Julius P. Kreier

Plasmodium berghei: Adherence and phagocytosis by rat macrophages in vitro

Abstract Comparative studies of the adherence and phagocytosis of Plasmodium berghei using macrophages obtained from normal rats, from rats with active parasitemias, and from recently recovered rats were conducted in vitro. Preliminary data indicated that the combination of immune macrophages with immune serum was more effective in phagocytosis of parasites than either component alone and that macrophages interacted differently with parasites isolated at different stages of infection. Immune macrophages were able to entrap and ingest parasites isolated both at the early stage and at the late stage of infection. Normal macrophages, without the help of immune serum, were able to entrap and ingest only parasites isolated at the early stage of infection. Immune serum enhanced the phagocytic capability of both normal and immune macrophages.

Autoimmune reactions in rats with Plasmodium berghei infection

Abstract An agglutinin for trypsinized autologous and homologous erythrocytes was demonstrated in rats with Plasmodium berghei infection. This agglutinin did not agglutinate nontrypsinized rat erythrocytes. The agglutinin was a cold type since it was eluted from erythrocytes of P. berghei infected rats at 37 °C. The antibody was susceptible to reductive cleavage by mercaptoethanol.

An experimental study of the pathogenesis of fowlpox infection in chickens.

SUMMARY The pathogenesis of experimental fowlpox in chickens infected with various strains of the virus by the intradermal, intravenous, and intratracheal routes was studied. The various strains all caused diseases which developed similarly if given by the same route. The clinical features of the disease produced were influenced more by route of infection than by the strain used. Lesions developed only at the site of injury when virus was given intradermally. Virus was not detected in the blood or internal organs of any of the 80 chickens infected intradermally. Lesions developed on many sites when infection was by the intravenous route. The disease in intravenously infected chickens was severe. Five of them died of the disease between the 13th and 18th days after inoculation and others would have if they had not first been killed for sample collection. Virus was detected in the liver, spleen, kidney, and lung but not in the brain. During the viremic stage, virus was detected only in the buffy coat portion of the blood. No lesions developed in chickens infected intratracheally. Virus was detected in the lungs of these birds but not in other organs. Growth curves of virus in tissues and organs were determined by the titration of samples on chorioallantoic membranes of 12-day-old developing chick embryos.

Plasmodium gallinaceum: fine structure by freeze-etch technique.

Abstract The fine structure of the obligate intracellular parasite Plasmodium gallinaceum was examined by the freeze-etching technique. The characteristics of this malarial parasite, from the early trophozoite stage to the segmenting schizont stage were studied, and structural aspects of each stage were examined. The malarial parasite was shown to have cytoplasmic and nuclear membrane characteristics which differ considerably from those of the host. Two types of discontinuities in the nuclear membranes of the parasites were noted. These were a small pore system in the inner nuclear membrane and a large pore system in the outer membrane. The cytoplasmic membranes of the parasite were more coarsely granular than those of the host. The outer parasite membrane which has been reported to be of host origin also differs considerably in structure from the erythrocyte membrane. This could be due to modifications after envelopment of the parasite, or could indicate another origin for the membrane.

Plasmodium berghei: Nucleic acid agglutinating antibodies in rats☆

Abstract The serum of rats infected with Plasmodium berghei was found to contain agglutinins for RNA and, at a lower titer, for DNA. Cross-absorption studies with RNA and trypsinized erythrocytes indicated that the anti-RNA agglutinin was not identical to the antitrypsinized erythrocyte agglutinin found in the same sera.

Erythrocyte destruction mechanisms in malaria.

Publisher Summary This chapter focuses on the erythrocyte destruction mechanisms in malaria. The mammalian hemopoietic system is a remarkable piece of biological machinery in terms of its productivity and its intrinsic regulatory processes. In humans, the erythropoietic system produces approximately 2.5 × 106 red cells/second or 2 × 1011 cells/day. This means that about 5 × 1015 cells are produced during an average human life span of 70 years. The erythropoietic system is positively regulated at the level of the committed stem cell by the glycoprotein hormone erythropoietin that is elaborated by the kidney at various rates depending on the oxygen requirements of the individual. Somewhat overlooked, but still equally impressive, is the ability of the reticuloendothelial system (RES) to recognize and eliminate numbers of senescent cells equal to the input to maintain a steady level of circulating red cells. The erythropoietic system can, under stress, increase its productivity approximately sevenfold. When red-cell loss exceeds the production rate, anemia occurs. The anemia is related to either decreased red-cell production or enhanced red-cell destruction.

Evaluation of Plasmodium berghei for endotoxin by the Limulus lysate assay.

The presence of endotoxin in the malaria parasite has long been suspected but has not been directly demonstrated by the Limulus amoebocyte lysate (LAL) assay or other assays. Tests for endotoxins have been done on plasma of infected individuals but not on the parasites themselves (Glew and Levin, 1975, Proc. Soc. Exp. Bio. Med. 148: 508-510). Recently, Tubbs (1980, Trans. R. Soc. Trop. Med. Hyg. 74: 121-123) detected endotoxin activity in the plasma of mice infected with Plasmodium berghei and in patients infected with P. falciparum using the LAL assay, and suggested that the endotoxin was derived from either the parasites or the endogenous bacteria in the gut. By assaying intact plasmodia washed free of contaminating host cells and plasma, it is possible to determine whether the plasmodia are the source of the endotoxin. We obtained free P. berghei using ultrasonic energy and tested the plasmodia for endotoxin activity using the LAL assay. The results obtained are the subject of this report. Groups of mice and rats were infected by the intraperitoneal injection of approximately 106 P. berghei parasites, and later the blood was collected aseptically by cardiac puncture when parasitemia reached 40%. The blood was transferred to pyrogen-free, sterile, heparinized tubes and washed three times in sterile, pyrogen-free 0.9% NaCl. Blood from uninfected mice was treated in an identical manner and served as a control. The blood specimens were then sonicated as previously described (Prior and Kreier, 1972, Exp. Parasitol. 32: 239-243). The continuous-flow chamber and sonicator probe tip were rendered pyrogen-free by heating at 160 C overnight. All equipment and solutions were tested for endotoxin contamination with the LAL test before use. Following sonication the specimens were centrifuged to harvest the plasmodia, and then adjusted to 25% packedcell volume. The specimens were lysed by three cycles of freeze-thawing and assayed quantitatively for the presence of endotoxin using the microdilution LAL assay (Prior and Spagna, 1979, J. Clin. Microbiol. 10: 394395). The minimum sensitivity of the Limulus lysate utilized in the microdilution assay (courtesy of Mallinckrodt Inc., St. Louis, Missouri) was 0.06 ng/ml of LPS using the E. coli reference standard (lot EC-2, Bureau of Biologics, Food and Drug Administration). Aliquots of free parasite and control samples were seeded with known quantities of the E. coli endotoxin and assayed. Because certain heat-labile protein substances may give positive LAL test results, portions of the samples were heated at 100 C for 5 min and then tested for endotoxin activity. Parasites and control samples were also cultured on blood agar aerobically and anaerobically to detect any possible bacterial contamination. The results obtained in four experiments using the LAL assay on the lysed parasite suspensions and controls are shown in Table I. A reaction suggestive of endotoxin was ob-

Plasmodium gallinaceum: Chicken erythrocyte survival as determined by sodium radiochromate51 and di-isopropylfluorophosphate32 labeling

Abstract The chromium-51 and di-isopropylfluorophosphate-32 labeling techniques were used to evaluate erythrocyte survival in healthy and Plasmodium gallinaceum -infected chickens. Analysis of the data obtained from noninfected chickens indicated that the chicken erythrocyte has a life span of about 34 days, that there is no physiological random destruction of chicken erythrocytes, and that 51 Cr elutes from chicken erythrocytes in the absence of disease at a fairly constant rate of about 2.5%/day. A comparison of curves of 51 Cr and DF 32 P disappearance from the circulations of Plasmodium gallinaceum -infected chickens indicated that the chromium elution rate increased greatly as the infection progressed. The amount of 51 Cr eluted from the erythrocytes of infected animals was sufficiently great that it must be assumed that the infection brought about increased elution from nonparasitized erythrocytes. Because of the effects of the infection upon elution of 51 Cr from erythrocytes it is suggested that DF 32 P is the better label for studies of erythrocyte destruction in malaria and that 51 Cr is not a suitable label for evaluating erythrocyte destruction in malaria except in studies of short duration.

Parasitemia and erythrocyte destruction in Plasmodium berghei-infected rats: II. Effect of infected host globulin☆

Abstract Rats were infected with Plasmodium berghei and erythrocytes collected at various stages of the infection. The erythrocytes were labeled with 51 Cr, suspended in an infected rat globulin solution, and transfused into compatible inbred rats protected from infection by chloroquine. The rat globulin solution agglutinated trypsinized erythrocytes when diluted 1 to 64. The number of labeled transfused erythrocytes lost during the first 24 hours after transfusion in the recipient rats was explainable as the total of losses from senescence, from the transfusion process, and from elimination of parasitized erythrocytes. The globulin from P. berghei -infected rats did not increase the efficiency of elimination of the parasitized erythrocytes over that previously observed by Kreier and Leste (1967) in the absence of injected globulin, nor did the injected globulin cause shortened survival of nonparasitized erythrocytes.

Malaria and babesiosis: similarities and differences

This inaugural paper presented by the senior author at the Second International Conference on Malaria and Babesiosis was chosen by the organizers of the conference to consider common features of the two diseases and their causative agents and thereby suggest the advantage of a comparative research approach. Our awareness of the similarities between the two diseases prompted us to plan the first conference in Mexico City in May of 1979. The proposal for this first conference arose during a review of babesiosis vaccine trials in Mexico among Drs. J.P. Kreier, Professor and malariologist of The Ohio State University; J. A. Pino, Director of the Agricultural Sciences of the Rockefeller Foundation and the senior author. At this meeting we concluded that findings from babesiosis research should be of interest to malariologists and that comparative analysis of many other aspects of the two diseases would be advantageous to each research group.