t‘ (‘1‘ v I" r- 4’3 {3‘ fl- ,2, 5-; ,3 .Lk‘wm; / 5-3.. e" ‘. E2 This is to certify that the thesis entitled PARTIAL CHARACTERIZATION OF THE PYROGEN FACTOR IN PLASMODIUM FALCIPARUM presented by Lenka Dimanina has been accepted towards fulfillment of the requirements for M. 3~ degree in fliflimLLabor‘atory Science Major professor Date November 20, T98] MSU ‘: “LIBRARIES ; m. 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. ‘. x i - “i ..-,‘-. . a \ ~4' 4“... . ‘ I. v - . .' ‘ . . . v I \ '_.. 1.- “.41 a >.b-/ -\~ ‘ -Ib . - v 4 .. 2.“: \I ' a ‘ ' n - | , .-: "i l P . v l‘ . l. '- \ ~ .‘ L". . , ‘. ‘ .‘ ‘ .- . _ t.“ 4' r , ; .| I _ ‘ ' ' -- ‘:.'-‘30 a _ n .3, . . ‘ - ‘ .'! .d g ',I ._ . . . ‘ I .d , . ' ‘ ' ‘ ' ‘9 1* ~. ‘ ~ . ._ _ -- i7 ' \‘ L. c ‘v \. "' I ‘ v 1.. ,. H, .c‘."."»‘~ .‘a-.r";‘= "T" "13*.“- n ,. _ PARTIAL CHARACTERIZATION OF THE PYROGEN FACTOR IN PLASMODIUM FALCIPARUM By Lenka Dimanina A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology I981 ABSTRACT PARTIAL CHARACTERIZATION OF THE PYROGEN FACTOR IN PLASMODIUM FALCIPARUM By Lenka Dimanina In an effort to clarify the genesis of fever in falciparum malaria, parasites were cultured jg_vitrg_and fractions assayed for pyrogenicity in rabbits or for "endotoxin-like” activity in the Limulus amebocyte lysate assay. Parasite post—schizont residual material, rich in the malarial pigment hemozoin, was found to have minimal activity in the Limulus assay. Extracts from schizont-infected red cells were positive in the assay and thus an "endotoxin-like” substance may be responsible for the febrile response in falciparum malaria infections. Treatment with pancreatic protease altered but did not significantly decrease the "endotoxin—like" activity in the parasite extract. Post-ribosomal super- natant, by ultracentrifugation of the parasite extract, as well as the subcelluler and membrane pellet, produced strong febrile responses in rabbits. ACKNOWLEDGEMENTS I wish to express my sincere appreciation and gratitude to Dr. James B. Jensen for his guidance, support and advice in my research. My sincere thanks are due to the other members of my guidance committee, Martha Thomas and Sue Estry for their helpful advice, encouragement and support throughout my graduate program. A special thanks is also due to Dr. Harland L. Verrill and Ruth Vrable for the many useful discussions and their skilled technical assistance in various aspects of this work. My thanks also go to my friends, graduate students, especially Alan Divo, for their assistance and encouragement which made the completion of this project a worthwhile and educational experience for me. I particularly wish to thank the late Dr. Frank V. Hodges, Director of Laboratory, Hurley Medical Center, and Rose Cusenza, Chief Medical Technologist, Hurley Medical Center, who contributed to my education in ways too numerous to possibly name. Most importantly, I would like to express my love, gratitude and appreciation to my son, Sashe, for being so supportive, patient and most important, believing in me which made it all possible. To my son, Sashe Dimitroff TABLE OF CONTENTS Page INTRODUCTION ........................................................ l Fever .......................................................... 2 The Parasite Life Cycle ........................................ 6 Malaria and Fever .............................................. 9 MATERIALS AND METHODS ............................................... l5 Limulus Amebocyte Lysate Assay (LALA) .......................... l5 Parasite Extracts in LAL Assay ................................. l6 Experiment 1 .............................................. 16 Experiment 2 .............................................. l6 Experiment 3 .............................................. l7 Fever-Testing of Parasite Extracts in Rabbits .................. l7 Experiment la ............................................. l8 Experiment 2a ............................................. l8 RESULTS ............................................................. 19 DISCUSSION .......................................................... 26 LIST OF REFERENCES .................................................. 28 iv LIST OF TABLES Table Page 1 Limulus Amebocyte Lysate Test 20 2 Limulus Amebocyte Lysate Test 21 3 Limulus Amebocyte Lysate Test 22 Figure LIST OF FIGURES Page Temperature Changes Before and After P. falciparum Extract Injections - Rabbit #l 24 Temperature Changes Before and After P. falciparum Extract Injections - Rabbit #2 25 vi INTRODUCTION Hippocrates, writing two millenia ago, categorized certain common fiavers according to their periodicity. We now recognize these "quotidian", u‘tertian" and “quartan” fevers as the different sorts of malaria, caused buy organisms of the genus Plasmodium. Our basic knowledge has come from tune work of Laveran and Golgi in the late 19th Century. Working independ- ently, Laveran correlated the disease malaria with the presence of ()rganisms in the blood, and Golgi attributed the periodicity of the fevers to the cyclic life cycle of the parasite within the host. But while the correlation of clinical fever spikes with the waves of [marasites released into the bloodstream was a great step forward in reationalizing this ancient affliction, the physiology associated with tliis correlation remains largely obscure. In this study, we will seek t1) examine some posSible mediators between fever and parasite. That the connection should still be unclear is not surprising, for i't is only in the past 40 years that what now seem the most basic processes crf fever, have begun to be elucidated. The exact mechanisms, the mediators ivivolved and even the physiological utility of fever all remain areas of (5) iritense research. - Moreover, malaria parasites, unlike the more iritensively studied bacterial pathogens, pose burdensome and sometimes ‘hisuperable obstacles to their exact observation and study. It is only iri the past five years that methods have become available for routine, ill vitro cultivation of P. falciparum, deadliest of the marlarial parasites.(45) 2 The new methods permit, to a degree unknown before, the study of the physiological, immunological and pharmaceutical interactions between host, parasite and chemotherapeutic agent. They also permit the isolation and study of substantial populations of the parasites at different stages of their life cycle.(17’ 25’ 39’ 41’ 45) We used these culture and separation techniques to isolate fractions ()f cultivated P, falciparum and subsequently tested their ability to FJYOdUCE fever in rabbits. The rabbit has been previously established as ain appropriate test subject with similar, if somewhat less sensitive, r~eactions to a number of common fever-producing agents.(23) Before discussing the febrile response of rabbits to our various F’lasmodium fractions, we will review briefly both fever and the parasite 1 ife cycle. FevJ Fever is the result of a complex process by which the thermoregulatory (:enter of the central nervous system (CNS) raises its "set point“. That ‘is, there is an elevation of the temperature which the body actively rnaintains by increasing or reducing heat production and heat dissipation. T'hat is in contrast to non-CNS-mediated causes of hyperthermia, such as e> blood monocytes and alveolar macrophages, certain fixed-cell macrophages, notably the hepatic Kupffer cells and the splenic sinusoidal cells, are capable of producing EP. FEVER 0 CONSUMPTION/HEAT CONSERVATION 2 MONOAMINES ' PROSTAGLANDINS CYCLIC AMP THERMOREGULATORY CENTER ENDOGENOUS PYROGEN NEW PROTEIN SYNTHESIS NEW m-RNA TRANSCRIBED KUPFFER CELLS 1‘ NEUTROPHILS SPLENIC AND PHAGOCYTIC LEUKOCYTES MONOCYTES ALVEOLAR _ MACROPHAGES [P EOSINOPHILS EXOGENOUS PYROGENS VIRUSES, BACTERIA, FUNGI, BACTERIAL PRODUCTS, ENDOTOXIN, ETIOCHOLANOLONE, Ag-Ab COMPLEXES, POLYNUCLEOTIDES, ANTIGENS (VIA LYMPHOKINES FROM SENSITIZED LYMPHOCYTES) . Figure 1. Proposed Mechanism of Fever Production 5 Repeated doses of exogenous pyrogens induce a state of tolerance, in which increasingly large doses are needed to produce a febrile response.(4) Endogenous pyrogen may act directly on the hypothalamus, or it may need to be mediated by prostaglandins or vasoacti‘ve monoamines. The prostaglandin level in the CNS fluid does not appear to rise and fall with febrile spikes, and the evidence seems to be leaning away from a mandatory role for prostaglandin mediators.(3’28) Endogenous pyrogens (EP) are' capable of producing fevers almost immediately upon injection into the experimental subjects, while exogenous pyrogens may take up to three hours or more to produce an unambiguous fever. The quantity of EP needed to produce fevers in both humans and rabbits has been estimated as between 30 and 50 nanograms, but the issue is clouded‘by the question of how well the EP has been purified. _ Atkins et al(2) outlined three general routes by which leukocytes appear to release EP. First is the release of EP upon phagocytosis of a stimulatory agent, such as bacterial cell walls. Phagocytosis does not necessarily result in EP production, as has been shown by experiments in which the leukocytes phagocytize latex balls without concomitant EP release.(5) The phagocytic mechanism is invoked to explain the fevers of septicemia and infection of deep tissues. The second general route is stimulation of the leukocytes by factors which induce other aspects of inflamation, including chemotactic factors and/or complement. The pyrogenicity of most inflammatory factors and the inflammatory capabilities of most pyrogens remain to be explored. The third basic mechanism of EP production cited by Atkins is its spontaneous rel ease by certain tumor and Hela cells. The EP-releasing leukocytes have receptors for both complement and Fe portions of antibodies, and antigen-antibody complexes have been shown by Bernheim, Francis and Atkins to stimulate EP production.(6) Interestingly, 10:1 excesses of either antigen or antibody, working by weight from equivalence zone, induced EP production, but the precipitated complexes themselves did not, even though they were phagocytized by the leukocytes. Cell-mediated immune responses are also believed to play a part in fever production, as T-cell products such as lymphokines are able to activate leukocyte EP production.(33) The Parasite Life Cycle The life cycle of the malarial parasite spans two hosts;the anopheline mosquito and either an avian or a marrmalian host. Human infection by _P_. falciparum usually begins with the bite of an 1‘ nfected .anopheline mosquito, which, as it takes its blood meal, injects the malarial sporozoite into the bloodstream. The sporozoite circulates through the blood for approximately 30 minutes before it absorbs to and invades a hepatic parenchymal cell. Within the hepatic cell, the parasite grows and its nucleus repeatedly divides until approximately 40,000 merozoites are produced, each about 0.7 microns in diameter. These are then released into the bloodstream. The prepatent period, which is the interval between injection of the sPorozoite and the appearance of parasites in the blood, ranges between six days to a month, most commonly two weeks or a little less.(10) The first fever above 101 F usually occurs within one to two days after the end of the prepatent period. 7 The merozoites released by the liver cell are quickly taken up by the red blood cells.- There is evidence for a specific protein or glycoprotein recognition site on the erythrocyte surface, a site which is sensitive to 'trypsin treatment but not chymotrypsin.(34) One hypothesis for the mechanism of parasite uptake is that a highly basic (70% histidine) protein, carried by the merozoite in terminal structures called rhoptries and micronemes, may cause a local disturbance iri the erythrocyte membrane.(29) During its uptake by the erythrocyte, the parasite comes to be enclosed hay a second membrane just overlaying its own plasma membrane. This second Iwuambrane, called the parasitiphorous vacuolar membrane (PVM), is erythrocyte- cjearived, but appears to come under increasing control by the parasite within. Inside the erythrocyte, the merozoite dedifferentiates, first appearing as a "ring" or "applique" in which the lumen of the ring is a large vacuole cxantaining cytoplasmic material of the host, and the "stone" of the ring is cfliromatin. As the trophozoite grows, "knobs" develop which appear to be beneath .the surface of the erythrocyte membrane, and seem to cause its iricreasing deformation as the parasites mature. These knobs have been ft>und to form focal connections with endothelial cells and may participate iri the sequestration in deep tissue of the later stages of parasite deevelopment.(]) The younger trophozoites appear to have smaller and more rquerous knobs while older, segmented forms appear to have larger, fewer kuiobs. There appears to be some shedding of knob material during the growth of the parasite within the red blood cell.(29) Antigenically, these knobs have been found to be distinct from the surrounding erythrocyte surface. Experiments in which parasite cultures were incubated With 328—labeled methionine showed greater radioactivity in membrane fractions derived from knobby erythrocytes than in non-knobby 8 fractions. In addition, knobby fractions showed altered binding characteristics with the glycoprotein probe, concanavalin A.(28) During its growth phase, the parasite utilizes the hemoglobin of its host as a source of amino acids. In addition, a substantial part of the hemoglobin is converted to the characteristic golden malarial pigment, hemozoin. Hemozoin has been found to consist largely of monomers and dimers of hematin. Some of it consists of ferriprotoporphyrin coupled to a plasmodial protein and some methemoglobin is also present. Hemozoin is inert insofar as respiration is concerned and otherwise nontoxic accord- ing to Maegraith, who studied the pigment in great detail during the 19503.92) The mature trophozoite nucleus undergoes a series of divisions which lead to the production of a schizont in which the several nuclei are each surrounded by a little bit of cytoplasm, to form merozoites. The usual number is about 16. When fully mature, the merozoites are released to infect more erythrocytes. The mechanism of release is unknown. After one or more generations of merozoite formation some of the parasite population will develop into the sexual stage within the infected red blood cell. The micro- or macrogametocyte formed may then be taken up by an anopheline mosquito to begin the sporogoni‘c part of its life cycle. In the gut of the mosquito the hametocytes develop into gametes, fuse to form a zygote and then migrate through the gut wall to form an oocyst on its outer surface. The oocyst then ruptures, releasing sporozoites into the insect's hemocel. Subsequently the sporozoites migrates into the mosquito's salivary glands and thus come to the injected into a mammalian host during feeding, starting a new cycle. 9 'Malaria and Fever It has been long recognized that many of the signs and symptoms of malaria resemble those of an acute general inflammation.(8) These include increased permeability of the vascular endothelium, permitting the loss of fluids and the ultimate blockage of circulation. The signs also include profound vasomotor disturbances and, of course, fever. Maegraith analyzed the blood of animals infected with E, knowlesi and found decreased levels of bradykininogen and kallidinogen, precursor forms of the vasoactive peptides bradykinin and kallidin, respectively.(37) He also found a rise in serun kinin and kallikrein levels. The latter enzyme, which converts kininogen to kinin, has considerable kinin-type activity. These findings were significant in helping to flesh out a model for the pathogenesis of malaria in which vascular collapse plays a central role. In addition, however, it is of interest because of the multiple feedback loops between the kinin system and Hageman Factor XII, and thence, to the fibrinolytic, clotting and complement systems. As mentioned earlier, complement may stimulate endogenous pyrogen production.(42) Complement levels have been shown to be lowered within a few hours of schizont rupture and peak fever in P, vivax_infection, apparently due to activation of the classical complement pathway.(36) Maegraith, as had others before him, pointed out the similarity between the malarial "inflammation" and the febrile paroxysm produced by bacterial infections. The gram—negative bacterial cell wall, and particu- larly the lipid A portion, in addition to being a good pyrogen, is also the cause of gram-negative toxicity, the symptoms of which are those of acute general inflammation. 10 The malarial parasite, a eukaryote, is very different from the bacterial prokaryotes -- the outer surface which it presents to the host immune system being fundamentally different. The bioactive part of the gram-negative bacterial cell wall, the lipopolysaccharide, and particularly its lipid A backbone, is without an obvious structural parallel in the membrane-enclosed parasite. This is true whether the membrane components of the parasite's outer surface are host-derived or produced by the parasite. It is possible, however, that there may be inserted in the parasite's outer membrane some component, whether lipid, protein, carbohydrate or some hybrid structural type, which mimics the biologic activity of the lipopoly- saccharide (LPS). To date, however, no such component has been found; LPS still appears to be uniquely a component of bacterial cells. There is tantalizing evidence that, however different they may be chemically, LPS and the malarial parasite's outer surface share at least some patterns of bioactivity. In 1973 Orsay and Heath(37) injected Plasmodium berghei into the pre- optic hypothalamic region of rabbits, testing for pyrogenicity. The test was positive and the rabbits showed a 1.5 to 3.0 C rise in temperature within four hours of injection. As the authors pointed out, it was impossible to tell whether the effect was due to some substance released by lysed erythrocytes or to a substance produced or stimulated by the parasites. However, the shape and timing of the febrile responses closely resembled those induced by Piromen who used a derivatiie of Pseudomonas LPS, and not those expected from endogenous pyrogen.(37) In the late 1960's and early 1970's, Levin et al(30’3]) published reports on the remarkable sensitivity of an extract of blood cells from the horseshoe crab, Limulus polyphemus, to the presence of lipopolysaccharide. The horseshoe crab has only one sort of blood cell, the amebocyte. A 11 protein extract of this cell will clot in the presence of as little as 0.5 ng. of chloroform-extracted LPS. Since the threshold for detectable fever production by E, 9911 LPS was shown to be in the range of 1 ng., the Limulus lysate test marked a great step forward in the early identification of infection by gram- negative organisms.(29’30) The test was used by Glew and Levin in 1974 to test for endotoxin activity in plasma from volunteers infected with either P, vivax or P, falciparum. The plasma samples used were taken from volunteer patients during periods of schizont rupture, i.e. rupture of erythrocytes infected by mature schizonts with release of merozoites. This event corresponds to the malarial paroxysm, which is the clinical response to schizont rupture in (21) humans. All samples obtained from volunteers infected with P, vivax or P, falciparum produced negative Limulus tests. The patients' temperatures had ranged as high as 40.7 C and the parasitemias as high as 7,000/mm3. In 1980 Tubbs repeated the Limulus assay, using plasma samples from mice infected with E, berghei and from children hospitalized with falciparum malaria. He obtained positive results in both tests, but found that the strength of the positive reaction was not directly related to the degree (46) of parasitemia. He noted that the levels of endotoxin observed were lower than those normally associated with endotoxin-related disease in man. Tubbs also speculated that perhaps his test equipment was more sensitive than that of Glew and Levin. He also suggested that the endotoxin was derived from either the parasites or the endogenous bacteria in the gut. Felton, Prior, Spagna and Kreier pointed out that, regardless of the results of plasma tests of infected individuals, the parasite should itself 46) be studied with the Limulus assay.( They used an ultrasonic device to free 3, berghei cells from their erythrocyte hosts in the method developed 12 ( earlier by Prior and Kreier. 40) The results were positive for endotoxic activity, using a system with minimum sensitivity for an E, gglj_standard of 0.06 ng/ml. of LPS. The level of activity corresponded with approximately 0.5 ng. equivalent of E, gglj_endotoxin per ml. of packed parasites. This ‘was approximately three log units below the threshold level (1 ng/kg) required for fever induction. The authors concluded that some other factor besides the parasites' endotoxin activity must be producing the malarial fever, and that the endotoxin detected by others in plasma from malarious patients is probably derived from endogenous bacteria in the gut. While it is extraordinarily sensitive to the presence of bacterial endotoxin, the Limulus assay is not uniquely specific for bacterial endo- toxin. Elin and Wolff showed in 1973 that the test would give a positive result for such diverse compounds as thrombin, thromboplastin, ribonuclease and the polynucleotides, polyriboadenylic-polyribouridylic acid (poly A/poly U) and polyriboinosinic-polyribocytidylic acid (poly I/poly C).(]9) Guarding against the possibility that these positive results were due to the contamination of samples by bacteria, Elin and Wolff showed the test compounds had very different biochemical and physiological properties than the endotoxin samples. For example, thrombin (but not thromboplastin) showed a lOD-fold drop in Limulus amebocyte lysate (LAL) activity upon extraction with chloroform (31) and usual work-up for LAL test samples while the bacterial standards showed no such drop. Ribonuclease showed a positive LAL reaction before extraction but not after (at a concentration of 64 Kunitz units/ml). The polynucleotides showed drops in LAL activity after extraction. In addition, the ratio of the minimum unextracted concentration of a compound needed to produce a positive LAL test to the amount needed to produce a 0.5 C fever 13 in rabbits was characteristic for the bacterial reference standards, and differed from the ratio of LAL activity/pyrogenic dose shown by the other samples. In view of the ability of such diverse substances to produce positive LAL results, the authors warn that it is essential to take the nonspecificity of the reaction into account when interpreting positive LAL tests of unknown solutions. In 1978 Clark offered the hypothesis that perhaps plasmodial parasites did in fact produce LPS at a very low level, below the levels of Limulus detection and that, in a very susceptible host, this may be enough to produce endotoxin shock.(9) He theorized that non-antibody mediators, released during endotoxin shock, would also be released during malarial attacks and that this would explain a nUmber of puzzling findings. These included the fact that exposure to BCG or Corynebacterium parvum offers some protection against malaria, and the fact that, during malarial infection, susceptibility is increased to bacterial LPS. Using tests with mice he found that after injection with any of four protozoa (P, vinckei petteri, E, berghei, B, inicroti and B, rodhaini) the effect of LPS was greatly enhanced. All of the mice which became ill or died appeared to be affected in the same way as normal mice given a large dose of endotoxin, though often more severely. The changes were pathognomonic for endotoxin shock in mice. He wrote that, if there is a corresponding increase in sensitivity of LPS in humans infected with malaria, there would be pyrexia at plasma concentrations several hundred times lower than that detected by the Limulus test.(9) The increased permeability of the vascular endothelium previously noted could be both cause and effect of a heightened endotoxin response. Another possible route of pyrogenicity, for the most part unexplored, is the non-specific stimulation of B cells noted in the crisis of malarial infection. As previously noted, antigen-antibody (Ag-Ab) complexes have 14 (22) pyrogenic activity. The mitogenic activity of malaria, however, is not unique; indeed, it has been found to be a property of LPS as well,(13) leading us back to the preceding discussion. MATERIALS AND METHODS Limulus Amebocyte Lysate Assay (LALA) The Limulus amebocyte assay was run according to the methods of Levin gt 31‘30) in pyrogen-free glassware baked at 180 C overnight. One-tenth milliliter of Limulus lysate was thoroughly mixed with 0.1 ml. of sample dilution to be assayed and placed in a waterbath at 37 C for one hour. Samples were examined after one hour and monitored for the next 24 hours. All samples were run in duplicate. The criterion for a positive test was the presence of solid gelation. The bacterial endotoxin standard was cell wall material from the organism Salmonella typhimurium, obtained from Difco Laboratories, Detroit, Michigan. The RPMI 1640 powder (Grand Island) media was prepared according to label instructions, with 10.4 mg. per liter of solution to which was added 5.94 g N-2Hydroxy-ethylpiperozine-Ny-Z-ethanesulfonic acid (HEPES). The RPMI 1640 media was tested for endotoxin activity before and after adding 5% sodium bicarbonate and 0.1 ml. gentamycin (GM). The media was also tested after adding 5% pooled serum obtained from the American Red Cross, Lansing, Michigan. Since blood usually gives false-positive reactions in the LAL assay the blood and sera used to grow the malarial parasites were tested before and after heat treatment at 100 C for 10 minutes. All components of the parasite culture medium, i.e., distilled water, sodium bicarbonate, culture 15 16 medium stock solutions, were tested for endotoxin in the LAL assay before and after medium preparation. Plasmodium falciparum, FCR3TC strain were grown using the candle-jar method according to Jensen and Trager§26) The parasite cultures were synchronized by washing them with 5% aqueous sorbitol which lyses all but the youngest parasite stages. The synchronized parasites were returned to cultures until they matured t0 segmenting schizonts (13-21% parasitemia). They were then concentrated using the gelatin-flotation technique described by Jensen.(25) Trophozoites were collected using the same procedures. The parasites obtained from the gelatin suspensions were diluted with equal amounts of RPMI 1640 medium (without serum) and stored frozen at -70 C tuitil used in the LAL assay or rabbit fever experiments. Parasite Extracts in LAL Assay Experiment 1 Extracts obtained from schizont-infected red cells were tested using 'thee LAL assay. In the first experiment an extract was prepared by repeated 'frweezing and thawing of the schizont-infected red cells obtained by the gelatin concentration method. The parasite extract was serially diluted frwanilzlo through 1:640, mixed with the Limulus amebocytes and incubated under the appropriate test conditions. Lipopolysaccharide from S. typhimurium (LPS) was tested at 10-f01d dilutions from 12.5 mg/ml down 0.00125 mg/ml. Bperiment 2 This experiment was conducted to determine whether the substance in the Parasite extract responsible for the positive LAL reaction was susceptible to protease digestion. Accordingly, a second portion of the schizont extract was treated with pancreatic protease (Type 1, Sigma Chemical (30., St. Louis, Missouri) 17 Seven milliliters of extract were mixed with phosphate buffer (pH 7.5) and to this was added 10 mg. of pancreatic protease followed by thorough mixing. The mixture was incubated at 37 C in a water bath for 60 minutes. After incubation the mixture was diluted as in Experiment 1 and tested for activity in the LAL assay. Salmonella typhimurium LPS was used as an endotoxin standard, as before. Experiment 3 Experiment 3 was conducted to determine whether the parasite residual debris, rich in the malarial pigment, hemozoin, was a source of LAL activity and malarial pyrogen. The hemozoin-rich debris was collected frxnn the culture medium aspirated from parasite cultures where the parasitemia was near 20%. This material was washed several times in pyrogen—free water and tested in the LAL assay for properties. All tests were run in duplicate. Fever-Testing_0f Parasite Extracts in Rabbits Since rabbits are routinely used in pyrogen testing. New Zealand white rabbits, weighing approximately 3 kg. were injected intravenously Ifl ‘the lateral ear vein with varying concentrations of parasite extract tC> (determine their fever-producing properties. The general procedure was to take a rectal temperature reading every 30 minutes using a digital thelr‘mometer (YSI Model 49 TA, Yellow Springs Inst. Co., 'Hirree or more temperature readings were taken on each rabbit until the r‘élbbi ts became accustomed to being handled and their body temperatures Stabilized. In each case, the rabbits were injected with a quantity of “U"Eiri red cells suspended in the same culture medium used to grow the Parasite cultures. i Several temperature readings were taken at 30 minute intervals until the temperature stabilized. These procedures were then 18 followed by injection of the parasite extract and again the body temperature monitored until stable. Experiment la Different amounts of parasite extracts were injected until it was (determined that 1.6 m1. consistently produced a fever in each rabbit tested. Experiment 2a In order to determine whether the pyrogenic substances in the parasite (extract were soluble, 1.6 ml. of the parasite extract was diluted 1:5 in 12PMI 1640 medium (to reduce the hemoglobin concentration and to allow the :sedimentation of the subcellular components) and centrifuged at 100,000 X G ‘for 90 minutes. This produced a post-ribosomal supernatant solution (:ontaining the soluble components of the extract and a pellet of the non- .soluble subcellular particles and membranes.. The supernatant fluid (8 m1) luas injected ultravenously into one rabbit and the resuspended pellet into arwother. The changes in body temperatures of the rabbits were then nuanitored. A few days later this experiemnt was repeated on the same rabbits, reversing the order of injection. RESULTS As can be seen in Table 1, all components of the culture system, except 'the parasite extract, were negative for the presence of endotoxin. The |_AL assay detected endotoxin activity in the S, typhimurium LPS suspension _uwmemm moamss mzpzemq .hmMH mh4 mh>oommz< magzzmg ._ m_oms 21 mpmuPFQ=u cw mcou mew: memo» "mpoz cowuuwws cm>wm op pcmpm>w=cm cwxououcm to pcaoe4 mh>uommz< mzazzmc .N mpnmh 22 mpwowpaan cw wcou mew: mommh umpoz I I m =5me em I I + use ovum: oemp exam - - m I I + 5mm: camp HZQm l I +6 I I +¢ XPCO m.0mm I I I +e +e +¢ mumoNosaoL» I I I +¢ +¢ +¢ :pwz m.uma - - - - - - - - mm as, I I I I I I I I + + +¢ +¢ we :woNOEm: - - - - - - - - mm a; I I I I I I I I + + +¢ +e Fe :WONoEo: mIo_ «Io_ mIo_ NIoF _Io_ emuspwuca mpazeoeaowm .<.z .~m\m_\m mmetaxm .wamose so; .Fs\me em_.o spw>wasmemm seamen mapsews .Hmmh mp4 mp>oommz< magzzHa .m mynmh 23 pooled schizonts produced a peak fever of approximately 1.5 C in one rabbit, with that peak occurring 90 minutes after injection. In the second rabbit, the peak was approximately 1 C, occurring 150 minutes after injection. . When the amount of pooled schizonts used was doubled, to 1.6 ml, the first rabbit showed approximately a 2.5 C temperature increase, with a peak at 360 minutes after injection. The second rabbit showed roughly a 2.25 C temperature increase, with the peak 180 minutes after injection. In each case, the baseline for fever was taken as the temperature mean established after injection of washed, uninfected red blood cells. (Figures 2, 3) The rabbits began to hyperventilate and shiver when their temperatures reached an average of 39.25 C after injection with 0.8 ml. of the schizonts, and they began to hyperventilate at 40.40 and 40.06 C respectively, when injected with 1.6 m1. In experiments to determine whether the fever-producing substance(s) . \were soluble, a post-ribosomal supernatant obtained by ultracentrifugation (of the parasite extract produced a significant temperature rise when injected into the latural ear vein of a rabbit.(Figures 2, 3) When 8 ml. of the ssupernatant, representing the soluble portion of 1.6 ml. of parasite extract, unis injected into rabbit #1, it produced a 3 C temperature increase, with a peak three hours after injection of the soluble fraction. In rabbit #2, 'trre insoluble fraction produced a 2 C temperature increase that peaked at three hours after injection. Four days later the injections were reversed wi‘tfi rabbit #1 receiving the insoluble pellet and rabbit #2 receiving the post-ribosomal supernatant. In this experiment the sedimental preparation Produced a 3.6 C temperature rise that peaked 3 hours post-injection and the soluble preparation a 1.5 C rise with a peak 2 hours post-injection. 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V533 on i .. «=00 coo—u no: I a «I... ..........I . 8.5 2:328 .VSaS wt :35. ..o u as .u .N mczmwc ( O) BUMVUMWBI 9: DISCUSSION The level of "endotoxin-type" activity shown in the LAL assay by falciparum schizonts is sufficient, of itself, to produce measurable fevers if the mechanism of fever production is identical to that of endotoxin and if the falciparum endotoxin-activity is operating by itself without the sort of synergism suggested by Clark.(]2) The "endotoxin-like" activity of the parasite extract is apparently susceptible to protease digestion. The curious absence after proteolysis of LAL activity until the extract was diluted 1:80 was probably due to a high concentration of assay-destroying proteases which interfered with the assay until sufficiently diluted. This is supported by the observation that most LAL positive substances are lipopolysaccharides and are chloroform extractable or protease resistant. Thus it appears that the source of the. "endotoxin-like" activity is not likely to be protein. Using the same reasoning, the scant LPS-like activity shown by the hemozoin-rich parasite firesidue also makes it unlikely to be the cause of malarial fevers. In the rabbit-fever assay, the time-course and duration of the fevers rnake it appear likely that the route of fever production includes the eelaboration of endogenous pyrogen and is not direct. The febrile response (3f the rabbits was considerably heightened and sustained as the amount of sschizont-extract injected was increased from 0.8 ml. to 1.6 ml. This amount ()f parasite material was equivalent to a parasitemia of 0.36% in the rabbits 'injected. This figure was derived by approximating the total number of Ibarasites injected (determined from the parasitemia from which the extract 26 27 was made) and the total blood volume calculated from the body weight of the rabbit (3.20 ml/kg), Biology Data Book, page 263. The fever-producing substance(s) were found to be soluble, since the post-ribosomal supernatant produced significant pyrexia when injected into rabbits. Although the volume was large (8 ml.) we purposely did not attempt to reconcentrate it since in so doing we might have lost the low- molecular weight components. In conclusion, extracts from schizonts of E, falciparium cultured jfl_yitrg contain "endotoxin-like" substances which produce a positive reaction in a LAL assay. The concentration of this material was equiva- lent to 50-100 ng/ml of bacterial endotoxin which is far in excess of the amount required to produce fevers in man. The injection of the parasite extract into rabbits produced a significant rise in body temperature Iaccompanied by hyperventilation and shivering, similar to the common Inalarial chills and fever. LIST OF REFERENCES 10. ll. 12. 13. LIST OF REFERENCES Aikawa, M. and Thompson, P.E.: Intracellular parasitic protozoa. New York, Academic Press, (1974). 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Proc., 38, (1979), 49-51. Dinarello, C.A.: Production of Endogenous pyrogen, Fed. Proc., 38, (1979), 52-56. Dinarello, C.A..and Wolff, S.M.: Exogenous and endogenous pyrogens, Brain Dysfunction in Infantile Febrile Confulsions, ed. by Brazier, M.A.B. and Coccani F., New York, Dixon, (1976), 117-128. Dinarello, C.A. and Wolff, S.M.: Partial purification of human leukocyte pyrogen, Inflammation, 2, (1977), 179-189. Dinarello, C.A. and Wolff, S.M.: Pathogenesis of fever in man, New Engl. J. Med., 298, (1978), 607-612. Elin, J.R., and Wolff, S.M.: Nonspecificity of the Limulus amebocyte lysate test, positive reactions and polynucleotides and proteins, The J. of Inf. Dis., 128, No. 3, Sept. (1973). Felton, S.C., Prior, R.B., Spagna, V.A., Kreier, J.P.: Evaluation of E, ber hei for endotoxin by the Limulus lysate assay, J. Parasit, . 66, 11980), 846-847. Glew, R.H. and Levin, J.: Failure to deomonstrate circulating endotoxin in malaria, Proc. Soc. Exp. Biol. Med., 148, (1975), 508-510. Greenwood, B.M., Vick, R.M.: Evidence for malaria mitogen in human malaria, Nature, 257, (1975), 592-594. Griesman, S.E. and Hornick, R.B.: Comparative pyrogenic reactivity of rabbit and man to bacterial endotoxin, Proc. Soc. Exp. Biol. Med., 131, (1969), 1154-1158. Hellon, R.F. et a1: Some tests of the prostaglandin hypothesis of fever, Fever, ed. by Lipton, J.M., New York, Raven Press, (1980), 159-164. IJensen, J.B.: Concentrations from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum, Am. J. Trop. Med. Hyg., V. 27, (1978), 1274-76. Ilensen, J.B., and Trager, W.: Plasmodium falciparum in culture, J. of Parasit., 63, No. 5, Oct. 11977). Kilejian, A. and Jensen, J.B.: A histidine-rich protein from Plasmodium falci arum and its interaction with membranes, Bull. W.H.0., 55, Hm ,' —191-197. Kilejian, A. and Olson, J.: Proteins and glycoproteins from human erythrocytes infected with Plasmodium falciparum, Bull. W.H.0., 57 (suppl. 1), (1979), 239-243. Langreth, S.G., Jensen, J.B., Reese, R.T. and Trager, W.: Fine structure of human malaria jfl_vitro, J. Protoz., 25, (1978), 443-453. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 44. 412. 4133. ‘1