Effects of folate deficiency and sex on carcinogenesis in a mouse model of oral cancer.

OBJECTIVES: To investigate the effects of dietary folate and sex on histopathology of oral squamous cell carcinoma in mice. MATERIALS AND METHODS: Mice (C57Bl/6, 30/sex) were fed either a deficient folate or sufficient folate diet. Vehicle or 4-nitroquinoline1-oxide (50 µg/mL) in vehicle were administered in drinking water for 20 weeks, followed by 6 weeks of regular drinking water. Oral lesions were observed weekly. Tongues were studied for histopathologic changes. Immunohistochemical techniques were used to measure cell proliferation (Ki67+), and to quantify expression of folate receptor, reduced folate carrier, and proton-coupled folate transporter. T cells, macrophages, and neutrophils were counted and normalized to area. RESULTS: All 4NQO-treated mice developed oral tumors. Dietary folate level did not affect tumor burden. More tumors were observed on the ventral aspect of the tongue than in other locations within the oral cavity. 4-nitroquinoline-1-oxide-treated mice displayed 27%-46% significantly lower expression of all three folate transport proteins; diet and sex had no effect on folate transporter expression. T-cell and neutrophil infiltration in tongues were 9.1-fold and 18.1-fold increased in the 4-nitroquinoline-1-oxide-treated mouse tongues than in controls. CONCLUSION: Treatment with 4NQO was the primary factor in determining cancer development, decreased folate transport expression, and lymphoid cell infiltration.

The microbiome of an outpatient rehabilitation clinic and predictors of contamination: A pilot study.

BACKGROUND: Understanding sources of microbial contamination in outpatient rehabilitation (REHAB) clinics is important to patients and healthcare providers. PURPOSE: The purpose of this study was to characterize the microbiome of an outpatient REHAB clinic and examine relationships between clinic factors and contamination. METHODS: Forty commonly contacted surfaces in an outpatient REHAB clinic were observed for frequency of contact and swiped using environmental sample collection kits. Surfaces were categorized based on frequency of contact and cleaning and surface type. Total bacterial and fungal load was assessed using primer sets specific for the 16S rRNA and ITS genes, respectively. Bacterial samples were sequenced using the Illumina system and analyzed using Illumina-utils, Minimum Entropy Decomposition, QIIME2 (for alpha and beta diversity), LEfSe and ANCOM-BC for taxonomic differential abundance and ADONIS to test for differences in beta diversity (p<0.05). RESULTS: Porous surfaces had more bacterial DNA compared to non-porous surfaces (median non-porous = 0.0016ng/µL, 95%CI = 0.0077-0.00024ng/µL, N = 15; porous = 0.0084 ng/µL, 95%CI = 0.0046-0.019 ng/µL, N = 18. p = 0.0066,DNA. Samples clustered by type of surface with non-porous surfaces further differentiated by those contacted by hand versus foot. ADONIS two-way ANOVA showed that the interaction of porosity and contact frequency (but neither alone) had a significant effect on 16S communities (F = 1.7234, R2 = 0.0609, p = 0.032). DISCUSSION: Porosity of surfaces and the way they are contacted may play an underestimated, but important role in microbial contamination. Additional research involving a broader range of clinics is required to confirm results. Results suggest that surface and contact-specific cleaning and hygiene measures may be needed for optimal sanitization in outpatient REHAB clinics.

Fat and exposure to 4-nitroquinoline-1-oxide causes histologic and inflammatory changes in murine livers.

Risk factors for liver cancer include tobacco use, alcohol consumption, obesity, and male sex. Administration of 4-nitroquinonline-1-oxide (4NQO) in drinking water mimics the effects of tobacco and leads to oral carcinoma in mice. This study compared the effects of diets high and low in saturated fat (HF and LF, respectively), and sex, on liver histopathology in 4NQO-treated mice and controls. We hypothesized that 4NQO would cause histopathological changes in liver, and that a HF diet would increase hepatic pathology when compared to the LF diet. Mice (C57Bl/6, 36/sex), were divided into a low fat (10 kcal% fat; LF) or high fat (60 kcal% fat, HF) diet. Mice were further subdivided into one of 3 water treatment groups for 17 weeks: water (control), vehicle (1.25% propylene glycol in water [PG]), or 4NQO in (50 µg/ml; 4NQO). All mice were subsequently given water alone for 6 more weeks. Upon euthanasia, livers were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E). H&E slides were graded for histopathology; frozen liver samples were analyzed for triglyceride content. Trichrome stained sections were graded for fibrosis. CD3+ T cells, CD68+ macrophages, and Ly6+ neutrophils were detected by immunohistochemistry. Compared to water controls, 4NQO-treatment caused mouse liver histopathological changes such as fibrosis, and increases in hepatic neutrophils, T cells, and macrophages. HF diet exacerbated pathological changes compared to LF diet. Male controls, but not females, demonstrated severe steatosis and increased triglyceride content. 4NQO treatment decreased hepatic fat accumulation, even in animals on a HF diet. In conclusion, this murine model of oral cancer may serve as a model to study the effects of tobacco and diet on liver.

The role of Nodal and Cripto-1 in human oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy of the oral cavity. Nodal and Cripto-1 (CR-1) are important developmental morphogens expressed in several adult cancers and are associated with disease progression. Whether Nodal and CR-1 are simultaneously expressed in the same tumor and how this affects cancer biology are unclear. We investigate the expression and potential role of both Nodal and CR-1 in human OSCC. Immunohistochemistry results show that Nodal and CR-1 are both expressed in the same human OSCC sample and that intensity of Nodal staining is correlated with advanced-stage disease. However, this was not observed with CR-1 staining. Western blot analysis of lysates from two human OSCC line experiments shows expression of CR-1 and Nodal, and their respective signaling molecules, Src and ERK1/2. Treatment of SCC25 and SCC15 cells with both Nodal and CR-1 inhibitors simultaneously resulted in reduced cell viability and reduced levels of P-Src and P-ERK1/2. Further investigation showed that the combination treatment with both Nodal and CR-1 inhibitors was capable of reducing invasiveness of SCC25 cells. Our results show a possible role for Nodal/CR-1 function during progression of human OSCC and that targeting both proteins simultaneously may have therapeutic potential.

Dietary fat and male sex increase histopathological changes in a mouse model of oral cancer.

OBJECTIVE: To compare the effects of dietary fat and sex on murine oral squamous cell carcinoma pathology. MATERIALS AND METHODS: Male and female C57Bl/6 mice (36/sex) received a low-fat (10 kcal%) or high-fat (60 kcal%) diet. Water (control), vehicle, or 4-nitroquinoline-1-oxide in vehicle (50 µg/ml) was provided for 17 weeks followed by six additional weeks of water. Oral lesion development was recorded weekly. Histopathologic changes in tongues were examined, and T cells (CD3+), macrophages (CD68+), and neutrophils (Ly6+) were quantified. RESULTS: All 4-nitroquinoline-1-oxide-treated mice developed oral tumors. High-fat diet exacerbated pathology, demonstrated by an increased final tumor burden (10.9 ± 4.5 vs. 7.9 ± 2.5, mm/mouse, p < .05; high-fat diet vs. low-fat diet, respectively), and a greater histopathology score. When dietary groups were combined, 4-nitroquinoline-1-oxide-treated males displayed higher histopathology scores than females (4.2 ± 0.3 vs. 3.6 ± 0.2, respectively, p < .05). Lymphoid cell infiltration was greater in the 4-nitroquinoline-1-oxide mouse tongues than controls: T cells (14.0 vs. 0.96 cells/mm2 ), macrophages (3.6 vs. 1.8 cells/mm2 ), and neutrophils (12.0 vs. 0.38 cells/mm2 ). CONCLUSION: High-fat diet and male sex increased the pathology of 4-nitroquinoline-1-oxide-induced oral cancer. Elevated lymphoid cell infiltration contributed to disease pathology.

Self-Perceived Knowledge, Skills, Attitudes, and Use of Evidence-Based Dentistry Among Practitioners Transitioning to Dental Educators.

Dental practitioners transitioning to dental educators (PTEs) have an integral role in dental education. While PTEs intrinsically apply some form of evidence-based dentistry (EBD) in patient care, it may not be a standardized, systematic approach. The aims of this study were to determine the self-perceived knowledge, skills, attitudes, and behaviors of PTEs regarding EBD at one U.S. dental school and to identify areas where formal calibration may be warranted to facilitate their competence and confidence as dental educators. Participants voluntarily completed a 32-question survey regarding their EBD training and self-perceived EBD skills in several areas: use of the clinical evidence pyramid; systematic, objective, and critical appraisal of the evidence; application of the evidence to patient care; and integrating clinical expertise, scientific evidence, and patient's preferences to formulate a treatment plan. The PTEs were invited to participate in the anonymous survey during regularly scheduled calibration sessions held between May and July 2014. After study information was distributed, 100% of the attendees (n=43) completed the survey. The percentage of total PTEs at the school could not be calculated. Of the responding PTEs, 69% rated themselves better than satisfactory (70% proficiency) in their knowledge, skills, and attitudes regarding EBD skills application. However, only 33-42% of the respondents indicated that they frequently used the evidence pyramid and systematically, objectively, and critically appraised the evidence, even though 65% indicated they applied the evidence to improve patient care over 70% of the time. In addition, the participating PTEs identified a need for more frequent use of formal EBD skills. Providing case-based EBD projects involving PTEs as mentors may provide more opportunities for the judicious and effective use of these important skills and may improve PTEs' self-perceived confidence.

Exercise prevents weight gain and alters the gut microbiota in a mouse model of high fat diet-induced obesity.

BACKGROUND: Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown. OBJECTIVE: Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO. METHODS: Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation. RESULTS: HF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p<0.05). Visualization of Unifrac distance data with principal coordinates analysis indicated clustering by both diet and Ex at week 12. Sequencing demonstrated Ex-induced changes in the percentage of major bacterial phyla at 12 weeks. A correlation between total Ex distance and the ΔCt Bacteroidetes: ΔCt Firmicutes ratio from qPCR demonstrated a significant inverse correlation (r2 = 0.35, p = 0.043). CONCLUSION: Ex induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO.

Erythropoietin protects intestinal epithelial barrier function and lowers the incidence of experimental neonatal necrotizing enterocolitis.

The impermeant nature of the intestinal barrier is maintained by tight junctions (TJs) formed between adjacent intestinal epithelial cells. Disruption of TJs and loss of barrier function are associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal diseases in preterm infants. Human milk is protective against NEC, and the human milk factor erythropoietin (Epo) has been shown to protect endothelial cell-cell and blood-brain barriers. We hypothesized that Epo may also protect intestinal epithelial barriers, thereby lowering the incidence of NEC. Our data demonstrate that Epo protects enterocyte barrier function by supporting expression of the TJ protein ZO-1. As immaturity is a key factor in NEC, Epo regulation of ZO-1 in the human fetal immature H4 intestinal epithelial cell line was examined and demonstrated Epo-stimulated ZO-1 expression in a dose-dependent manner through the PI3K/Akt pathway. In a rat NEC model, oral administration of Epo lowered the incidence of NEC from 45 to 23% with statistical significance. In addition, Epo treatment protected intestinal barrier function and prevented loss of ZO-1 at the TJs in vivo. These effects were associated with elevated Akt phosphorylation in the intestine. This study reveals a novel role of Epo in the regulation of intestinal epithelial TJs and barrier function and suggests the possible use of enteral Epo as a therapeutic agent for gut diseases.

Inducible heat shock protein 70 prevents multifocal flat dysplastic lesions and invasive tumors in an inflammatory model of colon cancer.

BACKGROUND: Heat shock protein 70 (Hsp70) regulates protein biosynthesis and refolding of denatured proteins. Since Hsp70 participates in recovery from stress injury, we examined the effect of Hsp70 genetic deletion in the azoxymethane (AOM)/dextran sulfate sodium (DSS) model of inflammation and colon cancer. METHODS: Hsp70 mutant mice (Hsp70.1(-/-)/70.3(-/-)) and wild-type (WT) littermates received AOM and three cycles of DSS and were killed 24 weeks later. Tumors were graded for histology and immunostained for p53, adenomatous polyposis coli, beta-catenin, cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS) and sequenced for p53 mutations. RESULTS: Elevated adenomas developed in 4/10 WT mice with no dysplasia in adjacent mucosa. In contrast, 7/8 Hsp70 knock out (KO) mice developed chronic mucosal inflammation and multifocal areas of flat dysplasia and 4/8 progressed to invasive carcinomas arising in a background of flat dysplastic mucosa. These differences in the incidence of flat dysplasia and invasive cancers were significant (P < 0.05). Nuclear p53 was stronger in Hsp70 KO tumors compared with WT tumors, and sequencing confirmed p53 mutations in 2/5 tumors from Hsp70(-/-) versus 0/5 in WT mice. In Hsp70 WT tumors, beta-catenin was predominantly nuclear, compared with membranous beta-catenin in Hsp70(-/-) tumors, suggesting that Hsp70 regulates beta-catenin in colonic tumorigenesis. Cox-2 and iNOS levels were increased in tumors from Hsp70(-/-) mice compared with Hsp70 WT tumors. CONCLUSIONS: Hsp70-deleted mice treated with AOM/DSS develop flat invasive colonic tumors that mimic many histological and molecular features of ulcerative colitis colon cancer. This model will be useful to dissect the role of Hsp70 in inflammatory bowel disease colon cancer.

Translational inhibition of colonic epithelial heat shock proteins by IFN-gamma and TNF-alpha in intestinal inflammation.

BACKGROUND & AIMS: Inducible heat shock proteins (iHsp), Hsp25/27 and Hsp70, play essential roles in protecting cells against stress and, in intestinal mucosal inflammation, potentially lessening the extent and severity of injury. We examined the expression and regulation of iHsp in human and experimental inflammatory bowel diseases (IBD) and in vitro. METHODS: iHsp expression and regulation were assessed in normal and IBD colonic biopsy specimens, IL-10(-/-) mice, and young adult mouse colonic epithelial cells by immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR). Phosphorylation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2alpha (eIF-2alpha) was determined by Western blot. RESULTS: Hsp25/27 and Hsp70 levels were selectively reduced in areas of active mucosal inflammation associated with human IBD and IL-10(-/-) mice with colitis. Wild-type mice treated in vivo with interferon (IFN)-gamma + tumor necrosis factor (TNF)-alpha also demonstrated reduced colonic Hsp25/27 and Hsp70. In young adult mouse colonic epithelial cells, IFN-gamma+TNF-alpha inhibited heat induction of Hsp25/27 and Hsp70, an effect not associated with changes in iHsp messenger RNA or protein half-lives but caused by suppressed de novo iHsp synthesis. IFN-gamma+TNF-alpha cotreatment activated PKR, resulting in phosphorylation and inactivation of eIF-2alpha, an essential factor in protein translation. These effects were not due to induced apoptosis and could be negated by PKR-inhibitor and short interfering RNA to PKR. Increased phosphorylation of PKR and eIF-2alpha were also observed in active IBD tissues. CONCLUSIONS: Mucosal inflammation is associated with iHsp down-regulation, an effect that appears mediated by translational down-regulation by proinflammatory cytokines. In the context of IBD, we propose that this mechanism contributes to the severity, extent, and persistence of inflammation-induced mucosal injury.

beta-Catenin activity negatively regulates bacteria-induced inflammation.

Wild-type (WT) Salmonella typhimurium causes acute intestinal inflammation by activating the nuclear factor kappa B (NF-kappaB) pathway. Interestingly, WT Salmonella infection also causes degradation of beta-catenin, a regulator of cellular proliferation. Regulation of beta-catenin and the inhibitor of NF-kappaB, IkappaBalpha, is strikingly similar, involving phosphorylation at identical sites, ubiquitination by the same E3 ligase, and subsequent proteasomal degradation. However, how beta-catenin directly regulates the NF-kappaB pathway during bacteria-induced inflammation in vivo is unknown. Using streptomycin-pretreated mice challenged with Salmonella, we demonstrated that WT Salmonella stimulated beta-catenin degradation and decreased the physical association between NF-kappaB and beta-catenin. Accordingly, WT Salmonella infection decreased the expression of c-myc, a beta-catenin-regulated target gene, and increased the levels of IL-6 and TNF-alpha, the NF-kappaB-regulated target genes. Bacterial infection directly stimulated phosphorylation of beta-catenin, both in vivo and in vitro. Closer examination revealed that glycogen synthase kinase 3beta (GSK-3beta) kinase activity was increased in response to WT Salmonella, whereas non-virulent Salmonella had no effect. siRNA of GSK-3beta was able to stabilize IkappaBalpha in response to WT Salmonella. Pretreatment for 24 h with LiCl, an inhibitor of GSK-3beta, reduced WT Salmonella induced IL-8 secretion. Additionally, cells expressing constitutively active beta-catenin showed IkappaBalpha stabilization and inhibition of NF-kappaB activity not only after WT Salmonella infection but also after commensal bacteria (Escherichia coli F18) and TNF-alpha treatment. This study suggests a new role for beta-catenin as a negative regulator of inflammation.

Recognition of intestinal epithelial HIF-1alpha activation by Pseudomonas aeruginosa.

Human intestinal epithelial cell monolayers (Caco-2) subjected to hypoxia and reoxygenation release soluble factors into the apical medium that activate the virulence of the opportunistic pathogen Pseudomonas aeruginosa to express the potent barrier-dysregulating protein PA-I lectin/adhesin. In this study, we defined the role of hypoxia-inducible factor (HIF)-1alpha in this response. We tested the ability of medium from Caco-2 cells with forced expression of HIF-1alpha to increase PA-I expression in P. aeruginosa and found that medium from Caco-2 cells overexpressing HIF-1alpha increased PA-I expression compared with medium from control cells (P < 0.001, ANOVA). To identify the components responsible for this response, medium was fractionated by molecular weight and subjected to mass spectroscopy, which identified adenosine as the possible mediator. Both adenosine and its immediate downstream metabolite inosine induced PA-I expression in P. aeruginosa in a dose-dependent fashion. Because inosine was not detectable in the medium of Caco-2 cells exposed to hypoxia or overexpressing HIF-1alpha, we hypothesized that P. aeruginosa itself might metabolize adenosine to inosine. Using mutant and parental strains of P. aeruginosa, we demonstrated that P. aeruginosa metabolized adenosine to inosine via adenosine deaminase and that the conditioned medium enhanced the extracellular accumulation of inosine. Together, these results provide evidence that P. aeruginosa can recognize and respond to extracellular end products of intestinal hypoxia that are released after activation of HIF-1alpha. The ability of P. aeruginosa to metabolize adenosine to inosine may represent a subversive microbial virulence strategy that deprives the epithelium of the cytoprotective actions of adenosine.

Role and regulation of intestinal epithelial heat shock proteins in health and disease.

Mucosal injury and inflammation are cardinal manifestations of inflammatory bowel diseases (IBD), arising when the effects of cytotoxic factors and conditions overwhelm the cell's capacity for defense (i.e. cytoprotection) or repair. To date, most research in this area has focused primarily on agents and processes involved in producing tissue injury, with less consideration given to inherent mechanisms of cytoprotection and cellular repair. Therapeutic approaches to IBD reflect this bias, being largely directed towards down-regulating the inflammatory process by inhibiting the production of immune and inflammatory mediators. This review will focus on the cell's inherent ability to defend itself against cellular stress and injury through the production of evolutionarily conserved stress proteins called heat shock proteins (HSP). The physiological role of these proteins in maintaining intestinal epithelial cell structure and function will be reviewed, with emphasis on studies that examine the role of HSP in IBD. A clearer understanding of the innate cytoprotective mechanisms inherent in intestinal epithelial cells will foster the development of new insights into basic epithelial cell biology, which ultimately can be used to establish target-specific therapies directed at reducing or alleviating mucosal injury, thereby promoting tissue healing and repair.