Arrest of cultured cells in the G1 phase of the cell cycle by indomethacin.

Indomethacin reversibly inhibited growth of rat hepatoma cells (HTC) and human diploid fibroblasts in the G1 phase of the cell cycle. Cytophotometric measurements showed that greater than 90% of cells incubated for 48 hr with indomethacin had a DNA content that corresponded to the G1 state. Synchronous growth of both the HTC and fibroblast cultures occurred after removal of drug as indicated by the sequence of changes in [3H]thymidine incorporation into DNA, cellular DNA content, mitotic index and cell number. Autoradiographs of HTC cell cultures incubated with [3H]thymidine indicated that all (98%) of the cells engaged in DNA synthesis following the removal of indomethacin. Since viability of the cells was not impaired, even by prolonged exposure to indomethacin, this drug provides a means of synchronizing growth. Suppression of cell proliferation could contribute to the therapeutic and/or toxic effects of indomethacin in vivo.     

Thymidine transport and metabolism in choroid plexus: effect of diazepam and thiopental

Choroid plexus contains an active transport (influx) and a facilitated diffusion (efflux) system for nucleosides. The ability of diazepam and thiopental to inhibit active transport or facilitated diffusion of thymidine in choroid plexus was measured in vitro under various conditions. When isolated rabbit choroid plexuses were incubated in artificial cerebrospinal fluid containing 1 microM [3H] thymidine for 10 min at 37 degrees C under 95% O2-5% CO2, diazepam (10 microM) and thiopental (500 microM) doubled the tissue-to-medium ratios of [3H] thymidine from 8 to 15 to 16. These results were not due to metabolism or intracellular binding but rather to inhibition of [3H] thymidine efflux from choroid plexus. Diazepam, unlike thiopental, inhibited [3H] thymidine efflux in a concentration-dependent manner. When isolated choroid plexuses were incubated in artificial cerebrospinal fluid containing low concentrations of [3H] thymidine (6 nM) to allow intracellular conversion of [3H] thymidine into [3H] thymidine phosphates and [3H] DNA, both diazepam (10 microM) and thiopental (500 microM) altered [3H] thymidine accumulation and metabolism consistent with inhibition of facilitated diffusion but not active transport of thymidine. These studies provide evidence that, at toxic but not therapeutic concentrations, diazepam and thiopental alter facilitated nucleoside transport in the choroid plexus.     

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin.

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of [3H]thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of [3H]thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.     

Comparative studies of humoral factors stimulating granulopoiesis in agar in vitro and in diffusion chambers.

The factor stimulating granulopoiesis in diffusion chambers (DCF) was compared with the CSA in post-EMS in two ways: (1) Mouse bone marrow cells were maintained in suspension cultures, containing DCF or EMS, which were assayed at intervals for colony-forming cells, both in agar in vitro and in diffusion chambers. (2) The enhanced growth seen in chambers incubated in pretreated mice was compared with the CSA of serum and DCF recovered from these animals. The results show that the cells forming granulocytic colonies in agar in vitro and in diffusion chambers are closely related, if not identical, and that different sources of CSA may differ in their ability to maintain precursor cell numbers in suspension culture. The CSA of DCF did not correlate with growth enhancement in diffusion chambers or with the levels of CSA in the serum.     

Influence of ageing on blastogenesis of mouse thymocytes in response to concanavalin A

The influence of ageing on the blastogenic response of mouse thymocytes to a mitogen was investigated. Thymocytes from mice of various ages (10-70 days) were incubated with the mitogen, concanavalin A, to induce blastogenesis. The thymocytes were then incubated with [3H]thymidine, and the radioactivity of [3H]thymidine taken up by the cells was measured. The stimulation index (ratio of radioactivity uptake by cells incubated with and without mitogen) was used as a measure of blastogenesis of thymocytes. The stimulation index of thymocytes from 10-day-old mice was 2.9; it increased with age, reaching a peak of 33.7 in 40-day-old mice and then gradually decreasing to 8.2 in 70-day-old mice. The blastogenesis of thymocytes, an indicator of thymus function, was thus found to change considerably with age.     

Identification of proliferating dendritic cell precursors in mouse blood.

While it has been known that dendritic cells arise from proliferating precursors in situ, it has been difficult to identify progenitors in culture. We find that aggregates of growing dendritic cells develop in cultures of mouse blood that are supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF) but not other CSFs. The dendritic cell precursor derives from the Ia-negative and nonadherent fraction. The aggregates of developing dendritic cells appear at about 1 wk of culture, with 100 or more such clusters being formed per 10(6) blood leukocytes. The aggregates can be dislodged and subcultured as expanding clusters that are covered with cells having the motile sheet-like processes ("veils") of dendritic cells. By about 2 wk, large numbers of single, major histocompatibility complex (MHC) class II-rich dendritic cells begin to be released into the medium. Combined immunoperoxidase and [3H]thymidine autoradiography show that the cells that proliferate within the aggregate lack certain antigenic markers that are found on mature dendritic cells. However, in pulse-chase protocols, the [3H]thymidine-labeled progeny exhibit many typical dendritic cell features, including abundant MHC class II and a cytoplasmic granular antigen identified by monoclonal antibody 2A1. The progeny dendritic cells are potent stimulators of the mixed leukocyte reaction and can home to the T-dependent areas of lymph node after injection into the footpads. We conclude that mouse blood contains GM-CSF-dependent, proliferating progenitors that give rise to large numbers of dendritic cells with characteristic morphology, mobility, phenotype, and strong T cell stimulatory function.     

Rapid, radiolabeled macrophage culture method for detection of dapsone-resistant Mycobacterium leprae.

Mycobacterium leprae cells extracted from the skin biopsies of 14 bacilliferous lepromatous patients were maintained in human-murine macrophage cultures for 3 weeks in the presence of [3H]thymidine and DDS (4,4'-diaminodiphenyl sulfone). All cultures except one containing freshly extracted viable bacilli showed significant incorporation of [3H]thymidine as compared to control cultures containing heat-killed bacilli of the corresponding strain. Six susceptible strains of M. leprae obtained from untreated, freshly diagnosed patients showed significant inhibition of the uptake of the radiolabel in the presence of 3 and 10 ng of DDS per ml per culture. Eight strains of M. leprae obtained from patients clinically suspected of DDS resistance were tested in a similar manner. These strains were also concurrently inoculated in the footpads of mice given orally 10(-2), 10(-3), and 10(-4) g of DDS per 100 g of body weight for 9 months. Concordant results were obtained by both methods: five strains were found to be resistant, one was susceptible, and one was partially resistant. Strain VIII did not incorporate [3H]thymidine in the macrophage cultures and proved to be resistant in the mouse footpad. The macrophage culture system provides a sensitive, rapid screening method for the early diagnosis of DDS resistance.     

Biochemical mechanisms in the Killmann experiment: critique of the deoxyuridine suppression test.

The degree of inhibition of [3H]thymidine incorporation into DNA by exogenous deoxyuridine is assayed in a procedure known as the deoxyuridine suppression test. We report studies of the biochemical basis of this phenomenon in phytohemagglutinin-stimulated lymphocytes, which suggest that its mechanism has not been fully understood. Results show that inhibition by deoxyuridine is caused only in part by expansion of the intracellular pools of nonradioactive dTMP and dTTP, which dilutes the specific radioactivity of the [3H]dTMP and [3H]dTTP derived from [3H]thymidine. Increased dTTP levels also inhibit thymidine kinase. In addition, thymidine kinase is competitively inhibited by intracellular deoxyuridine. Inhibition of thymidine kinase activity by both mebolites further decreases the specific radioactivity of [3H]dTMP and [3H]dTTP. Deoxyuridine also inhibits the incorporation of [3H]deoxyadenosine and [3H]deoxyguanosine into DNA in these cells. Exogenous deoxyuridine still inhibits [3H]thymidine incorporation in cells whose de novo thymidylate synthesis has been strongly inhibited by 5-fluorodeoxyuridine or methotrexate. In such drug-treated cells, exposure to high concentrations of exogenous deoxyuridine can partially overcome the inhibition of thymidylate synthetase with resulting increase in the severely depleted dTTP pools. This increase is associated with enhanced DNA synthesis, as measured by incorporation into DNA of labeled deoxyribonucleosides other than [3H]thymidine. We conclude that exogenous deoxyuridine has multiple effects on [3H]thymidine incorporation, which must be considered in interpretations of deoxyurindine suppression test results.     

Inhibition of lymphoproliferation by hyperlipoproteinemic plasma.

Plasma for patients with primary type IV or V hyperlipoproteinemia inhibited [3H]thymidine incorporation by cultured mononuclear leukocytes. This previously unreported abnormality affected mononuclear leukocytes from patients with type IV or V hyperlipoproteinemia and from normal subjects. Patient cells incorporated [3H]thymidine normally when washed and incubated in medium containing normal plasma. Both spontaneous incorporation and stimulated incorporation in response to various mitogens and antigens were inhibited. The inhibitory effect was identified with the chylomicron and very low density lipoprotein fractions isolated from plasma and was concentration-dependent. Lectin used to stimulate cultured cells and [3H]thymidine used to measure responses were not bound to the lipoproteins in appreciable amounts. [3H]-Thymidine incorporation correlated well with morphologic evidence of lymphoproliferation. The mechanism of the inhibitory effect of type IV or V hyperlipoproteinemic plasma upon the response tested was not identified by may be related to interaction between lipoproteins and the cell membranes. We suggest that these lipoproteins may also interfere with the function of other cells.     

Autonomy of growth and of iodine metabolism in hyperthyroid feline goiters transplanted onto nude mice.

Hyperthyroidism caused by nodular goiters is a common disease of aging cats. Growth and iodine metabolism were studied by autoradiography in normal and hyperfunctioning thyroid tissue obtained from cats injected with 125I before surgery, and in xenografts, grown in nude mice, after double-labeling with 131I and [3H]thymidine. Hyperthyroid cat goiters contain single or multiple hyperplastic nodules, consisting of highly cellular tissue with an iodine metabolism exceeding that of the surrounding normal tissue. Xenografts of hyperplastic hot tissue in thyroxine-treated nude mice retain their original histologic pattern and continue to accumulate radioiodine intensely. Autoradiographs assessed for [3H]thymidine incorporation reveal autonomously proliferating follicular cells within the hyperplastic foci but not within the normal tissue. Administration of sera from donor cats into host mice fails to stimulate the xenografts. Neither hyperfunction nor growth of toxic cat goiters depends on extrathyroidal stimulators. The basic lesion appears to be an excessive intrinsic growth capacity of some thyroid cells.     

Regulation of Glycosaminoglycan Synthesis by Thyroid Hormone in Vitro

Human skin fibroblasts synthesize and accumulate glycosaminoglycans (GAG). Recently, we reported that fibroblasts incubated in thyroid hormone-deficient media accumulate more GAG than do cultures incubated in the same media enriched with 0.1 μM triiodothyronine (T₃) (1981. Endocrinology. 108: 2397). The current study characterizes that enhanced accumulation. Confluent cultures were maintained in thyroid hormone-deficient media without or with added T₃, labeled with [³H]acetate and analyzed for total [³H]GAG and [³H]hyaluronic acid content.     

Antidepressant binding sites in brain: autoradiographic comparison of [3H]paroxetine and [3H]imipramine localization and relationship to serotonin transporter

Binding of two different antidepressant drugs, [3H]paroxetine and [3H]imipramine in 30 rat brain regions was visualized, compared and quantified by means of autoradiography and densitometry. Specific binding of [3H]paroxetine to coronal sections of diencephalon represented 85% of total binding and was saturable and of high affinity (KD, 0.36 +/- 0.07 nM) with a maximum number of binding sites of 276 +/- 41 fmol/mg protein. The autoradiograms showed a heterogenous distribution of [3H]paroxetine in brain with selective accumulation of label in brain regions known to contain serotonergic terminals, axons and cell bodies (amygdaloid and raphe nuclei, superior colliculus, substantia nigra and medial forebrain bundle). Binding was displaced selectively with other serotonin uptake inhibitors (clomipramine and fluoxetine) and almost abolished by lesioning the serotonergic neurons with p-chloroamphetamine. The desipramine-sensitive [3H]imipramine binding was more diffuse with relatively high density in cerebral cortex and hippocampus and was only decreased partially in animals treated with p-chloroamphetamine. The results indicate that [3H]paroxetine, but not [3H]imipramine, is a ligand of choice to selectively label serotonergic structures in brain.     

Suppression of human T-cell mitogenesis by prostaglandin. Existence of a prostaglandin-producing suppressor cell

Small amounts of PGE inhibit mitogen-induced [3H]thymidine incorporation in human peripheral lymphocytes. The 50% inhibitory concentration is approximately 10(-7) M, and this is reduced to approximately 10(-8) M when endogenous PGE production is blocked. PGE inhibits PHA- and Con A-stimulated cultures much better than PWM cultures, suggesting a differential effect of PGE on T-cell vs. B-cell function. In vitro blockade of PG synthesis results in approximately 50% increase in [3H]thymidine incorporation in PHA cultures. PGE is produced endogenously in PHA cultures by glass adherent suppressor cells.     

Effect of atmosphere of incubation on the isolation of group A streptococci from throat cultures

The optimal atmosphere of incubation for the isolation of group A beta-hemolytic streptococci from throat cultures has been the subject of considerable debate. To resolve this issue, we examined 5992 consecutive throat cultures performed at a private pediatric office in children with acute upper respiratory tract infections. All throat cultures were streaked onto duplicate blood agar plates, one of which was then incubated anaerobically and the other aerobically. beta-Hemolytic streptococci were isolated in cultures from 1885 (31.5%) of the patients; 1479 (24.7%) were identified as group A, and 406 (6.8%) were identified as non-group A. Group A streptococci were recovered significantly more often from the plates incubated anaerobically than from those incubated aerobically (1467 vs. 1054; anaerobic only, 425; aerobic only, 12; P less than 0.01). Non-group A streptococci were also recovered significantly more often from the plates incubated anaerobically than from the plates incubated aerobically (397 vs. 170; anaerobic only, 236; aerobic only, 9;P less than 0.01). Anaerobic incubation maximizes the yield of the throat culture. The additional cost and effort of anaerobic incubation are small, and would appear to be justified by the increased isolation rate of group A beta-hemolytic streptococci. The significance of the increased isolation rate of non-group A beta-hemolytic streptococci with anaerobic incubation needs to be investigated further.     

Epidermal growth factor accelerates functional recovery from ischaemic acute tubular necrosis in the rat: role of the epidermal growth factor receptor

1. Severe, ischaemic, acute tubular necrosis was induced in rats by bilateral occlusion of the renal arteries. The experimental group received exogenous epidermal growth factor infused directly into the renal arterial circulation. Serum creatinine concentration was measured daily for 1 week. Epidermal growth factor receptor binding was measured by autoradiography of whole kidney sections. Renal cell proliferation was measured by incorporation of [3H]thymidine into DNA. 2. Serum creatinine concentration increased after acute tubular necrosis with a peak at 48 h and remained elevated above control levels after 7 days. Binding of radiolabelled epidermal growth factor occurred in all regions of the kidney 48 h after ischaemia. Treatment with exogenous epidermal growth factor attenuated the rise in serum creatinine by 4 days after acute tubular necrosis and after 7 days serum creatinine was lower than in animals that did not receive epidermal growth factor. Infusion of epidermal growth factor also increased renal DNA synthesis. 3. The increase in epidermal growth factor binding in the kidney after acute tubular necrosis and the attenuation of the increase in serum creatinine concentration by administration of exogenous epidermal growth factor, suggest a role for epidermal growth factor in recovery from ischaemic damage. The increase in DNA synthesis in response to epidermal growth factor indicates that its effect may be due, at least in part, to accelerated tubular cell proliferation.     

Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis.

To understand the relationship between the proliferation of epidermis and its arachidonic acid metabolism, we studied human keratinocytes grown in vitro at confluent or nonconfluent densities. Keratinocyte cultures incubated with [14C]arachidonic acid synthesized prostaglandin (PG)E2 PGD2, PGF2 alpha, and small quantities of 6-keto-F1 alpha. Nonconfluent cultures, however, synthesized fourfold more PGE2 than did confluent cultures. When proliferation was studied using [3H]thymidine incorporation into DNA, it was found that this increased synthesis of PGE2 was accompanied by a fourfold increase in the rate of proliferation. When PGE2 synthesis was inhibited by indomethacin, the rate of proliferation of nonconfluent cultures was decreased 40%, while the rate of proliferation of confluent cultures was unchanged. Addition of 1 ng/ml of PGE2, but not PGF2 alpha, PGD2, or a stable analog of PGI2 to the indomethacin-treated nonconfluent cultures restored the initial rate of proliferation. These results suggest that PGE2 is a growth-promoting autocoid for epidermis. The synthesis of PGE2 by epidermis may be enhanced in wound healing and disease states where epidermal continuity is disrupted.     

Studies of endotoxin-induced decrease in lipoprotein lipase activity

A variety of invasive stimuli have been shown to induce hyperlipidemia due to impaired removal of triglyceride from the circulation. The mechanism by which endotoxin induces a deficiency in the activity of the key enzyme of triglyceride metabolism, lipoprotein lipase (LPL), has been studied. In C3H/HeN (endotoxin-sensitive) mice, LPL activity in adipose tissue was markedly suppressed 16 h after endotoxin administration. In contrast, the endotoxin-resistant C3H/HeJ mice were less sensitive to the suppressive effect of endotoxin on LPL activity. After endotoxin administration, a transferable factor had been detected in the blood of C3H/HeN mice 2 h after the injection of endotoxin that causes a suppression of adipose tissue LPL activity in C3H/HeJ mice as well as in C3H/HeN mice. Conditioned medium from the cultures of peritoneal exudate cells of C3H/HeN mice incubated in endotoxin also suppresses adipose tissue LPL in C3H/HeJ mice. These studies demonstrate that exudate cells produce a humoral factor in response to endotoxin, which suppresses adipose tissue LPL.     

肝素、高糖对人腹膜间皮细胞生长及合成细胞外基质的影响

目的 探讨肝素、高糖对体外培养的人腹膜间皮细胞(HMC)生长和合成细胞外基质影响。方法~3H-胸腺嘧淀核苷掺入法检测细胞增生;ELISA法检测细胞培养上清纤维连结蛋白(FN)、Ⅰ型胶原(COLⅠ)含量,细胞计数校正其浓度;RT-PCR检测FN基因表达。结果 1.36％葡萄糖介质中培养72h可见HMC~3H-TdR掺入量明显降低(P<0.05),呈时间和剂量依赖关系,高糖环境下肝素对HMC增殖率无明显影响;3.86％葡萄糖可明显促进FN和COLⅠ的分泌,肝素可明显降低高糖环境下FN和COLⅠ的分泌。结论 高糖可能是长期腹透致腹膜硬化的一个重要因素;肝素对保护腹膜功能可能有一定的作用。     

Immunological studies of aging. Decreased production of and response to T cell growth factor by lymphocytes from aged humans.

Human lymphocytes from elderly and young donors were cultured with phytohemagglutinin (PHA) or concanavalin A. Cultures from old donors produced less T cell growth factor (TCGF) and incorporated less tritiated thymidine (3H-Tdr) than did similar cultures from young donors in the presence of either mitogen. Furthermore, the response of lymphocytes from elderly donors to TCGF was impaired. Thus, PHA-activated T cells from aged donors showed no increase tritiated thymidine incorporation when incubated with exogenous human TCGF. In contrast, addition of exogenous human TCGF to PHA-activated peripheral blood leukocytes from younger individuals increased tritiated thymidine incorporation by 30-50%. The impaired response to TCGF was associated with decreased binding of TCGF by PHA-activated cells from old donors. TCGF production or responsiveness was not associated with the presence of "suppressor" activity in elderly T cell preparations. These studies suggest a possible molecular mechanism for the impaired proliferative response of elderly human T cells. These data lend support to the hypothesis that defects in the capacity to either produce or respond to TCGF may be a fundamental cause of immune deficiency.     

猫脑弥漫性轴索损伤扫描电镜观察

目的:探讨弥漫性轴索损伤(Diffuse axonal injury,DAI)的法医学诊断依据。方法:采用头颅半约束非撞击性旋转暴力致伤方法建立猫脑弥漫性轴索损伤模型。在致伤后不同时间对脑组织进行了HE染色、Bielshowsky Glee银染色和扫描电镜观察。结果:HE染色显示2米损伤组及3米损伤组均未见特征性的改变;Bielshowsky Glee银染色显示2米损伤组未见特征性改变,3米损伤组伤后6小时见收缩球形成,伤后12小时达到高峰,持续至24小时;扫描电镜显示2米损伤组未见特征性改变,3米损伤组伤后4小时可检见收缩球形成,随着时间延长其体积逐渐增大,数量逐渐增多,于12小时达到高峰,持续至24小时。结论:扫描电镜对弥漫性轴索损伤的诊断价值优于HE染色、Bielshowsky Glee银染色。扫描电镜形态学改变对弥漫性轴索损伤的诊断及损伤时间推断有一定的参考价值。