• Characterization of HSV-1 antigens expressed on human Langerhans cells

      Meiller, Timothy F.; Falkler, William A., Ph.D. (1992)
      Langerhans cells (LC) are immunocompetent antigen presenting cells (APC) which are involved with many infections including those caused by herpes simplex virus (HSV). Viral glycoproteins (g) on the surface of infected cells have been studied in established cell lines. The identification of which viral g participate with LC processing and antigen presentation on APC has not been studied. It is hypothesized that specific HSV-1 viral g species can be identified on the surface of LC very early after viral challenge of LC and are important antigens (Ag) involved in the human immune response to this virus. The purpose of this investigation has been to elucidate the expression of HSV-1 Ag, in vitro, using freshly isolated human LC. These experiments have established techniques for the isolation and enrichment of human LC that retain for up to 18 h post enrichment all of the ultrastructural characteristics and cell markers consistent with those of LC. These LC have been shown to adsorb and replicate HSV-1 virus at a low copy number. Immunofluorescence and ELISA have demonstrated the presence of viral antigens on the surface of membrane fractions of LC 8 h post infection using polyvalent antiserum. Specific viral epitopes related to gD and gE have been identified for the first time on HSV-1 challenged LC at 5 h post infection by two complementary techniques. SDS-PAGE of virally challenged LC membranes demonstrated the emergence of proteins consistent with the electrophoretic mobilities of gD, gE and possibly gH as early as 5 h post infection. Immunoblotting (IB) with monoclonal antibodies (Mab) confirmed the presence of these g in membrane preparations. Dual colloidal gold immunolabelling for transmission electronmicroscopy (TEM), utilizing Mab to LC markers and to epitopes found on viral g demonstrated visual confirmation of the presence of sites reactive with antibodies to gD and gE on the surface of LC 5 h after viral challenge. Although present later in infection larger proteins gB and gC do not appear to be involved in early membrane changes of LC. An epitope of gH was identified by IB, however, visual confirmation by TEM was not accomplished. The 5 h post infection LC, therefore, selectively express epitopes of gD, gE, and gH which may be involved in immunoactivation, particularly in light of the evidence supporting gD as a potent stimulator of neutralizing antibodies. Presentation of such viral antigens by LC would be an early step in this process.
    • 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.)
    • In vitro production of human antibodies reactive with periodontal pathogens, using Epstein-Barr virus immortalization of B lymphocytes derived from peripheral blood of periodontal patients, with an emphasis on the production of a human monoclonal antibody reactive with the heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans

      Raulin, Leslie A.; Falkler, William A., Ph.D. (1995)
      The purpose of this research was to develop cell line(s) that produce a human monoclonal antibody (huMAb) specific to an oral pathogenic microorganism using Epstein-Barr virus (EBV) transformation of B cells isolated from the gingiva and the peripheral blood of periodontal patients with active disease. The resulting huMAB was then characterized. Excised gingival tissue was obtained from 6 patients and peripheral blood was obtained from 3 of the same patients. To release cells, the gingival tissue was digested with collagenase. Gingival mononuclear cells (GMC) and peripheral blood mononuclear cells (PBMC) were then separated from the gingival extracts, patient peripheral blood, and leukophoresed blood from an anonymous donor by Ficoll centrifugation. The resulting cells were infected with EBV, cultured in 96-well plates, and observed for foci of transformation. Using a dot-immunobinding assay (DIB), the cell culture supernatant fluids (CCS) of transformation-positive wells were tested for Ab reactivity to a panel of 11 microorganisms. Continuously positive cultures were expanded and cloned by limiting dilution. Using an ELISA, clone CCS was tested for Ab reactivity to the microorganism to which the originating culture had exhibited reactivity (target organism). Positive responders were expanded and retested with the ELISA. Continuously producing clones were expanded further, CCS was collected, and excess cells were cryopreserved. The class of Ab in each CCS was determined with a DIB, IgG subclasses and concentration were determined with a commercial ELISA, and the light chain type was determined with a fixed cell indirect immunofluorescence assay (IFA). Using representative strains of the target microorganism, Western blots were performed (separation of bacterial proteins by SDS-PAGE, electroblotting onto nitrocellulose, and probing with concentrated CCS) to determine the MW of the band(s) to which the human antibodies (huAbs) bound. To exclude the possibility of non-specific Ig binding by the target organism, a Western blot was performed with a huMAb of the same IgG subclass and light chain type but with specificity toward a viral protein.;GMC did not transform. Of 517 wells receiving PBMC, 503 wells (97%) had foci of transformation. CCS from 71 of the 517 (14%) wells initially tested positive for Ab activity, most commonly to Eikenella corrodens, Actinobacillus actinomycetemcomitans (Aa), Bacteroides fragilis, Peptostreptococcus micros, and Campylobacter rectus, in order of decreasing frequency. Subsequent DIB during expansion of the cells demonstrated 28, 12, 7, 3, and finally 1 culture with Ab activity (II-24P, reactive with Aa ATCC 43718).;A similar (pilot) experiment was performed with PBMC from 2 healthy control volunteers and from leukophoresed blood. Although initial Ab-producing cultures were obtained, none of the clones produced significant amounts of Ab. (Abstract shortened by UMI).
    • Microbiological and serological studies of Peptostreptococcus micros

      Turng, Been-Foo; Falkler, William A., Ph.D. (1996)
      Peptostreptococcus micros, an anaerobic gram-positive coccus, has been associated with many polymicrobial infections at various locations of the human body, especially with periodontal and endodontic lesions of the oral cavity. Very little is known about the biology and pathogenicity of P. micros. In order to microbiologically characterize P. micros strains, the following studies were undertaken. P. micros was tested for its aerotolerance. P. micros was able to survive beyond 18 d of exposure to air losing only 2-4 logs in viability. A selective medium (PMM) for the primary isolation of P. micros was developed and evaluated. Using PMM, the prevalence of P. micros in periodontally diseased patients and the association of P. micros with halitosis patients were investigated. PMM was effective in differentiating 80 strains of P. micros from other oral microorganisms by the production of hydrogen sulfide after the utilization of glutathione resulting in a black precipitate directly under the colony. Significantly higher numbers and occurrences (P<0.05) of P. micros were isolated from the periodontal pockets of 12 adult or 18 early onset periodontitis patients than from healthy sites. In addition, P. micros was isolated from saliva, the surface of the tongue and supragingival plaque samples of 12 halitosis patients. The antibiotic susceptibility profiles of P. micros, detection of beta-lactamase and the effectiveness of mouthrinse products against P. micros were studied. Strains of P. micros tested were susceptible to 6 different antibiotics, higher MIC levels being observed with the cephems using three methods. There was no detectable beta-lactamase activity observed. A chlorhexidine gluconate mouthrinse solution inhibited the growth of P. micros. Hemolytic activity of P. micros was observed with only 2 out of 72 strains of P. micros tested. Using the CAMP test, the hemolytic activity of the P. micros strains was enhanced. P. micros attached to human buccal epithelial cells and to human, rabbit, horse, or sheep erythrocytes. The mechanisms involved in the coaggregation between P. micros and Fusobacterium nucleatum were studied. Coaggregation was inhibited by the addition of EDTA and 5 different amino acids but not by sugars. A higher affinity of coaggregation was observed between isolates of P. micros and F. nucleatum from the same periodontal lesions. All 21 human sera tested showed a positive reaction with P. micros by the dot-immunoblot assay. Cellular proteins of P. micros were separated using a 15% SDS-PAGE gel and the immunodominant antigens of P. micros and the subclass of human immunoglobulin G (IgG) reacting to these antigens determined. IgG2 was the major human IgG subclass reacting with the immunodominant antigens. The high aerotolerance, distribution throughout the human oral cavity, ability to attach to erythrocytes and human buccal epithelial cells and to coaggregate with F. nucleatum participate in allowing of P. micros to be involved in oral disease processes. (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.)
    • Role of cytokines in Campylobacter jejuni infection and immunity in mice

      Baqar, Shahida; Rollwagen, Florence M.; Falkler, William A., Ph.D. (1991)
      Cytokines incorporated into agarose blocks and implanted subcutaneously into mice established an in vivo gradient which can be used to mimic a local inflammatory process. A model was developed in which cellular influx into cytokine impregnated blocks paralleled the normal cellular reaction to infections or wounds. Agarose blocks containing antigens of the intestinal pathogen, C. jejuni, were implanted in normal and infected mice. Kinetics of cellular influx into the blocks showed an early influx of lymphocytes in infected mice only. The two groups showed similar but discrete patterns of cytokine secretion within the blocks. Infected, but not normal mice, were actively synthesizing antibodies to C. jejuni at the local site (within the blocks), whereas the levels of total immunoglobulin were similar for the two groups. The results suggested that the agarose block model can be successfully applied to study local cytokine production and the role of various cells in infectious diseases. The primary infection with C. jejuni in mice resulted in the generation of sensitized B and T cells which could be boosted by rechallenge with homologous bacteria. These results indicated the presence of a functional immunity to C. jejuni challenge in mice. An antibody secreting cell assay allowed the early detection of specific antibody producing cells in circulation. In addition, data suggested a homing pattern of B cells to spleen as well as Peyer's patches. The profile of cytokine production at a local site (intestine) and in the circulation is suggestive of a role of local IL-1 and local as well as systemic IL-6 in immunity and in the pathogenesis of Campylobacter. Crude cytokine suspensions, when fed orally before challenging with C. jejuni, reduced the bacterial load from the gut and also augmented both humoral and cellular immune responses to the bacterial antigens. Oral treatment of mice with rIL-6 had an immediate effect in reducing the bacterial load whereas this effect was delayed in mice treated with rIL-5. Although IL-6 and IL-5 enhanced local as well as systemic antibodies to C. jejuni, these provided only a partial protection from rechallenge with homologous bacteria. On the other hand, mice treated with rIL-2 before challenge had better protection and a faster clearance of bacteria from their guts. In none of the groups could DTH to bacterial antigens be demonstrated, however, the lymphocytes responded to in vitro by stimulation with the same antigens. The data suggested that various cytokines have potential for use as immune modifiers to enhance the immune response to various enteric pathogens. (Abstract shortened with permission of author.)