TO BARBARA RESISTANCE OF RATS TO TRYPANOSOMA LEWISI BY 8/ .{\ 4 \)t’/ Curtis LF'Patton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1966 ABSTRACT by Curtis L. Patton Resistance of Rats to Trypanosoma lewisi The presence of Egypanosoma lewisi in the blood of rats stimulates the development of ablastin which inhibits the reproduction of the parasite. Trypanocidal antibodies also develop which eliminate the trypanosomes from infected rats. Exceedingly large populations of g. lewisi which were fatal developed in rats treated with dexamethasone. Though sera from rats immune to T. lewisi agglutinated and lysed trypanosomes when injected together with the para- sites into the peritoneal cavities of dexamethasone treated rats, many of the trypanosomes escaped to the blood and established high levels of parasitemia which were fatal. Exudate cells from rats immune to g. lewisi also failed to protect dexamethasone treated rats from 2. lewisi infections. When sera from rats immune to g. lewisi and exudate cells either from immune or nonimmune rats‘were administered intra- peritoneally together with g. lewisi to dexamethasone treated rats, the trypanosomes were retained in the peritoneal cavi- ties where they were agglutinated, lysed and phagocytized. These rats did not develop parasitemias. It is concluded Curtis L. Patton from these observations that dexamethasone does not interfere with the activities of antibodies against 2. lewisi but rather inhibits the production of antibodies. Further, the control of 2. lewisi infections appears to be dependent upon a combination of antibodies and the presence and function of phagocytic cells. Autoradiography following injections of H3-thymidine into infected rats revealed that the labeled compound was incor— porated into the nucleus and kinetoplast of g. lewisi. The uptake and diminution of H3-thymidine indicated that the differences in levels of parasitemia between dexamethasone treated rats and untreated rats observed during the first 3 or 4 days of the infections were not attributable to differ- ent rates of reproductive activity. However, ablastic activity did interfere with the synthetic period of DNA in the reproductive cycle of the trypanosomes after the first 5 days of infection in untreated rats but not in dexamethasone treated rats. These data are interpreted to mean that dexa- methasone interferes with the development in rats of ablastin as well as trypanocidal antibodies. £2 33232 studies confirmed the results of in Ellg studies and also revealed that ablastic activity is titratable and that its effects are reversible as indicated by autoradio- graphy following assimilation of H3-thymidine by blood stream forms of T. lewisi in culture. ACKNOWLEDGEMENTS The author declares his most lasting thanks to Dr. David T. Clark, associate professor of Microbiology and Public Health, for his kind guidance and assistance throughout this study, and to very many generous and considerate friends and teachers for their inspiration and encouragement. TABLE Chapter INTRODUCTION . . . . . . . LITERATURE REVIEW . . . . MATERIALS AND METHODS . . RESULTS . . . . . . . . . DISCUSSION . . . . . . . . SUMMARY AND CONCLUSIONS . A. Summary . . . . . B. Conclusions . . . LITERATURE CITED . . . . . OF CONTENTS ii Page 11 27 73 92 92 95 96 LIST OF TABLES Table Page 1 In gitrg system of Experiment 4 . . . . . . . . . 16 Summary of means in Experiment 1 . . . . . . . . 34 Summary of data from Experiment 2 . . . . . . . . 35 Summary of data from Experiment 3 . . . . . . . . 37 UI-C'KDN Peak parasitemias of rats infected with g. lewisi Experiments0.000000000000000042 6 Results of the examination of tail blood in Ex- periment 6 for the presence of trypanosomes . . . 4h 7 Percent trypanosomes labeled with tritiated thymidine and trypanosomes/cmm cultured in vitro inExperimentll.o..............59 iii Figure LIST OF FIGURES Mean hemocytometer counts of trypanosomes per cmm of blood and weight gains of untreated rats and rats treated with dexamethasone . . . . Trypanosomes per cmm of blood and percent dividing forms of untreated rats and rats treated with dexamethasone every 3rd day of theinfeCtion0.000000000000000 Mean hemocytometer counts of trypanos mes per cmm blood of rats injected with H -thymidine and rats not given the compound . . . . . . . . Mean grain count over 100 labeled trypanosomes as a function of time following an injection of 500 no R3-thymidine into rats 3 days postinfection . . . . . . . . . . . . . . . . . Mean grain count over 100 labeled trypanosomes as a function of time following an injection of 500 no H3-thymidine into rats 5 days pOStinfGCtion0.000000000000000 Trypanosomes per cmm of blood, percent labeled trypanosomes and mean grain counts over trypano- somes from rats with late, subacute, and chronic infections. The rats were given two injections of H3-thymidine (500 pc each) separated by Zhours00000000000000.0000. Effect of increasing quantities of H3-thymidine on the uptake of the labeled compound in vitro by2018W131oooooooooooooooooo Effect of pooled ablastic sera on the uptake of H3-thymidine by T. lewisi in vitro . . . . . . . Effect of ablastic sera on incorporation of H3-thymidine in vitro after transfer to normal and before transfer to normal media . . . . . . iv Page 29 33 47 50 52 55 57 LIST OF FIGURES (continued) Figure Page 10 In vitro titration of pooled ablastic sera. The effect of increasing concentr tions of antibody on the assimilation of H -thymidine . . 68 INTRODUCTION ngganosoma lewisi (Kent, 1880), a blood parasite of rats, has held the interest of parasitologists for many years. The attractiveness of this hemoflagellate persists not only because of its accessibility in the peripheral blood stream where it is easily obtained for studies, but more because of the immune reactions that occur during parasitemia. The most remarkable characteristic of the infection is the development in the rat of an antibody called ablastin that inhibits the reproduction of the parasites without affecting their motility and viability (55). The host also produces more conventional antibodies which bring about the destruction of the trypanosomes. The latter antibodies are called trypanocidal and they differ fundamentally from ablastin (12, 13, 17, 18). The morphological changes from dividing to nondividing parasites under the influence of ablastin were observed many years ago (55), and in the past few years it has been shown that ablastin inhibits protein and nucleic acid synthesis of ‘2. lewisi (#2, 62). In addition, glucose utilization (38) and levels of lactic dehydrogenase (19) are reduced in try- panosomes inhibited by ablastin. However, nothing is yet known about the mechanism of ablastic action. 1 The purpose of this study was to affirm some of the evidence for the antibody nature of ablastin and to study the effectiveness of ablastin together with the trypanocidal antibodies as factors of resistance in the host. The study was approached by inhibiting antibody production in rats by treating them with a corticosteroid and passively immunizing rats which had been treated as well as untreated rats. In addition, the synthesis of DNA by 2. lewisi l£.Z£!2 and‘gg 33252 was studied utilizing autoradiography following incorporation of HB-thymidine by T. lewisi. LITERATURE REVIEW Trypanosoma lewisi is a blood protozoon of rats trans- mitted in nature by the rat flea Nosopsyllus fasciatus (69). In the laboratory the hemoflagellate can be maintained in rats and it also can be kept frozen at -195.8° C in liquid nitrogen. Trypanosoma lewisi reproduces in the blood stream of rats only during the first few days of infection, and it remains thereafter in the blood stream for several weeks or in some cases months as a non-reproducing population (57). Taliaferro (59) demonstrated that this behaviour is brought about by the development in the rat of a passively transfer- able serum component which inhibits reproduction of the trypanosomes and therefore limits population growth without producing detectable effects on their vitality or infectiv- ity. He named the serum component ablastin. He demonstrated that ablastin had many of the general properties of antibody except that it is not adsorbed from serum of immune rats by living parasites; a property which may result from unsatis- factory antigen preparation and/or very low avidity. The nonabsorption by living trypanosomes has proved to be very useful for separating ablastin from trypanocidal antibodies which also develop as a consequence of infection. III I ill: I ll I‘I I... )1 1 The term trypanocidal includes all serum elements which when injected into rats decrease the number of circulating organisms. There is a trypanocidal antibody that kills these trypanosomes early in the infection and gives rise to the first number crisis approximately 7 days after inocula- tion of the organisms. This antibody is usually referred to as Trypanocidal antibody I. A second trypanocidal antibody, Trypanocidal antibody II, acts on the surviving parasites to terminate the infection (13). This is the generally accepted interpretation advanced to explain the mechanism whereby the reproduction of‘g. lewisi is inhibited and is eventually eliminated from its host although other explanations have been offered. Chandler (9) postulated that ablastin inhibits reproduction when low in titer by reacting with metabolic products of trypanosomes. According to him, in high concen— tration the same antibody agglutinates and renders the trypanosomes more susceptible to phagocytosis. The antibodies which develop as a consequence of infec- tion with T, lewisi have been physically and chemically characterized by D'Alesandro (17). He found that the early trypanocidal antibody and ablastin are globulins of rela- tively small molecular size with a sedimentation constant of 68 whereas the late trypanocidal antibody is a globulin molecule with a sedimentation constant of 16S.' All three of the antibodies localized between the slow moving 8 globulins and the fast moving gamma globulins during starch zone electrophoresis. Ablastin-induced inhibition of reproduction causes the rapid disappearance of division in the nucleus as well as the duplication of organelles and the gradual disappearance of variability in the size of the organism associated with division and reproduction. Thus, an inhibited population consequently shows the remarkably low coefficient of varia- tion of total length of approximately 3% compared to a coefficient of variation of 25% or more in a population of trypanosomes whose reproduction is uninhibited (56). Changes in glucose metabolism.of g. lewisi can be cor- related with the inhibition of reproduction by ablastin during the course of the infection. Dividing parasites consume less oxygen but utilize more glucose per parasite than the adult forms. Their respiratory quotient and ratio of oxygen consumption to glucose consumption are lower than in non-reproducing trypanosomes. It is suggested that ablastin induces a shift in metabolism of the parasites from one of active oxidative assimilation to one of maintenance (38). It has been demonstrated that this normally benign organism may become pathogenic in pantothenate deficient animals (4). The reproduction phase of T. lewisi has been prolonged experimentally by treating the host with sodium salicylate (5, 49). Lysenko (30) demonstrated that the drug affects ablastic activity in a manner other than by combin- ing with the antibody. It has been clearly demonstrated that cortisone and related compounds affect the basic physiological mechanisms involved in diseases whose etiologies are quite different. By studying selected infectious diseases, it was possible to formulate some of the effects of the cortical hormones in infection and immunity, and these effects were excellently summarized by Kass and Finland (27). Thomas (67) also published an excellent review. Studies have been made of the effects of adrenocortical hormones on the course of diseases in patients caused by bacteria, rickettsia, fungi, protozoa, and helminths. In general the effects were anti-inflammatory. It was also realized that high dosages of cortisone disorganizes the natural host parasite relationship - the outcome being in favor of the parasite. It is thought that cortisone inhibi- tion of defense mechanisms is exerted early in the sequence of events which lead to the formation of antibody. Halpern gg‘gl. (22) concluded that cortisone tends to diminish the rate of antibody synthesis. I have found the relationship of parasite and host in this study to be alterable when the rat is administered corticosteroids. Phagocytosis, the whole act by which cells engulf and destroy particles, is a most important function of peritoneal exudate cells. Indeed, in the beginning stages in the development of immunology before this century, the migration of phagocytes to areas infected with bacteria and the engulf- ment of these organisms and other particulate matter was recognized to be a most important process by which animals defended their bodies against foreign invasion. Metchnikoff (36) described a general defense system of cells and empha- sized the importance of phagocytosis in immunity. It has been shown that in the peritoneal environment and also ig‘zitgg (49) that mouse peritoneal macrophages are able to kill many species of gram negative bacteria with great rapidity (48, 70). For any appreciable amount of phagocytosis to occur the bacteria must have previously been treated with serum containing opsonic factors. Once inside the macrophages, the bacteria were killed with equal rapid- ity, irrespective of the virulence for mice of the bacterial strain. Whitby and Rowley (70) concluded from their studies with P32-labe1ed Escherichia 22;; injected into the mouse peritoneum that the rapid decrease in viable bacteria which occurred was largely due to peritoneal events and not to the transport of bacteria elsewhere. In order that phagocytosis occur with efficiency, it has been established that opsonins or a coating of antibodies on the invading organisms is desirable (28). Laveran and Mesnil (29) considered the possibility that phagocytosis assumed an active role in T. lewisi infections. Depression of the reticuloendothelial system in infec- tions with T. lewisi has been demonstrated to be of import- ance. Regendanz and Kikuth (43) observed that in splenectom- ized rats division of the parasites continued several days longer than in intact controls. In some splenectomized rats the organisms continued their division until the death of the host. Taliaferro 33 El. (58) used blockade techniques as well as splenectomy to investigate the role of the reticulo- endothelial system. Tempelis and Lysenko (65) studied 2. lewisi infections in x-irradiated hosts. The results of these investigations confirmed the findings of Regendanz and Kikuth (43). All of the above studies might imply an im- portance of phagocytosis in the control of the infection but do not necessarily offer precise affirmative evidence of its participation. MacNeal (31), Manteufel (32), and Taliaferro (57) refuted the evidence for the role of phagocytosis in‘g. lewisi infections as offered by Laveran and Mesnil (29). Taylor and Becker (63) observed 2. lewisi in von Kupfer cells of the liver of pantothenate deficient infected rats. Lange and Lysenko (28) demonstrated in 13332 phagocytosis of 1. lewisi by peritoneal exudate cells. They found that phagocytosis was enhanced by the presence of immune serum. This enhanced effect was removed by absorption of immune rat serum by living trypanosomes. The intent of the experiments with peritoneal exudate cells in this study was to present evidence of (a) ig‘zitgg phagocytosis of g. lewisi, (b) in zigg phagocytosis of T. lewisi, and (c) the profound effect upon parasitemias of T. lewisi by phagocytic cells and specific antibodies when administered to normal rats and rats which have been made immunologically deficient. It is ablastin which has fascinated most of the workers who have studied 3. lewisi. It is the effect(s) of ablastin that occupies a great portion of the present study. Taliaferro and Pizzi (62) studied the inhibition by ablastin of protein and nucleic acid synthesis in‘g. lewisi by inject- ing rats which harbored infections with 335 amino acids and adenine-B-Clu. These authors reported that the inhibition of division and growth by ablastin in T. lewisi was accom- panied by an essentially complete inhibition of nucleic acid synthesis and a marked inhibition of protein synthesis. Autoradiography following the administration of tri- tiated thymidine has been used extensively in the study of various proliferating cells (15, 26, 54). Steinert and Steinert (53) studied the in vitro assimilation of HB-thymi- dine in Trypanosoma mega by autoradiographic methods. I have used autoradiographic labeling techniques to 10 study the question of ablastic immunity in T. lewisi infec- tions in the rat. The experiments are predicated on the assimilation of tritiated thymidine as an index of deoxy- ribonucleic acid synthesis (33, 45, 64). The validity of any conclusions which may result from this study depends on a number of premises which are widely held, but yet the subject of active discussion (11, 15, 54). The premises most relevant to the experiments in this study where HB-thy- midine was employed are: (a) the tritium label on thymidine is stable and does not exchange after incorporation into DNA; (b) DNA itself is metabolically stable; (c) H3-thymidine when injected into the peritoneal cavity of the rat is rapidly distributed throughout the body via the circulatory system and either promptly incorporated into DNA of‘g. lewisi and the cells of the rat or is eliminated (35); (d) H3-thymidine may be eliminated by repeated washing from the cultural environment; (e) labeled precursor mixes with unlabeled endogenous precursors in such a way that the cells are unable to distinguish labeled from unlabeled precursors; (f) H3-thymidine does not affect the normal behaviour of cells that have taken it up. This last premise has been challenged (21). MATERIALS AND METHODS Trypanosoma lewisi used in this study was maintained in white rats by weekly blood passage or frozen in liquid nitrogen. In preparation for freezing the hemoflagellates were washed free of serum and blood by alternate centrifuga- tion and suspension in Hanks' balanced salt solution (BSS). Then they were suspended in a 5% solution of glycerin, and one ml quantities were put into 2 m1 screw cap vials. The trypanosomes were frozen by lowering their temperature 1° C per second until the temperature was less than -20° C. This process was carried out by putting an insulated beaker con- taining 95$ ethyl alcohol into an insulated bath of dry ice and ethyl alcohol. The vials were mechanically swirled during the freezing operation in the beaker of alcohol. The vials containing the frozen organisms were then transferred to canisters which were kept in a liquid nitrogen refriger- ator at -295.8° C. In order to revive the trypanosomes the vials were withdrawn from the liquid nitrogen refrigerator and their contents rapidly thawed in a 37° C water bath. Rats used in this study were supplied by Spartan Research Animal Company, Williamston, Michigan. The rats were always white males and usually weighed between 65 and 75 s. 11 12 Trypanosomes and other cells were counted in the improved Neubauer hemocytometer. Disposable blood diluting pipettes, supplied by Becton, Dickinson and Company, contain— ing isosmotic saline were used for making dilutions. After introducing infected blood into these pipettes they were placed for at least two hours in the refrigerator at 4° C and afterwards brought to room temperature before counting the organisms. This treatment effected slow movement of the organisms to the point where their migration in and out of the microscopic field was negligible. Smears of blood or trypanosomes were stained with Wright's stain or Giemsa's stain with May Grflnwald's stain added. Measurements of the parasites were made with an ocular micrometer. The coefficient of variation (C.V.) was calculated by multiplying the standard deviation by 100 divided by the mean (C.V. = lgfifio. The number of divisions was calculated from the formula l°SloNl ' 1OsloNo. A total of 13 experiments will belgggcribed under the 4 major headings which follow: I. Influence of Dexamethasone on T. lewisi Infections in Rats. II. Effects of Sera and Peritoneal Exudate Cells from Immune and Nonimmune Rats on the Host Parasite Relationship of T. lewisi and the Rat. III. Reproductive Potential of T. lewisi IE;XEZR as 13 Indicated by Studies with Tritiated Thymidine. IV. Incorporation of Tritiated Thymidine by T. lewisi In Vitro. I. Influence of Dexamethasone on T. lewisi_;nfections in Rats Dexamethasone (9-a-f1uro-l6ed-methyl prednisolene) is a synthetic corticosteroid supplied by the Schering Corporation. In Experiments 1 and 2 white male rats which weighed between 65 and 75 g each were allotted to four groups each of which contained 5 rats. Rats in group A received intraperitoneally 0.5 mg of dexamethasone daily during the experiment but were not infected. Rats in group B received 0.5 mg of dexametha- sone 3 days prior to infection with T. lewisi and daily thereafter. Group C rats were infected with T. lewisi but were not treated with dexamethasone. Group D rats were uninfected and untreated. In Experiment 1 all treatment with dexamethasone ceased 5 days postinfection. In Experiment 2 treatment ceased 80 hours postinfection. Rats were allotted to groups in Experiment 3 just as they were in Experiments 1 and 2, how- ever, the drug was administered every third day during the infection and the experiment was terminated 16 days post- infection. 14 II. Effects of Sera and Peritoneal Exudate Cells from Immune and Nonimmune Rats on the Host Parasite Rela- tionship of T. lewisi and the Rat Hyperimmune sera were obtained from the blood of rats which had recovered from infections with T. lewisi and had been challenged on two occasions. The first challenge was given 45 days after the original infection and the second challenge was given 2 weeks later. Ten days after the second challenge blood samples were asceptically collected by cardiac puncture and pooled. Nonimmune sera were obtained from normal rats. Sera obtained from blood of rats infected with 2, lewisi for 5 days were designated acute sera. Peritoneal exudate cells were procured from normal rats, and rats which had been infected and challenged as above. In order to obtain the cells 5 ml of 1% glycogen in sterile physiologic saline were injected intraperitoneally into rats 48 hours before harvesting the cells. Just prior to collecting the cells, 20 ml of Hanks' BSS containing 100 USP units of heparin were injected into the peritoneal cavities of the rats. Their abdomens were massaged and they were anesthetized with ether. The abdominal skin was retracted and an incision of about 1 cm was made through the abdominal wall. The rats were then held over a sterile beaker to catch the fluid and cells which flowed through the incision. The viscera were washed with 20 ml more of Hanks' BSS. The 15 suspension of cells was centrifuged at 1000 rpm for 5 minutes in an International Centrifuge, Universal Model UV. The cells were washed twice in Hanks' B83 and then resuspended in it. The cells were counted in a hemocytometer. Trypanosoma lewisi used in Experiments 4 through 13 were obtained from rats which had been treated with dexamethasone. The organisms were washed twice in Hanks' BSS before use in the experiments. Egpgriment 4: In litre studies of peritoneal exudate cells and sera with T. lewisi. In,zit£g studies were carried out in 50 ml round bottom centrifuge tubes which contained Hanks' BSS with trypanosomes, peritoneal exudate cells, and/or serum (Table l). The tubes were incubated at 37° C. A drop of the contents of each tube was examined under the phase contrast microscope 3 times in a period of 3 hours. At the end of 3 hours, a drop of the contents of each tube was placed on a glass slide, dried and stained with Wright's stain. Experiment 5: Passive immunization in dexamethasonewtreated and untreated rats against T. lewisi using sera and peri- toneal exudate cells. Rats were administered intraperitoneally 0.5 mg dexa— methasone daily for 3 days prior to infection with 5x108 l6 oom.o oomeo oom.o ma oomec oofi.o oom.o ma ooW.o oom.o oom.o ma oom.o oom.o oom.o Ma oom.o oom.o oom.o NH oom.o oom.o oom.o HH cow.o oom.o oom.o 0a oom.o oom.o oom.o m oom.o oom.o oomeo m ooW.o oom.o oom.o m oom.o oom.o eom.o minim oom.o me.o oom.o oom.o oom.o oom.o .: oom.o oom.o oom.o m oom.o oomso oom.o N cow.o i oom.o a S 0958 m m r C S / Sd st 8 m le la t e o ai+e l.r a m r ace 6 r 0 pl nee c.e _ w 38 em em a a d w .leo "vi. v.m t p. e la .1 .1 o v. on p. esi eev. t.i e .n w.o Va 0 al a. f .1 r e de dr mt mn mt 1t m. new mic m.e o.a oil flea and v u n. r.r r air .011 or 11m m.c n.v. r. rive r. o N t1 7a 7h am a e “as x a. o. 0. mm m. we . .3 men 110 1.0 u m .mwi . o xai var ego .ec e a 9e n.n earl :19. non s_a .Sqi was : paoadhcawm mo aopmhm oped» mm .H oases 1? trypanosomes. The rats were treated daily with the drug for 5 days postinfection. Sera and peritoneal exudate cells were taken from normal rats and rats which had been made hyper- immune to T. lewisi. Infected rats were given these immune and nonimmune products immediately after infection and again 24 hours postinfection. Sixty rats were allotted to 10 groups of 6 rats per group. The rats were also separated into 2 sections. All the rats in Section A of the experiment received dexametha- sone. Rats in Section B did not receive the drug. Rats in Sections A and B were treated as follows: Group 1 uninfected 2 infected with.2. lewisi 3 infected plus 1x109 exudate cells from immune rats L; infected plus lxlO9 exudate cells from nonimmune rats U1 infected plus sera from immune rats infected plus sera from nonimmune rats 7 infected plus 1x109 exudate cells and sera from immune rats 8 infected plus 1x109 exudate cells from immune rats and nonimmune sera 9 infected plus lxlO9 exudate cells from nonimmune rats and immune sera lO infected plus 1x109 exudate cells and sera from nonimmune rats. 18 Peritoneal exudate cells and trypanosomes were counted in the hemocytometer. Experiment 6: Retention of T. lewisi in the peritoneal cavities of rats given peritoneal exudate cells and sera from immune rats. Ten normal rats and 10 rats which had for 3 days prior been treated daily with 0.5 m1 dexamethasone were injected each with 1 ml of a trypanosome suspension containing 3x1010 trypanosomes. Four rats in each group were immediately injected with a 1 ml suspension of exudate cells (6x1010) and 1 ml of sera from hyperimmune rats. Two rats in each group were injected with a 1 ml solution of exudate cells (6x1010) from hyperimmune rats and 1 ml of Hanks' BSS. Two rats from each group were injected with 1 m1 of sera from hyperimmune rats and 1 m1 of Hanks' BSS. The remaining two rats from each group were injected with 2 ml of Hanks' BSS. The tail blood of each rat was examined 30 minutes and 1 hour postinfection for the presence of trypanosomes. The rats were killed after 1 hour and the peritoneal cavities were washed with Hanks' BSS. The contents of the wash were examined for the presence of trypanosomes as well as the occurrence of phagocytosis by macrophages. 19 III; Reproductive Potential of T. lewisi In Vivo as Indi- cated by Studies with Tritiated Thymidine Tritiated thymidine used in the following experiments was supplied by Volk Radiochemical Co. Its specific activity was 3.5 c/mM in 10% 2-propanol. The labeled com- pound when used in‘zivg was injected intraperitoneally. The procedure for autoradiography of the labeled cells was as follows. Four smears were made for each time period in the experiments. In the dark room the slides were coated with NTBZ Kodak liquid emulsion. The slides were dried in an incubator at 28° C in which two large sponges had been placed. The wet emulsion coated slides were placed upright on flatbottom trays containing water soaked tissues. This procedure prevented buildup of emulsion at the base of the slide and resulted in a film of more uniform thickness. Thirty minutes were allowed for the slides to dry, and the slides were then placed in plastic slide boxes containing small bags of anhydrous CaSOh. The slide boxes along with more bags of anhydrous CaSOu were put into cardboard cigar boxes, wrapped with aluminum foil and sealed with masking tape. The boxes were then stored at 4° C for varying periods of time. Usually a set of slides was developed after 3, 5, 10, and 15 days exposure to the isotope. Data were collected from those film which resulted in the greatest clarity of autoradiography for an experiment. Underexposed and over- 20 exposed film were examined but no data were recorded. The slides were developed with Kodak Dektol Developer at 17° C for two minutes. The slides were rinsed in water, fixed for 5 minutes and finally washed for 15 minutes, all procedures at 17° C. After the slides were completely dry, they were stained with Giemsa and May Grfinwald stains in the same manner as for conventional blood smears. The silver grains in the film which have been exposed to 3 emissions of H3-thymidine appear as black grains under the microscope. Consequently, black grains appear over the organisms which have incorporated H3-thymidine. In the following experiments the percent trypanosomes over which there appeared black grains, and the mean number of grains over 100 organisms were recorded. Experiment 7: Uptake of tritiated thymidine inflzigg. Rats which weighed between 65 and 70 g were infected with T. lewisi (1x10lo trypanosomes) and were injected with tritiated thymidine according to the following schedule: at 48 and 72 hours postinfection rat 1 was injected with 100 no, at 96 and 120 hours postinfection rat 2 was injected with 130 yo, at 144 and 168 hours postinfection rat 3 was injected with 160 no and at 192 and 216 hours postinfection rat 4 was injected with 220 p0. Blood smears were made 8, 16, 20, 24, 28, 32, 40, 48, 56, and 64 hours after H3-thymidine was 21 injected. Experiment 8: Reproductive potential of 2. lewisi and effects of ablastic immunity on incorporation of tritiated . thymidine. In this experiment two rats which weighed 70 g were treated with 0.5 mg daily doses of dexamethasone 3 days prior to infection with T. lewisi. Treatment with 0.5 mg of dexamethasone was continued daily throughout the infection. Two untreated rats were similarly infected with T. lewisi. One rat of the dexamethasone-treated pair and one of the untreated pair were each injected with SOO/uC of the labeled compound on the 3rd day postinfection. The second rat of each pair was injected with 500 no of the isotope on the 5th day postinfection. Blood smears were made 8, 16, 20, 24, 28, 32, 36, no, 44, 48, 52, 56, 60, and 64 hours after H3-thymi- dine was injected. Experiment 9: Incorporation of tritiated thymidine by T. lewisi in subacute, late and chronic infections. To each of three rats which weighed approximately 75 g 500 uC of tritiated thymidine were given on two occasions separated by 2 hours. Hat 1 had been infected for 14 days and was designated as one with a late infection. Rat 2 had also been infected for 14 days but was in addition adminis- 22 tered 0.5 mg of dexamethasone every other day beginning with the day of the infection to produce a chronic trypanosome infection. The third rat had a subacute infection of 6 days. All three rats were infected with 0.5 m1 of a 1.15x10lo suspension of trypanosomes. The trypanosomes which were used in experiments 9 through 13 were harvested from dexamethasone treated rats, pooled, frozen and maintained as stabolites in liquid nitrogen. IV. Incorporation of Tritiated Thymidine by T. lewisi lemurs The culture medium used in experiments 10 through 13 was described by D'Alesandro (18). The medium consisted of the following: 40% Hanks' balanced salt solution, 40% rat serum and 20% lactalbumin hydrolysate-yeast extract medium. The composition of the lactalbumin hydrolysate-yeast extract medium was as follows: Lactalbumin hydrolysate 5.0 g Yeast extract 1.0 g NaHCO3 1.1 g Glucose 4.5 8 Phenol red, 1% solution 1.6 ml Hanks' balanced salt solution 1000 m1 In addition there were added per m1 of medium: 100 units 23 of penicillin, 100 pg of streptomycin, and 2.5 mg of glucose. Finally, for every 1.5 ml of medium, 0.05 ml rat blood lysate was added. The pH of the completed medium was approximately 7 as indicated by pH paper. To obtain rat blood lysate, rats were asceptically bled from their hearts. The blood was defibrinated with glass beads. The cells and serum were separated by centrifugation in the cold, and the cells were alternately frozen in an alcohol dry ice bath and thawed in cold tap water three times. The serum was added to the solution of lysed cells and then separated by centrifugation in the cold. The supernatant was withdrawn and used in the medium or frozen for future use. Sera containing ablastin were obtained from rats on the 14th day of infection with I. lewisi. Though trypanosomes were still present at this time, the first number crisis had already occurred. These rats, therefore, contained trypano- cidal antibodies in their sera in addition to ablastin. The trypanocidal antibodies found at this time are commonly designated trypanocidal antibody I (59). Trypanocidal anti- body I is adsorbable by live trypanosomes, but ablastin is not. To remove antibodies responsible for the trypanocidal effects 1x109 trypanosomes per ml of pooled sera were intro- duced into sterile screw cap tubes (120x13) together with 5 m1 of sera. The tubes were placed on their sides in a 370 C 24 incubator for 20 minutes and then refrigerated for approxi- mately 12 hours (18). The trypanosomes were removed from the sera by centrifugation. Sera taken from uninfected rats were similarly treated. Plastic, sterile tissue culture tubes (16x150mm) were used in all the Ip'nggg experiments. To each tube 1.5 ml of culture medium and 6x106 trypanosomes were added. The tubes were incubated on their sides at 37° C. Experiment 10: Determination of amount of tritiated thymi- dine required to label I. lewisi Ig‘nggg. In order to determine the quantity of tritiated thymi- dine sufficient to label at least half the trypanosomes in culture, the following amounts of isotope were added to culture tubes: 5 pc, 10 pC, 50 pc, 100 )lC, 200 p0. The cultures were smeared onto slides after 5 and 15 hours incubation. Egperiment 11: Determination of the DNA synthetic period in I. lewisi. To each culture 10 p0 of tritiated thymidine were added. The cultures were pulsed as soon as possible after the organisms were transferred to the medium according to the following schedule: 30 minutes, 1 hour, 2 hours, 3.5 hours, 5 hours, 13 hours, 20 hours, and 28 hours. At the appropri- 25 ate time the cultures were centrifuged (1000 rpm for 5 minutes in an International Centrifuge Universal Model U.V.), and the culture medium decanted. The trypanosomes were washed twice in 15 m1 of Hanks' balanced salt solution, and 1.5 ml of culture medium without the labeled compound were added. Viable trypanosomes were counted in the hemocytometer. Smears were made according to the time periods indicated in the results. Esperiment 12: The influence of ablastic serum on the uptake of tritiated thymidine 12.11232: In order to determine if ablastin interferred with synthesis of DNA, trypanosomes were pulsed with 10 no of HB-thymidine in culture medium containing 40% normal rat serum for various time periods. The organisms were then washed twice by suspending them in 15 m1 of Hanks' balanced salt solution. They were centrifuged at 1000 rpm for 5 minutes (International Centrifuge Universal Model U.V.) each time, and culture medium containing 40% ablastic sera without the labeled compound was added to the washed organisms. Trypanosomes were also pulsed with 10 pc of H3-thymidine in culture medium containing 40% ablastic sera and tritiated thymidine. The trypanosomes were washed as above. To these washed trypanosomes was added culture medium containing nor- mal rat sera without the labeled compound. The entire pro- 26 cedure above was repeated except that the culture medium added after the trypanosomes had been washed did contain 5‘uc of the isotope, and the time periods were more widely spaced as indicated in the results. Egperiment 12: Titration of ablastin. Titrations were made in order to determine the activity of sera containing ablastin. The concentration of ablastic sera in the culture medium ranged from 0.625% to 40%. Normal rat sera were used to bring the total concentration of sera to 40%. This was done by diluting the ablastic sera with sera from normal rats as follows: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64. Each culture contained 10 pc of H3~thymidine and was incubated at 37° C for 22 hours. RESULTS I. Influence of Dexamethasone on T. lewisi Infections in Rats Experiment 1: There was observed in Experiment 1 an increasingly higher parasitemia in the infected rats which received dexa- methasone (Table 2, Fig. 1) when compared with the infected but untreated rats. This difference between the two groups was observed to take place by the 2nd day of the infection. 0n the 5th day of infection, the treated infected rats appeared moribund and exhibited parasitemias of over a million parasites per cmm. On the same day, 3 rats in this group died. No rats in the treated infected group survived beyond the 6th day of infection. All dexamethasone treated but uninfected rats survived. However, they as well as the treated infected group gained little or no weight. The untreated uninfected rats and the untreated infected rats demonstrated growth and development as indicated by weight gains. The average peak parasitemia in the untreated rats was 410,000 trypanosomes per cmm. A sharp decline in parasitemia was noted after one week postinfection. Eventually the try- 27 Figure l. 28 Mean hemocytometer counts of trypanosomes per cmm of blood and weight gains of untreated rats and rats treated with dexamethasone TRYPANOSOMES PER cmm . I 071 Tj'U'IT I 0‘4 U r111“ [05-1 l I VTTT' lC’4‘ I UIF'U'U I |()3u -l 29 Figure 1 EXPERIMENT 1 9 Dexamethasone treated rats, infected e———e Untreated rats, infected o ------------ '0 Dexamethasone treated rats. uninfected O ------------ 0 Untreated rate, uninfected ’4 /°". If \\ I \\ qIOO I, ,9}? 70 3t: - .07" h‘0 - 60 ‘3 ,.-'l \ § I ".0; \ - so \ I ° ,4 \ ' a I, V ‘ 4° 3 I...-"./ \ . 3o 85 . O." \ k 9;", I \ 20 § eee o. . "on “M... a. ‘\ | 0 $1 . . - . \> d C) at 1 n l 4 1 1 m m n 1 xhl-|O O 23456789l0'lll2l3 DAYS POST INFECTION 30 panosomes disappeared from all the rats in this group; usually by the 3rd week postinfection. All rats in this group survived. Egperiment 2: The design of Experiment 2 was similar to that of the first experiment. The data were, however, collected earlier and more frequently during the infections. The parasitemias in the dexamethasone treated rats were not different from those in the untreated group until 21 hours postinfection (Table 3) when individual parasitemias and the mean parasi- temia was substantially greater in the treated rats than in the untreated rats. It was further observed that the number of dividing trypanosomes found in both groups was very simi- lar up to and including the 37th hour postinfection. Begin- ning with the 43rd hour postinfection, there was a sharp decline in the percentage of dividing forms found in the untreated rats; whereas the percentage of trypanosomes in a state of division continued to be relatively high in the treated group (Table 3). The variation in the length of the parasites was not different between the treated and untreated infected rats. Data from this experiment were not collected beyond the 80th hour of infection. 31 Egperiment 2: In this experiment the parasitemia declined in the un- treated rats beginning the 6th day postinfection while the parasitemia in the treated rats remained high with an average count of 800,000 or 900,000 trypanosomes per cmm of blood for 16 days (fig. 2). Four of the 6 rats died in the treated infected group, one each on the 7th, 9th, 10th and 13th day of the infection. None of the treated uninfected rats died. The percentage of dividing forms and the coefficient of variation remained relatively high during the 16 days of the experiment in the treated infected rats. Figure 2. 32 Trypanosomes per cmm of blood and percent dividing forms of untreated rats and rats treated with dexamethasone every 3rd day of the infection 1 33 I? 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.>.o e.m oe.m sm.m He.n om.n ea.o na.e u ea. om.o ao.ea am.mm ao.am s.mm mm.sa mm o.om o.om N.om m.om H.Hm n.mm m.om nausea use: oawo.a oaso.m onH.H oaxo.m oawm.n oaie.m sec o\hoso soreness m m m s e n n mn.ma so.ma s.ma ss.ma b.oa Hm.ma mn.sa .>.o aa.e no.2 an.m ms.m om.m oa.m m.n m No.oa mn.oa on.ma m.ea o.oa mm.na so.nm mm e.om H.mm m.om o.mm o.am n.om n.om nausea some moaso.e moaxo.m soaso.m eoaxa.s noHeH.n nous m. m sso\nosouosmaaae a mpmm copmoapab ew.ma o.nm on.mm .>.o um.m m.m mm.m m ew.mm ee.mm nm.mm mm moan n.mm m.mm o.mm rpmsoa cmm: moaxmo.a moaxm.m moaxw.m EEO\moaomocaaaaB m topmoaB oCOmQSDoEGKoD ma ca NH 0H m m : sofipoomaapmom mama u can dosaapaoo : manna on.s mn.m nm.o mm.na n.na m.am .>.o Hm.m sa.a o.H m.n m.n mo.o m em.n m.a Ho.m eo.nm eo.am nm.om mm o m.am H.om m.om m.om n.o~ m.mm nausea smog moawa moawo.m eoaxm.e noaea.n nonH.n sso\mosowosmaaae o am.m e.n an.a no.e m.na o.oa .>.o Ho.a n.N n.a on.e e.n n nn.m nm.n nm.m nm.om oa.om m o o m.om s.mm H.mm m.om e.mm sbmsoa cmm: :OHNH :OHNo.N moaxo.: moaxm.m aao\moaomosamhae m musm oopmoapza ma ea NH oa m im‘ e soaeoocsaamoa mama a com dosaapmoo : manna 40 II. Effects of Sera and Peritoneal Exudate Cells from Immune and Nonimmune Rats on the Host Parasite Rela- tionship of T. lewisi and the Rat ‘Experiment 4: IE zIEgg studies of peritoneal exudate cells and sera with I. lewisi Only when the trypanosomes were in combination with sera from hyperimmune rats was there obvious agglutination (see Table 1 Experiment 4). At the end of three hours, the agglutinated trypanosomes were very much less motile than those in any of the other tubes. Many of the organisms showed a lack of motility, a vital sign, and also appeared lysed. In all tubes which contained exudate cells the flagel- lates adherred to their surfaces and some were phagocytized. Those exudate cells which were without immune sera in the tubes showed less of this activity than in the presence of serum. Those tubes which contained sera from hyperimmune rats in combination with exudate cells showed the highest incidence of phagocytosis and the most active adherence of trypanosomes to the exudate cells. This was true when the peritoneal exudate cells were derived from normal rats, acutely infected rats, or hyperimmune rats. Trypanosomes and parts of trypanosomes could be clearly distinguished within exudate cells on Wright's stained 41 smears. No quantitation, such as phagocytic index, was attempted, but it appeared on examination of smears that exudate cells in combination with sera from hyperimmune rats showed more phagocytosis than any of the other systems. ‘Experiment 5: Passive immunization against I. lewisi using sera and peritoneal exudate cells in dexamethasone-treated and untreated rats. It was evident from Experiments 1 to 3 that rats which had been administered dexamethasone and infected with I. lewisi developed high and fatal parasitemias in all instances except when the rats were administered combinations of ex- udate cells from normal rats and sera from hyperimmune rats or exudate cells and sera from hyperimmune rats (Table 5). Rats which were infected with I. lewisi but were not treated with dexamethasone were absolutely protected when administered sera from hyperimmune rats, or a combination of sera from hyperimmune rats and cells from normal rats, or sera and cells from hyperimmune rats. Infections were established in untreated rats which were administered a com» bination of normal sera and exudate cells from hyperimmune rats, but the peak parasitemias were lower than those of rats which had been given sera and/or exudate cells from normal rats. .pcoeHaoQNo as» no Soapmasd esp oo>a>acm mpmm o .capmoaoca Ho>ea maaepammaad be dead mush oopoomsa oopmoap ozommnpoaaxon * HN Um>abhdm OHN o dopoomzacd eaaanm Hmaaoz a ooo.osm so ooo.nne.a seo\soasn sauce mgfifianofi .056 mHHmo mpwdfiunm OQSEEH OOH o oam o Bao\moaxm Bunch massaa can mHHoo opadsxo massed ooa o OHN o aao\noaxm SSHom massed one mHHoo omeSNo Hwahoa 00H 42 u ooo.om¢ so ooo.omm.a EEo\moaxm smash Hashes can mHHeo opadcwe Hanson ooa o 1mm ooo.oam.a sauce oscssa ooa n ooo.nom uMI ooo.ooo semen assaos cos a ooo.nae en ooo.ooo.a sso\soawn maaoo apmcsxo oases“ ooa n ooo.omn en ooo.ono.a sso\aoawn waaoo apadcxo Henson ooa n ooo.ooe en ooo.oom.a osaacm Hmeaoz soauoomaH aao\woSomocmmhae aoapoomaH sao\moaomoammaaa donopmdadad< uwom when ozomwspoamxom 02 neon when enomMSpoamNoQ HH coapoom H soHpoom amazoa meomoasmhae Spas vapoomaa much we mmHEopHmmawa xmom .m paoaahomxm .m canoe 43 Egppriment 6: Retention of I. lewisi in the peritoneal cavities of rats given peritoneal exudate cells and sera from immune rats (Table 6). Agglutinated trypanosomes and phagocytic cells contain- ing trypanosomes were recovered from the peritoneal cavities of all rats which were administered peritoneal exudate cells and sera. When dexamethasone treated rats were administered cells, sera, or Hanks' balanced salt solution separately, trypanosomes were infrequently found in the peritoneal cavities of the rats after 1 hour. When they were found, they were motile and not agglutinated except when immune sera were administered. In the case of the latter some few agglu- tinated trypanosomes were observed. When untreated rats were administered sera from hyper- immune rats, the peritoneal cavities revealed agglutinated trypanosomes. Many were in combination with peritoneal exudate cells. Many were non-motile and appeared lysed. The peritoneal cavities of these normal rats which had been administered Hanks' balanced salt solution revealed few try- panosomes. Though some of the trypanosomes were associated with peritoneal exudate cells it was unusual. The few try- panosomes which were recovered appeared quite viable. III. Reproductive Potential of T. lewisi as Indicated by Studies with Tritiated Thymidine In Vivo Table 6. 44 Results of the examination of tail blood in Experiment 6 for the presence of trypanosomes Treatment Dexamethasone Untreated 30 minutes 60 minutes 3Q minutes 60 minutes Exudate cells and sera from hyperimmune rats negative negative negative negative Exudate cells from hyperimmune positive positive negative positive rats Sera from hyper- negative ositive negative negative immune rats p I Hanks balanced positive positive positive positive salt solution 45 Experiment 7: Uptake of tritiated thymidine ble. lewisi maze- The parasitemias of rats injected with tritiated thymi- dine and rats not given this compound are graphed in Fig. 3. Both groups of rats appeared to have had similar infections and were unaffected by the administration of the compound. From autoradiographs of blood smears, it was determined that the thymidine was incorporated into the nuclei and kinetoplasts of trypanosomes, and the nuclei of white blood cells as well as some of the immature red blood cells. Less than 10 percent of the trypanosomes were labeled in the rats used in this experiment. When rat 1 was injected 48 and 72 hours postinfection, the labeled trypanosomes were 204p or less in length or they were in a state of division. The label was equally distri- buted among the dividing nuclei or the dividing kinetoplasts of reproducing trypanosomes which had incorporated the HB-thymidine during the synthetic period. Labeled trypano- somes were no longer observed in this rat 24 hours after the last injection of the radioactive compound. When H3-thymidine was given to rat 4 192 and 216 hours postinfection,none of the larger nondividing trypanosomes present at this time were labeled. However, injections which had been given rats 2 and 3 between 96 and 168 hours post- infection resulted in labeled trypanosomes which were non- 46 Figure 3. Mean hemocytometer counts of trypanosomes per cmm blood of rats injected with H3-thymidine and rats not given the compound .I r1111! 7' R YPANOSOMES PE R cmm. Figure 3 I. ll \ ’ \ I, . \Q—. I \ , \ I I I I l I I I I I I p e————e I H3 Thymidine \ . \\ I, Control \ I \ II ° L \ e RV ééééé-‘zééuofiu‘zu‘eufs DAYS POST INFECTION 48 dividing, dividing and of variable sizes including the larger nonreproducing trypanosomes. Labeled trypanosomes were observed from rats 2 and 3 for as long as 48 hours after the last injection of H3-thymidine. Ezperiment 8: Reproductive potential of‘I. lewisi and effects of ablastic immunity on its incorporation of tri- tiated thymidine. The mean grain counts of trypanosomes labeled on the third day of infection are shown in Fig. 4. The mean grain count of each labeled trypanosome was reduced by half every eight hours in untreated rats and dexamethasone treated rats. During the same period of the infection approximately 2.8 generations of trypanosomes occurred in 24 hours as indicated by hemocytometer counts. This amounted to one generation of trypanosomes every 8.5 hours. The mean grain counts of trypanosomes labeled on the 5th day of infection are shown in Fig. 5. Thirty-two hours were required before the mean grain count was halved over the trypanosomes of untreated rats. In the rats treated with the corticosteroid 11 hours were necessary to half the mean grain count. For this period of the infection the generation times for the trypanosomes as calculated from hemocytometer counts were 49 hours in the untreated rats and 14 hours in the dexamethasone treated rats. 49 Figure 4. Trypanosomes per cmm and mean grain count over 100 labeled trypanosomes as a function of time following an injection of 500 no H3-thymidine into rats 3 days postinfection TRYPANOSOMES PER cmm. 7 IXIO‘I IX l0°i IX I05- T I "V" IXIO 50 Figure 4 0—0 Untreated rate o-—---o Dexamethasone treated rats / /°’ ll / ’1 ’0 /’ [I ./. I I 50 I II I I e e ‘ 40 I II ‘ , . ‘ 30 I \ 1° ‘ ‘ I k 1 20 .4 . °"°\ « IO \1~. \_,\ .S—H o 8 IS 24 32 40 4e nouns Posrmascnow or H3—THYMIDINE MEAN GRAIN COUNT OVER I00 LABELED TRYPANOSOMES Figure 5. 51 Trypanosomes per cmm and mean grain count over 100 labeled trypanosomes as a function of time following an injection of 500 no H3-thymidine into rats 5 days postinfection 52 Figure 5 I X I073 )- I- p ’ e—e Untreated rate o----o Dexamethasone treated rate 0 ’/ ° 00 . I, E I XI ‘: I 0 E E . Q P- U) i‘ 0 8 g a 0: 1 g IX IO 33 k 0 " S b 0 u E Q S. 2 d I X lot 1 i l J 1 1 l - 0 § 0 8 I6 24 32 4O 48 56 64 nouns POSTINJECTION OF H3-mrmcms LABE L ED TRYPANOSOMES 53 Experiment 9: Incorporation of tritiated thymidine by I. lewisi in subacute, late and chronic infections. The percentage of trypanosomes labeled in subacute, late, and chronic infections may be seen in Fig. 6. Though trypanosomes were easily found in the late infection, as indicated by the hemocytometer counts in the same figure, only on one occasion were labeled trypanosomes found. Try- panosomes in the rat with a subacute infection and in the rat with a chronic infection were rapidly labeled. The grain counts over the nuclei and kinetoplasts of the trypano- somes in the chronic infection diminished more rapidly than those over the subacute infection. IV. Incorporation of Tritiated Thymidine py_I. lewisi In Vitro Egperiment 10: Determination of amount of tritiated thymi- dine necessary to label I. lewisi 12.22222: When trypanosomes were incubated (for 5 hours) in culture media containing tritiated thymidine, as many as 51% of the organisms were labeled using 5 no of the isotope. As shown in Fig. 7, increases in the amount of the H3-thymidine from 5 no to 50 no resulted in an increase in the percent of labeled trypanosomes when they were incubated with greater quantities of the isotope for 5 hours. Figure 6. 54 Trypanosomes per cmm of blood, percent labeled trypanosomes and mean grain counts over trypano- somes from rats with late, subacute, and chronic infe tions. The rats were given two injections of H -thymidine (500 us each) separated by two hours 2 x lo" 55 Figure 6 Sub-acute infection Chronic infection o .................. o - e—-e (WIII‘I dexamethasone) E' 5 Late infection e ----- e 95: s ' i q l X IQ ‘ i, 0) g E I.~“~q lfil Ir” _..a P . . ' Q -.. .f 8 l.__\\ hm. ? 5'5 I \ . E r ‘ ' \ x I ha’ I A ," E r | ’ \\-e”’ 5 L \../l 3 X IO 1 p . I ' .o.. *1 25 - ' [:4 20 - fig I5 _: ........ o' g l: E .' ...-a k St ;' u .0 x. s 0) l5 - Q g It: s u '0 - x m 0 °: 1 5 . k I .- o--- \° - - 1’" - - 0 F0 l_--I I I I I I I -l a. IO 20 30 4O 50 60 TO 80 90 HOURS AFTER THE INJECTION OF H3 THYMIDINE Figure 7. 56 Effect of increasing quantities of H3-thymid1ne on the uptake of the labeled compound lg vitro by 2. lewisi 57 .223 0.» $5.55 8.2.5: 01 08 8.8. 0:8. 099. on. 8. o: 8. cm 8 2. 8 cm 3 on 8 o. u d d d d u d 4 q d d d d d d d 3:2. n. 3:4 o lllll o 952. n .834 oIIIIIIo u 223m 03739177 SJWOS‘ONVdA a1 1N3? 33d 58 When the trypanosomes were in culture for 15 hours with H3-thymidine, as many as 80% of the trypanosomes were labeled using 5 pc. Trypanosomes cultured with lo‘pc for the same length of time resulted in 85% of them being labeled. Greater quantities of H3-thymidine appeared not to have effected a greater percentage of labeled trypanosomes. Experiment 11: Determination of the DNA synthetic period in 2. lewisi. Nine percent of the trypanosomes were labeled after they were pulsed for 30 minutes in culture medium containing 10 pc of tritiated thymidine. After the trypanosomes were transferred to medium which did not contain H3-thymidine (see Table 7 and Fig. 8) the percent of labeled trypanosomes increased to 56% after they had been in culture for 28 hours. When trypanosomes were pulsed for 1 hour, 22% of the organisms were labeled. Thirty-eight percent of the try- panosomes were labeled after they had been in culture for a total of 20 hours, only 1 hour of which was spent in medium containing the radioactive compound. As seen in Table 7, when the trypanosomes were pulsed in media containing tritiated thymidine for increasing amounts of times there were corresponding increases in the percentage of labeled organisms; however, more than 5 hours in the media containing the labeled compound did not result 59 ooow on as ow oooo om on om ooom ooo.oH so on ma ooo.oH ooo.oH ooo.oH mm mm no mm m oooo ooo.oH oooo am no no as mm mm m.m ooo.oH oooo ooom oo on mo mm m ooo.:H ooom oooo mm on on mm mm H ooo.oH oooo oomm on mm mm ma oa o m.o N: mm om Mw XW m.m N H n.o mason ca manodassp mmm mqa:HMpnoo Home ca made endos 2H onzuafio a“ mean Hmpoe ocadaamsp Umpdapanu Spas doHonH moaomozmqahp pnoonom Ha unusanomxm ca onpab mm.con5pH50 aao\moaomozmnhuu can .5 mHflwB 60 in an increase in percent of labeled trypanosomes. After the trypanosomes were recovered from media containing the H3-thymidine and were washed and put into media without the labeled compound, there was still an increase in the percent of labeled trypanosomes. If Table 7 is read diagonally, it is apparent that the trypanosomes double their number within 5 hours in zitgg. The second counts were not made on individual cultures until they had been incubating for a total of 13 hours. After 13 hours incubation, all cultures had at least doubled the number of trypanosomes in the original inoculum. Counts made 28 hours after incubation revealed that half of the cultures had increased their numbers 3 fold. The blank spaces in Table 7 are due to improperly prepared slides from which data could not be obtained. Experiment 12: The influence of ablastic sera on the uptake of tritiated thymidine in 31333. The graph in Fig. 8, representing trypanosomes pulsed with tritiated thymidine in normal rat sera, is constructed from data obtained in Experiment 11 (see Table 7). The try- panosomes which were pulsed in media containing sera in which trypanosomes had been previously incubated for 20 minutes, followed by storage in the refrigerator for 12 hours, served as a control for trypanosomes cultured in ablastic sera 61 which had been similarly treated. Trypanosomes cultured in normal rat sera, which had previously been treated in this way, incorporated the label after being pulsed for 15 minutes. Sixty-six percent of the trypanosomes had incorporated the label after 8 hours of incubation. The percentage of labeled organisms did not increase more than this with longer ex- posures (Fig. 8). The first indication of incorporation of H3-thymidine in ablastic media was after 2.5 hours. The largest percent of labeled organisms was eight which occurred after pulsation in ablastic media for 5 hours. Longer periods of pulsation in ablastic media did not result in an increase in percent of labeled trypanosomes (Fig. 8). When trypanosomes were pulsed in ablastic media and then transferred at various time intervals to culture media with normal sera and the labeled compound, there was an increase in the percentage of organisms labeled (Fig. 9). The earlier the transfer was made, the higher was the percentage of labeled trypanosomes. The percentage of labeled trypanosomes was unchanged when they were originally pulsed in media containing normal sera and the isotope followed by transfer to ablastic media also containing the isotope (Fig. 9). 62 Figure 8. Effect of pooled ablastic sera on the uptake of H3-thymidine by 2. lewisi in vitro PER CENT TRYPANOSOMES LABELED IOO 90 80 70- 60 50- 4O 30 20 63 Figure 8 D~——-_ Normal rat sera incu- D_-—-U bated with trypanosomes I I I, o——o Normal rat sera I I I I I o—. Ablastic sera I I 2 4 6 8 |0|2 l4l6 l8 202224 2628 HOURS IN MEDIUM CONTAINING H3 THYMIDINE (ac) on Figure 9. E fect of ablastic sera on incorporation of H -thymidine in vitro after transfer to normal media and before transfer to normal media 80 70 PER CENT TRYPANOSOMES LABEL [D on J> (n C) CD C) N (3 IO 0) C) 65 Figure 9 /, “.‘\~ \ _ \\\~._——/e I I I I - I a— —a Ablastic medium I Transferred from ablastic medium o——o to normal medium (total time in I culture 24 hours) O——O Normal medium 0. Transferred from normal medium H to ablastic medium (total time " ' in culture 24 hours) , 1| 0 O Ch.~ / \ \ x O haé 1 1 1 I 41 2 4 e 3 IO I2 l4 I6 I8 2022 HOURS CULTURED IN FIRST MEDIUM CONTAINING H3 THYMIDINE BEFORE BEING TRANSFERRED 66 Experiment 13: Titration of ablastin. When trypanosomes were grown in an increasing quantity of ablastic sera, the percentage of organisms labeled declined (Fig. 10). Though only 3#% of the trypanosomes were labeled in the presence of 0.625% ablastic sera, 84% were labeled when 1.25% of the sera were ablastic. Thirty- three percent of the organisms were labeled when 2.5% ablastic sera were present in the culture medium. This decreased assimilation of H3-thymidine continued until #% of the trypanosomes were labeled in the presence of “0% ablastic serae 67 Figure 10. In vitro titration of pooled ablastic sera. The {Effect of increasing congentrations of antibody on the assimilation of H -thymidine q 0 O O PEI? CENT TRYPANOSOMES LABELED a an o o 01 O 20 IO 68 In Figure 10 l IO 20 PER CENT ABLASTIC 3O 4O SERUM IN MEDIUM (IOpCI Plate 10 69 Pictures of trypanosomes in vivo. RBCS are approxi- mately 6 u. A. Labeled lymphocyte. B. Labeled trypanosomes of variable sizes. C. Two adjacent trypanosomes. The trypanosome located anteriorly is unlabeled. The trypanosome underneath it is in telephase and is labeled. D. Two labeled trypano- somes. The trypanosome in the lower left has a double nucleus. E - G. Variable sizes and shapes of T, lewisi. H and I. Large unlabeled non-repro- ducing trypanosomes. 71 Plate 2. Pictures of trypanosomes cultured in vitro. A and B. Unlabeled large non-reproducing trypano- somes. C. Unlabeled smaller trypanosomes. D and E. Unlabeled reproducing trypanosomes. F. Rosette formation of cultural or crithidial forms. G through I. Labeled trypanosomes. 72 Plate 2 DISCUSSION The susceptibility of the normal rat to Trypanosoma lewisi is well established. The formation of antibodies in the normal rat, one being powerful in its action against the reproduction of this parasite, other antibodies which are trypanocidal (12, 13, 17, 57), and the modification of this antibody response in dexamethasone treated rats cannot be doubted. The mechanisms responsible for this behaviour are not precisely expressed. Kass and Findland (27), Thomas (67), Taliaferro (61) all reported that the mechanisms involved in the decreased resistance to infections in animals which had been subjected to x-ray, cortisone and ACTH treat- ment are associated with the inhibition of inflammation and inhibition of antibody production. Although the normal rats in this study were certainly susceptible to infections with T. lewisi, once infected they did in time recover. The recovery was heralded by the decreased numbers of trypanosomes in a state of division, followed by a sharp decline in parasitemia, and eventual complete disappearance of the parasite from the peripheral blood stream. This response in the rat was altered in favor of T. lewisi when 0.5 mg doses of dexamethasone were admin- istered during the infection. 73 74 Becker (7) found a lower parasitemia level when rats were given 6 mercaptopurine. It was believed that 6 mer- captopurine was not only deleterious to the host but to the parasite as well. The results of this study are in agreement with those of other workers who successfully used x-irradia- tion of the rat (65), blockade and splenectomy of the rat (58) to raise parasitemia levels and prolong the multiplica- tion of T. lewisi infections. Though a part of the explanation of findings in the present study may be due to the failure of treated rats to develop humoral antibodies which act against the reproductive proclivities of T. lewisi as well as antibodies which are lethal to it, another explanation is necessary for the early events which occur in the infection. Experiment 2 clearly shows a higher parasitemia in treated rats beginning approximately the let hour of the infection. However, the reproduction of the trypanosomes, as indicated by the percentage of dividing forms found in normal rats and in treated rats, were similar though parasi— temias were different. It was not until 43 hours postinfecm tion that a remarkable difference was found in percentage of dividing forms between the two groups. Similar data were indicated in Experiment 1. The reticulo-endothelial system is a source of cells which are involved in inflammation. The arrival of phago- 75 cytic cells in inflammation is usual and one of their functions is to mechanically remove foreign substances (24). Dexamethasone inhibits processes associated with inflammation, and therefore, some of the differences in parasitemias between treated and untreated rats were thought to be due to the lack of certain cells and their ordinary activity in the earliest stages of infection and their increased activity later in the infection. The phagocytic activity might account for the lighter parasitemia in the untreated group which early in the infection demonstrated a percentage of dividing try- panosomes not unlike that of the treated group. Experiment 3 demonstrated that T. lewisi continues to reproduce at a high rate in rats treated with dexamethasone long after trypanosomes in normal rats have not only ceased to reproduce but have been mostly destroyed by trypanocidal antibodies. Apparently the formation of ablastin was inhibited by the periodic administration of the corticoster- oid. The results of this experiment are in good agreement with those of other workers who prolonged the reproduction phase of T. lewisi by treating infected rats with sodium salicylate (5, “9). It was observed that the rats which were treated with dexamethasone did not gain weight as did their untreated counterparts (Fig. 1). Similar findings were reported by Tadokoro gt §l° (55). According to the warnings in litera— 76 ture supplied with the drug, a negative nitrogen balance may occur in animals receiving dexamethasone. This alone would account for a lack of weight gain and reduced antibody pro- duction. It was discovered that tremendous numbers of trypano- somes could be harvested from a small number of dexamethasone treated rats. Such trypanosomes have been minimally influ- enced by antibodies, and they are from a population suitable for obtaining large numbers of trypanosomes in a high state of reproduction. By pooling such organisms and keeping them frozen in nitrogen, a source of trypanosomes with identical backgrounds were readily available; thus, unnecessary delay and the fret of dealing with widely differing populations of trypanosomes in each experiment was eliminated. As was anticipated from data reported by Lange and Lysenko (28), the effect of sera and cells from rats made hyperimmune to T. lewisi were lethal to the trypanosomes in zitgg. In all three of the experiments described where sera and cells from hyperimmune rats were used, the results indi_ cated that humoral antibodies act in conjunction with phago- cytes in order to effect protection in the rat against 3. lewisi infections. The protection so clearly evident in Experiment 5 (Table 4), where dexamethasone treated rats were administered sera from hyperimmune rats along with exudate cells, indi- 77 cates that dexamethasone does not interfere with antigen antibody reactions. Further evidence for the lack of inter- ference by this drug is indicated by the results from Experi- ment 6 where trypanosomes were found agglutinated, lysed, and phagocytised in the peritoneal cavities of rats which had been treated with the drug but had also been given sera and cells from hyperimmune rats. It appears that the role of dexamethasone was that of an anti-inflammatory agent, and thus it prevented the formation of cells and/or their function in phagocytosis and antibody formation. Corti- costeroids do not influence the metabolism of trypanosomes (68). Therefore, the differences noted in the infections in this study between treated and untreated rats are not due to the affects on the parasite. It was not anticipated that antisera which were so effective in agglutinating, lysing and eventually effecting death of the trypanosomes in vitro would be so inadequate in zlgg when given to dexamethasone treated rats. Whether administered in combination with exudate cells from hyper- immune or nonimmune rats, the sera from hyperimmune rats was then entirely effective (Table A) in preventing parasitemias in dexamethasone treated rats. These results are even more dramatic in Experiment 6 where the peritoneal contents were examined one hour after infecting the rats. The trypanosomes were retained in the peritoneal cavities of normal rats and 78 dexamethasone rats when both antisera and exudate cells were administered. When either cells or sera were administered singly to treated rats (Table 5) the trypanosomes found their way to the circulatory system within 60 minutes. Normal rats which were given antisera retained the trypanosomes in the peritoneal cavities where they were agglutinated, lysed, and ultimately phagocytised by the exudate cells normally present in the host. The finding that the trypanosomes were retained in the peritoneal cavities of rats passively immunized is in agree- ment with results reported by Cox (19) for naturally immune rats but differs in that he did not observe agglutination, lysis, or phagocytosis of trypanosomes recovered in peri- toneal washings. The trypanosomes, immune sera and peri- toneal exudate cells were put into contact with one another in the peritoneal cavities of rats in the study reported in this thesis which accounts for the agglutination, lysis and phagocytosis of the trypanosomes and possibly their retention in the peritoneal cavities of the rats. It was suggested that a sensitivity reaction between the immune rats and the trypanosomes might explain the retention of T. lewisi in the peritoneal cavities of naturally immune rats (14). Exudate cells from hyperimmune rats gave no indication of protection in dexamethasone treated rats (Tables h and 5), but they did afford a mild protection in normal rats. This 79 might be evidence in support of Nossal's (4) findings that among the heterogeneous population of exudate cells are apparently cells which contain specific antibodies. These results offer a basis for explaining in part the early higher parasitemias seen in rats treated with the corticosteroid as compared with rats not treated with the drug. Phagocytic cells are essential for the effective removal of trypanosomes as well as necessary for eventual antibody formation. Dexamethasone treated rats are deficient in phagocytes which are available and effect phagocytosis in normal rats. This phagocytic activity is sufficient apparent— ly in the normal rat to reduce the number of trypanosomes which initiate the parasitemias. Since for the first 48 hours postinfection the repro— ductivity of g. lewisi, as indicated by mensural data and presence of dividing forms (Table 3), is similar in normal rats and dexamethasone treated rats, it appears that only phagocytosis alone can account for the striking differences in parasitemias seen between the treated and untreated rats. The experiments dealing with passive immunity indicated by quantitation of parasitemias that neither trypanocidal antibodies nor reproduction inhibiting antibodies are effective when passively administered without the host's own normal responses to this parasite. Antisera were effective, however, in protecting rats when a phagocytic system was 80 provided either by the normal host or when peritoneal exudate cells were transferred to hosts made deficient in phagocytic cells by treatment with dexamethasone. In order to be more certain that reproduction of T. lewisi is affected by ablastin, experiments were pursued using H3—thymidine. The experiments were designed to elucidate the time during the infection and where in the parasite that ablastin was effective. The staining properties of the kinetoplast have attracted the attention of biologists for some time. The genetic continuity of the kinetoplast, the discovery of a Feulgen positive reaction in this structure (46), the sensitivity of the kinetoplast to DNAase and its faculty for incorporating tritiated thymidine (52, 53) led Steinert (53) to conclude that trypanosomes have two nuclei, one of which is absolutely permanent. The other, that is the kinetoplast, can be absent in certain species or certain races, naturally or experimentally (53). On occasion, it has been suggested that the kinetoplast is an endosymbiotic microorganism resembling bacteria. Electron microscopy of this organelle reveals it to be mito- chondrial in appearance (1, 10). Usually the kinetoplast is rigorously situated for a particular stage in the life cycle of the trypanosomatidae. It, by and large, is numerically constant, and it is thought to be synchronous with the 81 nucleus in the synthesis of the DNA and subsequent division (53). This evidence makes it difficult to admit that the kinetoplast is a symbiont, but it does not make it an im- possibility. Admittedly, it is more inviting to consider the kinetoplast as an organelle which transmits genetic information necessary for the synthesizing of respiratory enzymes generally associated with mitochondria. This work- ing hypothesis could be useful in further research with g. lewisi utilizing autoradiography in combination with electron microscopy. The hypothesis is favored by the fact that only some species of trypanosomes in which respiration is not sensitive to cyanide may have been able to give rise to some races devoid of kinetoplasts (25). The assimilation of tritiated thymidine into the nucleus and the kinetoplasts and its equal distribution between nuclei which were dividing as indicated by autoradiography is relevant to the experiments in this study. As a conse- quence of division, daughter cells share both old and newly synthesized nuclear material. Apparently the small forms of T. lewisi (20 u or less) which are so numerous the first 72 hours of the infection are the most active assimilators of H3~thymidine (Experiment 7) and in truth may be considered a reproducing population (57). More evidence for the small forms being truly reproducing forms is the reduction of the label from the small trypanosomes (Experiment 7) within 24 82 hours. Different conditions prevailed in rats given H3- thymidine between 96 and 168 hours postinfection. Large and small, dividing and non-dividing trypanosomes were labeled. Labeled trypanosomes were observed as long as 48 hours after the H3-thymidine had been injected. This data indicates less rapid division of the trypanosomes at this time. It follows that rapid division of labeled cells eventually results in fewer cells with sufficient H3-thymidine to cause autoradiography for the conditions of the experiments. The label was reduced less rapidly from those cells which repro- duced more slowly. Finally, when H3-thymidine was injected 192 and 216 hours postinfection, no trypanosomes were observed to be labeled, indicating they did not assimilate the H3-thymidine. It has been generally believed that ablastin becomes effective approximately 4 days postinfection (57). The observations in this experiment are in agreement with those made by Taliaferro and Pizzi (62). Apparently, ablastin not only inhibits directly or indirectly the assimilation of H3- thymidine, but it also interferes with the division processes after the synthetic period has passed. The calculation of generation time of T. lewisi ig‘vggg based on hemocytometer counts of trypanosomes in the peri- pheral blood is inaccurate because the parasitemia is affected by the hosts battery of defense mechanisms which, 83 if properly functioning, works towards the elimination of the trypanosomes. The hemocytometer counts of the parasitemia is an expression of the minimum increase in trypanosomes by reproduction which permits only the calculation of the maxi- mum time necessary for the organisms to complete one genera- tion. This problem was apparent in Experiment 8 where the generation time was 8.5 hours as indicated by hemocytometer counts and was 8 hours as indicated by reduction of grain counts over tritium labeled trypanosomes for the first 3 days of the infection of dexamethasone treated rats or untreated rats. When H3-thymidine was administered on the 5th day of the infection, 32 hours passed before the grain count was halved over the trypanosomes from untreated rats (Fig. 4). This is 28 hours more than was necessary at 3 days postinfection. The time required for the number of grains to be reduced by half over the trypanosomes from the dexamethasone treated rat 5 days postinfection was 11 hours. This is 3 hours longer than was required 3 days postinfection. The presence of ablastin by the 5th day explains the slower disappearance of the labeled trypanosomes in untreated rats. Some other explanation might be necessary for the data from dexametha- sone treated rats. Crowding, metabolic products and athrep- sis might account for the slower rate of reproduction in the dexamethasone treated rats by the 5th day postinfection. 84 It is observed in all these studies that the course of 1. lewisi infections may be correctly described by an ex- ponential curve for the first four days of infection. After 4 days, which is near the beginning of the effects of ablastin in untreated rats or near the end of the infection because of death in dexamethasone treated rats, the rate of increase diminishes. The point of inflection occurred when the concentration of trypanosomes reached approximately 7x105 to 1x106 per cmm in dexamethasone treated rats. The point of inflection in untreated rats corresponded with the development of ablastin in untreated rats. This inhibition of growth is indicated in both cases by the increased time necessary to halve the mean grain count of autoradiographed trypanosomes which had previously incorporated H3-thymidine during their synthetic period (Fig. 4). Though the departure from the exponential course of infection described by hemocytometer counts may be due to inhibition of reproduction (8), it also reflects the destruction of a portion of the trypanosomes. The experi- ments using H3—thymidine reflects only the rate of repro- duction and its inhibition, unless destruction is somehow selective rather than random. The one occasion on which labeled trypanosomes were observed from the rat with the late infection in Experiment 9 indicates that 2% of the trypanosomes were capable of 85 assimilating H3-thymidine (Fig. 6). This suggests that g. lewisi may still reproduce in late infections but rarely do so. This also suggests the heterogeneity in the population of trypanosomes some of which may have been more resistant to the antibodies formed against the majority of the popula- tion. It had been hoped in Experiment 9 that a rat which no longer exhibited an increasing course of infection with 2. lewisi had been selected for a subacute infection. This was not the case. The rat with the infection which had been designated subacute exhibited a rise in parasitemia at the beginning of the experiment. The uptake of the labeled com- pound by the trypanosomes reflected their reproduction. The two peaks seen in the parasitemias (Fig. 6) are reflected in the grain counts and in the percent of labeled trypanosomes of the rat which was designated as having a subacute infec- tion and the rat with the prolonged chronic infection due to dexamethasone administration. As in previous experiments, the curve depicting the grain counts reflected the inhibition of reproduction. Again the data indicated that mere parasi- temia does not give a complete picture of the reproductive activity of the trypanosomes. Later in in zitrg studies, it was discovered that the presence of duplicated organelles also may lead one to make questionable conclusions concerning trypanosomes and their reproductive activities. 86 Trypanosoma lewisi grew and survived at 37° C in D'Alesandro's medium for approximately 48 hours and survived for another 2 or 3 days. In agreement with D'Alesandro (18), most of the reproductive activity of g. lewisi occurred in this medium within the first 24 hours. The trypanosomes in the culture were morphologically indistinguishable from blood forms. The increased addition of HB-thymidine (Fig. 7) to cultures of T. lewisi did not result in an increased percent- age of labeled trypanosomes. The longer period of incuba- tion with the H3-thymidine did increase the percent of labeled trypanosomes which is in agreement with results of previous authors (53). When the cultures were supplied with more than 10 no of H3-thymidine, it was found to be in excess of the amount of the labeled compound needed to label the trypanosomes. Approximately 15% to 20% of the trypanosomes remained un- labeled in all the in 21232 experiments. These trypanosomes either did not synthesize DNA.$EIE$EEBI lacked enzymes necessary for the assimilation of H3-thymidine into their DNA, or at least did not utilize this pathway in their syn- thesis of DNA. It is difficult to explain the increase in the percent- age of labeled trypanosomes after the organisms were removed from the medium containing the label (Table 6). Assuming 87 that the washing technique was sufficient, it is possible that those trypanosomes which were capable of in zitgg assimilation of H3-thymidine were also more adaptable to in zitrg conditions than those which did not incorporate the compound or incorporated it more slowly. If this is true, then eventually the labeled trypanosomes would replace the unlabeled trypanosomes. Further studies would be necessary to test this hypothesis. The time required from the first signs of grain to where no further increase occurs approximates the DNA syn- thetic period (5). When the trypanosomes were incubated for as short a period as 15 minutes in the presence of H3-thymi- dine, some of the trypanosomes were labeled. The time required to where no further increase of grains over trypano- somes occurred was 1 hour. Therefore, 45 minutes approxi- mates the synthetic period (33). The generation time appeared to be less than 5 hours (Table 6). The synthetic period of T. lewisi and the generation time is considerably shorter than that of 2. mega. Steinert and Steinert (53) reported 7 hours for the synthetic period and 18.9 hours for the generation time. The first five hours appeared to be the most active period of reproduction for T. lewisi in culture, though there was some increase in numbers of organisms after 5 hours. Eighty percent of the trypanosomes were labeled after 5 88 hours incubation (Table 6) in the presence of the labeled compound. There were no substantial increases in labeled organisms after this time. It was observed throughout the in zitrg studies that there were present in all cultures trypanosomes with two or more nuclei or kinetoplasts which were not labeled. Their presence indicated that the mere observation of organisms with duplicated organelles may not be sufficient to assume that the individuals are actively involved in reproduction. Another interpretation is that some of the trypanosomes resist the incorporation of H3-thymidine because of other synthetic pathways. It has been recognized that the medium of D'Alesandro is less than adequate for the indefinite continuation of blood forms of T. lewisi i2:212£2 (18). However, reproduc- tion does occur for a limited time and in these experiments a substantial number of trypanosomes assimilated H3-thymidine. When trypanosomes were cultured in the presence of ablastic sera, the trypanosomes did not incorporate the H3- thymidine until 2.5 hours had passed. Maximum labeling required five hours. Apparently in the presence of ablastin the presynthetic period is lengthened as well as the synthe- tic period. The maximum percent of trypanosomes labeled in ablastic sera was eight. It can be presumed, therefore, that ablastin inhibits the incorporation of the compound by 89 most of the trypanosomes. Those few trypanosomes which did incorporate the isotope may have been sufficiently antigenic— ally different to be undisturbed by the presence of ablastin. Another explanation is that the concentration of ablastin was insufficient to completely inhibit the total number of trypanosomes in the culture. Trypanosomes incubated in control normal rat sera were labeled after 15 minutes (Fig. 8) incubation in the presence of H3—thymidine. The highest percent of labeled organisms in normal sera which had been previously treated allowing 2. lewisi to survive was 76 and this occurred after 8 hours of incubation in the media and in the isotope. This in 11232 demonstration of inhibition of assimila- tion of H3-thymidine is one of a number of demonstrations of the antibody nature of ablastin that began with the investi- gations of Taliaferro (59), who showed that ablastin could passively be transferred and is a globulin, the physico- chemical properties of which have been characterized (17). The in zitrg results are in agreement with the in‘zizg experiments in this study. This study also confirmed that ablastin is not absorbable with living trypanosomes (18, 59). Further, the data from Experiment 12 (Fig. 8) demonstrated that the effects of ablastin are reversible by simply removing the trypanosomes from ablastic media. When the trypanosomes were removed early from the 9O ablastic medium, a greater proportion of them incorporated the label. Trypanosomes which had remained for so long a time as 17 hours in the ablastic media, increased in percent labeled trypanosomes from 3 to 18 after only 5 hours in normal media which contained the labeled compound. Differences in antibody concentrations can be shown by titration methods (18). In this study, it is difficult to explain the observation that trypanosomes incubated in culture medium containing 0.625% ablastic sera incorporated less H3-thymidine than trypanosomes cultured in media con- taining 1.25% ablastic sera (Fig. 10). In general, the percent trypanosomes labeled decreased with the increased concentration of ablastic sera in the medium. Though a considerable body of knowledge is now available concerning the effects of ablastin on the metabolism.of T. lewisi, nothing is known about the mechanisms of action of the antibody. It is probable that ablastin exerts its effects upon different metabolic processes rather than directly at one point. Among the metabolic process of 2. lewisi affected by ablastin are glucose metabolism (38), protein synthesis (62), as well as nucleic acid synthesis. Recently D'Alesandro (19) demonstrated that dividing trypanosomes contain about three and one-half times as much lactic dehydrogenase than adult parasites. Apparently the antibody does not react with the enzyme directly. 91 Undoubtedly other enzymes and metabolites are affected by the antibody since there are changes in several general basic processes associated with ablastic immunity. SUMMARY Resistance of the rat to T. lewisi was studied by de- pressing the formation of antibodies in rats by treating them with dexamethasone, a corticosteroid. The dexamethasone treated rats provided an $2.3l22 system for studies of the agents in immune rats associated with protection against T. lewisi. Further, large numbers of T. lewisi which had not experienced the protective agents were obtained from the dexamethasone treated rats for $2.21322 experiments. The $2.1122 system consisted of rats treated with dexa- methasone and infected with T. lewisi. The resulting para- sitemia consisted of trypanosomes with a high coefficient of variation and percent dividing forms characteristic of populations of trypanosomes whose reproduction is uninhibited. Treated rats suffered severe levels of parasitemia, did not gain weight and died. Injections of dexamethasone into uninfected rats resulted in a lack of weight gain but none of them died. Of the rats treated with dexamethasone and infected with T; lewisi, only those which were administered sera from immune rats together with exudate cells from immune or non— immune rats were protected against the parasite. Rats not treated with dexamethasone and administered sera from immune 92 93 rats alone or in combination with exudate cells were protected against infection with T. lewisi. The use of autoradiography following injections of H3- thymidine into rats infected with T. lewisi revealed that the compound was incorporated into the nucleus and kinetoplast of the parasite. As indicated by reduction in the mean grain count, the generation time for T. lewisi was 8 hours $2.3l22 for trypanosomes of dexamethasone treated rats as well as those of untreated rats which had been infected for 3 days. The levels of parasitemia among the treated rats were higher than in the untreated rats. The lower levels of parasitemia in untreated rats were presumed due to the removal of trypano- somes by phagocytes. Thirty—two hours were required before the mean grain count was halved over the trypanosomes of un- treated rats when H3-thymidine was administered on the 5th day of the infection. Eleven hours were necessary to half the mean grain count of trypanosomes of treated rats when the labeled compound was given on the 5th day of the infec- tion. Tritiated thymidine was not incorporated by T. lewisi when it was administered to untreated rats which had been infected for 14 days, but the labeled compound was assimilated by trypanosomes of dexamethasone treated rats which had been infected with T. lewisi for 14 days. The influence of ablastin on the incorporation of H3~ 94 thymidine by blood stream forms of T. lewisi was studied T2 31233. The synthetic period of T. lewisi lasted 45 minutes 19. m in medium containing 40% normal serum with HB-thymi— dine added. Approximately 85% of the trypanosomes assimilated H3-thymidine during 5 hours incubation. Longer periods of incubation with the labeled compound did not increase the percentage of labeled trypanosomes. Not all trypanosomes which appeared to be reproducing over a period of time during which H3-thymidine was present assimilated the isotope. Culture medium containing 40% ablastic serum prevented the incorporation of H3-thymidine in more than 90% of the trypanosomes. Between 2 and 8% of the trypanosomes did in- corporate the isotope in the presence of ablastin. The pre- synthetic period was 2.5 hours and the synthetic period was between 2.5 and 5 hours. When the activity of ablastic serum was titrated the assimilation of H3-thymidine by the trypanosomes was inverse- ly proportional to the concentration of the ablastic serum. The effects of ablastin on T. lewisi were reversible when the ablastic medium was exchanged for normal medium. CONCLUSIONS The present study established that the formation of ablastin as well as trypanocidal antibodies are inhibited by dexamethasone, and that sera from rats immune to T. lewisi together with exudate cells are essential in the passive immunization of such treated hosts. Studies with H3-thymi- dine established that ablastin inhibits reproduction as indicated by inhibition of incorporation of the labeled compound and the inhibition of the reproductive sequences that usually result in the diminution of grain counts of autoradiographs. The l2.1$22 and $2.21322 demonstration of ablastic activity using H3-thymidine as a marker is one illustration of the antibody nature of ablastin. It is speculated that ablastin exerts its effects directly upon several different metabolic processes rather than directly at only one point and indirectly at others. Therefore, the use of metabolites involved in all these processes in order to obviate the inhibition may be necessary. 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