Journal:

Article Title: Expression of the Peptide Antibiotic Human ?-Defensin 1 in Cultured Gingival Epithelial Cells and Gingival Tissue

doi:

Figure Lengend Snippet: Expression of hBD-1 mRNA in cultured HGE cells stimulated with F. nucleatum cell wall extract (FN) as analyzed by 35 cycles of RT-PCR. HGE cells were cultured in serum-free keratinocyte growth medium and incubated overnight with 100-μg/ml F. nucleatum cell wall extract (+) or were unstimulated (−). DNase I was used in some samples to digest the genomic DNA possibly contaminating the RNA samples. PCR products, as described in Materials and Methods, were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. For the locations of the four primers, see Fig. ​Fig.2.2. −RT denotes a control in which the reverse transcriptase enzyme was omitted. The water lane was the negative control. The molecular sizes of PCR products from RNA samples and the hBD-1 plasmid (plasmid lane) were predicted from the sequences and were in accordance with the molecular size markers (φX174RF HaeIII fragments [not shown]).

Article Snippet: Anaerobic cultures of Fusobacterium nucleatum ATCC 25586 and Porphyromonas gingivalis ATCC 33277 were grown, and crude cell walls were prepared by differential centrifugation as previously described ( 24 ).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Incubation, Electrophoresis, Agarose Gel Electrophoresis, Staining, Negative Control, Plasmid Preparation

Journal:

Article Title: Expression of the Peptide Antibiotic Human ?-Defensin 1 in Cultured Gingival Epithelial Cells and Gingival Tissue

doi:

Figure Lengend Snippet: (A) Constitutive expression of hBD-1 mRNA in cultured HGE cells stimulated (Stim.) either with 10- or 100-μg/ml P. gingivalis (P.g.) or F. nucleatum (F.n.) cell wall extract in the presence or absence of 1% human serum or with 10-ng/ml PMA (P) overnight. C represents the control lane for PMA (only dimethyl sulfoxide, used as the vehicle for PMA, was added). PCR amplification was performed for 28 or 35 cycles by using primer pairs for hBD-1 (5.1-3.1 primer pair), IL-8 (a control marker indicating cellular response), and RPO, a control housekeeping gene for equivalent loading. It was evident that the apparent decrease in hBD-1 mRNA from HGE cells challenged by 100-μg/ml F. nucleatum cell wall extract in the absence of serum resulted from loading less of the RNA sample in the RT reaction mixture, since this lane gave a consistently lower signal. The sizes of PCR products were as predicted. Note the reduced signal for IL-8 with the higher dose of P. gingivalis. (B) Constitutive expression of hBD-1 mRNA in cultured HGE cells stimulated overnight either with various doses (1, 10, or 100 ng/ml) of E. coli LPS in the presence or absence of 1% human serum or with TNF-α (1, 10, or 100 ng/ml) in the absence of human serum. Three different primer pairs were included in the PCR amplification for 28 or 35 cycles, as for panel A. Note that hBD-1 expression is consistent in all lanes.

Article Snippet: Anaerobic cultures of Fusobacterium nucleatum ATCC 25586 and Porphyromonas gingivalis ATCC 33277 were grown, and crude cell walls were prepared by differential centrifugation as previously described ( 24 ).

Techniques: Expressing, Cell Culture, Amplification, Marker

Journal:

Article Title: Expression of the Peptide Antibiotic Human ?-Defensin 1 in Cultured Gingival Epithelial Cells and Gingival Tissue

doi:

Figure Lengend Snippet: hBD-1 is expressed in cultured HGE cells and not HGFs. No hBD-1 expression was detected in control (cont.) HGF cells and cells stimulated with 100-μg/ml F. nucleatum (F.n.) or P. gingivalis (P.g.) cell wall extract overnight (HGFs from passage 22). Unlike HGFs, both control and stimulated cultured HGE cells constitutively expressed hBD-1 mRNA. PCR amplification was done for 35 cycles to enhance the detection of a small signal in the HGFs. A housekeeping gene (RPO) served as a positive control for RT-PCR and RNA quality in samples. Similar results were obtained when primary HGFs were either left unstimulated or stimulated with E. coli LPS and TNF-α and then tested for hBD-1 expression.

Article Snippet: Anaerobic cultures of Fusobacterium nucleatum ATCC 25586 and Porphyromonas gingivalis ATCC 33277 were grown, and crude cell walls were prepared by differential centrifugation as previously described ( 24 ).

Techniques: Cell Culture, Expressing, Amplification, Positive Control, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Fusobacterium nucleatum Increases Collagenase 3 Production and Migration of Epithelial Cells

doi: 10.1128/IAI.73.2.1171-1179.2005

Figure Lengend Snippet: F. nucleatum preferentially binds to marginal cells of artificial epithelial wounds and increases their migration. Scratch wounds were created to confluent HaCaT cell cultures, and F. nucleatum cells were added to the culture medium (260 bacteria per cell) for different time periods. Epifluorescence of cultures detected with anti-F. nucleatum antibody after 6 h (A), 24 h (B) and 48 h (C). Light microscopic images of wounds at 48 h in the presence of TGF-β indicate faster closure of the epithelial wound sheets in F. nucleatum-treated cultures than in control cultures (D).

Article Snippet: When added to cultures over 90% of the bacteria were dead. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Bacterium Strain Source Porphyromonas gingivalis RHI 3610 (ATCC 33277) Gingival pocket Porphyromonas endodontalis AHN 4610 Tonsils RHI 3609 (ATCC 35406) Root canal Prevotella buccae AHN 10652 Gingival pocket Prevotella oris AHN 19550 Root canal Prevotella intermedia AHN 18240 Odontogenic abscess Prevotella denticola AHN 10695 Gingival pocket RHI 3606 (ATCC 33185) Maxillary atrium Prevotella nigrescens AHN 18826 Odontogenic abscess Prevotella loeshii AHN 10628 Saliva Actinobacillus actinomycetemcomitans JP-2 Gingival pocket Fusobacterium nucleatum AHN 19959 Odontogenic abscess RHI 4185 (ATCC 25586) Cervicofacial lesion Fusobacterium necrophorum AHN 12454 Odontogenic abscess RHI 3624 (ATCC 25286) Bovine liver abscess Mitsuokella dentalis AHN 12573 Root canal Campylobacter rectus AHN 19728 Odontogenic abscess Open in a separate window Bacteria used in this study and their sources

Techniques: Migration, Bacteria, Control

Journal:

Article Title: Fusobacterium nucleatum Increases Collagenase 3 Production and Migration of Epithelial Cells

doi: 10.1128/IAI.73.2.1171-1179.2005

Figure Lengend Snippet: Intracellular F. nucleatum colocalizes with lysosomal membrane structures in cells at the leading edge of the epithelial sheets. Scratched HaCaT cell cultures treated for 48 h with F. nucleatum (ATCC 25586; 260 bacteria per cell) were simultaneously immunostained for F. nucleatum and lysosomal membrane protein LAMP-1, followed by two fluorescent secondary antibodies, Alexa-488 and Alexa-546, respectively. Samples were analyzed by laser confocal microscopy, and optical z-axis sections were recorded. One optical section from the basal, mid-, and apical regions of the cells were presented together. Red channel (A and B) corresponding to Alexa-546 fluorescence reveals F. nucleatum invasion, and green channel (C), corresponding to Alexa-488 fluorescence, reveals LAMP-1 expression. Red and green images were merged into an RGB file (D) showing colocalization (yellow/orange) of the two signals.

Article Snippet: When added to cultures over 90% of the bacteria were dead. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Bacterium Strain Source Porphyromonas gingivalis RHI 3610 (ATCC 33277) Gingival pocket Porphyromonas endodontalis AHN 4610 Tonsils RHI 3609 (ATCC 35406) Root canal Prevotella buccae AHN 10652 Gingival pocket Prevotella oris AHN 19550 Root canal Prevotella intermedia AHN 18240 Odontogenic abscess Prevotella denticola AHN 10695 Gingival pocket RHI 3606 (ATCC 33185) Maxillary atrium Prevotella nigrescens AHN 18826 Odontogenic abscess Prevotella loeshii AHN 10628 Saliva Actinobacillus actinomycetemcomitans JP-2 Gingival pocket Fusobacterium nucleatum AHN 19959 Odontogenic abscess RHI 4185 (ATCC 25586) Cervicofacial lesion Fusobacterium necrophorum AHN 12454 Odontogenic abscess RHI 3624 (ATCC 25286) Bovine liver abscess Mitsuokella dentalis AHN 12573 Root canal Campylobacter rectus AHN 19728 Odontogenic abscess Open in a separate window Bacteria used in this study and their sources

Techniques: Membrane, Bacteria, Confocal Microscopy, Fluorescence, Expressing

Journal:

Article Title: Fusobacterium nucleatum Increases Collagenase 3 Production and Migration of Epithelial Cells

doi: 10.1128/IAI.73.2.1171-1179.2005

Figure Lengend Snippet: Time dependence of collagenase 3 secretion by epithelial cells treated with F. nucleatum. The bacteria were added to serum-free culture medium of semiconfluent HaCaT cells (260 bacteria per cell). Aliquots of the medium were analyzed for collagenase 3 at different time points by Western blotting. Control cells (CTL) were cultured for 24 h in the absence of the bacteria. Relative levels of pro-collagenase 3 protein were quantified by densitometric scanning.

Article Snippet: When added to cultures over 90% of the bacteria were dead. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Bacterium Strain Source Porphyromonas gingivalis RHI 3610 (ATCC 33277) Gingival pocket Porphyromonas endodontalis AHN 4610 Tonsils RHI 3609 (ATCC 35406) Root canal Prevotella buccae AHN 10652 Gingival pocket Prevotella oris AHN 19550 Root canal Prevotella intermedia AHN 18240 Odontogenic abscess Prevotella denticola AHN 10695 Gingival pocket RHI 3606 (ATCC 33185) Maxillary atrium Prevotella nigrescens AHN 18826 Odontogenic abscess Prevotella loeshii AHN 10628 Saliva Actinobacillus actinomycetemcomitans JP-2 Gingival pocket Fusobacterium nucleatum AHN 19959 Odontogenic abscess RHI 4185 (ATCC 25586) Cervicofacial lesion Fusobacterium necrophorum AHN 12454 Odontogenic abscess RHI 3624 (ATCC 25286) Bovine liver abscess Mitsuokella dentalis AHN 12573 Root canal Campylobacter rectus AHN 19728 Odontogenic abscess Open in a separate window Bacteria used in this study and their sources

Techniques: Bacteria, Western Blot, Control, Cell Culture

Journal:

Article Title: Fusobacterium nucleatum Increases Collagenase 3 Production and Migration of Epithelial Cells

doi: 10.1128/IAI.73.2.1171-1179.2005

Figure Lengend Snippet: Up-regulation of epithelial cell-associated collagenase 3 expression by F. nucleatum. HaCaT cells were cultured to full confluence and then wounded by scratching with a pipette tip. They were then cultured in the presence of F. nucleatum (ATCC 25586; 260 bacteria per cell) for 12 h. The cells were washed and simultaneously immunostained for F. nucleatum and collagenase 3, followed by two fluorescent secondary antibodies, Alexa-488 and Alexa-546, respectively. Alexa-546 fluorescence reveals F. nucleatum invasion in certain cells in the wound margin (A, arrows). Alexa-488 fluorescence reveals collagenase 3 staining in the infected cells (B, arrowheads). Combination of the two images shows no clear colocalization of the signals (C).

Article Snippet: When added to cultures over 90% of the bacteria were dead. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Bacterium Strain Source Porphyromonas gingivalis RHI 3610 (ATCC 33277) Gingival pocket Porphyromonas endodontalis AHN 4610 Tonsils RHI 3609 (ATCC 35406) Root canal Prevotella buccae AHN 10652 Gingival pocket Prevotella oris AHN 19550 Root canal Prevotella intermedia AHN 18240 Odontogenic abscess Prevotella denticola AHN 10695 Gingival pocket RHI 3606 (ATCC 33185) Maxillary atrium Prevotella nigrescens AHN 18826 Odontogenic abscess Prevotella loeshii AHN 10628 Saliva Actinobacillus actinomycetemcomitans JP-2 Gingival pocket Fusobacterium nucleatum AHN 19959 Odontogenic abscess RHI 4185 (ATCC 25586) Cervicofacial lesion Fusobacterium necrophorum AHN 12454 Odontogenic abscess RHI 3624 (ATCC 25286) Bovine liver abscess Mitsuokella dentalis AHN 12573 Root canal Campylobacter rectus AHN 19728 Odontogenic abscess Open in a separate window Bacteria used in this study and their sources

Techniques: Expressing, Cell Culture, Transferring, Bacteria, Fluorescence, Staining, Infection

Journal:

Article Title: Fusobacterium nucleatum Increases Collagenase 3 Production and Migration of Epithelial Cells

doi: 10.1128/IAI.73.2.1171-1179.2005

Figure Lengend Snippet: Effect of p38 MAP kinase inhibitor on F. nucleatum-induced collagenase 3 expression in epithelial cells. HaCaT cells were preincubated for 30 min with 10 μM SB203580 (inhibitor of p38) and then cultured in the presence of F. nucleatum (ATCC 25586; 160 bacteria per cell) for 24 h. Culture medium was analyzed by Western blotting, using anti-collagenase 3 antibodies, and RNA extracted from the cells (20 μg) was analyzed for collagenase 3 mRNA levels by Northern blotting.

Article Snippet: When added to cultures over 90% of the bacteria were dead. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Bacterium Strain Source Porphyromonas gingivalis RHI 3610 (ATCC 33277) Gingival pocket Porphyromonas endodontalis AHN 4610 Tonsils RHI 3609 (ATCC 35406) Root canal Prevotella buccae AHN 10652 Gingival pocket Prevotella oris AHN 19550 Root canal Prevotella intermedia AHN 18240 Odontogenic abscess Prevotella denticola AHN 10695 Gingival pocket RHI 3606 (ATCC 33185) Maxillary atrium Prevotella nigrescens AHN 18826 Odontogenic abscess Prevotella loeshii AHN 10628 Saliva Actinobacillus actinomycetemcomitans JP-2 Gingival pocket Fusobacterium nucleatum AHN 19959 Odontogenic abscess RHI 4185 (ATCC 25586) Cervicofacial lesion Fusobacterium necrophorum AHN 12454 Odontogenic abscess RHI 3624 (ATCC 25286) Bovine liver abscess Mitsuokella dentalis AHN 12573 Root canal Campylobacter rectus AHN 19728 Odontogenic abscess Open in a separate window Bacteria used in this study and their sources

Techniques: Expressing, Cell Culture, Bacteria, Western Blot, Northern Blot