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Background Biofilm-induced inflammatory osteolytic oral infections, such as periodontitis and peri-implantitis, have complex etiology and pathogenesis. A significant obstacle to research has been the lack of appropriate animal models where the inflammatory response to biofilms can be investigated. The aim of this study is to develop a novel animal model to study the host response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans)–biofilm colonizing titanium implants. Methods Titanium implants were inoculated in vitro with A. actinomycetemcomitans, establishing a biofilm for 1 to 3 days. Biofilm-inoculated and control implants were transmucosally placed into rat hard palate or alveolar ridge. Analysis included documentation of clinical inflammation, polymerase chain reaction, and culture detection of A. actinomycetemcomitans and microcomputed tomography quantitation of peri-implant bone volume. Results Viable A. actinomycetemcomitans biofilm was successfully established on titanium implants in vitro, detected by confocal laser scanning microscopy. An inflammatory response characterized by clinical inflammation, bleeding, ulceration, hyperplasia, and necrosis was observed around biofilm-inoculated implants. A. actinomycetemcomitans was detected by polymerase chain reaction and culture analysis on 100%of biofilm-inoculated implants for up to 3 weeks and 25%for up to 6 weeks. Microcomputed tomography analysis demonstrated significantly lower bone volume (P <0.05) around biofilm-inoculated implants (29.6% ± 7.6%) compared to non-inoculated implants (50.5% ± 9.6%) after 6 weeks. Conclusions These results describe a novel animal model where A. actinomycetemcomitans biofilm was established in vitro on titanium implants before placement in rat oral cavity, leading to an inflammatory response, osteolysis, and tissue destruction. This model may have potential use for investigation of host responses to biofilm pathogens and antibiofilm therapy.

Bone regeneration often requires harvesting of autologous bone with significant potential morbidity and cost. Recombinant human bone morphogenetic protein (rhBMP)-2 has been approved by the U.S. Food and Drug Administration for specific regenerative indications. However, administration of exogenous growth factors has many drawbacks. The objective of the present proof-of-concept study was to determine whether immobilized anti-BMP-2 antibodies (Abs) could capture endogenous BMP-2 in local sites to mediate osteogenesis, a strategy we refer to as antibody-mediated osseous regeneration (AMOR). We have generated a murine anti-BMP-2 monoclonal antibody library, which was tested along with commercially available Abs in vitro and in vivo for their ability to mediate AMOR. In vitro studies demonstrated that only some anti-BMP-2 Abs tested formed immune complexes with BMP-2, which can bind to BMP cellular receptor, whereas other BMP-2/anti-BMP-2 complexes failed to bind. To investigate whether anti-BMP-2 Abs were able to mediate AMOR in vivo, anti-BMP-2 Abs were immobilized on absorbable collagen sponge (ACS) and surgically placed in rat calvarial defects. Microcomputed tomography analysis of live animals at 2, 4, and 6 weeks demonstrated that some anti-BMP-2 Abs immobilized on ACS mediated significant bone regeneration, whereas other clones did not mediate any bone regeneration. In situ BMP-2 and osteocalcin expression was investigated by immunohistochemistry. Results demonstrated higher BMP-2 and osteocalcin expression in sites with increased bone regeneration. Results provide first evidence for the ability of anti-BMP2 Abs to form an immune complex with endogenous BMP-2 and mediate bone regeneration in vivo, suggesting a promising therapeutic method for tissue engineering.

Kocuria spp. are members of the Micrococcaceae family that are frequently found in the environment and on human skin. Few human infections have been reported. We describe what appear to be the first two cases of Kocuria marina peritonitis in patients undergoing continuous ambulatory peritoneal dialysis. CASE REPORTS Case 1.A 57-year-old man was admitted to the emergency department because of turbid dialysis effluent for 1 day. He had end-stage renal disease as a result of diabetic nephropathy and had been undergoing continuous ambulatory peritoneal dialysis (CAPD) for 6 years. Upon physical examination, he was afebrile, with a normal-appearing catheter exit site. However, the peritoneal dialysate fluid was straw colored and cloudy, with a total leukocyte count of 0.78 ϫ 10 9 leukocytes/ liter and a neutrophil count of 90%. No microorganisms were seen on a Gram stain. In the peripheral blood, the hemoglobin concentration was 10.1 g/dl, the white blood cell (WBC) count was 7.40 ϫ 10 9 cells/liter, and the platelet count was 171 ϫ 10 9 platelets/liter. The C-reactive protein concentration was 2.76 mg/dl (reference concentration, Ͻ0.5 mg/dl), and the serum urea and creatinine concentrations were 53 mg/dl and 11.2 mg/dl, respectively. Intraperitoneal administration of netilmicin and narrow-spectrum cephalosporin (ceftezole) was started for empirical treatment of CAPD peritonitis, which was changed to intraperitoneal ceftazidime and clindamycin when there was no response. Culture of the dialysate yielded a pure culture of grampositive cocci in pairs or clusters (strain M07-0128). After 48 h of incubation at 35°C in 5% CO 2 on sheep blood agar, the 1-to 2-mm colonies were nonhemolytic and yellow. The isolate was identified as Kocuria varians/Kocuria kristinae with a 50.28%/49.72% probability, respectively, by a Vitek 2 system (bioMérieux, St. Louis, MO) and as K. kristinae (code number 6714014) with a 99.3% probability by an API Staph system (bioMérieux, Marcy l'Etoile, France). We performed 16S rRNA gene sequencing as previously described (5) and compared the obtained sequence with sequences similar to those of the type strains using BLAST and EzTaxon (4). The result showed 99.86% homology with Kocuria marina; the second closest match was Kocuria carniphila, with 98.30% homology. This isolate was finally identified as K. marina by 16S rRNA gene sequence analysis. In spite of the start of administration of intravenous vancomycin on day 10, the response remained unsatisfactory. The Tenckhoff catheter in his abdomen was removed on day 17, and he was switched to hemodialysis with the placement of an arteriovenous shunt. The patient improved with antibiotic therapy for 7 days after catheter removal and was discharged.We performed antimicrobial susceptibility testing on the isolate using the agar dilution method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for Staphylococcus (4a). The isolate was susceptible to penicillin, ampicillin, ampicillin-sulbactam, gentamicin, cephalothin (cefaloti...

Bone engineering strategies often exploit modulation of the extracellular environment, including delivery of cell and growth factors to repair and regenerate damaged tissues. During bone healing, the expression of endogenous bone morphogenetic proteins is an essential component of the healing response. However, in some situations, the inherent reparative capacity available in the local microenvironment is exceeded by the requirements of the defects. We have recently reported on a novel strategy, that exploits the specificity of antibodies to capture and make available endogenous osteogenic growth factors, referred to as "antibody-mediated osseous regeneration" (AMOR). The objective of the present study was to identify some of the cellular and molecular events involved in AMOR in an effort to begin to elucidate the mechanism of AMOR. The rat critical-sized calvarial defect model was used, where anti-bone morphogenetic protein (BMP)-2 monoclonal antibody (mAb), isotype-control mAb, or recombinant human (rh)BMP-2 were immobilized on absorbable collagen calvarial sponge (ACS) by adsorption, and then implanted into calvarial defects. The results demonstrated persistence of implanted mAbs for short term from 1 to 2 weeks after implantation. Increased cell infiltration was found in defects treated with anti-BMP-2 mAb. Examination of proteins on ACS scaffolds retrieved from defect sites demonstration increased levels of BMP-2, BMP-4, and BMP-7 proteins in sites implanted with anti-BMP-2 mAb. Moreover, BMP-2, BMP-4, and BMP-7 gene expression levels were increased in sites implanted with anti-BMP-2 mAb. Micro-computed tomography and histological analysis demonstrated that the bone within calvarial defects was fully regenerated in sites implanted with either anti-BMP-2 mAb or rhBMP-2. However, rhBMP-2-regenerated bone exhibited aberrant histomorphology with dystrophic calcification and invasion of subjacent areas. Altogether, the results revealed evidence for anti-BMP-2 mAbs to form an immune complex with BMP-2, BMP-4, and BMP-7, and bind to cells to mediate osteogenesis bone regeneration in vivo. This approach suggests a significant role for antibodies in regenerative orthopedic medicine.

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Amino acid transport system L is a cell membrane protein that transports neutral amino acid and known to be the major route of neutral amino acids for cell proliferation.1-3) Kanai et al. identified the first isoform of amino acid transport system L (L-type amino acid transporter 1, LAT1) from C6 glioma cells.4) It is predicted to be 12-membrane-spanning proteins, and mediates Na ϩ -independent amino acid exchange and prefers large neutral amino acids with bulky or branched side chains for its substrates. [4][5][6][7][8] Following the molecular identification of LAT1, the second isoform of amino acid transport system L (L-type amino acid transporter 2, LAT2) has been identified. [9][10][11] It has the characteristics of transporting small neutral amino acids as well as large neutral amino acids. [10][11][12] LAT1 is only expressed in restricted organs such as brain, spleen, placenta and testis. 4,13) However, LAT2 is more ubiquitously expressed than LAT1. [10][11][12] Importantly, LAT1 is highly expressed in malignant tumors presumably to support their continuous growth and proliferation. 4,5,14,15) Based on the characteristics of LAT1 and LAT2, it is proposed that the manipulation of system L activity, particularly that of LAT1, would have therapeutic implications for cancer treatment. If the activity of LAT1 should be suppressed and thereby the depletion of intracellular neutral amino acids should be induced, this would be helpful for inducing the inhibition of cancer cell growth. However, the mechanism by which inhibition of LAT1 can cause cancer cell growth suppression or cytotoxicity of cancer cells is not entirely clear.2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is a model compound for the study of amino acid transporters, as it is a system L selective inhibitor. [4][5][6]11,16,17) Substrates for amino acid transport system L include several essential amino acids such as leucine, isoleucine, valine, phenylalanine, methionine and histidine.1-3) Therefore, the suppression of L-type amino transpoter activity using BCH could be effective in the retardation of tumor cell growth by depleting the intracellular essential amino acids. In this study, we attempted to clarify the effect of BCH on the expression of factors regulating the cell cycle to cause cell cycle arrest in KB human oral cancer cells. We report here that the BCH arrests KB cell growth at G1 phase of cell cycle through the inhibition of cyclin D3-cyclin-dependent protein kinase 6 (CDK6) complex expression while increasing the expression of p27, a CDK inhibitor. Cell Cultures KB cells were grown in Dulbecco's modi- The purpose of this study was to examine the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of L-type amino acid transporters, on the cell growth suppression in KB human oral cancer cells and to study the roles of cell cycle regulatory factors in the BCH-induced growth inhibition. The effect of BCH on cell growth suppression and the influence of BCH to cell cycle regulatory factors in KB cell gro...