Radiomarquage in vitro des leucocytes au [F]-fludésoxyglucose : première expérience à l’hôpital d’instruction des armées Val-de-Grâce

^99mTc-HMPAO (technetium^99m-hexamethylpropylene amine oxime) radiolabeled-leukocytes or Indium-111 oxine labeled leukocytes scintigraphy and positron emission tomography with [¹⁸F]-fludeoxyglucose (¹⁸F-FDG) are the reference techniques for infection imaging. These methods have some limits explaining the active research for an ideal infection tracer finding. Because of its potential advantages, leukocyte labeling with ¹⁸F-FDG have been developed but is not routinely used for clinical infection imaging. We report the results of our first experience of leukocyte radiolabeling with ¹⁸F-FDG, managed on 20 healthy subjects. Labeling efficiency, cellular viability and radiolabeling stability have been assessed. Our results exhibit the influence of different parameters on labeling efficiency: presence of glucose during the labeling reaction, number of cells and volumic activity of ¹⁸F-FDG. Stability assessment indicates that 60% of initial cellular activity persist in cells after 1 hour incubation. Our results are similar to literature data and permit us to consider a clinical use of radiolabeled leukocyte with ¹⁸F-FDG. Nevertheless, a clinical use of radiolabeled cells can’t be considered before the radiolabeling induced cellular effects have been assessed.     

Fate of intravenously administered rat lymphokine-activated killer cells labeled with different markers

Summary Rat lymphokine-activated killer (LAK) cells, generated by adhering rat splenocytes isolated from the 52% Percoll density fraction to plastic flasks, demonstrate restricted in vivo tissue distribution, localizing in the lungs and liver after 2 h, but redistributing into the liver and spleen 24 h after i.v. administration. However, a different pattern of distribution was observed when this population of LAK cells was labeled with one of four commonly used radioisotopes. For example, LAK cells showed a high distribution into the lungs 30 min after administration when labeled with⁵¹Cr,¹²⁵I-dUrd or¹¹¹In-oxine, whereas¹¹¹InCl-labeled LAK cells showed an equal distribution into the blood, lungs and liver at this time. Two hours after administration, cells labeled with¹¹¹In-oxine showed an equivalent distribution into the lungs and liver, those labeled with¹²⁵I-dUrd or⁵¹Cr showed a high accumulation in the lungs, whereas those labeled with¹¹¹In-Cl entered more into the liver and blood. The pattern of distribution of¹¹¹In-Cl- or¹¹¹In-oxine-labeled cells was confirmed using gamma camera imaging analysis. By 24 h, LAK cells labeled with¹¹¹InCl,¹¹¹In-oxine or⁵¹Cr distributed in the liver and spleen in variable concentrations. In contrast, cells labeled with¹²⁵I-dUrd were not detected in any organ tested.This study was paralleled by monitoring the distribution of LAK cells labeled with Hoechst 33342 (H33342) and analyzed for the presence of fluoresceinated cells in different organs either by flow cytometry analysis, or in frozen section. The data indicate that the distribution pattern of LAK cells labeled with¹¹¹In-oxine is the closest to the distribution of H33342-labeled cells. Of all the radioisotopes used,¹²⁵I-dUrd has the most disadvantages and is not recommended for monitoring the in vivo distribution of leukocytes.     

New 111In labeling of IgG: 111In-oxine mediated chelation

Current methods of ¹¹¹In chelate conjugation labeling of antibodies expose the protein to pH 5–6 during ¹¹¹In chelation. These conditions could be detrimental if the antibody is acid labile. We have successfully labeled human IgG via the cyclic anhydride of DPTA and ¹¹¹In-oxyquinoline(oxine). Chelation was achieved at pH 6.9–8.4 and was complete within 1 min at room temperature. The chelation was sensitive to trace metal contamination on labware and in some reagents (including commercial ¹¹¹In-oxine).     

Efficient radiolabeling of mammalian cells using 111In-tagged liposomes

When indium-111 oxine labeled neutral liposomes were incubated with Chinese hamster V79 cells in the presence of 100 mM calcium, the cell-associated radioactivity increased approximately 75-fold over that observed in the absence of calcium. This is considerably higher (~20 times) than the cellular uptake obtained when these cells are incubated in the presence of ¹¹¹In-oxine alone. The highest uptake of radioactivity occurred when no bovine serum albumin was present in the medium, while as little as 0.001% of the protein greatly reduced the cell-liposome association. These efficient cell labeling conditions were not found to affect the survival of the cells.     

A novel method for labelling of leukocytes with technetium-99m and its comparative evaluation for abscess scintigraphy

A new method, based on the pretreatment of leukocytes with glucoheptonate prior to treating with reduced ^99mTc, has been developed for the preparation of ^99mTc labelled leukocytes. The leukocytes labelled with a ^99mTc concentration (5.59%/g tissue) similar to that of ¹¹¹In-leukocytes (6.27%/g tissue), in the experimental abscess were in rat thigh. Concentration of ^99mTc-leukocytes in blood at 24 h was only about 35% as compared to that of ¹¹¹In-leukocytes. Biodistribution in the rat organs was similar in both cases, except in the liver where ^99mTc-leukocytes exhibited about 4-fold greater concentration. Images of experimental abscess in rat by using ^99mTc-leukocytes were comparable to those obtained with ¹¹¹In-leukocytes.     

Radiolabeling of lipid-based nanoparticles for diagnostics and therapeutic applications: a comparison using different radiometals

Radiolabeling of nanoparticles (NPs) has been performed for a variety of reasons, such as for studying pharmacokinetics, for imaging, or for therapy. Here, we describe the in vitro and in vivo evaluation of DTPA-derivatized lipid-based NP (DTPA-NP) radiolabeled with different radiometals, including ¹¹¹In and ^99mTc, for single-photon emission computed tomography (SPECT), ⁶⁸Ga for positron emission tomography (PET), and ¹⁷⁷Lu for therapeutic applications. PEGylated DTPA-NP with varying DTPA amounts, different composition, and size were radiolabeled with ¹¹¹In, ¹⁷⁷Lu, and ⁶⁸Ga, using various buffers. ^99mTc-labeling was performed directly and by using the carbonyl aquaion, [^99mTc(H₂O)₃(CO)₃]⁺. Stability was tested and biodistribution evaluated. High labeling yields (>90%) were achieved for all radionuclides and different liposomal formulations. Specific activities (SAs) were highest for ¹¹¹In (>4 MBq/μg liposome), followed by ⁶⁸Ga and ¹⁷⁷Lu; for ^99mTc, high labeling yields and SA were only achieved by using [^99mTc(H₂O)₃(CO)₃]⁺. Stability toward DTPA/histidine and in serum was high (>80 % RCP, 24 hours postpreparation).). Biodistribution in Lewis rats revealed no significant differences between NP in terms of DTPA loading and particle composition; however, different uptake patterns were found between the radionuclides used. We observed lower retention in blood (<3.3 %ID/g) and lower liver uptake (< 2.7 %ID/g) for ^99mTc- and ⁶⁸Ga, compared to ¹¹¹In-NP (blood, <4 %ID/g; liver, <3.6 %ID/g). Imaging potential was shown by both PET magnetic resonance imaging fusion imaging and SPECT imaging. Overall, our study shows that PEGylated DTPA-NP are suitable for radiolabeling studies with a variety of radiometals, thereby achieving high SA suitable for targeting applications.     

Clumping of labeled leukocyte suspension. A simple measure for avoiding it

Leukocytes in “mixed” suspensions can clump together, resulting in cell clusters which are responsible for false positive hot spots in lungs of patients, in the case of abscess localization studies using ¹¹¹In labeled leukocytes.Addition of extra ACD (acid-citrate-dextrose) in those labeled leukocyte suspensions prevented cell clumping and avoided occurrence of focal radioactivity accumulation in lungs. The acidification did not interfere in leukocyte migration under agar.     

DYNAMICS OF LEUKOCYTE MIGRATION INTO THE MOUSE ASCITES TUMOR

There is a marked increase in the number of peritoneal leukocytes (lymphocytes, monocytes and granulocytes) during the growth of Ehrlich ascites tumor in mice. No local proliferation (as indicated by a labeling at 1 hr following a single ³H-TdR injection) was observed in the normal peritoneal leukocytes or those in the ascites tumor, except for a very minor labeling of some tumor macrophages. Kinetics of peritoneal leukocytes was studied with a series of twelve injections of ³H-thymidine (20 μCi every 8 hr) in normal mice as well as mice injected with 10⁶ tumor cells i.p. 2 hr after the last ³H-TdR injection. Animals were sacrificed at intervals up to 6 days. Granulocyte labeling in the blood as well as peritoneal space was near 100% in both groups of animals at all the intervals. Temporal changes in the labeling of lymphocytes (from 10% at 0 day to 22% at day 6), and monocytes (from 20% at 0 day to 57% at day 6) were identical in the blood and peritoneal space of normal animals, indicating a free exchange of cells between these compartments. Higher labeling indices than those in the controls were attained in the blood of tumor-bearing hosts (viz 40% for lymphocytes and 80% for monocytes at 6 days) suggesting an increased turnover of these cells in the circulation. In addition, peritoneal mononuclear cells of tumor-bearing mice showed even a higher labeling than those in the blood (viz 65% for lymphocytes and 92% for monocytes at 6 days) indicating a selective migration and/or retention of newly formed cells within the tumor, in contrast to a random migration into the normal peritoneal cavity. Furthermore, an identical labeling of macrophages to that of monocytes within the tumor indicated a short monocyte-macrophage transition. The preferential accumulation of young mononuclear cells into the tumor may be of functional importance.     

Direct 99mTc labeling of monoclonal antibodies: Radiolabeling and in vitro stability

Direct labeling involves ^99mTc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind ^99mTc. The direct ^99mTc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability.Stable direct antibody labeling with ^99mTc requires the generation of sulfhydryl groups, which show high affinity binding sites for ^99mTc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the ^99mTc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution.Very good ^99mTc-antibody stability is obtained with activated IgG (IgGa) and Fab′ fragment, which makes these substances possible candidates for immunoscintigraphy use.     

Use of polyester leukocyte elimination filters in blood filterability research

We tested a new routine to eliminate leukocytes for blood rheology measurements using commercial leukocyte absorbing filters (here PALL RC400). These filters were punched out and fitted in smaller chambers through which blood was filtered under controlled suction pressure (< 30 mm Hg). This technique resulted in a very effective leukocyte elimination to 0.0022% but also a platelet reduction to 0.2%. The process causes a small but significant hemolysis with free hemoglobin, of the order of 0.06% of the filtered erythrocytes. A small fraction of the erythrocytes were retained in the filter, versus plasma, to reduce the hematocrit on the order of 1.4%. The leukocyte filtration did not cause any detectable functional trauma to the erythrocytes, measured as micro-pore filterability of normal and glutaraldehyde (GA) hardened erythrocytes. However, when 10% of the erythrocytes were hardened with GA, which caused an increase in pore clogging slope (p < 0.05), the additional passage through the leukocyte elimination filter removed this measured change in clogging. This observation suggests that the leukocyte elimination filter may selectively remove, not only leukocytes and platelets, but also hardened erythrocytes. Reticulocyte counting did not reveal any selective removal of young erythrocytes. In general, we find the presented method reproducible, efficient and easy for eliminating leukocytes for blood rheology research although the risk of removing undeformable erythrocytes must be considered.     

POST-MITOTIC AGE OF MONONUCLEAR CELLS MIGRATING INTO TA-3(St) SOLID TUMORS

Subcutaneous transplantation of 5 × 10⁶ TA-3(St) mammary carcinoma cells into A/J mice produced rapidly growing tumors that showed a substantial accumulation of lymphocytes, monocytes and macrophages. This must have resulted primarily from an influx of circulating cells, since none of the cell types exhibited any significant local proliferation as indicated by 1 hr ³H-TdR uptake. The post mitotic age of the various leukocyte types in circulation of normal and tumor bearing animals, and those appearing within the tumors, was evaluated by a repeated ³H-TdR labeling protocol designed to label newly formed leukocytes. Tumor transplantation (or injection of an equivalent volume of saline in control animals) was preceded by thirteen ³H-TdR injections (12.5 μCi every 8 hr) and followed by eight more injections at equivalent intervals. Animals were serially sacrificed at 5-14 days after tumor transplantation or saline injection. Total lymphocyte labeling in the blood during these intervals was higher in tumor bearing mice (approximately 25% between 5 and 12 days) than in the saline injected controls (approximately 20%), indicating that tumor transplantation resulted in an increase in the proportion of young lymphocytes in circulation. More significantly, a much higher labeling (57-60% at 5-12 days) was exhibited by lymphocytes isolated from the tumors, indicating selective migration (and/or retention) of newly formed lymphocytes within the tumor. This finding applied to lymphocytes in all size categories. Although blood monocyte labeling in the control and experimental animals was similar (e.g. 83% at day 5 and 52% at day 14), significantly higher labeling (e.g. 93% and 70% at the respective intervals) was exhibited by monocytes isolated from tumors. This suggested a preferential accumulation of young monocytes at the tumor site. An identical macrophage labeling pattern compared to that shown by monocytes within the tumor indicated a rapid monocyte-macrophage transition. Since these findings in the present strain-specific solid tumor are similar to those previously obtained in this laboratory in the strain-nonspecific Ehrlich ascites tumor, the phenomenon of selective localization of newly formed mono-nuclear leukocytes appears to be a general occurrence in transplanted murine tumors.     

Evaluation of the 111In-(Sn)-citrate method to label antibodies for radioimmunotargeting studies

Monoclonal antibody against carcinoembryonic antigen (CEA) was labeled by the ¹¹¹In-(Sn)-citrate technique and its characteristics were determined from the point of radioimmunotargeting studies. Antibodies of high specific radioactivity could be labeled with a high labeling yield of greater than 90% without any further purification step, and without loss of immunoreactivity. In biodistribution studies using nude mice bearing CEA-producing tumors, an extremely high tumor-to-blood ratio (40 on day 6) was obtained since ¹¹¹In in the blood cleared very rapidly, but nonspecific ¹¹¹In localized in the liver and spleen should be seriously considered.     

Simple and convenient radiolabeling of proteins using a prelabeling-approach with thiol-DOTA

Commonly applied methods for radiometal-labeling of proteins require complex and protracted derivatization reactions of the protein and the subsequent radiolabeling is time-consuming due to the low reaction temperatures applicable. Therefore, a convenient and efficient prelabeling technique for proteins using the DOTA derivative 2,2′,2′′-(10-(2-(2-mercaptoethylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (thiol-DOTA) containing a thiol moiety for rapid and selective introduction into maleimide-derivatized proteins was developed. Thiol-DOTA was labeled with ⁶⁸Ga, ⁹⁰Y and ¹⁷⁷Lu and subsequently introduced into bovine serum albumin and a human IgG with maximum radiochemical yields of 66%. The entire radiolabeling procedure was completed after only 30 min making this a favorable new labeling technique for proteins.     

Facile synthesis and evaluation of C-functionalized benzyl-1-oxa-4,7,10-triazacyclododecane-N,N',N″-triacetic acid as chelating agent for ¹¹¹In-labeled polypeptides

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N,N′,N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (DOTA). However, neither synthetic procedure for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-111 (¹¹¹In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated its ability as a chelating agent for radiolabeling peptides and proteins with ¹¹¹In. The synthetic procedure involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting material. The following intramolecular cyclization reaction was best performed (>73% yield) when Boc-protected linear compound and the condensation reagent, HATU, were simultaneously added to the reaction vessel at the same flow rate. The cyclic compound was then reduced with BH₃ and alkylated with tert-butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be required. However, most of the intermediate compounds were obtained easily in good yields, suggesting that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and cyclic peptides. In ¹¹¹In radiolabeling reactions, C-ODTA provided ¹¹¹In chelates in higher radiochemical yields at low ligand concentrations when compared with C-DOTA. The ¹¹¹In-labeled C-ODTA remained unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the ¹¹¹In-labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would be useful to prepare ¹¹¹In-labeled peptides of high specific activities in high radiochemical yields.     

Fructosyl Transfer between 1-Kestose and Sucrose in Wheat Leaves

The labeling pattern of the sugar moieties of 1-kestose after in vivo pulse labeling with ¹⁴CO₂ was not the same as that after in vitro labeling with ¹⁴C-sucrose. The two fructosyl residues of 1-kestose had similar specific radioactivities after in vitro synthesis, but after in vivo radiolabeling the specific radioactivity of the terminal fructosyl moiety was significantly less than the internal fructosyl moiety. Evidence is presented that the uneven specific radioactivity of in vivo radiolabeling results from enzymatic transfer of terminal fructosyl residue from 1-kestose to sucrose.     

Effect of albumin fusion on the biodistribution of interleukin-2

Purpose We investigated and compared the biodistribution of Albuleukin, a human serum albumin (HSA)-interleukin-2 (IL-2) fusion protein, with those of IL-2 and HSA. The objective was to determine whether Albuleukin distributes differently to normal organs and lymphoid tissues than IL-2 by virtue of its genetic fusion with HSA.Methods The chelating agent 2-(p-isothiocyanato-benzyl)-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-A^II was selected for radiolabeling with ¹¹¹In, and conjugation with CHX-A^II did not alter bioactivities of IL-2 and Albuleukin on proliferation of CTLL-2 cells. The radiolabeled proteins were injected intravenously into mice, uptake in organs was measured, and whole-body autoradiography was performed.Results Striking differences in the biodistribution of IL-2 and Albuleukin were noted. ¹¹¹In-IL-2 cleared from blood rapidly, with less than 1% ID/g (percentage of injected dose per gram of tissue) at 20 min after injection. At this time, the kidneys showed more than 120% ID/g uptake, and these high levels persisted through 6 h. ¹¹¹In-Albuleukin, by contrast, showed significantly longer circulation (14% ID/g at 6 h), lower kidney uptake (<6% ID/g), and higher localization in liver, spleen, and lymph nodes (maximal uptake ~22% ID/g for all three organs). Uptake in liver, spleen, and lymph nodes appears to be mediated in part by the IL-2 component of Albuleukin because ¹¹¹In-HSA showed significantly lower accumulation in those tissues despite more prolonged circulation in blood.Conclusion These data support the hypothesis that Albuleukin targets tissues where lymphocytes reside to a much greater extent than does IL-2, and suggest that Albuleukin may exhibit improved efficacy and reduced toxicity in the treatment of solid tumors. Clinical trials underway will determine whether the improved targeting in the mice translates into a better therapeutic index in humans.     

Observations on the Technique for the Study of Human Chromosomes-by the Culture of Leukocytes from Peripheral Blood

A critical analysis of the various features of blood leukocyte culture for the study of human chromosomes was carried out. The following observations were made: (1) Fasting blood was essential for effective separation of leukocytes. (2) These cells are easily obtained by differential centrifugation and RBC sedimentation. (3) Non-specific agglutination of leukocytes was prevented by the use of Eagle''s medium for suspension cultures. (4) No contamination occurred in a series of 50 leukocyte cultures to which antibiotics were not added. (5) Addition of an experimental Phaseolus vulgaris extract at concentration of 1 × 10^?4 to cultures resulted in a 12 to 15% mitotic index. (6) Desacetyl-methyl-colchicine (Colcemid) had optimal effect at concentration of 0.1 μg./ml. (7) Distilled water added to cell suspension in culture medium (5: 1) was an effective hypotonic agent.A simplified technique of leukocyte culture for chromosome preparations is proposed.     

Platelet subpopulation bearing leukocyte specific antigen and tissue factor

Platelets bearing leukocyte antigen CD45 were identified in the blood of patients with myocardial infarction (MI) and healthy donors by flow cytofluorimetry. Part of these platelets contained tissue factor (TF)–primary initiator of blood clotting. The number of CD45⁺ and CD45⁺/TF⁺ platelets in MI patients at the first day was comparable with their level in healthy donors, but was increased at 8–12 days after MI onset. At that time in some patients the amount of CD45⁺ and CD45⁺/TF⁺ platelets reached 5–6 and 2–3% of their total number. It is assumed that CD45⁺/TF⁺ platelets could be formed as a result of platelet interaction with leukocytes or leukocyte produced membrane microparticles.     

Radiolabeling Human Peripheral Blood Stem Cells for Positron Emission Tomography (PET) Imaging in Young Rhesus Monkeys

These studies focused on a new radiolabeling technique with copper (⁶⁴Cu) and zirconium (⁸⁹Zr) for positron emission tomography (PET) imaging using a CD45 antibody. Synthesis of ⁶⁴Cu-CD45 and ⁸⁹Zr-CD45 immunoconjugates was performed and the evaluation of the potential toxicity of radiolabeling human peripheral blood stem cells (hPBSC) was assessed in vitro (viability, population doubling times, colony forming units). hPBSC viability was maintained as the dose of ⁶⁴Cu-TETA-CD45 increased from 0 (92%) to 160 µCi/mL (76%, p>0.05). Radiolabeling efficiency was not significantly increased with concentrations of ⁶⁴Cu-TETA-CD45 >20 µCi/mL (p>0.50). Toxicity affecting both growth and colony formation was observed with hPBSC radiolabeled with ≥40 µCi/mL (p<0.05). For ⁸⁹Zr, there were no significant differences in viability (p>0.05), and a trend towards increased radiolabeling efficiency was noted as the dose of ⁸⁹Zr-Df-CD45 increased, with a greater level of radiolabeling with 160 µCi/mL compared to 0–40 µCi/mL (p<0.05). A greater than 2,000 fold-increase in the level of ⁸⁹Zr-Df-CD45 labeling efficiency was observed when compared to ⁶⁴Cu-TETA-CD45. Similar to ⁶⁴Cu-TETA-CD45, toxicity was noted when hPBSC were radiolabeled with ≥40 µCi/mL (p<0.05) (growth, colony formation). Taken together, 20 µCi/mL resulted in the highest level of radiolabeling efficiency without altering cell function. Young rhesus monkeys that had been transplanted prenatally with 25×10⁶ hPBSC expressing firefly luciferase were assessed with bioluminescence imaging (BLI), then 0.3 mCi of ⁸⁹Zr-Df-CD45, which showed the best radiolabeling efficiency, was injected intravenously for PET imaging. Results suggest that ⁸⁹Zr-Df-CD45 was able to identify engrafted hPBSC in the same locations identified by BLI, although the background was high.     

The predominant contribution of platelets to baseline and ascorbate-stimulated increments in cyclic GMP in human mononuclear leukocyte preparations.

In nine consecutive experiments with Ficoll-Hypaque-purified human mononuclear leukocytes containing 2.8 (range 1.1–4.3) platelets per leukocyte, 2–5 mM sodium ascorbate produced a 14-fold (range, 7- to 18-fold) rise in guanosine 3′: 5′-cyclic monophosphate (cyclic GMP) from baseline levels of 0.103 ± 0.056 pmol/10⁷ mononuclear leukocytes. In five experiments with mononuclear leukocytes prepared by the Ficoll-Hypaque method from human blood depleted of platelets by defibrination, 2–5 mM sodium ascorbate produced a twofold (range, one- to fourfold) rise in cyclic GMP from baseline levels of 0.030 ± 0.012 pmol/10⁷ mononuclear leukocytes. Thus, platelets contribute substantially to baseline and ascorbate-stimulated levels of cyclic GMP in standard Ficoll-Hypaque preparations of mononuclear leukocytes. The rise in cyclic GMP concentration in mononuclear leukocyte preparations elicited by ascorbate was independent of a calcium requirement, persisted for up to 3 hr in the presence of ascorbate, and was prevented by the introduction of nonsteroidal anti-inflammatory agents such as aspirin and indomethacin (ID₅₀ = 105 and 23.5 μM, respectively).