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.
Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO.
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.
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).
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Understanding sources of microbial contamination in outpatient rehabilitation (REHAB) clinics is important to patients and healthcare providers.
The purpose of this study was to characterize the ...microbiome of an outpatient REHAB clinic and examine relationships between clinic factors and contamination.
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).
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).
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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CMK, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Wild-type (WT) Salmonella typhimurium causes acute intestinal inflammation by activating the nuclear factor kappa B (NF-κB) pathway. Interestingly, WT Salmonella infection also causes degradation of ...β-catenin, a regulator of cellular proliferation. Regulation of β-catenin and the inhibitor of NF-κB, IκBα, is strikingly similar, involving phosphorylation at identical sites, ubiquitination by the same E3 ligase, and subsequent proteasomal degradation. However, how β-catenin directly regulates the NF-κB pathway during bacteria-induced inflammation in vivo is unknown. Using streptomycin-pretreated mice challenged with Salmonella, we demonstrated that WT Salmonella stimulated β-catenin degradation and decreased the physical association between NF-κB and β-catenin. Accordingly, WT Salmonella infection decreased the expression of c-myc, a β-catenin-regulated target gene, and increased the levels of IL-6 and TNF-α, the NF-κB-regulated target genes. Bacterial infection directly stimulated phosphorylation of β-catenin, both in vivo and in vitro. Closer examination revealed that glycogen synthase kinase 3β (GSK-3β) kinase activity was increased in response to WT Salmonella, whereas non-virulent Salmonella had no effect. siRNA of GSK-3β was able to stabilize IκBα in response to WT Salmonella. Pretreatment for 24 h with LiCl, an inhibitor of GSK-3β, reduced WT Salmonella induced IL-8 secretion. Additionally, cells expressing constitutively active β-catenin showed IκBα stabilization and inhibition of NF-κB activity not only after WT Salmonella infection but also after commensal bacteria (Escherichia coli F18) and TNF-α treatment. This study suggests a new role for β-catenin as a negative regulator of inflammation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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-2α (eIF-2α) 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)-γ + tumor necrosis factor (TNF)-α also demonstrated reduced colonic Hsp25/27 and Hsp70. In young adult mouse colonic epithelial cells, IFN-γ+TNF-α 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-γ+TNF-α cotreatment activated PKR, resulting in phosphorylation and inactivation of eIF-2α, 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-2α 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.
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