Reduced rate of DNA replication fork movement in megaloblastic anemia.

Chromatography on benzoylated naphthoylated DEAE-cellulose has been used to fractionate fully double-stranded from partially single-stranded DNA molecules. DNA was extracted from phytohemagglutinin-stimulated lymphocytes from patients with megaloblastic anemia resulting from vitamin B12 or folate deficiency after pulse-labeling the cells with [3H]thymidine for 5 min and chasing in unlabeled medium for 24 h. No gross accumulation of partially single-stranded material was observed in the DNA of these cells when compared with DNA from similarly labeled control cells obtained by the addition of 5-formyl tetrahydrofolic acid to the culture medium. When DNA from lymphocytes labeled with a 5-min pulse of [3H]thymidine and sheared to fragments of an average length of 18 micrometer was chromatographed on benzoylated naphthoylated DEAE-cellulose, approximately 80% of the label was recovered in the partially single-stranded fraction. After chasing in unlabeled medium the label was progressively transferred to the double-stranded fraction over a period of 2--3 h. The rate of transfer was slower in megaloblastic lymphocytes than in controls. The difference in rate suggested a slower rate of replication fork movement in megaloblastic lymphocytes and so the density shift technique of Painter and schaeffer (J. Mol. Biol. 45: 467--479, 1969) was used to measure the fork rate directly. [3H]Deoxycytidine was used as the labeled nucleoside to avoid possible complications arising from [3H]thymidine labeling of megaloblastic cells. Investigations on the lymphocytes from four patients showed that the replication fork rate in vitamin-treated control lyphocytes was about 1 micrometer/min. The fork rates in the corresponding untreated cells were invariably lower and rates ranging from 40 to 92% of those of controls were observed. Normal lymphocytes treated with the deoxynucleotide pool-depleting drugs methotrexate or hydroxyurea displayed defects in DNA synthesis similar to those of untreated megaloblastic lymphocytes. We propose that the delayed DNA replication fork movement in cells of patients with megaloblastic anemia results from impaired biosynthesis of DNA precursors.     

Evidence for a functional role of endogenously produced somatomedinlike peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells.

Cultured porcine aortic smooth muscle cells and human fibroblasts produce somatomedinlike peptides and secrete them into the surrounding microenvironment. This production has been linked to their ability to replicate. The objective of this study was to determine if a specific anti-somatomedin-C (Sm-C) monoclonal antibody that binds the somatomedinlike peptides could inhibit replication by porcine aortic smooth muscle cells and human fibroblasts. To determine if the antibody could inhibit the effect of endogenously produced somatomedinlike peptide, increasing concentrations of antibody were co-incubated with platelet-derived growth factor, a known stimulant of somatomedinlike peptide secretion, and Sm-C-deficient platelet-poor plasma. Addition of the antibody reduced fibroblast [3H]thymidine incorporation from 35,100 +/- 500 to 10,600 +/- 700 cpm (P less than 0.001), and in smooth muscle cells from 29,600 +/- 1,800 to 10,800 +/- 1,100 cpm (P less than 0.001). Co-incubation of exogenously added Sm-C (20 ng/ml) with maximally inhibitory dilutions of antibody increased [3H]thymidine incorporation in fibroblasts from 7,800 +/- 1,000 to 18,900 +/- 800 cpm (P less than 0.01), and in smooth muscle cells from 9,800 +/- 1,200 to 17,200 +/- 1,100 cpm (P less than 0.01). Insulin, which can substitute for Sm-C as a mitogen and does not bind to the antibody, stimulated DNA synthesis when co-incubated with the antibody, thereby excluding the possibility of nonspecific cytotoxicity. These results strengthen the hypothesis that the rate of DNA synthesis of these two cell types in vitro is directly linked to their capacity to produce somatomedinlike peptides. They further support the cellular production of somatomedinlike peptides as examples of the autocrine model of growth regulation.     

A mechanism for the stimulation by inorganic mercury of [3H] thymidine incorporation into DNA in cultured Molt-4F cells

Mercuric chloride at a narrow range of concentration (2 to 2.5 X 10(-5)M) facilitated [3H]thymidine incorporation into acid-insoluble material (DNA fraction) of cultured human T lymphoid cells, Molt-4F, after 72-hr culture with the metal. This effect by mercury was observed in spite of the decrease in growth rate and DNA contents of the cells. Thymidine kinase activity in Molt-4F cells treated with 2 X 10(-5)M mercury decreased to 50 to 60% of the control activity. The stimulation of [3H]thymidine incorporation into the cells by mercury, therefore, might be independent of the increase in thymidine kinase activity. 3H-Thymidine incorporation by the control cells decreased as culture time passed. In contrast to the control, [3H]thymidine incorporation by mercury-treated cells increased until 72-hr culture. [3H]Thymidine uptake by the control cells after 24, 48, or 72-hr culture increased until 20 min of incubation period, but thereafter no increase in the uptake was observed until 60 min. On the other hand, [3H]thymidine uptake by the cells treated with mercury for 24 to 72 hr increased linearly until 60 min of incubation period. These results seemed to indicate that the mercury stimulation of [3H]thymidine incorporation might be attributable not to the actual increase of DNA synthesis but to the suppression of the culture time-dependent decrease in the incorporation by the control cells.     

Selective release of excreted DNA sequences from phytohemagglutinin-stimulated human peripheral blood lymphocytes. Effects of trypsin and divalent cations.

We studied the synthesis of excreted DNA sequences and their release from phytohemagglutinin-stimulated human peripheral blood lymphocytes under conditions permitting optimal cell growth. Cells were labeled by constant exposure to low specific activity [3H]thymidine. Excreted DNA sequences were synthesized during the period of logarithmic cell growth and moved slowly from the high molecular weight chromosomal DNA fraction into the low molecular weight cell DNA fraction (Hirt supernate) from which they could be specifically released by treating the cells briefly with small amounts of various proteases; 1 microgram/ml trypsin for 5 min was optimal. On day 5 of culture, 13.3 +/- 6.9% of the total cellular acid-precipitable [3H]thymidine was released by this treatment. Trypsin-induced release was partially and reversibly inhibited by incubating the cells for 16 h with 5 mM dibutyryl-cyclic AMP. Cells incubated in the absence of divalent cations spontaneously released this Hirt supernatant DNA; after maximal release had occurred under these circumstances, additional trypsin treatment caused no further release of DNA. Trypsin-induced DNA release could be completely and reversibly inhibited by incubating the cells in the presence of 10 mM calcium. Trypsin-released DNA was isolated and analyzed by reassociation kinetics. A major component, representing 54% of the DNA, reassociated with a C0t1/2 of 68 mol.s/liter (the value at which DNA association is 50% complete). The reassociation of this DNA was studied in the presence of an excess of DNA isolated from stimulated lymphocytes on day 3 in culture, and in the presence of an excess of resting lymphocyte DNA. The high molecular weight fraction of day-3 cell DNA contained three times more copies of the trypsin-released DNA major component as compared to resting lymphocyte DNA. Hirt supernatant DNA isolated from day-5 stimulated lymphocytes reassociated in an intermediate component representing 34% of the DNA with a Cot1/2 of mol.s/liter; after cells were treated with trypsin, this component could no longer be identified in the Hirt supernatant fraction, presumably because it had been released into the incubation medium. These data describe a quantitatively reproducible system with which synthesis and release of excreted DNA sequences can be studied.     

Interleukin-10 inhibits apoptotic cell death in infectious mononucleosis T cells.

T lymphocytes from patients with acute EBV-induced infectious mononucleosis rapidly die by apoptosis in vitro. Because human and viral IL-10 are likely to be induced during acute EBV infection and display a variety of functions on human T cells, we examined IL-10 effects on infectious mononucleosis T cell death. After 12 h of incubation in medium alone, only 35.6 (+/- 8.2%) of the originally seeded infectious mononucleosis T cells were viable. Addition of human IL-10 (100 U/ml) to T cell cultures significantly improved recovery of viable cells (71.3 +/- 6.2%, P = 0.0156). Viral IL-10 had comparable effects to human IL-10 in this system. Protection from death by human and viral IL-10 (100 U/ml) was dose dependent and continued over a 6-d culture period. The human IL-10 effect was neutralized by the anti-human IL-10 mAb 19F1. Morphology and analysis of DNA after separation on agarose gels showed that IL-10 inhibits loss of cell volume, chromatin condensation, and DNA fragmentation, characteristics of death by apoptosis. As assessed by [3H]thymidine incorporation, the T cells were not induced to proliferate by IL-10 above the level exhibited when first removed from blood. T cells protected from death by IL-10 proliferated to IL-2 and spontaneously killed sensitive targets as effectively as medium-precultured T cells. Thus, IL-10 promotes the survival of infectious mononucleosis T cells otherwise destined to die by apoptosis and may be critical for the establishment of immunologic memory after resolution of the illness.     

Increased renal DNA synthesis in vivo after administration of low doses of gentamicin to rats

Kidney cortex DNA synthesis was studied in female rats treated with a low dose of gentamicin (10 mg/kg) up to 14 days. Synthesis was measured by incorporation of [3H]thymidine into DNA 1 h after intraperitoneal injection of the labeled precursor (200 muCi per animal). Gentamicin given in one injection per day resulted in a greater incorporation of [3H]thymidine into DNA after both 7 and 14 days of treatment as compared with control animals. When the daily dose was divided into three equal injections given at 8-h intervals, a statistically significant increase in thymidine incorporation was observed as early as 4 days after starting gentamicin administration. Excellent agreement was found between DNA specific radioactivity and kidney cortex nuclear labeling, as measured by histoautoradiography. The greatest amount of [3H]thymidine incorporation occurred within proximal tubular cells and interstitial cells. We conclude that a finite duration of gentamicin treatment at low dosage induces an increased DNA synthesis in vivo in rat kidney cortex. We suggest that this reaction results from cellular proliferation and could reflect a regenerative process after focal necrosis induced by gentamicin at low doses. The demonstrated early increase in DNA synthesis could be a useful tool to measure kidney cortex alterations caused by various aminoglycosides at low, therapeutic doses.     

Potent antiviral activity of north-methanocarbathymidine against Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS). Blocking lytic KSHV replication may hinder KS tumorigenesis. Here, we report potent in vitro anti-KSHV activity of 2'-exo-methanocarbathymidine [North-methanocarbathymidine (N-MCT)], a thymidine analog with a pseudosugar ring locked in the northern conformation, which has previously been shown to block the replication of herpes simplex virus types 1 and 2. N-MCT inhibited KSHV virion production in lytically induced KSHV-infected BCBL-1 cells with a substantially lower 50% inhibitory concentration (IC50) than those of cidofovir (CDV) and ganciclovir (GCV) (IC50, mean +/- standard deviation: 0.08 +/- 0.03, 0.42 +/- 0.07, and 0.96 +/- 0.49 microM for N-MCT, CDV, and GCV, respectively). The reduction in KSHV virion production was accompanied by a corresponding decrease in KSHV DNA levels in the N-MCT-treated BCBL-1 cells, indicating that the compound blocked lytic KSHV DNA replication. A time- and dose-dependent accumulation of N-MCT-triphosphate (TP) was demonstrated in lytically induced BCBL-1 cells, while uninfected cells showed virtually no accumulation. The levels of N-MCT-TP were significantly decreased in the presence of 5'-ethynylthymidine, a potent inhibitor of herpesvirus thymidine kinase, resulting in the abrogation of anti-KSHV activity of N-MCT. N-MCT-TP more effectively blocked in vitro DNA synthesis by KSHV DNA polymerase with an IC50 of 6.24 +/- 0.08 microM (mean +/- standard deviation) compared to CDV-diphosphate (14.70 +/-2.47 microM) or GCV-TP (24.59 +/- 5.60 microM). Taken together, N-MCT is a highly potent and target-specific anti-KSHV agent which inhibits lytic KSHV DNA synthesis through its triphosphate metabolite produced in KSHV-infected cells expressing a virally encoded thymidine kinase.     

Comparison of flow cytometric analysis and [3H]thymidine incorporation for measurement of the effects of drug toxicity on lymphocyte stimulation.

Human mononuclear cells were exposed to three antiviral agents, stimulated with phytohemagglutinin, and assayed for DNA synthesis with [3H]thymidine uptake and flow cytometric analysis. Cytosine-arabinoside demonstrated inhibition of blastogenic reactivity by both [3H]thymidine uptake and flow cytometric analysis, whereas acyclovir showed no significant suppression. In contrast, (E)-5-(2-bromovinyl)-2'-deoxyuridine, a thymidine analog, demonstrated a lack of correlation between the two methods. The competitive inhibition of some compounds with [3H]thymidine incorporation necessitates the use of other methods to measure DNA synthesis.     

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.     

Comparisons of anti-human immunodeficiency virus activities, cellular transport, and plasma and intracellular pharmacokinetics of 3''-fluoro-3''-deoxythymidine and 3''-azido-3''-deoxythymidine.

3'-Fluoro-3'-deoxythymidine (FLT), a candidate anti-AIDS compound in clinical trials, showed anti-human immunodeficiency virus type 1 (HIV-1) potency (50% effective concentration, 0.0052 microM) slightly better than or equal to that of 3'-azido-3'-deoxythymidine (AZT) in MT4 cells and was threefold more potent in H9 cells. There was no FLT resistance demonstrable in the AZT-resistant HIV-1 strains. Both FLT and AZT showed low cytotoxicity for MT4 cells, with selectivity indices (efficacy/toxicity ratio) of greater than 47,000 and greater than 33,000, respectively. Cellular permeation of FLT and thymidine (dThd) was greater than that of AZT, and FLT and dThd permeated the cell membranes by a carrier-mediated mechanism as well as by simple diffusion, as indicated by the existence of nitrobenzylthioinosine-5'-monophosphate-sensitive and -insensitive components. By contrast, transport of AZT into cells was by simple diffusion. The intracellular level of the triphosphate of FLT (FLTTP) in MT4 cells was two- to threefold higher than that of AZT (AZTTP) after exposure to 1.8 microM each compound for 12 h. The elimination kinetics of FLTTP and AZTTP in HIV-1-infected MT4 cells in fresh medium showed biphasic patterns, with initial half-lives of 1.03 and 1.09 h, respectively. In phytohemagglutinin-stimulated human peripheral blood lymphocytes, the FLTTP level was increased 59-fold compared with that in unstimulated cells at 12 h, was four- to sixfold higher than the level of AZTTP in stimulated cells at 12 h, and remained four- to fivefold higher during a 4-h elimination period in fresh medium and twofold higher at the end of a 12-h elimination period. Two- to eightfold more [3H]AZT than [3H]FLT was incorporated into the host cell DNA, and both [3H]AZT and [3H]FLT remained persistently incorporated for over 24 h. The incorporated [3H]AZT and [3H]FLT were alkali labile, whereas incorporated [3H]dThd was alkali stable. Pharmacokinetics of FLT in plasma of monkeys after intravenous (i.v.) administration showed that the FLT concentration in plasma declined, with a half-life of 1.19 +/- 0.1 h; the steady-state volume of distribution was 0.93 +/- 0.2 liter/kg of body weight, and total clearance was 0.56 +/- 0.15 liter/kg. Oral bioavailability of FLT was excellent and comparable to i.v. bioavailability in terms of areas under the concentration-time curves for three monkeys. Of the total dose, 41 to 61% was excreted in urine as unchanged FLT, and only 3.2 to 7.4% of the total dose was identified as glucuronide-conjugated FLT in urine 48 h after i.v. administration to monkeys. We conclude that FLT exhibits an anti-HIV-1 potency similar to that of AZT but with slightly better selectivity of effects and with higher intracellular active metabolite levels.     

Role of local prostaglandin synthesis in the modulation of proliferative activity of rat colonic epithelium.

The role of local prostaglandin (PG) synthesis in the modulation of the proliferative activity of colonic epithelium was examined in rat colon. Experimental rats were given either indomethacin (5 mg/kg s.c. every 8 h for three doses) or aspirin (0.5 g/100 g diet for 3 d). In rats treated with indomethacin or aspirin, the incorporation of [3H]thymidine (dThd) into DNA in vivo was increased approximately twofold over control in mucosal scrapings from distal colon, and approximately threefold over control in the proliferating pool of epithelial cells isolated from distal colon. [3H]dThd incorporation into DNA was also examined ex vivo immediately after distal colonic resection. It was approximately twofold higher in mucosa of colonic segments (1-h incubation) from rats treated with indomethacin or aspirin in vivo, compared with corresponding values of segments from control rats. Immunoreactive (i) prostaglandin E (PGE), the dominant PG product of colon segment incubates by high-performance liquid chromatography analysis of [14C]arachidonate metabolites, was markedly (95%) reduced in the media of 1-h colon incubates from indomethacin- or aspirin-treated rats, compared with control rats. Moreover, the cyclic (c)AMP content of mucosa of segments from indomethacin- or aspirin-treated rats was significantly lower than that of control rats. Prolonged incubation (4-24 h) of colonic segments from indomethacin-treated rats, in the absence of indomethacin in vitro, led to an eventual return of [3H]dThd incorporation into DNA, iPGE, and mucosal cAMP to control values. Conversely, inclusion of indomethacin (0.25 mM) in the incubations (6 h) of colonic segments from indomethacin-treated rats resulted in persistent suppression of iPGE and mucosal cAMP, as well as persistent enhancement of [3H]dThd incorporation into mucosal DNA. However, incubation of colonic segments from control rats (no in vivo drug exposure) with indomethacin or aspirin in vitro for periods up to 24 h failed to alter DNA synthesis, despite marked reduction in media iPGE and lower mucosal cAMP. The latter observations suggested that additional in vivo factors initiated the enhancement of DNA synthesis in indomethacin- or aspirin-treated rats. Exogenous PGE2, D2, I2, or F2 alpha, each of which increased the endogenous mucosal cAMP content of incubated colonic segments from control, indomethacin- or aspirin-treated rats, all suppressed [3H]dThd incorporation into mucosal DNA in vitro. Dibutyryl cAMP, but not dibutyryl cGMP, had an analogous suppressive effect on in vitro [3H]dThd incorporation into DNA. Thus, the present observations are consistent with an inhibitory action of endogenous colonic PG synthesis on the proliferative activity of colonic epithelium. This action may be mediated through cAMP.     

A neutrophil derived factor(s) stimulates [3H]thymidine incorporation by vascular smooth muscle cells in vitro

There is increasing evidence for the involvement of inflammatory cells in the development of intimal hyperplasia. This study examines the effect of human neutrophils on the incorporation of [3H]-thymidine into acid precipitable materials (DNA) in cultured rabbit vascular smooth muscle cells. Co-culture of intact neutrophils and vascular smooth muscle cells caused [3H]-thymidine incorporation by these cells to increase 209 +/- 22% (mean +/- SEM) of control. Sonication of the neutrophils abolished this effect (117 +/- 10%). Neutrophil conditioned medium prepared in the presence or absence of fetal calf serum had a similar effect on [3H]-thymidine incorporation by vascular smooth muscle cells (204 +/- 20% and 172 +/- 17% respectively). Stimulation of neutrophils with either Concanavalin A or serum-activated zymosan failed to increase production of the factor(s). The neutrophil derived factor(s) prepared in minimal essential medium was partially stable at 56 degrees C, but inactivated by boiling. Dialysis (pore size 12,000-14,000) did not remove the factor(s). Cyclooxygenase inhibition with indomethacin did not inhibit production of the factor(s) by neutrophils, but indomethacin caused an increase in [3H]-thymidine incorporation by vascular smooth muscle cells (181 +/- 11%). Indomethacin together with intact neutrophils had an additive effect on [3H]-thymidine incorporation (273 +/- 42%) into cellular DNA. These data show that neutrophils produce a factor(s) that stimulates [3H]-thymidine incorporation into DNA by vascular smooth muscle cells and suggests that vascular smooth muscle proliferation and intimal hyperplasia may be initiated by neutrophils adherent to sites of endothelial injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new type of vasodilator, HA1077, an isoquinoline derivative, inhibits proliferation of bovine vascular smooth muscle cells in culture

The effects of a newly developed vasodilator agent, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride], were investigated on the proliferation of cultured bovine aortic vascular smooth muscle cells (VSMC). HA1077 (10-100 microM) inhibited both fetal calf serum-induced proliferation and [3H]thymidine incorporation into DNA of the growth-arrested VSMC in a dose-dependent manner. When quiescent cells were stimulated with platelet-derived growth factor followed by insulin, HA1077 (1-30 microM), administered together with either stimulation, showed dose-dependent inhibition of [3H]thymidine incorporation. Further reduction of [3H]thymidine incorporation was observed when HA1077 was present at both stimulations, suggesting that HA1077 suppresses DNA synthesis acting in both competence and progression stages. After stimulation with fetal calf serum, quiescent VSMC started and ceased DNA synthesis in 15 to 18 hr and 24 hr, respectively. HA1077 inhibited [3H]thymidine incorporation when it was added either from 12 hr to 15 hr or from 21 hr to 24 hr after serum stimulation. In addition, when percent inhibition of [3H]thymidine incorporation by continuous exposure to HA1077 was examined as a function of the time it was added, reductions of the value were observed at 0 to 3 hr, 12 to 18 hr and 21 to 24 hr. Thus, we concluded that HA1077 suppresses DNA synthesis of bovine VSMC acting at the G0/G1 and the G1/S phase transitions and also in the S phase of the cell cycle. It is suggested that this agent may act as a potent inhibitor of VSMC proliferation as well as a vasodilator.     

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.     

Neutrophil preservation: the effect of short-term storage on in vivo kinetics.

A rabbit model was developed to study the in vivo viability of neutrophils stored in vitro for up to 72 h. Acid-citrate-dextrose anticoagulated whole blood was obtained from rabbits previously injected with tritiated thymidine ([3H]thymidine), stored under varying conditions, and then injected into recipient rabbits. Neutrophil viability and function were assessed by measuring the ability of the tagged neutrophils to circulate and to migrate into subcutaneous polyvinyl sponges. Unstored neutrophils disappeared exponentially from the circulation with a t1/2 of 3.2 h and gave a zero time recovery of 30.5%. Storage of cells at either room temperature or 4 degrees C for 24 h or longer resulted in temporary sequestration of cells from active circulation. With cells stored for up to 72 h at 4 degrees C, recovery returned to normal values after 1-2 h. Room temperature stored cells, in contrast, showed evidence of irreversible damage at 48-h storage with low recovery for the entire time span studied. With unstored blood, 8.1+/-0.9% of the injected neutrophil label was present in the subcutaneous sponges. The accumulated label progressively decreased as cell storage time increased reaching at 72 h 5.1+/-0.6 and 2.6+/-0.3% for 4 degrees C and room temperature-stored cells, respectively. The results of this study indicate that 4 degrees C storage of rabbit neutrophils is superior to storage at room temperature. The data suggest that it may be feasible to store neutrophils at least a few days without loss of in vivo functions.     

Inhibition of immune functions by antiviral drugs.

Immune functions were evaluated in vitro for PBMC isolated from healthy donors and cultured with the antiviral agents, 3'-azido-3'-deoxythymidine (AZT), ribavirin, ganciclovir, 2'3'-dideoxyinosine (ddI), or acyclovir. To identify methods for assessing the effects of antiviral drugs on immune cells, the PBMC response to mitogens, Con A, or phytohemagglutinin was evaluated from measurements of [3H]thymidine and [14C]-leucine incorporation, cell growth, cellular RNA, DNA, and protein levels, and the PBMC proliferative cycle (i.e., progression from G0----G1----S----G2 + M). At clinically relevant concentrations, AZT, ribavirin, or ganciclovir diminished PBMC responsiveness to mitogen. The numbers of proliferating cells in G1, S, and G2 + M phases of the cell cycle, DNA content, and [3H]thymidine uptake were decreased in cultures treated with AZT, ribavirin, or ganciclovir. AZT or ribavirin but not ganciclovir reduced RNA and protein in the cultures and inhibited cell growth. Whereas AZT, ribavirin, or ganciclovir were antiproliferative, ddI or acyclovir had little, if any, effect on PBMC mitogenesis. The inhibitory effects of antivirals on immune cells may contribute to the immune deterioration observed in patients following prolonged use of the drugs.     

Decreased sensitivity of old and progeric human fibroblasts to a preparation of factors with insulinlike activity.

To determine whether old cells have a reduced response to a preparation of factors from human plasma with insulinlike activity (ILA), we analyzed the response to ILA of early and late passage human fibroblasts from young, old, and progeric donors in the acute stimulation of [3H]2-deoxy-D-glucose (2dG) uptake and the delayed stimulation of [3H]thymidine (TdR) incorporation into DNA. The ILA concentration required to produce equivalent, relative stimulation of TdR incorporation was increased two- to three-fold in late passage cells and cells from old and progeric donors (P less than 0.01). 50 and 95% of maximal stimulation (ILA50, ILA95) was achieved by 0.26 +/- 0.07 and 1.38 +/- 0.13 ng insulin equivalents/ml (mean +/- SD) respectively, in cells from young adults at early passage. Corresponding values were 0.54 +/- 0.05 and 2.90 +/- 0.25 in cells from old donors; greater than 0.9 +/- 0.1 and greater than 3.1 +/- 0.1 in cells from a 9-yr-old progeric donor; and 0.4 +/- 0.05 and 1.1 +/- 0.04 in cells from normal children (9-13 yr). For two cell strains from young adults, ILA50 and ILA95 were 0.30 +/- 0.02 and 1.0 +/- 0.3 ng eq/ml at 30% of their in vitro lifespan completed (%LC) and these values increased at rates of 0.005 ng eq/ml per %LC and 0.04 ng eq/ml per %LC, respectively. The mean stimulation of 2dG uptake ratio (ILA/control) decreased from early to late passage from 2.1 +/- 0.6 to 1.3 +/- 0.1 in young adult donors (P less than 0.05), but there were no significant differences between young and old donors at either early or late passage. The mean stimulation ratio in progeric cells (1.2 +/- 0.2) did not change with in vitro passage, but was significantly lower than that of age-matched normal cells (2.1 +/- 0.8, P less than 0.001). In progeria cells, the reduced stimulation of 2dG uptake upon addition of ILA was due to an increased basal rate of uptake (0.19 +/- 0.01 pmol [3H]2dG/min per mg protein vs. 0.13 +/- 0.01 in age-matched normal cells), and not to a decline in the maximal rate of uptake (0.26 +/- 0.01 vs. 0.27 +/- 0.02, respectively). Similar results were found for in vitro aging in cells from an old donor.     

Antiviral activity of 2''-deoxy-2''-fluoro-beta-D-arabinofuranosyl-5-iodocytosine against human cytomegalovirus in human skin fibroblasts.

2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine (FIAC) was shown to be a selective anti-human cytomegalovirus agent in vitro with a 50% antiviral effective dose of 0.6 microM (J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother. 26:505-508, 1983) and a 50% cell growth inhibitory dose of 8 microM. Antiviral activity was more readily reversed with 10-fold excess thymidine, whereby the 50% effective dose was increased to 11.3 microM. FIAC-induced cytotoxicity was more readily reversed with 10-fold excess of deoxycytidine, whereby the 50% inhibitory dose was increased to greater than 100 microM. Thymidine was unable to reverse completely the antiviral activity of FIAC. Although, the extent of phosphorylation of thymidine, deoxycytidine, and deoxyuridine was 6-, 4-, and 4-fold greater, respectively, in human cytomegalovirus-infected cell lysates than in uninfected cell lysates, the extent of phosphorylation of FIAC was only 1.3-fold greater in human cytomegalovirus-infected cell lysates than in uninfected cell lysates. By comparison, the extent of FIAC phosphorylation was 500 times greater in herpes simplex virus type 1-infected cells than in uninfected cell lysates. Methotrexate was 400 times more effective against human cytomegalovirus replication than it was against herpes simplex virus type 1 replication, indicating that thymidylate synthetase may be important for human cytomegalovirus replication. However, 10 microM FIAC did not inhibit thymidylate synthetase activity in uninfected or virus-infected cells as determined by their metabolism of [6-3H]deoxyuridine in the presence or absence of drug. FIAC at 1 microM suppresses and FIAC at 10 microM completely inhibits human cytomegalovirus DNA replication as indicated by Southern blot analysis. This inhibition was reversible. FIAC incorporation into the DNA of human cytomegalovirus strain AD169-infected cells was stimulated relative to that in nondividing, uninfected cells.     

Effects of growth factors on cytosolic free calcium concentration and DNA synthesis in cultured rat aortic smooth muscle cells

Proliferation of vascular smooth muscle cells (SMCs) has been considered to be an important process in the development of atherosclerosis. This study was conducted to investigate the role of cytosolic free calcium in DNA synthesis of SMCs stimulated by growth factors. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and somatomedin-C (Sm-C) increased [3H]thymidine incorporation, an index of DNA synthesis, and cell number of rat aortic SMCs after 36 hr of incubation. Cytosolic free calcium concentration [( Ca++]i) in quiescent SMCs, measured by using quin 2, was 178 +/- 18 nM (n = 15). Both PDGF and EGF provoked a rapid and transient rise in [Ca++]i, while Sm-C did not alter [Ca++]i. Nifedipine (3 X 10(-6) M) suppressed the rise in [Ca++]i provoked by PDGF and EGF. On the other hand, nifedipine suppressed the enhancement of DNA synthesis provoked by EGF, but did not suppress those by PDGF and Sm-C. These results suggest that the transient rise in [Ca++]i plays an important role in the proliferation of SMCs stimulated by EGF, while the rise in [Ca++]i is not involved in the mechanism of proliferation of SMCs provoked by Sm-C. The role of cytosolic free calcium in the proliferation of SMCs provoked by Sm-C. The role of cytosolic free calcium in the proliferation of SMCs provoked by PDGF was not definitive.