Evaluation method for the cytotoxicity of cigarette smoke by in vitro whole smoke exposure

An in vitro whole smoke (WS) exposure method was established to evaluate the toxicological effects of fresh cigarette smoke using the VITROCELL^® system associated with the neutral red uptake (NRU) cytotoxicity assay. The VITROCELL^® system is a newly representative culture and exposure system for in vitro studies of gases or complex mixtures. The impacts of two factors on cytotoxicity measurements of cigarette smoke were investigated using this WS exposure system. The factors include synthetic air exposure and optimal time to perform the NRU assay after smoke exposure. Results showed that synthetic air exposure used in the system did not significantly alter cell survival; 24 h after smoke exposure appeared to be an optimal time-point to assess the cytotoxicity of cigarette smoke. A clear dose–response relationship between smoke exposure and cell viability was demonstrated using this system, and the evaluation method was sensitive to distinguish the differences in smoke-induced cytotoxic effects from different cigarettes. In addition, we tried converting the values of EC₅₀ from WS exposure testing into the values in unit used in total particulate matter (TPM) testing for a purpose of comparison, and the data indicate that the cytotoxicity of smoke measured by WS exposure is greater than that measured by TPM exposure.     

Pivotal role of MUC1 glycosylation by cigarette smoke in modulating disruption of airway adherens junctions in vitro

Cigarette smoke increases the risk of lung cancer by 20‐fold and accounts for 87% of lung cancer deaths. In the normal airway, heavily O‐glycosylated mucin‐1 (MUC1) and adherens junctions (AJs) establish a structural barrier that protects the airway from infectious, inflammatory and noxious stimuli. Smoke disrupts cell–cell adhesion via its damaging effects on the AJ protein epithelial cadherin (E‐cad). Loss of E‐cad is a major hallmark of epithelial–mesenchymal transition (EMT) and has been reported in lung cancer, where it is associated with invasion, metastasis and poor prognosis. Using organotypic cultures of primary human bronchial epithelial (HBE) cells treated with smoke‐concentrated medium (Smk), we have demonstrated that E‐cad loss is regulated through the aberrant interaction of its AJ binding partner, p120‐catenin (p120ctn), and the C‐terminus of MUC1 (MUC1‐C). Here, we reported that even before MUC1‐C became bound to p120ctn, smoke promoted the generation of a novel 400 kDa glycoform of MUC1's N‐terminus (MUC1‐N) differing from the 230 kDa and 150 kDa glycoforms in untreated control cells. The subsequent smoke‐induced, time‐dependent shedding of glycosylated MUC1‐N exposed MUC1‐C as a putative receptor for interactions with EGFR, Src and p120ctn. Smoke‐induced MUC1‐C glycosylation modulated MUC1‐C tyrosine phosphorylation (TyrP) that was essential for MUC1‐C/p120ctn interaction through dose‐dependent bridging of Src/MUC1‐C/galectin‐3/EGFR signalosomes. Chemical deglycosylation of MUC1 using a mixture of N‐glycosylation inhibitor tunicamycin and O‐glycosylation inhibitor benzyl‐α‐GalNAc disrupted the Src/MUC1‐C/galectin‐3/EGFR complexes and thereby abolished smoke‐induced MUC1‐C‐TyrP and MUC1‐C/p120ctn interaction. Similarly, inhibition of smoke‐induced MUC1‐N glycosylation using adenoviral shRNA directed against N‐acetyl‐galactosaminyl transferase‐6 (GALNT6, an enzyme that controls the initiating step of O‐glycosylation) successfully suppressed MUC1‐C/p120ctn interaction, prevented E‐cad degradation and maintained cellular polarity in response to smoke. Thus, GALNT6 shRNA represents a potential therapeutic modality to prevent the initiation of events associated with EMT in the smoker's airway. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Pulmonary function assessment in the early phase of patients with smoke inhalation injury from fire

Objectives

Fire smoke contains toxic gases and numerous chemical compounds produced by incomplete combustion, and may cause injury to the airways. Increased airway reactivity, as well as a decrease in lung function, has been reported as a sequela of smoke inhalation injury. This study was undertaken to assess lung functions in the early phase of patients with smoke inhalation damage from fires.

Methods

A total of 15 patients with fire smoke inhalation (fire smoke group) and 15 subjects with chronic cough but no previous history of lung disease (chronic cough group) were enrolled. For diagnosis of inhalation injury, we performed bronchoscopy, high-resolution computed tomography (HRCT), as well as arterial carboxyhemoglobin (COHb) at admission. Clinical characteristics, pulmonary function tests (PFTs) and mannitol bronchial provocation tests (BPTs) were analyzed and compared between the two groups.

Results

In fire smoke group, initial COHb levels and the PaO₂/FiO₂ ratio were (14.8±18.49)% and 425.7±123.68, respectively. Of seven patients performing HRCT, 4 (57.1%) showed the CT findings compatible with lung involvement of inhalation injury. Post bronchodilator value of the percent of forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV₁) were (76.0±24.27)% and (79.8±27.82)%, respectively. Pre-and post- bronchodilator forced expiratory flow between 25% and 75% of the FVC (FEF_25-75) and the percent predicted FEF_25-75 were 2.41±1.47 vs. 2.65±1.45 L (P=0.045), and (68.7±37.29)% vs. (76.4±36.70)% (P=0.031), respectively. Two patients (13.3%) had positive bronchodilator response (BDR). In fire smoke and chronic cough group, all the subjects showed mannitol BPTs within normal limits.

Conclusions

Fire smoke inhalation leads to mild obstructive small airway disease pattern of pulmonary function in the early phase of patients with fire smoke damage. Further studies, however, need to be followed to identify the relationship between airway narrowing to inhaled mannitol and smoke inhalation injury.     

Cigarette smoking during an N-acetylcysteine-assisted cannabis cessation trial in adolescents

Background and objectives: Tobacco and cannabis use are both highly prevalent worldwide. Their co-use is also common in adults and adolescents. Despite this frequent co-occurrence, cessation from both substances is rarely addressed in randomized clinical trials. Given evidence that tobacco use may increase during cannabis cessation attempts, and additionally that tobacco users have poorer cannabis cessation outcomes, we explored tobacco outcomes, specifically cigarette smoking, from an adolescent cannabis cessation trial that tested the efficacy of N-acetylesteine (NAC). Methods: Cannabis-dependent adolescents (ages 15–21; n?=?116) interested in cannabis treatment were randomized to NAC (1200?mg bid) or matched placebo for 8 weeks. Participants did not need to be cigarette smokers or be interested in smoking cessation to qualify for inclusion. Results: Approximately 59% of enrolled participants were daily and non-daily cigarette smokers, and only differed from non-smoking participants on the compulsion sub-scale of the Marijuana Craving Questionnaire. Among cigarette smokers who were retained in the study, there was no change in cigarettes per day for either NAC or placebo groups during the eight-week treatment phase. Being a cigarette smoker did not appear to influence the effects of NAC on cannabis abstinence, though there was a trend in the placebo group of poorer cannabis outcomes for cigarette smokers vs. non-smokers. Conclusions: No evidence was found of compensatory cigarette smoking during this cannabis cessation trial in adolescents. Further work assessing interventions to reduce both cannabis and tobacco use in this population is greatly needed.     

AECOPD患者呼出气冷凝液中分泌性白细胞蛋白酶抑制剂的检测及其临床意义

目的分析慢性阻塞性肺疾病急性加重期(AECOPD)患者治疗前后呼出气冷凝液(EBC)中分泌性白细胞蛋白酶抑制剂(SLPI)浓度的变化,及其与AECOPD各临床指标的相关性。方法随机选择AECOPD患者30例,正常健康者18例,采用ELISA方法检测AECOPD组治疗前后及健康对照组EBC中SLPI浓度,应用Pearson直线相关分析AECOPD组治疗前EBC中SLPI浓度与COPD临床特征、肺功能(PEF、FEV1%预计值)、血气分析、血WBC计数等指标的相关性。结果经抗感染、氧疗、解痉、激素等常规治疗7天后,AECOPD组EBC中SLPI浓度明显高于治疗前(P0.05);AECOPD组治疗前EBC中SLPI浓度与患者FEV1%预计值、痰量、呼吸困难症状密切相关(P0.05),与患者PEF、血白细胞(WBC)计数、动脉血pH值、PaO2/FiO2、PaCO2无相关性(P均0.05)。结论 AECOPD患者EBC中SLPI浓度可作为反映病情的严重程度和临床转归的指标。     

香烟烟雾提取物对CD4^＋T细胞向Th17细胞和调节性T细胞分化的影响

目的观察香烟烟雾提取物（CSE）对CD4^＋T细胞向Th17细胞和调节性T细胞分化的影响,为探索香烟烟雾暴露导致气道慢性炎症的机制提供实验依据。方法采用免疫磁珠法分离纯化健康志愿者外周血CD4^＋T细胞,以1×10^6／ml细胞密度接种于96孔培养板,分为以下8个组①空白对照组;②T细胞刺激剂组：加入T细胞刺激剂抗人CD3／28抗体微磁珠;③T细胞刺激剂CSE干预组：CD3／28＋2%CSE;④细胞因子组：CD3／28＋细胞因子;⑤细胞因子CSE干预组：CD3／28＋细胞因子＋2％CSE;⑥芳香烃受体（AHR）激动剂6-甲酰基吲哚[3,2-b]咔唑（FICZ）组CD3／28＋细胞因子＋FICZ;⑦AHR拮抗剂白藜芦醇组：CD3／28＋细胞因子＋2％CSE＋白藜芦醇;⑧溶剂对照组：CD3／28＋细胞因子＋DMSO。细胞因子为含TGF-β/IL-1β／IL-6／IL～23的混合细胞因子,以诱导Th17细胞和调节性T细胞分化。培养5d后,收集细胞,采用流式细胞术检测细胞表面分子CD4^＋CD25^＋Foxp3^＋细胞（Treg细胞）,以及胞内细胞因子IL-17＋的CD4^＋T细胞（Th17细胞）,计算各种刺激培养条件下,诱导生成的Th17及Treg细胞占CD4^＋T细胞的百分比。结果①CSE对Th17细胞分化的影响：空白对照组Th17细胞比例为（0．69±0．12）％,加入T细胞刺激剂后为（1．32±0．12）％,在此基础上加入CSE的干预组IL-17＋细胞比例升高为（2．17±0．24）％,（t=3．21,P〈0．01）;细胞因子组IL-17＋细胞比例为（1．35±0．08）％,而在此基础上加入CSE的干预组Th17细胞升高为（2．58±0．39）%（t=3．13,P〈0．01）。②CSE对Treg细胞分化的影响：在空白对照组中,Treg细胞占CD4^＋T细胞中的比例为（0．21±0．19）％,在加入T细胞刺激剂后,Treg细胞比例升高（3．59±0．37）％,加入细胞因子后,Treg细胞比例明显增加（5．85±0．76）％;在继续加入CSE干预后,Treg细胞比例明显减少（3．07±0．33）％（t=3．74,P〈0．01）;同样,T细胞刺激剂CSE干预组也出现Treg细胞比例减少（2．19±0．19）％,（t=2．71,P〈0．05）。③AHR活化对Treg细胞和Th17细胞分化的影响：AHR激动剂FICZ组的Treg细胞比例明显低于细胞因子组[（2．60±0．40）％,（5．85±0．76）％]（t=4．18,P〈0．01）,但Th17细胞比例升高[（2．86±0．43）％,（1．35±0．08）％]（t=3．65,P〈0．01）。AHR阻断剂白藜芦醇组中的Treg细胞比例和Th17细胞比例均低于细胞因子组,分别为[（0．33±0．14）％,（5．85±0．76）％],（t=7．71,P〈0．01）和[（0．42±0．07）％,（1．35±0．08）％],（t=8．87,P〈0．001）,并且和空白对照组接近,在细胞培养第5天通过7-AAD-AnnexinV对该组细胞检测并未发现细胞异常凋亡或死亡情况,结合该组细胞培养过程中的镜下形态推测该实验组CD4^＋T细胞的分化增殖受到白藜芦醇抑制。结论CSE可促进CD4^＋T细胞向Th17细胞分化,并抑制Treg细胞分化,这一过程可能通过AHR诱导。     

Salivary Cotinine Concentrations and Prevalence of Periodontal Disease in Young Japanese Women: The Kyushu Okinawa Maternal and Child Health Study

Background: The authors investigated the relationship between objectively assessed tobacco smoke exposure and periodontal disease. Methods: This cross‐sectional study included 1,103 women with a mean age of 31.5 years. Information on potential confounding factors was obtained through a self‐administered questionnaire. Periodontal disease was defined as positive if a woman had at least one tooth with a probing depth of ≥3.5 mm. Exposure to tobacco smoke was determined based on salivary cotinine concentration. Adjustment was made for age, region of residence, household income, education, toothbrushing frequency, and use of an interdental brush. Results: The prevalence of periodontal disease was 11.3%. Salivary cotinine concentration was independently positively associated with the prevalence of periodontal disease: the adjusted odds ratio for every 1‐unit (ng/mL) increase in salivary cotinine was 1.004 (95% confidence interval: 1.000 to 1.007). Conclusion: Salivary cotinine concentrations were positively associated with the prevalence of periodontal disease among young women.     

Pepsin levels and oxidative stress markers in exhaled breath condensate of patients with gastroesophageal reflux disease

Aim

To evaluate the pepsin and oxidative stress markers in exhaled breath condensate (EBC) in patients with gastroesophageal reflux disease (GERD).

Patients and Method

Patients with a presumptive diagnosis of GERD with recurrent respiratory and gastrointestinal problems aged between 2 and 14 years were included in the study. All patients underwent pH monitoring. Patients with a reflux index (RI) ≥ 4 were assessed as the reflux group, and those with an RO < 4 were assessed as the non-reflux group. Pepsin levels and oxidative stress markers [NO metabolites (NOX) and total sulphydrile (TSH) levels] were measured in the EBC.

Results

There were 24 patients in the reflux group [RI 17.6 (6.6–46.4)] [median, interquartile range] and 23 in the non-reflux group [RI 0.8 (0.5–1.9) (p < 0.001). Pepsin levels in the EBC were below the level of detection. The median levels of NOx in the EBC of children with reflux [13.7 μmol/L (7.3–24.5)] were lower in than non-reflux group [21.0 μmol/L (14.0–25.2)] (p = 0.034). There was a negative correlation between reflux index and NOX levels in EBC (rs: − 0.331, p = 0.023). In contrast, there was no difference in TSH levels between the reflux and non-reflux groups [37.4 μmol/L (30.2–44.6) vs 40.1 μmol/L (37.4–44.9), respectively, (p > 0.05)].

Conclusion

Decreased levels of NOX in patients with GER disease suggest increased oxidative stress in airways of these patients.