Changes in Cigarette Smoking and Smokeless Tobacco Use During the Coronavirus Disease 2019 Lockdown Period Among Youth and Young Adults in Denmark.

INTRODUCTION: Cigarettes and smokeless tobacco (snus and nicotine pouches) are prevalent among youth and young adults in Denmark. Here, we examined the extent of changes in the use of cigarettes and smokeless tobacco during the first Coronavirus Disease 2019 (COVID-19) lockdown in March and April 2020 in Denmark as well as reasons for changed behavior. AIMS AND METHODS: This study used data from a nationwide survey conducted among 15- to 29-year-olds from January to March 2021 including 13 530 respondents (response rate = 36.0%). Logistic regression analyses assessed the associations between sociodemographic characteristics and the odds of initiating or increasing as well as trying to stop or decreasing cigarette smoking and smokeless tobacco use. RESULTS: The prevalence of cigarette smoking was 17.8% and 10.5% reported using smokeless tobacco. Around 40% of those currently smoking cigarettes reported smoke on par during the COVID-19 lockdown as before, 24.5% started to smoke or increased their smoking, and 27.4% tried to stop or smoked less. Approximately 37% used smokeless tobacco on the same level as, before the COVID-19 lockdown, 38.8% initiated or used more, and 14.1% tried to stop or used less. Females were more prone to initiate smokeless tobacco use and increase their level of smoking during the lockdown, and younger participants smoked less. More females compared with males changed their smoking behaviors because of their mood, and more younger participants did so because of fewer social gatherings. CONCLUSION: Although most youths and young adults' tobacco behaviors remained the same during the COVID-19 lockdown, many also increased or decreased their behaviors-especially females and younger participants. IMPLICATIONS: This study enables the possibility of detecting new tendencies in smoking and the use of smokeless tobacco among subgroups of the population during the COVID-19 lockdown. This knowledge is crucial for identifying which groups of youths are vulnerable to increasing their tobacco use in other pandemic situations and which groups call for special attention after the lockdown period. Future efforts may focus on vulnerable groups affected by the COVID-19 pandemic, such as females, and there is a need to monitor closely whether youth tobacco use changes as society becomes more normalized.

Evaluating the Effects of Denmark's New Tobacco Control Act on Young People's Use of Nicotine Products: A Study Protocol of the §SMOKE Study.

(1) Background: In December 2020, a broad majority of political parties in Denmark agreed on a new tobacco control act. In addition, price increases on tobacco in 2020 and 2022 became part of the Danish Finance Act. This study protocol describes the study "§SMOKE-A Study of Tobacco, Behavior, and Regulations" designed to monitor and evaluate the implementation and effect of the new strengthened tobacco control acts. The overall aim is to monitor tobacco use among young people before, during, and after implementation of the new tobacco control legislation, including an increased price on tobacco, a ban on point-of-sale tobacco displays, and plain packaging. Subgoals are to monitor overall use of nicotine products, attitudes, and norms. (2) Methods: This study is designed as a five-year impact evaluation with repeated cross-sectional survey data collections. The baseline survey was conducted before implementing an increased price on tobacco, the first step in the new legislation, initiated 1 April 2020. Study participants (n = 37,500) were a random sample of individuals living in Denmark aged 15 to 29 years. (3) Conclusions: This study examines the impact of the new strengthened tobacco control legislation in Denmark from 2020 to 2025. The findings of this study are relevant to other countries facing implementation of similar measures to explore intended and unintended consequences of the legislation and help to identify how the legislation could be further improved.

Use of Tobacco and Nicotine Products among Young People in Denmark-Status in Single and Dual Use.

Lots of new tobacco or nicotine products are being launched, e.g., e-cigarettes and smokeless tobacco, which appeal especially to the youngest part of the population. For example, the use of smokeless tobacco among Danish youth rose from approx. 2% in 2010 to 9% in 2020. Hence, there is an urgent need to follow and intervene against youth tobacco or nicotine product use. This study explored the current use of cigarettes, e-cigarettes, heated tobacco, and smokeless tobacco among Danish 15- to 29-year-olds. Further, we examined the concurrent use of two products or more. We used a nationwide survey conducted among 15- to 29-year-olds in February and March 2020. Overall, approx. 35,700 individuals received the questionnaire of which 35.5% responded (n = 13,315). One out of five (20.1%) smoked cigarettes, half of them daily, the other half occasionally. About one in twenty (3.9%) used e-cigarettes (daily or occasionally), and more than one in three (31.6%) had tried e-cigarettes. The use of heated tobacco among Danish youth is still relatively limited (0.3%). In comparison, about 9% used smokeless tobacco (daily or occasionally). Overall, 27.0% stated that they use at least one type of tobacco or nicotine product, while 5.6% used more than one product. Monitoring tobacco-related behavior in youth provides extremely important information for, e.g., policymakers and health professionals.

Desensitization of human breast progenitors by a transient exposure to pregnancy levels of estrogen.

Full term pregnancy at an early age is the only factor known to consistently protect against breast cancer. Because hormone receptor positive progenitors in the human breast relay endocrine signaling, we here sought to determine whether an experimental mimicry of the third trimester surge of hormones would change their susceptibility to growth stimulation. Hormone receptor positive, reduction mammoplasty-derived human breast epithelial progenitors were exposed to a short-term, pregnancy-level of estradiol, and their subsequent response to estradiol stimulation was analyzed. Exposure to pregnancy-level of estradiol results in subsequent lower sensitivity to estrogen-induced proliferation. Expression array and immunoblotting reveal upregulation of S100A7 and down-regulation of p27, both associated with parity and epithelial differentiation. Notably, we find that the epithelial differentiation is accompanied by upregulation of E-cadherin and down-regulation of vimentin as well as by diminished migration and more mature luminal epithelial differentiation in a mouse transplantation model. Our findings are in support of a de-sensitization mechanism for pregnancy-induced prevention against breast cancer.

Proof of region-specific multipotent progenitors in human breast epithelia.

The human breast parenchyma consists of collecting ducts and terminal duct lobular units (TDLUs). The TDLU is the site of origin of most breast cancers. The reason for such focal susceptibility to cancer remains poorly understood. Here, we take advantage of a region-specific heterogeneity in luminal progenitors to interrogate the differentiation repertoire of candidate stem cells in TDLUs. We show that stem-like activity in serial passage culture and in vivo breast morphogenesis relies on the preservation of a myoepithelial phenotype. By enrichment for region-specific progenitors, we identify bipotent and multipotent progenitors in ducts and TDLUs, respectively. We propose that focal breast cancer susceptibility, at least in part, originates from region-specific myoepithelial progenitors.

Establishment of a normal-derived estrogen receptor-positive cell line comparable to the prevailing human breast cancer subtype.

Understanding human cancer increasingly relies on insight gained from subtype specific comparisons between malignant and non-malignant cells. The most frequent subtype in breast cancer is the luminal. By far the most frequently used model for luminal breast cancer is the iconic estrogen receptor-positive (ERpos) MCF7 cell line. However, luminal specific comparisons have suffered from the lack of a relevant non-malignant counterpart. Our previous work has shown that transforming growth factor-ß receptor (TGFßR) inhibition suffices to propagate prospectively isolated ERpos human breast luminal cells from reduction mammoplasties (HBEC). Here we demonstrate that transduction of these cells with hTERT/shp16 renders them immortal while remaining true to the luminal lineage including expression of functional ER (iHBECERpos). Under identical culture conditions a major difference between MCF7 and normal-derived cells is the dependence of the latter on TGFßR inhibition for ER expression. In a breast fibroblast co-culture model we further show that whereas MCF7 proliferate concurrently with ER expression, iHBECERpos form correctly polarized acini, and segregate into proliferating and ER expressing cells. We propose that iHBECERpos may serve to shed light on hitherto unappreciated differences in ER regulation and function between normal breast and breast cancer.

Evidence of two distinct functionally specialized fibroblast lineages in breast stroma.

BACKGROUND: The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human breast. We here demonstrate the impact on epithelium of two lineages of breast stromal fibroblasts, one of which accumulates in the TDLU while the other resides outside the TDLU in the interlobular stroma. METHODS: The two lineages are prospectively isolated by fluorescence activated cell sorting (FACS) based on different expression levels of CD105 and CD26. The characteristics of the two fibroblast lineages are assessed by immunocytochemical staining and gene expression analysis. The differentiation capacity of the two fibroblast populations is determined by exposure to specific differentiating conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271low/MUC1high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation of morphological development. Epithelial structure formation and polarization is shown by immunofluorescence and digitalized quantification of immunoperoxidase-stained cultures. RESULTS: Lobular fibroblasts are CD105high/CD26low while interlobular fibroblasts are CD105low/CD26high. Once isolated the two lineages remain phenotypically stable and functionally distinct in culture. Lobular fibroblasts have properties in common with bone marrow derived mesenchymal stem cells and they specifically convey growth and branching morphogenesis of epithelial progenitors. CONCLUSIONS: Two distinct functionally specialized fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that lobular fibroblasts constitute a specialized microenvironment for human breast luminal epithelial progenitors, i.e. the putative precursors of breast cancer.

Propagation of oestrogen receptor-positive and oestrogen-responsive normal human breast cells in culture.

Investigating the susceptibility of oestrogen receptor-positive (ER(pos)) normal human breast epithelial cells (HBECs) for clinical purposes or basic research awaits a proficient cell-based assay. Here we set out to identify markers for isolating ER(pos) cells and to expand what appear to be post-mitotic primary cells into exponentially growing cultures. We report a robust technique for isolating ER(pos) HBECs from reduction mammoplasties by FACS using two cell surface markers, CD166 and CD117, and an intracellular cytokeratin marker, Ks20.8, for further tracking single cells in culture. We show that ER(pos) HBECs are released from growth restraint by small molecule inhibitors of TGFß signalling, and that growth is augmented further in response to oestrogen. Importantly, ER signalling is functionally active in ER(pos) cells in extended culture. These findings open a new avenue of experimentation with normal ER(pos) HBECs and provide a basis for understanding the evolution of human breast cancer.

ADAM12 is expressed in the tumour vasculature and mediates ectodomain shedding of several membrane-anchored endothelial proteins.

ADAM (a disintegrin and metalloproteinase) 12 is a metalloprotease implicated in cancer progression. ADAM12 can activate membrane-anchored proteins, such as sonic hedgehog, Delta-like 1 and certain epidermal growth factor receptor ligands, through a process called ectodomain shedding. We screened several membrane-anchored proteins to further dissect the substrate profile of ADAM12-mediated ectodomain shedding, and found shedding of five previously unreported substrates [Kitl1, VE-cadherin (vascular endothelial cadherin), Flk-1 (fetal liver kinase 1), Tie-2, and VCAM-1 (vascular cell adhesion molecule 1)], of which the latter four are specifically expressed by endothelial cells. We also observed that ADAM12 expression was increased in the tumour vasculature of infiltrating ductal carcinoma of the human breast as compared with little to no expression in normal breast tissue vasculature, suggesting a role for ADAM12 in tumour vessels. These results prompted us to further evaluate ADAM12-mediated shedding of two endothelial cell proteins, VE-cadherin and Tie-2. Endogenous ADAM12 expression was very low in cultured endothelial cells, but was significantly increased by cytokine stimulation. In parallel, the shed form of VE-cadherin was elevated in such cytokine-stimulated endothelial cells, and ADAM12 siRNA (small interfering RNA) knockdown reduced cytokine-induced shedding of VE-cadherin. In conclusion, the results of the present study demonstrate a role for ADAM12 in ectodomain shedding of several membrane-anchored endothelial proteins. We speculate that this process may have importance in tumour neovascularization or/and tumour cell extravasation.