Thymidine Assay Research Articles

Increase by tri-iodothyronine of endothelin-1, fibronectin and von Willebrand factor in cultured endothelial cells.

Hyperthyroidism is associated with elevated plasma levels of endothelium-derived proteins such as von Willebrand factor (vWF), fibronectin (FN) and endothelin-1 (ET-1). This study was designed to characterize the mechanisms involved in this phenomenon at the cellular level. vWF, FN and ET-1 secretion and mRNA expression were measured in human umbilical vein endothelial cells (HUVECs) exposed to tri-iodothyronine (T3) for 13 +/- 1 days, using ELISA, Western blot, RIA and Northern blot analysis respectively. Exposure of HUVECs to T3 significantly increased vWF secretion (50 ng T3/ml: 117 +/- 5%, P < 0.01; 100 ng T3/ml: 127 +/- 26%, P < 0.01) as well as vWF mRNA expression (50 ng/ml: 116 +/- 13%, P < 0.001; 100 ng/ml: 136 +/- 30%, P < 0.002) (results are means +/- S.D. analysed by the Wilcoxon signed rank test). FN secretion was significantly affected by 50 (145 +/- 42% of control, P < 0.05) and 100 (116.8 +/- 16% of control, P < 0.05) ng T3/ml, and FN mRNA expression by 50 ng T3/ml (123 +/- 20%, P < 0.05). Long-term incubation with T3 increased both ET-1 secretion (25 ng/ml: 124 +/- 25%, P < 0.001; 50 ng/ml: 165 +/- 53%, P < 0.05; 100 ng/ml: 116 +/- 17%, P < 0.05) and prepro-ET-1 mRNA expression (25 ng/ml: 112 +/- 16%, P < 0.05; 50 ng/ml: 134 +/- 43%, P < 0.02; 100 ng/ml: 120 +/- 20%, P < 0.02). Protein kinase C (PKC) isoforms epsilon and beta II were not significantly affected by T3, whereas PKC alpha was increased in whole cell lysates and in membrane fractions of cells incubated with 100 but not 50 ng T3/ml. Prepro-ET-1 mRNA stability, cell numbers and proliferation, measured by [3H]thymidine assays, remained unaffected in HUVECs after exposure to T3. These data indicate thyroid hormone-induced upregulation of mRNA expression and protein synthesis of vWF, FN and ET-1, by PKC alpha-, beta II- and epsilon-independent pathways, explaining, at least in part, increased plasma concentrations of endothelial proteins and peptides in the hyperthyroid state.

Microtubule dynamics regulates the level of endothelin-B receptor in rat cultured astrocytes.

We investigated the effect of cytoskeleton modulators on endothelin-B (ET(B)) receptor expression in rat primary cultured astrocytes. Northern blot analysis and a binding study revealed that colchicine and nocodazole, microtubule-disrupting agents, decreased the levels of both ET(B) receptor mRNA and the number of ET-1 binding sites in quiescent astrocytes. Down-regulation of both ET(B) receptor mRNA and the number of binding sites for ET-1 was also observed in quiescent astrocytes treated with taxol, a microtubule-stabilizing agent. In contrast, neither beta-lumicolchicine, an inactive isomer of colchicine, nor cytochalasin D, a microfilament-disrupting agent, influenced ET(B) receptor expression. The level of ET(B) receptors in astrocytes was affected by the cell state, namely, proliferative, quiescent, or differentiated state. The order of ET(B) receptor expression according to the cell state was proliferative state < quiescent state << differentiated state induced by dibutyryl cyclic AMP. Also, in proliferative astrocytes and differentiated astrocytes, colchicine significantly down-regulated both ET(B) receptor mRNA and the number of binding sites for ET-1. However, thymidine assay revealed that colchicine did not change quiescent astrocytes and differentiated astrocytes to a proliferative state. Furthermore, the increase in glutamine synthetase activity in differentiated astrocytes was not affected by colchicine. These results suggest that microtubule dynamics possibly regulates ET(B) receptor expression in astrocytes without affecting the cell state.

Measuring in situ bacterial specific growth rates and population dynamics in wastewater

Reliable design and operation of biological wastewater treatment systems demands robust modelling of the degradation processes. However, methods to directly measure key bacterial growth parameters have not been available to the wastewater industry. Here, we directly measure bacterial specific growth rates without changing the treatment environment. Bacterial growth (with the thymidine assay) and biomass (by direct counts) measurements were used to compare the dynamics of the microbial community in the different compartments of an activated sludge process. The aerobic compartment specific growth rate μ was 0.5 d −1. In this work, we explain the concept of the thymidine growth assay, the traps for the novice and how to apply the method to biological nutrient removal processes. These methods now allow us to distinguish bacterial growth from metabolic activity and observe how the cell allocates its resources between the two. This represents the first application of a very powerful tool to directly measure population dynamics and kinetics of bacteria in wastewater treatment processes.

In situ measurement of archaeal and bacterial specific growth rates in two stage high rate anaerobic treatment systems

The thymidine assay is applied and evaluated as a direct measure of archaeal and bacterial volumetric growth rate in the first and second stages of a full scale High Rate Anaerobic Treatment system (HRAT). By coupling the method to an epifluorescent microscopic technique we show how to measure microbial specific growth rates in situ. In the first stage reactor, the acidogenic (hydrolytic-fermentative) apparent bacterial specific growth rate measurements (2.2 d –1 ) agreed with that predicted by the hydraulic residence time. For the methanogenic archaeal populations in the second stage upflow anaerobic sludge blanket (UASB) reactor the measurement was the same as published values. The results also showed that in the UASB reactor the archaeal growth dynamics in the liquid phase and 'sludge' granules were the same.

Inhibition of Human Transitional Cell Carcinoma in Vitro Proliferation by Fluoroquinolone Antibiotics

Purpose The in vitro effects of the fluoroquinolone antibiotics ciprofloxacin and ofloxacin upon 3 human transitional cell carcinoma cell lines were investigated at concentrations that are attainable in the urine of patients taking these drugs orally. Materials and Methods Cell lines TCCSUP, T24, and J82 were exposed in culture to either ciprofloxacin or ofloxacin at concentrations ranging from 0 to 800 microgram/ml. and at durations ranging from 24 to 120 hours. Inhibition of proliferation and DNA synthesis were assessed via MTT and tritiated thymidine assays, respectively. Results From the MTT assay ciprofloxacin, at concentrations of 25 to 800 microgram/ml., produced proliferation inhibition in the TCCSUP line ranging from 8.1 percent to 90.2 percent at 24 hours, 25.1 percent to 94.9 percent at 72 hours, and 53.8 percent to 96.9 percent at 120 hours. Inhibition of proliferation for the T24 line ranged from 8.0 percent to 85 percent, 31.5 percent to 96.5 percent, and 27.3 percent to 98.2 percent. Inhibition of proliferation of the J82 line ranged from 20.8 percent to 84.8 percent, 22.8 percent to 92.7 percent, and 37.4 percent to 97.1 percent. Inhibition of DNA synthesis (due to ciprofloxacin at the concentrations above) as measured by the tritiated thymidine assay was also significant for each of the 3 cell lines. Inhibition of proliferation and DNA synthesis due to ofloxacin was lower but not overall statistically different from that due to ciprofloxacin. In a separate experiment, enhanced cytotoxicity was observed at lower concentrations of ciprofloxacin when the initial media pH was approximated to 5.5. Conclusions Ciprofloxacin and ofloxacin inhibit proliferation and DNA synthesis of these 3 human TCC lines in vitro. Inhibition occurred in a concentration- and time-dependent manner. The concentrations that were assessed are attainable in the urine of patients taking these agents orally.

Bacterivory by heterotrophic nanoflagellates and bacterial production in sediments of a freshwater littoral system

We made simultaneous measurements of benthic bacterial production, using [3H]thymidine assays, and bacterivory by benthic heterotrophic nanoflagellates (HNAN), using fluorescently labeled bacteria. Sediment samples were collected at two stations in a eutrophic freshwater littoral in different seasons. Bacterial production ranged from 19.7 to 263.0 × 109 bacteria dm‐3 h‐1, with the highest values measured in summer. HNAN grazing rates (3.8–64.2 bacteria flagellate‐1 h‐1) varied significantly with season and station, and rates were correlated with flagellate biovolume. At both stations, there were large discrepancies between bacterial production and HNAN grazing. The estimated bacterivory ranged from 0.4 to 5.2% of bacterial production. The minor impact of HNAN as grazers on bacteria during summer months was caused by high bacterial production and low HNAN abundances. During winter, bacterial production decreased, whereas HNAN abundance increased. However, due to the low grazing rate of these relatively small flagellates, the total HNAN community still consumed only an insignificant amount of the bacterial production. The results of our measurements show that bacterivory by HNAN cannot account for the fate of bacterial production in the littoral studied.

High-Glucose–Triggered Apoptosis in Cultured Endothelial Cells

High ambient glucose concentration, linked to vascular complications in diabetes in vivo, modulates mRNA expression of fibronectin, collagen, tissue-type plasminogen activator, and plasminogen activator inhibitor and induces delayed replication and excess cell death in cultured vascular endothelial cells. To determine the role of high ambient glucose (30 mmol/l) in apoptosis, paired cultures of individual isolates of human umbilical vein endothelial cells (HUVECs) were exposed to both high (30 mmol/l) and low (5 mmol/l) concentrations of glucose for short-term (24, 48, and 72 h) and long-term (13 +/- 1 days) experiments. Incubation of HUVECs with high glucose for > 48 h increased DNA fragmentation (13.7 +/- 6.5% of total DNA, mean +/- SD) versus cultures kept in 5 mmol/l glucose (10.9 +/- 5.6%, P < 0.005), as measured by [3H]thymidine assays. Data were confirmed by apoptosis-specific fluorescence-activated cell sorter analysis of confluent HUVEC cultures, which displayed after long-term exposure to 30 mmol/l glucose a 1.5-fold higher prevalence of apoptosis than control cultures exposed to 5 mmol/l glucose (P < 0.005). In contrast, no increase in DNA fragmentation in response to 30 mmol/l glucose was seen for standardized cell lines (K 562, P 815, YT) and fibroblasts. Expression of clusterin mRNA, originally reported to be a molecular marker of apoptosis, was only slightly affected by short-term (24-h) high-glucose exposure but was significantly reduced after long-term incubation in 30 mmol/l glucose (82.2 +/- 13.8% of control) versus 5 mmol/l glucose, which questions the role of clusterin gene expression as a marker of apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonradioactive alternative to clinical mixed lymphocyte reaction

The MLR is used clinically as a functional assay for genetic HLA disparity. Traditionally this test relies on [ 3H]thymidine incorporation to detect T-cell proliferation as an indicator of alloantigenicity. Environmental and administrative concerns regarding radioisotope use and disposal have encouraged the development of sensitive, nonradioactive assay for T-cell alloactivation. We describe a nonradioactive alternative to the clinical MLR which uses an IL-2-dependent cell line, CTLL20.3, and MTT reduction to detect IL-2 accumulation in MLC SNs as an index of T-cell alloactivation. We first determined (a) the optimal number of CTLL20.3 cells for the assay, (b) the optimal time of SN analysis for IL-2, and (c) additional manipulations that significantly increase the assay sensitivity for IL-2. Using this assay system with patient lymphocyte combinations, we demonstrated that the CTLL20.3-MTT assays correlate well with the [ 3H]thymidine assays of T-cell proliferation for the detection of MHC incompatibility. Indeed, the CTLL20.3-MTT assay may be slightly more sensitive than the traditional clinical MLR. Human Immunology 43:38–44 (1995)

Human eosinophils stimulate DNA synthesis and matrix production in dermal fibroblasts.

Fibrosis is frequently found in diseases exhibiting tissue eosinophilia, such as some parasitic worm infections, eosinophilia-myalgia syndrome, and eosinophilic fasciitis. Previously, eosinophil extracts have been shown to induce proliferation in neonatal foreskin fibroblasts in vitro. To determine if living eosinophils can induce synthesis of DNA and components of the extracellular matrix in dermal fibroblasts, we cultured purified human eosinophils for 2 or 7 days in the presence of the eosinophil-active cytokines granulocyte/macrophage colony stimulating factor, interleukin-3, or interleukin-5, and added eosinophil-conditioned medium to cultures of dermal fibroblasts. Using flow cytometry, we found that eosinophil-conditioned medium increased by two-fold the percentage of fibroblasts in S-phase. This stimulation of fibroblast DNA synthesis was corroborated using a standard tritiated thymidine assay and the two methods were shown to correlate well with each other. Eosinophil-conditioned medium stimulation of DNA synthesis was dose dependent and conditioned medium from eosinophils treated with any one of the three cytokines induced increased DNA synthesis. Treatment of fibroblasts with cytokines alone did not induce enhanced DNA synthesis. Eosinophil-conditioned medium also affected fibroblast matrix production. Eosinophil-conditioned medium induced a two-fold increase in soluble and cell-associated fibroblast glycosaminoglycan production and a 76% increase in collagen production. These observations support the concept that eosinophils may be active contributors to the pathophysiology of eosinophil-associated fibrotic disease.

A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [ 3H]thymidine incorporation assay

A one-step non-radioactive assay to determine the proliferation of murine lymphocytes, lymphoid tumor cells and hybridoma cells is described. This assay requires the addition of Alamar Blue dye to cell cultures and the degree of change in its color, which is reflective of the extent of cellular proliferation, can be determined by an ELISA plate reader. Alamar Blue must be added during the initial phase of cell culture. The pattern of concanavalin A (ConA) or anti-CD3 antibody-induced proliferative response of murine lymphocytes as assessed by Alamar Blue was similar to that of a [ 3H]thymidine assay. Similarly, the spontaneous proliferation curve of anti-CD3 antibody secreting cell line (YCD3-1), monocytic macrophage cell lines (PU5-1.8, P388D1, J774.A1) and myeloma cells (Sp2/0) as determined by Alamar Blue closely resembled that of the [ 3H]thymidine assay. The minimum detectable number of proliferating cells was comparable in Alamar Blue and [ 3H]thymidine assays. Since cell lysis/extraction and washing procedures are not involved in the Alamar Blue assay, this approach has several distinct advantages over currently available assays (eg. [ 3H]thymidine). First, it allows daily monitoring of proliferation without compromising the sterility of cultures. An indication of proliferation can be evaluated (spectrophotometrically or visually) as early as 24 h after ConA stimulation. Second, unlike previously reported assays, Alamar Blue permits further analysis of proliferating cells by other methods. Analysis of cells in culture with Alamar Blue for various surface antigens (CD44, CD45RB, CD4, heat stable antigen) by flow cytometry revealed that the fluorescent profile and relative percentage of cells in cultures with the Alamar Blue were comparable to those without this reagent. The salient advantages of Alamar Blue assay over the [ 3H]thymidine assay include: (i) non-radioactivity; (ii) simplicity; (iii) less costly; (iv) non-labor intensive; (v) rapidity of assessment of proliferation of large number of samples; (vi) non-toxicity; (vii) usefulness in determining the kinetics of cell growth of hybridomas; and (viii) non-interference of secretion of antibodies by a hybridoma cell line.

A new, rapid colorimetric assay for quantitative determination of cellular proliferation, growth inhibition, and viability

A sensitive spectrophotometric test has been developed to rapidly quantitate cell growth and viability on monolayer cultures. The method consists of staining fixed cells with the supravital dye Janus green, extraction of the dye from these cells with absolute alcohol, spectrophotometric measurement of the eluant, and referring optical density (OD) readings to a previously established standard curve such that measurements can be expressed directly as number of surviving cells. Staining of cells with the same colorant before fixation allows microscopical or colorimetric quantitation or both of damaged cells still adherent to the substratum. Both steps can be conveniently carried out on the same cell culture plate. More rapid and easier to perform than the usual cell counting or tritiated thymidine assays, this technique may prove to be useful in evaluation of the effect of growth promoting substances, growth inhibitors, or cytotoxic agents on monolayer cultures.

Proliferation of spleen colony forming units (CFU-S8, CFU-S13) and cells with marrow repopulating ability.

The practicalities of gene transfer therapy using retroviral vector systems require both that host cells be as primitive as possible and that those cells be proliferating. Here, the kinetics of hemopoietic stem cells with marrow repopulating ability (MRA) have been studied with a view to defining the timescale over which these normally quiescent cells can be triggered into cell cycle. Mice were injected with hydroxyurea (1 g/kg) four times over a period of 26 h and assayed at intervals up to eight days for 8-day and 13-day spleen colony-forming units (CFU-S) and for generation of 12-day CFU-S in the bone marrow (MRA assay). The proliferative activity of these cell populations was measured by in vitro tritiated thymidine assays. CFU-S were reduced rapidly to 11% of normal and induced into cycle. Their number and proliferative quiescence recovered by four to five days. Cells with MRA reached their nadir after four days and only then started to proliferate. For each of these progenitor cell subclasses, the proliferative activity inversely reflects their numbers and indicates regulation by negative feedback processes.

Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity

Platelet-derived endothelial cell growth factor (PD-ECGF), a protein which stimulates angiogenesis in vivo, is shown to have a 39.2% amino acid sequence similarity over a 439 amino acid region with the thymidine phosphorylase of Escherichia coli (E. coli). Using recombinant human PD-ECGF, we show that PD-ECGF has thymidine phosphorylase activity. Analysis by gel chromatography revealed that recombinant human PD-ECGF occurs as a 90 kDa homodimer, similar to other thymidine phosphorylases. In addition to a possible effect on DNA synthesis, PD-ECGF was shown to affect [3H]thymidine assays in a manner which is not related to cell proliferation. The in vitro and in vivo effects of PD-ECGF may thus occur by an indirect mechanism through its enzymatic activity.

Bacterioplankton production and protozoan bacterivory in a mesotrophic reservoir

Protozoan bacterivory [via uptake of fluorescently labelled bacteria (FLB)] and production of bacteria ([H]thymidine assay) were simultaneously measured in the mesotrophic Rimov Reservoir (Southern Bohemia) from April to November, 1988. Heterotrophic nanoflagellates (HNF) were mostly responsible for a greater fraction of protozoan bacterivory during the spring period. From 10 to 23% of bacterial production was grazed daily with the only exception of the spring peak of ciliate abundance (up to 60%). Protozoans decreased significantly during the clearwater phase (ciliates disappeared), and thus their role in bacterivory was negligible. Through the summer-fall period ciliates, not HNF, were the most important bacterial micrograzers. Protozoan community grazing balanced or even exceeded the daily bacterial production in August and September. Alternate fates of bacterial production besides protozoan grazing during the spring period are discussed.

A fluorometric assay to monitor mitogenic stimulation of human lymphocytes

A fluorometric assay using Hoechst 33342 to measure DNA in cell culture has been applied to detect mitogenic stimulation of human lymphocytes. Mononuclear cells from the peripheral blood of healthy donors and of HIV infected patients were stimulated with mitogens (PHA and ConA, respectively) in serum-free culture medium (5 × 10 4 or 1 × 10 5 cells/well) and incubated with Hoechst 33 342. Fluorescence intensities were read using an automated fluorescence reader against the dye solution as a blank. The stimulation index (fluorescence units of stimulated vs. unstimulated cell culture) was calculated and compared to the stimulation index obtained in the [ 3H]thymidine assay. A sensitivity of 94.6% and a specificity of 97.4% was observed for the fluorometric assay. The intra-assay variability (12.3–15.6%) and the interassay variability (9.6–21.8%) were calculated using 1 × 10 5 cells/culture for PHA stimulation. We recommend the inexpensive and rapid fluorometric Hoechst 33342 assay as an alternative procedure, for monitoring mitogen induced stimulation of human lymphocytes in the diagnosis of immunodeficiencies.

A rapid non-radioactive fluorescence assay for the measurement of both cell number and proliferation

Measuring the incorporation of radioactive thymidine into the cell nucleus gives important information as to cell activation and proliferation. In this study the DNA-intercalating fluorochromes, Hoechst 33342 and Hoechst 33258, were tested as an alternative to the classical [ 3H]thymidine assay. Mitogen and alloantigen stimulated lymphocytes as well as FK 506 and CsA inhibited lymphocytes were treated with the two dyes, and the cell number and proliferation rates by means of measured fluorescence values. Of these tested fluorochromes H33342 appears to be an appropriate alternative to the [ 3H]thymidine assay. It mirrors the cell number in a fast and convenient manner without any pretreatment of the cell suspension which can remain in the culture plates. The complete assay procedure including data analysis can be performed rapidly and the standard deviations are small. This dye may also prove to be of value in other assay procedures, e.g., adhesion experiments.

The effects of five hematopoietic growth factors on human small cell lung carcinoma cell lines: Interleukin 3 enhances the proliferation in one of the eleven cell lines : Vellenga E, Biesma B, Meyer C, Wagteveld L, Esselink M, De Vries EGE. Department of Hematology, University Hospital, Oostersingel 59, 9713 EZ Groningen. Cancer Res 1991;51:73-6

Eleven small cell lung carcinoma cell lines of human origin were exposed to different colony stimulating factors (CSFs) to study whether CSFs could enhance the spontaneous cell proliferation and modify the action of cytotoxic drugs. In ten cell lines no suppressive or stimulative effect was observed when measured in a [3H]thymidine assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. However, one cell line (GLC-20) could be stimulated by interleukin 3 (IL-3) when measured with a proliferative as well as a clonogenic assay. This enhancing effect was cell concentration dependent in the [3H]thymidine assay. Additional CSFs such as granulocyte-macrophage-CSF, granulocyte-CSF, IL-4, IL-6, insulin, or bombesin could not further augment the IL-3 supported proliferation. In addition, IL-3 binding studies demonstrated the presence of IL-3 receptors on the GLC-20 cells. Two types of receptors were demonstrated by Scatchard analysis: high affinity receptors (59 +/- 4 sites/cell) with a dissociation constant (Kd) of 31 +/- 9 pmol/liter; and low affinity receptors (1915 +/- 91 sites/cell) with a Kd of 2.0 +/- 0.8 nmol/liter. Finally, it was shown that the toxic effects of adriamycin and cisplatin on the proliferation of the GLC-20 cell line could partially be abrogated in the presence of IL-3. These data indicate that in some cases CSFs can modulate the proliferation of small cell lung carcinoma cell lines and interfere with the effects of chemotherapeutic drugs.

Induction of cytotoxicity from fresh splenocytes after in vivo administration of cyclophosphamide. Importance of long-term culture with high-dose recombinant interleukin-2.

Cyclophosphamide, combined with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2), is known to mediate regression of tumors, but the effects of cyclophosphamide on the subsequent generation of LAK cells are unclear. It was the aim of the experiments in this paper to determine whether fresh splenocytes cultured with rIL-2 would maintain or regain their cytotoxicity in vitro after being exposed to the cytotoxic agent cyclophosphamide in vivo. Functional monitoring of splenocytes after in vitro incubation with rIL-2 was performed at various times through chromium-release assays, thymidine assays and cell-cycle analysis. Chromium-release assays determined that the cytotoxicity of cultured splenocytes returned to normal after 12 days of in vitro culture with rIL-2. The thymidine assays indicated a normal rate of uptake of thymidine after 7 days in culture, while the cell cycle was still abnormal by day 12 of culture. The growth and expansion of rIL-2-activated splenocytes after different times of in vitro culture indicated a return to normal compared to control animals after 7 days of continuous in vitro exposure to rIL-2. It is concluded that murine splenocytes can demonstrate cytotoxicity after exposure to cyclophosphamide, through prolonged continuous in vitro culture with rIL-2. Since cyclophosphamide did not jeopardize the production of splenocyte cytotoxic effectors generated with rIL-2, it appears to be a strong contender for use in chemoimmunotherapy protocols.

DNA-synthesis and tumor growth inhibitions by AGGA, a bleomycin-amsacrine hybrid derivative

AGGA, [[(amino-2-ethyl)-2-aminomethyl]-2-pyridine-6-carboxylhistidyl-amino-4-butyrylglycylamino]-4-phenyl-1-amino-9-acridine is a synthetic model gathering the simplified metal-chelating part of bleomycin and the intercalating moiety of amsacrine. This molecule was found to possess the metal-complexing and intercalative properties of both antitumor parent drugs. On the basis of results obtained on L1210 and HeLa S 3 cells growth inhibition studies and labeled thymidine assay, AGGA clearly indicates a cytostatic activity. On the other hand, the oxygenated free radicals produced in the presence of iron and oxygen do not seem to be able to cleave DNA as BLM does. This lack of cytotoxicity is analyzed in terms of fundamental differences between BLM and AGGA-DNA complexes.

Modulation of fibroblast proliferation by postsurgical macrophages

Macrophages and fibroblasts are major components of postsurgical peritoneal repair. In order to understand the interaction between these two cell types, we studied the effects of spent macrophage culture media on fibroblast proliferation. Rabbits underwent resection and reanastomosis of their small intestine. Peritoneal exudative cells (PEC) were then collected from these animals on postoperative Days 4, 7, and 28 and from nonsurgical controls. PEC (5 × 10 5 cells/ml) were cultured in M-199 with 3% fetal calf serum. After 48 hr the spent media from the cultured PEC were harvested, centrifuged (200g for 10 min), and stored (medium: M-D 0, D 4, D 7, D 28). A second group of rabbits underwent peritoneal wall abrasion followed by collection of fibroblasts directly from the site of injury on postoperative Days 1, 4, and 7. After Day 7 of culture, fibroblasts were resuspended and seeded into dishes (1 × 10 5 cells in 1 ml medium), to which was added 1 ml of spent PEC culture medium. After 24 hr of incubation, 1 μCi [ 3H]thymidine was added for an additional 18 hr. Fibroblasts were then collected and the amount of [ 3H]thymidine incorporated into trichloroacetic acid-precipitable material was quantitated. In one protocol, fresh M-199 with 3% fetal calf serum was used, and in another protocol, “U-medium” (which was previously incubated for 48 hr with fibroblasts) was used. The cytology of the PEC was determined by Wright's staining, nonspecific esterase activity, and phagocytosis. At least 80% of the peritoneal exudative cells were identified as macrophages. Postsurgical Day 7 fibroblasts demonstrated greater [ 3H]thymidine incorporation compared to fibroblasts from postoperative Days 1 and 4. Thymidine incorporation into fibroblasts cultured with spent medium from macrophage cultures (M-D 0, D 4, D 7, or D 28) was suppressed compared to the control group, but the M-D 4 and D 7 medium group showed greater [ 3H]thymidine incorporation compared to the M-D 0, D 28 group. This pattern was repeated when the assay was performed with U-medium. In addition, M-D media obtained from various concentrations of PEC was investigated to determine the effect of cell dose on the [ 3H]thymidine assay. Thymidine incorporation was gradually decreased with increasing concentrations of macrophages, but at each macrophage concentration M-D 4 and D 7 media demonstrated greater activities compared to M-D 0 medium. These data suggest that macrophages act as both positive and negative modulators of fibroblast proliferation. Postsurgical macrophages may therefore accelerate the reconstruction of injured tissue as well as moderate the activity of fibroblasts once tissue repair is complete.