rMfiSfl mm “a a $5 ' U . This is to certify that the dissertation entitled Characterization of various effects of crisis form induction on Plasmodium falciparum in vitro presented by Joseph M. Carlin has been accepted towards fulfillment of the requirements for Ph. D. degreein Microbiology M jor professor Date fl 3/; /9?5 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 MSU LIBRARIES M v RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. CHARACTERIZATION OF VARIOUS EFFECTS OF CRISIS FORM INDUCTION ON PLASMODIUM FALCIPARUM IN VITRO BY Joseph M. Carlin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1985 ABSTRACT CHARACTERIZATION OF VARIOUS EFFECTS OF CRISIS FORM INDUCTION ON PLASMODIUM FALCIPARUM IN VITRO BY Joseph Michael Carlin The various effects of crisis form induction on Plas- modium falciparum ‘in ‘vitro has been examined. Sudanese parasite isolates were analyzed for chloroquine and meflo- quine sensitivity by two techniques differing in serum source. 4A 72%, test failure rate was noted when parasites were analyzed in the patient's serum as compared to a 28.8% rate ix: nonimmune serum. In subsequent experiments, sera containing crisis form factor (CFF) were used to supplement standard test plates containing parasites of Iknown drug sensitivities. These sera retarded parasite development in the presence or absence of drug. Inhibition of growth by drug and Sudanese serum combinations was additive. A variety of known or suspected inducers of crisis form parasites: Sudanese CFF sera, TB patient sera, rabbit tumor necrosis sera, and human y-interferon, were compared in vitro for cytotoxic effects on g. falciparum and mouse L-M cell cultures. Inhibition was determined by measurement of incorporation of radiolabeled nucleic acid precursors. When compared to normal serum, parasites cultivated in a: 1:4 dilution of rabbit tumor necrosis sera, TB patient sera, or CFF sera were metabolically inhibited 73%, 75% and 95%, respectively. Human y-interferon tun} no direct effect on parasite growth. However, cnflqr rabbit tumor necrosis sera were inhibitory to L-M cells, inhibiting incorporation by 80% at a 1:1000 serum dilution. These findings suggest that tumor necrosis factor is apparently not responsible for induction of parasite crisis forms by the inhibitory human sera tested. CFF was examined for stage- and time-dependent effects. The erythrocytic cycle was divided into 8 h intervals, and synchronized parasites of each interval were exposed to CFF for various time periods.‘ At cell harvest, hypoxanthine and phenylalanine incorporation and glucose consumption were compared to values obtained in normal serum. The most pro- found derangement of metabolism occurred in parasites 0—8 h post-invasion. Inhibition decreased in tests started with progressively older parasites.‘7“3 Cultivation in CFF serum for 8 h caused maximal inhibition of precursor incorpora- tion: longer exposure did not increase inhibition. However: the effect on glucose consumption varied inversely to the duration of exposure, decreasing as parasites matured, showing little reduction as they entered schizogony. This dissertation is dedicated to my wife, Dawn, for her constant encouragement, sup- port, understanding, and love: and to our daughter, Lindsey, for the incredible joy she has added to our lives. I love you both dearly. ii ACKNOWLEDGMENTS I would like to thank Dr. James B. Jensen for serving as my research advisor, for guidance throughout this study, and for concern for my professional growth. Special thanks are extended to John A. Vande Waa, Alan A. Divo and Michael T. Boland for their friendship, discussion and encouragement. I would also like to thank my parents, Joseph and Marian: and my sister, Susan, for all the love and support they have provided through the years. And finally, I would like to thank my wife, Dawn, for typing this manuscript. I could not have finished without her assistance. iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION Incidence and control of Malaria Roles of various components of immunity in malaria Crisis form induction Relationship of CFF to mononuclear cell products Purpose of dissertation List of references CHAPTER 1. AFRICAN SERUM INTERFERENCE IN THE DETERMINATION OF CHLOROQUINE SENSITIVITY IN PLASMODIUM FALCIPARUM Abstract Introduction Materials and methods Field study area Collection of parasitized blood and sera Drug sensitivity assays and parasite culture Serum histories Results Discussion Acknowledgments List of References iv vii ix wmww 10 17 18 19 21 21 21 22 24 25 33 37 38 Chapter 2. 'COMPARISON OF INDUCERS OF CRISIS FORMS IN PLASMODIUM FALCIPARUM IN VITRO Abstract Introduction Materials and methods Parasite and cell cultivation Collection of human serum Production of BCG-LPS sera Serum preparation Parasite inhibition assay L cell cytotoxicity assay Results Toxicity of various sera to P. falciparum Toxicity of various sera to L-M cells BCG-LPS serum ICSOs Effect of HuIFN-y on P. falciparum Discussion Acknowledgments List of references Chapter 3. STAGE- AND TIME-DEPENDENT EFFECTS OF CRISIS FORM FACTOR ON PLASMODIUM FALCIPARUM £5 VITRO Abstract Introduction Materials and methods Parasite cultivation Serum preparation Metabolic inhibition assays 41 42 43 45 45 46 46 47 47 49 51 51 56 58 58 6O 63 64 67 68 69 71 71 71 72 Results Stage-dependency Time-dependency Discussion Acknowledgments List of references SUMMARY vi 76 76 78 83 86 87 9O Table Table Table Table Table Table Table LIST OF TABLES CHAPTER 1 Chloroquine minimum inhibitory concentra- -tions (MIC) obtained in the 48-h drug sensitivity assay Chloroquine/mefloquine minimum inhibitory concentrations (MIC) obtained using the 24-h drug sensitivity assay CHAPTER 2 Percent inhibition of P. falciparum [3H]hypoxanthine incorporation by rabbit BCG-LPS serum Percent inhibition of P. falciparum [38]hypoxanthine incorporation by sera from humans with TB Percent inhibition of P. falciparum [3H]hypoxanthine incorporation by Sudanese sera with high CFF activity P. falciparum and L-M cell ICSOs of BCG-LPS sera CHAPTER 3 Inhibition of hypoxanthine incorporation in P. falciparum by CFF vii 26 27 52 53 55 59 79 CHAPTER 3 (Continued) Table 2 Inhibition of phenylalanine incorporation in P. falciparum by CFF 81 Table 3 Inhibition of glucose utilization in P. falciparum by CFF 82 viii Figure 1 Figure 2 Figure 3 LIST OF FIGURES CHAPTER 1 Results of the 24-h drug sensitivity tests with various sera, using parasite strains differing in clinical drug resistance. a. Counts per minute due to incorporation of [3H]hypoxanthine by FCN-l, a drug-sensitive parasite, at various chloroquine concentra- tions. b,c. Data obtained with strains FCR-BTC, an RI parasite, and FCR-l, an RIII parasite, respectively 29 The data are plotted as % inhibition in com- parison to the homologous serum control. a-c. Data obtained with strains FCN-l, FOR-3TC and FCR-l, respectively 31 Comparison of results obtained with the 48—h test and 24-h test conducted in the presence of pooled nonimmune serum (PNS). The 48-h test was quantified by measuring incorporation of [BH]hypoxanthine: the 24-h test results were determined by incorporation of the radiolabel and calculating the percentage inhibition of development to the schizont stage. a. Results of a comparison with FCN-l. b. Results obtained with FCR-3TC 32 ix Figure 1 Figure 1 Figure 2 CHAPTER 2 Effects of sera from a variety of sources on [3H]thymidine incorporation by L-M cells during a 24-h period. Results are expressed as percent inhibition : standard error of the mean. Symbols: BCG-LPS sera,.: TB sera, ‘ : Sudanese sera, . 57 CHAPTER 3 Periods of exposure of P. falciparum to CFF at various times post-invasion 73 Effect of CFF on various stages of g. falcip- arum. a. Effect of 8 h exposure_to CFF serum on three metabolic processes in parasites har- vested at various times. Results are expressed as percent inhibition compared to control cul- tures. Symbols: hypoxanthine incorporation, I: phenylalalanine incorporatiom‘: glucose consumption, . . b. Differential distribution of various stages expressed as percentage of total parasites present at cell harvest. Symbols: ring stages, .: trophozoite stages, . : schizont stages, ‘ 77 INTRODUCTION Incidence and control of malaria Malaria remains the most lethal of the major parasitic diseases. The World Health Organization estimates a world— wide incidence of over 150 million cases per year.46 In Africa alone, annual mortality in children is greater than one million.42 Although eradication programs had reduced malaria incidence, the world is presently experiencing an alarming resurgence in this often fatal disease, due in part to the spread of drug-resistant strains of Plasmodium fal- ciparum and insecticide-resistant mosquito vectors. Para- site drug resistance in humans has been demonstrated for the anti-folates, the quinolines and quinine.52 In relevant mosquito vectors, insecticide resistance has been documented for the organochlorines, organophosphates, carbamates and pyrethroids.23 Clearly, the need exists for alternative control measures, including the development of malaria vac- cines. Research is currently in progress on the creation of effective vaccines against potentially susceptible stages in the parasite life cycle, including sporozoite, merozoite and gametocyte stages.19'21’42’45’48 It has been demonstrated that monoclonal antibody to the circumsporozoite protein in malaria sporozoites is able to specifically block attachment and .entry of sporozoites into cultured human hepatoma cells.29 Furthermore, poly- clonal antibodies raised in rats against sporozoites of P. falciparum exhibit strain cross-reactivity.49 Limited suc- cess has also been achieved in blocking merozoite invasion into erythrocytes with antibodies produced in response to 19,20,21,65 merozoite preparations. Despite the progress that has been made in these areas, serious problems remain to be solved, including the completeness of protection pro- vided by sporozoite immunization (successful invasion of one 42,45 sporozoite can lead to fatal disease ), and the effect 5,6 of antigenic variation and diversity among strains and 39,43,65 geographic isolates on merozoite-induced immunity. Roles of various components of immunity in malaria Although much time and money has been invested in anti- malarial vaccines, questions remain as to the relationship of vaccine-induced immunity to acquired immunity in man, a process generally accepted as being multifactorial, includ- ing components of both antibody-based and cell-mediated immunity.54 It is clear that antibody plays several roles in effective immunity to malaria. Protection has been pas— sively transferred in) West African children by inoculation of purified IgG from immune adults.22 Specific immune serum can agglutinate merozoite stages, inhibiting dispersal from mature schizonts and thus interfering with subsequent rein— vasion.27 Purified IgG specific to a 155,000 Mr ring-stage- infected erythrocyte surface antigen strongly inhibits rein- vasion in yi£52.66 Immune serum also enhances phagocytosis of parasite-infected erythrocytes by neutrophils,4’ll’12 monocytes,lo'11'30"37'6l’64 and eosinophils:61 and antibody- dependent cellular cytotoxicity has been demonstrated in Plasmodium falciparum in vitro.3 Several lines of evidence suggest the importance of 54 First, many manifes— T—cell mediated immunity to malaria. tations of T-cell activation correlate with protective immu- nity, including delayed-type hypersensitivity to parasitized erythrocytes, T-cell production of lymphotoxin and macro- phage activation.31 Second, transfer of immunity has been accomplished with T lymphocytes from animals immune to 26,32,53 malaria. And, third: although T-cell or B—cell deprived mice cannot control normally non-lethal E. yoelii 31'56 those B-cell deficient mice drug-rescued infections, from infection resisted reinfection with the same para- site.56 This indicates that some function of T-cells other than that of helper cells in antibody formation is important in malaria immunityl, and that this function may interact nonspecifically with the malaria parasite, possibly leading to intraerythrocytic deterioration (crisis forms) and para- site death. Crisis form induction Crisis form malaria parasites were originally observed in P. brasilianum infection of Cebus capucinus, Ateles geof— . . . 58 . . froyi, and A. dar1ens1s monkeys. These crisis forms were characterized by reduction in average merozoite number per segmenter, retardation of development to maturity,anuiintra- erythrocytic deterioration. Similar observations have been 4 described for g. knowlesi infections of immunized Macaca mulatta,8 g. berghei infections of albino rats,2 g. vinckei and P. chabaudi infections of mice previously innoculated with killed Propionibacterium acnes (formerly' Corynebac— terium parvum)15 and P. vinckei and P. berghei Loelii infections of Mycobacterium bovis strain Bacille Calmette- Guérin (BCG) stimulated mice.l4 Although these deteriorat- ing parasites have been observed in various animal models, they had not been described in human infections due to the sequestration and adherence of maturing stages of E. falcip- §£u_m_ to venous capillary endothelium, and the paucity of 40,44 these stages in peripheral blood. It was not until after the 311 vitro technique for cultivation of falciparum malaria was developed35’62 that crisis forms of human para- sites were seen. The existence of crisis form factor (CFF), a non-antibody serum factor able to induce crisis form pro- duction in cultivated P, falciparum, has been demonstrated in the blood of Sudanese adults functionally immune to malaria,34 and has been associated with histories of clini- cal immunity.33 In general, parasite retardation and growth inhibition were greatest when parasites were cultivated with sera from individuals both from endemic regions and with no clinical histories of malaria. Moreover, greater parasite inhibition was observed in sera from holoendemic versus hyperendemic regions with no inhibition noted in sera from hypoendemic areas. Furthermore, in an extensive survey of individuals in a: hyperendemic region experiencing seasonal transmission of malaria, significant, nearly threefold, increases in CFF activity were found in wet-season sera as 63 compared to dry-season sera. Crisis form activity has also been observed in sera from patients diagnosed as suf— 7’60 Parasites fering from cerebral malaria and meningitis. grown in these sera and examined ultrastructurally exhibited loss of internal organelles and parasite membranes, and cytoplasmic vacuolation, all clear signs of parasite stress. Relationship of CFF to mononuclear cell products The focus of much research into the induction of crisis parasite formation has been on products of mononuclear cells. Rodents vaccinated with agents known to activate cells of the reticuloendothelial system, BCG and killed 3. acnes, have enhanced resistance against malaria and other hemopro- 14,15,18,47 tozoan diseases. Parasites observed inside the erythrocytes of these stimulated animals appear degenerate, much like the crisis forms described in the Plasmodium-Cebus mode1.58 Similar studies in athymic nude mice have shown that prior injection of P. acnes, but not BCG, can protect mice from Plasmodium infections,25 indicating that the effector phase of nonspecific immunity activated by g. acnes bypasses a: T-cell requirement. Several in vitro parasite cultivation studies have been performed with sera obtained from animals previously vaccinated with ECG or P. acnes and subsequently inoculated Vfilfll endotoxin.28’59’67 Not only were these stimulated animal sera rich in macrophage secre- tory products, including tumor necrosis factor (TNF)9 inter- leukin 1 (IL U41 and type I interferon,57 but they were 6 also quite toxic to P. falciparum and other Plasmodium spp., producing typical crisis forms in vitro. Further studies have shown that mice infected with Plasmodium spp. and then inoculated with endotoxin also have detectable TNF, IL 1, 17,59 Type I interferon and antiparasitic activity, demon- strating that Plasmodium infection is able to substitute for vaccination with BCG (n: P. acnes in this immunostimulatory treatment. Another approach to crisis form induction research involves examining the production of reactive oxygen species by monocytic cells undergoing respiratory burst and the effect of these oxygen radicals on malaria parasites. In one report,51 g. yoelii were cultivated adjacent to lympho- kine-activated macrophages, separated by a.CL45 um filter. When phorbol myristate acetate (PMA) or g. yoelii antigen were added to the macrophage cultures, H202 was elaborated, destroying the parasite cultures. When catalase was added to the cultured macrophages prior to H release, the anti- 202 parasitic activity was abrogated, indicating the toxic effect on malaria cultures was due to [-1202 production. Another study demonstrated that human y-interferon-activated macrophages induced crisis form production when co-cultured with P. falciparum.50 Parasite killing correlated with the magnitude of the oxidative response of the macrophages. The effects of reactive oxygen species on malaria para- sites were also examined in systems where the free radicals were generated with enzyme-substrate reactions. Parasites cultivated with xanthine-xanthine oxidase or glucose-glucose oxidase combinations were killed by the resultant production of H202.24'50’67 This effect was inhibited with catalase but not other reactive oxygen scavengers. In: addition, inoculation of alloxan in g. vinckei infected mice caused hemolysis and parasite death in 1119, most likely due to the production of hydroxyl radicals.16 The relationship of CFF to reactive oxygen species appears remote. First, no leukocytes are present to gener- ate oxidative intermediates le the cultivation system used to detect CFF activity.35 Second, all serum tested is pre- viously dialysed, and any free radicals present in the serum wouLd be expected to be removed by this procedure. Third, in experiments to determine if CFF itself could generate reactive oxygen, a variety of oxygen scavengers, anti-oxi- dants and reducing agents, including catalase, superoxide dismutase, a-tocopherol and reduced glutathione have been included in assays for CFF, with no effect on inhibition (T. G. Geary, M. T. Boland, and J. B. Jensen, submitted for publication). CFF may however, be related to various lymphokines and monokines elaborated by the immune system. Non-oxidative mechanisms of immune protection have been described for other parasites.13’38 In addition, it has been demonstrated that lymphokine-activated macrophages isolated from a patient with chronic granulomatous disease, in which oxi- dative metabolism is impaired, were able to inhibit g. fal- ciparum growth in a reactive oxygen-independent manner.50 Furthermore, crisis forms have been observed when parasites have been cultivated with PMA-stimulated neutrophils.36 The addition of various oxygen scavengers to the cultures was ineffective in blocking the induction of crisis forms, indicating the possibility of non—oxidative inhibitory activity functioning in this system. It has been suggested that CFF may be TNF.55 TNF has tumoricidal activity, and is produced in large quantities in animals vaccinated with BCG with subsequent lipopolysaccharide (LPS) injection.9 Sev- eral reports have indicated that BCG-LPS serum i1: quite toxic to malaria parasites, inducing crisis forms lg vitro.28’59’67 It has not however, been clearly demon- strated that TNF is responsible for crisis form induction in these sera. Since the immuno-stimulatory treatment of the animals releases many macrophage secretory products into circulation, the possibility exists that both CFF and TNF are present in BCG-LPS sera. Despite the report that anti- body raised to partially-purified TNF blocks the anti-para- sitic activity of BCG-LPS serum,28 CFF and TNF may be anti- genically related molecules, and antisera generated against one may inhibit both activities. Indeed, one study has demonstrated the cross-reactivity of antibody generated against guinea pig lymphotoxin to guinea pig TNF and macro- phage cytotoxic factor.68 Thus the relationship of CFF to other lymphokines and monokines remains unclear. Purpose of dissertation The purpose of this dissertation is to further charac- terize the effects of CFF on parasite cultures and to begin to clarify the relationship of CFF to other inducers of malaria parasite crisis forms. Chapter 1 is concerned with the effect of CFF on $2 vitro field tests of drug resistance in isolates of P. falciparum. In addition, parasites culti— vated with both CFF and the antimalarial drugs chloroquine and mefloquine are examined for serum-drug interactions. In Chapter 2, known or suspected inducers of crisis form para- sites in cultivated P. falciparum are compared. Sera from Sudanese residents of malaria endemic areas, sera from American tuburculosis patients, human y-interferon and rab- bit sera containing tumor necrosis factor are assayed _i_r_1_ vitro for cytotoxic activities against falciparum malaria and mouse L-M cell cultures. And finally, Chapter 3 con- tains eyi analysis of stage— and time-dependent effects of CFF on malaria parasite metabolism. Highly“ synchronous cultures were exposed to CFF for increasing lengths of time. At cell harvest, hypoxanthine and phenylalanine incorpora- tion into nucleic acids and protein, respectively, and glu- cose consumption by parasites cultivated in CFF serum are compared to values obtained in normal human serum. 10 LIST OF REFERENCES Allison, A. C., and Eugui, E. M., 1983. The role of cell-mediated immune responses in resistance to 'malaria, with special reference to oxidant stress. Ann. Rev. Immunol., 1:361-392. Barnwell, J. W., and Desowitz, R. S., 1977. Studies on parasitic crisis in malaria: I. Signs of impending crisis in Plasmodium berghei infections of the white rat. Ann. Trop. Med. Parasitol., 21:429- 433. Brown, J., and Smalley, M. E., 1980. Specific anti- body-dependent cellular cytotoxicity in human malaria. Clin. Exp. Immunol., 41:423-429. Brown, J., and Smalley, M. E., 1981. Inhibition of the in vitro growth of Plasmodium falciparum by human polymorphonuclear neutrophil leukocytes. Clin. Exp. Immunol., 4§:106-109. Brown, K. N., 1977. Antigenic variation in malaria. In Immunity to Blood Parasites of Animals and Man. Miller, L. H., Pino, J. A., and McKelvey, J. J., (Eds.) Plenum Press, New York. pp. 5-25. Brown, K. N., and Hills, L. A., 1974. Antigenic varia- tion and immunity to Plasmodium knowlesi: anti- bodies which induce antigenic variation and anti- bodies which destroy parasites. Trans. R. Soc. Trop. Med. Hyg., §§:lB9-l42. Butcher, G. A., Maxwell, L., Cowen, M., Clancy, R. L., and Stace, J. D., 1985. The development and ultra- structure of Plasmodium falciparum damaged i5 vitro by human "crisis” sera and by chloroquine. Aust. J. Exp. Biol. Med. Sci., 63:9-18. Butcher, (I. A., Mitchell, G. H., and Cohen, S., 1978. Antibody mediated mechanisms of immunity to malaria induced by vaccination with Plasmodium knowlesi merozoites. Immunology, 34:77-86. 10. 11. 12. 13. 14. 15. 16. 17. 18. 11 Carswell, E. A., 01d L. J., Kassel, R. L., Green, S., Fiore, N., and Williamson, B., 1975. An endotoxin- induced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. U.S.A., 12:3666- 3670. Celada, A., Cruchaud, A., and Perrin, 1;. H., 1982. Opsonic activity of human immune serum on $2 vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes. Clin. Exp. Immunol., 42:635-644. - Celada, A., Cruchaud, A., and Perrin, 13. H., 1983. Assessment of immune phagocytosis of Plasmodium falciparum infected red blood cells by human mono- cytes and polymorphonuclear leukocytes. A method for visualizing infected red blood cells ingested by phagocytes. J. Immunol. Meth., p3:263-271. Celada, A. Cruchaud, A., and Perrin, In. H., 1983. Phagocytosis of Plasmodium falciparum-parasitized erythrocytes by human polymorphonuclear leuko— cytes. J. Parasitol., g2:49-53. Chinchilla, M., and Krenkel, J. K., 1984. Specific mediation of cellular immunity to Toxoplasma gondii in somatic cells of mice. Infect. Immun., 4§:862-866. Clark, I. A., Allison, A. C., and Cox, F. E., 1976. Protection of mice against Babesia and Plasmodium with BCG. Nature (London), 259:309-311. Clark, I. A., Cox, F. E. G., and Allison, A. C., 1977. Protection of mice against Babesia spp. and Plasmodium spp. with killed Corynebacterium parvum. Parasitology, 13:9-18. Clark, L A., and Hunt, N. H., 1983. Evidence for reactive oxygen intermediates causing hemolysis and parasite death in malaria. Infect. Immun., Clark, I. A., Virelizier, J. L., Carswell, E. A., and Wood, P. R., 1981. Possible importance of macro- phage-derived mediators in acute malaria. Infect. Immun., 32:1058-1066. Clark, I. A., Wills, E. J., Richmond, J. E., land Allison, A” (2., 1977. Suppression of babesiosis in BCG-infected mice and its correlation with tumor inhibition. Infect. Immun., 11:430-438. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 12 Cohen, S., 1982. Progress in malaria vaccine develop- ment. Brit. Med. Bull., 38:161-165. Cohen, 8., Butcher, G. A., and Crandall, R. B., 1969. Action of malarial antibody i2 vitro. Nature (London), 223:368-371. Cohen, 5., Butcher, G. A., and Mitchell, G. H., 1977. Immunization against erythrocytic forms of malaria parasites. In Immunity' to Blood Parasites of Animals and Man. Miller, L. H., Pino, J. A., and McKelvey, J} .J., (Eds.) Plenum Press, New York. pp. 89-112. Cohen, 8., McGregor, I. A., and Carrington S., 1961. Gamma-globulin and acquired immunity to human malaria. Nature (London), 192:733-737. Davidson, G., 1982. Developments in malaria vector control. Brit. Med. Bull., 38:201-206. Dockrell, H. M., and Playfair, J. H. L., 1984. Killing of Plasmodium yoelii by enzyme-induced products of the oxidative burst. Infect. Immun., 42:451-456. Eugui, E. M., and Allison, A. C., 1982. Natural cell- mediated immunity and interferon in malaria and babesia infections. In NK Cells and Other Natural Effector Cells. Herberman, R., (Ed.) Academic Press, New York. pp. 1491-1502. Gravely, 8., and Kreier, J. P., 1976. Adoptive trans- fer of immunity to Plasmodium berghei with immune T and B lymphocytes. Infect. Immun., lfl:lB4-190. Green, T. J., Morhardt, M., Brackett, R. G., and Jacobs, R. L., 1981. Serum inhibition of merozoite dispersal from Plasmodium falciparum schizonts: indicator of immune status. Infect. Immun., 31:1203-1208. Haidaris, C. G., Haynes, J. D., Meltzer, M. S., and Allison, A. C., 1983. Serum containing tumor necrosis factor is cytotoxic for the human malaria parasite Plasmodium falciparum. Infect. Immun., 42:385-393. Hollingdale, M. R., Nardin, E. H., Tharavanij, S., Swartz, A. L., and Nussenzweig, R. S., 1984. Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells: an vitro assay of protective antibodies. Immunol., 132:909-913. 1.9. l; 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 13 Hunrer, K. W., Winkelstein, J. A., and Simpson, T. W., 1979. Serum opsonic activity in rodent malaria: functional and immunochemical characteristics. _i_r_1_ vitro. J. Immunol., 123:2582-2587. Jayawardena, A. N., 1981. Immune responses in malaria. In Parasitic Diseases, The Immunology. Mansfield, J. M., (Ed.) Marcel Dekker, Inc., New ‘York. pp. 85-136. Jayawardena, A. N., Target, G. A. T., Leuchars, E., and Davies, A. J. S., 1978. The immunological response of CBA mice to P. yoelii. II. The passive trans- fer of immunity with serum and cells. Immunology, 33:157-165. Jensen, J. B., Boland, M. T., Allan, J. S., Carlin, J. M., Vande Waa, J. A., Divo, A. A., and Akood, M. A. S., 1983. Association between human serum- induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan. Infect. Immun., 41:1302-1311. Jensen, J. B., Boland, M. T., and Akood, M., 1982. Induction of crisis forms in cultured Plasmodium falciparum with immune serum from Sudan. Science, 2I6:1230-1233. Jensen, J. B., and Trager, W., 1977. ' Plasmodium falciparum in culture: use of outdated eryth- rocytes and description of the candle jar method. J. Parasitol., 93:883-886. Kharazmi, A., and Jepsen, S., 1984. Enhanced inhibi- tion of in vitro multiplication of Plasmodium falciparum by stimulated human polymorphonuclear leucocytes. Clin. Exp. Immunol., 51:287-292. Khusmith, S., Druilhe, P., and Gentilini, M., 1982. Enhanced Plasmodium falciparum merozoites phago- cytosis by monocytes from immune individuals. Infect. Immun., 35:874-879. Kierszenbaum, E., Zenian, A., and Wirth, J. J., 1984. Macrophage activation by cord .factor (trehalose 6,6'-dimycolate): enhanced association with and intracellular killing of Trypanosoma cruzi. Infect. Immun., 43:531-535. Knowles, G., Davidson, W. L., McBride, QL. S., and Jolley, 13., 1984. Antigenic diversity found in isolates of Plasmodium falciparum from Papua New Guinea by using monoclonal antibodies. Am. (J. Trop. Med. Hyg., §§:204-211. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 14 Luse, S. A., and Miller, L. H., 1971. Plasmodium falciparum malaria. Ultrastructure of parasitized erythrocytes :hi cardiac vessels. Am. .J. Trop. Mannel, 11. N., Farrar, J. J., and Mergenhagen, S. E., 1980. Separation of a: serum-derived tumoricidal factor from a helper factor for plaque-forming cells. J. Immunol., 134:1106-1110. Maugh, T. H., 1977. Malaria: resurgence in research brightens prospects. Science, 196:413-416. McBride, J. S., Walliker, D., and Morgan, G., 1982. Antigenic diversity in the human malaria parasite Plasmodium falciparum. Science, 217:254-257. Miller, L. H., 1969. Distribution of mature tropho- zoites and schizonts of Plasmodium falciparum in the organs of Aotus trivirgatus, the night monkey. Am. J. Trop. Med. Hyg., 18:860-865. Miller, L. H., 1977. A critique of merozoite and spor- ozoite vaccines in malaria. In Immunity to Blood .Parasites of Animals and Man. Miller, L. H., Pino, (I. A., and McKelvey, J. J., (Eds.) Plenum Press, New York. pp. 113-120. Noguer, A., Wernsdorfer, W., Kouznetsov, R., and Hemple, J., 1978. The malaria situation in 1976. Won-O. Chronol 2:9-170 Nussenzweig, N. S., 1967. Increased nonspecific resis- tance to malaria produced by administration of killed Corynebacterium parvum. Exp. Parasitol., 21:224-231. Nussenzweig, IL. 8., 1977. Immunoprophylaxis of mal- aria: sporozoite induced immunity. In Immunity to Blood Parasites of Animals and Man. Miller, L. H., Pino, J. A., and McKelvey, J. J., (Eds.) Plenum Press, New York. pp. 75-87. Nussenzwehg, R. S., and Chen, D., 1974. The antibody response in) sporozoites of simian and human mal- aria parasites: its stage and species specificity and strain cross-reactivity. Bull. 'W.H.O., 29:293-297. Ockenhouse, C. F., Schulman, S. and Shear, H. L., 1984. Induction of crisis forms in the human malaria parasite Plasmodium falciparum by y-interferon- activated, monocyte derived macrophages. J; Immunol., 133:1601-1608. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 15 Ockenhouse, (L. F., and Shear, H. L., 1984. Oxidative killing of the intraerythrocytic malaria parasite Plasmodium ypelii by activated macrophages. J_._ Immunol., 132:424-431. Peters, W., 1982. Antimalarial drug resistance: an increasing problem. Brit. Med. Bull., 38:187-192. Phillips, R. S., 1970. Plasmodium berghei: passive transfer of immunity by antisera and cells. Exp. Parasitol., 21:479-495. Playfair, J. H. L., 1982. Immunity to malaria. Brit. Med. Bull., 38:153-159. Playfair , J. H. L . , Taverne, J . , and Matthews, N. , 1984. What is tumor necrosis factor really for? Immunol. Today, §:165-166. Roberts, D. W., and Weidanz, W. P., 1979. T-cell immunity to malaria in the B-cell deficient mouse. Am. J. Trop. Med. Hyg., 28:1-3. Salvin, S. B., Younger, J. S., and Lederer, W. H., 1973. Migration inhibitory factor and interferon in the circulation of mice with delayed hyper- sensitivity. Infect. Immun., 1:68-75. Taliaferro, W. H., and Taliaferro, L. G., 1944. The effect of immunity on the asexual reproduction of Plasmodium brasilianum. J. Infect. Dis., 15:1-32. Taverne, .J., Dockrell, H. M., and Playfair, J. H. L., 1981. Endotoxin-induced serum factor kills mal- aria parasites in vitro. Infect. Immun., 33:83-89. Tharavanij, S., Warrell, M. J., Tantivanich, S., Tapchaisri, P., Chongsa-Nguan, M., Prasertsiriroj, V., and Patarapotikul, J., 1984. Factors contrib- uting to the development of cerebral malaria: I. Humoral immune responses. Am. J. Trop, Med. Hyg., 33:1-11. Tosta, C. E., and Wedderburn, N., 1980. Immune phago- cytosis of Plasmodium yoelii-infected erythrocytes by macrophages and eosinophils. Clin. Exp. Immunol., 42:114-120. Trager, W., and Jensen, J. B., 1976. Human malaria parasites in continuous culture. Science, 193:673—675. 63. 64. 65. 66. 67. 68. 16 Vande Waa, J. A., Jensen, J. B., Akood, M. A. S., and Bayoumi, R., 1984. Longitudinal study on the in vitro immune response to Plasmodium falciparum in Sudan. Infect. Immun., 22:505-510. Vernes, A., 1980. Phagocytosis of g. falciparum para- sitised erythrocytes by peripheral monocytes. Lancet, 33:1297-1298. Vernes, A., Haynes, J. D., Tapchaisri, P., Williams, J. L., Dutoit, E., and Diggs, C. L., 1984. Plasmo- dium falciparum strain-specific human antibody inhibits merozoite invasion of erythrocytes. Am; J. Trop. Med. Hyg., 33:197-203. Wahlin, B., Wahlgren, M., Perlmann, H., Berzins, K., Bj6rkman, A., Patarroyo, M. E., and Perlmann, P., 1984. Human antibodies to a M 155,000 Plasmodium falciparium antigen efficientfy inhibit merozoite reinvasion. Proc. Natl. Acad. Sci. U.S.A., 33:7912-7916. Wozencraft, A. 0., Dockrell, H. M., Taverne, J., Targett, (L. A. T., and Playfair, J. H. L., 1984. Killing of human malaria parasites by macrophage secretory products. Infect. Immun., 43:664-669. Zacharchuk, C. M., Drysdale, B., Mayer, M. M., and Shin, H. S., 1983. Macrophage-mediated cyto- toxicity: role of a soluble macrophage cytotoxic factor similar to lymphotoxin and tumor necrosis factor. Proc. Natl. Acad. Sci. U.S.A., 39:6341-6345. CHAPTER ONE AFRICAN SERUM INTERFERENCE IN THE DETERMINATION OF CHLOROQUINE SENSITIVITY IN PLASMODIUM FALCIPARUM Joseph M. Carlin and James B. Jensen Reprinted from Zeitschrift ffir Parasitenkunde 70: 589-597, 1984. 17 18 ABSTRACT Isolates of Plasmodium falciparum from villagers in central Sudan were tested for chloroquine and mefloquine sensitivity using the W.H.0. microtechnique test procedure and a modi- fied 48-h 33 vitro test for drug resistance. No drug-resis- tant strains were noted. In the W.H.0. procedure, in which parasites were cultivated in the presence of the patient's plasma, 72% of the isolates failed to mature to the schizont stage, tun: when infected erythrocytes were washed free of the patient's plasma and cultivated in pooled nonimmune serum only 28.8% of the isolates failed to develop to the schizont stage. In subsequent experiments, sera from g. falciparum-infected patients or from noninfected "immune" adults were used to supplement standard ip'yipgp test plates which contained parasites of known chloroquine sensitivi- ties. Sera from malaria-infected patients, or from immune adults, retarded parasite development in the presence or absence of drug. The effect of these humoral factors and the antimalarial drugs was additive. The replacement of the patient's plasma with nonimmune serum in drug sensitivity tests performed with African isolates is recommended. 19 INTRODUCTION The spread of chloroquine-resistant Plasmodium falcip- arum presents a potentially severe problem to the nations of Africa. An increasing number of studies have described chloroquine-resistant malaria acquired :hi East Africa. by . . . . 1,2,4,15 nonimmune European and North American ViSitors, although chloroquine resistance in indigenous Africans has 16,19,20,22 not been well documented. To monitor the spread of chloroquine-resistance in Africa, both the Rieckmann/ 24,26 for World Health Organization (W.H.O.) microtechnique ip_ ylggp. determination of chloroquine sensitivity (24-h test) and the 48-h test described by Nguyen-Dinh and Trager18 have been developed as field tests requiring minimal labora- tory equipment and which can be performed by most individ- uals after modest training. These standard ifl.!£££2 assays differ primarily in the source of serum/plasma for culture, the former utilizing the patient's plasma whereas the latter employing sera pooled from nonimmune individuals. Since serum from Africa has been previously shown to retard para- site development lp ylpgp,ll it is possible that this inhi- bition could interfere with assays for drug sensitivity in Africans tested by the 24-h microtechnique. Reports are available suggesting that a high percentage of such tests are ineffective due to failure of controls to grow20 (Walter Wernsdorfer, personal communication). Thus, in an attempt to characterize the effects of African serum on interpreta- tion of drug sensitivity, parasite isolates from central 20 Sudan were assayed for chloroquine sensitivity by both pro- cedures. In addition, the effects of African serum on chlo- roquine sensitivity of well characterized laboratory strains were examined. 21 MATERI ALS AND METHODS Field study area Field studies were conducted in the Blue Nile Province, Sudan, a region hyperendemic for P. falciparum malaria, with peak transmission during the months of October and November, following annual rains. Villages in the area rely on local health clinics for malaria treatment where microscopic con- firmation of infection is unavailable. Thus, nearly all febrile episodes are treated with chloroquine and. those which fail to respond are considered drug resistant. Chlo- roquine is essentially the only antimalarial drug used in this area of Sudan. Sudanese serum, frozen to -70°C, was transported on blue-ice gel to our laboratory in Michigan13 to be tested with cultivated parasite strains of known chloroquine sensi- tivities. Sera from individuals living in Bahr E1 Ghazal Province ix: Southern Sudan where P. falciparum malaria is holoendemic and treatment with antimalarial drugs is limited were also examined. Collection of parasitized blood and sera Febrile patients seen in local clinics were screened for malaria parasites with the aid of Giemsa-stained thick blood films and individuals with P. falciparum infections who denied a recent history of chloroquine treatment were asked to donate a blood sample for i_Q vitro chloroquine sensitivity assay. Blood, collected in siliconized vac- utainers with and without citrate-phosphate-dextrose-adenine 22 anti-coagulant (CPD-A)3 was kept on wet ice 2 to 4 h during transport to the laboratory before being processed. Samples drawn with CPD-A were divided into two parts. One remained as whole blood, while the other was washed free of plasma with RPMI 1640 medium. Samples drawn without anti-coagulant were allowed to clot at 40C overnight before centrifugation and separation of the serum from the cellular elements. Drug sensitivity assays and parasite culture Parasite isolates were tested for drug sensitivity by two techniques. The first, the 24-h test,26 was used according to the W.H.O. protocol. Whole blood containing CPD-A was diluted 1:9 with RPMI 1640 and dispensed into 96-well microtiter plates prepared by W.H.O. with either chloroquine or mefloquine (W.H.O. plates). Results were obtained 24 h later by determining in Giemsa-stained thin blood films the number of parasites that matured to the schizont stage /200 asexual parasites. In the second assay, a modification of the 48-h test of Nguyen-Dinh and Trager,18 infected erythrocytes were washed free of plasma with RPMI 1640 and suspended in fresh medium containing pooled AB+ serum obtained in Michigan from American Red Cross. Parasitized erythrocytes were dispensed, 3 ul/well, into 96-well microtiter plates to which 200 ul of RPMI 1640 containing various concentrations of chloroquine was added, then cultivated in a candle jar for 48 h at 37°C. Sensitiv- ity was determined by comparing the parasitemias in drug- containing wells with untreated control wells by examination 23 of stained thin films. Some assays were conducted by meas- uring incorporation of [3H]hypoxanthine into parasite nucleic acid using scintillation spectrometry.5'7’12 In these experiments, 1-2 uCi of [3H]hypoxanthine were added to each well during the final 24 h of cultivation. The 96-well plates were subsequently harvested onto glass-fiber filter strips using a Bellco Microharvester cell harvester, and label incorporation was determined with a Beckman LS-7500 scintillation spectrometer. Each test well was compared to the control well and percent inhibition was calculated for each drug concentration using the formula: % inhibition = CPM of control well - CPM of test well x 100 CPM of control well In some experiments, sera obtained from infected‘ patients and from healthy Sudanese adults, both nonimmune and semi-immune, were examined for possible serum factor- drug interactions in cultivated laboratory strains of P. falciparum with well documented in vivo and in vitro sensi- tivities to chloroquine. .All of these serum samples were heat-inactivated, dialysed 1:106 against RPMI 1640, and sterilized by filtration as previously described.12 Parasite strains, grown in type 0+ erythrocytes, were synchronized tx: the ring stage using a modification of the sorbitol method of Lambros and Vanderberg17 described previ- ously.12 The synchronized ring stages, used in serum-drug interaction experiments, were mixed with undialyzed pooled . + . . nonimmune A serum (PNS) and dialyzed Sudanese serum to give final concentrations of 5% PNS and 5% Sudanese serum in RPMI 24 1640 at 5% hematocrit. PNS was included to compensate for possible nutritional deficiencies in the Sudanese sera. This concentration of PNS has been shown previously6 to be the minimum serum requirement for optimal growth, and serum concentrations of up to 10% do not further increase the growth of parasites. This cell suspension was then added to W.H.O. plates. Parasite strains employed :hi serum-drug interaction experiments included FCN-l, a chloroquine sensitive iso- late,18 FCR-3TC7'10 7’9 and FCR-l which exhibit RI and RIII chloroquine resistance,25 respectively. These strains have been maintained in continuous culture by the candle jar technique.8 Serum histories Sera W-1260, W-1262, and W-1278 were obtained from 50-, 11- and 30-year-old males respectively in the village of Besselia, located in Bahr El Ghazal Province. No recent malaria histories were available for these individuals. Sera S-lSS and S-157 were from 10- and 14-year-old females residing in Umm Shoka, Blue Nile Province. Both young women had patent P. falciparum infections when their sera were obtained. 25 RESULTS Sixty-six parasite isolates were tested in Sudan for chloroquine sensitivity using the modified 48-h test in which the patient's plasma was replaced in the cultures with PNS (Table 1). It is evident from Table 1 that the minimum inhibitory concentration (MIC), i.e. the drug concentration at which the parasitemia is 550% of that obtained in wells containing no drugs, was at most 5.62 x 10.2 ug. More than half of the isolates were sensitive to chloroquine at even lower concentrations. Since no parasites developed in 10-1 uM chloroquine, all isolates were considered tn: be chloroquine sensitive. Nineteen parasite isolates (28.8%) failed to grow in any wells, including those without chloroquine. Using the 24-h microtechnique, 25 of the above isolates were tested for chloroquine and mefloquine resistance in W.H.O. plates (Table 2). As shown in Table 2, all parasite isolates were chloroquine and mefloquine sensitive. In contrast to the results of the 48-h test, however, 72% of the cultivated parasites failed to grow in any of the wells. Even in the drug-free wells, parasites were retarded in development, and appeared shrunken, pyknotic, and karyorrhexic. All parasite isolates that developed to schizonts in control wells of the 24-h test underwent schizogony, produc- ing new ring stages in control wells of the 48-h test. How- ever, 61% of the isolates that grew in the 48-h test failed to develop in the 24-h test wells. For example, parasites 26 TABLE 1 Chloroquine minimum inhibitory concentrations (MIC)a obtained in the 48-h drug sensitivity assay. MIC Isolates % of (uM) Number Total No. 1.00 x 10'1 0 0.0 5.62 x 10‘2 29 43.9 3.16 x 10"2 16 24.2 1.00 x 10‘2 2 3.0 Controlb 19 28.8 aConcentration of the drug at which parasitemia i 50% of that in drug-free control wells. bParasites failed to grow in control wells (Tables 1, 2). 27 TABLE 2 Chloroquine/mefloquine minimum inhibitory concentrations (MIC)a obtained using the 24-h drug sensitivity assay. Chloroquine Mefloquine MIC Isolates MIC Isolates (uM) Number % of (uM) Number % of Total no. Total no. 1.14 x 10'1 0 0.0 8.00 x 10‘2 0 0.0 8.00 x 10‘2 0 0.0 4.00 x 10‘2 3 12.0 4.00 x 10‘2 3 12.0 2.00 x 10‘2 2 8.0 2.00 x 10‘2 4 16.0 1.00 x 10‘2 2 8.0 Controlb 18 72.0 Control 18 72.0 aConcentration of the drug at which the number of schizonts/ 200 asexual parasites : 50% of that in drug-free control wells. bSee Table 1. 28 obtained from patients S-155 and S-157 did not mature in the 24-h test, but increased in number in the 48-h test, with MIC values for chloroquine of 3.16 x 10'2 and 5.62 x 10‘2 up respectively. The failure of so many of the Sudanese parasite iso- lates to develop in drug—free wells of the W.H.O. plates suggested strongly that: (a) the patients' plasmas were preventing parasite maturation 33 11339: or (b) the test plates were in some way defective. To test these two possi- bilities, sera from some of the patients whose parasites were field-tested for chloroquine sensitivity were reexam- ined in the U.S.A. in a new lot of W.H.O. plates against parasite strains of known drug sensitivities. Three labora- tory strains were tested by the 24-h microtechnique in W.H.O. plates using either dialyzed PNS or various dialyzed Sudanese sera. In tests utilizing sera from semi-immune or malaria-infected Sudanese, parasite structure was identical to that seen in field tests conducted in Sennar, making stage determination difficult and unreliable. Assays util- izing only PNS or serum from nonimmune Sudanese (data not shown) resulted in structurally normal parasites, indicating that the semi-immune serum itself, rather than the test plates, was the cause of test failure. To examine further the role of African serum in drug sensitivity assays, quantification of parasite development was determined by measuring incorporation of [3H]hypoxan- thine into parasite nucleic acid (Figure 1). Parasites grown 29 c) FCR-l Nahum-lamb v v. vvvv. 3 CHMXK) NUOU‘O‘NQ‘D — - V A A 2 4 8 b x'E4 o-Loaoours CONCENTRATION (102w) 01 N 5 Q at g 9 C Figure 14 Results of the 24-h drug sensitivity tests with various sera, using parasite strains differing in clinical drug resistance. a. Counts per minute due to incorporation of [3thypoxanthine by FCN-l, a drug-sensitive parasite, at various chloroquine concentrations. b,c. Data obtained with strains FCR-3TC, an RI parasite, and FCR-l, an RIII parasite, respectively 30 in inhibitory Sudanese sera incorporated much less of the radiolabel, e.g. FCN-l grown in the presence of serum S-155 without chloroquine incorporated less than 60% of the [3H]hypoxanthine incorporated by parasites grown in PNS (Figure la). Differences were even greater with chloroquine resistant strains FCR-3,IIC and FCR-l. Parasites grown in S-155 serum incorporated less than 25% of the label than when grown in PNS (Figure lb, c). To determine if the inhibition altered parasite sensi- tivity to the drugs, data were expressed as percent inhibi- tion in comparison to that noted for the homologous serum control value: chloroquine sensitivity remained unchanged in every strain (Figure 2). Although tests conducted in semi- immune Sudanese sera incorporated less label, parasites were still inhibited by the same concentration of drug as in PNS. Thus, the results illustrated in Figure 1 reflect additive effects of the sera and drugs, without synergistic interac- tion. This pattern also held true for experiments performed with mefloquine (data run: shown). In addition, with PNS alone employed in sensitivity assays, no differences were found between the results of 48-h and 24-h tests, irrespec- tive of whether [3H]hypoxanthine incorporation or micro- scopic determination of parasitemia and parasite development was used as the basis for parasite quantification (Figure 3). 31 0% a) FCN-I .00, b) FCR-3TC , c) FCR-l 01012000015 CONCENTRATION (102w) Figure 2. The data are plotted as % inhibition in compar- ison to the homologous serum control. a-c. Data obtained with strains FCN-l, FCR-3TC and FCR-l, respectively 32 0) FCN-I 100 :00 90 90 80 80- 70 70 60 50 z 50 50 Q l— 40 40 Q :1: . Z 30 30 o\° 20 20 48"r TEST (’H-HYPmANerE) A -IO- 0 24hr TESTl’H-HYPOXANI‘HNE) -|0 I 24hr TEST(SCH20NT COUNT) 0248l163126‘4 0248163264 CH_0RO0UINE CONCENTRATION (Id2 11M) Figure 3. Comparison of results obtained with the 48-h test and 24-h test conducted in the presence of pooled nonimmune serum (PNS). The 48-h test was quantified by measuring incorporation of [3H]hypoxanthine: the 24-h test results were determined by incorporation of the radiolabel and cal- culating the percentage inhibition of development to the schizont stage. a. Results of a comparison with FCN-l. b. Results obtained with FCR-BTC 33 DISCUSSION Our experience using W.H.O. plates in Sudan indicates that some yet undefined components of the patient's plasma inhibit parasite development 33 y3££p_even in the absence of chloroquine. It might be argued that these failures were due to the presence of chloroquine in the patient's plasma, as suggested by Onorizo who observed unexplainable failure of development in control wells. This may have been the case for a few of the samples tested in the field, since we did not test for the presence of chloroquine in the patients' urine. However, the cause of most test failure appears to be undialysable serum factors in the patients' serum, since we have demonstrated that dialysis quantita- tively removes chloroquine.12 Furthermore» the Sudanese sera were more inhibitory to chloroquine-resistant strains FCR-3TC and FCR-l than to FCN—l, a chloroquine-sensitive strain. The fact that FCN-l grew better in inhibitory serum than the others may be a strain specific phenomenon. The retardation of intraerythrocytic development seen in para- sites cultivated with the patients' sera probably represents serum-induced crisis forms.ll’23 The failures of the 24-h microtechnique in i_n 2E2 determination of chloroquine sensitivity were due to the presence of factors in the patients' plasma/serum and not to defects in the test plates themselves, since: (a) the plates sustained parasite growth when the patients' plasmas were replaced with PNS: and (b) supplementing the patient's serum with PNS still resulted in crisis forms. 34 It should be noted that the high failure rate in 24-h field tests might have been due in part to an age bias in selecting parasite and serum donors. Since large blood samples were needed to conduct this comparative study and serum analysis, only older patients, with presumably greater immunity to malaria, were chosen. Furthermore, most studies on chloroquine sensitivity in Africa report only successful tests, with no mention of the rate of test failures which range between 30 and 60% (Harrison Spencer: Phuc Nguyen- Dinh: Walter Wernsdorfer: et a1., personal communication). We conclude from our experience with the 48-h test that strains of P. falciparum isolated in central Sudan were chloroquine sensitive. These observations agree with previ- ous reports.7'16 Furthermore, since few of the malaria infections 1J1 the Blue Nile Province are confirmed micro- scopically and since the area is also endemic for typhoid fever, a febrile disease whose clinical manifestations are often confused with malaria, we believe that the chloroquine resistance reported by the clinicians in Blue Nile Province (M. Akood, personal communication) has yet to be demon- strated by 33 vitro testing of parasite isolates. However, with the spread of proved chloroquine resistance from Kenya and Tanzania, the situation in Sudan may soon change. It is possible that chloroquine-resistant malaria in Africa is transmitted to nonimmune European and North Ameri- cans from semi-immune indigenous populations. Since sera from Sudanese residents can contain factor(s) that inhibit intracellular parasite development, one may hypothesize that 35 such factors may be masking the incidence of chloroquine- resistant malaria in Africans. Indeed, Spencer22 and Schwartz et al.21 have demonstrated malaria infections that by _i_p_ vitro sensitivity assay proved to be chloroquine- resistant, but were subsequently cleared in the patients during the course of the standard W.H.O. 32 yiyp test. Both investigators have speculated that immune responses may play a role in modifying the expression of drug resistance in 33 yiyp and 33 vitro drug sensitivity tests. More experiments are required 1x) test this hypothesis. .It is of interest that 1J1 an Indonesian population we have recently studied, despite high ‘titers (ME parasite-specific antibody, there existed no serum crisis forming activity: unlike the situa- tion among Africans, more than 80% of the P. falciparum . . . . l4 infections were chloroquine-reSistant. Notwithstanding the extensive use of chloroquine in Africa, most infections are probably eliminated, or at least controlled, by immunity to the disease. Nevertheless, as control programs increase, population immunity will undoubt- edly decrease, creating a situation where: (a) control of infections must rely more heavily on chemotherapy, and (b) drug resistant parasites already present in the indigenous population will no longer be controlled by immune responses. We postulate that these conditions favor the spread of drug-resistant parasites. In summary, we have demonstrated that serum factors from Sudanese individuals interfeee with determination of chloroquine sensitivity 33 vitro. After extensive dialysis 36 to remove chloroquine, the only antimalarial drug used in the field test area, the sera remained inhibitory to para- site development. If our findings are confirmed in other regions cu? Africa, 33 vitro drug sensitivity test results may be questionable due to antiparasitic factors present in semi-immune sera. Under such. circumstances, it: may' be advantageous to replace the patient's plasma with PNS in 32 vitro field tests of drug sensitivity in Africa. 37 ACKNOWLEDGMENTS It is a pleasure to acknowledge the assistance of Abdalla Hassan Bashier and of the staff of. the Malaria Training Center, including Salah Eldin A/Rahim, and Abdel Moniem Mohmed Fazaa. This investigation was supported by Research Grant AI 16312, from the National Institute of Allergy and Infectious Diseases, 0.5. Public Health Service, under the auspices of the Michigan State University-Sudan Ministry of Health "Collaborative Research on Parasitic Diseases in Sudan" project. This report is listed as paper number 10764 of the Michigan Agricultural Experiment Station. 38 LIST OF REFERENCES Aronsson,B., Bengtsson,E., BjOrkman,A., Pehrson,P. O., Rombo, L., and Wahlgren, M., 1981. Chloroquine- resistant falciparum malaria in Madagascar and Kenya. Am. Trop. Med. Parasitol., 33: 367-373. Campbell, C. C., Chin, W., Collins, W. E., Tentsch, S. M., and Moss, D. M., 1979. Chloroquine-resistant Plasmodium falciparum from East Africa. Cultiva- tion and sensitivity of the Tanzania I/CDC strain from an American tourist. Lancet, 3: 1151-1154. Capps, T. C., and Jensen, J. B., 1983. Storage require- ments for erythrocytes used to culture Plasmodium falciparum. J. Parasitol., 93: 158-162. Center for Disease Control, 1978. Chloroquine-resistant malaria acquired in Kenya and Tanzania-Denmark, Georgia, New YOrk. CDC Morbidity and Mortality Weeklnyep., 31: 463-464. Desjardins, R. D., Canfield, C. J., Haynes, J. D., and Chulay, J) 1)., 1979. Quantitative assessment of antimalarial activity 33 vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother., 33: 710-718. Divo, A. A., and Jensen, J. B., 1982. Studies on serum requirements for the cultivation of Plasmodium falciparum. 1” Animal Sera. Bull W.H.O., 39; 565-569. Geary, T. G., and Jensen, J. B., 1983. Lack of cross- resistance to 4-aminoquinolines in chloroquine- resistant Plasmodium falciparum 32. vitro. ‘3. Parasitol., 33: 97-105. Jensen, J; B., and Trager, W., 1977. Plasmodium falcip- arum in culture. Use of outdated erythrocytes and description of the candle jar method. _3. Parasitol., 33: 883-886. Jensen, J; B., and Trager, W., 1978. Plasmodium falcip- arum in culture: Establishment of additional strains. Am. J. Trop. Med. Hyg., 33: 743-746. 10. ll. 12. 13. 14. 15. 16. 17. 18. 19. 39 Jensen, J. B., Capps, T. C., and Carlin, J. M., 1981. Clinical drug-resistant falciparum malaria acquired from cultured parasites. Am. J. Trop. Med. Hyg., 33: 523-525. Jensen, J. B., Boland, M. T., and Akood, M. A., 1982. Induction of crisis forms in cultured Plasmodium falciparum with human immune serum from Sudan. Science, 216: 743-746. Jensen, J. B., Boland, M. T., Hayes, M., and Akood, M. A.,-1982. Plasmodium falciparum: A rapid assay for 33 vitro inhibition due to human serum from residents of malarious areas. Exp. Parasitol., 33: 416-424. Jensen, J. B., Boland, M. T., Allan, J. 8., Carlin, J. M., Vande Waa, J. A., Divo, A. A., and Akood, M. A., 1983. Association between human serum-induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan. Infect. Immun., 33: 83-89. Jensen, J. B., Hoffman, S. L., Boland, M. T., Akood, M. A., Laughlin, IL. W., Kurniawan, L., and Marwoto, H. A., 1984. Comparison of immunity to malaria in Sudan and Indonesia: Crisis-form versus merozoite-invasion inhibition. Proc. Natl. Acad. Sci. U.S.A., 33: 922-925. Kean, B. H., 1979. Chloroquine-resistant falciparum malaria from Africa. J. Am. Med. Assoc., 241: 395-396. Kouznetsov, R. L., Rooney, W., Wernsdorfer, W. H., El Gaddal, A. A., Payne, D., and Abdalla, R. E., 1980. Use of the 33 vitro microtechnique for the assess- ment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan. Bull. W.H.O. 33: 785-789. Lambros, C., and Vanderberg, J. P., 1979. Synchroniza- tion of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol., 33: 418-420. Nguyen-Dinh, P., and Trager, W., 1980. Plasmodium fal- ciparum i_n_ vitro: Determination of chloroquine sensitivity of three new strains by a modified 48- hour test. Am. J. Trop. Med. Hyg., 33: 339-342. Omer, A.IL.S., 1978. Response of Plasmodium falciparum in Sudan tn) oral chloroquine. Am. J. Trop. Med. Hyg., 33: 853-857. 20. 21. 22. 23. 24. 25. 26. 4O Onori, E., Payne, D., Grab, B., Horst, H. I., Almeida Franco, J., and Joia, H., 1982. Incipient resis- tance of Plasmodium falciparum to chloroquine among a semi-immune population of the United Republic of Tanzania. I. Results of 33 vivo and 33 vitro studies and of an ophathalmological survey. Bull W.H.O., 33: 77-87. Schwartz, I.K., Campbell,<2.C., Payne, D., and Khatib, O. J., 1983. 33 vivo and 33 vitro assessment of chloroquine-resistant Plasmodium falcipgrum malaria in Zanzibar. Lancet, 3: 1003-1005. Spencer, H. C., Masaba, S. C., and Kiaraho, D., 1982. Sensitivity of Plasmodium falciparum isolates to chloroquine in Kisumu and Malindi, Kenya. Am. J. Trop. Med. Hyg., 3;: 902-906. Taliaferro, W.H. and Taliaferro, L.G., 1944. The effect of immunity on the asexual reproduction of Plasmodium brasilianum. J3 Infect. Dis., 13: 1-32. Wernsdorfer, W. H., 1980. Field evaluation of drug resistance in malaria. 33 vitro micro-test. Acta. Tropica., 31: 222-227. World Health Organization, 1973. Chemotherapy of malaria and resistance to antimalarials. W.H.O. Tech. Rep. Ser. No. 529. Yisunsri, L., and Rieckmann, K., 1980. 33 vitro micro- technique for determining the drug susceptibility of cultured parasites of Plasmodium falciparum. Trans. R. Soc. Trop. Med. Hyg., 13: 809-810. CHAPTER TWO COMPARISON OF INDUCERS OF CRISIS FORMS IN PLASMODIUM FALCIPARUM 13 VITRO Joseph M. Carlin and James B. Jensen Reprinted from American Journal of Tropical Medicine and Hygiene 34: 668-674, 1985. 41 42 ABSTRACT A variety of known or suspected inducers of crisis form parasites in cultivated Plasmodium falciparum were examined. Sera from Sudanese residents of malaria-endemic areas, sera from American tuberculosis patients, and rabbit sera con- taining tumor necrosis factor were assayed i_3 vitro for cytotoxic activities against E. falciparum and mouse L-M cell cultures. Inhibition was determined by measurement of incorporation of radiolabeled nucleic acid precursors. When compared to normal serum, parasites grown in the presence of a 1:4 dilution of rabbit sera containing tumor necrosis factor, TB patient sera, or Sudanese sera were metabolically inhibited 73%, 75%, and 95% respectively. However, only the rabbit sera containing tumor necrosis factor were cytotoxic to L-M cells, inhibiting radiolabel incorporation by 80% at a 1:1000 serum dilution. These findings suggest that tumor necrosis factor is apparently not responsible for the induc- tion of parasite crisis forms by the inhibitory human sera tested. In addition, human gamma-interferon had no effect on parasite growth. 43 INTRODUCTION The term "crisis form" was applied originally by Taliaferro and Taliaferro to idescribe abnormal parasites observed in Plasmodium brasilianum infections of Cebus capucinus monkeys.20 These parasites were characterized by reduction in average merozoite number per segmenter, retar- dation of development to maturity, and intraerythrocytic deterioration. Since their initial study, several other investigators have reported induction of crisis forms of Plasmodium spp. by a variety of methods. Jensen 33 33. have demonstrated the existence of a human non-antibody serum factor that induces crisis forms in cultivated Plasmodium falciparum.12 This crisis form factOr (CFF), found in serum from Sudanese adults functionally immune to malaria, is both positively correlated with malaria endemicity22 and associ- ated with clinical immunity to malaria in Sudan,11 and thus is probably part of acquired immunity to g. falciparum. The metabolic activity of parasites cultivated in the presence of sera containing high titers of CFF is inhibited by greater than 90% as determined by scintillation spectrometry of incorporation of [3H]hypoxanthine into parasite nucleic acids. Rodents vaccinated with agents known to activate macro- phages, such as Mycobacterium bovis strain Bacille Calmette Guérin (BCG) and Propionibacterium acnes (formerly Corynebacterium parvum), have enhanced resistance against 3,4 malaria and other hemoprotozoan diseases. Parasites 44 examined from these animals appear morphologically similar to the crisis forms described by Taliaferro and Taliaferrozo. Mechanisms postulated for intracellular parasite killing include secretion of non-specific effector molecules by activated macrophages into the serum,6 and damage due tn) the production of free radical intermediates of the reduction of oxygen by effector cells which bind to the surface of parasitized erythrocytes.l Sera from animals vaccinated with BCG and subsequently inoculated with a bacterial lipopolysaccharide (LPS) contain tumor necrosis factor (TNF)2, a monokine cytotoxic to mouse L cells. These TNF containing sera (BCG-LPS sera) are also inhibitory to Plasmodium spp. 33 vitro, and promote the formation of 8,21 crisis form parasites. In addition, investigations have shown that the generation of 02-derived free radicals within 33 vitro and 33 vivo Plasmodium cultivation systems leads to 5,24 parasite death. In the present study, we have examined various known or suspected inducers of crisis form malaria parasites 33 vitro (CFF sera from Sudan, BCG-LPS sera from rabbits: and sera from tuberculosis patients, human gamma-interferon [HuIFN-y] respectively). In order to clarify the relationship between these agents, they were assayed for their ability to induce crisis form parasites in cultivated E. falciparum and tested for.tumoricidal activity with mouse L-M cell cultures. 45 MATERI ALS AND METHODS Parasite and cell cultivation g. falcipgrum strain FCR3TCl4 was routinely cultivated by the candle-jar system,15 using RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 5% pooled human A+ sera (PHS), 25m3 lT—Z-hydroxyethylpiperazine-N-Z- ethanesulfonic acid (HEPES) and 0.2% sodium bicarbonate (RP-5). We have previously found minimal differences in parasite growth rates when normal human serum was used at 10% v/v concentration in RPMI 1640 medium.9 The use of human sera pooled from 20 individuals allows for the reduc- tion of required serum in culture medium to 5% v/v with parasite growth equal to nonpooled sera used at 10%.7 Para- sites to be employed in cytotoxicity assays were synchro- nized to a six hour age differential with a modification of the sorbitol technique.16 Resultant ring stages were allowed to mature to the late schizont-stage before concen- tration by the gelatin flotation method10 and subcultivation to 0.5% parasitemia in freshly washed, aged human O+ erythrocytes devoid of viable leukocytes. L-M cells (American Type Culture Collection No. CCL 1.2-derivative of NCTC clone 929) were used as target cells for tumoricidal activity. These cells were maintained in Medium 199 (Gibco Laboratories, Grand Island, NY) supple- mented with 0.5% Bacto-peptone (Difco Laboratories, Detroit, MI) and 0.22% sodium bicarbonate (M-199BP) in an atmosphere of 5% 002, 95% air at 37°C. 46 Collection of human serum The procedure for collection of sera from Sudan has been described previously.13 Briefly, blood was drawn in siliconized vacutainers (Becton-Dickinson, Rutherford, NJ) from healthy adult volunteers residing in Blue Nile Province, Sudan, a region hyperendemic for E. falciparum. Blood from tuberculosis patients was obtained through the Ingham Medical Center Chest Clinic, Lansing, MI. TQ, CJ, DW, and SG were classified as type III tuberculosis patients (positive for THE) skin tests, acid-fast bacillus stain, and chest X-ray): MS was a class II TB patient (posi- tive for PPD skin test, with no other evidence of tubercu- losis). All patients were undergoing antibiotic therapy at the time of blood drawing. Production of BCG-LPS sera BCG-LPS sera were prepared by the method of Matthews and Watkins.19 Female New Zealand white rabbits (2-2.5 kg) were bled for control serum and subsequently inoculated with 4-16 1: 107 viable BCG organisms (University of Illinois at Chicago Medical Center, Chicago, IL). After two weeks, the rabbits were inoculated i.v. with 100 ug of lipopolysaccha- ride B from Escherichia coli 055:35 (Difco Laboratories, Detroit, MI). Blood for BCG-LPS serum was collected two hours after LPS treatment. Control sera were also obtained from rabbits inoculated with LPS only, BCG only, or saline only. 47 Serum preparation All blood samples were allowed to clot at 4°C overnight before centrifugation and separation of the serum from the cellular elements. Serum samples obtained in Sudan were immediately frozen and stored at -20°C until transported on blue-ice gel to our laboratory' in Michigan, where they arrived still frozen. Other sera were kept at -20°C until use. All sera were heat-inactivated at 56°C for 30 min and dialysed 1:106 against RPMI 1640 or Medium 199 in Spectrapor 12000-14000 MWCO tubing (Spectrum Medical Industries, Inc., Los Angeles, CA) to remove all drugs and equilibrate the sera nutritionally for radiometric studies. Previous stud- ies have shown that dialysis removes chloroquine13 and anti- tubercular drugs from serum (T.G. Geary, East Lansing, MI, personal communication). After dialysis, sera were steril- ized by filtration through 0.45 um pore membranes (Schleicher & Schuell, Keene, NH). Parasite inhibition assay This assay was performed as described in detail else- where.13 Synchronized schizont-stage parasites were dis- pensed into 96-well microtiter plates, 3 ul/well. Various concentrations of test sera in RPMI 1640 containing 40 ug/ml of gentamicin sulfate (Valley Biologics, Inc., State College, PA) and 5% PHS were assayed for total inhibition (merozoite invasion inhibition and growth retardation) by exposing triplicate wells to 200 ul of each serum concentra- tion for 48 h, and for intraerythrocytic parasite growth 48 inhibition by allowing merozoite invasion to occur in normal serum before cultivating the parasite in test serum. The presence of 5% background PHS in all sera tested provides completely for the nutritional requirements of the para- sites. Control wells were set up with PHS or matched indi- vidual pre-treatment rabbit sera at the same concentration as test sera. Metabolic inhibition was determined by scin- tillation spectrometry. Each microtiter well was pulsed with l uCi of [3H]hypoxanthine (sp. activity = 10.0 Ci/mmol, New England Nuclear, Boston, MA) for the final 24 h of cultivation, before harvesting the parasites onto glass- fiber filter strips using a cell harvester (Bellco Glass, Inc., Vineland, NJ). Incorporation of radiolabel into para- site nucleic acid was measured with a Beckman LS 7500 scin- tillation spectrometeru Results obtained with parasites grown in non-inhibitory control sera usually ranged from 10,000-20,000 CPM. All values were corrected for background (incorporation of label by uninfected erythrocytes) and expressed as percent inhibition 3 standard deviation. Per- cent inhibition was calculated with the following equation: % inhibition = CPM of control well - CPM of test well x 100% CPM of control well Statistical comparisons were made using Student's 3 test. Giemsa-stained thin films were prepared from identical wells for visual assessment of parasite inhibition. 49 In experiments assessing the effect of HuIFN-y on para- site metabolism, HuIFN-y or interferon diluent were serially diluted in RP-S containing gentamicin sulfate. Concentra- tions of 10, 102, 103, and 104 U/ml were added to ring-stage parasites and assayed for intraerythrocytic parasite inhibi- tion as above. HuIFN-y (Immunomodulators Laboratories, Staffort, TX) was a gift from Dr. Harold C. Miller, Michigan State University. It was obtained from culture supernatants of leukocytes stimulated with staphylococcal enterotoxin B and phytohemagglutinin, and was supplied lyophilized, 106 U/vial, with diluent (0.05 3 Tris and 0.5 3 lysine, pH 7.5). 6.5 23 Activity was 10 U/ml in a Sindbis virus assay. L cell cytotoxicity assay Cytotoxicity was determined by a modification of an assay described by Matthews and Watkins.19 Microtiter plates were seeded with 75 ul of L-M cells at a concentration of 2.5 x 105 cells/m1 M-l99BP and incubated at 37°C in 5% CO2 for 3 In After cells had adhered to the plates, 75 ul of various concentrations of test sera in M-l99BP containing 100 U penicillin and 100 ug streptomycin/ml (Gibco Laborato- ries, Grand Island, NY) were added to triplicate wells. Control wells were set up with PHS or matched individual pre-treatment rabbit sera at the same concentration as test sera. Plates were then incubated for either 24 or 72 in During the final 24 h of cultivation, cells were pulsed with 0.5 uCi of [methyl-3H]thymidine/well (sp. activity = 2.0 Ci/mmol, Research Products International Corp., Mount 50 Prospect, IL). At the end of cultivation, medium was replaced with distilled water, and the plates were refrig- erated at 4°C overnight. Cells were then harvested and incorporated radiolabel was determined as in the parasite inhibition assay. 51 RESULTS Toxicity of various sera to P. falciparum BCG-LPS sera, sera from patients infected with TB, and CFF sera from Sudan were all found to be toxic to intra- erythrocytic stages of g. falciparum in comparison to normal rabbit or human sera (Tables 1, 2 and 3, respectively), producing typical crisis form parasites 33 33333. The anti- malarial effects of the sera were assayed in two ways: by measuring total inhibitory activity (both merozoite invasion inhibition and growth retardation) and by measuring retarda- tion of intraerythrocytic parasite growth. To determine total inhibition, parasites were cultivated in test sera 48 h, from schizont to schizont-stage, and pulsed with [3H]hypoxanthine during the final 24 h of ‘cultivation. Intracellular growth retardation was determined by measuring radiolabel incorporation by parasites grown in test sera for 36 h, from ring to schizont-stage, and pulsed during the final 24 h. With BCG-LPS sera, total inhibition in a 48 h period was significant (p<.001) with averages of 73% inhibition at a 1:4 dilution and 47% at a 1:8 concentration (Table 1). Control sera (from BCG, LPS, or saline inoculated control rabbits) showed no toxic effects on malaria cultures. Simi- lar to results obtained with BCG-LPS sera, TB patient sera significantly inhibited parasite cultures (p<.01), averaging 75% at a 1:4 serum dilution (Table 2). Serum concentrations 52 Table 1 Percent inhibition of E. falciparum [3H]hypoxanthine incorporation by rabbit BCG-LPS serum. Rabbit Dilution % Inhibition Serum 36 ha 48 hb Rb-l 1:4 68.0 1 7.8 (3)C 94.7 1 3.9 (6) 1:8 16.8 1 1.6 (3) 55.4 1 12.3 (6) Rb-2 1:4 36.1 1 10.6 (3) 79.1 1 4.9 (6) 1:8 19.9 1 6.8 (3) 26.8 1 9.6 (5) Rb 3 1:4 86.8 1 3.7 (5) 98.8 1 2.0 (6) 1:8 49.1 1 30.9 (5) 92.5 1 3.4 (8) Rb 4 1:4 25.2 1 12.2 (7) 61.9 1 16.2-(8) 1:8 18.8 1 10.1 (7) 24.2 1 7.2 (10) Rb 5 1:4 20.7 1 10.2 (8) 31.2 1 15.6 (6) 1:8 19.7 1 1.5 (6) 22.0 1 16.2 (6) Rb 6 1:4 58.6 1 21.2 (10) 76.0 1 10.8 (7) 1:8 41.1 1 23.2 (8) 57.6 1 19.6 (6) Totald 1:4 46.5 1 27.0 (36) 73.1 1 24.4 (39) 1:8 29.2 1 20.9 (32) 47.0 1 28.8 (41) aIntraerythrocytic parasite development retardation, rings to schizonts, b 48 h to serum. CPercent inhibition 1 standard deviation. cates in parentheses. dMean of six rabbits. Total parasite retardation, exposed 36 h to serum. schizont to schizont, exposed Number of repli- 53 Table 2 Percent inhibition of g. falciparum [3H]hypoxanthine incorporation by sera from humans with TB. Serum Dilution % Inhibition Donor 36 ha ‘ 48 hb TQ 1:4 83.6 1 2.7 (2)C 93.8 1 0.4 (2) 1:8 57.1 1 2.1 (2) 77.5 1 1.7 (2) 1:16 36.9 1 4.2 (2) 57.6 1 3.0 (2) MS 1:4 38.0 1 1.0 (2) 59.3 1 21.9 (2) 1:8 14.4 1 0.7 (2) 18.0 1 8.5 (2) 1:16 8.1 1 1.4 (2) 18.6 1 3.3 (2) CJ 1:4 89.0d (1) 96.5 (1) 1:8 79.8 (1) 91.6 (1) 1:16 64.3 (1) 81.7 (1) so 1:4 47.5 (1) 66.5 (1) 1:8 40.5 (1) 53.6 (1) 1:16 36.2 (1) 34.8 (1) 0w 1:4 43.2 (1) 53.8 (1) 1:8 25.2 (1) 38.1 (1) 1:16 20.6 (1) 35.6 (1) 54 Table 2 (Continued) Serum Dilution % Inhibition Donor 36 ha 48 nb Totale 1:4 60.5 1 23.6 (7) 74.7 1 21.1 (7) 1:8 41.2 1 24.8 (7) 53.5 1 30.1 (7) 1:16 30.2 1 19.9 (7) 43.3 1 23.3 (7) aSee Table 1. bSee Table l. CSee Table 1. 6Only one well tested due to eMean of five tested sera. insufficient sera. 55 Table 3 Percent inhibition of 3. falciparum [3H]hypoxanthine incorporation by Sudanese sera with high CFF activity. Serum Dilution % Inhibition Donor 36 ha 48 hb 830 1:4 70.0 1 1.4 (2)C 89.1 1 2.8 (2) 1:8 54.0 1 4.4 (2) 76.8 1 4.0 (2) s-81-15 1:4 88.1 1 2.8 (2) 98.5 1 0.3 (2) 1:8 64.9 1 1.8 (2) 94.6 1 1.1 (2) S-81-55 1:4 72.0 1 4.4 (2) 91.4 1 2.7 (2) 1:8 50.1 1 1.6 (2) 74.8 1 1.3 (2) S-81-132 1:4 83.5 1 7.8 (2) 97.9 1 0.3 (2) 1:8 64.4 1 2.8 (2) 92.3 1 0.1 (2) S-81-139 1:4 79.4 1 4.2 (2) 96.2 1 1.7 (2) 1:8 56.2 1 3.1 (2) 88.4 1 2.1 (2) Total6 1:4 78.6 1 7.9 (10) 94.6 1 4.1 (10) 1:8 57.9 1 6.6 (10) 85.8 1 8.2 (10) aSee Table l. b See Table 1. CSee Table 1. 6Mean of five Sudanese sera. 56 of 1:8 resulted in 54% parasite inhibition. Total inhibi- tion due to cultivation in Sudanese sera was significant (p<.001) and averaged 95% at a 1:4 concentration and 85% at 1:8 (Table 3). These sera were selected for their high levels of CFF for purposes of comparison. When parasites were cultivated 36 h, from ring to schizont-stages in the presence of various sera, hypoxan- thine incorporation was still significantly reduced. At a 1:4 concentration, BCG-LPS sera produced an average of 46% inhibition (p<.001), TB sera inhibited by 60% (p<.05), and Sudanese CFF sera reduced incorporation by 79% (p<.001). Since no reinvasion took place» during this part of the malaria cycle, all inhibition observed was “due to serum effects on intracellular stages. Microscopic examination of inhibited cultures demonstrated crisis form parasites, with retarded development and shrunken cytoplasm when compared to control cultures. Toxicity of various sera to L-M cells The three groups of sera (BCG-LPS, TB, and Sudanese) were assayed for tumor cell cytotoxicity. Data from these experiments were pooled for Figure l, a graphic representa- tion of the effects of various sera during a 24 h period on thymidine incorporation by L-M cells. In contrast to results obtained with malaria parasites, only BCG-LPS sera were inhibitory to the target cells. BCG-LPS sera were cytolytic at serum concentrations greater than 1:32000. As serum concentration was decreased, a corresponding decrease in residual cell bodies was observed. In the case of TB and 57 I00 80‘ Z 60- O i l: 90 . I Z o\° 40. 204 1 1M , r r 1 - --—- I 2 3 4 5 L06 ( l/ SERUM DILUTION ) Figure 1. Effects of sera from a variety of sources on [3H]thymidine incorporation by L-M cells during a 24-h period. Results are expressed as percent inhibition 1 standard error of the mean. Symbols: BCG-LPS sera, I: TB sera, ‘ : Sudanese sera, . 58 Sudanese CFF sera, no cell lysis was observed at any concen- tration. In fact, incorporation of radiolabel in the pres- ence of these sera was slightly higher than that seen when cells were cultivated either with PHS or in the absence of serum. Results obtained when cells were grown for 72 h in the presence of various sera were similar to those from 24 h cultures (data not shown). BCG-LPS serum ICSOS The concentration of BCG-LPS serum required for 50% inhibition of radiolabel incorporation in Ind! cells was nearLy 700 times less than that needed to produce the same effect in malaria parasites (Table 4). The ICSOs for para- sites ranged from 1:1.0 to 1:30.3, averaging 1:6.6: whereas, IC50s for tumor cells ran from 1:2449 to 1:6281, with a mean of 1:4414. Effect of HuIFN-y on P. falciparum Previous studies had shown that human alpha-interferon had no effect on growth of E. falciparum.11 It was observed that HuIFN-y also had no effect on the incorporation. of hypoxanthine into parasite nucleic acids. Parasites culti- vated in various amounts of HuIFN-y were not inhibited sig- nificantly when compared ix) parasites cultivated :hl the diluent control (data not shown). 59 Table 4 E. falciparum and L-M cell ICSOS of BCG-LPS sera. Rabbit IC50 Serum Parasitea Tumor cellb Rb-l l: 8.8 1:5408 Rb-2 l: 5.9 1:2449 Rb-3 1:30.3 1:5188 Rb-4 l: 5.0 1:5248 Rb-S 1: 1.0 1:3266 Rb-6 1:10.7 1:6281 Totalc 1: 6.6 1:4414 aConcentration at which parasite incorporation of [3H]hypoxanthine is reduced by 50%. bConcentration at which cell line incorporation of [3H]thymidine is reduced by 50%. cMean of six rabbits. 60 DISCUSSION The present studies were conducted to clarify the rela- tionship between various inducers of malaria parasite crisis forms. We found that HuIFN-y had no effect on parasite incorporation of purine label. However, BCG-LPS sera, TB patient sera, and Sudanese CFF sera were all able to induce crisis forms in i_3 vitro cultures of P_. falciparum. This induction was observed in synchronous parasites cultivated) from ring to schizont-stages, demonstrating that merozoite invasion was not required for the serum factors to have an effect on parasite development. Although all three types of sera were active against E. falciparum, only TNF-containing rabbit BCG-LPS serum was cytotoxic to L-M cells. Since other investigators have demonstrated that TNF generated in rabbit,19 human18 and mouse17 systems are all active at high dilutions against the standard L cell target, and since we have shown that rabbit BCG-LPS serum is about 700 times more active against L-M cells than malaria parasites, it appears likely that TB and Sudanese CFF sera contained no appreciable amounts of TNF, and that TNF was not responsible for crisis form induction in these human sera. In addition, the production of TNF usually requires both treatment with macrophage activating agents and subsequent stimulation with LPS. Minnel e_t_ _a_l. have shown that tumor necrotic activity peaks 2 hours after LPS treatment of BCG-infected mice, and is absent after 6 3,4 hours.l7 Alternatively, studies by Clark 33 33. have 61 shown that mice injected with BCG or killed 3. acnes alone are protected from lethal Plasmodium and Babesia infections. Since the TB and Sudanese sera used in our study were obtained without endotoxin treatment of the donors, and the anti-parasitic activity in their sera remains relatively stable over time, crisis form induction by these sera would appear more closely related to the rodent model described by C1ark3'4 than to those models requiring LPS injection. One might argue that the antimalarial activity in Sudanese CFF sera is due to TB infection. However, the Sudanese serum donors did not have any clinical evidence of infections, and in previous studies we have noted that many of the inhibitory sera were from PPD skin test-negative donors (unpublished observations). Thus, the relationship between TNF and CFF remains unclear. ‘They may be entirely different molecular species both capable of inducing crisis forms of malaria parasites. Alternatively, BCG-LPS sera may contain both TNF and CFF. Despite the fact that Haidaris 33 33.8 were able to block the cytotoxic activities of mouse BCG-LPS serum with anti- body made to partially purified rabbit TNF, CFF and TNF may be antigenically related molecules, and antibody generated against one may inhibit the other. Indeed Zacharchuk E 33.25 have shown that antibody prepared against purified guinea pig lymphotoxin neutralizes not only lymphotoxin, but also guinea pig TNF as well as macrophage cytotoxic factor, indicating that these lymphokines and monokines have strong 62 antigenic relationships. Studies on the effect of highly purified TNF and CFF on malaria cultures are required to answer these questions. 63 ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of Michael T. Boland and the cooperation of Dr. Gauresh Kashyup of the Ingham Medical Center Chest Clinic. This investiga- tion was supported by Research Grant AI 16312, from The National Institute of Allergy and Infectious Diseases, U.S. Public Health Service, under the auspices of the Michigan State University-Sudan Ministry of Health "Collaborative Research ("1 Parasitic Diseases in Sudan" project. This is article no. 11362 of the Michigan Agricultural Experiment Station. 64 LIST OF REFERENCES Allison, A.C., and Eugui, E.M., 1982. A radical inter- pretation of immunity to malaria parasites. Lancet, 3: 1431-1433. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., and Williamson, B., 1975. An endotoxin induced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. U.S.A., 13: 3666- 3670. Clark, I. A., Allison, A. C., and Cox, F. E., 1976. Protection of mice against Babesia and Plasmodium with BCG. Nature (London), 259: 309-311. Clark, I. A0! COX: F. E0! and Allison, A. Col 1977. Protection of mice against Babesia spp. and Plasmodium spp. with killed Corynebacterium parvum. Parasitology, 13: 9-18. Clark, I. A., and Hunt, N.Ii., 1983. Evidence for reac- tive oxygen intermediates causing hemolysis and parasite death in malaria. Infect. Immun., 33: 1-6. Clark, I.A., Virelizier, J., Carswell, E. A., and Wood, P. R., 1981. Possible importance of macrophage- derived mediators in acute malaria. Infect. Immun., 33: 1058-1066. Divo, A. A., and Jensen, J. B., 1982. Studies on serum requirements for the cultivation of Plasmodium falciparum. 1. Animal sera. Bull. W.H.O., 33: 565-569. Haidaris, C. G., Haynes, J. D., Meltzer, M. S., and Allison, A. C., 1983. Serum containing tumor necrosis factor is cytoxic for the human malaria parasite Plasmodium falciparum. Infect. Immun., 33: 385-393. 10. 11. 12. 13. 14. 15. l6. 17. 18. 19. 65 Jensen,.I.B., 1976. Some aspects of serum requirements for continuous cultivation of Plasmodium falciparum. 8111].. W.H.Oo I él: 27-310 Jensen, J. B., 1978. Concentration from continuous cul- ‘ ture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum. Am. J. Trop. Med. Hyg., 31: 1274-1276. Jensen, J. B., Boland, M. T., Allan, J. S., Carlin, J. M., Vande Waa, J. A., Divo, A. A., and Akood, M. A. S., 1983. Association between human serum- induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan. Infect. Immun., 33: 1302-1311. Jensen, J. B., Boland, M. T., and Akood, M. A., 1982. Induction of crisis forms in cultured Plasmodium falciparum with human immune serum from .Sudan. Science, 216: 743-746. Jensen, J.B., Boland, M.T., Hayes, M., and Akood, M.A., 1982. Plasmodium falciparum: Rapid assay for 33 vitro inhibition due to human serum from residents of malarious areas. Exp. Parasitol., 33: 416-424. Jensen, J. B., Capps, T. C., and Carlin, J. M., 1981. Clinical drug-resistant falciparum malaria acquired from cultured parasites. Am. J. Trop. Med. Hyg., 33: 523-525. Jensen, J. B. , and Trager, W. , 1977. Plasmodium falciparum in culture. Use of outdated erythrocytes and description of the candle jar method. 33 Parasitol., 33: 883-886. Lambros,<2.,and Vanderberg,.J.P., 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol., 33: 418-420. Mannel, D. N., Meltzer, M. S., and Mergenhagen, S. E., 1980. Generation and characterization of a lipo- polysaccharide-induced and serum-derived cytotoxic factor for tumor cells. Infect. Immun., 33: 204-211. Matthews, N., 1981. Production of an anti-tumor cyto- toxin by human monocytes. Immunology, 33: 135-142. Matthews, N., and Watkins, J. F., 1978. Tumor-necrosis factor from the rabbit. I. Mode of action, specificity, and physicochemical properties. 333 J. Cancer, 33: 302-309. 20. 21. 22. 23. 24. 25. 66 Taliaferro, W. H., and Taliaferro, L. G., 1944. The effect of immunity on the asexual reproduction of Plasmodium brasilianum. J. Infect. Dis., 13: 1-32. Taverne, J., Dockrell, H. M., and Playfair, J. H. L., 1981. Endotoxin-induced serum factor kills malaria parasites 33 vitro. Infect. Immun., 33; 83-89. Vande Waa, J. A., Jensen, J. B., Akood, M. A. S., and Bayoumi, R., 1984. Longitudinal study on the 33 vitro immune response to Plasmodium falcipgrum in Sudan. Infect. Immun., 33: 505-510. Weigent, D. A., Stanton, G. J., Langford, M. P., Lloyd, R. E., and Baron, S., 1981. Virus yield-reduction assay for interferon by titration of infectious virus. Methods Enzymol., 13: 346-351. Wozencraft, A.O., Dockrell, H.M., Taverne, J., Targett, <3. A. T., and Playfair, J. H. L., 1984. Killing of human malaria parasites by macrophage secretory products. Infect. Immun., 33: 664-669. Zacharchuk, C. M., Drysdale, B., Mayer, M. M., and Shin, H. S., 1983. Macrophage-mediated cytotoxicity: Role of a soluble macrophage cytotoxic factor' similar to lymphotoxin and tumor necrosis factor. Proc. Natl. Acad. Sci. U.S.A., 33: 6341-6345. CHAPTER THREE STAGE- AND TIME-DEPENDENT EFFECTS OF CRISIS FORM FACTOR ON PLASMODIUM FALCIPARUM 33 VITRO Joseph M. Carlin and James B. Jensen Submitted to the Journal of Parasitology 67 68 ABSTRACT To determine the stage- and time-dependent effects of crisis form factor (CFF) on Plasmodium falciparum metabolism E vitro, the parasite erythrocytic cycle was divided into sequential 8 h time intervals, and highly synchronous para- sites were exposed to CFF for various lengths of time. Hypoxanthine anui phenylalanine incorporation into parasite nucleic acids auui proteins, respectively, and glucose con- sumption by the parasites were compared in cultures grown in CFF-containing or nonimmune sera. The most profound derange- ment of metabolism occurred in parasites 0-8 h post-inva- sion. Inhibition correspondingly decreased in tests started with progressively older parasites. Cultivation 1h: CFF serum for 8 h caused maximal inhibition of purine and amino acid incorporation: longer periods of exposure did not increase inhibition In contrast, CFF's effect on glucose consumption varied inversely to the duration of exposure to CFF. As the parasites matured in the presence of CFF, inhi- bition of the rate of glucose utilization decreased, with little or no reduction in consumption observed as parasites entered schizogony. Of the three metabolic parameters studied, hypoxanthine was the most sensitive indicator of metabolic inhibition throughout the cycle. 69 INTRODUCTION "Crisis form" malaria parasites were originally described22 as abnormal parasites characterized by their reduced average number of merozoites per segmenter, retarded development to maturity and intraerythrocytic deterioration. Although crisis form parasites had been observed in several 1,5,6 3,22 rodent and simian models, it was not until after the 33 vitro technique for cultivation of Plasmodium falciparum was developed24 that crisis forms of human parasites were seen. Jensen 33 33.13 have demonstrated the existence of a human serum factor able to induce crisis forms in malaria parasites. This factor (CFF) has been found to be both associated with clinical immunity to malaria in Sudan,15 and positively correlated with malaria endemicity:25 thus, it is probably part of acquired immunity 2,23 to g. falciparum. Furthermore, other investigators have demonstrated intraerythrocytic parasite growth inhibition by human sera from malarious regions. Despite the fact that crisis form parasites had been described as early as 1944,22 little is known about how crisis form inducers affect cell metabolism. Jensen 1:33 al.14 have shown that parasites cultivated in the presence of sera containing high titers of CFF are inhibited in incor- poration of [3H]hypoxanthine by greater than 90% during a 48 h period. However, since certain aspects of Plasmodium 7,11,19,20,21,27 metabolism have demonstrable stage dependence, the possibility exists that CFF may affect the i_n vitro 70 development of 3. falciparum in a stage-specific manner. The experiments reported here demonstrate stage- and time- dependent effects of CFF, characterized by several metabolic criteria, on synchronous cultures of g. falciparum in vitro. 71 MATERI ALS AND METHODS Parasite cultivation l6 3TC cultivated by the candle jar method17 using RPMI 1640 medium Plasmodium falciparum strain FCR was routinely (Gibco, Grand Island, NY) supplemented with 5% pooled human A+ sera (PHS), 25 m3 N-Z-hydroxyethyl-piperazine-N-2-ethane- sulfonic acid (HEPES) and 0.2% sodium bicarbonate (RP-5). However, in metabolic inhibition assays, RPMI 1640 was replaced with the ndnimal medium described by Divo 33 33.8 This substitution enhances parasite incorporation of radio- labeled phenylalanine, facilitating measurement of CFF inhi- bition of this process. Parasites to be employed in cytotoxicity assays were synchronized .to a 4 h age differential with the following procedure. Late stage schizont-infected cells were concen- trated by the gelatin flotation method,12 diluted to 25% parasitemia in freshly washed O+ erythrocytes, suspended in RP-S at a 2.5% hct, and incubated in a candle jar atmosphere at 37°C to allow merozoites to invade fresh cells. After 4 h, invasion was terminated by sorbitol lysis18 of all remaining schizonts, resulting in young ring-staged para- sites 0-4 h old. Parasitemia was then adjusted to 10% with additional red blood cells (RBC). Serum preparation The procedure for collection of sera and the region of Sudan from which the sera were obtained has been described previously.4 Both normal human sera (NHS) and sera 72 containing enough CFF to reduce parasite incorporation of hypoxanthine by 50% when used at 25% serum concentration in culture were each pooled from 20 individuals. Following heat-inactivation at 56°C for 30 min, the serum lipoproteins were removed by precipitation with 0.05% w/v dextran sul- fate26 (500,000 nw, Sigma Chemical Co., St. Louis, MO). Residual dextran sulfate was precipitated with 5% v/v satu- rated BaClZ. The serum pools were then dialysed 1:106 against minimal medium in Spectrapor 12,000-14,000 MWCO tubing (Spectrum Medical Industries, Inc., Los Angeles, CA) to remove any remaining BaCl and equilibrate the sera nutri- 2 tionally for metabolic studies. After dialysis, sera were sterilized by filtration through 0.45 um pore membranes (Schleicher & Schuell, Keene, NH). Metabolic inhibitiOn assays Inhibition was determined by a modification of a tech- nique described previously.14 The CFF and NHS pools were diluted to 25% serum concentration in minimal medium supple- mented with 5% undialysed PHS. The presence of 5% back- ground PHS 1J1 each pool tested provided completely for the nutritional requirements of the parasites.9 Synchronized parasites 0, 8, 16, 24, and 32 h post-invasion (p.i.) were dispensed into 96-well microculture plates, 1 ul packed cell volume/well, and exposed to CFF serum for various lengths of time (Figure 1). Since all experiments used parasites synchronized to a 4 h age differential, these times actually represent parasites 0-4, 8-12, 16-20, 24-28, and 32-36 h 73 WWW ' i6 24 32 40 HOURS POST-INVASION falciparum to CFF at C31 OD Figure 1d Periods of exposure of 3, various times post-invasion 74 old, but for convenience and clarity, the age spread shall not be given throughout the report. Metabolic inhibition was determined after exposure of triplicate wells to 200 ul of each serum pool and cultivating parasites of various ages for 8, 16, 24, 32 or 40 h. Inhibition of purine and amino acid incorporation were determined by scintillation spectrometry. Each microculture well was pulsed with 2 uCi of [3H]hypoxanthine (sp. activity = 10.0 Ci/mmol, New England Nuclear, Boston, MA) and 0.2 uCi of [U-14C]phenyla1anine (sp. activity = 450 mCi/mmol, ICN Radiochemicals, Irvine, CA) for the final 8 h of cultivation, before harvesting the parasites onto glass-fiber filter strips using a cell harvester (Bellco Glass, Inc., Vineland, NY). Parasite incorporation of radiolabel was measured with a Beckman LS 7500 scintillation spectrometer. All values were corrected for background incorporation of label by uninfected erythrocytes and expressed as percent inhibition 3 standard error of the mean. Percent inhibition was calculated with the following equation: % inhibition = CPM of NHS well - CPM of CFF well x 100% CPM of NHS well Inhibition of glucose consumption was determined by UV spectrophotometry. At the time of cell harvest, 100 111 aliquots of culture medium were removed from each well for measurement of glucose concentration using a glucose assay kit (Glucose No. lS-UV, Sigma Chemical Co., St. Louis, MO). The assay procedure is based on the quantitative conversion 75 of glucose to glucose-6-phosphate and then to 6-phosphogluconate by hexokinase and glucose-6-phosphate dehydrogenase, respectively. The latter reaction generates NADPH from NADP, of which the absorbance at 340 nm was measured with a Gilford Model 2000 spectrophotometer. Change in absorbance (AA), proportional to parasite-specific glucose consumption, was determined by subtracting the absorbance of culture medium from parasite-infected erythrocyte wells from that of uninfected RBC control wells. All values were expressed as percent inhibition 3 standard error of tin: mean. Percent inhibitino was calculated with the following equation: % inhibition = AA of NHS well - AA of CFF well x 100% AA of NHS well Statistical comparisons were made using Student's 3 test. Giemsa-stained thin films were prepared from identical wells for visual assessment of parasite inhibition. 76 RESULTS St33e-dependency The parasite erythrocytic cycle~ was divided into 5 sequential 8 h time periods. Synchronized parasites corre- sponding ix) each period were exposed to CFF serum, and the serum's effect on each of three parasite metabolic proc- esses: hypoxanthine and phenylalanine incorporation into nucleic acids and proteins, respectively, and glucose con- sumption, was determined (Figure 2). The greatest inhibition in the measured metabolic parameters occurred during the first time period, the 0 h to 8 h p.i. ring-stage parasites. Glucose utilization, purine incorporation and amino acid incorporation were significantly inhibited 63.0% (p<0.05), 38.6% (p<0.001) and 21.9% (p<0.05), respectively. Percent inhibition progressively decreased in parasites exposed to CFF during later periods. In parasites cultivated from 8 h to 16 h p.i. in CFF, glucose consumption was reduced 38.8%, while hypoxanthine and phenylalanine incorporation were inhibited 28.9% (p<0.01) and 17.9% (p<0.05), respectively. During the third time period examined (16 h to 24 h p.i.), corresponding to the transition from ring to trophozoite- stage parasites, CFF serum continued to inhibit purine and amino acid incorporation by 26.8% (p<0.01) and 19.2% (p<0.05), respectively, but glucose utilization was no longer affected. Parasites exposed to CFF during the fourth or fifth time intervals (24 h to 32 h and 32 h to 40 h p.i., respectively) were not significantly' inhibited 1J1 any' of their observed metabolic processes. 77 60 % INHIBITION is O % DISTRIBUTION v r a— 8 IS 24 32 40 AGE (HOURS POST-INVASION) Figure 2. Effect of CFF on various stages of 3. falciparum. 8. Effect of 8 h exposure to CFF serum on three metabolic processes in parasites harvested at various times. Results are expressed as percent inhibition compared to control cultures. Symbols: hypoxanthine incorporation,-: phenyl- alanine incorporatiom‘: glucose consumption, .. b. Differential distribution of various stages expressed as percentage of total parasites present at cell harvest. Sym- bols: ring stages, .: trophozoite stages,.: schizont stages, ‘ 78 Time-dependency Data given above demonstrated that parasites exposed to CFF for 8 h at various times post-invasion were more sensi- tive as younger stages, but different metabolic processes were selectively affected. In experiments conducted to determine the effects of longer exposure to CFF, the various metabolic parameters exhibited either time-dependent or time-independent sensitivity to the action of CFF. Para- sites of various ages were exposed to CFF for increasing duration and the degree of inhibition observed was compared in cultures initiated at the same parasite age. The sensi- tivity of incorporation of radiolabeled hypoxanthine to CFF (Table 1) was the greatest (significant at p<0.001) in cul- tures initiated with early ring stages (0 h p.i.). In those cultures, inhibition determined after continuous exposure to CFF for one, two, three, four, and five 8 h pulses was 38.6%, 41.5%, 52.6%, 48.4% and 47.1% respectively: thus remaining relatively constant, independent (ME the duration of CFF exposure. Parasites initially exposed to CFF 8 h and 16 h p.i. and maintained in CFF serum for longer duration were also significantly inhibited (p<0.001) in purine incor- poration, with mean inhibitions of 31.2% and 30.9%, respec- tively. As seen before, the length of time during which the parasites were grown in the presence of CFF did not signifi- cantly affect the amount of inhibition seen. Parasite cul- tures initiated with 24 h trophozoite stages were still 79 TABLE 1 Inhibition of hypoxanthine incorporation in g. falciparum by CFF Cultivation Hypoxanthine Incorporation % Inhibition Starta Endb Normal Serum CFF Serum 0 8 1272 1 76C 781 1 26 38.6 (6)d 16 2255 1 165 1319 _ 28 41.5 (5) 24 8424 1 632 3997 1 199 52.6 (6) 32 23608 1 1152 12172 1 534 48.4 (6) 40 66552 1 1231 35219 1 778 47.1 (6) 8 16 2391 1 67 1701 1 114 28.9 (6) 24 8137 1 714 5862 1 508 28.0 (5) 32 25592 1 1479 16210 1 669 36.7 (6) 40 80154 1 1178 55066 1 2070 31.3 (6) 16 24 7551 1 263 5528 1 459 26.8 (6) 32 22530 1 1451 13536 1 615 39.9 (6) 40 77818 1 957 57570 1 2314 26.0 (6) 24 32 18345 1 1003 16641 1 937 9.3 (6) 40 77913 1 1737 66327 1 3635 14.9 (6) 32 40 65368 1 1216 59988 1 3299 8.2 (6) LT aParasite age post-invasion (h) at initiation of assay bParasite age post-invasion (h) at termination of assay CIncorporation (CPM/well) 1 standard error of the mean dNumber of replicates in parentheses 80 sensitive to CFF (p<0.05), but much less so than with cul- tures started with younger parasites. Inhibition was 9.3% after one 8 ll pulse and 14.9% after two 8 h pulses, aver- aging 12.1%. Hypoxanthine incorporation was not inhibited by CFF when parasites were exposed 32 h p.i. Similar data were seen in inhibition assays using phenylalanine incorporation (Table 2): however, inhibition was less than that observed in assays measuring hypoxanthine incorporation. Although the effect of CFF on both hypoxanthine and phenylalanine incorporation appeared independent of the number of time intervals during which the parasites were exposed to CFF, such was not the .case for CFF's effect on glucose utilization by the parasite. Early ring stages cultivated for one and two 8 h pulses were significantly inhibited 63.0% (p<0.05) and 69.8% (p<0.01), respectively (Table 3). However, when these parasites were continuously exposed to CFF for three, four, and five 8 h periods, inhi- bition progressively decreased to 39.7% (p<0.05), 24.4% (p<0.05) and 11.1% (p<0.01), respectively. A similar pattern was observed in tests initiated with 8 h p.i. parasites. As the duration of exposure of parasites to CFF serum was increased, inhibition of glucose consumption decreased, from 38.8% after one 8 ll pulse to 7.3% after four 8 h periods. Furthermore, no inhibition of glucose utilization was seen in cultures begun with parasites 3 16 h p.i. 81 ' TABLE 2 Inhibition of phenylalanine incorporation in g. falciparum by CFF Cultivation Phenylalanine Incorporation % Inhibition Starta Endb Normal Serum CFF Serum 0 8 1849 1 90C 1444 1 58 21.9 (6)d 16 2912 1 199 2219 1 77 23.8 (5) 24 5134 1 262 3055 1 141 40.5 (6) 32 7748 1 447 4945 1 208 36.2 (6) 40 13644 1 235 9258 1 434 32.2 (6) 8 16 3050 1 105 2505 1 92 17.9 (6) 24 4732 1 274 3702 1 228 21.8 (5) 32 7880 1 514. 6073 1 237 22.9 (6) 40 15709 1 196 12472 1 696 20.6 (6) 16 24 4829 1 109 3900 1 292 19.2 (6) 32 7606 1 471 5501 1 258 27.7 (6) 40 15331 1 517 12419 1 503 19.0 (6) 24 32 7137 1 381 7013 1 330 1.7 (6) 40 15679 1 448 14767 1 604 5.8 (6) 32 40 15378 1 249 15459 1 737 0.0 (6) aParasite age post-invasion (h) at initiation of assay bParasite age post-invasion (h) at termination of assay CIncorporation (CPM/well) 1 standard error of the mean dNumber of replicates in parentheses WV ‘4. 82 TABLE 3 Inhibition of glucose utilization in B. falciparum by CFF Cultivation Glucose Utilization % Inhibition Starta Endb Normal Serum CFF Serum 0 8 3.5 1 0.6c 1 3 1 0 6 63.0 (9)d 16 6.3 1 1.0 1.9 1 0 7 69.8 (10) 24 11.2 1 1 6 6.8 1 0 9 39.7 (7) 32 21.5 1 1.5 16.2 1 l 3 24.4 (6) 40 38.7 1 0.5 34.3 1 0 8 11.1 (4) 8 16 4.1 1 0 9 2.5 1 0.9 38.8 (10) 24 8.3 1 l l 6.3 1 l 3 24.0 (8) 32 20.0 1 0.8 17.8 1 1 9 11.3 (6) 40 38.8 1 1.2 36.0 1 1.5 7 3 (4) 16 24 4.2 1 0.8 4 3 1 0 6 0.0 (8) 32 15.9 1 1.2 15.5 1 0.8 2.6 (6) 40 35.1 1 1.1 34.1 1 0.8 2 8 (4) 24 32 12.5 1 1.4 11.6 1 1.0 7 9 (6) 40 32.2 1 0.6 30.6 1 1.4 5 2 (4) 32 40 19.1 1 1.1 18.1 1 1.7 4.9 (4) aParasite age post-invasion (h) at initiation of assay bParasite age post-invasion (h) at termination of assay CGlucose utilized (nM/well) 1 standard error of the mean dNumber of replicates in parentheses 83 DISCUSSION Since certain aspects of Plasmodium metabolism have 7,11,19,20,21,27 demonstrable stage dependence, the present studies were conducted to determine if CFF affected the 33 vitro development of 3. falciparum in a stage- and time- dependent manner. A moderately inhibitory serum pool was used in these experiments to determine the effects of CFF on parasite metabolism since more inhibitory sera quickly kill the parasites, rendering longer exposure times to CFF mean- ingless. The erythrocytic cycle was divided into sequential 8 h time periods and highly synchronous parasites correspon- ding to each period were exposed to CFF serum for various lengths of time. The effects of CFF on hypoxanthine and phenylalanine incorporation into nucleic acids and proteins, respectively, and glucose consumption were then examined for stage- and time-dependency. In contrast to the activity reported for the antimalarial drug chloroquine,27 in which ring stages were the least sensitive, it was found that early ring-staged parasites were the most sensitive to an 8 h exposure to CFF, regardless of the metabolic parameter examined. Assays begun with older parasites and cultivated for 8 ll in CFF serum showed progressively less inhibition. Although inhibition of glucose utilization was not observed in tests initiated with parasites 3 16 h p.i., purine and amino acid incorporation remained' inhibited in parasites first exposed to CFF at this age. However, in experiments begun with parasites 3_24 ll ELI”, CFF had little or no effect on any of the three measured metabolic parameters. 84 Both hypoxanthine anui phenylalanine incorporation exhibited no time—dependent effects. Regardless of the number of 8 h periods that parasites were continuously exposed tn) CFF, percent inhibition in tests initiated with parasites of identical ages remained relatively constant. However, in contrast to the time-independent inhibition observed in the incorporation studies, CFF's effect on glu- cose consumption varied inversely to the duration of expo- sure to cmTu As the parasites matured in the presence of CFF, inhibition of glucose utilization decreased, with lit- tle or no reduction or consumption observed as parasites entered schizogony. It appears likely that the effect of CFF on glucose utilization is not persistent, and once the parasite is past the .16 h p.i. ring stage, it becomes refractory to CFF inhibition of this process. A possible explanation of this observation may be found in the studies of Sherman and Tanigoshi21 who showed that the permeability of g. lophurae-infected erythrocytes was markedly enhanced for a glucose analog, and this enhancement was due to increases in both carrier-mediated and simple diffusion components of transport of the sugar. Furthermore, the size of the parasite positively correlated with this observed enhancement. The increased erythrocyte permeability observed 111 maturing parasites might then account for the decrease 1J1 CFF inhibition of glucose utilization seen in maturing parasites. If glucose transport was being inhib- ited by CFF, the increased glucose permeability of maturing 85 g. falciparum-infected erythrocytes could overcome this effect, allowing glucose utilization to proceed at normal rates. Further experimentation will be required to deter- mine if such is the case. It is important to note the implications these results have on determination of CF]? titers in malaria-immune sera using cultured parasites. First, although our results have shown that hypoxanthine incorporation is a more sensitive indicator of CFF inhibition than phenylalanine incorpora- tion, this observation does not lead to the conclusion that CFF affects nucleic acid more than protein synthesis. Hypo- xanthine is an exogenously required medium constituent,lo whereas phenylalanine is not,8’ thus protein synthesis may continue without incorporation of the radiolabeled amino acid, even though it is readily incorporated if available to the parasite. Second, despite the fact that CFF affected the three metabolic parameters differently, metabolism in young ring stages was always inhibited more than in other stages. Thus, the amount of inhibition any given immune serum may have on cultured g. falciparum will depend upon the degree of synchrony of the parasites, and their post- invasion age at the time of initial CFF exposure. 86 ACKNOWLEDGMENTS This investigation was supported by Research Grant AI l6312, from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service, under the auspices of the Michigan State University-Sudan Ministry of Health "Collaborative Research on Parasitic Diseases in Sudan" project. 87 LIST OF REFERENCES Barnwell, J. W., and Desowitz, R. S., 1977. Studies on parasitic crisis in malaria: I. Signs of impending crisis in Plasmodium berghei infections of the white rat. Ann. Trop. Med. Parasitol., ll: 429-433. Butcher, G. A., Maxwell, L., Cowen, N., Clancy, R. L., and Stace, J. D., 1985. The development and ultra- structure of Plasmodium falciparum damaged in vitro by human ”crisis" sera and by chloroquine. Aust. J. Exp. Biol. Med. Sci., £3: 9-18. Butcher, (I. A., Mitchell, G. H., and Cohen, 3., 1978. Antibody mediated mechanisms of immunity to malaria induced by vaccination with Plasmodium knowlesi merozoites. Immunology, 23: 77-86. Carlin, J. M., Vande Waa, J. A., Jensen, J. B., and Akood, M. A. S., 1984. African serum interference in the determination of chloroquine sensitivity in Plas- modium falciparum. z. Parasitenkd., 12: 589-597. Clark] 10 A0] Allison! A. CD] and COX] F. E., 1976. Protection of udce against Babesia and Plasmodium with BCG. Nature (London), 259: 309-311. Clark, I.A., Cox, F. E., and Allison, A. C., 1977. Protection of mice against Babesia spp. and Plas- modium spp. with killed Corynebacterium parvum. Parasitology, 13: 9—18. Deans, J. A., Thomas, A. W., and Cohen, 5., 1983. Stage- specific protein synthesis by asexual blood stage parasites of Plasmodium knowlesi. Mol. Biochem. Parasitol., g: 31-44. Divo, A. A., Geary, T. G., Davis, N. L., and Jensen, J. B., 1985. Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth. J. Protozool., g3: 59-64. Divo, A. A., and Jensen, J. B., 1982. Studies on serum requirements for the cultivation of Plasmodium falciparum. 1. Animal sera. Bull. W.H.O., £9: 565-569. 10. ll. 12. 13. 14. 15. 16. 17. 18. 19. 88 Divo, A. A., and Jensen, J. B., 1982. Studies on serum requirements for the cultivation of Plasmodium falciparum. 2. Medium enrichment. Bull. W.H.O., g9: 571-575. Gritzmacher, C. A., and Reese, R. T., 1984. Protein and nucleic acid synthesis during synchronized growth of Plasmodium falciparum. .J. Bacteriol., légz 1165-1167. Jensen, J. B., 1978. Concentration from continuous cul- ture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum. Am. J. Trop. Med. Hyg., 31: 1274-1276. Jensen, J. B., Boland, M. T., and Akood, M. A., 1982. Induction of crisis forms in cultured Plasmodium falciparum with human immune serum from Sudan. Science, 216: 743—746. Jensen, J. B., Boland, M. T., Hayes, M., and Akood, M. . A., 1982. Plasmodium falciparum: Rapid assay for in vitro inhibition due to human serum from resi- dents of malarious area. Exp. Parasitol., £3: 416-424. Jensen, J. B., Boland, M. T., Allan, J. S., Carlin, J. M., Vande Waa, J. A., Divo, A. A., and Akood, M. A. S., 1983. Association between human serum-induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan. Infect. Immun., 31: 1302-1311. Jensen, J. B., Capps, T. G., and Carlin, J. M., 1981. Clinical drug-resistant falciparum malaria acquired from cultured parasites . Am. J . Trcm. M96. 329.: 29: 523-525. Jensen, J. B., and Trager, W., 1977. Plasmodium falcip- arum in culture. Use of outdated erythrocytes and description of the candle jar method. J. Parasitol., §§: 883—886. Lambros, C., and Vanderberg, J. P., 1979. Synchroniza— tion of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol., §§: 418-420. Newbold, C. I., Boyle, D. B., Smith, C. C., and Brown, K. N., 1982. Stage specific protein and nucleic acid synthesis during the asexual cycle of the rodent malaria Plasmodium chabaudi. Mol. Biochem. Parasitol., é: 3§;44. “5r 20. 21. 22. 23. 24. 25. 26. 27. 89 Polet, H., and Barr, C. F., 1968. DNA, RNA, and protein synthesis in erythrocytic forms of Plasmodium knowlesi. Am. J. Trop. Med. Hyg., 11: 672-679. Sherman, I.W., and Tanigoshi, L., 1974. Glucose trans- port in the malarial (Plasmodium lophurae) infected erythrocyte. J. Protozool., 21: 603-607. Taliaferro, W. H., and Taliaferro, L. G., 1944. The effect of immunity on the asexual reproduction of Plasmodium brasilianum. .I.Infect. Dis., 1;: 1-32. Tharavanij, S., Warrell, M. J., Tantiavanich, S., Tapchaisri, P., Chongsa-Nguan, M., Prasertsiriroj, V., and Pataradotikul, J., 1984. Factors contrib- uting to the development of cerebral malaria. I. Humoral immune responses. Am. J. Trop. Med. Hyg., gg: 1-ll. Trager, W., and Jensen, J. B., 1976. Human malaria parasites ix: continuous culture. Science, 193: 673-675. Vande Waa, J. A“, Jensen, J. B., Akood, M. A. S., and Bayoumi, R., 1984. Longitudinal study on the i_n vitro immune response to Plasmodium falciparum in Sudan. Infect. Immun., 5;: 505-510. Warnick, G. R., Nguyen, T., and Albers, A. A., 1985. Comparison of improved precipitation methods for quanitification of high-density lipoprotein cho- lesterol. Clin. Chem., £1: 217-222. Yayon, A., Vande Waa, J. A., Yayon, M., Geary, T. G., Jensen, (I. B., 1983. Stage—dependent effects of chloroquine on Plasmodium falciparum in vitro. :1. Protozool., g9: 642-647. um:— _..- T I F- 90 SUMMARY The objective of this research was to further charac- terize the various effects of crisis form induction on gigg- modium falciparum. As a result of this effort, the fol- lowing conclusions have been reached: 1) The presence of CFF in patient serum interferes with field tests of parasite drug sensitivity, resulting in a test failure rate > 70%. . 2) Inhibition of parasite development due to CFF and the antimalarial drugs chloroquine and mefloquine are addi— tive in effect. 3) Although human CFF and TB sera and rabbit BCG-LPS sera all induce crisis forms in cultivated P. falciparum, only BCG-LPS sera are cytotoxic to L-M cell cultures, indi- cating that TNF is most likely not responsible for crisis form induction in the human sera tested. 4) Human y-interferon has no direct effect on parasite growth. 5) CF]? exhibits stage-dependent inhibition as deter- mined by various metabolic criteria. Hypoxanthine and phenylalanine incorporation into parasite nucleic acids and protein, respectively, and glucose consumption are most ; a. u'BaJ- 91 inhibited in cultures initiated with parasites O h post- invasion. Inhibition correspondingly decreases in tests started with progressively older parasites. 6) Variation in length of exposure to CFF has little effect on inhibition of parasite incorporation of hypoxan- thine and phenylalanine. However, in contrast to the time- independent inhibition observed in purine and amino acid incorporation, inhibition of glucose consumption varies inversely to the time of cultivation in the presence of CFF. As the parasites mature in the presence of CFF, inhibition of glucose utilization decreases, with little or no reduction of consumption observed as parasites enter schizogony. I.