T. J. Sims

Effects of prostaglandin on the antigen- and mitogen-driven responses of peripheral blood lymphocytes from patients with adult and juvenile periodontitis☆

Abstract Peripheral blood lymphoid cells were obtained from individuals with juvenile periodontitis or the adult form of the disease and from normal control subjects and were cultured with and without bacterial substances or polyclonal activators. Blastogenic response and cytotoxic factor production were measured and the effectiveness of prostaglandin- E 2 in inhibiting these cell functions was evaluated. The data show that cells obtained from individuals with juvenile periodontitis in general respond to stimulation in vitro more vigorously and are suppressed by prostaglandin E 2 less effectively than cells from individuals with the adult form of the disease or cells from normal control individuals. Measurement of activated lymphoid cell responses in the presence and absence of PGE 2 may have diagnostic usefulness in distinguishing individuals with the juvenile form from those with adult forms of the disease.

Effects of cell concentration and exogenous prostaglandin on the interaction and responsiveness of human peripheral blood leukocytes.

Abstract The role of cell-to-cell interactions and the effects of endogenous and exogenous prostaglandin on the mitogenic and antigenic responsiveness of peripheral blood leukocytes from five adult human subjects were evaluated. Cells were activated by phytohemagglutinin (PHA) or a homogenate of Actinomyces viscosus (AVIS), a gram-positive suspected pathogen, in the presence or absence of prostaglandin E2 (PGE2) or indomethacin. A cell concentration range from 0.1 to 10 × 105 was studied using flat-bottom microtest wells. Cells from all five donors, who ranged in age from 33 to 46 years and who were healthy and clinically and radiographically free of inflammatory periodontal disease, could be induced to respond to AVIS blastogenically by increasing the cell concentration. PGE2 at 10 μM suppressed responsiveness to AVIS at all cell concentrations, while the PHA response was suppressed at some concentrations and enhanced at others. Regression analysis of the log-log transformation of the data indicated that a single cell type may be responsible for the PHA response. In contrast, the responses to AVIS appeared to be more complex. In order to obtain a significant DNA synthetic response, 5- to 10-fold more cells were required than for PHA, and two cell types participated.

An improved multimembrane microassay for quantitating the motility of granulocytes and monocytes labeled with chromium-51☆

Various modifications of the Boyden chamber chemotaxis assay have been used to screen patients for abnormalities in granulocyte or monocyte motility. In most cases, cell motility has been assessed by quantitating the fraction of cells that migrates from an upper chamber through a filter toward a lower chamber containing chemoattractant. Existing versions of the assay have several shortcomings. They are labor-intensive, require relatively large numbers of cells and lengthy incubation, or they require visual cell counting and do not permit assessment of cells which may drop off the filter into the attractant medium. We have improved the accuracy and efficiency of existing microchamber assays by using 51Cr-labeled cells to eliminate microscopic cell counting, shortening the incubation time, adjusting the assay sensitivity, and accounting for cells which drop off into the attractant well. The modified method uses Neuroprobe multiwell microchambers and two 10 microns polycarbonate filters with 3 microns pores on top of one 100 microns nitrocellulose filter. The optimal incubation period is 60 min, and the assay requires about one-fifth as many cells as the standard Boyden chamber methods. Cell drop-off can be measured accurately by harvesting the attractant wells with detergent, and the assay sensitivity is comparable to that of existing radiometric assays using large chambers. The data indicate that the range of chemotactic and random motility of normal granulocytes and monocytes measured in the modified assay system is comparable to that reported for studies which have used established motility assays.

Antigenic variation and cross-reactivity in Bacteroides forsythus clinical isolates detected by western blot

Bacteroides forsythus is one of the etiologic agents of destructive periodontal diseases. Determining which antigenic components of the bacterium are recognized in the immune response of periodontitis patients is an important step in assessing strategies for vaccine development. The aim of this study was to identify the major strain-variable and cross-reactive antigens of B. forsythus clinical isolates recognized by serum IgG from patients with early-onset rapidly progressive periodontitis. Ten patient sera with measurable IgG against antigenic components of the species were identified by Western blot. Positive sera were tested by checkerboard ELISA to identify those most responsive to strain-variable antigens in nine clinical isolates and ATCC strain 43037. Correlation analysis of the ELISA data suggested that different subsets of isolates were preferentially recognized by different sera. Western blots revealed that certain sera also recognized major shared components across all the isolates, but preferential recognition of different isolate subsets by different patients was clearly confirmed. To determine if the variable antigens recognized were nonprotein, proteinase K-digested isolates were compared to undigested controls by Western blot. The main strain-variable antigens were proteinase resistant, while proteins at 200 and 210 kDa were identified as the major shared components. Two-dimensional SDS-PAGE revealed that these proteins are the quantitatively dominant heat-modifiable components of the cell envelope. Even though variable antigens are prominent in the immune response of patients, a cross-protective vaccine based on the shared envelope proteins of B. forsythus seems feasible in light of these observations.

An improved method for assessing the incorporation of labeled precursors into DNA by human mononuclear cells

The blastogenic responsiveness of activated lymphoid cells is usually assessed in vitro by measuring the incorporation of radioactive thymidine or iododeoxyuridine, a thymidine analog, into DNA. The accuracy of this method is compromised by the presence in activated and unactivated lymphocytes and in some of the substances used to activate them, of degradative enzymes which compete with DNA synthetase, the incorporation efficiency of exogenous precursor is inherently low. We have done studies aimed at improving both the efficiency and the accuracy of the assay system by selectively inhibiting the enzymes responsible for thymidylate synthesis de novo and DNA precursor degradation. Culture conditions were investigated and potential inhibitors were tested using human peripheral blood mononuclear cells activated with phytohemagglutinin. Nucleoside-degrading activity of mammalian and bacterial cells is due largely to nucleoside phosphorylases, enzymes that require orthophosphate for activity. We partly inhibited DNA precursor degradation by lowering the phosphate concentration in the culture medium and lowering the pH, thereby reducing the orthophosphate concentration. To reduce precursor degradation further, we tested several potential nucleoside phosphorylase and thymidylate synthetase inhibitors at various concentrations. Our data show that the addition of 1 mM fluorouracil and 1 mM deoxyuridine to the culture medium largely prevents degradation of radioactive thymidine and iododeoxyuridine without unduly compromising the DNA-labeling efficiency of cells activated with mitogens or bacterial homogenates. Under these conditions, label incorporation increases linearly as the number of blast cells or the labeling time increases.