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Biomedical subjects

J G Tew

Publications and source records attributed to J G Tew.

At least 109 records · Page 6Linked to original sources
PubMed1986

Antigenic phenotyping of isolated and in situ rodent follicular dendritic cells (FDC) with emphasis on the ultrastructural demonstration of Ia antigens.

The antigenic phenotype of mouse lymph node follicular dendritic cells (FDCs) was studied by immunocytochemical techniques. Indirect fluorescence was used in conjunction with monoclonal antibodies to localize FDC surface antigens on FDC-enriched cell preparations and in cryostat sections. Lymph nodes from rats and mice were also labeled directly for Ia antigens with fluorescein- or peroxidase-conjugated Ia-specific monoclonal antibodies (i.e., MRC Ox4 and 10-2.16, respectively). Lymphoid tissue was also prepared for electron microscopy to allow clear distinction between Ia antigens of B lymphocytes and FDCs in situ. In these experiments, gold-labeled antigen was used to clearly identify FDCs and their processes among the Ia-positive cells of lymph node follicles. The labeling observed by light and electron microscopy showed that FDCs expressed Ia in situ and in vitro. Additional surface determinants shown to be expressed by FDCs included H2-K, common leukocyte antigen, and the receptor for the Fc portion of IgG1 and IgG2b. Neither macrophage antigens, such as Mac-1, Mac-2, Mac-3, and F4/80, nor the lymphocyte markers Ly-1, Ly-2, and Thy-1 were expressed by FDCs. Thus, the antigenic phenotype of FDCs, along with their distinctive dendritic morphology, their nonphagocytic and nonadherent nature, and their ability to trap and retain immune complexes on their plasma membrane, identifies them as a unique cell population.

Animals↗
PubMed1986

Increased leukocyte diversity and responsiveness to B-cell and T-cell mitogens in cell suspensions prepared by enzymatically dissociating murine lymph nodes.

The isolation of viable follicular dendritic cells (FDCs) from murine lymph nodes requires that the nodes be enzymatically dissociated with collagenase and the protease dispase. The present study was undertaken to compare leukocyte populations derived from enzymatically dissociated and mechanically disrupted lymph nodes. We examined cell viability, cell number, cell types, and the proliferative response to the mitogens LPS, PHA, and Con A. Cells were prepared by taking the inguinal, brachial, axillary, and popliteal lymph nodes from one side of the animals and mechanically disrupting them. The corresponding lymph nodes from the other side of the same animals were digested with enzymes. The enzyme-treated lymph nodes yielded substantially more cells and had a higher percentage of viable cells. Over 40% more viable cells were available for cell culture using the enzyme dissociation method. The numbers of FDCs, macrophages, plasma cells, and fibroblasts were clearly increased. The cells dispersed by enzymatic means responded better than the mechanically dispersed cells to both B-cell and T-cell mitogens. This was especially striking in cultures at lower cell densities. We conclude that the method of cell dissociation has a marked effect on the types of viable cells released and available for culture, as well as on the ability of the cells to respond. We believe the cell types released by enzymatic dissociation and their response in culture more accurately reflect conditions in vivo.

Animals↗
PubMed1986

Localization of antibody-forming cells in draining lymphoid organs during long-term maintenance of the antibody response.

Previous studies on the "spontaneous antibody response" have included in vitro steps and it is possible that the response is an in vitro artifact. The objective of the present study was to induce a spontaneous antibody response entirely in vivo and determine if the response is localized and if the magnitude of the response is related to the location of persisting antigen. Antigen was injected into the right hind footpads of mice, and lymph nodes on the right side were draining and lymph nodes on the left side were controls. Antibody-forming cells (AFCs) were enumerated in both draining and nondraining nodes 2 weeks, 2 months, and 1 year after secondary immunization. Four days prior to determining AFC number, the mice were severely bled to stimulate AFC production. Thousands of AFCs were found in the draining lymph nodes and the numbers were dramatic in nodes closest to the injection site that retain the most antigen. In contrast, the vast majority of nondraining nodes lacked any AFCs. One year after immunization, the response was almost exclusively in the popliteal node, draining the foot where antigen was administered a year earlier. These results are consistent with previous data on the spontaneous response and support the hypothesis that antigen retained on FDCs is essential in the maintenance of serum antibody levels.

Animals↗
PubMed1986

Human serum antibody response against Streptococcus mutans antigens.

Antigens from Streptococcus mutans were examined to identify specific polypeptides that may have stimulated antibody responses and possibly play some role in caries immunity. A group of 10 adult human subjects was screened for serum antibodies reactive with antigens from S. mutans. Extracellular and cellular protein preparations from S. mutans LM7 (Bratthall serotype e) and V403 (biotype c) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western electrophoretic transfer and immunoblotting analysis. Antibodies reactive with polypeptides ranging from 34 to 400 kilodaltons in apparent molecular mass were detected by these means. Radioimmunoassay competition experiments revealed that the cellular and extracellular antigens did not compete with each other for serum antibodies. Preabsorption of sera with extracellular proteins from other oral streptococcal species prior to immunoblotting indicated that the antigens unique to S. mutans have molecular masses greater than 100 kilodaltons, and each individual produced antibodies against different antigens of high molecular mass. Examination of sera from young children also indicated heterogeneous responses against S. mutans LM7 antigens.

Animals↗
PubMed1985

Serum antibody reactive with predominant organisms in the subgingival flora of young adults with generalized severe periodontitis.

In the present study we sought to determine whether serum antibody was present against microorganisms which predominate in the subgingival flora of young adults with generalized severe periodontitis (SP). Subjects with SP were often seropositive for Eubacterium brachy, Fusobacterium nucleatum E3C22, and Peptostreptococcus micros, whereas subjects with juvenile periodontitis (JP) and subjects with healthy periodontium (HP) were not. Both SP and JP subjects were more frequently seropositive for Bacteroides gingivalis, F. nucleatum D52B16, and F. nucleatum E1D1 than were HP subjects. The data were most striking for B. gingivalis, for which both the incidence and the magnitude of specific antibody was clearly elevated for SP and JP subject groups. However, SP subjects generally had either a high antibody titer or no detectable titer. In contrast, JP and HP subjects generally had at least very small amounts of antibody. Except at very low levels of antibody, neither SP nor JP groups differed significantly from the HP group for antibody to Eubacterium nodatum, Bacteroides intermedius (homology group 4197 or 8944), or Lactobacillus minutus antibody. There was a high frequency of antibody to E. nodatum, with very high titers in all groups despite the fact that this organism is rarely found in HP subjects. For Eubacterium timidum, the JP group was clearly more frequently seropositive than the HP group. Despite high levels of L. minutus in subgingival flora, none of the 50 SP subjects had a detectable antibody titer, and only four of the HP and JP subjects had detectable antibody. These results indicate that many organisms in the subgingival flora elicit antibody responses. B. gingivalis is probably the best example among the species tested. However, some organisms that are present in high concentration, e.g., L. minutus, apparently fail to induce significant antibody responses.

Adolescent↗
PubMed1985

Relationship between gingival crevicular fluid and serum antibody titers in young adults with generalized and localized periodontitis.

The objective of the present study was to determine the relationship between concentrations of antibodies in serum and those in gingival crevicular fluid (GCF) of patients with juvenile periodontitis and severe periodontitis. Most antigens used to quantitate antibodies were obtained from a panel of bacteria associated with juvenile periodontitis or severe periodontitis. We further investigated variation in antibody titer among different periodontal sites and the extent to which antibody in GCF is locally derived. Titers of antibody, total immunoglobulin G (IgG), and human serum albumin were determined with sensitive radioimmunoassays. The relationship between serum and GCF antibody was complex. Both person-to-person variability and marked variability within the same subject were found among different sites of similar clinical status. The site-to-site variability was found not only for antibody reactive with periodontal organisms, but also for antitetanus toxoid, total IgG, and even human serum albumin. Generally the variability was in the degree of depression of the level in GCF relative to that in serum. However, anti-Bacteroides gingivalis and anti-Actinobacillus actinomycetemcomitans in GCF often exceeded the level in serum. When antibody titers in serum and GCF were calculated per milligram of human serum albumin, most of the apparent depressions of antibody in GCF disappeared. The ratio of antibody in serum to that in GCF approached unity for all organisms except B. gingivalis and A. actinomycetemcomitans Y4, which were markedly elevated. Furthermore, the level of IgG per milligram of human serum albumin in GCF was about twice the level in serum. We believe that human serum albumin reflects serum contribution to the GCF, and we therefore attribute the increased level of IgG per milligram of albumin in GCF to local synthesis. It appears that anti-B. gingivalis and anti-A. actinomycetemcomitans represent an important portion of this local antibody synthesis, since most seropositive patients with severe or juvenile periodontitis had at least one site elevated, and the magnitudes of the elevations were large in many sites. Those sites yielding elevated antibody exhibited no obvious differences in clinical parameters of probeable depth or attachment level as compared with sites in which antibody levels in GCF were similar to serum levels. Elevated antibody in GCF may relate to changes in disease activity that are not detectable by usual clinical measures.

Actinobacillus↗
PubMed1985

Isolated follicular dendritic cells: cytochemical antigen localization, Nomarski, SEM, and TEM morphology.

The objectives of the present study were to determine the cytological features of isolated follicular dendritic cells (FDC), which distinguish them from other leukocytes or dendritic cell types. Consequently, we have developed methods for the fixation, peroxidase cytochemistry, and visualization of FDC, which are applicable to cytological evaluations by Nomarski optics, scanning, and transmission electron microscopy. A functionally supported identification of FDC in vitro was made possible by utilizing, in conjunction with the dendritic morphology, the cytochemically identifiable antigen, horseradish peroxidase (HRP), and the known capacity of FDC to sequester immune complexes (i.e. HRP-anti-HRP) on their plasma membranes. The observations showed that FDC constitute a relatively pleomorphic, nonphagocytic group, distinct from other dendritic type cells such as lymphoid dendritic cells, Langerhans cells, and interdigitating cells (LDC, LC, and IDC), as well as typical leukocytes. Morphologically distinct FDC were identified as cells either with filiform dendrites or with "beaded" dendrites. FDC possessed a single or sometimes a double, lymphocyte-size cell body, which contained an irregular, lobated nucleus, Golgi apparatus, numerous small vesicles, and some mitochondria. Mitochondria were not abundant in the dendritic processes. Filiform dendrites tended to branch and anastomose near the cell body and form a radiating "sunburst"-like pattern. On the average, dendrites measured 15-20 microns in length and 0.1-0.3 micron in diameter. Occasional dendrites were extremely elongated, reached several hundred microns in length, and terminated in an enlargement measuring nearly a micron in diameter. Other filiform dendrites usually had a club-shaped terminal enlargement. The microspheres of "beaded" dendrites ranged between 0.3 and 0.6 micron in diameter. The dendritic processes were also shown to have a highly ordered pattern of immune complex attachment on their surface, suggestive of a periodic arrangement of receptor sites.

Animals↗
PubMed1985

Follicular dendritic cells in suspension: identification, enrichment, and initial characterization indicating immune complex trapping and lack of adherence and phagocytic activity.

Antigen-retaining follicular dendritic cells (FDC) have been identified and studied in sections of lymph nodes and spleen, but studies of these cells in culture have been extremely limited. The purpose of this study was to establish techniques to release these fragile cells from mouse lymph nodes in a viable state and to identify these cells routinely in lymph node cell suspensions. FDC were obtained from passively or actively immunized popliteal lymph nodes of mice injected in the footpads with 125I-labeled human serum albumin (HSA) or horseradish peroxidase (HRP). Lymph nodes were removed 1 hr after the footpads had been injected with collagenase. After another hour of incubation in vitro with collagenase, protease, and deoxyribonuclease, FDC were released by gentle teasing and enriched by centrifugation on a low density bovine serum albumin (BSA) or Percoll gradient. Most FDC with the associated radiolabel floated at densities greater than 1.06 g/ml on BSA or Percoll gradients. Slides of the FDC-enriched fraction were prepared, using a cytobucket which allowed the cells to be affixed to glass slides by centrifugation in a less disruptive manner than by cytocentrifugation. FDC that were air-dried and fixed with 3% glutaraldehyde had a characteristic pink acidophilic cytoplasm after Wright's staining, and had a faintly basophilic euchromatic nucleus frequently with peripherally-clumped chromatin. In addition, these cells were large and irregularly shaped (up to 60 micron long). Fixation of FDC with 0.6% paraformaldehyde/ 0.9% glutaraldehyde on poly-L-lysine-coated slides resulted in a preservation of FDC which made possible visualization of long dendritic processes by Nomarski optics. Antigen presence on the cell surface was confirmed by autoradiography and, in the case of HRP, was also visualized enzymatically using diaminobenzidine. In contrast to resident peritoneal macrophages or some contaminating lymph node macrophages present on the same slides, FDC did not phagocytize opsonized sheep red blood cells (SRBC) or adhere to plastic surfaces although they did form rosettes with opsonized SRBC. Cell marker studies indicated FDC have a distinctive phenotype. They were positive for Ia, Fc receptor, and leukocyte common antigen, but negative for Thy-1, Ly-1, Ly-2, endogenous Ig, Mac-1, Mac-2, Mac-3, and F4/80, and negative to weakly positive for nonspecific esterase. Cultured FDC remained viable and retained radiolabeled antigen-antibody complexes on their surfaces and were significantly enriched for FDC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Tissue localization and retention of antigen in relation to the immune response.

Antigen persists for months or even years in lymphoid tissues of immune animals and this antigen is believed to participate in the induction and maintenance of B-cell memory as well as in the maintenance of serum antibody levels. In the present report we describe the phenomenon of antigen localization and long-term retention on mouse follicular dendritic cells (FDCs). The antigens used were injected in the hind footpads of immune mice and the popliteal lymph nodes were the lymphoid organs generally studied. In addition to presenting the morphological features of mouse FDCs, we report the results of a study of the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus to the regions of follicular retention in the cortex. The migration was followed by light and electron microscopy. The results support the concepts that immune complexes are trapped in the subcapsular sinus and are transported by a group of nonphagocytic cells to follicular regions. The mechanism of transport may involve either migration of pre-FDCs with a concomitant maturation into FDCs, or cell-to-cell transport utilizing dendritic cell processes and membrane fluidity; or a combination of the two mechanisms may be in operation.

Animals↗
PubMed1984

Follicular dendritic cells in the regulation and maintenance of immune responses.

The objectives of this report are to explore known and potential regulatory roles of follicular dendritic cells (FDCs) in immune responses. FDCs are found in B-cell areas of lymphatic tissue and are especially rich around germinal centers where B-cell proliferation is occurring. Recent advances in isolating FDCs in our laboratories made it possible to begin investigating interactions of FDCs with other cells in vitro. In a series of experiments, we sought to determine whether a low concentration of FDC-enriched cells (2-10%) could enhance B-cell and/or T-cell mitogenesis. The results showed that an enriched population of FDCs markedly enhanced lipopolysaccharide-induced stimulation of nude mouse spleen cells, while an equivalent number of resident peritoneal cells or thymocytes had no effect. Depletion of Thy 1.2 positive cells or an exposure to 2400 rads of gamma radiation did not abrogate the enhancing capacity of FDC-enriched cells. FDC-enriched cells also increased phytohemagglutinin-induced proliferation. Scanning electron microscopy of FDC preparations showed that antigen-bearing dendritic processes of Type 2 FDCs have a "beaded" appearance. It was proposed that these immune complex coated microspheres may represent an important mechanism for exposing persisting antigen to the immune system. The gradual shedding of these microspheres may play an important role in regulating and maintaining immune responses within lymphoid tissues.

Animals↗
PubMed1984

B-lymphocyte blastogenesis in response to periodontitis-associated bacteria. Kinetics and proportion of total response.

A role for activated B-lymphocytes in mediating the initiation and/or progression of periodontal diseases has been proposed in previous work. The present study was conducted to: (1) assess the proportion of total lymphocyte blastogenic response to selected oral bacteria which is composed of T-cell and B-cell activation, respectively, and (2) to determine whether different kinetic patterns exist for the T-cell vs. the B-cell responses to these bacteria. Using lymphocyte rosetting and culturing techniques, rosette-positive and rosette-negative lymphocyte preparations were examined for blastogenic responsiveness following stimulation with a variety of both Gram-positive and Gram-negative periodontitis-associated bacteria. Results of these studies indicated that both peripheral blood lymphocytes (PBL) from healthy adults and cord blood lymphocytes (CBL) from placental afterbirths responded to these bacteria with similar kinetic patterns. The net PBL blastogenic response appeared to consist of an early B-cell response, which peaked at Days 2 or 3 of culture, followed by a later T-cell response, which peaked at Days 5 to 6 of culture. The B-cell response appeared to be T-cell-dependent in that B-cells cultured alone showed minimal thymidine uptake over the entire 6-day period, but the addition of irradiated T-cells to these B-cell cultures resulted in a greatly enhanced B-cell response.

Actinomyces↗
PubMed1984

Anamnestic responses induced by antigen persisting on follicular dendritic cells from cyclophosphamide-treated mice.

In contrast to most mouse lymph node cells, follicular dendritic cells (FDCs) resist cyclophosphamide (Cy; 300 mg/kg)-mediated destruction in vivo. In this study we sought to determine if antigen-bearing FDCs from Cy-treated animals maintained biological activity. We were especially interested in whether FDCs from Cy-treated animals could stimulate an antibody response when combined with primed spleen cells and whether the FDCs needed to be intact and viable for stimulation to occur. The effect of Cy treatment on lymph node histology, number of T cells and B cells, and the 'spontaneous antibody response' was determined. Cy treatment resulted in a massive depletion of the lymph node cortex and a loss of follicles and germinal centres. Over 90% of B cells in the lymph node were eliminated. The paracortex was more resistant although nearly 80% of T cells were eliminated. Cy treatment also eliminated the 'spontaneous antibody response' as established by in vitro culture or after adoptive transfer. The addition of primed spleen cells to antigen-bearing FDCs including sonicated non-viable FDCs from Cy-treated animals resulted in an anamnestic antibody response. Memory lymphocytes, injected into the hind foot pads of Cy-treated animals, migrated to the follicular area of popliteal lymph nodes and cells from these reconstituted nodes spontaneously responded upon subsequent adoptive transfer. It was concluded that antigen retained on Cy-treated FDCs maintains its immunogenicity and is capable of inducing a 'spontaneous antibody response' or an anamnestic response. Furthermore antigen on FDCs or on fragments of FDCs from one animal can interact with memory cells from another animal to induce a productive antibody response. Lymph nodes enriched for FDCs by Cy treatment should be a good source of FDCs for isolation and further study of the nature of this interaction.

Animals↗
PubMed1983

Evidence of mitogenic activity in periodontitis-associated bacteria.

This study examines several periodontitis-associated bacterial isolates for the presence of mitogenic activity, as indicated by their capacity to stimulate unsensitized lymphocytes to undergo blastogenesis. Germfree mouse spleen cells responded vigorously to all of the bacterial sonic extracts tested. The kinetics and dose responses to these activators in germfree mouse spleen cell cultures paralleled those seen with the standard murine B-cell mitogen, Escherichia coli lipopolysaccharide. In contrast, Streptokinase-Streptodornase (Varidase; Lederle Laboratories) antigen elicited no response. Human cord blood lymphocytes also responded upon stimulation with these same bacterial isolates but failed to respond to Streptokinase-Streptodornase. The frequency, magnitude, and kinetics of these cord blood lymphocyte responses were remarkably similar to those seen with adult peripheral blood lymphocytes. However, in this and previous studies, individuals with unresponsive peripheral blood lymphocytes have been observed. Studies were initiated to determine whether these unresponsive leukocyte preparations truly lacked the capacity to respond to these bacteria or whether unresponsiveness reflected the presence of a regulatory cell population in these cultures. After the removal of the adherent cells from unresponsive peripheral blood lymphocyte cultures, the nonadherent cells were found to be responsive. Therefore, peripheral blood lymphocyte responsiveness appears to be regulated via an adherent cell population. The removal of adherent cells from unresponsive cord blood lymphocyte preparations resulted in a less consistent alteration to responsiveness. However, cord blood lymphocyte preparations unresponsive at a standard cell density were shown to be responsive at altered cell densities.

Animals↗
PubMed1983

Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology.

The objective of the present study was to investigate the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus (SS) to regions of follicular retention in the cortex. The migration of horseradish peroxidase (HRP), used as a histochemically identifiable antigen, was followed by light and electron microscopy in C3H mouse popliteal lymph nodes obtained 1, 5, 15, and 30 min, and 5 and 24 hr after hindfoot pad injection of HRP. The observations showed that as early as 1 min after HRP injection, localization of antigen occurred at distinct sites in the SS and subjacent areas of the cortex on the afferent side. At these sites, between 1 min and 24 hr, the antigen formed light microscopically identifiable trails, which reached progressively deeper into the cortex with time toward individual follicular regions. By 24 hr this apparent migration of antigen was complete, and HRP was localized in follicles. This migration pattern did not occur on the efferent sides of lymph nodes, and it was dependent on the systemic presence of specific antibodies since it was observable only in passively immunized but not in nonimmune mice. Temporary retention of antigen by typical macrophages was also observed in the SS on the efferent side. This was minimal in nonimmune mice and was significantly enhanced in passively immunized mice. Electron microscopy indicated that the apparent migration of immune complexes was mediated by a group of cells observed in the migration path that had immune complexes sequestered on their surface or in plasma membrane infoldings. These antigen transporting cells (ATC) were relatively large nonphagocytic cells, with lobated or irregular euchromatic nuclei and cell processes of various complexity. ATC observed in or near the SS appeared to be less differentiated, were monocyte-like, and resembled non-Birbeck granule-containing Langerhans cell precursors or veiled cells. Others, located deeper in the cortex, appeared more differentiated, interdigitated with antigen-retaining dendritic cells, and shared morphologic characteristics with follicular dendritic cells (FDC). The results support the concepts that immune complexes are trapped in the SS and are transported by a group of non-phagocytic cells, other than lymphocytes, to follicular regions. The mechanism of transport may involve the migration of ATC with a concomitant maturation into FDC, or by a mechanism of ATC to FDC transport utilizing dendritic cell processes and membrane fluidity, or by a combination of the two mechanisms.

Animals↗