• Characterization of the response of GM-CSF supplemented THP-1 human monocytes to LPS of oral microorganisms

      Baqui, A. A. M. Abdullahel; Falkler, William A., Ph.D.; Meiller, Timothy F. (1996)
      The effect of granulocyte macrophage colony stimulating factor (GM-CSF) on the differentiation and activation of a human monocyte cell line (THP-1) in the presence of lipopolysaccharide (LPS) from oral organisms has not been investigated. It was hypothesized that GM-CSF treated THP-1 cells are immunologically and functionally hyperactivated in the presence of LPS of oral microorganisms. A study was undertaken to elucidate the immunological expression of activation antigens and production of cytokines by THP-1 cells after treatment with GM-CSF and in response to LPS of the putative periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). LPS of F. nucleatum and P. gingivalis was prepared, characterized by SDS PAGE, standardized by protein concentration and tested for endotoxin content. Morphological changes in THP-1 cells were observed with light, immunofluorescence (IF) and transmission electron microscopy (TEM), following treatment with LPS/PMA (phorbol-12-myristate-13 acetate) and/or GM-CSF at various concentrations and time intervals. The expression of seven different activation antigens namely, CD-11b, CD-11c, CD-14, CD-35, CD-68, CD-71 and HLA-DR, in THP-1 cells was evaluated in this experimental model. Direct labeling of THP-1 cell activation antigens was performed using the Vectastain ABC-AP staining kit with light microscopy. Alternatively, LPS/PMA and/or GM-CSF stimulated and unstimulated cells were stained with FITC labelled antibodies for IF and gold-labelled antibodies were used for TEM. To evaluate chemotaxis as a functional variation in THP-1 cells, an assay was performed using a microchemotaxis chamber and polyvinylpyrolidone free polycarbonate membrane filters. To determine phagocytic activity in the experimental model, an assay was performed using FITC labelled S. cereviseae. Phagocytic uptake of cells was determined with the use of a fluorescence microscope. Production of extracellular cytokines, TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12, by THP-1 cells in the experimental model, was measured by ELISA. Reverse transcription polymerase chain reaction (RT-PCR) of the of TNF-alpha, IL-1 beta and IL-6 cytokines was performed to correlate the cytokine gene transcription with cytokine gene translation (ELISA). Three percent of untreated THP-1 cells expressed HLA-DR; 9%, CD-11b; 8%, CD-11c; 22%, CD-14; 9%, CD-35 and 7%, CD-68 antigens. CD-71 was not expressed in untreated THP-1 cells. Treatment with LPS of F. nucleatum and P. gingivalis and PMA increased the expression of activation antigens. Following treatment with combined GM-CSF and LPS of P. gingivalis and F. nucleatum there was a significant (p<0.05) up-regulation and expression of HLA-DR, CD-11b, CD-11c, CD-35 and CD-71 activation antigens over baseline values. Expression of the LPS receptor, CD-14, was significantly (p<0.05) down-regulated by this treatment for 1-2 d and then up-regulated at 2-4 d. Antigens important in phagocytosis, CD-11b and CD-35, were significantly (p<0.05) up-regulated by GM-CSF. The up-regulation was further demonstrated in phagocytosis functional assays. Stimulation with combined GM-CSF and oral LPS resulted in a significant (p<0.05) escalation in phagocytosis by the THP-1 cells. There was a two-fold increase in chemotactic response with GM-CSF treatment by 4 d, which decreased after 7 d. RT-PCR data indicated that TNF-alpha transcripts were constituitively produced in the THP-1 cell but that translation to a high level of production of functional cytokines required the LPS/GM-CSF stimulus. Gene transcription for IL-6 was detected as early as 5 min post stimulation. (Abstract shortened by UMI.)
    • Molecular characterization and epidemiological analysis of Fusobacterium nucleatum and the Fusobacterium species

      George, Kimberly Sue; Falkler, William A., Ph.D. (1995)
      Fusobacterium nucleatum is the most frequently isolated organism from the subgingival plaque of periodontally diseased patients. DNA-DNA homology, SDS PAGE analysis, and enzyme electrophoretic migration patterns have resulted in the creation of four distinct subspecies of F. nucleatum, however, additional subgroups may exist. Experiments using AP-PCR were performed to determine if the members of the genus Fusobacterium could be differentiated and the results revealed that each of the nine species tested demonstrated a unique fingerprint using two different random primers. Common amplicons were observed among the Fusobacterium species and F. nucleatum which may represent conserved regions of Fusobacterium genomes. Experiments screening seven of the Fusobacterium species against a panel of eleven periodontal microorganisms for coaggregating ability revealed that F. periodonticum and F. necrophorum shared coaggregation activity with F. nucleatum and that coaggregating ability may be a useful criterion in differentiating members of the Fusobacterium species. Dot immunobinding studies revealed that antigens were shared among most of the Fusobacterium with the exception of F. ulcerans. The type strains of each subspecies of F. nucleatum were readily differentiated using their unique AP-PCR fingerprints, however, additional clinical F. nucleatum isolates demonstrated heterogeneous AP-PCR patterns and the isolates could not be assigned to one of the subspecies based on AP-PCR fingerprints. Similar genotypes were observed between F. nucleatum isolates from different states of health and disease, from different individuals, and from different geographical locations indicating that F. nucleatum isolates demonstrated characteristics associated with a clonal species. Up to four different genotypes were distinguished from isolates from the same oral cavity and up to three different genotypes were observed within an single site. An intense amplicon at approximately 450 bp generated in AP-PCR amplifications with primer C2 was associated with F. nucleatum subsp. nucleatum (ATCC 25586; the type strain that has been associated with disease) and with several F. nucleatum isolates from diseased sites suggesting that this amplicon may be an indicator of F. nucleatum genotypes associated with disease. The AP-PCR patterns of F. nucleatum isolates from dogs, cats, and monkeys were distinct from the AP-PCR patterns of F. nucleatum isolates from humans, however, a strong similarity was observed among the amplification patterns generated by monkey and human isolates suggesting that monkey isolates are genetically similar to human F. nucleatum isolates and that monkey isolates may have their own genetic cluster. Common amplicons were observed among all F. nucleatum isolates regardless of their source of isolation which may reflect conserved regions of F. nucleatum genomes. Unique amplicons were observed among the F. nucleatum isolates from humans, dogs, cats, and monkeys, suggesting that these fragments may be useful in differentiating F. nucleatum isolates from different animal species. Pulsed field gel electrophoresis (PFGE) of isolates from patient JMG6 confirmed the AP-PCR genotypes and indicated that PFGE offered increased discriminatory power over AP-PCR. A repeated DNA sequence was observed among F. necrophorum strains but was absent from F. nucleatum strains. (Abstract shortened by UMI.)