The Oral Mouse Microbiome Promotes Tumorigenesis in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck worldwide. Dysbiosis of the microbiome has increasingly been linked to the development of different kinds of cancer. Applying 16S rRNA gene sequence analysis and metatranscriptomic analyses, we characterized the longitudinal changes in the profiles and the function of the oral microbiome in a 4-nitroquinoline-1-oxide (4-NQO)-induced model of OSCC in gnotobiotic mice. We characterized the dynamics of the oral microbiome in this model using two different microbiome inocula: one from healthy mice and the other from mice bearing a 4-NQO-induced tumor. Mice colonized with different oral microbiomes and exposed to 4-NQO had increased tumor numbers and sizes compared to controls exposed to 4-NQO but lacking a microbiome. We observed an overall increase in diversity in the tumorigenic samples compared to that in the nontumor group not exposed to 4-NQO. Despite the variability in community dynamics, specific patterns emerged during the progression of the disease. In the two groups that were inoculated with the OSCC-associated microbiome, we observed opposite profiles of abundance in Parabacteroides and Corynebacterium While the percentage of Parabacteroides bacteria decreased in the control group, it increased in the OSCC group, and the opposite was observed for Corynebacterium The metatranscriptomic analysis revealed overexpression of the same metabolic signatures associated with OSCC regardless of the community profile. These included nitrogen transport, response to stress, interspecies interactions, Wnt pathway modulation, and amino acid and lipid biosynthesis. Thus, these results seem to suggest that certain collective physiological activities are critical for microbiome-mediated OSCC progression.IMPORTANCE There is growing evidence that changes in the microbiome are associated with carcinogenesis. To date, no consistent oral microbiome composition associated with OSCC has been identified. Longitudinal and functional studies like the study presented here should yield a better understanding of the role that the oral microbiome plays in OSCC. Our findings, obtained using a germ-free mouse model, indicate that the presence of different oral microbiomes enhances tumorigenesis and increases the final number of tumors in mice. By studying community-wide expression profiles, we found that regardless of the phylogenetic composition of the microbiome, the same metabolic activities were consistently associated with OSCC. Therefore, due to the functional redundancy of the microbiome, the critical element in explaining the contribution of the microbiota in OSCC is the collective physiological activity of the community, thus accounting for the previous inability to identify a consensus community profile or etiologic agents for OSCC.

Endodontic Infection-induced Inflammation Resembling Osteomyelitis of the Jaws in Toll-like Receptor 2/Interleukin 10 Double-knockout Mice.

INTRODUCTION: In general, mice develop chronic and nonhealing periapical lesions after endodontic infection. Surprisingly, we recently found that toll-like receptor 2 (TLR2)/interleukin 10 (IL-10) double-knockout (dKO) mice exhibited acute but resolving osteomyelitislike inflammation. In this study, we examined the kinetics of endodontic infection-induced inflammation in TLR2/IL-10 dKO mice and explored a potential mechanism of periapical wound healing mediated by the hypoxia-inducible factor 1 alpha (HIF-1α) subunit and arginase 1. METHODS: TLR2/IL-10 dKO and wild-type C57BL/6J mice were subjected to endodontic infection in the mandibular first molars. Mice were sacrificed on days 0 (noninfected), 10, and 21 postinfection. The extent of bone destruction, inflammation, bone deposition, and gene expression were determined by micro-computed tomographic imaging, histology, bone polychrome labeling, and microarray analysis. In addition, the effect of blocking endogenous HIF-1α was tested in infected TLR2/IL-10 dKO mice using the specific inhibitor YC-1. RESULTS: Infected TLR2/IL-10 dKO mice exhibited extensive bone destruction and inflammation on day 10 followed by spontaneous periapical wound healing including bone formation and resolution of inflammation by day 21 postinfection. In contrast, WT mice developed increasing chronic periapical inflammation over the 21-day observation period. Gene expression analyses and immunohistochemistry revealed that HIF-1α and arginase 1 were up-regulated in spontaneous wound healing in TLR2/IL-10 dKO mice. Blocking of HIF-1α in TLR2/IL-10 dKO mice using YC-1 resulted in significant inhibition of regenerative bone formation. CONCLUSIONS: The TLR2/IL-10 dKO mouse is a novel model resembling osteomyelitis of the jaws in which HIF-1α and arginase 1 appear to be crucial factors in spontaneous wound healing and bone repair.

IL-17 receptor A signaling is protective in infection-stimulated periapical bone destruction.

IL-17 is a pleiotropic cytokine produced by Th17 T cells that induces a myriad of proinflammatory mediators. However, different models of inflammation report opposite functional roles of IL-17 signal in terms of its effects on bone destruction. In this study we determined the role of IL-17RA signal in bone resorption stimulated by dentoalveolar infections. Infrabony resorptive lesions were induced by surgical pulp exposure and microbial infection of mouse molar teeth. IL-17 was strongly induced in periapical tissues in wild-type (WT) mice by 7 d after the infection but was not expressed in uninfected mice. Dentoalveolar infections of IL-17RA knockout (KO) mice demonstrated significantly increased bone destruction and more abscess formation in the apical area compared with WT mice. Infected IL-17RA KO mice exhibited significantly increased neutrophils and macrophages compared with the WT littermates at day 21, suggesting a failure of transition from acute to chronic inflammation in the IL-17RA KO mice. The expression of IL-1 (both α and ß isoforms) and MIP2 were significantly upregulated in the IL-17RA KO compared with WT mice at day 21 postinfection. The development of periapical lesions in IL-17RA KO mice was significantly attenuated by neutralization of IL-1ß and MIP2. Taken together, these results demonstrate that IL-17RA signal seems to be protective against infection-induced periapical inflammation and bone destruction via suppression of neutrophil and mononuclear inflammation.

Severe Spinal Cord Injury Causes Immediate Multi-cellular Dysfunction at the Chondro-Osseous Junction.

Spinal cord injury is associated with rapid bone loss and arrested long bone growth due to mechanisms that are poorly understood. In this study, we sought to determine the effects of severe T10 contusion spinal cord injury on the sublesional bone microenvironment in adolescent rats. A severe lower thoracic (vertebral T10) spinal cord injury was generated by weight drop (10 g×50 mm). Severely injured and body weight-matched uninjured male Sprague-Dawley rats were studied. At 3 and 5 days post-injury, we performed histological analysis of the distal femoral metaphysis, TUNEL assay, immunohistochemistry, real-time PCR, and western blot analysis compared to uninjured controls. We observed severe hindlimb functional deficits typical of this model. We detected uncoupled remodeling with increased osteoclast activity in the absence of osteoblast activity. We detected osteoblast, osteocyte, and chondrocyte apoptosis with suppressed osteoblast and chondrocyte proliferation and growth plate arrest due to spinal cord injury. We also detected altered gene expression in both whole bone extracts and bone marrow monocytes following spinal cord injury. We conclude that spinal cord injury results in altered gene expression of key regulators of osteoblast and chondrocyte activity. This leads to premature cellular apoptosis, suppressed cellular proliferation, growth plate arrest, and uncoupled bone remodeling in sublesional bone with unopposed osteoclastic resorption.

Nanoemulsions of an anti-oxidant synergy formulation containing gamma tocopherol have enhanced bioavailability and anti-inflammatory properties.

The present study investigated whether MicroFluidizer Processor-based nanoemulsions of an antioxidant synergy formulation (ASF), containing delta, alpha and gamma tocopherol influenced inflammation and bioavailability in CD-1 mice. Croton oil was applied to all animals' right ear lobe to induce inflammation. Auricular thickness was measured after 2 and 6h after the various treatments. The animal plasma and ear lobes were collected and frozen for bioavailability and cytokine analyses. The ASF nanoemulsions of alpha, delta, or gamma tocopherol significantly reduced auricular thickness compared to control (57, -57, and -71%, respectively) and blank nanoemulsion (-50, -50, -67%, respectively). Relative to the suspensions of ASF, only the nanoemulsion of ASF containing gamma tocopherol significantly reduced auricular thickness (-60%), whereas the 40% reduction with nanoemulsions of delta tocopherol compared to suspension was not statistically significant. Auricular concentrations of cytokines TNF-alpha and IL-1 alpha were significantly reduced in mice treated only with ASF nanoemulsions of gamma tocopherol compared to control (-53, -46%, respectively) and blank nanoemulsion (-52, -46%, respectively). Auricular thickness was significantly associated with tissue TNF-alpha (r=0.539, p<0.001) and IL-1 alpha concentrations (r=0.404, p=0.01). Bioavailability for gamma and delta was dramatically enhanced (2.2- and 2.4-folds) with the nanoemulsion compared to suspensions. Only the plasma gamma tocopherol concentration was significantly associated with auricular thickness (r=-0.643, P=0.001). In conclusion, nanoemulsions of ASF containing gamma, alpha, and delta tocopherol, have enhanced anti-inflammatory properties and increased bioavailability, with gamma tocopherol, in particular compared to their suspensions.

Enhancement of anti-inflammatory property of aspirin in mice by a nano-emulsion preparation.

Aspirin, a non-steroidal anti-inflammatory drug, widely used for its anti-inflammatory properties is associated with several systemic side effects including gastro-intestinal discomfort. Inflammation can be mediated by pro-inflammatory cytokines and, along with various other host factors eventually give rise to edema at the inflamed site. Because of the adverse side effects oftentimes associated with systemic exposure to aspirin, the aim of the present study was to investigate whether the anti-inflammatory property of aspirin would enhance if delivered as nano-emulsion preparation. Nano-emulsion preparations of aspirin prepared with a Microfluidizer Processor were evaluated in the croton-oil-induced ear edema CD-1 mouse model using ear lobe thickness and the accumulation of specific in situ cytokines as biomarkers of inflammation. The results showed that particle size (90 nm) populations of nano-emulsion preparations of aspirin compared to an aspirin suspension (363 nm), significantly decreased (p<0. 05) ear lobe thickness approximately 2 fold greater than the aspirin suspension. In addition, the aspirin nano-emulsion further reduced the auricular levels of IL-1alpha (-37%) and TNFalpha (-69%) compared to the aspirin suspension preparation (p<0.05). The reductions in ear lobe thickness were also significantly associated with accumulated tissue levels of IL-1alpha (r=0.5, p<0.009) and TNFalpha (r=0.7, p<0.0004), respectively. In conclusion, these studies indicate that a nano-emulsion preparation of aspirin significantly improved the anti-inflammatory properties of an aspirin suspension in a CD-1 mouse model of induced inflammation.

Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice.

Fibroblast growth factor-23 (FGF-23), a recently identified molecule that is mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR), appears to be involved in the regulation of phosphate homeostasis. Although increased levels of circulating FGF-23 were detected in patients with different phosphate-wasting disorders such as oncogenic osteomalacia (OOM) and X-linked hypophosphatemia (XLH), it is not yet clear whether FGF-23 is directly responsible for the abnormal regulation of mineral ion homeostasis and consequently bone development. To address some of these unresolved questions, we generated a mouse model, in which the entire Fgf-23 gene was replaced with the lacZ gene. Fgf-23 null (Fgf-23-/-) mice showed signs of growth retardation by day 17, developed severe hyperphosphatemia with elevated serum 1,25(OH)2D3 levels, and died by 13 weeks of age. Hyperphosphatemia in Fgf-23-/- mice was accompanied by skeletal abnormalities, as demonstrated by histological, molecular, and various other morphometric analyses. Fgf-23-/-) mice had increased total-body bone mineral content (BMC) but decreased bone mineral density (BMD) of the limbs. Overall, Fgf-23-/- mice exhibited increased mineralization, but also accumulation of unmineralized osteoid leading to marked limb deformities. Moreover, Fgf-23-/- mice showed excessive mineralization in soft tissues, including heart and kidney. To further expand our understanding regarding the role of Fgf-23 in phosphate homeostasis and skeletal mineralization, we crossed Fgf-23-/- animals with Hyp mice, the murine equivalent of XLH. Interestingly, Hyp males lacking both Fgf-23 alleles were indistinguishable from Fgf-23/-/ mice, both in terms of serum phosphate levels and skeletal changes, suggesting that Fgf-23 is upstream of the phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and that the increased plasma Fgf-23 levels in Hyp mice (and in XLH patients) may be at least partially responsible for the phosphate imbalance in this disorder.

Supramolecular assemblies based on copolymers of PEG600 and functionalized aromatic diesters for drug delivery applications.

A chemoenzymatic approach has been developed to synthesize poly(ethylene glycol)-based amphiphilic copolymers under mild reaction conditions that self-assemble in aqueous media to form polymeric nanomicelles in the range of 20-50 nm. The supramolecular organization of polymeric nanomicelles was studied by 1H NMR longitudinal relaxation time (T1) and light scattering techniques (static and dynamic). Interestingly, the enzyme novozyme-435 plays an important role in controlling the polymerization and distribution of polymer chains, which is critical for the formation of nanomicelles with unimodal distributions. The methodology developed is highly flexible as it allows the introduction of various functionalities in the polymeric nanomicelles. These self-organized nanomicelles are highly efficient drug delivery vehicles for hydrophobic and partially hydrophilic drugs, both transdermally and orally, as they have the ability to encapsulate guest molecules during self-organization. In vivo studies by encapsulating anti-inflammatory agents (aspirin and naproxen) in these polymeric nanomicelles and by applying topically resulted in significant reduction in inflammation. The % reduction in inflammation using polymeric nanomicelles containing aspirin and naproxen was 62 and 64%, respectively.

Antagonism of croton oil inflammation by topical emu oil in CD-1 mice.

Emu oil is derived from the emu (Dromaius novaehollandiae), which originated in Australia, and has been reported to have anti-inflammatory properties. Inflammation was induced in anesthetized CD-1 mice by applying 50 microL of 2% croton oil to the inner surface of the left ear. After 2 h, the area was treated with 5 microL of emu, fish, flaxseed, olive, or liquified chicken fat, or left untreated. Animals were euthanized at 6 h postapplication of different oils, and earplugs (EP) and plasma samples were collected. Inflammation was evaluated by change in earlobe thickness, increase in weight of EP tissue (compared to the untreated ear), and induction in cytokines interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha) in EP homogenates. Although reductions relative to control (croton oil) were noted for all treatments, auricular thickness and EP weights were significantly reduced (-72 and -71%, respectively) only in the emu oil-treated group. IL-1alpha levels in homogenates of auricular tissue were significantly reduced in the fish oil (-57%) and emu oil (-70%) groups relative to the control group. The cytokine TNF-alpha from auricular homogenates was significantly reduced in the olive oil (-52%) and emu oil (-60%) treatment groups relative to the control group. Plasma cytokine levels were not changed by croton oil treatment. Although auricular thickness and weight were significantly correlated with each other (r = 0.780, P < 0.003), auricular thickness but not weight was significantly correlated with cytokine IL-alpha (r = 0.750, P < 0.006) and TNF-alpha (r = 0.690, P < 0.02). These studies indicate that topical emu oil has anti-inflammatory properties in the CD-1 mouse that are associated with decreased auricular thickness and weight, and with the cytokines IL-1alpha and TNF-alpha.

Efficacy of niclosamide as a potential antipenetrant (TAP) against cercariae of Schistosoma mansoni monkeys

A 1% (W/V) formulation of Niclosamide (2', 5-Dichloro-4-nitrosalicylanilide) (TAP) was tested on Cebus apella monkeys as a topical prophylatic against schistosomiasis mansoni. Two experiments were conducted using the same formulation. In the first experiment, the TAP provided complete protection against schistosomiasis for 3 days. Of the 4 monkeys treated with TAP 7 days before exposure to Schistosoma mansoni cercariae, 2 were completely protected. The remaining 2 monkeys of the 7 day treatment group had a 78% or greater reduction in adult worm buderns when compared to the placebo treated monkeys. The second experiment was designed determine the time between day 3 and 7 when the TAP no longer provided complete protection. However, all of the TAP treated monkeys in this experiment were completely protected, even the monkeys treated 7 days earlier. In both experiments, all monkeys used as infection controls and those receiving only the placebo became infected and showed typical experimental schistosomiasis. These results demonstrate that the TAP could provide fast acting, short-term protection to people who must enter cercariae infested water