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Autologous grafts are superior to their synthetic counter-parts for grafting arteries smaller than 6-mm diameter both in terms of acute thrombogenicity and chronic intimal hyperplasia. Endothelial cell (EC) coating of the blood contacting surface may reduce thrombogenicity of synthetic small diameter vascular prostheses. In this study, the survival of EC monolayers on synthetic 4-mm diameter arterial prostheses over short-term implantations (< or = 6 weeks) was examined. Graft types examined were expanded polytetra-fluoroethylene (ePTFE) and microporous polyurethane (PU). Lumenal coverage with ECs was achieved by culturing ovine ECs on prostheses treated by either physical adsorption or covalent binding of ovine fibronectin (Fn). An ovine carotid interposition model was used to examine the performance of EC coated ePTFE and microporous PU over implantation periods of 1, 3, and 6 weeks. Outcomes assessed at the end of each experiment were graft patency, area covered by ECs, and thrombus free surface area (TFSA). Fn concentration, cell density at the time of coating and prostacyclin production in vitro were similar for both graft types. Occlusion occurred more frequently in unseeded grafts compared with EC coated grafts over 3 and 6 week implantation periods; however, the difference was not significant (p = 0.099). In prostheses precoated with ECs, approximately 40-60% of the surface area remained covered with endothelial-like cells following the first postoperative week. Recovery of EC layers occurred rapidly thereafter with 80-90% coverage at 3 weeks. TFSA remained low in comparison to EC cover in these prostheses until between 3 and 6 weeks postoperatively, suggesting a lag phase in recovery of EC function of seeded cells. In contrast, EC cover of unseeded prostheses only achieved 10-30% at 3 weeks, primarily by pannus EC ingrowth from the adjacent artery. TFSA of unseeded grafts increased in direct proportion to EC cover over time suggesting that there was no lag phase in function of these ingrowing cells.

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Cell affinity separations are based on the selective attachment of cell phenotype using antibody or lectins specific for cell surface markers. The major physicochemical factors which influence ligand-mediated cell adhesion dynamics and the efficiency of cell affinity separation have been examined. Uniform cell detachment forces were generated with a parallel-plate flow cell (plate separation 100 microns, surface area 3 cm2). Hydrodynamic shear stress was used to measure cell adhesion strength and to separate cells on the basis of surface affinity. Human cell lines grown in tissue culture were separated on a flat derivatised glass immunoadsorbent which formed the floor of the flow chamber. Flow-cell residence time, detachment shear stress, temperature, and ligand density were shown to influence cell attachment probability. An understanding of the physical basis of ligand-mediated cell adhesion provided a rationale for optimisation of affinity cell separation. At room temperature attachment of positive cells was rapid (< 2 min) and adhesion strength was directly related to immunoadsorbent ligand density. Purity and recovery of enriched fractions were dependent on the separation shear stress and could be optimised using this parameter. Enrichment factors were greater than 100-fold, with at least 90% of positive cells recovered in enriched fractions. Enrichment purity and yields did not decline at higher loading densities (10(5) cells/cm2). Selective immunoadsorbent surface chemistry is a prerequisite for efficient affinity cell separation. Purity and recovery may be optimised by fractionating enriched and depleted cell populations with uniform fluid shear stress.

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The performance of small-diameter vascular prostheses may be improved by implantation of grafts lined with endothelial cells. Expanded polytetrafluoroethylene (ePTFE) prostheses (4 mm x 40 mm) were coated with fibronectin (20 micrograms/ml), seeded with endothelial cells, and cultured for 48 h to produce a confluent, autologous endothelial cell lining. They were implanted as carotid interposition grafts in sheep. Seeded ePTFE grafts were compared with nonseeded ePTFE grafts and autologous carotid artery grafts. No anticoagulant or antiplatelet therapy was administered, making this a stringent test model for the thromboresistance of a small-diameter prosthesis. After 13 weeks the patencies of seeded, nonseeded, and autologous artery grafts were 16% (1/6), 0% (0/6), and 100% (6/6), respectively. The one seeded graft that was patent was fully lined with endothelial cells and showed no stenosis. The remaining five seeded grafts were occluded by fibrous tissue and displayed substantial spindle cell hyperplasia. There was no apparent difference between the autologous artery grafts and normal arterial tissue, and the anastomoses showed no stenosis. The ovine model provides a conservative test of prosthesis survival and may be useful for study of graft failure.

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The kinetics of beta 2-microglobulin (beta 2m) were studied in five anephric or anuric hemodialysis patients. Human beta 2m was isolated from peritoneal dialysate using ion-exchange and gel chromatography and radiolabeled with 125I. Patients were injected with 10 microCi labeled beta 2m. In one study (N = 4), plasma activity was measured over 72 hours. In a second study (N = 4), patients received low-flux dialysis 24 hours after injection and high-clearance dialysis (Bellco BL655) at 48 hours. Plasma activities were fitted to a three-compartment, variable volume model. Endogenous beta 2m levels (radioimmunoassay) were 56 +/- 6 mg/liter. The beta 2m distribution volume was 12.7 +/- 2.0 liter (0.20 +/- 0.03 liter/kg) and the non-renal clearance was 3.0 +/- 0.4 ml/min. The generation rate, 9.9 +/- 1.7 mg/hr (0.16 +/- 0.04 mg/kg/hr), was similar to that measured in subjects with normal renal function. The three compartment model derived from the turnover data gave an adequate fit of the arterial concentrations of endogenous and exogenous beta 2m during low-flux (nil beta 2m clearance) and high-clearance (beta 2m clearance of 19 ml/min) dialysis. Simulations based on this model indicate that extracorporeal treatment can at best remove about 50% of weekly production. These results suggest that beta 2m production is not increased in dialysis patients, that there is substantial non-renal beta 2m clearance, and that the amount of beta 2m that can be removed by extracorporeal therapy is therefore limited.

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This study examines, under flow conditions, the adhesion of endothelial cells to 3 mm diameter fibronectin (Fn)-coated expanded polytetrafluoroethylene (PTFE) vascular grafts. Cultured ovine carotid artery endothelial cells were labelled with 35S-methionine. The grafts were seeded with endothelial cells (1.5 x 10(6)/ml) by rolling for 1 h at 37 degrees C and then either cultured to confluence for 48 h or flow tested immediately. Cell attachment to grafts (n = 5) was evaluated in an in vitro flow circuit, using flows of up to 330 ml/min. Ex vivo studies (n = 5 grafts) were conducted without anticoagulant using autologous cells in a sheep model. Grafts were inserted into an externalized carotid-jugular shunt and exposed to blood flows of approximately 150 ml/min for 3 h. One hour seeded and 48 h cultured grafts demonstrated greater than 95% cell retention following in vitro flow studies. Ex vivo studies of 48 h cultured grafts gave endothelial cell retention of 81% with no sign of thrombogenicity. Furthermore, a preliminary 24 h ex vivo study has shown greater than 95% retention. This study demonstrates the firm attachment of seeded endothelial cells to Fn-coated PTFE grafts in the sheep model.

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We have studied the antiproliferative effects of gallium nitrate in cultured CCRF-CEM lymphoblasts. The 50% inhibitory dose for these cells was 120 microM, and after 24 h at a cytostatic concentration (480 microM) S-phase arrest was observed by DNA flow cytometry. Deoxyribonucleoside triphosphate pools were all reduced (dATP, dGTP, and dCTP by 50%, dTTP by 25%), suggesting inhibition of ribonucleotide reductase. Administration of tracer amounts (0.5 microM) of either [3H]uridine or [3H]deoxyuridine confirmed that DNA synthesis had been inhibited to 20% of control rates by gallium. Further, the flow of the ribonucleoside into the dTTP pool and DNA was selectively reduced compared to that of the deoxyribonucleoside. Gallium decreased the specific activity of dTTP labeled from uridine by 50%, whereas the specific activity of dTTP labeled from deoxyuridine was increased 2.5-fold. Thus counts in DNA derived from [3H]uridine were decreased by more than 80%, while counts in DNA derived from [3H]deoxyuridine were virtually unaltered. Uridine incorporation into RNA was not affected. Gallium did not significantly alter the capacity of permeabilized naive cells to incorporate [3H]dTTP into DNA, while 24-h gallium pretreatment (which increased the percentage of S-phase cells) produced a modest increase in [3H]dTTP incorporation, indicating that any effect of gallium on DNA polymerase alpha is minor. Gallium treatment did not induce or inhibit the repair of DNA single strand breaks. These data demonstrate that gallium inhibits replicative DNA synthesis, with the major specific enzyme target probably being ribonucleotide reductase.

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In vitro studies in exponentially growing L1210 cells utilizing DNA flow cytometry and cell proliferation measurements indicate enhancement of methotrexate effects by Dipyridamole provided: Methotrexate concentrations exceed those required to shut off maximally de novo pathways of purine and pyrimidine synthesis (i.e. 30 nM for 48 h), and Dipyridamole concentrations exceed 3 microM. In 10% fetal calf serum, this concentration inhibits tritiated thymidine uptake by about 80%. These data should prove helpful in the planning of clinical studies with dipyridamole or other inhibitors of nucleoside transport used to potentiate inhibitors of de novo pathways.

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Three methotrexate (MTX)-resistant cell lines and their MTX-sensitive counterparts have been used to examine 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methyl-pyrido[2,3-d]pyrimidine (BW301U), a novel lipophilic antifolate, and compare its cytotoxicity with MTX and metoprine. Collateral sensitivity for both BW301U and metoprine was observed in CCRF-CEM/MTX R-cells, where MTX resistance appeared to be primarily due to a deficiency in drug uptake. This was particularly pronounced with BW301U which proved to be as effective in killing CCRF-CEM/MTX R as was MTX with the parental CCRF-CEM cell line. This effect was not seen in other cell lines, L5178Y/MTX or L1210/MTX R, where resistance to MTX was correlated with either an overproduction of 5,6,7,8-tetrahydrofolate:nicotinamide adenine dinucleotide phosphate oxidoreductase EC 1.5.1.3 (DHFR) or with combined uptake defect and increased DHFR levels, respectively. In each case, however, BW301U and metoprine, especially at high concentrations, were more effective than MTX in treating MTX-resistant cells.

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The deoxyuridine suppression test and labeled deoxyuridine incorporation studies assume a stable level of deoxyuridine phosphorylase (thymidine:orthophosphate deoxyribosyltransferase, EC 2.4.2.4) activity. We report a large increase in deoxyuridine phosphorylase activity in three of five cultured lymphoblast lines treated with 10(-7) M methotrexate. In two of these lines, a parallel increase in tritiated deoxyuridine incorporation into RNA was seen following methotrexate treatment. The high basal deoxyuridine phosphorylase activity in another lymphoblast line was associated with 80% incorporation of tritiated deoxyuridine into RNA in untreated cells. Since methotrexate-induced changes in deoxyuridine phosphorylase activity were time dependent, the reliability of the deoxyuridine suppression test and labeled deoxyuridine incorporation into DNA as measures of thymidylate synthetase (EC 2.7.4.6) inhibition would also vary with time. Moreover, increases in deoxyuridine phosphorylase activity in methotrexate-treated cells may influence the metabolism of fluorouracil, a drug frequently used in combined treatment regimens.

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The selective toxicity of purine deoxynucleoside to specific lymphocyte cell populations and recent evidence that purine nucleosides are important extracellular modulators of neurotransmission and coronary blood flow have prompted measurement of extracellular purines in man. By using a highly sensitive fluorimetric assay and collecting specimens into an inhibitor of adenosine deaminase, we have accurately measured purine nucleoside and hypoxanthine-xanthine levels in arterial and venous blood, in cerebrospinal fluid and in bone marrow aspirates. In peripheral venous plasma from normal volunteers, purine levels average 2.7 +/- 1.2 microM (mean +/- S.D.) with 38% in the form of adenosine and 47% as hypoxanthine and xanthine. Arterial purine levels are similar to those in mixed venous plasma; however, the hypoxanthine-xanthine component is reduced compared to simultaneously drawn mixed venous specimens (p less than 0.005). Hepatic venous plasma tends to have higher purine levels than does peripheral venous plasma (not significant), whereas bone marrow aspirates have 10-fold higher hypoxanthine-xanthine levels, suggesting that bone marrow may be a major source of plasma purines. Cerebrospinal fluid hypoxanthine-xanthine is twofold to eightfold higher than mixed venous levels, whereas adenosine levels are lower (p less than 0.01 and p less than 0.025, respectively).

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The modulation of MTX cytotoxicity by purines has been studied in a number of mammalian cell lines. In each case, it was found that exogenous purines (guanosine, deoxyguanosine, adenosine, deoxyadenosine, and hypoxanthine) both reduced and potentiated MTX cytotoxicity depending on the MTX concentration. At low MTX concentrations (less than 6 X 10(-8) M), purines reduced MTX toxicity and at higher concentrations they potentiated MTX toxicity. The reduction of low-concentration MTX cytotoxicity by purines was associated with the reversal of MTX-induced changes in deoxyribonucleotide pools. On the other hand, potentiation of MTX toxicity by purines was associated with substantial increases in deoxyadenosine 5'-triphosphate levels in conjunction with low deoxythymidine 5'-triphosphate levels. The magnitude of increase in deoxyadenosine 5'-triphosphate levels tended to correlate with the degree of potentiation which varied between 5-fold and 200-fold, depending on cell type and the exogenous purine.

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The rescue of lymphocytes from methotrexate (MTX) growth inhibition by 5-methyltetrahydrofolate (5-methyl-THF) and 5-formyltetrahydrofolate (5-formyl-THF) has been studied. Rescue by 5-methyl-THF is selective for cells with high levels of homocysteine:5-methyl-THF methyl-transferase (methyltransferase). At MTX concentrations which inhibited growth greater than or equal to 85% in both leukemic T-lymphocytes (CCRF-CEM) and Epstein-Barr-transformed B-lymphocytes (LAZ-007), 5 micro M 5-formyl-THF rescued more effectively than did 5-methyl-THF, in either the presence or absence of the methyltransferase inhibitor, nitrous oxide. At less inhibitory MTX concentrations, both reduced folates rescued equally, except when methyltransferase was inhibited by nitrous oxide in which case 5-formyl-THF was clearly superior. In the absence of nitrous oxide, both cell lines contained approximately equal amounts of methyltransferase. Some apparent differences in the rescue of these cell lines with 5-methyl-THF were attributable to their different sensitivity to MTX. When metabolism of reduced folates was severely impaired by MTX and nitrous oxide, lymphocytes were rescued with 5-[methyl-14C]methyl-THF, and the uptake of 14C into DNA was measured. In corporation was very low, indicating that cellular oxidation of 5-methyl-THF to 5,10-methylene-tetrahydrofolate is minimal even under forcing conditions. MTX selectively in vivo will be influenced by the level of methyltransferase in tumor and normal tissues.

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The effects of methotrexate (MTX) in the presence or absence of exogenous thymidine (dThd, 10(-5) M) or hypoxanthine (Hx, 10(-4) M) on cell cycle kinetics and deoxyribonucleoside triphosphate pools (dNTP) were studied in cultured human leukemic T-cells (CCRF-CEM). MTX cytotoxicity was found to increase linearly with drug dose for MTX concentrations between 10(-9) M and 10(-7) M. No further increase in cytoxicity was observed with much higher MTX concentrations (10(-7) M-10(-4) M). A similar dose-response relationship was found for both MTX-induced inhibition of DNA synthesis and changes in dTTP and dGTP pools but not for either MTX-induced inhibition of purine synthesis or changes in dATP and dCTP pools. Exogenous dThd reduced MTX cytotoxicity, at all MTX concentrations examined, but Hx reduced cytotoxicity only at MTX concentrations less than 6 X 10(-8) M and potentiated toxicity with higher MTX concentrations. This potentiation of cytotoxicity was accompanied by substantial elevation of dATP pools. In all instances where dThd or Hx reduced MTX cytotoxicity, a concomitant increase in both dTTP and dGTP levels and in the rate of DNA synthesis was observed. These results suggest a close correlation between MTX-induced alterations of dNTP and inhibition of DNA synthesis and subsequent MTX cytotoxicity. The possible modulation of MTX cytotoxicity by purines is discussed.

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