Browsing by Subject "tobacco"
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Item EFFECT OF TOBACCO-TREATED MG63 OSTEOBLAST ON HUMAN PULP CELLS(Office of the Vice Chancellor for Research, 2012-04-13) Gebreslassie, Seret; Huang, Ruijie; Li, Mingyun; Song, Fengeyu; Gregory, Richard L.Objective: The objective of this study is to determine the effects of to-bacco products on protein concentration and growth of MG63 osteoblasts and the effects of the bacterial cells and culture supernatants on human pulp cells. The study was designed to observe the effects of P.gingivalis grown in four different tobacco solutions such as CSC (cigarette smoked condensate), nicotine (chewing tobacco), and DST (dissolvable smokeless tobacco) strips, and in the media control only without tobacco products. Methods: MG63 os-teoblast was grown in BHI-YE (Bacteria Heart Infusion-Yeast Extract) and hemin-vitamin K. In addition, MG63 osteoblast was grown in BHI-Y-E con-taining nicotine, CSC, and DST. Human pulp cells were grown in media con-taining BGS (Bovine Growth Serum) and washed. The pulp cell cultures will be assayed for cytotoxicity and the supernatants will be assayed for cyto-kines and MMP expression. Results: The protein assays was performed us-ing a microplate spectrophometer and SoftMax Pro 5.2, and we observed that nicotine and DST treated cells had significantly less protein than control cells, however, CSC treated cells had significantly more protein. The undilut-ed control had significantly less protein than the tobacco-treated superna-tants. Conclusion: Based on the previous experiments, we speculate that the additional protein in the undiluted CSC cells and tobacco-treated super-natant may stimulate more effect on human pulp cells than the control, nico-tine or DST treated cells or the control supernatant.Item Effects of casein phosphopeptide-amorphous calcium phosphate crème on nicotine-induced Streptococcus mutans biofilm in vitro(Springer, 2020) Alawadhi, Naser B.; Lippert, Frank; Gregory, Richard L.; Biomedical Sciences and Comprehensive Care, School of DentistryObjectives The aim of this study was to test the effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) crème, or MI Paste™ (MIP), on nicotine-induced Streptococcus mutans biofilm. The experiment utilized S. mutans biofilm assays with varying concentrations of nicotine and MIP aqueous concentrate levels. First hand exposure to nicotine has been demonstrated to significantly increase S. mutans biofilm formation, while the active component, CPP-ACP, in MIP has been shown to reduce S. mutans biofilm formation. Materials and methods A 24-h culture of S. mutans UA159 in microtiter plates were treated with varying nicotine concentrations (0–32 mg/ml) in Tryptic Soy Broth supplemented with 1% sucrose (TSBS) with or without MIP aqueous concentrate. A spectrophotometer was used to determine total growth absorbance and planktonic growth. The microtiter plate wells were washed, fixed, and stained with crystal violet dye and the absorbance measured to determine biofilm formation. Results The presence of MIP aqueous concentrate inhibits nicotine-induced S. mutans biofilm formation at different concentrations of nicotine (0–32 mg/ml). Conclusion The results demonstrated nicotine-induced S. mutans biofilm formation is decreased in the presence of MIP. This provides further evidence about the cariostatic properties of CPP-ACP, the active soluble ingredient in the MIP, and reconfirms the harmful effects of nicotine. Clinical significance Smokers may gain dual benefits from the use of MIP, as a remineralization agent and as a cariostatic agent, by inhibiting nicotine-induced S. mutans biofilm formation.Item THE EFFECTS OF TOBACCO TREATED PORPHYROMONAS GINGIVALIS ON HUMAN EPITHELIAL CELLS(Office of the Vice Chancellor for Research, 2012-04-13) Tursunova, Roziya H.; Al-Shibani, Nouf; Windsor, L. Jack; Gregory, Richard L.Bacteria and tobacco are risk factors for periodontal diseases. Bacteria-host interactions play a critical role in disease development and progression. The effects of tobacco-treated bacteria such as Porphyromonas gingivalis on epithelial cells have not yet been examined. Therefore, P. gingivalis were treated with different tobacco products (nicotine, cigarette smoke conden-sate (CSC), and dissolvable smokeless tobacco (DST) strips) to determine the effects that they have on epithelial cells. P. gingivalis were grown with or without the products for 24 hours at 37◦C. The cells were separated from the supernatant, washed with 0.9% NaCl and incubated at 60◦C to kill the bacte-ria. Protein assays was performed to determine the protein concentration in the cell pellets and supernatants. Lactate dehydrogenase (LDH) assays are being used to measure the cytotoxicity of the cells and supernatants on epi-thelial cells in a dose dependent manner. Non-toxic amounts of the cell pel-lets and supernatants will be used to treat epithelial cells for 72 hours and the media analyzed by cytokine/growth factor protein arrays. The protein assays showed that CSC and nicotine treated P. gingivalis cells had less pro-tein than the others. The total protein in the supernatant for the CSC treated bacteria was less compared to others. The protein data suggests that CSC and nicotine affect protein expression in and by the P. gingivalis cells. To-bacco-treated bacteria are hypothesized to increase the expression of pro-inflammatory cytokines/growth factors by the epithelial cells, thereby con-tributing to the inflammation seen in periodontal diseases. This research was funded by Indiana University-Purdue University Indianapolis, Multidisci-plinary Undergraduate Research Institute (MURI).Item Exome chip meta-analysis fine maps causal variants and elucidates the genetic architecture of rare coding variants in smoking and alcohol use(Elsevier, 2018) Brazel, David M.; Jiang, Yu; Hughey, Jordan M.; Turcot, Valérie; Zhan, Xiaowei; Gong, Jian; Batini, Chiara; Weissenkampen, J. Dylan; Liu, MengZhen; Barnes, Daniel R.; Bertelsen, Sarah; Chou, Yi-Ling; Erzurumluoglu, A. Mesut; Faul, Jessica D.; Haessler, Jeff; Hammerschlag, Anke R.; Hsu, Chris; Kapoor, Manav; Lai, Dongbing; Le, Nhung; de Leeuw, Christiaan A.; Loukola, Anu; Mangino, Massimo; Melbourne, Carl A.; Pistis, Giorgio; Qaiser, Beenish; Rohde, Rebecca; Shao, Yaming; Stringham, Heather; Wetherill, Leah; Zhao, Wei; Agrawal, Arpana; Bierut, Laura; Chen, Chu; Eaton, Charles B.; Goate, Alison; Haiman, Christopher; Heath, Andrew; Iacono, William G.; Martin, Nicholas G.; Polderman, Tinca J.; Reiner, Alex; Rice, John; Schlessinger, David; Scholte, H. Steven; Smith, Jennifer A.; Tardif, Jean-Claude; Tindle, Hilary A.; van der Leij, Andries R.; Boehnke, Michael; Chang-Claude, Jenny; Cucca, Francesco; David, Sean P.; Foroud, Tatiana; Howson, Joanna M. M.; Kardia, Sharon L. R.; Kooperberg, Charles; Laakso, Markku; Lettre, Guillaume; Madden, Pamela; McGue, Matt; North, Kari; Posthuma, Danielle; Spector, Timothy; Stram, Daniel; Tobin, Martin D.; Weir, David R.; Kaprio, Jaakko; Abecasis, Gonçalo R.; Liu, Dajiang J.; Vrieze, Scott; Medical and Molecular Genetics, School of MedicineBackground Smoking and alcohol use have been associated with common genetic variants in multiple loci. Rare variants within these loci hold promise in the identification of biological mechanisms in substance use. Exome arrays and genotype imputation can now efficiently genotype rare nonsynonymous and loss of function variants. Such variants are expected to have deleterious functional consequences, and contribute to disease risk. Methods We analyzed ∼250,000 rare variants from 16 independent studies genotyped with exome arrays and augmented this dataset with imputed data from the UK Biobank. Associations were tested for five phenotypes: cigarettes per day, pack years, smoking initiation, age of smoking initiation, and alcoholic drinks per week. We conducted stratified heritability analyses, single-variant tests, and gene-based burden tests of nonsynonymous/loss of function coding variants. We performed a novel fine mapping analysis to winnow the number of putative causal variants within associated loci. Results Meta-analytic sample sizes ranged from 152,348-433,216, depending on the phenotype. Rare coding variation explained 1.1-2.2% of phenotypic variance, reflecting 11%-18% of the total SNP heritability of these phenotypes. We identified 171 genome-wide associated loci across all phenotypes. Fine mapping identified putative causal variants with double base-pair resolution at 24 of these loci, and between 3 and 10 variants for 65 loci. 20 loci contained rare coding variants in the 95% credible intervals. Conclusions Rare coding variation significantly contributes to the heritability of smoking and alcohol use. Fine mapping GWAS loci identifies specific variants contributing to the biological etiology of substance use behavior.Item INTERACTIONS OF HUMAN GINGIVAL FIBROBLASTS WITH TOBACCO TREATED PORPHYROMONAS GINGIVALIS(Office of the Vice Chancellor for Research, 2012-04-13) Lanier, Branden; Al-Shibani, Nouf Khider; Windsor, L. Jack; Gregory, RichardPorphyromonas gingivalis (P. gingivalis) and tobacco are risk factors for periodontal disease. The objective of this study was to determine the effects that tobacco treated P. gingivalis cells have on human gingival fibroblasts (HGFs). The study was conducted to examine the effects that cigarette smoke condensate (CSC), nicotine, and dissolvable smokeless tobacco (DST) strips treated P. gingivalis has on cell cytotoxicity and the expression of cy-tokines and growth factors from HGFs. The P. gingivalis was grown at 37°C and then the cells and supernatant were separated. P. gingivalis cells were then washed and killed. The concentration of protein in the cell pellet and supernatant were determined by protein assay using the Bradford method. The lowest non-toxic levels of the cell pellet and supernatant will be used to treat the HGFs for 72 hours and then cytotoxicity was determined by lactate dehydrogenase (LDH) assays. The cytokine/growth factor expression will be determined by antibody protein arrays. The protein assays showed that the tobacco products reduced the protein amounts as compared to untreated bacteria. The results should show an increase in cytotoxicity with increasing protein concentrations, along with increased pro-inflammatory cyto-kine/growth factors expression by the HGFs treated with tobacco treated P. gingivalis compared to P. gingivalis that was not treated with tobacco prod-ucts. A better understanding of the detrimental effects that tobacco has on the underlining causes of periodontal disease can advance the quest of con-trolling the disease. This study was funded by the Indiana University–Purdue University Indianapolis Multidisci-plinary Undergraduate Research Institute (MURI).Item INTERACTIONS OF HUMAN ORAL CELLS WITH ORAL BACTERIAL(Office of the Vice Chancellor for Research, 2012-04-13) HEEKE, K.S.; GREGORY, R.L.; SONG, F.; WINDSOR, L.J.Introduction: Streptococcus mutans is the main etiological cause of den-tal caries, and it has been shown that individuals who smoke have increased dental caries. S. mutans is known to bind to or interact with MG63 osteo-blasts. However, very little is known about the effects of tobacco directly on these bacteria on their ability to affect human pulp MG63 osteoblasts. We are hypothesizing that tobacco upregulates the expression of pro-inflammatory cytokines and MMPs to increase the pathogenic potential of S. mutans. The objective of this research project is to investigate the effects that nicotine, cigarette smoke condensate (CSC), and dissolvable smokeless tobacco (DST)-extract treated bacterial cells have on humanMG63 osteo-blasts, in respect to their release of pro-inflammatory and anti-inflammatory cytokines, as well as MMP expression. In addition, the effects of the S. mutans cells will be examined for the ability to affect MG63 osteoblast growth. The long-term goal is to develop treatment modalities to reduce the effects of smoking on dental caries. Materials and Methods: S. mutans UA159 was incubated in Tryptic Soy Broth (TSB), with the following concentrations: 2 mg/mL nicotine, 0.125 mg/mL CSC, 100 uL/3 mL DST-extract, and a 0 mg/mL control group. The cultures were grown in the presence of the tobacco products for 8 h at 37oC in 5% CO2, and centrifuged to isolate cells and supernatants. The cells were washed and heat-killed for 1 h at 60oC. Human MG63 osteoblasts were iso-lated from extracted teeth, and cell passages 3-8 will be used. The tobacco-treated S. mutans cells and supernatants will be incubated with the osteo-blasts in culture plates for 72 h and cytokine expression evaluated by re-verse transcriptase polymerase chain reaction. Results: The protein concentration of each tobacco-treated sample was found. The undiluted concentrations of the nicotine- and CSC-treated cells were slightly lower and the DST-treated cells was slightly higher than the control cells. The undiluted nicotine (p<0.05) and DST-treated supernatants were higher than the control, while the CSC supernatant protein concentra-tion was lower. From our previous studies, it was found that nicotine in-creases bacteriocin production of S. mutans, so we might hypothesize that nicotine induces bacteriocin secretion, thus increasing dental caries.Item INTERACTIONS OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS WITH TOBACCO TREATED STREPTOCOCCUS MUTANS(Office of the Vice Chancellor for Research, 2013-04-05) Lanier, Branden; Windsor, L. Jack; Gregory, Richard L.Streptococcus mutans and tobacco are risk factors for atherosclerosis. The objective of this study was to determine the ability that a spaP isogenic defective mutant of S. mutans UA 159 has on binding to Human Umbilical Vein Endothelial Cells (HUVEC) when treated with tobacco products and what second messenger signals are involved. The study was conducted to examine the effects that various concentrations of cigarette smoke condensate (CSC)- and nicotine have on S. mutans cell cytotoxicity and expression of cytokines and growth factors from HUVECs. S. mutans was grown at 37°C and planktonic and biofilm cells were separated from the culture supernatant. The supernatant was discarded the cells were washed, sterilized with formaldehyde and washed again to remove the formaldehyde. The concentrations of the various S. mutans cells were standardized to the same concentration (absorbance of 0.50 ± 0.01) by spectroscopy at a wavelength of 600 nm. The lowest non-toxic levels of the sterilized bacterial cells were used to treat HUVECs for 72 hours and cytotoxicity was determined by lactate dehydrogenase (LDH) assays. The cytokine/growth factor expression will be determined by antibody protein arrays. The results are expected to indicate an increase in cytotoxicity with increasing cell concentrations, along with increased pro-inflammatory cytokine/growth factors expression by the HUVECs treated with tobacco treated S. mutans compared to S. mutans that was not treated with tobacco products. Second messenger signaling pathways will be analyzed with ERK and JNK inhibitors and specific antibodies to ERK and phospho-JNK. Immunoblots using HUVECs will be done to determine expression of ERK/JNK. A better understanding of the detrimental effects that tobacco has on the underlining causes of atherosclerosis can advance the quest of controlling the disease.Item Mechanisms of Attachment of Tobacco-Treated Streptococcus mutans to Human Endothelial Cells(Office of the Vice Chancellor for Research, 2013-04-05) Miller, Alyssa R.Smoking has been proven to cause increased dental caries, which is an infectious disease caused by Streptococcus mutans, a gram-positive bacteria commonly found in the oral cavity. S. mutans is also known for its contribution to atherosclerosis, specifically the accumulation of plaque in the coronary arteries. This is facilitated by the interaction and binding of S. mutans to local endothelial cells (HUVEC). This study was conducted to explore the direct effects that tobacco has on the ability of S. mutans to affect endothelial cells that might lead to atherosclerosis. S. mutans were treated with different concentrations of nicotine and cigarette smoke condensate (CSC) to test if they affect the binding capabilities of S. mutans to endothelial cells. Blocking reagents, enolase antibody and purified DnaK, were also used to treat the HUVEC to observe the effects these reagents have on the ability of S. mutans to bind to the cells. Binding was measured by preforming a binding assay that incorporated these reagents and reading the absorbance using a spectrophotometer at 450 nm. To do this, sonicated HUVEC were added to a 96-well microtiter plate with 1% bovine serum albumin (BSA), followed by treated S. mutans, extra-avidin labeled horseradish peroxidase, and O-phenylenediamine (OPD). The experiment is still in progress; therefore, no results have been obtained thus far. However, it is expected that the nicotine/CSC treatment of S. mutans will increase binding to the endothelial cells thereby providing a possible mechanism of S. mutans contributing to atherosclerosis. The knowledge obtained from this experiment will be significant in developing treatment modalities to decrease the effects of smoking on cardiovascular disease.Item Nicotine Kill Time of Streptococcus Mutans(Office of the Vice Chancellor for Research, 2015-04-17) Cavazos, Ana; Gregory, Richard L.Cigarettes have thousands of components aside from tobacco and nicotine that are harmful to the smoker’s body. Smoking is considered a significant risk factor for cardiovascular disease (CVD) and periodontal disease. Yet smoking also plays a significant role in the buildup of plaque in the mouths of smokers. This is in part due to the formation of biofilm by Streptococcus mutans. S. mutans is an oral bacterium found in most humans that is considered to be the causative agent for dental caries. Particularly, S. mutans UA159 was used in this experiment. Biofilm formation regarding S. mutans and nicotine concentrations has previously been studied. It was found that at high concentrations of nicotine, biofilm formation of S. mutans decreased significantly. One of the aims of this study is to determine the time required to kill S. mutans.Item Nicotine Regulates Streptococcus mutans Extracellular Polysaccharide and Related Protein Expression(Office of the Vice Chancellor for Research, 2013-04-05) Huang, Ruijie; Li, Mingyun; Gregory, Richard L.Streptococcus mutans, a gram-positive facultatively anaerobic bacterium, is considered as the primary contributor to caries due to its high acidogenicity and aciduricity. Smoking is one of the risk factors of periodontal disease and dental caries. Nicotine is one of the alkaloid pharmacologically active agents in tobacco. Previous studies indicated nicotine stimulated S. mutans biofilm formation and metabolism. However, the detailed mechanism is still unknown. Thus, the aim of this study is to investigate how nicotine facilitates S. mutans biofilm formation focused on extracellular polysaccharide synthesis. S. mutans UA159 (ATCC 700610) was used in the present study. Confocal laser scanning microscopy (CLSM) was used to investigate the effect of 0, 1, 2 and 4 mg/ml nicotine on 24 h S. mutans biofilm extracellular polysaccharide (EPS) expression (red fluorescentlabeled) and nucleic acid expression (green fluorescent-labeled). Western blot assays were used to investigate the effect of 0, 1, 2 and 4 mg/ml nicotine on the expression of glucosyltransferase (Gtfs), glucan-binding protein A (Gbp-A) and Gbp-B in 24 h S. mutans biofilm cells. CLSM results indicated nicotine increased both EPS and nucleic acid, and the ratio of EPS/nucleic acid was also increased. It implied EPS synthesis in single S. mutans cells was stimulated by nicotine treatment. Biofilm thickness was thicker in nicotine-treated groups than the non-treated group. Western blot assay results indicated that nicotine stimulated GtfC, Gbp-A and Gbp-B expression, but decreased GtfB expression. In conclusion, nicotine stimulates S. mutans cell proliferation and EPS synthesis partially by increasing GtfC, Gbp-A and Gbp-B.