Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and Pepstatin A (aspartyl protease inhibitor).

We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

Plasmodium falciparum: Differential Sensitivity In Vitro to E-64 (Cysteine Protease Inhibitor) and Pepstatin A (Aspartyl Protease Inhibitor)

ABSTRACT We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 μM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 μM) or 12 h at the inhibitory concentration 50% (12 μM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 μM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

Culture of Plasmodium falciparum: the role of pH, glucose, and lactate

Yields of P. falciparum in intraerythrocytic in vitro cultures were maximized when extracellular pH was maintained between 7.2 and 7.45, and extracellular lactate was kept below 12 mM. Host erythrocytes metabolized 4.6 +/- 1.5 microM glucose/10(9) RBC/24 hr and produced 7.9 +/- 1.8 microM lactate/10(9) RBC/24 hr. Asynchronous parasite cultures used 122 +/- 34 microM glucose/10(9) parasitized RBC/24 hr and produced 143 +/- 47 microM lactate/10(9) parasitized RBC/24 hr. Synchronous cultures that were 80 to 100% ring forms after 24 hr in culture exhibited significantly lower glycolysis per 10(9) parasitized RBC than cultures that were 0 to 25% ring forms after 24 hr. The percent of glucose utilization accounted for by lactate production by parasites was significantly less than that of uninfected erythrocytes. These optimum ranges and metabolic rates can be used in the development of parasite culture techniques.     

Kinetics of the initial rounds of cell division of Toxoplasma gondii

RH strain Toxoplasma gondii tachyzoites that had naturally lysed their host cells were allowed to infect new host cells for a limited amount of time; subsequent parasite cell divisions were observed closely. On the basis of 4 independent trials, the estimated time to first cell division was 9.8 hr postinfection (PI) and was quite variable (95% confidence interval [CI]: 3.1-16.5 hr PI). The estimated time to second cell division was 14.9 hr PI and was less variable (95% CI: 12.1-17.7 hr PI). Few parasites divided before 6 hr PI in these 4 trials. When tachyzoites were derived by forced lysis (scraping an infected host cell culture and passing it through 27-gauge needles), the first parasite cell division occurred much more rapidly than had been observed in any of the trials with parasites derived by natural lysis. When parasites derived by forced lysis were held away from host cells for 3 hr PI, the first cell division was delayed in a manner similar to that seen in parasites derived by natural lysis. No differences were observed in the timing of the second cell division of parasites derived by forced lysis whether or not they had been held away from cells. These studies demonstrate that the conditions to which tachyzoites are exposed during transit from one host cell to another can affect the kinetics of parasite cell division in the new host cell.     

Plasmodium falciparum: synchronization of asexual development with aphidicolin, a DNA synthesis inhibitor

The asexual development cycle of Plasmodium falciparum, a malarial parasite of humans, has been synchronized in culture by treating ring-stage parasites with aphidicolin, an inhibitor of DNA synthesis. Optimization of both the concentration of drug added to ring stage containing red blood cells and the duration of exposure of parasites to drug led to a reversible block of their maturation at the early trophozoite stage. Release of the aphidicolin block led to a synchronous development of parasites that was manifested by about 80% of the new ring stages being produced within a 2- to 3-hr interval.     

Trypanosoma cruzi: morphogenesis in diffusion chambers in the mouse peritoneal cavity and attempted stimulation of host immunity

Sequential morphologic changes and antigen producing capacity of Trypanosoma cruzi in peritoneally implanted diffusion chambers were studied. Diffusion chambers were equipped with two Nuclepore filters (0.20 μm pore size) sandwiched between three Lucite rings. Epimastigotes or trypomastigotes and amastigotes were placed in diffusion chambers and surgically implanted into the peritoneal cavity of mice, or placed in in vitro cell culture, or in various types of culture media and incubated at 26 or 37 C.Epimastigotes maintained in diffusion chambers in mice changed into trypomastigotes as evidenced by the presence of numerous transitional stages and the concomitant decrease in the percentage of the former and increase in the percentage of the latter in chambers removed and examined at 16, 24, 36, 48, 72, and 84 hr after implantation. The maximum of 68% trypomastigotes was noted in chambers examined at 84 hr. Amastigotes subsequently appeared, apparently arising from trypomastigotes and reached the highest percentage (49%) obtained at 132 hr. The total number of parasites in chambers decreased slightly during the first 36 hr (20%). Little change in the total number of parasites was noted during the interval of 36–108 hr. A subsequent decrease in numbers of parasites was noted until by 280 hr after implantation, chambers contained less than 2% of the original number of organisms present in the chambers. No similar transformation of epimastigotes was noted in diffusion chambers maintained in cell culture at 37 C or in a cell culture growth medium or LIT medium at 37 or 26 C.No detectable morphological change was noted when trypomastigotes and amastigotes were implanted in diffusion chambers in the peritoneal cavity of mice. The total number of these parasites decreased notably (82%) after 24 hr.Mice receiving diffusion chambers containing epimastigotes implanted at two different intervals (21 days apart), developed only marginal protective immunity when challenged with virulent T. cruzi three weeks after the second implant of chambers, and no protection was afforded those mice implanted with chambers containing trypomastigotes and amastigotes. Sera collected from mice 6 wk after the second implantation of diffusion chambers containing parasites were observed to have antibody titers to T. cruzi as demonstrated by the fluorescent antibody technique and direct agglutination procedure.     

In vitro culture of Cryptosporidium muris in a human stomach adenocarcinoma cell line

We investigated the optimal culture conditions for Cryptosporidium muris in a human stomach adenocarcinoma (AGS) cell line by determining the effects of medium pH and of selected supplements on the development of C. muris. The optimum pH of the culture medium required for the development of C. muris was determined to be 6.6. The number of parasites significantly increased during cultivation for 72 hr (p < 0.05) at this level. On the other hand, numbers decreased linearly after 24 hr of incubation at pH 7.5. When cultured in different concentrations of serum, C. muris in media containing 5% FBS induced 4-7 times more parasites than in 1% or 10% serum. Of the six medium supplements examined, only 1 mM pyruvate enhanced the number of C. muris in vitro. Transmission electron microscopic observation showed the developmental stages of C. muris in the cytoplasm of the cells, not in an extracytoplasmic location. The growth of C. muris in AGS cells provides a means of investigating its biological characteristics and of testing its response to therapeutic agents. However, a more optimized culture system is needed for the recovery of oocysts on a large scale in vitro.     

Plasmodium falciparum: synchronization of cultures with DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum. Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr. Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.     

Stimulation by granulocyte-macrophage colony-stimulating factor of Leishmania tropica killing by macrophages.

The intracellular protozoan parasite Leishmania tropica was found to survive unharmed and to multiply for several days in normal mouse peritoneal macrophages. In contrast, when infected monolayers were treated with GM-CSF, there was a continuous decrease in the percentage of infected cells, reaching less than 10% on day 4 in culture, compared to about 30% in normal controls. Microscopic observations showed an increased number of dead parasites in GM-CSF treated infected cells. Within 5 hr of incubation with GM-CSF, almost 40% of intracellular parasites showed morphologic damage, compared to less than 10% in untreated cells. Pretreatment of macrophage monolayers with pure GM-CSF before infection led to an increased level of phagocytosis of L. tropica parasites as reflected by the percentage of infected cells and the increased number of parasites in each infected cell. GM-CSF treated cultures showed 73% infected cells containing a mean of five parasites per cell, as compared to controls in which only about 50% of macrophages were infected with only two parasites per cell. The number of dead parasites per cell was 5-fold higher in the GM-CSF treated cultures at 2 hr. After 24 hr the percentage of infected GM-CSF treated cells was less than one-third that in the control cultures.     

Growth in vitro and metabolism of Plasmodium vinckei chabaudi.

An in vitro system, based on the rocker dilution technic, has been developed that supports intraerythrocytic growth of a rat-adapted strain of Plasmodium vinckei chabaudi from ring to schizont stages; some reinvasion was obtained, although invariably, this was associated with a decrease in parasite numbers. Pertinent features were the very high buffer content of the medium and the low oxygen tension of the gaseous phase. Lactate production, glucose utilization, and 3H-leucine and 3H-adenosine incorporations were investigated for their suitability to monitor parasite growth. Throughout an 18-hr incubation there was a continuous and increasing production of lactate and utilization of glucose, which correlated well with the development of the parasites from ring to schizont stages. During the same period, there was a low but continuous and increasing incorporation of 3H-leucine into parasite protein. However, 3H-adenosine was incorporated only for the 1st hr of incubation, after which time no net incorporation occurred. Parasites grew normally from ring to schizont stages even in the absence of adenosine from the dilution medium.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Development of resistance to coccidiosis in the absence of merogonic development using X-irradiated Eimeria acervulina oocysts

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic development in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.     

The site of action of the anticoccidial salinomycin (Coxistac).

The anticoccidial salinomycin has a cidal effect against chicken coccidia. Restricted and unrestricted medication studies and histopathological examinations of chicks infected with Eimeria acervulina, E. maxima, or E. tenella showed that parasites were destroyed within host cells during asexual development. Most sporozoites failed to become trophozoites and were destroyed 30--72 hr after ingestion of oocysts. The drug also affected schizonts during initial nuclear replication by either destroying or significantly delaying their maturation. Parasites affected by the drug were distorted grossly. Drug action against gametogony was not observed histologically, but when medication was restricted to this period of the life cycle, subsequent oocyst shedding of all 3 species was reduced by 20--70% compared to unmedicated controls. When drug was provided during the entire parasite life cycle, activity against asexual stages was so complete that only a limited number of parasites survived to form gamonts, and oocyst shedding was reduced by 80--90% relative to controls. As with other ionophores, salinomycin had no effect upon rate of oocyst sporulation.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Mechanism of escape of exoerythrocytic forms (EEF) of malaria parasites from the inhibitory effects of interferon-gamma

We have studied the mechanism of inhibition by interferon-gamma (IFN-gamma) of the development of exoerythrocytic forms (EEF) of Plasmodium berghei in the livers of rats. At the time corresponding to the maximum development of EEF (44 hr after injection of sporozoites), the livers of the IFN-gamma-treated rats contained less parasite DNA as compared with controls. Twenty-four to 72 hr later, the livers of both groups of animals were free of parasites; that is, IFN-gamma treatment does not delay the development of the EEF. The decrease in parasite DNA observed in the IFN-gamma-treated rats was due to a diminution in the number, but not the size, of EEF. It appears, therefore, that treatment with the lymphokine either destroys the parasites or does not affect their replication. To study the mechanism of resistance to IFN-gamma of a small population of EEF, we subjected the parasites to four cycles of selection by IFN-gamma. The parasites from the "selected" and "nonselected" populations were equally susceptible to inhibition by IFN-gamma, indicating that the escape from IFN-gamma activity is not inherited.     

Localization of a low molecular weight antigen of Eimeria tenella by use of hybridoma antibodies.

Three monoclonal antibodies (Mabs), found by western blot analysis to recognize 10-kDa bands of Eimeria tenella sporozoite preparations, were used with immunoelectron (IE) microscopy, immunogold-silver staining (IGSS), and indirect immunofluorescent antibody (IFA) light microscopy to determine the location and distribution of the antigens in or on extra- and intracellular parasites. All 3 of the Mabs (designated C3, E5, and 1231) were found by IE microscopy to label amylopectin granules of extracellular sporozoites. Additionally, these Mabs extensively gold-labeled the sporocyst wall. In cultured primary chicken kidney cells inoculated with sporozoites of E. tenella, IGSS showed surface labeling of the parasite and intense labeling of the infected host cells by 6 hr postinoculation (PI). At 24 hr PI, host cell vacuoles in infected and uninfected cells were labeled by the 3 Mabs by IFA. The E5 and C3 Mabs also were seen to label the host cell membrane of newly infected cells. The C3 and 1231 Mabs showed little label of the host cells by 48 hr PI, but the parasites still were labeled up to 96 hr PI. The E5 Mab had intense IFA labeling of infected host cells at 48 hr PI. The results of this study indicate that parasites apparently release antigenic material during the early stages of parasite development and that this material is found internally and/or on the surface of the infected host cells.     

Trypanosoma cruzi: productive infection is not allowed by chorionic villous explant from normal human placenta in vitro

We hypothesize that a sustained infection of Trypanosoma cruzi into placental tissue might be diminished. Human placental chorionic villi and VERO cells as controls were co-cultured with T. cruzi. Parasites occupied 0.0137% at 3h, 0.0224% (24h), and 0.0572% (72 h) of the total chorionic villi area analyzed and some few placental samples were negative to parasite DNA, whereas 52% of VERO cells were infected at 3h and parasites occupied 0.57%, at 24h the parasite area was of 2.78% and at 72 h was of 3.32%. There were no live parasites in placenta-T. cruzi culture media at 72 h of co-culture. There were significantly increased dead parasites when T. cruzi was treated with unheated culture media coming from placental explants and fewer dead parasites when pre-heated culture media were employed. CONCLUSION: Low productive infection by T. cruzi into placental tissue associated with no viable parasites in the culture media partially due to placental thermo labile substances.     

Leishmania major and Leishmania donovani: effect of LPG-containing and LPG-deficient strains on monocyte chemotaxis and chemiluminescence.

Lipophosphoglycan (LPG) is a major glycolipid present on the membrane of Leishmania promastigotes and amastigotes. We have previously shown that preincubation of peripheral blood monocytes with purified LPG inhibits IL-1 production, chemotactic locomotion, and luminol-dependent chemiluminescence (LDCL). In the present study we tested the effect of LPG present on live parasites on monocyte activity. For this purpose, we used two mutant strains deficient in LPG and two LPG-containing strains. One pair was Leishmania major and the other Leishmania donovani. Monocytes in suspension were infected with the different parasite strains and tested for chemotactic locomotion and LDCL at different times between 1 and 72 hr after infection. In parallel, the percentage of infected monocytes was measured in stained cytospin preparations. The results obtained showed that at 1 hr of incubation only the LPG-containing strains inhibited chemotaxis, while the mutant strains showed a normal response. From 4 hr of incubation onwards, the mutant strains also inhibited monocyte chemotactic locomotion. LDCL was only slightly inhibited by the LPG-containing strains after 1 hr, because of a high level of spontaneous stimulation, probably due to phagocytosis. At 24 and 72 hr all strains inhibited LDCL. These results suggest that LPG is responsible for early inhibition of macrophage activity, but that other factors are responsible for inhibition at later stages of in vitro infection. The model described here might represent a useful tool to further analyze the mechanisms involved in immune evasion of Leishmania parasites.     

Short-term in vitro culture of field isolates of Plasmodium vivax using umbilical cord blood

Plasmodium vivax research has been hampered by the lack of technology for culturing this parasite. Culturing P. vivax is difficult because the parasite selectively invades reticulocytes. Here we describe a modified procedure to establish and maintain short-term cultures of freshly collected P. vivax parasites using reticulocyte-enriched cord blood. Using this method, parasites could be cultured for a month. Manipulation of the culture allowed procurement of synchronized stages of the parasite. This short-term culture method can be easily adapted to study various aspects of the parasite biology.     

Growth in vitro and Metabolism of Plasmodium vinckei chabaudi*

SYNOPSIS. An in vitro system, based on the rocker dilution technic, has been developed that supports intraerythrocytic growth of a rat-adapted strain of Plasmodium vinckei chabaudi from ring to schizont stages; some reinvasion was obtained, although invariably, this was associated with a decrease in parasite numbers. Pertinent features were the very high buffer content of the medium and the low oxygen tension of the gaseous phase. Lactate production, glucose utilization, and ³H-leucine and ³H-adenosine incorporations were investigated for their suitability to monitor parasite growth. Throughout an 18-hr incubation there was a continuous and increasing production of lactate and utilization of glucose, which correlated well with the development of the parasites from ring to schizont stages. During the same period, there was a low but continuous and increasing incorporation of ³H-leucine into parasite protein. However, ³H-adenosine was incorporated only for the 1st hr of incubation, after which time no net incorporation occurred. Parasites grew normally from ring to schizont stages even in the absence of adenosine from the dilution medium.