The Effects of Continual Intermittent Parathyroid Hormone Treatment on Human Periodontal Ligament Fibroblasts in an In Vitro Wound Repopulation Model.

INTRODUCTION: Periodontal ligament fibroblasts (PDLFs) play a vital role in periodontal regeneration. Parathyroid hormone (PTH) is important in catabolic regulation on osteoclasts; it also has anabolic effects on hard tissue formation. Using an in vitro wound repopulation model, this study investigated the effect of continual intermittent administration of PTH on PDLFs wound repopulation. Methods and Materials: PDLFs were grown in 12-well plates and divided into 0 (control), 5, 10, 20, 40, and 80 nM of PTH treatments. A 3-mm wound was created on confluent and synchronized cells. Six PTH treatments were initiated using serum-free medium with supplements. Cell repopulation was measured at four time points: 5, 10, 15, and 20 days. RESULTS: A 5% increase wound repopulation showed an enhancement on day 10 for all treatment groups as compared to control groups. On days 15 and 20, treatment groups showed a decrease in proliferation and migration compared to controls with significant decreases at concentrations of 40 and 80 nM. CONCLUSION: Continual intermittent treatment with PTH has the potential to enhance proliferation and migration of PDLFs for wound repopulation at early time points. A dose-dependent correlation was seen with a positive trend on day 10 while a significant decrease on day 20.

Effect of Sodium Lauryl Sulfate (SLS) on Primary Human Gingival Fibroblasts in an In Vitro Wound Healing Model.

OBJECTIVES: Sodium lauryl sulfate (SLS) is a surfactant used to decrease the surface tension of water. Most commercially available dentifrices contain 0.5-2.0% SLS. This study investigated the potential effect of SLS on oral wound healing using primary human gingival fibroblasts (HGFs). METHODS: HGFs cells were grown in12-well culture plates in DMEM medium. A 3 mm wound was created on confluent HGFs. The cells were challenged with 0 (the control group), 0.01, 0.02, 0.03, 0.04, or 0.05% SLS-containing media once daily for 2 minutes. The cells were stained on day 0, 2, 4, 6 and 8. The percent of wound fill area was measured. RESULTS: On day 2, 4, 6, and 8, the wound fill of the control group (0% SLS) was 15, 35, 67 and 98%, respectively; at 0.01% SLS, it was 10, 20, 65 and 84%; at 0.02%, it was 7, 10, 15 and 25%; at 0.03% SLS, it was only 5% and 8% on day 2 and 4. CONCLUSION: Our results showed a dose- and time-dependent inhibition on HGFs wound fill by SLS; however, future in vivo studies are needed to validate if our in vitro findings using SLS-free dentifrices to avoid the potential delay of wound healing.

Evaluation of Virulence Gene Expression Patterns in Acinetobacter baumannii Using Quantitative Real-Time Polymerase Chain Reaction Array.

According to the Centers for Disease Control's recently devised National Strategy for Combating Antibiotic-Resistant Bacteria, Acinetobacter baumannii is a "serious" threat level pathogen. A. baumannii's notoriety stems from the fact that a large number of modern strains are multidrug resistant and persist in the hospital setting, thus causing numerous deaths per year. It is imperative that research focus on a more fundamental understanding of the factors responsible for the success of A. baumannii. Toward this end, our group investigated virulence gene expression patterns in a recently characterized wound isolate, AB5075, using quantitative real-time polymerase chain reaction array. Notably, several genes showed statistically significant upregulation at 37°C compared to 25°C; MviM, Wbbj, CarO, and certain genes of the Bas, Bar, and Csu operons. Additionally, we found that in vitro biofilm formation by Csu transposon insertion mutant strains is attenuated. These findings validate previous reports that suggest a link between the Csu operon and biofilm formation. More importantly, our results demonstrate a successful method for evaluating the significance of previously identified virulence factors in a modern and clinically relevant strain of A. baumannii, thereby providing a path toward a more fundamental understanding of the pathogenicity of A. baumannii.

In vitro study of the adverse effect of nicotine and physical strain on human gingival fibroblasts as a model of the healing of wounds commonly found in the military.

BACKGROUND: Significant adverse effects on fibroblast growth and metabolism are observed with nicotine. We investigated the synergistic effects of nicotine and cyclical mechanical strain (CMS) on human gingival fibroblasts (HGFs) in a wound-healing model. METHODS: HGFs were isolated and grown in Dulbecco's modified Eagle's medium. Three-millimeter wounds were created on a confluent cell monolayer grown in a media containing 0, 1, 2, or 4 mM nicotine, with or without CMS. The applied deformation regimen remains constant for 6 days. On days 1, 2, 4, and 6, the cells were stained with hematoxylin and eosin Y for the evaluation of wound repopulation. RESULTS: The application of CMS alone demonstrates a biphasic response, with an initial stimulatory effect on wound repopulation (days 1-2) and less repopulation during the later phase (days 4-6). The addition of nicotine clearly demonstrated a time and inverse dose-dependent relationship on wound repopulation, with no effect during the early phase and reduced wound repopulation during the later phase. CONCLUSIONS: Initial treatment of HGF wounds with CMS resulted in faster wound repopulation regardless of nicotine presence. By day 6, wound healing of HGF exposed to both nicotine and CMS is delayed. These findings suggest that CMS and nicotine may affect fibroblasts and delay wound healing at other sites in the body as well.

The effectiveness of endodontic solvents to remove endodontic sealers.

Dental emergencies negatively affect troop readiness, especially during combat. Endodontic retreatment, when required, is especially challenging when the removal of endodontic sealer is required. In this study, we investigated the effectiveness of synthetic endodontic solvents to remove endodontic sealers. Fifty capillary tubes (2.7 mm ID×22 mm L), each filled to 15 mm with either Roth 801, AH Plus, MetaSEAL, or gutta-percha, were stored at 75% humidity for 14 days at 37°C. Ten capillary tubes containing each sealer were treated with either chloroform, xylene, EndoSolv R, EndoSolv E, or no solvent, and then penetrated with D3 ProTaper Universal Retreatment file on the same day. The time for the file to penetrate the length of each sealer was recorded, and the data statistically analyzed. Roth 801 failed to set and was not tested. The file took 3.4±0.1, 4.8±0.3, 5.7±0.4, 4.5±0.2, and 10.6±1.0 seconds (mean±SD) to penetrate gutta-percha using chloroform, xylene, EndoSolv R, EndoSolv E, or no solvent, respectively, and was performed by one endodontic resident at one sitting. The time for penetration of gutta-percha with any solvent was significantly faster (p≤0.05) than for AH Plus or MetaSEAL.The time for AH Plus ranged from 23.1±1.0 to 81.5±4.5 seconds. The time for MetaSEAL ranged from 97.2±6.1 to >180 seconds. EndoSolv E was the most effective solvent for AH Plus. It took significantly more time to remove MetaSEAL than AH Plus, regardless of the solvent used. Our study indicated that the use of the proper endodontic solvent makes complete removal of a sealer much more effective during retreatment.

Evaluating the molecular basis for acute mountain sickness: hypoxia response gene expression patterns in warfighters and murine populations.

Acute mountain sickness (AMS) is an illness that affects many individuals at altitudes above 2,400 m (8,000 ft) resulting in decreased performance. Models that provide quantitative estimates of AMS risk are expanding, but predictive genetic models for AMS susceptibility are still under investigation. Thirty-four male U.S. Army Soldier volunteers were exposed to baseline, 3,000 m, 3,500 m, or 4,500 m altitude conditions in a hypobaric chamber and evaluated for onset of AMS symptoms. In addition, mice were evaluated at extreme hypoxia conditions equivalent to 7,600 m. Real-time polymerase chain reaction hypoxia response array was used to identify 15 genes that were activated in Soldiers and 46 genes that were activated in mice. We identified angiopoietin-like 4 (ANGPTL4) as a gene that is significantly activated in response to hypoxia (5.8-fold upregulated at 4,500 m in humans). The role of ANGPTL4 in high-altitude response has not been explored. Pretreatment of mice with fenofibrate, an ANGPTL4-activating pharmaceutical, had a considerable effect on overall hypoxia response gene expression and resulted in significantly decreased cerebral edema following exposure to hypoxia. Activation of ANGPTL4 may protect against cerebral edema by inhibiting vascular endothelial growth factor and therefore serve as a potential target for AMS prevention.

Purification and characterization of a recombinant Yersinia pestis V-F1 "Reversed" fusion protein for use as a new subunit vaccine against plague.

We previously developed a unique recombinant protein vaccine against plague composed of a fusion between the Fraction 1 capsular antigen (F1) and the V antigen. To determine if overall expression, solubility, and recovery of the F1-V fusion protein could be enhanced, we modified the original fusion. Standard recombinant DNA techniques were used to reverse the gene order such that the V antigen coding sequence was fused at its C-terminus to the N-terminus of F1. The F1 secretion signal sequence (F1S) was subsequently fused to the N-terminus of V. This new fusion protein, designated F1S-V-F1, was then co-expressed with the Y. pestis Caf1M periplasmic chaperone protein in BL21-Star Escherichia coli. Recombinant strains expressing F1-V, F1S-F1-V, or F1S-V-F1 were compared by cell fractionation, SDS-PAGE, Western blotting, and suspension immunolabelling. F1S-V-F1 exhibited enhanced solubility and secretion when co-expressed with Caf1M resulting in a recombinant protein that is processed in a similar manner to the native F1 protein. Purification of F1S-V-F1 was accomplished by anion-exchange and hydrophobic interaction chromatography. The purification method produced greater than 1mg of purified soluble protein per liter of induced culture. F1S-V-F1 polymerization characteristics were comparable to the native F1. The purified F1S-V-F1 protein appeared equivalent to F1-V in its ability to be recognized by neutralizing antibodies.

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague.

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.

Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum.

The type III secretion system (YscC) protein of Yersinia pestis plays an essential role in the translocation of Yersinia outer proteins (Yops) into eukaryotic target cells through a type III secretion mechanism. To assess the immunogenicity and potential protective efficacy of YscC against lethal plague challenge, we cloned, overexpressed, and purified YscC using two different bacterial expression and purification systems. The resulting expression plasmids for YscC, pETBlue-2-YscC and pTYB11-YscC, were regulated by robust T7 promoters that were induced with isopropyl-beta-D-thiogalactopyranoside. The intein-fusion pTYB11-YscC system and the six-histidine-tagging pETBlue-2-YscC system were both successful for producing and purifying YscC. The intein-mediated purification system produced about 1mg of soluble YscC per liter of bacterial culture while the YscC-His(6)-tag method resulted in 16mg of insoluble YscC per liter of bacterial culture. Protein identity for purified YscC-His(6) was confirmed by ion trap mass spectrometry. Antisera were produced against both YscC and YscC-His(6). The specific immune response generated in YscC-vaccinated mice was relative to the particular purified protein, YscC or YscC-His(6), which was used for vaccination as determined by Western blot analysis and ELISA. Regardless of the purification method, either form of the YscC protein failed to elicit a protective immune response against lethal plague challenge with either F1 capsule forming Y. pestis CO92 or the isogenic F1(-)Y. pestis C12.

Characterization of human ribosomal S3a gene expression during adenosine 3':5' cyclic monophosphate induced neuroendocrine differentiation of LNCaP cells. Regulation of S3a gene expression in LNCaP.

Elevation of intracellular cAMP levels in the human prostatic adenocarcinoma cell line LNCaP results in the induction of reversible neuroendocrine differentiation and cell growth arrest. We have used the differential display technique to identify genes that are differentially expressed during cAMP induced neuroendocrine differentiation in LNCaP cells. We identified the human ribosomal S3a gene as being down regulated in response to LNCaP differentiation. The S3a gene is known to be expressed at high levels in both tumors and cancer cell lines. It has also been shown that down regulation of S3a is associated with a loss of the transformed phenotype. In order to better ascertain the mechanism by which S3a gene expression is decreased during differentiation, the promoter region for this gene was analyzed. Electrophoretic mobility shift assay, antibody supershift assays, site-directed mutagenesis, and luciferase reporter gene analysis were employed to authenticate the roles of several transcription factors in the regulation of the S3a gene. We found that two cyclic AMP response elements, a Sp1 element, and a GA-binding protein element were involved in the regulation of S3a gene expression. The CRE elements were found to be necessary for high level expression of the 53a gene in undifferentiated LNCaP cells. Mutations in the CRE elements abolished CREB-1 binding and resulted in a 57% decrease in S3a gene expression. The addition of cAMP elevating agents to LNCaP cells in sufficient quantity to induce differentiation generated a 50% decrease in S3a gene expression. These results suggest that the CRE elements participate in cAMP-induced down regulation of gene expression. Furthermore, our experiments demonstrate that occupation of the GABP binding site results in a substantial decrease in S3a promoter activity.

Identification of differentially expressed genes during cyclic adenosine monophosphate-induced neuroendocrine differentiation in the human prostatic adenocarcinoma cell line LNCaP.

The LNCaP cell line is a versatile and useful model suitable for the study of human prostate cancer in vitro. It has been determined that the elevation of LNCaP intracellular cyclic adenosine monophosphate (cAMP) levels through the addition of membrane-permeable cAMP analogs, phosphodiesterase inhibitors, adenylate cyclase activators, or components of the cAMP signal-transduction pathway can induce reversible neuroendocrine (NE) differentiation. Elucidation of those genes that are differentially expressed between undifferentiated prostate cancer cells and prostate cancer cells that have been induced to differentiate may present new insights into the molecular mechanisms governing NE differentiation, early detection of prostate cancer, and potential targets for gene therapy. In this study, differential display polymerase chain reaction (PCR) was used to identify 226 differentially expressed PCR products. Twelve of the differential display PCR products were confirmed by Northern blot analysis and were cloned. DNA sequencing and database comparisons were performed. To our knowledge, this is the first report of the use of differential gene expression techniques to analyze gene expression during cAMP-induced NE differentiation in LNCaP cells. Confirmation of NE differentiation reversibility also was accomplished.