Visual Attention to Health Warning Labels on Waterpipe Venue Menus in Immersive Virtual Reality.

INTRODUCTION: This study examined how health warning labels (HWL) on a waterpipe venue menu captured and held the attention of consumers and influenced waterpipe tobacco smoking (WTS) attitudes, beliefs, and behaviors. AIMS AND METHODS: A randomized experiment (N = 96) of young adult waterpipe smokers was conducted in an immersive virtual reality laboratory. Participants viewed one of two virtual reality scenarios, a menu with an HWL and nicotine concentration or menu without an HWL and nicotine concentration. Eye-tracking metrics were collected, and participants completed posttest questionnaires on demographics, tobacco use history, and WTS attitudes, beliefs, and behaviors. T-tests were used to assess group differences, and a mediation analysis conducted to examine the relationship between the HWL and intention to quit WTS. RESULTS: Participants in the HWL group demonstrated greater visual attention to the warning and nicotine areas and less visual attention to the flavor and ingredients areas of the menu compared to the control group. The HWL group demonstrated greater negative attitudes toward WTS (p = .002), greater perceived risk of decreased lung function (p = .026), and greater intention to quit WTS (p = 0.003). The mediation model indicated the relationship between the HWL on a menu and intention to quit WTS was mediated by an increase in negative attitudes toward WTS. CONCLUSIONS: The HWLs on a menu captured and held the attention of consumers and increased negative attitudes, perceptions of health risk, and intention to quit WTS indicating potential benefit of including a warning label or nicotine concentration on menus to correct misperceptions of WTS. IMPLICATIONS: The study contributes to the broader literature on communicating the harms and risks of WTS. The findings suggest that HWL and nicotine concentration on waterpipe venue menus attract attention from consumers in environments comparable to the real world and the strategy warrants further exploration as a targeted policy intervention to educate the public and reduce the health burden of WTS.

'We're not gonna have a big quit if loose ones are around': urban, African American smokers' beliefs concerning single cigarette use reduction.

Single cigarette use (i.e. loosies, loose ones, singles) poses risks for smoking continuation among urban, African American smokers. There is, however, limited research to inform health education interventions addressing this behavior. We conducted 25 in-depth interviews with urban, African American users (ages 20-58 years) from Baltimore, MD and the District of Columbia in June and July 2018 to assess their beliefs about reducing single cigarette use. Interviews were guided by the Health Belief Model and its constructs of perceived benefits, perceived barriers, perceived susceptibility, perceived severity and self-efficacy. We analyzed qualitative data using framework analysis. Perceived benefits of reducing single cigarette use involved the avoidance of health risks, including concerns about buying fake cigarettes and exposure to unknown personal hygiene practices from sellers. Perceived barriers were the convenience of buying singles due to their availability, accessibility and low cost. Participants shared they were willing to use cognitive behavioral strategies to reduce their purchasing and use of singles. This study provides insights on potential intervention targets related to beliefs towards reducing single cigarette use. These findings can inform enforcement policies and health education interventions targeting single cigarette use among urban, African American smokers who use singles.

The Dilemma of Correcting Nicotine Misperceptions: Nicotine Replacement Therapy versus Electronic Cigarettes.

Many people overestimate the health risks associated with nicotine, mistakenly perceiving nicotine as the main carcinogen in cigarettes and a leading cause of smoking-related diseases. Health professionals have been calling for public education programs to correct nicotine misperceptions in the hope that a lower risk perception of nicotine could encourage the use of nicotine replacement therapy (NRT). However, a lower risk perception of nicotine could also lower perceived risk of electronic cigarettes (e-cigarettes). This paper evaluated the necessity of correcting nicotine misperceptions and explored possible intervention strategies to increase use of NRT and decrease use of e-cigarettes. In Study 1, smokers were surveyed about their perceptions of nicotine harm, and attitudes and intention toward using NRT and e-cigarettes. Results showed that overestimation of nicotine harm was associated with e-cigarette attitude and intention, but not with NRT. Informed by the survey results, three correction messages (a nicotine message, an NRT message, and an e-cigarette message) were developed and experimentally tested in Study 2 on both tobacco users and non-tobacco users. The nicotine message lowered people's perception of nicotine harm but it did not change attitude and intention toward tobacco product use. The NRT message also failed to influence NRT attitudes and intentions. The e-cigarette message significantly lowered attitudes and intentions to use e-cigarette.

Identification and Analysis of Islet Antigen-Specific CD8+ T Cells with T Cell Libraries.

Type 1 diabetes (T1D) is most likely caused by killing of ß cells by autoreactive CD8+ T cells. Methods to isolate and identify these cells are limited by their low frequency in the peripheral blood. We analyzed CD8+ T cells, reactive with diabetes Ags, with T cell libraries and further characterized their phenotype by CyTOF using class I MHC tetramers. In the libraries, the frequency of islet Ag-specific CD45RO+IFN-γ+CD8+ T cells was higher in patients with T1D compared with healthy control subjects. Ag-specific cells from the libraries of patients with T1D were reactive with ZnT8186-194, whereas those from healthy control recognized ZnT8186-194 and other Ags. ZnT8186-194-reactive CD8+ cells expressed an activation phenotype in T1D patients. We found TCR sequences that were used in multiple library wells from patients with T1D, but these sequences were private and not shared between individuals. These sequences could identify the Ag-specific T cells on a repeated draw, ex vivo in the IFN-γ+ CD8+ T cell subset. We conclude that CD8+ T cell libraries can identify Ag-specific T cells in patients with T1D. The T cell clonotypes can be tracked in vivo with identification of the TCR gene sequences.

Impacts of Nicotine and Flavoring on the Sensory Perception of E-Cigarette Aerosol.

INTRODUCTION: To examine the interaction between an added flavoring (cherry) and nicotine on the perception of electronic cigarette (e-cigarette) aerosol and how this impacts the appeal of flavored liquids for e-cigarette (e-liquids). METHODS: A total of 19 subjects (13 male, 6 female) vaped six commercially available e-liquids with varying contents of nicotine (0, 6, 12 mg/mL) and cherry flavor (4.7% or 9.3% vol/vol). For each e-liquid, subjects first rated overall liking/disliking of the aerosol using the Labeled Hedonic Scale, followed by perceived intensities of sweetness, bitterness, harshness (irritation), and cherry flavor of the aerosol using the general version of Labeled Magnitude Scale. RESULTS: The main findings were that (1) added nicotine increased perceived irritation and bitterness, and decreased the perceived sweetness of the e-cigarette aerosol; (2) cherry flavoring added a characteristic "cherry flavor" and an increase in the flavoring concentration from 4.7% to 9.3% tended to increase perceived intensities of sweetness, harshness, and bitterness; and (3) hedonic ratings of the e-cigarette aerosol decreased as nicotine level increased, but were not affected by flavor level. CONCLUSIONS: Our findings indicate that the appeal of the e-cigarette aerosol decreases as nicotine concentration increases. Conversely, perceived sweetness improved liking. An increase in the concentration of cherry flavoring did not appear to impact any of the measured attributes to a significant degree. IMPLICATIONS: This work demonstrates that the perception of specific sensory attributes of e-cigarettes and their overall appeal are affected by the e-liquid constituents. Most significantly, the results suggest that nicotine decreases the sensory appeal of e-cigarettes by contributing to the perceived irritation and bitterness of the aerosol. These data have implications for the role that nicotine plays in the sensory perception and appeal of e-cigarettes aerosol and further how these sensory factors can be modulated by sweet flavoring.

The Application of Commercially Available Mobile Cigarette Topography Devices for E-cigarette Vaping Behavior Measurements.

INTRODUCTION: The ability to reliably measure real-world vaping behavior is critical to understand exposures to potential toxins. Commercially available mobile topography devices were originally designed to measure cigarette puffing behavior. Information regarding how applicable these devices are to the measurement of electronic cigarette (e-cigarette) vaping topography is needed. METHODS: Clinical Research Support System (CReSS; Pocket) and Smoking Puff Analyzer Mobile (SPA-M) topography devices were tested against the calibrated laboratory-based smoking puff analyzer duplicator (SPA-D) device combined with an analytical smoking machine that generates programmable puffs with high precision. Puff topography of e-cigarettes was measured over a range of puff volumes (10-130 mL) at 2 and 5 s puff durations (using bell- and square-shaped puffs). "Real-world" topography data collected from 10 participants during 1 week of at-home vaping were also analyzed. Recording anomalies and limitations of the devices, such as accuracy of detection of the puff end, flow rate dropouts, unreported puffs, and abandoned vaping sessions for the CReSS, and multi-peak puffs for the SPA-M were defined. RESULTS: The accuracy of puff volumes and durations was determined for both devices. The error for SPA-M was generally within ±10%, whereas that for the CReSS varied more widely. The CReSS consistently underestimated puff duration at higher flow rates. CONCLUSIONS: CReSS and SPA-M topography devices can be used for real-world e-cigarette topography measurements, but researchers have to be aware of the limitations. Both devices can provide accurate measurements only under certain puff parameter ranges. The SPA-M provided more accurate measurements under a wider range of puffing parameters than the CReSS. Summary data reported by both devices require thorough analysis of the raw data to avoid misleading data interpretation. IMPLICATIONS: Results of this study provide researchers with valuable information about the capability of commercially available cigarette topography devices to measure real-world vaping behaviors. The differing measurement ranges of the two devices and puff recording limitations and anomalies should be taken into account during analysis and interpretation of real-world data.

Mentholation triggers brand-specific shifts in the bacterial microbiota of commercial cigarette products.

Bacterial communities are integral constituents of tobacco products. They originate from tobacco plants and are acquired during manufacturing processes, where they play a role in the production of tobacco-specific nitrosamines. In addition, tobacco bacterial constituents may play an important role in the development of infectious and chronic diseases among users. Nevertheless, tobacco bacterial communities have been largely unexplored, and the influence of tobacco flavor additives such as menthol (a natural antimicrobial) on tobacco bacterial communities is unclear. To bridge this knowledge gap, time series experiments including 5 mentholated and non-mentholated commercially available cigarettes-Marlboro red (non-menthol), Marlboro menthol, Newport menthol box, Newport menthol gold, and Newport non-menthol-were conducted. Each brand was stored under three different temperature and relative humidity conditions. To characterize bacterial communities, total DNA was extracted on days 0 and 14. Resulting DNA was purified and subjected to PCR of the V3V4 region of the 16S rRNA gene, followed by sequencing on the Illumina HiSeq platform and analysis using the QIIME, phyloseq, metagenomeSeq, and DESeq software packages. Ordination analyses showed that the bacterial community composition of Marlboro cigarettes was different from that of Newport cigarettes. Additionally, bacterial profiles significantly differed between mentholated and non-mentholated Newports. Independently of storage conditions, tobacco brands were dominated by Proteobacteria, with the most dominant bacterial genera being Pseudomonas, unclassified Enterobacteriaceae, Bacillus, Erwinia, Sphingomonas, Acinetobacter, Agrobacterium, Staphylococcus, and Terribacillus. These data suggest that the bacterial communities of tobacco products differ across brands and that mentholation of tobacco can alter bacterial community composition of select brands. KEY POINTS: • Bacterial composition differed between the two brands of cigarettes. • Mentholation impacts cigarette microbiota. • Pseudomonas and Bacillus dominated the commercial cigarettes. Graphical abstract.

Evidence of compensation among waterpipe smokers using harm reduction components.

OBJECTIVES: We examined two waterpipe tobacco smoking components advertised to reduce harm to determine if they result in lower levels of biomarkers of acute exposure. METHODS: We conducted a crossover study of 34 experienced waterpipe smokers smoking a research-grade waterpipe in three configurations ad libitum in a controlled chamber: control (quick-light charcoal), electric (electric heating) and bubble diffuser (quick-light charcoal and bubble diffuser). We collected data on smoking topography, environmental carbon monoxide (CO), subjective effects, heart rate, plasma nicotine and exhaled CO and benzene. RESULTS: Smokers' mean plasma nicotine, heart rate, and exhaled benzene and CO boost were all significantly lower for electric compared with control. However, smokers puffed more intensely and took significantly more and larger volume puffs for a larger total puffing volume (2.0 times larger, p<0.0001) when smoking electric; machine yields indicate this was likely due to lower mainstream nicotine. Smokers rated electric smoking experience less satisfying and less pleasant. For charcoal heating, the mean mass of CO emitted into the chamber was ~1 g when participants smoked for a mean of 32 minutes at a typical residential ventilation rate (2.3 hr-1). CONCLUSION: Waterpipe smokers engaged in compensation (i.e., increased and more intense puffing) to make up for decreased mainstream nicotine delivery from the same tobacco heated two ways. Waterpipe components can affect human puffing behaviours, exposures and subjective effects. Evidence reported here supports regulation of waterpipe components, smoking bans in multifamily housing and the use of human studies to evaluate modified or reduced risk claims.

Treatment of type 1 diabetes with teplizumab: clinical and immunological follow-up after 7 years from diagnosis.

AIMS/HYPOTHESIS: The long-term effects of successful immune therapies for treatment of type 1 diabetes have not been well studied. The Autoimmunity-Blocking Antibody for Tolerance (AbATE) trial evaluated teplizumab, an Fc receptor non-binding humanised anti-CD3 monoclonal antibody in individuals with new-onset type 1 diabetes, and ended in 2011. Clinical drug-treated responders showed an increased frequency of 'partially exhausted' CD8+ T cells. We studied the clinical, immunological and metabolic status of participants after an average follow-up of 7 years. METHODS: Participants with detectable C-peptide at year 2 of AbATE returned for follow-up. C-peptide responses were assessed by 4 h mixed-meal tolerance test. Autoantibodies and HbA1c levels were measured and average daily insulin use was obtained from patient logs. Peripheral blood mononuclear cells were analysed by flow cytometry and cytokine release. RESULTS: Fifty-six per cent of the original participants returned. Three of the original control group who did not return had lost all detectable C-peptide by the end of the 2 year trial. The C-peptide responses to a mixed-meal tolerance test were similar overall in the drug vs control group of participants but were significantly improved, with less loss of C-peptide, in drug-treated responders identified at 1 year. However, the improvements in C-peptide response were not associated with lower HbA1c levels or insulin use. Drug-treated responders showed a significantly increased frequency of programmed cell death protein 1-positive central memory and anergic CD8+ T cells at follow-up. CONCLUSIONS/INTERPRETATION: These findings suggest there is reduced decline in C-peptide and persistent immunological responses up to 7 years after diagnosis of diabetes in individuals who respond to teplizumab. TRIAL REGISTRATION: ClinicalTrials.gov NCT02067923; the protocol is available at www.immunetolerance.org (ITN027AI).

Unmethylated Insulin as an Adjunctive Marker of Beta Cell Death and Progression to Type 1 Diabetes in Participants at Risk for Diabetes.

Islet autoantibody (iAb)-positive individuals have a high risk of progression to type 1 diabetes (T1D), although the rate of progression is highly variable and factors involved in the rate of progression are largely unknown. The ratio of unmethylated/methylated insulin DNA levels (unmethylated INS ratio) has been shown to be higher in participants at high risk of T1D compared to healthy controls. We aimed to evaluate whether an unmethylated INS ratio may be a useful biomarker of beta cell death and rate of progression to T1D. In TrialNet participants who were followed in the Pathway to Prevention Study and progressed to diabetes (n = 57, median age of onset 15.3 years), we measured unmethylated INS ratio and autoantibodies by electrochemiluminescence (ECL) assays (ECL-IAA, ECL-GADA, and ECL-IA2) and radioimmunoassays (RIA) (mIAA, GADA, IA2A, and ZnT8A) longitudinally for 24 months prior to diagnosis. Linear models were used to test the association between unmethylated INS ratio and the age at T1D diagnosis and unmethylated INS ratio and iAb over time. Close to diabetes onset, the unmethylated INS ratio was associated with mIAA (p = 0.003), ECL-IAA (p = 0.002), and IA2A (p = 0.01) levels, but not with GADA, ECL-GADA, ECL-IA2, or ZnT8A levels. No significant associations were found at baseline (24 months prior to T1D diagnosis). Only mIAA levels were significantly associated with an unmethylated INS ratio over time, with a 0.24 change in the ratio for each 0.1 change in mIAA z-score (p = 0.02). Adjusting for a baseline unmethylated INS ratio, an increased rate of change in unmethylated INS ratio from baseline to diabetes onset was associated with a five-year decrease in age at T1D diagnosis (p = 0.04).

Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+ NIK+ signalosome on Rab5+ endosomes.

Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB-inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5(+)endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC(+) endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt(+)NIK(+) signalosome on Rab5(+) endosomes.

E-Cigarettes Use Behavior and Experience of Adults: Qualitative Research Findings to Inform E-Cigarette Use Measure Development.

OBJECTIVES: To gain a better understanding of electronic cigarette (e-cigarette) use behavior and experience among adult e-cigarette users, with the goal of informing development of future e-cigarette use measures. METHODS: Between August and October 2014 six focus groups were conducted in Seattle. Participants (63% male; 60% >35 years old; 60% White): e-cigarette users who used combustible tobacco products either currently or in the past. E-cigarette discussion topics covered: their daily use pattern (eg, frequency), product-related characteristics (eg, nicotine levels), and perceptions about health risks and benefits. RESULTS: Participants' descriptions of daily use were so varied that no common "unit" of a "session" easily summarized frequency or quantity of typical e-cigarette use. Most users had difficulty in tracking their own use. Participants reported nicotine craving relief when using e-cigarettes, but described e-cigarettes use as less satisfying than combustible cigarettes. Valued characteristics included "ready availability" and the possibility of using indoors. A unique aspect of the e-cigarette use experience is the option of adding flavors and having the ability to exhale "big clouds" of vapor/aerosol. Most perceived e-cigarettes as a better and safer alternative to conventional cigarettes, yet still sought further information about health consequences and safety of e-cigarettes from trusted sources. CONCLUSIONS: E-cigarettes users are far from homogeneous in their behavior and motivation for adopting e-cigarettes. A range of use patterns arising from both hedonic and utilitarian factors, along with product characteristics (eg, variable nicotine levels and flavors) extending beyond those of conventional cigarettes, suggest that new, specific e-cigarette use measures must be developed. IMPLICATIONS: The current study provides timely information on adult e-cigarette use behavior, which is a crucial step in measuring this new phenomenon and assessing the risks associated with using e-cigarette products. Our findings reveal that vaping is not a mere replacement for combustible cigarette smoking and that many users of e-cigarettes enjoy product characteristics such as flavors and "clouds" that are unavailable in combustible cigarettes. Therefore, commonly available cigarette smoking measures are not well suited to measurement of e-cigarette use behavior, and indication that new measures need to be developed to accurately track e-cigarette use.

Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice.

AIMS/HYPOTHESIS: Type 1 diabetes is caused by the immunological destruction of pancreatic beta cells. Preclinical and clinical data indicate that there are changes in beta cell function at different stages of the disease, but the fate of beta cells has not been closely studied. We studied how immune factors affect the function and epigenetics of beta cells during disease progression and identified possible triggers of these changes. METHODS: We studied FACS sorted beta cells and infiltrating lymphocytes from NOD mouse and human islets. Gene expression was measured by quantitative real-time RT-PCR (qRT-PCR) and methylation of the insulin genes was investigated by high-throughput and Sanger sequencing. To understand the role of DNA methyltransferases, Dnmt3a was knocked down with small interfering RNA (siRNA). The effects of cytokines on methylation and expression of the insulin gene were studied in humans and mice. RESULTS: During disease progression in NOD mice, there was an inverse relationship between the proportion of infiltrating lymphocytes and the beta cell mass. In beta cells, methylation marks in the Ins1 and Ins2 genes changed over time. Insulin gene expression appears to be most closely regulated by the methylation of Ins1 exon 2 and Ins2 exon 1. Cytokine transcription increased with age in NOD mice, and these cytokines could induce methylation marks in the insulin DNA by inducing methyltransferases. Similar changes were induced by cytokines in human beta cells in vitro. CONCLUSIONS/INTERPRETATION: Epigenetic modification of DNA by methylation in response to immunological stressors may be a mechanism that affects insulin gene expression during the progression of type 1 diabetes.

Identification of New and Distinctive Exposures from Little Cigars.

Little cigar mainstream smoke is less well-characterized than cigarette mainstream smoke in terms of chemical composition. This study compared four popular little cigar products against four popular cigarette products to determine compounds that are either unique to or more abundant in little cigars. These compounds are categorized as new or distinctive exposures, respectively. Total particulate matter samples collected from machine-generated mainstream smoke were extracted with methylene chloride, and the extracts were analyzed using two-dimensional gas chromatography-time-of-flight mass spectrometry. The data were evaluated using novel data-processing algorithms that account for characteristics specific to the selected analytical technique and variability associated with replicate sample analyses. Among more than 25 000 components detected across the complete data set, ambrox was confirmed as a new exposure, and 3-methylbutanenitrile and 4-methylimidazole were confirmed as distinctive exposures. Concentrations of these compounds for the little cigar mainstream smoke were estimated at approximately 0.4, 0.7, and 12 µg/rod, respectively. In achieving these results, this study has demonstrated the capability of a powerful analytical approach to identify previously uncharacterized tobacco-related exposures from little cigars. The same approach could also be applied to other samples to characterize constituents associated with tobacco product classes or specific tobacco products of interest. Such analyses are critical in identifying tobacco-related exposures that may affect public health.

Real-Time Measurement of Electronic Cigarette Aerosol Size Distribution and Metals Content Analysis.

INTRODUCTION: Electronic cigarette (e-cigarette) use is increasing worldwide and is highest among both daily and nondaily smokers. E-cigarettes are perceived as a healthier alternative to combustible tobacco products, but their health risk factors have not yet been established, and one of them is lack of data on aerosol size generated by e-cigarettes. METHODS: We applied a real-time, high-resolution aerosol differential mobility spectrometer to monitor the evolution of aerosol size and concentration during puff development. Particles generated by e-cigarettes were immediately delivered for analysis with minimal dilution and therefore with minimal sample distortion, which is critically important given the highly dynamic aerosol/vapor mixture inherent to e-cigarette emissions. RESULTS: E-cigarette aerosols normally exhibit a bimodal particle size distribution: nanoparticles (11-25nm count median diameter) and submicron particles (96-175nm count median diameter). Each mode has comparable number concentrations (10(7)-10(8) particles/cm(3)). "Dry puff" tests conducted with no e-cigarette liquid (e-liquid) present in the e-cigarette tank demonstrated that under these conditions only nanoparticles were generated. Analysis of the bulk aerosol collected on the filter showed that e-cigarette emissions contained a variety of metals. CONCLUSIONS: E-cigarette aerosol size distribution is different from that of combustible tobacco smoke. E-cigarettes generate high concentrations of nanoparticles and their chemical content requires further investigation. Despite the small mass of nanoparticles, their toxicological impact could be significant. Toxic chemicals that are attached to the small nanoparticles may have greater adverse health effects than when attached to larger submicron particles. IMPLICATIONS: The e-cigarette aerosol size distribution is different from that of combustible tobacco smoke and typically exhibits a bimodal behavior with comparable number concentrations of nanoparticles and submicron particles. While vaping the e-cigarette, along with submicron particles the user is also inhaling nano-aerosol that consists of nanoparticles with attached chemicals that has not been fully investigated. The presence of high concentrations of nanoparticles requires nanotoxicological consideration in order to assess the potential health impact of e-cigarettes. The toxicological impact of inhaled nanoparticles could be significant, though not necessarily similar to the biomarkers typical of combustible tobacco smoke.

Design and Validation of a Research-Grade Waterpipe Equipped With Puff Topography Analyzer.

INTRODUCTION: Worldwide, commercially available waterpipes vary widely in design and durability, including differences in fabrication materials, degree of leak-tight fit, and flow path diameter. Little is known about how the components of the waterpipe may influence puffing behavior and user's exposure to toxins. To systematically evaluate exposure, it is necessary to use a standardized research-grade waterpipe (RWP) when conducting clinical and laboratory-based trials. METHODS: We developed a RWP that is configured with an in-line topography system which allows real-time measurement and recording of the smoke volume drawn through the RWP. The RWP was calibrated across the flow rate range expected for waterpipe tobacco smoking and the calibration was verified for known puff volumes using a smoking machine. Operation of the RWP was qualified in a cohort of experienced waterpipe smokers, each smoker using the RWP ad libitum in a laboratory setting while smoker topography and subjective effects data were collected. RESULTS: RWP machine smoking was highly reproducible and yielded puff volumes that agreed well with true values. User acceptance was comparable, and puffing behavior was similar in pattern, with more frequent puffing in the beginning of the session, but significantly different in intensity from that used to estimate the majority of toxicant exposure reported in the literature. CONCLUSIONS: The RWP operates with known precision and accuracy and is well accepted by experienced smokers. This tool can be used to determine the extent to which puffing behaviors are affected by the waterpipe design, components, and/or accessories, tobacco nicotine content, sweet flavorings and/or additives known to increase addictiveness. IMPLICATIONS: This study describes a standardized RWP, equipped with a puffing topography analyzer, which can operate with known precision and accuracy, and is well-accepted by experienced smokers in terms of satisfaction and reward. The RWP is an important tool for determining if puffing behaviors, and thus estimated toxin exposures, are affected by the waterpipe design, components, and/or accessories, tobacco nicotine content, sweet flavorings, and/or additives that are known to increase addictiveness.

Using Electrophysiological Measures to Assess the Consumer Acceptability of Smokeless Tobacco Products.

INTRODUCTION: Adequate evaluation of novel tobacco products must include investigation of consumers' psychological response to such products. Traditionally, subjective scales of product liking have been used to assess consumer acceptability of tobacco products. However, subjective scales may miss cognitive changes that can only be captured by direct neurophysiological assessment. The present investigation explored the viability of using electroencephalography (EEG), in combination with traditional subjective measures, to assess consumer acceptability of five smokeless tobacco products. Given previous work linking product liking to arousal/attentional (executive function) enhancement, we focused on EEG measures of attention/arousal to objectively characterize cognitive changes associated with tobacco product use. METHODS: During five separate laboratory visits, smokeless tobacco users used Verve discs, Ariva dissolvables, Skoal snuff, Camel snus, or Nicorette lozenges. The N2 and P3b event-related potential components elicited by an oddball task were used to index attentional changes before/after product usage. Additionally, resting state alpha band EEG activity was analyzed before/after product usage to index cortical arousal. RESULTS: Although analyses of the subjective results provided limited inference, analyses of the electrophysiological measures, particularly the alpha suppression measure, revealed robust differences between products. Skoal elicited significantly enhanced alpha suppression compared to all four other products tested. Additionally, alpha suppression was found to correlate positively with subjective measures of satisfaction and psychological reward, but was unrelated to perceived aversion. CONCLUSIONS: The present results provide evidence that electrophysiological measures can yield important insights into consumer acceptability of novel tobacco products and are a valuable complement to subjective measures. IMPLICATIONS: This study is the first to employ a combination of electrophysiological measures and traditional subjective assays in order to assess the consumer acceptability of smokeless tobacco products. The results highlight the importance of adopting a multidimensional/multi-method approach to studying the consumer acceptability of tobacco products.

Role of sweet and other flavours in liking and disliking of electronic cigarettes.

OBJECTIVE: To examine the extent to which the perception of sweet and other flavours is associated with liking and disliking of flavoured electronic cigarettes (e-cigarettes). METHODS: 31 participants (13 females/18 males; 12 sole/19 dual users) vaped 6 commercially available flavours of blu Tanks: Classic Tobacco (CT), Magnificent Menthol (MM), Cherry Crush (CC), Vivid Vanilla (VV), Piña Colada (PC) and Peach Schnapps (PS); all 'medium' strength, 12 mg/mL nicotine concentration. For each flavoured e-cigarette, participants first rated liking/disliking on the Labeled Hedonic Scale, followed by perceived intensities of sweetness, coolness, bitterness, harshness and specific flavour on the generalised version of the Labeled Magnitude Scale. The psychophysical testing was conducted individually in an environmental chamber. RESULTS: PC was perceived as sweetest and liked the most; CT was perceived as least sweet and liked the least. Across all flavours, liking was correlated with sweetness (r=0.31), coolness (r=0.25), bitterness (r=-0.25) and harshness (r=-0.29, all p<0.001). Specifically, liking was positively correlated with sweetness of PS (r=0.56, p=0.001) and PC (r=0.36, p=0.048); and with coolness of MM, CT and VV (r=0.41-0.52, p<0.05). In contrast, harshness was negatively correlated with liking for CC, PC and PS (r=0.37-0.40, p<0.05). In a multivariate model, sweetness had the greatest positive impact on liking followed by coolness; harshness had the greatest negative impact on liking. CONCLUSIONS: Our findings indicate that bitterness and harshness, most likely from nicotine, have negative impacts on the liking of e-cigarettes, but the addition of flavourants that elicit sweetness or coolness generally improves liking. The results suggest that flavours play an important role in e-cigarette preference and most likely use.

Comparison of True and Smoothed Puff Profile Replication on Smoking Behavior and Mainstream Smoke Emissions.

To estimate exposures to smokers from cigarettes, smoking topography is typically measured and programmed into a smoking machine to mimic human smoking, and the resulting smoke emissions are tested for relative levels of harmful constituents. However, using only the summary puff data--with a fixed puff frequency, volume, and duration--may underestimate or overestimate actual exposure to smoke toxins. In this laboratory study, we used a topography-driven smoking machine that faithfully reproduces a human smoking session and individual human topography data (n = 24) collected during previous clinical research to investigate if replicating the true puff profile (TP) versus the mathematically derived smoothed puff profile (SM) resulted in differences in particle size distributions and selected toxic/carcinogenic organic compounds from mainstream smoke emissions. Particle size distributions were measured using an electrical low pressure impactor, the masses of the size-fractionated fine and ultrafine particles were determined gravimetrically, and the collected particulate was analyzed for selected particle-bound, semivolatile compounds. Volatile compounds were measured in real time using a proton transfer reaction-mass spectrometer. By and large, TP levels for the fine and ultrafine particulate masses as well as particle-bound organic compounds were slightly lower than the SM concentrations. The volatile compounds, by contrast, showed no clear trend. Differences in emissions due to the use of the TP and SM profiles are generally not large enough to warrant abandoning the procedures used to generate the simpler smoothed profile in favor of the true profile.

Alloantibody and complement promote T cell-mediated cardiac allograft vasculopathy through noncanonical nuclear factor-κB signaling in endothelial cells.

BACKGROUND: Cardiac allograft vasculopathy is the major cause of late allograft loss after heart transplantation. Cardiac allograft vasculopathy lesions contain alloreactive T cells that secrete interferon-γ, a vasculopathic cytokine, and occur more frequently in patients with donor-specific antibody. Pathological interactions between these immune effectors, representing cellular and humoral immunity, respectively, remain largely unexplored. METHODS AND RESULTS: We used human panel reactive antibody to form membrane attack complexes on allogeneic endothelial cells in vitro and in vivo. Rather than inducing cytolysis, membrane attack complexes upregulated inflammatory genes, enhancing the capacity of endothelial cells to recruit and activate allogeneic interferon-γ--producing CD4(+) T cells in a manner dependent on the activation of noncanonical nuclear factor-κB signaling. Noncanonical nuclear factor-κB signaling was detected in situ within endothelial cells both in renal biopsies from transplantation patients with chronic antibody-mediated rejection and in panel-reactive antibody--treated human coronary artery xenografts in immunodeficient mice. On retransplantation into immunodeficient hosts engrafted with human T cells, panel-reactive antibody--treated grafts recruited more interferon-γ--producing T cells and enhanced cardiac allograft vasculopathy lesion formation. CONCLUSIONS: Alloantibody and complement deposition on graft endothelial cells activates noncanonical nuclear factor-κB signaling, initiating a proinflammatory gene program that enhances alloreactive T cell activation and development of cardiac allograft vasculopathy. Noncanonical nuclear factor-κB signaling in endothelial cells, observed in human allograft specimens and implicated in lesion pathogenesis, may represent a target for new pharmacotherapies to halt the progression of cardiac allograft vasculopathy.