice-Based Research Network (DPBRN), a consortium of participating practices and dental organizations., Results: Sixty-nine percent of DPBRN dentists perform CRA on their patients. Recently graduated dentists, dentists with busier practices, and those who believe a dentist can predict future caries were the most likely to use CRA. The association between CRA and individualized prevention was weaker than expected (r = 0.21). Dentists who perform CRA provide ICP to 57% of their patients, compared with 42% for dentists who do not perform CRA. Based on their responses to radiographic and clinical scenarios in the questionnaire, dentists who use CRA appear to use this information in restorative decisions., Conclusion: A substantial percentage of DPBRN dentists do not perform CRA, and there is not a strong linkage between its use and use of individualized preventive regimens for adult patients. More progress in the implementation of current scientific evidence in this area is warranted.

Many temperate animals spend half their lives in a non-active, overwintering state, and multiple adaptations have evolved to enable winter survival. One notable vertebrate model is Chrysemys picta, whose hatchlings display dichotomous overwintering strategies: some hatchlings spend their first winter aquatically after nest emergence in the autumn, whereas others overwinter terrestrially within their natal nest with subsequent emergence in the spring. The occurrence of these strategies varies among populations and temporally within populations; however, factors that determine the strategy employed by a nest in nature are unknown. We examined potential factors that influence intra-population variation in the overwintering strategy of C. picta hatchlings over two winters in Algonquin Park, Ontario. We found that environmental factors may be a trigger for the hatchling overwintering strategy: autumn-emerging nests were sloped towards the water and were surrounded by a relatively higher percentage of bare ground compared with spring-emerging nests. Autumn-emerging hatchlings were also relatively smaller. Overwintering strategy was not associated with clutch oviposition sequence, or mammalian or avian predation attempts. Instead, autumn emergence from the nest was associated with the direct mortality threat of predation by sarcophagid fly larvae. Body condition and righting response, measured as proxies of hatchling fitness, did not differ between overwintering strategies. Costs and benefits of overwintering aquatically versus terrestrially in hatchling C. picta are largely unknown, and have the potential to affect population dynamics. Understanding winter survival has great implications for turtle ecology, thus we emphasize areas for future research on dichotomous overwintering strategies in temperate hatchling turtles., (© 2014. Published by The Company of Biologists Ltd.)

Background: The authors' objective was to evaluate the long-term performance of a resin-based composite restorative material (Beautifil, Shofu, Kyoto, Japan) in combination with a self-etching primer (FL-Bond, Shofu) for posterior restorations., Methods: Two clinicians placed 61 restorations, 26 Class I and 35 Class II, in 31 patients. They placed restorations while using rubber dam isolation. Two other clinicians examined the restorations according to the modified U.S. Public Health Service (USPHS) criteria, observing color match, marginal adaptation, anatomy, surface roughness, marginal staining, interfacial staining, proximal and occlusal contacts, secondary caries, postoperative sensitivity and luster. Clinicians examined restorations at baseline as well as at one-, two-, four-, eight- and 13-year recall visits., Results: All restorations were examined at one year, 58 (95 percent) at two years, 39 (64 percent) at four years and 41 (67 percent) at eight years; at the 13-year recall examination, 41 (67 percent) either were examined or had a known outcome. Of the 41 restorations seen at the 13-year examination, 25 restorations (14 Class I and 11 Class II) were intact and acceptable, two had secondary caries and 14 either were not present or had failed (two were missing, 10 had received crowns and two had been replaced). No changes were observed in the modified USPHS criteria for 12 of the 25 restorations that were intact (48 percent). Areas of change observed in 13 of the 25 intact restorations included color match (12 percent), marginal adaptation at the occlusal (20 percent) and proximal surfaces (4 percent), marginal staining on occlusal (24 percent) and proximal surfaces (8 percent), and interfacial staining on occlusal (4 percent) and proximal surfaces (12 percent)., Conclusion: The study results showed that most of the restorations observed at the 13-year recall examination maintained acceptable clinical qualities., Practical Implications: Beautifil restorative material demonstrated long-term successful results for restoration of posterior teeth.

Conservation biology integrates multiple disciplines to expand the ability to identify threats to populations and develop mitigation for these threats. Road ecology is a branch of conservation biology that examines interactions between wildlife and roadways. Although the direct threats of road mortality and habitat fragmentation posed by roads have received much attention, a clear understanding of the indirect physiological effects of roads on wildlife is lacking. Chronic physiological stress can lower immune function, affect reproductive rates and reduce life expectancy; thus, it has the potential to induce long-lasting effects on populations. Reptiles are globally in decline, and roads are known to have negative effects on reptile populations; however, it is unknown whether individual responses to roads and traffic result in chronic stress that creates an additional threat to population viability. We successfully extracted reliable measures of corticosterone (CORT), a known, commonly used biomarker for physiological stress, from claw trimmings from painted turtles (Chrysemys picta) captured at three study sites (road-impacted site, control site and validation site). Corticosterone levels in claws were evaluated as a measure of chronic stress in turtles because CORT is deposited during growth of the claw and could provide an opportunity to examine past long-term stress levels. While male turtles had higher CORT levels on average than females, there was no difference in the level of CORT between the road-impacted and control site, nor was there a relationship between CORT and turtle body condition. In validating a novel approach for non-invasive measurement of long-term CORT levels in a keratinized tissue in wild reptiles, our study provides a new avenue for research in the field of stress physiology.

Purpose: Immunotherapy using vaccines or adoptively transferred tumor-infiltrating lymphocytes (TIL) is limited by T-cell functional inactivation within the solid tumor microenvironment. The purpose of this study was to determine whether a similar tumor-induced inhibition occurred with genetically modified cytotoxic T cells expressing chimeric antigen receptors (CAR) targeting tumor-associated antigens., Experimental Design: Human T cells expressing CAR targeting mesothelin or fibroblast activation protein and containing CD3ζ and 4-1BB cytoplasmic domains were intravenously injected into immunodeficient mice bearing large, established human mesothelin-expressing flank tumors. CAR TILs were isolated from tumors at various time points and evaluated for effector functions and status of inhibitory pathways., Results: CAR T cells were able to traffic into tumors with varying efficiency and proliferate. They were able to slow tumor growth, but did not cause regressions or cures. The CAR TILs underwent rapid loss of functional activity that limited their therapeutic efficacy. This hypofunction was reversible when the T cells were isolated away from the tumor. The cause of the hypofunction seemed to be multifactorial and was associated with upregulation of intrinsic T-cell inhibitory enzymes (diacylglycerol kinase and SHP-1) and the expression of surface inhibitory receptors (PD1, LAG3, TIM3, and 2B4)., Conclusions: Advanced-generation human CAR T cells are reversibly inactivated within the solid tumor microenvironment of some tumors by multiple mechanisms. The model described here will be an important tool for testing T cell-based strategies or systemic approaches to overcome this tumor-induced inhibition. Our results suggest that PD1 pathway antagonism may augment human CAR T-cell function., (©2014 American Association for Cancer Research.)

Objective/background: Prior work has not addressed sex differences in the incidence of severe postoperative pain episodes. The goal of this study was to examine sex differences in clinical postoperative pain scores across an array of surgical procedures using direct comparisons of numeric rating scale pain scores as well as using the incidence of severe pain events (SPEs)., Design/setting: Retrospective cohort study of over 300,000 clinical pain score observations recorded from adult patients undergoing nonambulatory surgery at a tertiary care academic medical center over a 1-year period., Methods/patients: To test the hypothesis that the number of SPE on postoperative day (POD) 1 differed by sex after controlling for procedure, we calculated Cochran-Mantel-Haenszel statistics of sex by count of SPE, controlling for type of surgery., Assessment Tools/outcomes: Pain scores were collected from clinical nursing records where they were documented using the numeric rating scale., Results: In female patients, 10,989 (25.09%) of 43,806 POD 1 pain scores were considered SPE compared with 10,786 (22.45%) of 48,055 POD 1 pain scores in male patients. This produced an overall odds ratio of 1.16 (99% confidence interval 1.11-1.20) for females vs males to report an SPE for a pain score on POD 1. Estimates of the odds that a given pain observation represents an SPE for female vs male patients after controlling for type of surgery yielded an odds ratio of 1.14 (99% confidence interval, 1.10-1.19)., Conclusion: Female patients experience greater mean pain scores, as well as a higher incidence of SPE, on POD 1 for a variety of surgical procedures., (Wiley Periodicals, Inc.)

Objectives: We examined whether health literacy was associated with self-rated oral health status and whether the relationship was mediated by patient-dentist communication and dental care patterns., Methods: We tested a path model with data collected from 2 waves of telephone surveys (baseline, 2009-2010; follow-up, 2011) of individuals residing in 36 rural census tracts in northern Florida (final sample size n = 1799)., Results: Higher levels of health literacy were associated with better self-rated oral health status (B = 0.091; P < .001). In addition, higher levels of health literacy were associated with better patient-dentist communication, which in turn corresponded with patterns of regular dental care and better self-rated oral health (B = 0.003; P = .01)., Conclusions: Our study showed that, beyond the often-reported effects of gender, race, education, financial status, and access to dental care, it is also important to consider the influence of health literacy and quality of patient-dentist communication on oral health status. Improved patient-dentist communication is needed as an initial step in improving the population's oral health.

Objective: Knee osteoarthritis (OA) contributes significantly to disability in older individuals, and racial/ethnic minorities are disproportionately affected. The present study aimed to characterize differences in clinical and experimental pain, including pain inhibition, among older African American (AA) and non-Hispanic white (NHW) subjects with knee OA., Methods: AA and NHW subjects with knee OA (n = 267) completed clinical and functional pain assessments, including quantitative sensory testing (QST). We hypothesized that, when compared to NHW subjects, AA subjects would display 1) lower pain tolerance and higher ratings of heat-, mechanical-, and cold-induced pain, 2) greater temporal summation of pain, 3) reduced pain inhibition, and 4) greater clinical pain and poorer function. In addition, we hypothesized that the findings from QST would significantly predict the severity of clinical pain within each race/ethnicity., Results: AA subjects with knee OA displayed increased pain sensitivity, greater temporal summation, and reduced pain inhibition when compared to NHW subjects with knee OA. Moreover, AA subjects reported greater clinical pain and poorer function. Racial/ethnic differences in clinical pain became nonsignificant when the analyses were controlled for education and annual income, whereas differences in QST findings remained highly significant. Although the extent of pain inhibition predicted the severity of clinical pain in both groups, different QST measures were additionally predictive of clinical pain within each group., Conclusion: The results of this study establish that there are racial/ethnic differences in experimental and clinical pain and function in older individuals with knee OA. Our findings indicating that different QST measures were associated with clinical pain within the 2 racial/ethnic groups, whereas reduced pain inhibition was important in all participants, warrant further study in order to elucidate the common and group-specific pathophysiologic mechanisms contributing to clinical pain in OA., (Copyright © 2014 by the American College of Rheumatology.)

Rhesus (rh) but not human (hu) TRIM5α potently restricts human immunodeficiency virus (HIV)-1 infection. It is not clear why huTRIM5α fails to effectively block HIV infection, but it is thought to have a lower affinity for the viral core. Using primary human CD4 T cells, we investigated the ability of huTRIM5α, rhTRIM5α, and the huTRIM5αR323-332 B30.2/SPRY patch-mutant to form cytoplasmic bodies, postulated as key components of the HIV-1 restriction apparatus. Both rhTRIM5α and huTRIM5αR323-332 formed pronounced cytoplasmic bodies, whereas cytoplasmic bodies in T cells overexpressing huTRIM5α were present but more difficult to detect. As expression of all three TRIM5α orthologs was similar at the RNA level, we next investigated the role of protein stability in conferring TRIM5α-mediated HIV-1 restriction. Both steady-state and pulse-chase experiments revealed that the huTRIM5α protein was much less stable than rhTRIM5α, and this difference correlated with higher self-ubiquitination activity. Using a stabilized form of huTRIM5α in which the steady-state expression level was more similar to rhTRIM5α, we observed comparable HIV-1 restriction activity in multi-round HIV-1 challenge assays. Lastly, primary human CD4 T cells expressing a stabilized huTRIM5α were protected from HIV-1-mediated destruction in vivo, indicating that efforts to stabilize huTRIM5α should have significant long-term therapeutic value.

Introduction: Despite many studies investigating exercise-induced hypoalgesia, there is limited understanding of the optimal intensity of aerobic exercise in producing hypoalgesic effects across different types of pain stimuli. Given that not all individuals are willing or capable of engaging in high-intensity aerobic exercise, whether moderate-intensity aerobic exercise (MAE) is associated with a hypoalgesic response and whether this response generalizes to multiple pain induction techniques needs to be substantiated., Purpose: This study's purpose is to test for differences in the magnitude of pressure and heat pain modulation induced by MAE and vigorous-intensity aerobic exercise (VAE)., Methods: Twelve healthy young males and 15 females completed one training session and three testing sessions consisting of 25 min of 1) stationary cycling at 70% HR reserve, 2) stationary cycling at 50% HR reserve, or 3) quiet rest (control). Pain testing was conducted on both forearms before and immediately after each condition and included the following tests: pressure pain thresholds, suprathreshold pressure pain test, static continuous heat test, and repetitive pulse heat pain test. Repeated-measures ANOVA was conducted on each pain measure., Results: VAE and MAE reduced pain ratings during static continuous heat stimuli and repetitive heat pulse stimuli, with VAE producing larger effects. VAE also increased pressure pain thresholds, whereas neither exercise influenced suprathreshold pressure pain ratings., Conclusion: These results suggest that MAE is capable of producing a hypoalgesic effect using continuous and repetitive pulse heat stimuli. However, a dose-response effect was evident as VAE produced larger effects than MAE.

Objectives: In this study, the authors examined the dentist's view of the patient's experience and concordance with the patient's rating of satisfaction., Methods: Practitioners from 197 practices in The National Dental Practice-Based Research Network recruited consecutively seen patients who had defective restorations that were replaced or repaired. At the end of the dental visit, the treating dentist and 5,315 patients completed and returned a survey that asked about the patient's satisfaction., Results: Most dentists viewed their patients as having been satisfied with the treatment experience (n = 4,719 [89 percent]) and as having perceived them as friendly (n = 5,136 [97 percent]). Dentists had less strong feelings about whether patients had a preference for the restorative material (n = 2,271 [43 percent]) or an interest in obtaining information about the procedure (n = 1,757 [33 percent]). Overall, patients were satisfied, and most of the time dentists correctly predicted this outcome. Among patients who were less than satisfied, there was a substantial subset of cases in which dentists were not aware of this dissatisfaction., Conclusion: For improved patient-centered care, dentists should assess patients' desires, expectations and perceptions of the dental care experience and then manage or correct the expectations and perceptions as needed., Practical Implications: By taking a patient-centered approach, dentists should seek to understand how patients evaluate and rate the services provided, thereby enabling them to focus on what each patient values most.

Objective: Little is known regarding whether exercise-induced hypoalgesia (EIH) produced by isometric exercise is influenced by psychological factors or systematically varies across multiple experimental psychophysical pain tests. Thus, this study sought to determine the influence of experimental pain test, psychological factors, and sex on the hypoalgesic response of submaximal isometric exercise., Methods: Healthy young males (N = 12) and females (N = 15) completed one training and two testing sessions consisting of quiet rest (control condition) or a 3-minute isometric handgrip performed at 25% of maximum voluntary contraction. Pain testing was conducted on both forearms prior to and following exercise and quiet rest. The pain tests included: pressure pain thresholds (PPT), suprathreshold pressure pain test, static prolonged heat test, and temporal summation of heat pain. Participants completed the Pain Catastrophizing Scale during the training session and the State-Trait Anxiety Inventory-State version prior to each session. The data were analyzed with mixed model analyses of variance, partial Pearson correlations, and hierarchical regression analyses., Results: Isometric exercise increased PPTs for men and women, reduced pain perception during static prolonged heat stimuli for women, and reduced temporal summation of pain for men and women. Greater pain catastrophizing was associated with smaller reductions in temporal summation following isometric exercise., Conclusions: These findings demonstrate that the hypoalgesic response to submaximal isometric exercise is partially a function of sex and experimental pain test. Furthermore, the relationship between EIH and pain catastrophizing was psychophysical pain test specific, with greater pain catastrophizing predicting diminished EIH only during the temporal summation of pain trials., (Wiley Periodicals, Inc.)