Increased Thymidine Uptake Research Articles

Changes in thymidine uptake in the gastrointestinal tract of the rat following treatment with 16,16-dimethyl prostaglandin E 2

Thymidine uptake in the organs of the gastrointestinal tract of the rat was studied to determine if cell synthesis was involved in the increases in weight of the stomach, small intestine and colon which result from treatment with 16,16-dimethyl prostaglandin E 2 (16,16-dimethyl PGE 2). Animals were treated for 2 days with 16,16-dimethyl PGE 2. They were injected with the 3H-thymidine, sacrificed and the organs of interest were removed. The total amount of tritium in the stomach, duodenum, jejunum, ileum, and colon was determined. Thymidine uptake was significantly increased in the duodenum (1.50 times), jejunum (1.53 times), and colon (1.40 times) but not in the stomach and ileum. The increases were dose related in the duodenum and jejunum. The colon showed a similar dose response pattern but the changes with dose did not reach significance. These results confirm and extend a previous report that 16,16-dimethyl PGE 2 increased thymidine uptake in the duodenum but not the stomach (1). This is different from gastrin which has been shown by others to increase thymidine uptake in the stomach, duodenum, ileum and colon (2,3).

Carrageenan-mediated suppression or augmentation of mitogen-induced lymphocyte proliferation

The effects of carrageenan on mitogen-induced lymphocyte proliferation were studied in the guinea pig. According to the dose used, carrageenan displayed opposite effects on thymidine uptake by spleen cells or peripheral blood leukocytes stimulated by Concanavalin A or phytohemagglutinin (25 μg ml −1 carrageenan increased whereas 0.25 μg ml −1 depressed thymidine incorporation). Carrageenan, at the high concentration which increased thymidine uptake by mitogen-stimulated spleen cells, potentiated the enhancing activity of macrophages that was observed with cell suspensions containing 20% macrophages. Conversely, low concentrations of carrageenan abolished the enhancing effect of macrophages. These effects of carrageenan on lymphocyte proloferation could be explained by its activities on a macrophage functional subset rather than on the whole macrophage population.

Effect of porcine relaxin and estradiol-17β on the incorporation of tritiated thymidine by fibroblasts isolated from guinea pig uteri

Fibroblasts isolated from the uteri of 5 guinea pigs were cultured in vitro to determine the effect of relaxin and estradiol. Both relaxin and estradiol increased thymidine uptake by fibroblasts. Thymidine incorporation with 1.5 μg/ml relaxin and no estradiol was 107% of control while 600 pg/ml estradiol and no relaxin gave 112% of control. Thymidine incorporation in the presence of 1.5 μg/ml relaxin and 600 pg/ml estradiol was 128% of control. This indicated a significant interaction between relaxin and estradiol.

Circulating growth factor studies in growth plate versus resting cartilage in vitro: tissue responsiveness.

We attempted to develop a system in which cartilage growth-plate responses could be examined in vitro. In vivo, rat osteochondral growth-plate width was decreased by hypophysectomy and restored toward normal by GH. Subsequently, segments of resting costal cartilage and osteochondral growth plate from hypophysectomized rats were incubated in vitro with [35S]sulfate and [3H]thymidine. Within ribs, growth plate generally had basal uptake greater than resting cartilage and a greater increase in activity on addition of normal rat serum. Across ribs 3-9, there was little change in growth plate or resting cartilage sulfate uptake, and only a modest increase in thymidine uptake caudally. Increasing concentrations of normal rat serum provided parallel stimulation for both growth plate and resting cartilage, with significant responses to as little as 1 microliter (sulfate uptake) or 5 microliters (thymidine uptake). Stimulation for both growth plate and resting cartilage by hypophysectomized rat serum was significantly less than by normal rat serum; activity was restored by GH in vivo, establishing growth plate responsiveness to GH- and pituitary-dependent factors. Growth plate and resting cartilage were generally unaffected by exposure in vitro to supraphysiological concentrations of GH, cortisol, testosterone, estradiol, or T3. Use of osteochondral junction tissue appears to yield an assay system that is sensitive and sufficiently reproducible to allow study of growth factor action at the growth plate in vitro.

Interleukin-2 in the ontogeny of human lymphoid tissues

Cells from different fetal and adult lymphoid organs were tested for the capacity to (a) react to the T-cell growth factor interleukin-2 (IL-2) and (b) to produce IL-2 under appropriate conditions. IL-2 was determined as growth promoting activity for mouse T blasts. Optimal conditions for IL-2 production were: 5 × 10 6 cells/ml; 4–6 µg mitogen, 24 h incubation time. Concanavalin A was preferable for fetal thymus, whereas phytohemagglutinin was the appropriate mitogen for lymphocytes from blood and lymph node. Fetal and adult spleen cells produced equal activities of IL-2 irrespective of the mitogen used. Cells from thymus and spleen of 17–21-week-old fetuses produced IL-2 activities like adult cells. Fetal liver cells from the same fetuses produced no IL-2. A comparison of IL-2 activities released from cells of 26 donors showed that within individual variations there was no decrease of the capacity to produce IL-2 with old age. An activity present in culture media of mitogen treated lymphocytes which increased thymidine uptake in fetal liver cells could be distinguished from IL-2 by its different molecular weight and by a heat treatment which abolished IL-2 but not fetal liver cell growth promoting activity. This latter activity is discussed to be colony-stimulating activity. It is concluded that IL-2 is produced by and reacts with T cells only after they have reached or passed the thymus.

The effect of folate analogues on the thymidine utilization by human and rat marrow cells and the effect on the deoxyuridine suppression test.

Summary The deoxyuridine (dU) suppression test assesses the capacity of marrow cells or activated lymphocytes to convert exogenous dU into thymidine. In addition, the effect of the added cobalamin or folate on the result is used to determine the nature of the deficiency in megaloblastic marrows. This study shows that folates have a considerable effect on the uptake of labelled thymidine by marrows from patients with megaloblastic anaemia in the absence of added dU. Less thymidine was taken up by megaloblastic marrow cells if they were first incubated with 5-formyltetrahydrofolate, 10-formyltetrahydrofolate or tetrahydrofolate. 5-Methyltetrahydrofolate increased thymidine uptake in cobalamin-deficient marrows and reduced thymidine uptake in folate-deficient marrows. These results could be explained if added folates facilitated utilization of endogenous dU. However, the addition of folates or cobalamin did not affect the validity of a dU suppression test with human marrow. More marked changes were present on the addition of folates to rat marrow cells both from control and nitrous oxidetreated animals.

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: I. Study of proliferative response

Culture of normal CBA lymphocytes on monolayers of syngeneic thyroid epithelial cells leads to significant thymidine incorporation. The specificity of this model was demonstrated by depletion of the CBA lymphocytes binding to syngeneic thyroid cells and the increase of thymidine uptake after secondary exposure on syngeneic thyroid monolayers. Removal of B cells (by treatment with anti-Ig serum plus complement) or of adherent cells does not modify the proliferative response whereas T-cell depletion strongly diminishes the response. Thus T cells are stimulated to undergo DNA synthesis and are sensitized when exposed to syngeneic thyroid epitelial cells. The nature of the antigens recognized by T cells (native autoantigen, enzyme, or virus-modified autoantigen) is not yet determined. Whether such autoreactive T cells play a role in the onset of experimental autoimmune thyroiditis as regulatory T-cells or cytotoxic effector cells is discussed.

Kinetics of human lymphocyte responses in vitro: the phase of exponential growth.

The kinetics of thymidine uptake in human peripheral lymphocytes stimulated by allogenic cells, antigen E (ragweed allergen) and a variety of mitogens can generally be divided into four consecutive phases. First, a lag period with no increase in thymidine uptake, then a short period of rapid change in uptake, followed by a log-linear growth period and finally a decay phase. In this report we examine in detail the characteristics of the third, log-linear growth phase. Since, as discussed in the preceding paper, thymidine uptake is proportional to the number of cells acumulating thymidine, we can calculate from the log-linear growth period an apparent doubling time. We show that for five different stimulating agents the cells reach a log-linear growth phase of varying length and that the doubling times show little variation. This invariance indicates that, despite possible variation in cell death and recruitment rates, the rate of proliferation is in all cases dominated by the generation time of human lymphocytes.

Stimulation by a hydroxythiazolobenzimidazole of enhanced formation of antibodies to sheep erythrocytes in vitro and in vivo

A hydroxythiazolobenzimidazole, a low molecular weight compound, was found to have an immunoenhancing effect on both the in vivo and in vitro antibody response of mouse spleen cells to an optimum immunizing dose of sheep red blood cells. At noncytotoxic concentrations the optimum range of 25 to 50 micrograms per 5 x 19(6) spleen cells was most effective in vitro. Concentrations greater than 100 micrograms per culture were toxic in vitro and reduced cell viability as well as antibody responsiveness. The compound enhanced to an even greater degree the antibody response of spleen cell cultures immunized with suboptimum doses of SRBC. The background PFC response, in the absence of SRBC, also was stimulated by the benzimidazole. These immunoenhancing responses were not related to mitogenic effects, since increased thymidine uptake did not occur when normal mouse spleen cells were incubated with graded doses of the compound. Therefore, the immunostimulatory properties of the compound, both in vivo and in vitro, were not due to mitogenic stimulation of lymphoid cells.

Endothelium-lymphocyte interactions in vitro: II. Adherence of allergized lymphocytes

Peripheral blood lymphocytes from skin graft-sensitized pigs will adhere in vitro to fresh donor-type large vessel endothelium, but do not spread out or migrate. Similar cells will however spread out on and migrate through monolayers of cultured donor-type aortic endothelium to a significantly greater extent than nonallergized lymphocytes. Cells sensitized in mixed lymphocyte culture at first exhibit a nonspecific increase in adherence and migration correlated with increased thymidine uptake, but after more prolonged incubation adherence becomes specific for stimulator-type endothelium. It is suggested that lymphocyte infiltration of an allograft in the presence of circulating sensitized cells involves a combination of nonspecific lymphocyte adhesion to endothelium, antigenic stimulation of “primed” cells to increased motility, endothelial penetration and lymphokine production, and soluble-factor-mediated stimulation of migration by nonsensitized cells.

Stimulation by amphotericin B of uridine transport, RNA synthesis and DNA synthesis in density-inhibited fibroblasts

Amphotericin B is known to enhance the permeability of biological membranes through binding to membrane cholesterol. When added to density-inhibited 10T1/2 cells, this antibiotic stimulated uridine transport into the cells and its incorporation into RNA. This effect was not due to non-specific stimulation of the uptake of nutrients into the cells, since the antibiotic treatment of the cells brought about no change in either adenosine transport, nor its incorporation into RNA. A similar increase in uridine uptake caused by serum addition was observed within 1 h, whereas the increase stimulated by amphotericin B was preceded by a long lag of 10 h. Furthermore, amphotericin B increased uptake of thymidine into DNA and also caused a 20% increase in number of nuclei labeled with thymidine. The increase of thymidine uptake was inhibited by hydroxyurea. The results suggest that amphotericin B altered membrane permeability and allowed the initiation of DNA replication in density-inhibited fibroblasts.

Immunologic dysfunction in the myeloproliferative disorders

Forty patients with various types of myeloproliferative disorders were evaluated immunologically. Serum immunoglobulin levels were within the normal range in most patients and no monoclonal gammopathies were detected. Serum C'3 levels were decreased in 19 of 40 (48%) patients. The response of peripheral blood lymphocytes to phytohemagglutinin was decreased in 26 of 40 (65%) and to pokeweed mitogen in 18 of 28 (64%) patients studied. Lymphocytes from patients with polycythemia vera were least affected. Unstimulated lymphocytes from some patients demonstrated markedly increased thymidine uptake activity. Despite the diminished mitogenic response, only 2 of 33 patients (6%) were anergic by intradermal skin testing. There was no association between depressed lymphocyte response and recent chemotherapy except in chronic myelogenous leukemia where 6 of 8 patients were receiving cytotoxic therapy when studied. These observations suggest that most of our patients with myeloproliferative disorders have abnormal cellular responses in vitro, but that delayed hypersensitivity and humoral responses are minimally affected.

Cell-mediated immunity in lichen planus. In vitro tests with extracts from lichen planus lesions.

Extracts were prepared from lichen planus lesions and from healthy epidermis of normal controls. Their effect on the migration and lymphocyte transformation of autologous leukocytes was studied. On the whole, the ranges of variation and mean values of the autologous leukocyte migration indices obtained in the presence of LP skin extract or sediment from 10 LP patients did not significantly differ from those recorded in the presence of similar extracts prepared from the epidermis of 12 normal controls. Two assays with LP skin sediment antigen and one assay with LP supernatant antigen, however, fell within the range of significant changes in migration. Lymphocyte transformation, as indicated by increased thymidine uptake, was significantly stimulated by LP epidermis in 1 case of 5 LP patients. Extracts from normal epidermis did not affect the transformation of autologous lymphocytes. The phytohemagglutinin response of lymphocytes from LP patients was the same as that of normal controls. The results do not indicate that cell-mediated immunity toward epidermal antigens play any significant role in the etiopathogenesis of lichen planus.

Increased thymidine uptake by methylcholanthrene-treated C3H/10T1/2 cells.

The uptake of 3H-thymidine in post-confluent cultures of methylcholanthrene-transformed C3H/10T1/I mouse embryo cells was markedly higher than in their non-transformed counterparts. In a reconstruction experiment as few as 2% transformed cells could be detected by increased thymidine uptake. Measurements made at various times up to 110 days in multi-chambered plates revealed that after 25 days methylcholanthrene-treated cultures incorporated significantly more thymidine than the acetone-treated controls. This increased uptake correlated with the appearance of Type III transformed foci.

The lipoprotein of the outer membrane of Escherichia coli: a B-lymphocyte mitogen.

The lipoprotein of the outer membrane of Escherichia coli is a B-cell mitogen in mice. Polyclonal activation of B lymphocytes was measured by an increase in thymidine uptake, by the development of plaque-forming cells against densely coupled trinitrophenylated sheep red cells, and by selectively increased rates of synthesis and secretion of leucine-labeled IgM. Murein-free and muropeptides-containing lipoprotein are effective in B-cell activation, while free murein is inactive. Removal of ester-linked fatty acids from the amino-terminal end of the lipoprotein by alkaline hydrolysis abolishes the mitogenicity of the lipoprotein. B lymphocytes from high responder (C3H/Tif and BALB/c nu/nu) or from low responder (C3H/HeJ) mice to the mitogen lipopolysaccharide (LPS) both respond well to the lipoprotein. Anti-immunoglobulin antibodies inhibit the mitogenic stimulation of B cells by lipoprotein. A complex of structures including the Ig-receptor molecules, the LPS receptor, and the lipoprotein receptor appear involved in the regulation of mitogenic stimulation of B cells to proliferation and differentiation to IgM-secreting cells.

Cytokinetics of human acute leukemia before and after chemotherapy

In order to evaluate the relationship of relative and absolute blast size to blast proliferative activity, both in treated and untreated human acute leukemia, blasts from peripheral blood of patients with acute leukemia were labeled in vitro with tritiated thymidine and then separated as to size by the 1 g sucrose gradient technique for subsequent evaluation of thymidine uptake and DNA content. Results suggested: (1) Leukemic lymphoblasts were smaller and less variable in size than leukemic myeloblasts; (2) thymidine uptake was excluded from relatively small cells and concentrated in relatively large cells, with some medium cell uptake; (3) DNA content of blasts increased with cell size; (4) Cytosine arabinoside and 6-thioguanine in AML caused an early increase in thymidine uptake and a shift of the cell size distribution to larger cells followed by a rapid depletion of all blasts with preferential large cell loss. This is consistent with early recruitment of small cells into cycle and S-phase specific cell kill (5) l-asparaginase in ALL caused a rapid depletion of blasts with a preferential loss of both large and medium cells and with decreased thymidine uptake, consistent with cycle nonspecific kill plus a G 1 S block inhibiting recruitment. The data support a cytokinetic model of human acute leukemia in which relative cell size correlates with cell cycle stage. Changes in size distribution may reflect drug effect and may be of value in evaluating response to chemotherapy, both of proliferating and resting cells, and in planning and sequencing multiple drug chemotherapy.

Production by murine spleen cells of an activity stimulating the PHA-responsiveness of thymus lymphocytes

An increase in thymidine uptake was found when murine thymus cells were cultured together with spleen cells in the presence of phytohemagglutinin (PHA). The results of experiments with γ-irradiated and mitomycin C-treated cells indicated that this effect was due to enhancement of the PHA responsiveness of thymus cells by spleen cells (Thymocyte Stimulating Activity: TSA). TSA was found to be present in supernatants of cultures of spleen cells but not of bone marrow or thymus cells from normal or hydrocortisone-treated mice. Some TSA were present in cultures of lymph node cells. TSA did not enhance the PHA responsiveness of spleen or lymph node cells. The PHA-responsiveness of bone marrow cells was stimulated only to a small degree. PHA was a necessary requirement for the production of TSA. TSA was not mitogenic for thymus cells in the absence of PHA. The absence of adherent cells did not prevent production of TSA.

Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes.

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.

The participation of thymus-derived and of bone marrow-derived lymphocytes of sensitized mice, in the proliferative response to specific antigen, in vitro.

AbstractSpleen cells of mice primed with sheep red cells (SRC) responded with increased thymidine uptake when stimulated with SRC in vitro. Depletion of bone marrow‐derived (B) cells by filtering the spleen cells through columns of plastic beads coated with anti‐mouse immunoglobulin serum resulted in a drastic loss of the proliferative response. A similar loss occurred on depleting the thymus‐derived (T) cells by treatment with anti‐Θ serum and complement. Addition of peritoneal exudate cells failed to restore the reactivity of the purified T cells to SRC. The proliferative response could be restored to a level exceeding the sum of the individual responses, by mixing the purified T with the purified B cells. A synergistic interaction between T and B cells is suggested.

IN VITRO LYMPHOCYTE STUDIES IN HYDATID DISEASE

Top of pageAbstract Diagnosis of hydatid disease becomes a clinical problem because of cross reactivity of the serological and delayed hypersensitivity tests among echinococci and tapeworms such as tenia and hymenolepis nana. In this study in vitro lymphocyte response to the echinococcus antigen (cyst fluid) in patients with hydatid disease, teniasis and hymenolepiasis is investigated in tissue cultures by measuring the incorporation of tritiated thymidine (H3Tdr) into DNA. Lymphocytes from the patients with hydatid disease showed increased H3Tdr uptake to echinococcus antigen. Also a cross reaction with teniasis and hymenolepiasis is detected in some patients by increased thymidine uptake on the lymphocytes from these patients after stimulation by the echinococcus antigen.