Potentiation of fibroblast DNA synthesis by a phenytoin-induced mononuclear cell derived factor in vitro.

Human mononuclear cells purified by Lymphoprep flotation were incubated with phenytoin (PHT) (20 micrograms/ml) or its metabolite p-HPPH (2 micrograms/ml) in the presence of Concanavalin A (10 micrograms/ml) in vitro. The results indicate that phenytoin and its metabolite p-HPPH induce the release of a mononuclear cell factor(s) that activates quiescent human gingival fibroblast to synthesize DNA.

Potentiation of fibroblast spreading by extracellular matrix from fibroblasts derived from phenytoin-induced gingival overgrowth.

Cell attachment and spreading appear when a cell, on contact with an appropriate substratum, adheres and changes its shape and accommodates to the substratum. The transition from a non-spreading to a spreading state is a prerequisite for growth. Cell-free extracellular matrix (ECM) was produced by fibroblast-like cells from normal gingiva (N-ECM) and phenytoin-induced gingival overgrowth (PHT-ECM). The effect of the ECM on cell attachment and spreading of human gingival fibroblasts was studied in the presence of 2% serum. Within 30 min after seeding 40% of the normal fibroblast cells showed an advanced flattening on PHT-ECM-prepared dishes, compared with 10% on normal ECM-prepared dishes and 5% on uncoated plastic dishes. The results indicate that cells derived from PHT-induced gingival overgrowth produce an ECM with special properties, which could regulate cell functions such as cell attachment and spreading.

Induction of lymphocyte locomotor capacity by a substratum-dependent factor from mononuclear cells.

Media conditioned by human mononuclear cells contain a factor which renders freshly purified T lymphocytes motile and which is distinct from interleukin 1 and interleukin 2. The motility induced by this factor is morphologically indistinguishable from spontaneous lymphocyte motility. The factor is most effective when allowed to attach to the substratum. The effect of the factor on lymphocyte motility is detectable within 30 min but increases over a period of several hours. The relatively rapid action taken together with the ability to interact with both lymphocytes and the substratum point to the possibility that the factor rendering lymphocytes motile is a ligand which mediates cell-substrate adhesion. Attempts to characterize this putative ligand using gel filtration chromatography yielded activity peaks at 500 to 600 kDa.

Motility and infiltration capacity of lymphoid tumour cells: disturbance of motile behaviour in TCLL-lymphocytes.

The capacity of T-cells from 6 normal individuals and 4 cases of TCLL to develop motile forms and migrate within a collagen matrix was compared. In order to exclude possible inhibitory or cooperative cellular interactions the lymphocytes were plated at low density. Only a small proportion of fresh T-lymphocytes from healthy individuals developed motile forms after plating on substrata of collagen or plastic. During a 2 day culture period on collagen 50 to 90% of the T enriched cells from separate normal individuals developed motile forms and migrated into the collagen. Under the same conditions on plastic 30 to 50% of the lymphocytes developed motile forms. Virtually every single lymphocyte from one TCLL case (T3+, T4-, T8-) showed motile behaviour immediately after purification on both collagen and plastic and migrated into the collagen. This patient was demonstrated to exhibit lymphocyte infiltration in non-lymphoid tissues. The majority of the leukemic lymphocytes from 2 other patients (T3+, T4+, T8- and T3+, T4-, T8+ respectively) developed motile forms on collagen immediately after purification. On plastic the development of motile forms by cells from these 2 patients was slightly delayed but within 24 hours the percentage motile lymphocytes was the same as on collagen. In the fourth case (T3+, T4-, T8+) only a few lymphocytes showed motile behaviour on collagen and plastic and no migration within collagen was observed. The low number of motile T-cells in this patient did not increase with time. This patient exhibited an aggressive clinical course but no apparent lymphocyte infiltration into non-lymphoid tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

A labile antigen complex at the lymphocyte surface: the effect of the substratum on its expression.

Human T lymphocytes carry a surface antigen, detectable by a monoclonal antifibronectin antibody, which appears to consist of 150 and 55 kd components as revealed by SDS-PAGE. After in vitro culture of the lymphocytes on a plastic substratum for 48 hr comparatively few cells (40 +/- 18% in separate individuals) express the antigen. In contrast, the vast majority of lymphocytes cultured on a collagen matrix for the same time period maintains surface expression of the antigen (76 +/- 14% in separate individuals). Conditioned media from lymphocytes on plastic contain substantial amounts of antigenicity detectable by the same antibody, whereas conditioned media from lymphocytes on collagen are devoid of such antigenicity. The expression at the cell surface of other T lymphocyte antigens (Leu-4, Leu-3, and OKT8) is identical during culture on plastic and collagen for 48 hr. Collagen does not activate the cells to DNA synthesis or expression of IL 2 receptors, and consequently the potentiation of antigen expression by this substratum cannot be attributed to a mitogenic effect. The composition of subsets of T lymphocytes and the viability of the cells are the same on plastic and collagen, which excludes that the substratum-dependent variations in antigenicity reflect selection or loss of antigen-bearing cells. Thus, substratum-dependent regulation of the expression at the cell surface appears to be a unique property of the 150/55 kd T cell surface antigen. Culture on collagen substrata augments the number of lymphocytes showing motile behavior two to four times compared with culture on plastic.

Anchorage and lymphocyte function: collagen and the maintenance of motile shape in T cells.

When cultured on a collagen matrix at a density of 7 X 10(4) cells/cm2 for 48 hr, 80 +/- 10% of unfractionated and 70 +/- 14% of T-enriched human blood lymphocytes from eight healthy individuals developed a motile morphology defined as the presence of lamellar surface activity and cytoplasmic flattening. During culture on plastic for 48 hr, 32 +/- 5% of unfractionated and 38 +/- 6% of T-enriched lymphocytes from the same individuals developed a motile morphology. The motile morphology was not a rigid state but a series of oscillations in cell shape. The conversion from a spherical into a motile morphology was independent of cell density. After preculture on plastic at 'high' density (1.5 X 10(6) cells/cm2), and subsequent transfer to a plastic surface, 54 +/- 12% of the cells from separate individuals exhibited a motile morphology within 2 hr. These motile forms were, however, transient and approximately 50% disappeared within 12 hr. Collagen augmented the motile behaviour of unfractionated and T-enriched lymphocytes by two to four times when fresh from the blood compared with glass or plastic. During culture on plastic, the lymphocytes lost this prompt responsiveness to collagen contact. Thus, during culture on plastic, the ratio between percentage motile lymphocytes after subsequent transfer to collagen and plastic, respectively decreased from values between 2 and 4 immediately after purification to close to 1 within 2 days. However, when retransferred to collagen, the majority of the lymphocytes within another 2-day period acquired responsiveness to collagen measured as potentiation of motile cell shape on this substrate compared with on plastic. These data suggest that the variation in motile behaviour in the T lymphocyte reflects a labile property which is enhanced by contact with a collagen matrix.

Phenytoin potentiates accessory cell dependent DNA-synthesis in human lymphocytes in vitro.

The effect of phenytoin and its major metabolite p-HPPH on concanavalin A (ConA) induced DNA-synthesis of human lymphocytes was studied in vitro. In lymphocyte cultures depleted of phagocytizing cells by iron treatment PHT and p-HPPH, in pharmacologic concentrations, caused a depression of the mitogen response measured as uptake of 3H-thymidine. In contrast, the presence of phagocytizing mononuclear cells during ConA-stimulation in the presence of PHT and p-HPPH caused a potentiation of ConA induced DNA-synthesis. These effects of phenytoin on immunocompetent cells may be of significance in the pathogenesis of the PHT-induced gingival overgrowth, since earlier we have reported the presence of large infiltrates of lymphocytes, mainly T-lymphocytes in gingival biopsies from clinically non-inflamed PHT-induced gingival overgrowth.

Compartment dependence of T-lymphocyte motility.

Fresh human T lymphocytes from the blood of healthy individuals exhibited few motile forms, i.e. nonspherical shape and lamellar surface activity, when allowed to settle on a plastic surface. This poor motility of 'normal' blood T lymphocytes is most likely physiological, since under the same conditions more than 75% of the blood lymphocytes from T-cell chronic lymphocytic leukaemia (TCLL) cases showed motile forms. In contrast to blood T cells, a large proportion of fresh human splenic T lymphocytes from separate individuals generally showed motile behaviour within 1 h when plated on a substrate. The rate of migration of spleen T cells into a collagen matrix was higher than that of blood T cells. The poor motile behaviour therefore appeared to be a limiting factor for translocation and migration of blood T cells within a collagen matrix. Culture on a collagen matrix at 'low' cell density in the presence or absence of serum for 2 days augmented the percentage of motile blood T cells to the same level as for fresh spleen T cells, whereas culture on plastic caused a relatively moderate increase in motility. This collagen-mediated potentiation probably does not reflect polyclonal T-cell activation, since it occurred in serum-free medium and appeared independent of cell interactions, and since collagen did not induce DNA synthesis. These results demonstrate two major factors regulating the 'spontaneous' motility of T lymphocytes, namely the location of the cell within the body and the nature of the substratum.

Subpopulations of lymphocytes in connective tissue from phenytoin-induced gingival overgrowth.

The presence of mononuclear cells was studied in gingival biopsies from seven children exhibiting phenytoin(PHT)-induced gingival overgrowth, three children with gingivitis and a control group consisting of three children without clinical signs of inflammation. The mononuclear cells were detected using monoclonal antibodies defining functional T-lymphocyte subpopulations, B-lymphocytes and monocytes. Gingival biopsies from the individuals in the PHT-group showed a substantial number of mononuclear cells. The distribution of mononuclear cells in separate individuals were as follows: 69-95% OKT3 +/Leu4+ cells (T-lymphocytes), 50-64% OKT4 +/Leu3+ cells (T-helper phenotype) and 29-46% OKT8+ cells (T-suppressor/cytotoxic phenotype). None of the biopsies in the PHT-group contained more than a few scattered plasma cells. The vast majority of all mononuclear cells present in the biopsies reacted with OKIa1, a monoclonal antibody defining the HLA-DR framework. In contrast, biopsies from the control group and the gingivitis group contained few mononuclear cells, the majority of which were T-cells. This suggests that immunologic reactions mediated by T-cells may play a role in the pathogenesis of the PHT-induced lesion.

Anchorage and lymphocyte function. Acquisition of spontaneous motile behaviour by human blood lymphocytes and its modulation by concanavalin A.

The majority of splenic lymphocytes were motile, showing lamellipodial activity almost immediately after purification. In contrast, fresh blood lymphocytes were non-motile and maintained their spherical suspension morphology. The number of motile blood lymphocytes increased markedly during a 2-day in vitro culture period. This increase was enhanced by high cell density and required a metabolically active cell with protein synthesis but not exogenous mitogens. The spontaneous development of motility in different subpopulations of blood lymphocytes was analysed by means of monoclonal antibodies. The results indicated that cells which were motile immediately after purification were almost exclusively non-T lymphocytes. Lymphocytes which became motile during in vitro culture included both T and non-T cells. Substrate adhesion mediated by concanavalin A (Con A) changed the morphology of motile T lymphocytes and instead of being polar, the cells flattened over the substratum and acquired a non-polar shape. Furthermore, the morphogenetic response induced by Con A-mediated substrate adhesion appeared to distinguish T and non-T lymphocytes. Thus, the length of the cell perimeter showing lamellar activity was greater in T than in non-T lymphocytes, and the degree of polarity was greater in non-T (with and without B-cell markers) than in T lymphocytes.

Scanning electron microscopic study of Streptococcus mutans BHT lysed by lysozyme.

The effects of purified salivary lysozyme (HSL) and hen egg white lysozyme (HEWL) on the surface structure of Streptococcus mutans BHT were studied with the aid of scanning electron microscopy. In parallel experiments bacteriolysis was monitored by liberation of 3H-thymidine incorporated into the bacteria. Control cells maintained their shape and had intact cell walls during the experimental period. Exposure of the cells to HSL (5.0 U/ml) or HEWL (5.0 micrograms/ml) for 3 and 18 h resulted in progressive destruction of cell structure. Some cells exhibited ruptures of the cell walls on top of spherical swellings, predominantly located at the ends of the bacteria. After 18 h the majority were disrupted in the septal area leaving numerous empty cup shaped cell walls in the preparations. The findings of the electron microscopic examination were confirmed in the biochemical assay.

Anchorage and lymphocyte function: pattern of spreading distinguishes T- and B-cell chronic lymphocytic leukaemia.

T and B lymphocytes from normal individuals and patients with T and B chronic lymphocytic leukaemia (TCLL and BCLL) were induced to spread on a solid surface in the presence of Con A. The proportion of cell circumference exhibiting lamellar activity was considerably greater in T than in B cells. This difference also applied to T and B cells with a single leading lamellipodium. The different pattern of formation of active cell edges implied that the degree of polarity measured as the ratio between the largest and the shortest diameter (over the nucleus) was significantly greater in B than in T cells. This was also obvious when T and B cells, both with a single leading lamellipodium, were compared. The formation of active cell edges in T lymphocytes was generally accompanied by nuclear flattening, even in polar cells with a single leading lamellipodium. B cells, with the exception of one BCLL case, did not exhibit nuclear flattening. Thus, during the entire course of a contact-induced morphogenetic response, T- and B-cell leukaemias were easily distinguishable on the basis of the following criteria: (i) the proportion of the lymphocyte perimeter showing active cell edges, (ii) the degree of polarity and (iii) nuclear flattening.

Anchorage and lymphocyte function. Antibodies as adhesion and spreading factors for human T lymphocytes.

When attached to a solid surface coated with protein A various antibodies reacting with lymphocyte membrane antigens (anti-beta 2m, OKT3, OKT8, Leu2, 3, 4 and certain patient sera) catalyse the formation of peripheral lamellar activity, i.e. an active spreading process in human T lymphocytes. In contrast, binding only of the same antibodies to the cells or allowing antibody-coated cells to settle and bind to a protein A-coated surface did not induce spreading although the number of cells attached to the solid surface was virtually the same as in the former case. The peripheral lamellar activity markedly facilitated short-range lymphocyte interactions and appeared to constitute the region of the lymphocyte that actively contacts other cells. These results show that antibodies can act as spreading factors, and indicate that this function is critically dependent on the presentation of the inducing ligand. The asymmetry in the induction of active cell edges may influence functional lymphocyte interactions with environmental surfaces.

A clinical and radiographic evaluation of cultivated and autotransplanted human teeth.

A vital periodontal membrane (PDM) is of ultimate importance for a successful periodontal healing of auto-transplanted teeth. It has been suggested that a damaged PDM may heal during an intermediate tissue culture period. In the present study, 26 canines, bicuspids and third molars were surgically removed and cultivated in a modified Eagle's medium for 3 to 17 weeks. The teeth were then transplanted to their new positions. 11 out of 18 (61%) transplanted teeth with complete root formation and 7 out of 8 (88%) transplanted teeth with incomplete root formation healed with an apparently normal periodontal ligament. 5 teeth, all canines, became ankylotic. Tissue cultivation of teeth to be transplanted resulted in approximately the same healing rate as has been reported for autotransplanted teeth without the tissue culture procedure.

Characterization of the spreading process in human T lymphocytes.

Substratum-bound concanavalin A (conA) caused attachment and spreading of human T lymphocytes identified by monoclonal anti-T cell antibodies and sheep erythrocyte rosette formation. The simultaneous presence of conA in the medium increased the spreading, whereas preincubation of the cells with conA inhibited spreading. The tendency of conA to induce spreading was dependent on the concentration used, the higher the conA concentration the more pronounced was the spreading. For example, conA at 10 micrograms ml-1 triggered the formation of prominent substratum-attached filopodia with a length of 1-10 micron in 60-80% of T-enriched lymphocytes obtained from separate individuals. At the same conA dose the filopodia were, in 10-20% of the lymphocytes, accompanied by development of lamellipodia. With conA at 100 micrograms ml-1 the number of cells that underwent pronounced spreading was 55-90% in separate individuals. Observation of T-enriched cells fixed at different times after initiation of spreading induced by conA at 100 micrograms ml-1 indicated that filopodia formation represented the initial morphological alteration during the spreading process. This process thereafter proceeded with development of lamellipodia, extensive cytoplasmic spreading and flattening of the central cell mass. Quiescent and mitogen-activated cells exhibited the same sequence of changes during spreading. Spreading led to disappearance of the microvilli with a length of 0.1-0.7 micron present on lymphocytes in suspension, although some microvilli persisted over the cell center.

Anchorage and lymphocyte function. Spreading-capacity distinguishes common thymocytes and peripheral T lymphocytes.

Contact of T-enriched human blood lymphocytes with an adhesive surface in the presence of Concanavalin A (Con A) almost immediately induced a sequence of motile changes in virtually all cells. The initial event in this spreading process was the formation of filopodia distinct from the microvilli of lymphocytes in suspension. The filopodia were accompanied by lamellipodia, ruffles and flattening of the nucleus. Contact with a nonadhesive substratum in the presence of Con A did not trigger this sequence of changes. Cytochalasin B and D or low temperature inhibited the contact-induced changes. With the exception of a small number of cells (5-15%), T-enriched lymphocytes that were allowed to settle in the absence of Con A showed a radius of action (area occupied by the cells/translational movement per hr) of 39 micrometers 2/ less than 1 micrometer. The small 'motile' population showed a radius of action of 74 micrometers 2/8 micrometers. The Con-A-mediated spreading-process yielded a radius of action of the lymphocytes of 117 micrometers 2/6 micrometers. This augmented radius of action markedly facilitated cell-cell interaction in a high frequency of the cells and appeared to be a prerequisite for such interactions at 'low' cell density. Thymocytes reactive with OKT 6 antibodies or belonging to the 'high-density' fraction of cells attached to a Con-A-coated surface to the same extent as peripheral OKT 3 positive lymphocytes, but did not exhibit the morphological changes characteristic of a spreading-process. In contrast, OKT 6 negative thymocytes or thymocytes with a relatively low density showed spreading indistinguishable from that of OKT 3 positive peripheral lymphocytes. These results characterize the spreading-process in human T lymphocytes and demonstrate its functional importance for interactions with the environment. Spreading-capacity appears to reflect the stage of maturation of T cells.

Cytochalasins distinguish by their action resting human T lymphocytes from activated T-cell blasts.

In the majority of resting human peripheral T lymphocytes obtained from separate individuals cytochalasin B (CB) and D (CD) cause a disappearance of microvilli and induce a rapid formation of prominent sac and bleb-like projections with a length of 1-10 microns randomly distributed over the cell surface. During mitogen stimulation the cells lose the tendency to develop such projections when subsequently exposed to CB and CD. By contrast, in activated T lymphocytes the cytochalasins provoke an asymmetric localization of microvilli including cell surface antigens and actin to a prominent protuberance often separated from the cell body by a constriction. This protuberance is distinct from conventional spontaneous uropods formed by conA-stimulated lymphocytes in relation to contact with other cells and with non-cellular surfaces. The cytochalasins therefore in their action distinguish resting small lymphocytes from activated T-cell blasts.

The effect of the phenytoin metabolite p-HPPH on proliferation of gingival fibroblasts in vitro.

The effect of the major phenytoin metabolite-5-(parahydroxyphenyl)-5-phenylhydantoin (p-HPPH) was studied on cultures on human fibroblast-like cells grown out from explanted gingival biopsies. The explants were taken from children undergoing phenytoin medication. The results showed that the number of cells per culture decreased whereas the protein and DNA-contents remained relatively unaffected. This effect was most pronounced at the concentrations of 0.20 micrograms/ml p-HPPH. The results indicate that the metabolite interfere with cell division without affecting protein or DNA synthesis.