Fine structure of Plasmodium berghei exoerythrocytic forms in cultured primary rat hepatocytes

Sporozoites of the rodent malaria parasite Plasmodium berghei have been grown in primary cultures of hepatocytes from Brown Norway rats. The ultrastructure of in vitro grown exoerythrocytic forms was compared with that of parasites in vivo. Peculiar vesicles, previously not described in vivo, were identified and their possible origin is discussed. Otherwise, the fine structure of the hepatocytic stages grown in vitro was shown to be grossly similar to those in vivo. Therefore, electron microscopy of cultured exoerythrocytic parasites will contribute to the understanding of the cell biology and drug sensitivity of this elusive stage.     

The influence of cell type and culture medium on the in vitro cultivation of exoerythrocytic stages of Plasmodium berghei

Plasmodium berghei sporozoites successfully entered and developed into exoerythrocytic schizonts in a variety of cell types cultured in vitro, but segmentation and release of merozoites was only observed in human embryonic lung cells. Exoerythrocytic development was generally not influenced by the culture medium, and NCTC-135 was used routinely. In vitro infectivity of P. berghei sporozoites was unaffected by the serum type used for isolation.     

Plasmodium berghei: preparation of rat hepatic cell suspensions that include infective exoerythrocytic schizonts.

Employing an enzymatic method to dissociate rat liver, we prepared suspensions of liver cells from rats infected with sporozoites of Plasmodium berghei 3 to 10, 18 to 28, or 29 to 36 hr prior to liver dissociation. These suspensions of liver cells included hepatocytes, Kupffer cells, fibroblasts, and unidentified cells, as well as hepatocytes infected with exoerythrocytic schizonts (HEX) of P. berghei. These HEX were infective for recipient rodents when inoculated intraperitoneally into the recipients. The number of infective HEX present in the liver cell suspensions was quantitated by varying the number of HEX inoculated into recipients. This infectivity assay made it possible to compare the numbers of HEX in suspensions of liver cells from different donor rats. Infective HEX were obtained from donor rats in 35 of 41 experiments. The greatest number of infective HEX was obtained from donors injected with sporozoites 18 to 28 hr prior to liver dissociation. For morphological observation of mature HEX in cell suspensions, hepatic cells were prepared from donors infected with sporozoites 48 hr prior to liver dissociation. For experimental purposes, the preparation of infective HEX in suspensions of liver cells is superior to the preparation of infective HEX in liver fragments, because it is possible to quantitate the number of HEX which are present either visually or by means of the infectivity assay.     

Immunity to exoerythrocytic forms of malaria. I. Course of infection of Plasmodium fallax in turkeys

Turkeys inoculated intravenously with Plasmodium fallax parasitized erythrocytes developed an initial parasitemia. After the parasitemia crisis, the number of exoerythrocytic forms increased and caused the death of the bird about a week later. When the size of the erythrocytic-form inoculum was decreased tenfold, the day of maximum parasitemia and the day of death due to a high level of exoerythrocytic-form parasitism was delayed approximately 1 day.Turkeys inoculated intravenously with exoerythrocytic forms obtained from erythrocyte-free tissue cultures of parasitized turkey embryo brain cells developed an initial exoerythrocytic-form infection. The growth of exoerythrocytic forms in the poults was not affected by daily drug treatment with chloroquine; the number of exoerythrocytic forms/1000 cerebral cell nuclei was not significantly different in chloroquinetreated or untreated poults. Following the exoerythrocytic-form crisis, the parasitemia increased for several days in nondrug-treated birds. In chloroquine-treated birds, the erythrocytic forms were only detected during the period when exoerythrocytic forms were prevalent. Erythrocytic-form schizonts were not observed in chloroquinentreated birds. The poults stopped gaining body weight when either the exoerythrocytic forms or the erythocytic forms were prevalent. A tenfold decrease in the exoerythrocytic-form inoculum size delayed the exoerythrocytic-form infection 1 day. The development of exoerythrocytic forms was not synchronous in turkeys inoculated with exoerythrocytic forms and examined prior to the exoerythrocytic-form crisis.     

The Morphology and Behavior of Living Exoerythrocytic Stages of Plasmodium gallinaceum and P. fallax and Their Host Cells

The morphology and behavior of living exoerythrocytic stages of Plasmodium gallinaceum and P. fallax were studied by the use of tissue cultures, phase contrast microscopy, and time-lapse cinephotomicrography. The morphology of exoerythrocytic stages of these two species was essentially that previously observed in fixed, stained material, with the following exceptions: (1) the presence of a filament on one end of the merozoite, (2) the absence of clefts in the cytoplasm of the large schizonts, and (3) the absence of a vacuole-like space around the parasite. The following behavior was observed either directly or in time-lapse sequences: (1) emergence of merozoites from mature schizonts, (2) progressive motility of free merozoites, (3) entry of merozoites, both actively and passively, into host cells, (4) nuclear division in the parasite, (5) the various stages of schizogony, including final production of merozoites, (6) massive infection of host cells, and (7) phagocytosis of merozoites and attempted phagocytosis of mature schizonts by macrophages. Exoerythrocytic stages of P. fallax differed from those of P. gallinaceum in that the merozoites of the former were (1) somewhat more curved in shape and (2) present in fewer numbers in mature schizonts. The use of tissue culture, phase contrast microscopy, and time-lapse cinephotomicrography promises to solve many of the remaining problems concerning exoerythrocytic stages of malarial parasites and their interrelationships with host cells.     

Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers

The exoerythrocytic stage of Plasmodium falciparum has remained a difficult phase of the parasite life-cycle to study. The host and tissue specificity of the parasite requires the experimental infection of humans or non-human primates and subsequent surgical recovery of parasite-infected liver tissue to analyze this stage of the parasites development. This type of study is impossible in humans due to obvious ethical considerations and the cost and complexity in working with primate models has precluded their use for extensive studies of the exoerythrocytic stage. In this study we assessed, for the first time, the use of transgenic, chimeric mice containing functioning human hepatocytes as an alternative for modeling the in vivo interaction of P. falciparum parasites and human hepatocytes. Infection of these mice with P. falciparum sporozoites produced morphologically and antigenically mature liver stage schizonts containing merozoites capable of invading human red blood cells. Additionally, using microdissection, highly enriched P. falciparum liver stage parasites essentially free of hepatocyte contamination, were recovered for molecular studies. Our results establish a stable murine model for P. falciparum that will have a wide utility for assessing the biology of the parasite, potential anti-malarial chemotherapeutic agents and vaccine design.     

Characterization of the antigen SO7 during development of Eimeria tenella

The developmental expression of the antigen SO7, which has been previously shown to protect chickens against infection by several Eimeria species, was investigated. Using RT-PCR, mRNA for SO7 was found to be restricted primarily to unsporulated oocysts (0 hr). Western blot (WB) analysis with an antibody to recombinant SO7 (rbSO7) revealed expression of the protein from 6 to 72 hr (fully sporulated) of sporulation and in sporozoites (SZ). SO7 was absent in host-derived second-stage merozoites (MZ) and was present in culture-derived first-stage MZ but at a level of only 25% of that exhibited by SZ. During invasion of Madin-Darby bovine kidney (MDBK) cells by SZ in vitro, the level of SO7 within cells, as determined by WB analysis, remained relatively constant until 48 hr of development and then decreased by about 40% at the next time point (72 hr). The SO7 secreted into the culture media during in vitro development increased to a relative maximum at 48 hr and then decreased to about 20% of maximum at 72 hr. Immunostaining with anti-rbSO7 indicates that SO7 is highly concentrated in both refractile bodies (RB) of SZ, with some limited distribution in the apical complex. Anti-rbSO7 intensively stained the intracellular parasites and the first-stage schizonts during in vitro development of E. tenella in MDBK cells. Upon release from the schizonts, the first-stage merozoites stained with 1 or 2 bright spots typically at each end. The results suggest that SO7 is closely associated with the SZ RB and is developmentally regulated but may not play a direct role in cellular invasion.     

Plasmodium berghei: infective exoerythrocytic schizonts in primary monolayer cultures of rat liver cells.

Suspensions of rat liver cells which included hepatic exoerythrocytic schizonts (HEX) of Plasmodium berghei were used to initiate primary monolayer cultures of rat hepatic cells. These cell suspensions were prepared by using an enzymatic method for the dissociation of the livers of rats that had been infected with sporozoites of P. berghei 3 to 10, 18 to 28, and 29 to 36 hr prior to the liver dissociation procedure. These cell suspensions included HEX which were infective for recipient rodents when inoculated intraperitoneally into the recipients. HEX were considered to have been successfully maintained if they retained their infectivity for rodents following the cultivation period. The relative number of infective HEX present in the liver cell suspensions before and after cultivation was determined by use of an infectivity assay. Using this infectivity assay, it was observed that less infective HEX were present in the cell population following cultivation than were present before cultivation. Infective HEX were recovered from culture in experiments in which the time in vitro ranged from 3 to 44 hr. Twelve of fifteen (80%) attempts to maintain infective HEX in culture for 21 to 28 hr were successful, while one of eight (12.5%) attempts to maintain HEX in culture for 36 to 48 hr were successful. Thus, these experiments have provided an 80% success rate for maintaining HEX for a period equivalent to over 50% of the incubation period of HEX of this parasite. This technique should be sufficient for studying in vitro the factors which influence the development of HEX, as well as for testing methods of causal prophylaxis.     

Plasmodium fallax: phagocytosis of exoerythrocytic stages in tissue culture

Wandering phagocytes in tissue cultures were attracted to the exoerythrocytic stages, both intracellular and extracellular, of Plasmodium fallax. They phagocytized free merozoites or schizonts that had been freed from host cells. They attempted to phagocytize large intracellular parasites.     

Eimeria tenella: use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development

A monoclonal antibody, which recognizes the refractile body of Eimeria sporozoites, was used to study the developmental fate of this organelle during asexual development of E. tenella and to determine the effect of this monoclonal antibody on in vitro development of the parasite. Through use of immunofluorescent antibody and gold-labeling techniques at the light and electron microscopy level, the refractile body at 48 to 96 hr postinoculation was found to separate into 6 to 10 small globules, then diffuse throughout the schizont cytoplasm, and eventually reconcentrate as a small dot of material in each of the mature first-generation merozoites. The schizont did not develop to maturity if diffusion of the refractile body did not occur. The refractile body material was quickly lost as the merozoite left the schizont and invaded new cells and was not detected in any later developmental stages. The in vitro development of first- and second-generation schizonts of E. tenella was greatly inhibited (up to 100%) with exposure to the monoclonal antibody. There was an increase in the number of schizonts with nondispersed refractile body in the monoclonal antibody-treated cells when compared to the untreated controls, and the few mature schizonts seen had up to a 50-fold decrease in the number of merozoites. Immunofluorescent antibody labeling of the refractile body of intracellular sporozoites and schizonts treated in vitro with the monoclonal antibody for 24-96 hr postinoculation indicated that the antibody had crossed the host cell and parasite plasma membrane during incubation.     

Eimeria tenella: parasite-specific incorporation of 3H-uracil as a quantitative measure of intracellular development

An assay has been developed using parasite-specific incorporation of 3H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, 3H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional 3H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of 3H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.     

Plasmodium vivax: exoerythrocytic schizonts recognized by monoclonal antibodies against blood-stage schizonts

Exoerythrocytic parasites of Plasmodium vivax grown in human hepatoma cells in vitro were probed with monoclonal antibodies raised against other stages of P. vivax. Monoclonal antibodies specific for four independent antigens on blood-stage merozoites all reacted with exoerythrocytic schizonts and merozoites by immunostaining. The characteristic staining pattern of each monoclonal antibody was similar on both blood- and exoerythrocytic-stage parasites and appeared only in mature schizont segmenters. In contrast, a monoclonal antibody specific for the caveolar-vesicle complex of the infected host cell membrane and a second monoclonal antibody reacting with an unknown internal antigen did not appear to react with exoerythrocytic parasites. We confirm prior reports that monoclonal antibodies against the sporozoite immunodominant repeat antigen react with all exoerythrocytic-stage parasites, but note that as the exoerythrocytic parasite matures the immunostaining is concentrated in plaques reminiscent of germinal centers and apparently distinct from mature merozoites. These results indicate that mature merozoites from either exoerythrocytic or blood-stage parasites are antigenically very similar, but that stage-specific antigens may be found in specialized structures present only in a specific host cell type.     

A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration

Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions. A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J. C. Y. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J. 3, 174, 1989). Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me2SO to -70 degrees C, and stored at approximately -100 degrees C. Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing. Comparison was made with cryopreservation of liver cells in single cell suspensions. Cryopreservation of liver cells in suspension resulted in only a 2% yield of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes. Freezing cultured liver cells in the sandwich configuration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively. Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented approximately 30% of the cells in culture. These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function.     

Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.     

Plasmodium falciparum: characterization of a 33-kDa soluble antigen

A 33-kDa soluble antigen identified in the culture supernatant by patient serum and monoclonal antibodies was present in rings, trophozoites, schizonts, and merozoites of Plasmodium falciparum. The antigen which is released into the culture supernatant by growing parasites was also observed in the host cells of trophozoites and schizonts and could be localized on the host cell surface. Its specificity for the surface of trophozoites and schizonts was observed to decrease with increased duration without subculture. The antigen could then be detected on the surface of noninfected erythrocytes. The antigenicity of the 33-kDa antigen was destroyed by heating at 65 degrees C. Monoclonal and polyclonal specific antibodies weakly inhibited parasite growth in vitro. The antigen was present in both knob positive and knob negative parasites in all the P. falciparum isolates tested.     

Hormonal inducibility of liver-specific enzymes in cultured rat embryos

In monolayer cultures, hepatocyte-specific enzymes are inducible by hormones as soon as hepatocytes differentiate from the embryonic foregut (15-somite stage). Though offering an excellent opportunity for quantitative studies, several features of a normal cell environment are lost in such a model system. To determine the inducibility of such tissue-specific enzymes in intact organisms, rat embryos were cultured in vitro for 48 h and exposed to the hormonal factors that had been found effective in monolayer culture, viz. dexamethasone, triiodothyronine and dibutyryl cyclic AMP. Normal development of the embryos during culture in vitro was assessed by general criteria reflecting growth, morphogenesis and cytodifferentiation. Development of external features, organogenesis, the distribution of cell divisions and the appearance of tissue-specific proteins such as alpha-fetoprotein and glutamate dehydrogenase served as parameters. Despite undisturbed development of the embryos as judged by these criteria, irrespective of whether the culture was started at day 10 or at day 11 of gestation (just before, respectively after the appearance of the liver primordium), induction of hepatocyte-specific enzymes like carbamoylphosphate synthetase by hormones could not be demonstrated immunohistochemically. However, induction of this enzyme by hormones could be demonstrated in monolayers of hepatocytes isolated from such embryos after 48 h of culture, providing yet another demonstration of the adequate culture conditions. In addition, an adequate uptake of hormones by the embryo during culture could be shown with radio-actively labeled dexamethasone and triiodothyronine and with a radioreceptor assay for cyclic AMP. Therefore, the presence of factors in young embryos that inhibit tissue-specific enzyme synthesis has to be postulated.     

Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats.

Vascular endothelial growth factor (VEGF) plays a major role in angiogenesis, which is essential for both healing of injured tissue and proliferation of carcinoma cells. In this study we elucidated the expression and role of VEGF in rat liver regeneration after partial hepatectomy. VEGF expression was mainly detected in periportal hepatocytes and reached a maximal level 48-72 hr after partial hepatectomy by both immunohistochemistry and in situ hybridization. Similarly, immunohistochemistry for Ki-67 showed that the proliferative activity of sinusoidal endothelial cells was highest in the periportal area and reached a maximal level 72 hr after partial hepatectomy. Moreover, neutralization of VEGF significantly inhibited proliferative activity of hepatocytes (p<0. 0001), as well as sinusoidal endothelial cells (p<0.001), at 48 and 96 hr after partial hepatectomy. Conversely, injection of VEGF significantly promoted proliferative activity of hepatocytes (p<0. 0001) as well as sinusoidal endothelial cells (p<0.0005) at 48 hr after partial hepatectomy. These results suggest that VEGF promotes proliferation of hepatocytes through reconstruction of liver sinusoids by proliferation of sinusoidal endothelial cells. Furthermore, these data point to a new therapeutic strategy, the use of VEGF and other hepatocyte growth factors in fulminant or severe acute hepatitis.     

In Vitro Development of Exoerythrocytic Forms of Plasmodium Gallinaceum Sporozoites In Avian Macrophages

ABSTRACT 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 Ac end of the primary exoerythrocytic schizogony.     

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.     

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc.