ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1990, p. 1672-1677
Vol. 34, No. 9
0066-4804/90/091672-06$02.00/0 Copyright © 1990, American Society for Microbiology
Effects of Antiretroviral Dideoxynucleosides on Polymorphonuclear Leukocyte Function EMMANUEL ROILIDES,1 DAVID VENZON,2 PHILIP A. PIZZO,'* AND MARC RUBIN't Infectious Diseases Section, Pediatric Oncology Branch,' and Biostatistics and Data Management Section,2 National Cancer Institute, Bethesda, Maryland 20892 Received 21 March 1990/Accepted 12 June 1990
Dideoxynucleosides (zidovudine [AZT], dideoxycytidine [ddC], and dideoxyinosine [ddI]) are promising new agents for the management of human immunodeficiency virus type 1 (HIV-1) infections. In light of recent data demonstrating defects in the polymorphonuclear leukocyte (PMN) bactericidal activity of HIV-1-infected patients and since many chemotherapeutic agents affect PMN function, we examined their effects on the function of PMNs from both healthy and HIV-1-infected individuals in vitro. AZT (0.1 to 25 PM), ddC (0.01 to 1 ,uM), and ddl (0.2 to 50 ,uM) had no effect on viability, chemotaxis to N-formylmethionyl leucyl phenylalanine, phagocytosis of Candida albicans or Staphylococcus aureus, or superoxide production following stimulation by N-formylmethionyl leucyl phenylalanine. Killing of C. albicans was not affected by AZT but was enhanced by 0.1 and 1 ,uM ddC (at 1 ,uM, killing was 26.0 ± 2.02% compared with 17.0 + 0.73% for controls; P = 0.006) and 0.2 to 50 ,uM ddl (at 10 ,M, killing was 25.0 ± 0.68% compared with 17.8 ± 0.91% for controls; P = 0.002). Killing of S. aureus was unchanged by AZT and ddC but was significantly enhanced by ddl at 0.2 to 20 ,M (at 2 ,M, killing was 71.2 + 5.57% compared with 51.4 ± 6.29% for controls; P = 0.0045). In addition, the preexisting defective bactericidal capacity of PMNs from HIV-1-infected patients was enhanced by ddI (P < 0.025). Potential enhancement by these dideoxynucleosides of certain PMN functions of HIV-1-infected patients deserves further study.
With the discovery that human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immunodeficiency syndrome, a number of antiretroviral compounds targeted at HIV-1 have entered preclinical and clinical trials. Of these, the dideoxynucleosides zidovudine (azidothymidine [AZT]), dideoxycytidine (ddC), and dideoxyinosine (ddI) are being studied most extensively (6, 14, 21, 28-30). Infections remain a major cause of morbidity and mortality in patients infected with HIV-1 (9) as a result of impairment in both cellular and humoral immunities (13). In addition to parasitic, viral, and fungal pathogens (e.g., Pneumocystis carinii, cytomegalovirus, Toxoplasma gondii, and Cryptococcus neoformans), a number of bacteria may cause significant infections, particularly in HIV-1-infected children (12). Polymorphonuclear leukocytes (PMNs) are the most important component of the host defense against bacterial and fungal infections. Recent studies have documented significant defects in the function of PMNs from both HIV-1-infected adults and children prior to antiretroviral therapy. The most consistent defects that have been found are impairments in the chemotactic and bactericidal activities of PMNs (4, 15; E. Roilides, S. Mertins, J. Eddy, T. J. Walsh, P. A. Pizzo, and M. Rubin, J. Pediatr., in press). Although antiretroviral agents like AZT can improve some features of the HIV-1 syndrome (e.g., neuropsychological function), bacterial and other infections still occur (21). Previous studies have shown that a number of chemotherapeutic agents, including antineoplastic and antimicrobial agents, may significantly impair PMN function, as measured by in vitro assays (1, 3, 17, 20, 22). To determine whether antiretroviral therapy itself might contribute to the risk for bacterial infections, we evaluated the effects of AZT, ddC, *
and ddl on PMN function from healthy donors and HIV-1infected patients in vitro. (Preliminary results of this study were presented at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 17 to 20 September 1989 [E. Roilides, P. A. Pizzo, and M. Rubin, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 1180, 1989].) MATERIALS AND METHODS Subjects. Ten healthy volunteers served as donors for testing the effect of the dideoxynucleosides on normal PMN function. Some of them donated blood more than once. In addition, PMNs with preexisting bactericidal defects were obtained from six HIV-1-infected patients (four adults and two children) who were not receiving any antiretroviral therapy, to assess the effects of ddI on their bactericidal
activity. Drugs. AZT was kindly provided by Steven Good, Burroughs Wellcome Co., Research Triangle Park, N.C. ddC and ddl were obtained from the Investigational Drug Branch of the National Cancer Institute, Bethesda, Md. Stock solutions were prepared by dissolving the dideoxynucleosides in distilled H20 and were kept frozen at -20°C. Final dilutions were made in Hanks balanced salt solution (HBSS) before each experiment. The solutions used for the experiments contained 95% purity and >95% viability, as judged by trypan blue exclusion. Drug pretreatment of cells. Prior to performing the assays, 2.5 x 106 or 1 x 106 PMNs in HBSS were incubated at 37°C for 30 min in the presence of buffer or various concentrations of AZT in the range of 0.1 to 25 ,uM, ddC in the range of 0.01 to 1 ,uM, and ddl in the range of 0.2 to 50 ,uM. The true therapeutic level of a dideoxynucleoside in plasma has not been well defined, but based on in vitro activity, we chose the following target concentrations: AZT, 1 to 5 ,uM (28); ddC, 0.1 to 0.5 ,uM (30); and ddl, 2 to 10 ,uM (29). The cells remained exposed to each of the respective drugs during the performance of the assays. Chemotaxis. Chemotaxis of PMNs was assessed by the method of Harvath et al. (10). Forty-five microliters of suspensions containing 1 x 106 PMNs per ml in HBSS (4.5 x 104 PMNs per well) was added to the upper wells of a 48-multiwell chemotaxis chamber (Neuroprobe, Inc., Cabin
John, Md.). Polyvinylpyrrolidone coating-free polycarbonsize, 3 ,um; 25 by 80 mm) separated the upper
ate filters (pore
and lower chambers so that the entire population of the
chemotactically active cells could be measured. Migrating cells were adherent to the lower surface of the polyvinylpyr-
rolidone-free filter and did not fall into the lower chamber
(10). The wells of the lower chambers were filled with either 10-8 M N-formylmethionyl leucyl phenylalanine (FMLP; Sigma Chemical Co., St. Louis, Mo.) or control buffer (HBSS). To assess PMN migration, the chamber was incubated at 37°C in a humidified atmosphere with 5% CO2 for 60 min. The filters were then removed, air-dried, fixed, and stained with modified Wright-Giemsa stain (Diff-Quick; Diagnostic Systems Inc., Gibbstown, N.J.). The cells on the lower surface of the filters were counted by direct microscopy by using a computer-facilitated counter (Optomax System IV; Ion Track Instruments, Burlington, Mass.). A
chemotactic index was calculated as the ratio of the number of cells which were attracted to FMLP divided by the number of randomly migrating cells. The percentage of the chemotactic indices of the PMNs in the presence of various
1673
concentrations of the compounds divided by that of the control PMNs was then calculated. Fungicidal assay. The fungicidal activity of PMNs was assessed by the methylene blue staining technique by the method described by Lehrer and Cline (16). Briefly, C. albicans blastoconidia were mixed with 106 PMNs in a 1/1 ratio in 1 ml of HBSS containing 10% human type AB serum (GIBCO Laboratories, Grand Island, N.Y.) that was kept frozen at -40°C in aliquots and incubated at 37°C for 1 h on a shaker. Methylene blue (0.01%) was then added, and the cells were left at 37°C for 3 min. Wet mounts were prepared, and the number of methylene blue-stained (dead) versus the number of unstained (alive) intracellular blastoconidia was counted (a total of 300 blastoconidia were counted for each preparation). Intracellular killing (IK) was estimated by the following formula: IK (%) = [stained (dead) blastoconidia/ (stained + unstained (alive) blastoconidia)] x 100. Bactericidal assay. The bactericidal activity of PMNs was assessed by a CFU assay. S. aureus was mixed with 2.5 x 106 PMNs in a 1/1 ratio in 1 ml of HBSS containing 10% normal human type AB serum. Preparations were incubated at 37°C for 1 h on a shaker, and samples were obtained at time zero and after 45 and 90 min. The PMNs were lysed in sterile H20 containing 0.01% bovine serum albumin, and serial dilutions were made and plated in duplicate on Trypticase-soy agar plates and Were incubated for 18 h at 37°C. Colonies were counted with an automated colony counter (Optomax IV). Bacterial killing was calculated by the following formula: percent killing of S. aureus at Tx = [1 (CA/C0)] x 100, where C. is the number of CFU obtained after 45 or 90 min (T,,), and CO is the number of CFU in the initial inoculum. Phagocytosis. PMN phagocytosis was assessed by direct microscopy by using C. albicans or S. aureus as targets according to previously described methods (7, 24). Briefly, C. albicans blastoconidia were mixed with 106 PMNs in a 1/1 ratio in 1 ml of HBSS containing 10% human type AB serum and incubated at 370C for 15 min on a shaker. Samples were then obtained, cytocentrifuged, and stained with modified Wright-Giemsa stain (Diff-Quick). Percent phagocytosis was calculated as the proportion of PMNs containing 1 or more blastoconidia after 100 PMNs were counted. A phagocytic index (PI) was also calculated as the average number of blastoconidia per phatocytosing PMN. To assess the phagocytosis of bacterial targets, preopsonized S. aureus bacteria (incubation for 30 min at 37°C with 50% [vol/vol] human type AB serum) were mixed with 106 PMNs in a 1/100 effector/target ratio in 1 ml of HBSS and incubated at 37°C on a shaker. Samples were obtained after 5 and 15 min and were immediately mixed with cold N-ethylmaleimide (0.1 mM; Sigma) to inhibit additional phagocytosis. They were then incubated with lysostaphin (20 U/ml; Sigma) for an additional 10 min at 37°C to lyse the nonphagocytosed, extracellular bacteria. Preparations were cytocentrifuged and stained with modified Wright-Giemsa stain. Percent phagocytosis was calculated as the proportion of PMNs containing 1 or more bacteria after 100 PMNs were counlted. The weighted PI was calculated by multiplying the number of PMNs with 1 to 10, 11 to 20, 21 to 30, 31 to 40, or >40 ingested organisms by 1, 2, 3, 4, or 5, respectively, and dividing the total score by the number of PMNs examined
(usually 100). Superoxide anion assay. Superoxide anion production in
response to FMLP was assessed spectrophotometrically. A total of 1 x 106 PMNs were mixed with 5 x 10-5 M cytochrome c (Sigma) and 5 x 10' M FMLP in 1 ml of
1674
ROILIDES ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Chemotaxis of neutrophils (PMNs) pretreated with various concentrations of dideoxynucleosides AZT
% Chemotactic indexa ddC
103 ± 9
105 ± 7 97 ± 6
105 ± 7
100 ± 6
Drug concn
(>M) 0.01 0.1 0.2 1.0 2.0 10.0
ddl 94 ± 6 102 ± 7
95 ± 11
20.0
100 ± 11
Chemotaxis in response to FMLP (10-8 M); values are percentages of chemotactic indices of PMNs in the presence of dideoxynucleosides divided by the chemotactic index of PMNs without the drugs and are means + standard errors of three experiments. a
HBSS at 371C. The change in absorption at 550 nm was measured on a spectrophotometer (Gilford 260) equipped with a thermostated cuvette holder (CIBA-Coming Diagnostics Corp., Oberlin, Ohio) (18). Superoxide produced by 106 PMNs in 5 min was then calculated by using the millimolar extinction coefficient for reduced cytochrome c. Statistics. The effects of dideoxynucleosides on PMN function were analyzed by a variant of the method of Tukey et al. (27), in which repeated-measures analysis of variance was used to account for the same donors being tested repeatedly at different concentrations. Linear trends in each functional parameter over increasing concentrations of a dideoxynucleoside were evaluated by this method. Since the distributions of the parameters were consistent with normality, the changes from the base line at individual concentrations were assessed by Student's t test. No adjustment for the multiplicity of tests was made because of the correlations between them. All the P values reported are two-tailed. RESULTS Toxicity. The viability of the PMNs remained >95% by trypan blue exclusion when they were incubated with AZT, ddC, or ddI for as long as 120 min at concentrations of 0. 1 to 25 ,uM, 0.01 to 1 ,uM, and 0.2 to 50 ,uM, respectively. Chemotaxis. None of the dideoxynucleosides tested had any significant effect on PMN chemotaxis in response to
AZT Co c to
C.) .0
ddl
ddC 30
30 r
30 r
25 I-
25p
C
Co
FMLP (Table 1). The chemotactic index of the untreated PMNs was 1.50 ± 0.08 (mean ± standard error of eight determinations), and this remained unchanged even at concentrations two to five times higher than those achieved when these agents are used clinically. Random migration of PMNs was also unaffected by the presence of drugs. Fungicidal activity of PMNs from healthy donors. Figure 1 shows the effects of the three compounds on the fungicidal activity of PMNs. The overall base-line intracellular killing of C. albicans after 1 hour incubation was 17.2 ± 0.47%. AZT did not significantly affect fungicidal activity in the donors tested (Fig. 1). In contrast, ddC and ddl, in six experiments performed with different donors, significantly enhanced the fungicidal activity of normal PMNs within and above the range of therapeutically achievable concentrations (P values of the linear trends, LM) Control 0.01 0.1 0.2 1.0 2.0 5.0 10.0 25.0 a
b
c
ddl
ddC
AZT
Drug concn
PIc
%
PI
%
PI
3.4
1.77 ± 0.10
1.82 ± 0.18
1.88 ± 0.10
1.77 ± 0.79 1.70 ± 0.09 1.78 ± 0.03
59 ± 4.0
66 ± 2.8
61 ± 3.8 61 ± 3.3 60 ± 2.9
63 ± 5.5
1.78 ± 0.07
69 ± 2.1
1.92 ± 0.17
65 ± 3.8
1.70 ± 0.08 61 ± 7.1
1.67 ± 0.06
64 ± 3.2
1.60
63 ± 7.4
1.80 ± 0.13
62 ± 5.9 57 ± 6.1
1.70 ± 0.03 1.96 ± 0.12
ob 64
+
0.09
Values are means ± standard errors of four experiments. Percentage of phagocytosing PMNs divided by the total number of PMNs counted. PI is the total number of phagocytosed C. albicans blastoconidia divided by the number of PMNs that phagocytosed them.
1676
ROILIDES ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
AZT
ddC
ddl
5r
5
LZz 4 I-
4
4
0~CL
3,
3
ONC~ 2
2 .
2.
0
1
1
*
.E
0 r ;
3
0 v-
-
-
1
E O
L. .1
0
-
__
1
10 100
a) 0
.01 .01
.1. 1 .1 1
1 10
0
0
.1
.1
10 1O
Concentration (MM) FIG. 2. Superoxide anion production by neutrophils (PMNs) pretreated with various concentrations of dideoxynucleosides. FMLP (5 x 1l-7 M) was used as a stimulus. The results are expressed as the means ± standard errors of three experiments.
appear to have adverse effects on T- and B-lymphocyte functions in vitro at concentrations of the drug that protect cells from HIV-1 (19), and in this study, we did not demonstrate that there are any adverse effects of AZT on PMN function. Similarly, neither ddC nor ddI had an inhibitory effect on PMN function. Rather, ddC significantly enhanced fungicidal activity, and ddI increased both bactericidal and fungicidal activities at therapeutically achievable concentrations. Although AZT has been shown to have antibacterial activity against some gram-negative organisms (e.g., E. coli, Shigella flexneri, and Salmonella typhimurium) (5, 11), S. aureus and C. albicans were not directly affected by any of the dideoxynucleosides we tested (also cited in reference 5). Thus, the enhancement of killing appears to be related either to an interaction between dideoxynucleosides and the PMNs or to an indirect interaction between dideoxynucleosides and the microorganisms, making them more susceptible to the PMN action. A direct growth inhibitory or microbicidal effect of either ddC or ddl, however, was excluded in the control experiments. The increased microbicidal activity observed with ddC and ddI does not appear to be a consequence of increased phagocytosis or increased oxidative metabolism. Nonoxidative killing (e.g., lysozyme and defensins) (8) might be responsible for the increased microbicidal activity of PMNs when they are exposed to those agents, although the current data do not address this. Also, intracellular killing of either bacteria or fungi by PMNs depends on the way the organism is presented and processed by the oxidative and nonoxidative metabolic products (e.g., phagosome-lysosome fusion or secretion of microbicidal products in response to the particular organisms). How the dideoxynucleosides affect each of these intracellular processes in response to either bacteria or fungi is not known. The basis for the differences we observed among these three dideoxynucleosides is also unclear. It is possible that they are related to differences in the intracellular uptake and phosphorylation of these dideoxynucleosides. Further studies assessing the in vivo effects of these agents on PMN function are in progress. ACKNOWLEDGMENTS We acknowledge the help of Frank Balis in obtaining the investigational drugs evaluated in this study. We thank the following people who contributed in providing blood specimens from patients: Robert Yarchoan, Rose Thomas, and Janie Eddy. We also thank Robert Danner for testing our reagents for endotoxin.
LITERATURE CITED 1. Amano, M., and H. Tanaka. 1986. Effect of antibiotics on phagocytosis and bactericidal activity of human leukocytes. Chemotherapy 34:157-163. 2. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Suppl.
97:77-89. 3. Daschner, F. D. 1985. Antibiotics and host defence with special reference to phagocytosis by human polymorphonuclear leukocytes. J. Antimicrob. Chemother. 16:135-141. 4. Ellis, M., S. Gupta, S. Galant, S. Hakim, C. VandeVen, C. Toy, and M. S. Cairo. 1988. Impaired neutrophil function in patients with AIDS or AIDS-related complex: a comprehensive evaluation. J. Infect. Dis. 158:1268-1276. 5. Elweil, L. P., R. Ferone, G. A. Freeman, J. A. Fyfe, J. A. Hill, P. H. Ray, C. A. Richards, S. C. Sieger, V. B. Knick, J. L. Rideout, and T. P. Zimmerman. 1987. Antibacterial activity and mechanism of action of 3'-azido-3'-deoxythymidine (BW A509U). Antimicrob. Agents Chemother. 31:274-280. 6. Fischl, M. A., D. D. Richman, M. H. Grieco, M. S. Gottlieb, P. A. Volberding, 0. L. Laskin, J. M. Leedom, J. E. Groopman, D. Mildran, R. T. Schooley, G. G. Jackson, D. T. Durack, D. King, and the AZT Collaborative Working Group. 1987. The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. A double-blind, placebocontrolled trial. N. Engl. J. Med. 317:185-191. 7. Fleischmann, J., D. W. Golde, R. H. Weisbart, and J. C. Gasson. 1986. Granulocyte-macrophage colony-stimulating factor enhances phagocytosis of bacteria by human neutrophils. Blood 68:708-711. 8. Ganz, T., M. E. Selsted, D. Szklarek, S. S. L. Harwig, K. Daher, D. F. Bainton, and R. I. Lehrer. 1985. Defensins: natural peptide antibiotics of human neutrophils. J. Clin. Invest. 76:1427-1435. 9. Glatt, A. E., K. Chirgwin, and S. H. Landesman. 1988. Treatment of infections associated with human immunodeficiency virus. N. Engl. J. Med. 318:1439-1448. 10. Harvath, L., W. Falk, and E. J. Leonard. 1980. Rapid quantitation of neutrophil chemotaxis: use of a polyvinylpyrrolidonefree polycarbonate membrane in a multiwell assembly. J. Immunol. Methods 37:39-45. 11. Keith, B. R., G. White, and H. R. Wilson. 1989. In vivo efficacy of zidovudine (3'-azido-3'-deoxythymidine) in experimental gram-negative bacterial infections. Antimicrob. Agents Chemother. 33:479-483. 12. Krasinski, K., W. Borkowsky, S. Bonk, R. Lawrence, and S. Chandwani. 1988. Bacterial infections in human immunodeficiency virus-infected children. Pediatr. Infect Dis. 7:323-328. 13. Lane, H. C., and A. S. Fauci. 1985. Immunologic abnormalities in the acquired immunodeficiency syndrome. Annu. Rev. Im-
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EFFECTS OF ANTIRETROVIRAL DIDEOXYNUCLEOSIDES ON PMNs
munol. 3:477-500. 14. Langtry, H. D., and D. M. Campoli-Richards. 1989. Zidovudine. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy. Drugs 37:408-450. 15. Lazzarin, A., C. Uberti Foppa, M. GaU, A. Mantovani, G. Poli, F. Franzetti, and R. Navati. 1986. Impairment of polymorphonuclear leukocyte function in patients with acquired immunodeficiency syndrome. Clin. Exp. Immunol. 65:105-111. 16. Lehrer, R. I., and M. J. Cline. 1969. Interaction of Candida albicans with human leucocytes and serum. J. Bacteriol. 98: 996-1009. 17. Malawista, S. E. 1971. Vinblastine: colchicine-like effects on human blood leukocytes during phagocytosis. Blood 37:519529. 18. Metcalf, J. A., J. I. GalUla, W. M. Nauseef, and R. K. Root. 1987. Laboratory manual of neutrophil function, p. 109-114. Raven Press, New York. 19. Mltsuya, H., K. J. Weinhold, P. A. Furman, M. H. St. Clair, S. Nusinoff-Lehrman, R. C. Gallo, D. Bolognesi, D. W. Barry, and S. Broder. 1985. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type-III/lymphadenopathy-associated virus in vitro. Proc. Natl. Acad. Sci. USA 82:70967100. 20. Pickering, L. K., C. D. Ericsson, and S. Kohl. 1978. Effect of chemotherapeutic agents on metabolic and bactericidal activity of polymorphonuclear leukocytes. Cancer 42:1741-1746. 21. Pizzo, P. A., J. Eddy, J. Failoon, F. M. Balis, R. F. Murphy, H. Moss, P. Wolters, P. Brouwers, P. Jarosinski, M. Rubin, S. Broder, R. Yarchoan, A. Brunetti, M. Maha, S. NusinoffLehrman, and D. G. Poplack. 1988. Effects of continuous intravenous infusion of zidovudine (AZT) in children with symptomatic HIV-1 infection. N. Engl. J. Med. 319:889-896. 22. Pruzanski, W., S. Saito, and G. Deboer. 1983. Modulatory activity of chemotherapeutic agents on phagocytosis and intracellular bactericidal activity of human polymorphonuclear and mononuclear phagocytes. Cancer Res. 43:1420-1425.
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23. Richman, D. D., M. A. Fischl, M. H. Grieco, M. S. Gottlieb, P. A. Volberding, 0. L. Laskin, J. M. Leedom, J. E. Groopman, D. Mildran, M. S. Hirsch, G. G. Jackson, D. T. Durack, S. N. Lehrman, and the AZT CoUlaborative Working Group. 1987. The toxicity of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. A double-blind, placebocontrolled trial. N. Engl. J. Med. 317:192-197. 24. Roilides, E., T. J. Walsh, M. Rubin, D. Venzon, and P. A. Pizzo. 1990. Effects of antifungal agents on the function of human neutrophils in vitro. Antimicrob. Agents Chemother. 34:196201. 25. Sommadossi, J.-P., and R. Carlisle. 1987. Toxicity of 3'-azido3'-deoxythymidine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine for normal human hematopoietic progenitor cells in vitro. Antimicrob. Agents Chemother. 31:452-454. 26. Steele, R. W., D. L. Crosby, R. W. Steele, N. S. Pilkington, and R. K. Charlton. 1988. Effects of ribavirin on neutrophil function. Am. J. Med. Sci. 295:503-506. 27. Tukey, J. W., J. L. Ciminera, and J. F. Heyse. 1985. Testing the statistical certainty of a response to increasing doses of a drug. Biometrics 41:295-301. 28. Yarchoan, R., and S. Broder. 1989. Anti-retroviral therapy of AIDS and related disorders: general principles and specific development of dideoxynucleosides. Pharmacol. Ther. 40:329348. 29. Yarchoan, R., H. Mitsuya, R. V. Thomas, J. M. Pluda, N. R. Hartman, C. F. Perno, K. S. Marczyk, J. P. Allain, D. G. Johns, and S. Broder. 1989. In vivo activity against HIV and favorable toxicity profile of 2',3'-dideoxyinosine. Science 245:412-415. 30. Yarchoan, R., C. F. Perno, R. V. Thomas, R. W. Klecker, J.-P. Allain, R. J. Wills, N. McAtee, M. A. Fischl, R. Dubinsky, M. C. McNeely, H. Mitsuya, J. M. Pluda, T. J. Lawley, M. Leuther, B. Safai, J. M. Collins, C. E. Myers, and S. Broder. 1988. Phase I studies of 2',3'-dideoxycytidine in severe human immunodeficiency virus infection as a single agent and alternating with zidovudine (AZT). Lancet :76-81.
Vol. 34, No. 9
0066-4804/90/091672-06$02.00/0 Copyright © 1990, American Society for Microbiology
Effects of Antiretroviral Dideoxynucleosides on Polymorphonuclear Leukocyte Function EMMANUEL ROILIDES,1 DAVID VENZON,2 PHILIP A. PIZZO,'* AND MARC RUBIN't Infectious Diseases Section, Pediatric Oncology Branch,' and Biostatistics and Data Management Section,2 National Cancer Institute, Bethesda, Maryland 20892 Received 21 March 1990/Accepted 12 June 1990
Dideoxynucleosides (zidovudine [AZT], dideoxycytidine [ddC], and dideoxyinosine [ddI]) are promising new agents for the management of human immunodeficiency virus type 1 (HIV-1) infections. In light of recent data demonstrating defects in the polymorphonuclear leukocyte (PMN) bactericidal activity of HIV-1-infected patients and since many chemotherapeutic agents affect PMN function, we examined their effects on the function of PMNs from both healthy and HIV-1-infected individuals in vitro. AZT (0.1 to 25 PM), ddC (0.01 to 1 ,uM), and ddl (0.2 to 50 ,uM) had no effect on viability, chemotaxis to N-formylmethionyl leucyl phenylalanine, phagocytosis of Candida albicans or Staphylococcus aureus, or superoxide production following stimulation by N-formylmethionyl leucyl phenylalanine. Killing of C. albicans was not affected by AZT but was enhanced by 0.1 and 1 ,uM ddC (at 1 ,uM, killing was 26.0 ± 2.02% compared with 17.0 + 0.73% for controls; P = 0.006) and 0.2 to 50 ,uM ddl (at 10 ,M, killing was 25.0 ± 0.68% compared with 17.8 ± 0.91% for controls; P = 0.002). Killing of S. aureus was unchanged by AZT and ddC but was significantly enhanced by ddl at 0.2 to 20 ,M (at 2 ,M, killing was 71.2 + 5.57% compared with 51.4 ± 6.29% for controls; P = 0.0045). In addition, the preexisting defective bactericidal capacity of PMNs from HIV-1-infected patients was enhanced by ddI (P < 0.025). Potential enhancement by these dideoxynucleosides of certain PMN functions of HIV-1-infected patients deserves further study.
With the discovery that human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immunodeficiency syndrome, a number of antiretroviral compounds targeted at HIV-1 have entered preclinical and clinical trials. Of these, the dideoxynucleosides zidovudine (azidothymidine [AZT]), dideoxycytidine (ddC), and dideoxyinosine (ddI) are being studied most extensively (6, 14, 21, 28-30). Infections remain a major cause of morbidity and mortality in patients infected with HIV-1 (9) as a result of impairment in both cellular and humoral immunities (13). In addition to parasitic, viral, and fungal pathogens (e.g., Pneumocystis carinii, cytomegalovirus, Toxoplasma gondii, and Cryptococcus neoformans), a number of bacteria may cause significant infections, particularly in HIV-1-infected children (12). Polymorphonuclear leukocytes (PMNs) are the most important component of the host defense against bacterial and fungal infections. Recent studies have documented significant defects in the function of PMNs from both HIV-1-infected adults and children prior to antiretroviral therapy. The most consistent defects that have been found are impairments in the chemotactic and bactericidal activities of PMNs (4, 15; E. Roilides, S. Mertins, J. Eddy, T. J. Walsh, P. A. Pizzo, and M. Rubin, J. Pediatr., in press). Although antiretroviral agents like AZT can improve some features of the HIV-1 syndrome (e.g., neuropsychological function), bacterial and other infections still occur (21). Previous studies have shown that a number of chemotherapeutic agents, including antineoplastic and antimicrobial agents, may significantly impair PMN function, as measured by in vitro assays (1, 3, 17, 20, 22). To determine whether antiretroviral therapy itself might contribute to the risk for bacterial infections, we evaluated the effects of AZT, ddC, *
and ddl on PMN function from healthy donors and HIV-1infected patients in vitro. (Preliminary results of this study were presented at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 17 to 20 September 1989 [E. Roilides, P. A. Pizzo, and M. Rubin, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 1180, 1989].) MATERIALS AND METHODS Subjects. Ten healthy volunteers served as donors for testing the effect of the dideoxynucleosides on normal PMN function. Some of them donated blood more than once. In addition, PMNs with preexisting bactericidal defects were obtained from six HIV-1-infected patients (four adults and two children) who were not receiving any antiretroviral therapy, to assess the effects of ddI on their bactericidal
activity. Drugs. AZT was kindly provided by Steven Good, Burroughs Wellcome Co., Research Triangle Park, N.C. ddC and ddl were obtained from the Investigational Drug Branch of the National Cancer Institute, Bethesda, Md. Stock solutions were prepared by dissolving the dideoxynucleosides in distilled H20 and were kept frozen at -20°C. Final dilutions were made in Hanks balanced salt solution (HBSS) before each experiment. The solutions used for the experiments contained 95% purity and >95% viability, as judged by trypan blue exclusion. Drug pretreatment of cells. Prior to performing the assays, 2.5 x 106 or 1 x 106 PMNs in HBSS were incubated at 37°C for 30 min in the presence of buffer or various concentrations of AZT in the range of 0.1 to 25 ,uM, ddC in the range of 0.01 to 1 ,uM, and ddl in the range of 0.2 to 50 ,uM. The true therapeutic level of a dideoxynucleoside in plasma has not been well defined, but based on in vitro activity, we chose the following target concentrations: AZT, 1 to 5 ,uM (28); ddC, 0.1 to 0.5 ,uM (30); and ddl, 2 to 10 ,uM (29). The cells remained exposed to each of the respective drugs during the performance of the assays. Chemotaxis. Chemotaxis of PMNs was assessed by the method of Harvath et al. (10). Forty-five microliters of suspensions containing 1 x 106 PMNs per ml in HBSS (4.5 x 104 PMNs per well) was added to the upper wells of a 48-multiwell chemotaxis chamber (Neuroprobe, Inc., Cabin
John, Md.). Polyvinylpyrrolidone coating-free polycarbonsize, 3 ,um; 25 by 80 mm) separated the upper
ate filters (pore
and lower chambers so that the entire population of the
chemotactically active cells could be measured. Migrating cells were adherent to the lower surface of the polyvinylpyr-
rolidone-free filter and did not fall into the lower chamber
(10). The wells of the lower chambers were filled with either 10-8 M N-formylmethionyl leucyl phenylalanine (FMLP; Sigma Chemical Co., St. Louis, Mo.) or control buffer (HBSS). To assess PMN migration, the chamber was incubated at 37°C in a humidified atmosphere with 5% CO2 for 60 min. The filters were then removed, air-dried, fixed, and stained with modified Wright-Giemsa stain (Diff-Quick; Diagnostic Systems Inc., Gibbstown, N.J.). The cells on the lower surface of the filters were counted by direct microscopy by using a computer-facilitated counter (Optomax System IV; Ion Track Instruments, Burlington, Mass.). A
chemotactic index was calculated as the ratio of the number of cells which were attracted to FMLP divided by the number of randomly migrating cells. The percentage of the chemotactic indices of the PMNs in the presence of various
1673
concentrations of the compounds divided by that of the control PMNs was then calculated. Fungicidal assay. The fungicidal activity of PMNs was assessed by the methylene blue staining technique by the method described by Lehrer and Cline (16). Briefly, C. albicans blastoconidia were mixed with 106 PMNs in a 1/1 ratio in 1 ml of HBSS containing 10% human type AB serum (GIBCO Laboratories, Grand Island, N.Y.) that was kept frozen at -40°C in aliquots and incubated at 37°C for 1 h on a shaker. Methylene blue (0.01%) was then added, and the cells were left at 37°C for 3 min. Wet mounts were prepared, and the number of methylene blue-stained (dead) versus the number of unstained (alive) intracellular blastoconidia was counted (a total of 300 blastoconidia were counted for each preparation). Intracellular killing (IK) was estimated by the following formula: IK (%) = [stained (dead) blastoconidia/ (stained + unstained (alive) blastoconidia)] x 100. Bactericidal assay. The bactericidal activity of PMNs was assessed by a CFU assay. S. aureus was mixed with 2.5 x 106 PMNs in a 1/1 ratio in 1 ml of HBSS containing 10% normal human type AB serum. Preparations were incubated at 37°C for 1 h on a shaker, and samples were obtained at time zero and after 45 and 90 min. The PMNs were lysed in sterile H20 containing 0.01% bovine serum albumin, and serial dilutions were made and plated in duplicate on Trypticase-soy agar plates and Were incubated for 18 h at 37°C. Colonies were counted with an automated colony counter (Optomax IV). Bacterial killing was calculated by the following formula: percent killing of S. aureus at Tx = [1 (CA/C0)] x 100, where C. is the number of CFU obtained after 45 or 90 min (T,,), and CO is the number of CFU in the initial inoculum. Phagocytosis. PMN phagocytosis was assessed by direct microscopy by using C. albicans or S. aureus as targets according to previously described methods (7, 24). Briefly, C. albicans blastoconidia were mixed with 106 PMNs in a 1/1 ratio in 1 ml of HBSS containing 10% human type AB serum and incubated at 370C for 15 min on a shaker. Samples were then obtained, cytocentrifuged, and stained with modified Wright-Giemsa stain (Diff-Quick). Percent phagocytosis was calculated as the proportion of PMNs containing 1 or more blastoconidia after 100 PMNs were counted. A phagocytic index (PI) was also calculated as the average number of blastoconidia per phatocytosing PMN. To assess the phagocytosis of bacterial targets, preopsonized S. aureus bacteria (incubation for 30 min at 37°C with 50% [vol/vol] human type AB serum) were mixed with 106 PMNs in a 1/100 effector/target ratio in 1 ml of HBSS and incubated at 37°C on a shaker. Samples were obtained after 5 and 15 min and were immediately mixed with cold N-ethylmaleimide (0.1 mM; Sigma) to inhibit additional phagocytosis. They were then incubated with lysostaphin (20 U/ml; Sigma) for an additional 10 min at 37°C to lyse the nonphagocytosed, extracellular bacteria. Preparations were cytocentrifuged and stained with modified Wright-Giemsa stain. Percent phagocytosis was calculated as the proportion of PMNs containing 1 or more bacteria after 100 PMNs were counlted. The weighted PI was calculated by multiplying the number of PMNs with 1 to 10, 11 to 20, 21 to 30, 31 to 40, or >40 ingested organisms by 1, 2, 3, 4, or 5, respectively, and dividing the total score by the number of PMNs examined
(usually 100). Superoxide anion assay. Superoxide anion production in
response to FMLP was assessed spectrophotometrically. A total of 1 x 106 PMNs were mixed with 5 x 10-5 M cytochrome c (Sigma) and 5 x 10' M FMLP in 1 ml of
1674
ROILIDES ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Chemotaxis of neutrophils (PMNs) pretreated with various concentrations of dideoxynucleosides AZT
% Chemotactic indexa ddC
103 ± 9
105 ± 7 97 ± 6
105 ± 7
100 ± 6
Drug concn
(>M) 0.01 0.1 0.2 1.0 2.0 10.0
ddl 94 ± 6 102 ± 7
95 ± 11
20.0
100 ± 11
Chemotaxis in response to FMLP (10-8 M); values are percentages of chemotactic indices of PMNs in the presence of dideoxynucleosides divided by the chemotactic index of PMNs without the drugs and are means + standard errors of three experiments. a
HBSS at 371C. The change in absorption at 550 nm was measured on a spectrophotometer (Gilford 260) equipped with a thermostated cuvette holder (CIBA-Coming Diagnostics Corp., Oberlin, Ohio) (18). Superoxide produced by 106 PMNs in 5 min was then calculated by using the millimolar extinction coefficient for reduced cytochrome c. Statistics. The effects of dideoxynucleosides on PMN function were analyzed by a variant of the method of Tukey et al. (27), in which repeated-measures analysis of variance was used to account for the same donors being tested repeatedly at different concentrations. Linear trends in each functional parameter over increasing concentrations of a dideoxynucleoside were evaluated by this method. Since the distributions of the parameters were consistent with normality, the changes from the base line at individual concentrations were assessed by Student's t test. No adjustment for the multiplicity of tests was made because of the correlations between them. All the P values reported are two-tailed. RESULTS Toxicity. The viability of the PMNs remained >95% by trypan blue exclusion when they were incubated with AZT, ddC, or ddI for as long as 120 min at concentrations of 0. 1 to 25 ,uM, 0.01 to 1 ,uM, and 0.2 to 50 ,uM, respectively. Chemotaxis. None of the dideoxynucleosides tested had any significant effect on PMN chemotaxis in response to
AZT Co c to
C.) .0
ddl
ddC 30
30 r
30 r
25 I-
25p
C
Co
FMLP (Table 1). The chemotactic index of the untreated PMNs was 1.50 ± 0.08 (mean ± standard error of eight determinations), and this remained unchanged even at concentrations two to five times higher than those achieved when these agents are used clinically. Random migration of PMNs was also unaffected by the presence of drugs. Fungicidal activity of PMNs from healthy donors. Figure 1 shows the effects of the three compounds on the fungicidal activity of PMNs. The overall base-line intracellular killing of C. albicans after 1 hour incubation was 17.2 ± 0.47%. AZT did not significantly affect fungicidal activity in the donors tested (Fig. 1). In contrast, ddC and ddl, in six experiments performed with different donors, significantly enhanced the fungicidal activity of normal PMNs within and above the range of therapeutically achievable concentrations (P values of the linear trends, LM) Control 0.01 0.1 0.2 1.0 2.0 5.0 10.0 25.0 a
b
c
ddl
ddC
AZT
Drug concn
PIc
%
PI
%
PI
3.4
1.77 ± 0.10
1.82 ± 0.18
1.88 ± 0.10
1.77 ± 0.79 1.70 ± 0.09 1.78 ± 0.03
59 ± 4.0
66 ± 2.8
61 ± 3.8 61 ± 3.3 60 ± 2.9
63 ± 5.5
1.78 ± 0.07
69 ± 2.1
1.92 ± 0.17
65 ± 3.8
1.70 ± 0.08 61 ± 7.1
1.67 ± 0.06
64 ± 3.2
1.60
63 ± 7.4
1.80 ± 0.13
62 ± 5.9 57 ± 6.1
1.70 ± 0.03 1.96 ± 0.12
ob 64
+
0.09
Values are means ± standard errors of four experiments. Percentage of phagocytosing PMNs divided by the total number of PMNs counted. PI is the total number of phagocytosed C. albicans blastoconidia divided by the number of PMNs that phagocytosed them.
1676
ROILIDES ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
AZT
ddC
ddl
5r
5
LZz 4 I-
4
4
0~CL
3,
3
ONC~ 2
2 .
2.
0
1
1
*
.E
0 r ;
3
0 v-
-
-
1
E O
L. .1
0
-
__
1
10 100
a) 0
.01 .01
.1. 1 .1 1
1 10
0
0
.1
.1
10 1O
Concentration (MM) FIG. 2. Superoxide anion production by neutrophils (PMNs) pretreated with various concentrations of dideoxynucleosides. FMLP (5 x 1l-7 M) was used as a stimulus. The results are expressed as the means ± standard errors of three experiments.
appear to have adverse effects on T- and B-lymphocyte functions in vitro at concentrations of the drug that protect cells from HIV-1 (19), and in this study, we did not demonstrate that there are any adverse effects of AZT on PMN function. Similarly, neither ddC nor ddI had an inhibitory effect on PMN function. Rather, ddC significantly enhanced fungicidal activity, and ddI increased both bactericidal and fungicidal activities at therapeutically achievable concentrations. Although AZT has been shown to have antibacterial activity against some gram-negative organisms (e.g., E. coli, Shigella flexneri, and Salmonella typhimurium) (5, 11), S. aureus and C. albicans were not directly affected by any of the dideoxynucleosides we tested (also cited in reference 5). Thus, the enhancement of killing appears to be related either to an interaction between dideoxynucleosides and the PMNs or to an indirect interaction between dideoxynucleosides and the microorganisms, making them more susceptible to the PMN action. A direct growth inhibitory or microbicidal effect of either ddC or ddl, however, was excluded in the control experiments. The increased microbicidal activity observed with ddC and ddI does not appear to be a consequence of increased phagocytosis or increased oxidative metabolism. Nonoxidative killing (e.g., lysozyme and defensins) (8) might be responsible for the increased microbicidal activity of PMNs when they are exposed to those agents, although the current data do not address this. Also, intracellular killing of either bacteria or fungi by PMNs depends on the way the organism is presented and processed by the oxidative and nonoxidative metabolic products (e.g., phagosome-lysosome fusion or secretion of microbicidal products in response to the particular organisms). How the dideoxynucleosides affect each of these intracellular processes in response to either bacteria or fungi is not known. The basis for the differences we observed among these three dideoxynucleosides is also unclear. It is possible that they are related to differences in the intracellular uptake and phosphorylation of these dideoxynucleosides. Further studies assessing the in vivo effects of these agents on PMN function are in progress. ACKNOWLEDGMENTS We acknowledge the help of Frank Balis in obtaining the investigational drugs evaluated in this study. We thank the following people who contributed in providing blood specimens from patients: Robert Yarchoan, Rose Thomas, and Janie Eddy. We also thank Robert Danner for testing our reagents for endotoxin.
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