There are challenges that require collaboration among researchers to ensure that tobacco harm reduction strategies are evidence-based. A key challenge is evaluating the safety of flavors in e-cigarettes. Many flavorings are deemed “generally recognized as safe” (GRAS) for ingestion, but this does not guarantee their safety when inhaled. The Replica international research group is dedicated to the replication of studies on tobacco alternatives. As part of this effort, the group conducted a conceptual replication of the vanillin-specific experiments from the study by Fetterman et al. (2018), with the goal of validating their conclusions regarding this compound. We used Aspire Zelos 3 e-cigarette and prepared e-liquids containing propylene glycol, vegetable glycerin, and vanillin. The e-liquids were vaporized under regular (1 Ohm coil, 14 watts) and sub-Ohm (0.3 Ohm coil, 200 °C) settings using a vaping machine, following ISO20768:2018 puffing regime. The vapor was collected into a trapping solution to prepare aqueous extracts for treating human aortic endothelial cells. We evaluated cytotoxicity, oxidative stress, nitric oxide bioavailability, and inflammation addressing some gaps reported in the original study. We observed some harmful effects mostly attributable to ethanol used to trap vanillin, but no harmful effects from vanillin. Our results confirm the endothelial cell dysfunction observed in the original paper, but clarify that these effects are mainly attributable to ethanol and not to vaporized vanillin, indicating that, under the experimental conditions applied, vanillin does not elicit significant adverse effects on vascular endothelial cells. However, further research is needed to confirm its toxicological profile. Created in BioRender. https://BioRender.com/rn8dt1m .

AbstractBackgroundCisplatin chemoresistance is a critical challenge in the treatment of head and neck squamous cell carcinoma (HNSCC). Since previous research has suggested that nicotine and e-cigarette (e-cig) aerosol might increase cisplatin resistance in oral cancer cells, this multicenter replication study aimed to replicate the work by Manyanga et al. (2021) and evaluate the oncologic implications of e-cigarette exposure during chemotherapy.MethodsThis in vitro study involved standardized and harmonized protocols in international laboratories to examine the effects of cigarette smoke (1R6F) and e-cig aerosols with different concentrations of nicotine (0, 12, and 20 mg/ml nicotine) on cisplatin sensitivity in HNSCC cell lines (SCC-25, FaDu, and UM-SCC-1). Aerosols from 1R6F smoke and e-cig vapor were collected using a smoking and vaping machine, following ISO20778:2018 and ISO20768:2018 puffing regimes. The smoke and vapor were collected in PBS and diluted to 10 puffs/5L for HNSCC cell treatment. Chemosensitivity, clonogenicity, expression of gene for repair of cisplatin-induced DNA damage and gene and protein expression of cisplatin transporters were assessed by MTS, NRU, trypan blue, PCR, and Western blot assays, respectively.ResultsContrary to previous findings, exposure to e-cig aerosols did not significantly modulate cisplatin sensitivity in all cell lines. IC50 values, cytotoxicity assays, and clonogenic survival rates remained similar between e-cig treatments and cisplatin alone. Analysis of gene and protein expression revealed sporadic changes in the levels of transporters and repairs of cisplatin-induced DNA damage.ConclusionsThis study did not fully substantiate previous claims of increased cisplatin resistance due to e-cigarette aerosols and nicotine. The variability in gene and protein expression among different cell lines underscores the need for cautious interpretation and further investigation of the role of e-cigarette components in cancer treatment. These findings provide a critical perspective for shaping public health policies and clinical practices related to e-cigarette use during chemotherapy.

Detrimental effects of smoking on mesenchymal stem cell (MSC)-dependent immunosuppression and hepatoprotection are unknown. Herewith, by using α-galactosylceramide (α-GalCer)-induced liver injury, a well-established murine model of fulminant hepatitis, we examined molecular mechanisms which were responsible for negative effects of cigarette smoke on MSC-dependent immunomodulation. MSC which were grown in cigarette smoke-exposed medium (MSCWS-CM) obtained pro-inflammatory phenotype, were not able to optimally produce hepatoprotective and immunosuppressive cytokines (TGF-β, HGF, IL-10, NO, KYN), and secreted significantly higher amounts of inflammatory cytokines (IFN-γ, TNF-α, IL-17, IL-6) than MSC that were cultured in standard medium never exposed to cigarette smoke (MSCCM). In contrast to MSCCM, which efficiently attenuated α-GalCer-induced hepatitis, MSCWS-CM were not able to prevent hepatocyte injury and liver inflammation. MSCWS-CM had reduced capacity for the suppression of liver-infiltrated inflammatory macrophages, dendritic cells (DCs) and lymphocytes. Although significantly lower number of IL-12-producing macrophages and DCs, TNF-α, IFN-γ or IL-17-producing CD4 + and CD8 +T lymphocytes, NK and NKT cells were noticed in the livers of α-GalCer+MSCCM-treated mice compared to α-GalCer+saline-treated animals, this phenomenon was not observed in α-GalCer-injured mice that received MSCWS-CM. MSCWS-CM could not induce expansion of anti-inflammatory IL-10-producing FoxP3 +CD4 + and CD8 + T regulatory cells and were not able to create immunosuppressive microenvironment in the liver as MSCCM. Similarly as it was observed in mice, MSCWS-CM were not able to optimally inhibit production of inflammatory and hepatototoxic cytokines in activated human Th1/Th17 and NKT1/NKT17 cells, confirming the hypothesis that cigarette smoke significantly attenuates therapeutic potential of MSC in cell-based immunotherapy of inflammatory liver diseases.

Concerns have recently increased that the integrity of some scientific research is questionable due to the inability to reproduce the claimed results of some experiments and thereby confirm that the original researcher's conclusions were justified. This phenomenon has been described as 'reproducibility crisis' and affects various fields from medicine to basic applied sciences. In this context, the REPLICA project aims to replicate previously conducted in vitro studies on the toxicity of cigarette smoke and e-cigarette aerosol, sometimes adding experiments or conditions where necessary, in order to verify the robustness and replicability of the data. In this work the REPLICA Team replicated biological and toxicological assessment published by Rudd and colleagues in 2020. As in the original paper, we performed Neutral Red Uptake (NRU) assay for the evaluation of cytotoxicity, Ames test for the evaluation of mutagenesis and In Vitro Micronuclei (IVMN) assay for the evaluation of genotoxicity on cells treated with cigarette smoke or e-cigarette aerosol. The results showed high cytotoxicity, mutagenicity and genotoxicity induced by cigarette smoke, but slight or no cytotoxic, mutagenic and genotoxic effects induced by the e-cigarette aerosol. Although the two studies presented some methodological differences, the findings supported those previously presented by Rudd and colleagues.

Previous research yielded conflicting results on the association between cigarette smoking and risk of SARS-CoV-2 infection. Since the prevalence of smoking is high globally, the study of its impact on COVID-19 pandemic may have considerable implications for public health. This study is the first to investigate the association between the SARS-CoV-2 antibody sero-positivity and biochemically verified smoking status, to refine current estimates on this association. SARS-CoV-2-specific IgG and serum cotinine levels (a well-known marker of tobacco exposure) were assessed in a large sero-epidemiological survey conducted in the town of Troina (Sicily, Italy). A propensity score matching was carried out to reduce the effect of possible factors on SARS-CoV-2 infection risk among study participants. Of the 1785 subjects included in our study, one-third was classified as current smokers, based on serum cotinine levels. The overall proportion of subjects with positive serology for SARS-CoV-2 IgG was 5.4%. The prevalence of SARS-CoV-2 antibody positivity and previous COVID-19 diagnosis were reduced in smokers. This reduced prevalence persisted after adjusting for possible confounders (such as sex, age, previous infection, chronic conditions, and risk group) at regression analyses, and the point estimates based on the PS-matched models resulted consistent with those for the unmatched population. This study found a lower proportion of positive SARS-CoV-2 serology among current smokers, using direct laboratory measures of tobacco exposure and thus avoiding possible bias associated with self-reported smoking status. Results may also serve as a reference for future clinical research on potential pharmaceutical role of nicotine or nicotinic-cholinergic agonists against COVID-19.

Cigarette smoking is associated with impairment of repair mechanisms necessary for vascular endothelium homeostasis. Reducing the exposure to smoke toxicants may result in the mitigation of the harmful effect on the endothelium and cardiovascular disease development. Previous investigations performed by the tobacco industries evaluated in vitro the effect of electronic cigarette (e-cig) compared to cigarette smoke demonstrating a significant reduction in endothelial cell migration inhibition following e-cig aerosol exposure. In the present study, we replicated one of these studies, evaluating the effects of cigarette smoke on endothelial cell migration compared to e-cig and heated tobacco products. We used a multi-center approach (ring-study) to verify the robustness and reliability of the results obtained in the replicated study. Consistently with the original study, we observed a substantial reduction of the effects of e-cig and tobacco heated products on endothelial cell migration compared to cigarette smoke. In conclusion, our study further confirms the importance of e-cig and tobacco heated products as a possible harm reduction strategy for cardiovascular diseases development in smokers. Highlights Cigarette smoking is strictly related to impairment of vascular repair mechanisms Reducing the exposure to toxicants in smoke, could reduce the harm to endothelium ENDS showed a reduced effect on endothelial cell migration compared to cigarette These data demonstrated the reduced toxicity of ENDS compared to cigarettes.

Electronic nicotine delivery systems (ENDS) can reduce the health risks associated with chronic smoke exposure, and their potential benefits are the subject of intense scientific debate. The international "Replica Study Group" replicated independently three relevant studies from the tobacco industry on the cytotoxic and inflammatory effects of cigarette smoke and ENDS aerosol. Our primary goal was to establish the reliability of the results and the robustness of the conclusions. In order to assess cytotoxicity of smoke and aerosol, we exposed human bronchial epithelial cell (H292) to cigarette smoke and to ENDS aerosol at air-liquid interface (ALI). Moreover, we aimed to assess different inflammatory and remodeling mediators release (IL-6, IL-8 and MMP-1) from cells exposed to whole smoke (WS) and to smoke deprived of total particulate matter (vapor phase; VP). We were able to replicate the results obtained in the original studies on cytotoxicity confirming that almost 80% of the cytotoxic effect of smoke is due to the vapor phase of smoke. Moreover, our results substantiated the significantly reduced cytotoxic effects of ENDS aerosol vs. cigarette smoke. However, our data were notably different in terms of inflammatory and remodeling activity triggered by smoke. Altogether, the data obtained independently in different laboratories clearly confirm the reduced toxicity of ENDS products compared to smoke, thus providing a valuable tool to the harm reduction strategies in smokers. Otherwise, we were not able to replicate the results on inflammatory and remodeling mediators released from smoke exposed cells. This could be due to the lack of normalization of the quantity of cytokines released, with the number of viable cells, in the original paper, since the production of cytokines itself requires the metabolic capability of cells.

Cigarette smoke is associated to severe chronic diseases. The most harmful components of cigarette smoke derive from the combustion process, which are significantly reduced in the electronic cigarette aerosol, thus providing a valid option in harm reduction strategies. To develop safer products, it is therefore necessary to screen electronic cigarette liquids (e-liquids) to meet high safety standards defined by government regulations. The aim of the present study was to evaluate the presence of metal- and plastic-derived contaminants in four different commercial e-liquids with high concentration of nicotine and their cytotoxic effect in normal human bronchial epithelial cells by a number of in vitro assays, in comparison with the 1R6F reference cigarette, using an air-liquid interface (ALI) exposure system. Moreover, we evaluated the effect of aerosol exposure on oxidative stress by measuring the production of reactive oxygen species and mitochondrial potential. Our results showed no contaminants in all e-liquids and a significantly reduced cytotoxic effect of e-liquid aerosol compared to cigarette smoke as well as a maintained mitochondria integrity. Moreover, no production of reactive oxygen species was detected with e-cigarette aerosol. In conclusion, these results support the reduced toxicity potential of e-cigs compared to tobacco cigarettes in an in vitro model resembling real life smoke exposure.

Inter-laboratory comparison is widely used to ensure quality control among laboratories. In in vitro toxicology studies for tobacco harm reduction (THR), exposure system performance and laboratory proficiency along with product smoke and aerosol stream are tested for variability to assess accuracy. Here we aim to test a novel inter-laboratory setup created in a new collaborative research group using identical and small footprint systems- in order to minimize variability factors and increase reproducibility. Seven independent laboratories from different geographical areas tested the aerosol and smoke stream and exposure system performance (LM1 and LM4E) using Cambridge Filter Pad (CFP) trapping techniques. We tested 1R6F reference cigarettes, two electronic cigarettes (Vype e-Pen and Vype e-Stick Maxx), and two tobacco heating products (IQOS and Glo™) under the appropriate ISO and/or HCI regimes. Nicotine quantification was performed by GC-FID at the laboratory of the leading center. The performance of participant laboratories was assessed by z-score values obtained from results either in relation to the mean and standard deviation of total participants or in relation to the reference leading center. Z-Scores were satisfactory when |z| ≤ 2, questionable when 2 < |z| < 3 and, unsatisfactory when |z| ≥ 3. In the first evaluation, for all the tested devices, Z- scores values generated by dosimetry data ranged from -2 to +2. However, high intra-laboratory variability (RSD> 10%) was observed for almost all laboratories. In the second, data showed borderline and unsatisfactory exposure performances versus LAB-A. Particularly, Z-scores ≥ 3 were observed once for LAB-B (e-Stick exposure) and LAB-G (e-Pen exposure), twice for LAB-C (1R6F-ISO and e-Stick exposures) and LAB-E (e-Pen and e-Stick exposures), and three times for LAB-F (1R6F-HCI, e-Pen, and Glo exposures). This study demonstrates that nicotine dosimetry is a fundamental method for quality assurance of smoke/vapor run exposure in the early stage of an interlaboratory study, allowing the identification and possibly the resolution of gaps. Extended practice sessions on exposure runs and several rounds of nicotine dosimetry testing should be planned to keep in check overall equipment and operator performance. ### Competing Interest Statement Riccardo Polosa is full tenured professor of Internal Medicine at the University of Catania (Italy) and Medical Director of the Institute for Internal Medicine and Clinical Immunology at the same University. In relation to his recent work in the area of respiratory diseases, clinical immunology, and tobacco control, RP has received lecture fees and research funding from Pfizer, GlaxoSmithKline, CV Therapeutics, NeuroSearch A/S, Sandoz, MSD, Boehringer Ingelheim, Novartis, Duska Therapeutics, and Forest Laboratories. Lecture fees from a number of European EC industry and trade associations (including FIVAPE in France and FIESEL in Italy) were directly donated to vaper advocacy no-profit organizations. RP has also received grants from European Commission initiatives (U-BIOPRED and AIRPROM) and from the Integral Rheumatology & Immunology Specialists Network (IRIS) initiative. He has also served as a consultant for Pfizer, Global Health Alliance for treatment of tobacco dependence, CV Therapeutics, Boehringer Ingelheim, Novartis, Duska Therapeutics, ECITA (Electronic Cigarette Industry Trade Association, in the UK), Arbi Group Srl., Health Diplomats, and Sermo Inc. RP has served on the Medical and Scientific Advisory Board of Cordex Pharma, Inc., CV Therapeutics, Duska Therapeutics Inc, Pfizer, and PharmaCielo. RP is being paid textbook royalties from ELSEVIER. RP is also founder of the Center for Tobacco prevention and treatment (CPCT) at the University of Catania and of the Center of Excellence for the acceleration of Harm Reduction (CoEHAR) at the same University, which has received support from Foundation for a Smoke Free World to conduct 8 independent investigator-initiated research projects on harm reduction. RP currently involved in a patent application concerning an app tracker for smoking behaviour developed for ECLAT Srl. RP is also currently involved in the following pro bono activities: scientific advisor for LIAF, Lega Italiana Anti Fumo (Italian acronym for Italian Anti-Smoking League), the Consumer Advocates for Smoke-free Alternatives (CASAA) and the International Network of Nicotine Consumers Organizations (INNCO); Chair of the European Technical Committee for standardization on Requirements and test methods for emissions of electronic cigarettes (CEN/TC 437; WG4). Giovanni Li Volti is currently elected Director of the Center of Excellence for the acceleration of HArm Reduction. Konstantinos Poulas has received service grants and research funding from a number of Vaping Companies. He is the Head of the Institute of Research and Innovations, which has received a grant from the Foundation for a Smoke Free World. All other authors declare no competing interests.

Biorąc pod uwage, ze wielu pacjentow z przewleklą obturacyjną chorobą pluc (POChP) pali papierosy pomimo objawow, wazne jest zrozumienie dlugofalowego wplywu zdrowotnego zastepowania tradycyjnych papierosow poprzez podgrzewane produkty tytoniowe (HTP, heated tobacco products). Przez 3 lata monitorowano parametry zdrowotne pacjentow z POChP, ktorzy znacznie ograniczyli palenie lub zaprzestali palenia papierosow po przejściu na HTP. Zmiany w liczbie codziennie wypalanych papierosow, rocznej czestości zaostrzen choroby, wskaźnikach czynności pluc, wyniku kwestionariusza ciezkości POChP wedlug oceny wlasnej pacjentow (wskaźnik CAT) i wyniku testu 6-minutowego marszu (6MWD) w stosunku do wartości wyjściowych byly mierzone u pacjentow z POChP stosujących HTP w 12., 24. i 36. miesiącu. Porownano je z grupą pacjentow z POChP dopasowanych pod wzgledem wieku i plci, ktorzy nadal palili papierosy. Kompletne zestawy danych byly dostepne u 38 pacjentow (po 19 w kazdej grupie). U osob stosujących HTP odnotowano znaczne zmniejszenie średniej (± SD) rocznej czestości zaostrzen POChP z 2,1 (± 0,9) na początku badania do 1,4 (± 0,8), 1,2 (± 0,8) i 1,3 (± 0,8) po 12, 24 i 36 miesiącach obserwacji (p < 0,05 dla wszystkich wizyt). Ponadto we wszystkich trzech punktach czasowych w kohorcie HTP stwierdzono znaczną i klinicznie istotną poprawe wynikow CAT i 6MWD. Nie zaobserwowano istotnych zmian u pacjentow z POChP, ktorzy kontynuowali palenie. Niniejsze badanie jestpierwszym, w ktore opisano dlugoterminowe skutki zdrowotne stosowania HTP u pacjentow z POChP. Poprawe objawow ze strony ukladu oddechowego, tolerancji wysilku, jakości zycia i czestości zaostrzen choroby zaobserwowano konsekwentnie u pacjentow z POChP, ktorzy powstrzymali sie od palenia lub znacznie ograniczyli palenie papierosow poprzez przestawienie sie na stosowanie HTP. Slowa kluczowe: zaprzestanie palenia, podgrzewane produkty tytoniowe, IQOS, glo, POChP, zmniejszenie szkod związanych z paleniem tytoniu

Background After the global spread of SARS-CoV-2, research has highlighted several aspects of the pandemic, focusing on clinical features and risk factors associated with infection and disease severity. However, emerging results on the role of smoking in SARS-CoV-2 infection susceptibility or COVID-19 outcomes are conflicting, and their robustness remains uncertain. Objective In this context, this study aims at quantifying the proportion of SARS-CoV-2 antibody seroprevalence, studying the changes in antibody levels over time, and analyzing the association between the biochemically verified smoking status and SARS-CoV-2 infection. Methods The research design involves a 6-month prospective cohort study with a serial sampling of the same individuals. Each participant will be surveyed about their demographics and COVID-19–related information, and blood sampling will be collected upon recruitment and at specified follow-up time points (ie, after 8 and 24 weeks). Blood samples will be screened for the presence of SARS-CoV-2–specific antibodies and serum cotinine, being the latter of the principal metabolite of nicotine, which will be used to assess participants’ smoking status. Results The study is ongoing. It aims to find a higher antibody prevalence in individuals at high risk for viral exposure (ie, health care personnel) and to refine current estimates on the association between smoking status and SARS-CoV-2/COVID-19. Conclusions The added value of this research is that the current smoking status of the population to be studied will be biochemically verified to avoid the bias associated with self-reported smoking status. As such, the results from this survey may provide an actionable metric to study the role of smoking in SARS-CoV-2 infection and COVID-19 outcomes, and therefore to implement the most appropriate public health measures to control the pandemic. Results may also serve as a reference for future clinical research, and the methodology could be exploited in public health sectors and policies. International Registered Report Identifier (IRRID) DERR1-10.2196/32285

ABSTRACT Despite the growing numbers of studies with cigarettes and other electronic nicotine delivery products (ENDs), there is no standard covering nicotine dosimetry and its stability in various matrix. The aim of the present study was to provide a protocol to normalize nicotine concentration adsorbed in Cambridge Filter PADs (CFPs) and their storage method. Smoke/vapor generated by a reference tobacco cigarette (1R6F) and ENDs with different exposure regimes (ISO, HCI and CRM81) was collected in CFPs. For each exposure, some CFPs were analyzed at time zero, whereas the others were stored under different conditions for nicotine assessment after 30 days. Principal Component Analysis (PCA) was also performed to establish the best parameter for nicotine normalization. PCA showed the best correlation between nicotine in CFPs and TPM. Our results showed differences between products and puffing regimes, but storage of CFPs at −80°C was always effective in maintaining the nicotine content. In conclusion, this study highlights that different exposure regimens and products can affect the preservation of nicotine titer in CFPs and samples storage at −80°C may prevent the loss of nicotine. These conditions are recommended and should be adopted for Inter-laboratory comparison studies on ENDs to ensure harmonization between participating laboratories.

Given that many patients with chronic obstructive pulmonary disease (COPD) smoke despite their symptoms, it is important to understand the long-term health impact of cigarette substitution with heated tobacco products (HTPs). We monitored health parameters for 3 years in COPD patients who substantially attenuated or ceased cigarette consumption after switching to HTPs. Changes in daily cigarette smoking, annualized disease exacerbations, lung function indices, patient-reported outcomes (CAT scores) and 6-minute walk distance (6MWD) from baseline were measured in COPD patients using HTPs at 12, 24 and 36 months. These were compared to a group of age- and sex-matched COPD patients who continued smoking. Complete data sets were available for 38 patients (19 in each group). Subjects using HTPs had a substantial decrease in annualized COPD exacerbations within the group mean (± SD) from 2.1 (± 0.9) at baseline to 1.4 (± 0.8), 1.2 (± 0.8) and 1.3 (± 0.8) at 12-, 24- and 36-month follow-up (p < 0.05 for all visits). In addition, substantial and clinically significant improvements in CAT scores and 6MWD were identified at all three time points in the HTP cohort. No significant changes were observed in COPD patients who continued smoking. This study is the first to describe the long-term health effects of HTP use in COPD patients. Consistent improvements in respiratory symptoms, exercise tolerance, quality of life, and rate of disease exacerbations were observed in patients with COPD who abstained from smoking or substantially reduced their cigarette consumption by switching to HTP use.

Electronic Nicotine Delivery Systems (ENDS), i.e., electronic cigarettes (e-cigs) and Tobacco Heating Products (THPs), are rapidly growing in popularity. The marketing of these products is regulated by specific rules in the European Union and in the US, which permit their legal sales. Nonetheless, comprehensive quality and safety requirements for regulatory purposes are still under development. Cytotoxicity studies are an important initial step in appraising the potential toxicity of ENDS. The aim of the present study was to screen a battery of different in vitro cytotoxicity methods for the assessment of toxicity induced by ENDS. We evaluated different cytotoxicity assays, including neutral red uptake (NRU), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin V apoptosis, High Content Screening (HCS) assays and Real Time Cell Analysis (RTCA), to compare two e-cigs (Vype ePen 3 and Vype eStick Maxx) and two THPs (IQOS and GLO™) with the 1R6F reference tobacco cigarette. Human bronchial epithelial cells (H292) were exposed to 1R6F smoke (5 puffs by HCI regime), ePen vapor (10 puffs by modified HCI regime), eStick vapor (25 puffs by CRM81 regime), IQOS vapor (7 puffs by HCI regime) and GLO vapor (8 puffs by HCI regime) at air-liquid interface. All tests showed reduced cell viability following 1R6F smoke exposure and slight or no reduction with ENDS at 24 hours compared to controls. In addition, Annexin V and RTCA exhibited a further significant reduction in cell viability following 1R6F exposure compared with other assays. Furthermore, Annexin V allowed to discriminate viable cells from those in early/late apoptosis. Finally, RTCA and HCS being time-resolved analyses allowed also to determine the kinetic dependency parameter for toxicity of smoke/vapor chemicals on cell viability. In conclusion, NRU assay may be considered a suitable test, especially when combined with a time-resolved test, for assessing the kinetic of cytotoxicity induced by these products.

Electronic nicotine delivery systems (ENDS) may reduce health risks associated with chronic exposure to smoke and their potential benefits have been the matter of intense scientific debate. We aimed to replicate three published studies on cytotoxic and inflammatory effects of cigarette smoke and ENDS aerosol in an independent multi-center ring study. We aimed to establish the reliability of results and the robustness of conclusions by replicating the authors' experimental protocols and further validating them with different techniques. Human bronchial epithelial cells (NCI-H292) were exposed to cigarette whole smoke and vapor phase and to aerosol from ENDS. We also assessed the inflammatory cytokines interleukin-6 and interleukin-8 and the remodeling mediator matrix metalloproteinase-1. We replicated cell viability results and confirmed that almost 80% of cytotoxic effects are due to volatile compounds in the vapor phase of smoke. Our findings substantiated the reduced cytotoxic effects of ENDS aerosol. However, our data on inflammatory and remodeling activity triggered by smoke differed significantly from those in the original reports. Taken together, independent data from multiple laboratories clearly demonstrated the reduced toxicity of ENDS products compared to cigarettes.

Prevalence studies of current smoking, among hospitalized COVID-19 patients, demonstrated an unexpectedly low prevalence among patients with COVID-19. The aim of the present study was to evaluate the effect of smoke from cigarettes on ACE-2 in bronchial epithelial cells. Normal bronchial epithelial cells (H292) were exposed to smoke by an air-liquid-interface (ALI) system and ACE-2 membrane protein expression was evaluated after 24 h from exposure. Our transcriptomics data analysis showed a significant selective reduction of membrane ACE-2 expression (about 25%) following smoking exposure. Interestingly, we observed a positive direct correlation between ACE-2 reduction and nicotine delivery. Furthermore, by stratifying GSE52237 as a function of ACE-2 gene expression levels, we highlighted 1,012 genes related to ACE-2 in smokers and 855 in non-smokers. Furthermore, we showed that 161 genes involved in the endocytosis process were highlighted using the online pathway tool KEGG. Finally, 11 genes were in common between the ACE-2 pathway in smokers and the genes regulated during endocytosis, while 12 genes with non-smokers. Interestingly, six in non-smokers and four genes in smokers were closely involved during the viral internalization process. Our data may offer a pharmaceutical role of nicotine as potential treatment option in COVID-19.