LncRNA SNHG1 enhances cartilage regeneration by modulating chondrogenic differentiation and angiogenesis potentials of JBMMSCs via mitochondrial function regulation.

BACKGROUND: Cartilage is a kind of avascular tissue, and it is difficult to repair itself when it is damaged. In this study, we investigated the regulation of chondrogenic differentiation and vascular formation in human jaw bone marrow mesenchymal stem cells (h-JBMMSCs) by the long-chain noncoding RNA small nucleolar RNA host gene 1 (SNHG1) during cartilage tissue regeneration. METHODS: JBMMSCs were isolated from the jaws via the adherent method. The effects of lncRNA SNHG1 on the chondrogenic differentiation of JBMMSCs in vitro were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Pellet experiment, Alcian blue staining, Masson's trichrome staining, and modified Sirius red staining. RT-qPCR, matrix gel tube formation, and coculture experiments were used to determine the effect of lncRNA SNHG1 on the angiogenesis in JBMMSCs in vitro. A model of knee cartilage defects in New Zealand rabbits and a model of subcutaneous matrix rubber suppositories in nude mice were constructed for in vivo experiments. Changes in mitochondrial function were detected via RT-qPCR, dihydroethidium (DHE) staining, MitoSOX staining, tetramethyl rhodamine methyl ester (TMRM) staining, and adenosine triphosphate (ATP) detection. Western blotting was used to detect the phosphorylation level of signal transducer and activator of transcription 3 (STAT3). RESULTS: Alcian blue staining, Masson's trichrome staining, and modified Sirius Red staining showed that lncRNA SNHG1 promoted chondrogenic differentiation. The lncRNA SNHG1 promoted angiogenesis in vitro and the formation of microvessels in vivo. The lncRNA SNHG1 promoted the repair and regeneration of rabbit knee cartilage tissue. Western blot and alcian blue staining showed that the JAK inhibitor reduced the increase of STAT3 phosphorylation level and staining deepening caused by SNHG1. Mitochondrial correlation analysis revealed that the lncRNA SNHG1 led to a decrease in reactive oxygen species (ROS) levels, an increase in mitochondrial membrane potential and an increase in ATP levels. Alcian blue staining showed that the ROS inhibitor significantly alleviated the decrease in blue fluorescence caused by SNHG1 knockdown. CONCLUSIONS: The lncRNA SNHG1 promotes chondrogenic differentiation and angiogenesis of JBMMSCs. The lncRNA SNHG1 regulates the phosphorylation of STAT3, reduces the level of ROS, regulates mitochondrial energy metabolism, and ultimately promotes cartilage regeneration.

Inhibition of experimental periodontitis by a monoclonal antibody against Porphyromonas gingivalis HA2.

It is known that complexes of the multi-protein, gingipain, possess heme binding domains (hemagglutinin 2, HA2) that bind hemoglobin to provide heme and iron to the bacterium, Porphyromonas gingivalis. The DHYAVMISK peptide sequence was proposed to act as an inhibitor of hemin binding, and thus, it might be used to control or prevent periodontal disease. In this study, we created a monoclonal antibody (mAb) that targeted the DHYAVMISK peptide, aimed to determine whether it could inhibit the growth of P. gingivalis in vitro, and block its induction of experimental periodontitis and subsequent bone loss. Peptide DGFPG-DHYAVMISK conjugated to KLH (DK-KLH) was synthetic, and injected subcutaneously into BALB/c mice to generate specific mAbs with the hybridoma technique. We isolated mAb 1H11, which showed specific binding to DK. When we incubated these mAbs with P. gingivalis in vitro for 18 h, bacterial growth was significantly lower in cultures treated with mAb 1H11 compared to those treated with control (PBS; P < 0.05). Next, we induced experimental periodontitis in mouse models with a silk ligature and a P. gingivalis infection. When we injected the mAbs into the gingival sulcus, the group treated with mAb 1H11 displayed a reduction in bone loss compared to the other treatment groups. Thus, mAb 1H11 might provide protection against a P. gingivalis infection. Accordingly, this antibody could serve as a candidate therapy for periodontitis or other infections caused by P. gingivalis.

Identification of bacteria associated with periapical abscesses of primary teeth by sequence analysis of 16S rDNA clone libraries.

OBJECTIVE: This study aims to detect the predominant bacteria in acute periapical abscesses of primary teeth using culture-independent molecular methods based on 16S ribosomal DNA cloning. METHODS: Purulent material was collected from nine children diagnosed with abscesses of endodontic origin. DNA was extracted and the 16S rRNA gene amplified with universal primer pairs 27F and 1492R. Amplified genes were cloned, sequenced by Applied Biosystems, and identified by comparison with known 16S rRNA gene sequences. RESULTS: Bacterial DNA was present in all nine purulence samples. A total of 681 clones were classified into 8 phyla, 78 genera, and 125 species/phylotypes. The phyla were Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes, Actinobacteria, Tenericutes, Deinococcus-Thermus, and Spirochaetes. The most dominant genera were Streptococcus (13.3%), Fusobacterium (11.8%), Parvimonas (7.8%), Prevotella (6.7%), Sphingomonas (5.8%), and Hafnia (5.2%). Fusobacterium nucleatum (11.5%), Parvimonas micra (7.8%), Streptococcus intermedius (6.6%), Sphingomonas echinoides (5.3%), Hafnia alvei (5.2%), and Citrobacter freundii (4.9%) were the most common species/phylotypes. Among these species/phylotypes, F.nucleatum was the most prevalent (7/9). C. freundii, Carnobacterium maltaromaticum, and H. alvei were seldom detected species in acute periapical abscesses but had relatively high abundance and prevalence. CONCLUSION: Acute periapical abscesses are polymicrobial with certain prevalent bacteria, especially anaerobic bacterium. The most predominant and prevalent bacteria of acute periapical abscesses in children was F. nucleatum.

Application of Denaturing Gradient Gel Electrophoresis to the Analysis of Bacterial Communities Associated With Asymptomatic and Symptomatic Pericoronitis.

PURPOSE: Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities associated with asymptomatic and symptomatic pericoronitis. The aim of the study was to compare the fingerprinting patterns of these 2 clinical conditions. MATERIALS AND METHODS: The microbiota of mandibular third molar pockets associated with asymptomatic or symptomatic pericoronitis cases were collected and profiled by the polymerase chain reaction DGGE method. Banding patterns were compared by cluster analysis techniques. RESULTS: Thirteen symptomatic pericoronitis and 7 asymptomatic pericoronitis samples were collected. Comparative analysis of the 2 clinical conditions showed bands that were common to the symptomatic and asymptomatic cases, but most DGGE bands appeared to be unique to the clinical condition. No single band occurred in all profiles. The mean number of bands detected in the 16S rDNA community profiles was 23.8 ± 4.2 (range, 19 to 34) for samples from symptomatic cases and 24.1 ± 2.4 (range, 21 to 29) for those from asymptomatic cases. Cluster analysis and multidimensional scaling analysis of the DGGE banding pattern showed a distinction in the similarity of banding patterns according to the presence or absence of symptoms. CONCLUSIONS: These results suggest that the diversity of pericoronal pocket microbiota in asymptomatic pericoronitis cases differs markedly from that of symptomatic cases.

Preliminary molecular analysis of bacterial composition in periapical lesions with primary endodontic infections of deciduous teeth.

BACKGROUND: The bacterial composition of periapical lesions in deciduous teeth has not been well documented. This study was designed to explore the bacterial compositions, especially the dominant bacteria in periapical lesions using 16S rRNA sequencing. METHODS: Tissue samples were collected from 11 periapical lesions in deciduous teeth with primary endodontic infections. DNA was extracted from each sample and analyzed using 16S rRNA cloning and sequencing for the identification of bacteria. RESULTS: All DNA samples were positive for 16S rRNA gene PCR. One hundred and fifty-one phylotypes from 810 clones were identified to eight phyla, and each sample contained an average of 25.9 phylotypes. In addition, 59 phylotypes were detected in more than two samples, and Fusobacterium (F.) nucleatum (8/11), Dialister (D.) invisus (8/11), Campylobacter (C.) gracilis (7/11), Escherichia (E.) coli DH1 (6/11), Aggregatibacter (A.) segnis (6/11), and Streptococcus (S.) mitis (6/11) were the most prevalent species. Furthermore, 45 as-yet-uncultivated phylotypes were also identified. CONCLUSIONS: Chronic periapical lesions in deciduous teeth contained polymicrobial infections. F. nucleatum, D. invisus, C. gracilis, E. coli DH1, A. segnis, and S. mitis were the most prevalent species detected by 16S rRNA sequencing.

Polymerase chain reaction-denaturing gradient gel electrophoresis, cloning, and sequence analysis of bacteria associated with acute periapical abscesses in children.

INTRODUCTION: Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequencing were applied to the microbiologic study of acute periapical abscesses of endodontic origin in children to examine the predominant bacteria. METHODS: Purulent material was collected from 11 children diagnosed with acute abscesses of endodontic origin, and DNA was extracted to evaluate the predominant bacteria by using PCR-DGGE, cloning, and sequence analysis. RESULTS: Bacterial DNA was present in all of the 11 purulence samples. The microflora of clinical purulence samples were profiled by the PCR-DGGE method, and overall 17 bacterial genera were identified. The number of bacterial phylotypes in the purulence samples ranged from 1-8 (mean, 5.5). The most dominant genera found were Prevotella (24%), Fusobacterium (17.7%), Porphyromonas (13.9%), Lactobacillus (11.3%), Peptostreptococcus (8.3%), Streptococcus (6.4%), Eubacterium (3.8%), Campylobacter (3.3%), Treponema (2.6%), and Bulleidia (2.6%). CONCLUSIONS: The DGGE allowed visualization of the bacterial qualitative composition and revealed the major bacteria in the samples. The dominant bacteria associated with acute periapical abscess examined by PCR-DGGE, cloning, and sequencing methods are similar to those of culture-dependent studies. Although PCR-DGGE, cloning, and sequencing methods detected some bacteria at lower proportions than are unreported by culture methods, the method has the disadvantage of low resolution and is too time-consuming and laborious and more expensive.

[Sequence analysis of hemin-binding peptide derived from recombinant hemagglutinin-2 of Porphyromonas gingivalis].

OBJECTIVE: To determine the sequence of the active peptide derived from recombinant hemagglutinin (rHA-2) of Porphyromonas gingivalis (Pg). METHODS: The HA-2 gene from PgATCC33277 was cloned, expressed in Escherichia coli (Ec) BL21 (DE3), and purified. The purified recombinant protein was evaluated for its ability to bind hemin-linked agarose. The active peptide was subjected to endoproteinase-mediated sequence analysis. RESULTS: The protein expressed in Ec BL21 (DE3) was identified as PgHA-2 by plasmid sequence analysis, Western blotting, and mass spectrometry. The recombinant protein was confirmed functional by its ability to bind hemin. The sequence of the active peptide of rHA-2 was determined to be DHYAVMISKTGTNAG. CONCLUSIONS: The availability of sequence of the active peptide of rHA-2 provides a foundation for the development of immunoprophylactic and therapeutic agents against this human pathogen.

[Construction of the hemagglutinin-2-deficient mutant of Porphyromonas gingivalis].

OBJECTIVE: To construct and identify the Porphyromonas gingivalis(Pg)ATCC33277 hemagglutinin-2(HA-2)-deficient mutant. METHODS: The genomic DNA of Pg was isolated from PgATCC33277. The up/down stream genes of HA-2-HA(u), HA(l) were amplified by PCR, and inserted into pSY118 separately which contains a 2.1 kb antibiotic resistance ermF-ermAM cassette. The resultant recombinant plasmid-pSY118-HA was linearized as the gene targating fragment HA-ermF-ermAM and used in the electroporation of PgATCC33277. The Pg HA-2-deficient mutant was screened by allelic exchange. The test of aggregation of red blood cells was used to investigate the function change between PgHA2-deficient mutant and the wild type of PgATCC33277. RESULTS: The PgHA-2-deficient mutant was identified by PCR. The ability of Pg HA-2-deficient mutant to aggregate red blood cell was significantly decreased compared with the wild type. CONCLUSIONS: HA-2-deficient mutant of Pg ATCC33277 was constructed successfully, which lays a foundation for further study of its biological function.

Effectiveness of the B subunit of cholera toxin in potentiating immune responses to the recombinant hemagglutinin/adhesin domain of the gingipain Kgp from Porphyromonas gingivalis.

The hemagglutinin/adhesin HArep domain is present in the gingipains HRgpA and Kgp and in the hemagglutinin HagA of Porphyromonas gingivalis and is felt to be important in the virulence of this bacterium. In the present study, we determined the immunogenicity of recombinant HArep from the gingipain Kgp (termed Kgp-rHArep) and the effectiveness of the B subunit of cholera toxin (CTB), compared to other adjuvants in potentiating a specific response to Kgp-rHArep following intranasal (i.n.) immunization of mice. Furthermore, we determined the effectiveness of anti-Kgp-rHArep antibodies in protection against P. gingivalis invasion of epithelial cells. Evidence is provided that Kgp-rHArep was effective in inducing immune responses following systemic or mucosal immunization. Kgp-rHArep induced both a Th1- and Th2-type response following i.n. immunization. Immunization of mice with Kgp-rHArep and CTB, either admixed or chemically conjugated to the antigen, via the i.n. route, resulted in a significant augmentation of the systemic and mucosal immune response to Kgp-rHArep, which was similar to or higher than the responses seen in mice immunized with antigen and the other adjuvants tested. CTB and the heat-labile toxin of Escherichia coli potentiated a Th1- and Th2-type response to Kgp-rHArep, whereas the adjuvant monophosphoryl lipid A preferentially promoted a Th1-type response to the antigen. Furthermore, anti-Kgp-rHArep antibodies were shown to protect against P. gingivalis invasion of epithelial cells in an in vitro system. These results demonstrate the effectiveness of certain mucosal adjuvants in potentiating and in altering the nature of the immune response to Kgp-rHArep following i.n. immunization, and provide evidence for the potential usefulness of Kgp-rHArep for the development of a vaccine against periodontal disease.

Role of B7 costimulatory molecules in immune responses and T-helper cell differentiation in response to recombinant HagB from Porphyromonas gingivalis.

In addition to antigen-specific signals mediated through the T-cell receptor, T cells also require antigen nonspecific costimulation for activation. The B7 family of molecules on antigen-presenting cells, which include B7-1 (CD80) and B7-2 (CD86), play important roles in providing costimulatory signals required for development of antigen-specific immune responses. Hemagglutinin B (HagB) is a nonfimbrial adhesin of the periodontopathic microorganism Porphyromonas gingivalis and is thought to be involved in the attachment of the bacterium to host tissues. However, the immune mechanisms involved in responses to HagB and their roles in pathogenesis have yet to be elucidated. Therefore, the purpose of this study was to determine the role of B7 costimulatory molecules on T-helper-cell differentiation for the induction of immune responses to HagB. Mice deficient in either or both of the costimulatory molecules B7-1 and B7-2 were used to explore their role in immune responses to HagB after subcutaneous immunization. B7-1(-/-) mice had levels of immunoglobulin G (IgG) anti-HagB antibody activity in serum similar to those of wild-type mice, whereas lower serum IgG anti-HagB antibody responses were seen in B7-2(-/-) mice. Moreover, significantly lower numbers of IgG antibody-secreting cells and lower levels of CD4(+)-T-cell proliferation were observed in B7-2(-/-) mice compared to wild-type mice. No serum IgG response to HagB was detected in B7-1/B7-2(-/-) mice. Analysis of the subclass of the serum IgG responses and the cytokines induced in response to HagB revealed that B7-2(-/-) mice had significantly lower IgG1 and higher IgG2a anti-HagB antibody responses compared to wild-type mice. The B7-2(-/-) mice also had significantly reduced levels of interleukin-4 (IL-4) and IL-5 and enhanced level of gamma interferon. Furthermore, assessment of B7-1 and B7-2 expression on B cells and macrophages derived from wild-type BALB/c mice after in vitro stimulation with HagB revealed a predominant upregulation in the expression of the B7-2 costimulatory molecule on B cells and macrophages. Essentially no change was seen in the expression of B7-1. Taken together, these results suggest a critical role for B7, especially B7-2, for the preferential induction of a Th2-like response to HagB.

Mechanisms of monophosphoryl lipid A augmentation of host responses to recombinant HagB from Porphyromonas gingivalis.

Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection to P. gingivalis infection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 microg) alone or with MPL (25 microg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P < 0.05) and serum IgG (P < 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P < 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was approximately 1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.

Hydrolysis of epithelial junctional proteins by Porphyromonas gingivalis gingipains.

Porphyromonas gingivalis has been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivalis can degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis (i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivalis cells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivalis cells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis.