Browsing by Subject "Oral Biology"

Now showing 1 - 7 of 7
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    Accelerating development of treatments for trigeminal neuralgia using intranasal delivery and a novel behavioral screening device for rats.
    (2011-08) Johnson, Neil James
    Orofacial pain disorders are challenging to treat because of poor targeting to the trigeminal, orofacial and brain structures involved. Intranasal administration targets these structures while minimizing systemic exposure and side effects. Lidocaine was intranasally administered to rats, and the trigeminal nerve and trigeminally-innervated structures (teeth, temporomandibular joint (TMJ), and masseter muscle) accumulated up to 20-fold higher tissue concentrations of lidocaine than the brain and blood as measured by ELISA. Intranasally administered infrared dye reached the trigeminal nerve and brain within 10 minutes. Intranasal administration distributed the dye to the trigeminal nerve through three regions with high drug concentrations in the nasal cavity: the middle concha, the maxillary sinus and the choana. These findings indicate that intranasal delivery has the potential to target and treat trigeminal pain disorders with fewer side effects. Such disorders include: tooth pain, TMJ disorder, trigeminal neuralgia, headache, and even certain brain diseases. Although intranasal delivery may aid in targeting approved medications, better novel treatments need to be developed for orofacial pain disorders, specifically trigeminal neuralgia. A new testing device (TrigeminAir) was developed to assist in the screening of trigeminal neuralgia models and treatments. The TrigeminAir device assesses orofacial sensitivity in rats over time by measuring the sip rate of sweetened condensed milk in the presence of a normally non-painful air puff stimulus on the whisker pad. This device detected, 5 to 12 hours after infraorbital nerve injection, that carrageenan (2% or 4%) reduced sip rate by inflaming the whisker pad. During the same time period, intranasal 10% lidocaine (8mg) reversed this reduced sip rate by anesthetizing the inflamed whisker pad. In the first four hours after 4% carrageenan injection, intranasal lidocaine (4% and 10%) decreased neuronal activity in nerves innervating the maxillary teeth, palate, whisker pad and snout. Sensory and motor activity was unaffected after intranasal lidocaine administration, except for anesthesia of the whiskers affecting balance. In conclusion, these results suggest the TrigeminAir device is an efficient and reliable screening method of preclinical models and treatments for orofacial pain and demonstrate that intranasal lidocaine effectively reduces tactile allodynia in rodents. Intranasal delivery and the TrigeminAir device could accelerate the development of new orofacial pain treatments by improving targeting and screening of treatments.
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    Gene expression profiling in peripheral blood of Sjögren’s syndrome patients.
    (2010-05) Emamian, Eshrat Sadat
    Sjögren’s syndrome (SS) is a common autoimmune disorder characterized by lymphocytic infiltration into exocrine glands. Disruption of target organ function, such as salivary and lacrimal glands may lead to irreversible manifestations such as severe dry eyes and mouth. A complex genetic architecture combined with the influence of environmental factors is thought to contribute to the etiology of SS; however, the pathophysiological basis of SS is poorly understood. To identify important molecular pathways involved in SS and define biomarkers for clinical features of the disease, we have used high-density oligonucleotide microarrays to compare global gene expression profiles in peripheral blood samples of SS patients and controls. We first analyzed mononuclear cells of 21 SS patients and 23 controls and identified a prominent pattern of over-expressed genes that are inducible by interferons (IFNs). We then repeated the analysis in whole blood of a second independent dataset of 17 SS patients and 22 controls and observed the same pattern with respect to IFN inducible genes. Furthermore, we observed that gene expression of IFN-inducible genes was positively correlated with titers of anti-Ro/SSA (P<0.001) and anti-La/SSB (P<0.001) autoantibodies and negatively correlated to salivary flow or tear production. Additional inflammatory and immune-related pathways with altered expression patterns in SS cases included B and T cell receptor, IGF-1, GM-CSF, PPARα/RXRα, and PI3/AKT signaling. Our results strongly support innate and adaptive immune processes in the pathogenesis of SS and provide numerous candidate disease markers for further study. We also compared gene expression profiles between patients with SS and systemic lupus erythematosus (SLE). Systemic manifestations in SS patients are common and may include clinical and serological overlap with other autoimmune disease features such as SLE. The pathophysiology of common and distinct features of SS and SLE are poorly understood. In this section of the study, we have directly compared gene expression profiles in whole blood of SS patients and SLE patients to determine which genes are differentially expressed in both phenotypes, and which genes distinguish the two patient groups. Approximately 22,000 RNA transcripts were interrogated using the Affymetrix U133A GeneChip®. Differentially expressed genes were defined using t-tests with nominal significance of p<0.001 and average fold change of >1.5. Our gene expression datasets included 35 Caucasian female SLE patients, 36 Caucasian female SS patients, and 62 Caucasian female controls. We compared each group of SS (n=36) and SLE (n=35) patients separately to the 62 controls. A total of 349 genes were differentially expressed in SS patients and 625 genes were differentially expressed in SLE patients. Of these two gene lists, 95 genes overlapped and were differentially expressed in both SS and SLE patients, the majority of which are IFN-inducible. Among the non-overlapping genes, ribosomal proteins were highly overexpressed in SLE patients but underexpressed in SS patients relative to controls. These results show that SS and SLE patients have identifiable gene expression signatures that are either common or distinct between the two patient populations. Characterization of these profiles has significant potential to facilitate development of improved diagnostic approaches and targeted therapies.
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    A magnetoencephalographic (MEG) study of brain mechanisms in temporomandibular disorder.
    (2009-12) Alonso DDS, Aurelio Abdalla MS.
    The main goal of this study was to investigate, using MEG, the dynamic neural mechanisms underlying facial tactile stimulation in two groups of subjects, namely a control group (without pain) and a TMD pain group (arthromyalgia), by stimulating the facial skin with a non-painful air-driven plastic membrane. Our first specific aim was to investigate and compare the spatial and temporal features of the ECDs following innocuous tactile stimuli in both groups. And t he second specific aim was to investigate the differences in dynamic brain function between these two groups using a time-frequency analysis of the MEG data. In summary, innocuous tactile stimulation proved to be a successful way to measure brain spatio-temporal dynamics in two group population. We were able to demonstrate very clear the differences in brain organization and dynamics between these two groups using an innocuous stimulus and without causing an unpleasant feeling. The results obtained allow for a paradigm shift in future research of brain mechanisms in pain by the use of non-painful tactile stimuli to evaluate brain function in various orofacial (or other) pain conditions, including neurovascular and neuropathic pains and other complex orofacial pain disorders.
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    Regulatory roles of calprotectin in head and neck squamous cell carcinogenesis.
    (2011-07) Khammanivong, Ali
    Malignant transformation in squamous cell carcinomas (SCC) such as those of the head and neck (HNSCC) remains an enigmatic process that results in abnormal cellular differentiation, loss of growth and cell cycle regulation, gain of replicative immortality and resistance to apoptosis, activation of cellular migration and invasion, increase in energy metabolism, and evasion of immune destruction. These abnormalities in cell functions emerged as the hallmarks of cancer. Calprotectin, a heterodimeric protein complex of calcium regulating S100A8 and S100A9 encoded by genes mapped to the chromosomal locus 1q21.3 of the epidermal differentiation complex (EDC), may play essential roles in the regulation of cell differentiation, cell cycle progression, cellular survival and cell migration that are part of the cancer hallmarks. While highly upregulated in a variety of cancers, calprotectin is down-regulated in squamous cell carcinomas of the cervix, esophagus and the head and neck. Using microarray analysis for gene expression we found that the S100A9 subunit of calprotectin was significantly down-regulated along with other EDC genes in human primary HNSCC cases, suggesting a loss of functional S100A8/A9 protein complex and differentiation during carcinogenesis. Expression of S100A9 correlated strongly with a set of HNSCC downregulated genes putatively involved in loss of cytodifferentiation and control of cell cycle. To probe its role in carcinogenesis, S100A8/A9 was stably expressed in a calprotectin-negative human carcinoma cell line (KB cells). Expression of S100A8/A9 in KB cells up-regulated differentiation and cell-cell contact growth inhibition signaling pathways, re-established epithelial actin microfilament cytoskeletal structures and v cellular adhesion to the extracellular matrix, down-regulated anti-apoptosis gene networks, and suppressed anchorage-independent survival and stress-induced (by serum starvation) cell migration. Calprotectin appeared to induce growth suppression by signaling a reactivation of G2/M cell cycle checkpoint regulators, Chk1 and PP2A, and inactivation of mitotic activators Cdc25C and Cdc2. As a result, we saw a marked increase in Cdc2-PThr14/Tyr15 phosphorylation and down-regulation of cyclin B1, suggesting an inactivation of the mitotic entry promoting Cdc2/cyclin B1 complex, resulting in cell cycle and mitotic arrest at the G2/M checkpoint. When inoculated into nude mice, KB cells producing calprotectin showed reduced tumor growth when compared to sham-transfected control KB cells. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increased carcinoma growth and survival and reduced Cdc2 phosphorylation at Thr14/Tyr15. Calprotectin expression in KB and TR146 cells also down-regulated expression of putative HNSCC marker genes, INHBA, PTGS2 (Cox-2) and SULF1, found to be expressed only in HNSCC samples. Calprotectin-mediated control of cellular differentiation and G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may point to new molecular targets for therapy.
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    Role of twisted gastrulation and matrix gamma-carboxyglutamic acid protein in bone homeostasis.
    (2012-05) Sotillo Rodriguez, Julio Eliezer
    The regulation signaling activity of osteoblasts and osteoclasts has been linked to extracellular proteins, including twisted gastrulation (TWSG1) and matrix gamma-carboxyglutamic acid protein (MGP). TWSG1 is a regulator of bone morphogenetic proteins (BMPs) signaling activity. Previous studies have shown that TWSG1 is an essential modulator of not only skeletal development but also soft tissue development (i.e. salivary gland). MGP is a vitamin K-dependent protein previously characterized as a potent inhibitor of biomineralization. Numerous cells types produce MGP, including osteoblasts, chondrocytes, vascular smooth muscle cells and endothelial cells. Despite current research, little is known about the effects of TWSG1 and MGP on the regulation of osteoblasts and osteoclasts. Both of which are cells that maintain bone remodeling. In this dissertation, we assessed the role of both TWSG1 and MGP in postnatal bone homeostasis as well as in vitro functional analysis of TWSG1 and MGP on osteoblasts and osteoclasts. We were able to determine that the C57BL/6 MGP deficient mice and 129Sv/Ev TWSG1 deficient mice exhibited an osteopenic skeletal phenotype. This phenotype was explained by an imbalance in the cellular regulatory patterns associated with the regulation of homeostatic bone remodeling. Further, we characterized cellular pathways which explained how, in both TWSG1 and MGP deficient mice, excessive osteoclastic activity was the name culprit in the osteopenic phenotypes. Taken together, our results highlight the importance in the modulation of osteoblastic and osteoclastic activity by TWSG1 and MGP.
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    Streptococcus cristatus modulates epithelial innate immune response through regulating nuclear factor-kappa B pathway.
    (2009-12) Zhang, Guizhen
    Streptococci are the dominant oral commensal organisms. They, along with putative periodontal pathogens, normally colonize multiple oral tissue sites. An emerging paradigm indicates that the host can distinguish between pathogenic and nonpathogenic stimuli, and commensal bacteria could modulate expression of host genes to contribute to mucosal tolerance. Here, I hypothesized that an oral commensal Streptococcus cristatus can attenuate epithelial proinflammatory response to Fusobacterium nucleatum via regulation of Toll-Like Receptor (TLR) signaling, as a mechanism for oral mucosal tolerance to polymicrobial infection. I first demonstrated that S. cristatus itself did not provoke IL-8 production in epithelial cells, and it was able to inhibit IL-8 responses to several putative oral pathogens including F. nucleatum. The inhibitory effect of S. cristatus on IL-8 was independent of its viability and its coaggregation with F. nucleatum, was not related to soluble bacterial products, and appeared to require bacterial contact with epithelial cells. Similar effects were seen with several other species of oral streptococci. Next, I performed pathway-specific microarray analysis to specifically monitor host gene modulation by S. cristatus on a broad scale. I found that S. cristatus altered the F. nucleatum–induced expression of a number of proinflammatory cytokine genes. Profiling of TLR signaling related genes revealed that S. cristatus most significantly impacted the downstream pathways, especially NF-κB, rather than altering TLRs and their adaptors and interacting proteins. Lastly, I examined the underlying mechanism(s) involved in the modulating effect of S. cristatus by looking specifically at its impact on the nuclear factor kappaB (NF-κB) pathway under the Toll-like receptor signaling background. I found that the IL-8 suppression by S. cristatus was coincident with the inhibition of NF-κB activation and IκB-α degradation. Pre-incubation with TLR2 and TLR4 antibodies, however, did not affect the epithelial response to either species alone or in combination. I thus conclude that the oral commensal S. cristatus is not only tolerated by the host, but also able to modulate host inflammatory response to pathogenic species through inhibiting IκB-NF-κB signaling module. The anti-inflammatory effect of S. cristatus might represent a regulatory mechanism present at the epithelial surface to tolerate polymicrobial colonization.
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    Streptococcus sanguinis Ecto-5'-nucleotidase modulates platelet aggregation.
    (2011-05) Fan, Jingyuan
    Streptococcus sanguinis, an oral commensal bacterium, is the leading cause of infective endocarditis (IE). In an animal model, the abilities of S. sanguinis to adhere to and activate platelets are correlated with the increased severity of IE. In response to S. sanguinis, platelet activation is associated with secretion of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) from dense granules. The extracellular ADP is a potent platelet agonist and amplifies platelet aggregation induced by other pro-thrombotic agonists, whereas, the final product of hydrolysis of adenine nucleotides, adenosine, is a platelet aggregation antagonist. Here, we show that cell surface ecto-5'-nucleotidase (NT5E) of S. sanguinis can hydrolyze adenine nucleotides ATP to ADP, adenosine monophosphate (AMP) and finally adenosine. Therefore, we hypothesize that S. sanguinis ecto-5'-nucleotidase modulates platelet aggregation. A nt5e deletion mutant of S. sanguinis 133-79 showed significantly shorter lag time to onset of platelet aggregation than the wild-type strain (wt). However, the nt5e deletion mutant adhered to human platelets indistinguishably from the wild-type and complemented strains. By hydrolyzing the released ATP and ADP from dense granules of activated platelets, therefore, NT5E modulates S. sanguinis-induced platelet aggregation in vitro. In addition, strains of S. sanguinis showed different cell surface enzymatic activities for hydrolysis of adenine nucleotides, which may contribute to the determination of the platelet interactivity phenotypes. To further elucidate the mechanism, we distinguished the roles of ADP and adenosine receptors on streptococcal-platelet interactions using specific antagonists. We showed that the ADP receptors, P2Y1 and P2Y12, and the adenosine receptor A2a were all involved in S. sanguinis-induced platelet aggregation. Downstream of P2Y12, platelet activation involved two waves of Akt phosphorylation in response to S. sanguinis. NT5E also modulates platelet aggregation by indirectly signaling Rap1 activity. Through these pathways, S. sanguinis NT5E slows down platelet aggregation by removing ADP and generating adenosine. Using a rabbit endocarditis model, we found that in the absence of nt5e, the mass of the vegetations and recovered bacterial loads were greatly decreased, suggesting a contribution of NT5E to the virulence of S. sanguinis in vivo. Similar to the release of ADP, activated platelets secrete platelet microbicidal proteins (PMPs), which antagonize a broad range of pathogens. These data, therefore, indicate that NT5E-mediated inhibition of platelet aggregation might delay presentation of PMPs to infecting bacteria on heart valves. The delay would enable the infecting bacteria to colonize in the absence of this innate immune effector. Extracellular adenine nucleotides are also important signaling molecules that mediate both inflammatory and anti-inflammatory processes. By hydrolyzing ATP, a pro-inflammatory molecule, and generating adenosine, an immunosuppressive molecule, NT5E might inhibit phagocytic monocyte/macrophages associated with valvular vegetations, promoting the survival of infecting S. sanguinis. In conclusion, we now show for the first time that streptococcal NT5E modulates S. sanguinis-induced platelet aggregation and contributes to the virulence of streptococci in IE. These findings expand our knowledge of bacterial-host interactions and may suggest novel therapeutics for cardiovascular infectious diseases.