In vitro development of exoerythrocytic forms of Plasmodium gallinaceum sporozoites in avian macrophages

Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. The host cells were macrophage precursors from chicken bone marrow. At various times after introduction of sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). The time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until the end of the primary exoerythrocytic schizogony.     

Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry

The study of the liver stage of malaria has been hampered by limitations in the experimental approaches required to effectively dissect and quantify hepatocyte infection by Plasmodium . Here, we report on the use of flow cytometry, in conjunction with GFP-expressing Plasmodium sporozoites, to assess the various steps that constitute a successful malaria liver infection: cell traversal, hepatocyte invasion and intrahepatocyte parasite development. We show that this rapid, efficient and inexpensive method can be used to overcome current limitations in the independent quantification of those steps, facilitating routine or large-scale studies of host–pathogen molecular interactions.     

Transformation of sporozoites of Plasmodium berghei into exoerythrocytic forms in the liver of its mammalian host

Summary Intrahepatocytic transformation in vivo of the rodent malaria sporozoite of Plasmodium berghei, into the young trophic exoerythrocytic tissue stage was studied by immunofluorescence, light- and electron microscopy. The first 20 h of intracellular life were involved entirely in dedifferentiation with limited proliferation of organelles. From about 20 h onwards nuclear division commenced, rough endoplasmic reticulum became markedly expanded, and mitochondria increased in numbers. However, remains of the sporozoite pellicle (i.e., inner membranes and subpellicular microtubules) persisted for at least 28 h, which correlates with the persisting reaction of young exoerythrocytic forms with antisporozoite antibodies. In general, the basic mechanism of transformation resembles that of the ookinete into oocyst and that of the merozoite into erythrocytic trophozoite.     

Observation on complete schizogony of Plasmodium vivax in vitro

Attempts to grow Plasmodium vivax in vitro were made on 43 isolates in three different culture media. Complete schizogony occurred in the new medium SCMI 612 in which 34 out of 43 isolates produced merozoites. The RPMI 1640 and Waymouth media suitable for the cultivation of P. falciparum were also used with markedly less success. Results of the experiments indicate differences in nutritional requirements between the two species of Plasmodium.     

Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells; an in vitro assay of protective antibodies

Plasmodium falciparum and P. vivax sporozoites were observed to invade cultured human hepatoma cells in vitro. Monoclonal antibodies to the circumsporozoite (CS) protein of each of these malarial species blocked invasion. Inhibition was species-specific, but was independent of the geographic origin of each strain. Because these monoclonal antibodies have been shown to diminish or abolish sporozoite infectivity to susceptible primate hosts, it is suggested that inhibition of invasion of sporozoites (ISI) into cultured cells may represent in in vitro assay for protective antibodies. This was confirmed by the finding that serum taken from volunteers immune to sporozoite challenge also totally blocked sporozoite invasion. The ISI assay also detected naturally acquired invasive-neutralizing antibodies in areas endemic for malaria. This ISI assay may therefore be useful in determining the incidence of inhibitory anti-sporozoite antibodies in general populations, and allow the monitoring of the effect of an anti-malarial vaccine using sporozoite-derived antigens.     

Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.     

d-glucose uptake in human liver cell cultures

Summary The kinetic parameters ford-glucose uptake were studied in human liver cell cultures under strictly defined experimental conditions. Using a wide concentration range (0.005 to 30 mmol/l), the kinetic data obtained suggested strongly thatd-glucose in human liver cell cultures can be transported by two separate systems. For the high-affinity system, the apparentK _m was 0.645±0.21 mmol/l and the V_max, 12.49±3.74 nmol/mg protein per min. For the low-affinity system, the apparentK _m was 6.91±0.58 mmol/l and the V_max, 79.90±5.27 nmol/mg protein per min. At a concentration of 2.1×10⁻⁷ mol/l, cytochalasin B preferentially inhibited the high-affinityd-glucose site or transport system. The time course ofd-glucose uptake, studied in two cell lines from patients with hereditary fructose intolerance, was significantly higher than for the control lines. This work was supported by Grant I.N.S.E.R.M. CRL 77-5-210-4.     

Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species

Plasmodium sporozoites can enter host cells by two distinct pathways, either through disruption of the plasma membrane followed by parasite transmigration through cells, or by formation of a parasitophorous vacuole (PV) where the parasite further differentiates into a replicative exo-erythrocytic form (EEF). We now provide evidence that following invasion without PV formation, transmigrating Plasmodium falciparum and Plasmodium yoelii sporozoites can partially develop into EEFs inside hepatocarcinoma cell nuclei. We also found that rodent P. yoelii sporozoites can infect both mouse and human hepatocytes, while human P. falciparum sporozoites infect human but not mouse hepatocytes. We have previously reported that the host tetraspanin CD81 is required for PV formation by P. falciparum and P. yoelii sporozoites. Here we show that expression of human CD81 in CD81-knockout mouse hepatocytes is sufficient to confer susceptibility to P. yoelii but not P. falciparum sporozoite infection, showing that the narrow P. falciparum host tropism does not rely on CD81 only. Also, expression of CD81 in a human hepatocarcinoma cell line is sufficient to promote the formation of a PV by P. yoelii but not P. falciparum sporozoites. These results highlight critical differences between P. yoelii and P. falciparum sporozoite infection, and suggest that in addition to CD81, other molecules are specifically required for PV formation during infection by the human malaria parasite.     

Ani immunomodulator Hepon inhibits hepatitis C virus replication in human cell cultures in vitro]

Antiviral activity of immunomodulator "Hepon" was evaluated in human cells culture infected with hepatitis C virus. "Hepon" presence protected human cells SW-13 from cytopathogenic effect of hepatitis C virus. Maximum antiviral effect was demonstrated by "Hepon" at concentration 1 mcg/mL. Control antiviral agent reaferon (interferon alfa-2a) was more potent as vitality protecting agent in the case of SW-13 human cells culture. "Hepon" activity is based on changes of cytokins and interferons spectrum so this immunomodulator is expected to be effective against different viruses including herpes virus and encephalocarditis virus.     

Formation of heterosymplasts in human reticular cell cultures]

The cell fusion has been studied in human reticular cell cultures J-96 and J-41 treated with the Sendai virus or with polyethylene glycol 1000 and 6000. The J-96 cells have a high alkaline phosphatase activity, in J-41 cells the enzyme is not detectable. No heterogenous alkaline phosphatase activity was seen in the protoplasm of symplasts 18 hours after virus cell fusion. It has been shown with polyethylene glycol treatment that during the fusion of cells J-96 and J-41 the enzyme activity was spreading over the symplast protoplasm.     

Production of erythropoietic cells in vitro for continuous culture of Plasmodium vivax

Plasmodium vivax cannot be maintained in a continuous culture. To overcome this major obstacle to P. vivax research, we have developed an in vitro method to produce susceptible red blood cell (RBC) precursors from freshly isolated human cord hematopoietic stem cells (HSCs), which were activated with erythropoietin to differentiate into erythroid cells. Differentiation and maturation of erythroid cells were monitored using cell surface markers (CD71, CD36, GPA and Fy6). Duffy⁺ reticulocytes appeared after 10 days of erythroid cell culture and exponentially increased to high numbers on days 14–16. Beginning on day 10 these erythroid cells, referred to as growing RBCs (gRBCs), were co-cultured with P. vivax-infected blood directly isolated from patients. Parasite-infected gRBCs were detected by Giemsa staining and a P. vivax-specific immunofluorescence assay in 11 out of 14 P. vivax isolates. These P. vivax cultures were continuously maintained for more than 2 weeks by supplying fresh gRBCs; one was maintained for 85 days before discontinuing the culture. Our results demonstrate that gRBCs derived in vitro from HSCs can provide susceptible Duffy⁺ reticulocytes for continuous culture of P. vivax. Of particular interest, we discovered that parasites were able to invade nucleated erythroid cells or erythroblasts that are normally in the bone marrow. The possibility that P. vivax causes erythroblast destruction and hence inflammation in the bone marrow needs to be addressed.