Exoerythrocytic merozoites of Plasmodium berghei in rat hepatic Kupffer cells.

Liver biopsies of white rates infected by Plasmodium berghei sporozoites were examined by electron microscopy. Intrahepatocytic schizont development was confirmed. In addition, at 60 and 70 h after sporozoite inoculation, exoerythrocytic merozoites were noted in Kupffer cells of liver sinusoids. Although it is theoretically possible that this observation may be of merozoite development in Kupffer cells, the authors suspect that this example of phagocytosis would be one of the host's natural defenses against sporozoite-transmitted malaria.     

Exoerythrocytic Merozoites of Plasmodium berghei in Rat Hepatic Kupffer Cells

SYNOPSIS. Liver biopsies of white rates infected by Plasmodium berghei sporozoites were examined by electron microscopy. Intrahepatocytic schizont development was confirmed. In addition, at 60 and 70 h after sporozoite inoculation, exoerythrocytic merozoites were noted in Kupffer cells of liver sinusoids. Although it is theoretically possible that this observation may be of merozoite development in Kupffer cells, the authors suspect that this example of phagocytosis would be one of the host's natural defenses against sporozoite-transmitted malaria.     

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.     

Direct infection of hepatocytes by sporozoites of Plasmodium berghei

To identify the unknown liver cell type initially invaded by sporozoites of mammalian malaria, young rats were inoculated intravenously with large numbers of Plasmodium berghei sporozoites obtained from infected Anopheles stephensi mosquitoes. Fine structural studies of liver specimens obtained from the rats within 2 min after inoculation demonstrated the presence of morphologically unaltered sporozoites in the cytoplasm of hepatocytes. Many sporozoites were also observed undergoing cytolysis within the lysophagosomes of Kupffer cells, as well as other phagocytic cells. These observations strongly suggest direct infection of the hepatocyte by the sporozoite.     

Sexual and Asexual Development of Cryptosporidium parvum in Five Oocyst– or Sporozoite-Infected Human Enterocytic Cell Lines

ABSTRACT. The human enterocytic cell lines Caco-2, HT29, HCT8 and the Caco-2 clones TC7 and PF11 were studied for their ability to support Cryptosporidium parvum development. Following the addition in cultures of either oocysts or excysted sporozoites, immunofluorescent and transmission electron microscopy revealed the presence of all stages of the parasite life cycle by both procedures, and no difference in the ration of infected cells was found among cell lines. More oocysts were seen in cell monolayers infected with oocysts than with sporozoites (p < 0.0001). The number of meronts observed was the same after either oocysts or sporozoites inoculation. Data suggest that the two methods yield a same cell infection rate.     

Direct Infection of Hepatocytes by Sporozoites of Plasmodium berghei1

ABSTRACT. To identify the unknown liver cell type initially invaded by sporozoiles of mammalian malaria, young rats were inoculated intravenously with large numbers of Plasmodium berghei sporozoites obtained from infected Anopheles stephensi mosquitoes. Fine structural studies of liver specimens obtained from the rats within 2 min after inoculation demonstrated the presence of morphologically unaltered sporozoites in the cytoplasm of hepatocytes. Many sporozoites were also observed undergoing cytolysis within the lysophagosomes of Kupffer cells, as well as other phagocytic cells. These observations strongly suggest direct infection of the hepatocyte by the sporozoite.     

Changes in the cytoplasmic elements of cultured cells infected with Eimeria vermiformis sporozoites

Epithelial-type (PK-15) and fibroblast-type (MDBK) mammalian cell cultures were inoculated with purified Eimeria vermiformis sporozoites. Matched samples from 0 to 93 h after inoculation (HAI) were processed for electron microscopy; half of the sample preparations were extracted with non-ionic detergent prior to fixation. Specimens were examined by both transmission and scanning electron microscopy. Numerous sporozoites were attached to the cultured cells from 2 to 93 HAI, usually near the cell periphery. Some host cell microvilli extended up and appeared attached to the sporozoites. Sporozoites fixed during the penetration process were markedly constricted at the site of entry; however, no noticeable changes occurred in the host cell membrane or surface microvilli during sporozoite invasion or in sporozoite-infected cells. In cells extracted with 1% Triton X-100, the host cytoskeleton was progressively reorganized about the parasites but changes were limited to the immediate area of the sporozoite. Around resident sporozoites, the cytoskeleton became less dense but also more ordered, which contrasted with adjacent cell areas. Cytoskeletal elements passed both over and under the parasites. The appearance of the cytoskeleton suggested that the host cell formed a loose, basket-like net of cytoskeletal elements about the parasite.     

Changes in the Cytoplasmic Elements of Cultured Cells Infected with Eimeria vermiformis Sporozoites

Epithelial-type (PK-15) and fibroblast-type (MDBK) mammalian cell cultures were inoculated with purified Eimeria vermiformis sporozoites. Matched samples from 0 to 93 h after inoculation (HAI) were processed for electron microscopy; half of the sample preparations were extracted with non-ionic detergent prior to fixation. Specimens were examined by both transmission and scanning electron microscopy. Numerous sporozoites were attached to the cultured cells from 2 to 93 HAI, usually near the cell periphery. Some host cell microvilli extended up and appeared attached to the sporozoites. Sporozoites fixed during the penetration process were markedly constricted at the site of entry; however, no noticeable changes occurred in the host cell membrane or surface microvilli during sporozoite invasion or in sporozoite-infected cells. In cells extracted with 1% Triton X-100, the host cytoskeleton was progressively reorganized about the parasites but changes were limited to the immediate area of the sporozoite. Around resident sporozoites, the cytoskeleton became less dense but also more ordered, which contrasted with adjacent cell areas. Cytoskeletal elements passed both over and under the parasites. The appearance of the cytoskeleton suggested that the host cell formed a loose, basket-like net of cytoskeletal elements about the parasite.     

Development of First-Generation Schizonts of Eimeria bovis in Cultured Bovine Cells

SYNOPSIS. Monolayer primary and secondary cultures of embryonic bovine kidney, spleen, intestinal and testicle cells, and secondary cultures of embryonic bovine thymus, maintained in lactalbumin hydrolysate, Earle's balanced salt solution and ovine serum were observed for a maximum of 21 days after inoculation of E. bovis sporozoites. The sporozoites entered the cells in all of these cultures but underwent development only in primary cultures of kidney and intestinal cells and in secondary cultures of kidney, spleen, thymus, intestinal, and testicle cells. In acellular media, the sporozoites retained motility no longer than 21 hr. In the cell cultures, free motile sporozoites were seen for as long as 18 days after inoculation. Sporozoites entered cells anterior end first; the process of penetration required a few seconds to about a minute. Sporozoites were also observed leaving host cells. Intracellular sporozoites were first seen 3 min after inoculation; they were observed at various intervals up to 18 days after inoculation. In transformation of sporozoites into trophozoites a marked change in size and appearance of the nucleus took place before the change in shape of the body occurred. Trophozoites were first found 7 days after inoculation, multinucleate schizonts after 8 days, and schizonts with merozoites after 14 days. Schizonts containing merozoites were seen only in kidney, spleen, and thymus cells. The mature schizonts were smaller and represented a much lower proportion of the total number than in comparable stages of infections in calves. Schizonts with many nuclei occurred in intestinal cells; the most advanced stage seen in testicle cells was the binucleate schizont. Nuclear and cytoplasmic changes were observed in the infected cells.     

Extraintestinal stages fo Isospora ohioensis from dogs in mice

The development of Isospora ohioensis was studied in mice by feeding tissues of mice inoculated with oocysts to coccidia-free dogs and by the examination of mesenteric lymph nodes using light and electron microscopes. Extraintestinal organs of mice became infectious to dogs within 1 day after ingesting oocysts and remained infectious for at least 211 days after inoculation (DAI). Isospora ohioensis sporozoites were found in lymphoreticular cells of mesenteric lymph nodes of mice from 1-374 DAI. Intracellular sporozoites were located in parasitophorous vacuoles. Sporozoites grew from 5--6 to 11--16 micron in length on the 39th DAI but never lost the 2 crytalloid bodies typical for coccidian sporozoites. PAS-positive granules accumulated gradually in intracellular sporozoites with duration of infection in mice. The appearance of parasitophorous vacuoles varied with duration of infection. Beginning with 7 DAI, the vacuole contained a marginal zone of electron-dense material (up to 0.8 micron wide), giving the appearance of a cyst wall or sheath under the light microscope; a true cyst wall was was not found.     

Reduced Invasion of Cultured Cells Pretreated With A Monoclonal Antibody Elicited Against Refractile Body Antigens of Avian Coccidial Sporozoites

The effect of a monoclonal antibody (1209-C2) elicited against sporozoite refractile-body antigen on invasion of cultured baby hamster kidney cells by avian Eimeria species was examined in vitro. Pretreatment of sporozoites with 1209-C2 for 45 min before inoculation into cultures or simultaneous introduction of sporozoites and 1209-C2 into cultures had no significant effect on invasion. However, pretreatment of cultures for 45 min with 1209-C2 (also with media from other cloned 1209 cell lines) significantly inhibited invasion by sporozoites of Eimeria tenella and E. adenoeides. Pretreatment of cultures with 2 unrelated monoclonal antibodies with the same isotype as 1209-C2 did not inhibit invasion by E. tenella. There was a significant correlation between time of exposure of the cultures to 1209-C2 and invasion (r = -0.80924; p = 0.0001), with inhibition of invasion occurring after 20 min exposure, but not after 10 min. There was also a significant correlation between the titer of 1209-C2 and invasion (r = 0.62291; p = 0.0305). Monoclonal antibody 1209-C2 cross-reacted with epitopes of baby hamster kidney cells by both immunofluorescence and Western blot. The fluorescent labeling of the cells differed according to the fixative that was used. In formalin-fixed cultures labeled with 1209-C2, fluorescent foci were distributed over the entire cell; after methanol fixation, 1209-C2 reacted with only discrete foci in the nucleus. On Western blots of sporozoites, 1209-C2 reacted with antigens having molecular sizes of approximately 8, 17, 23, 30, and 45-60 kDa, and with several minor bands. On baby hamster kidney cells, the antibody reacted primarily with bands of approximately 30, 45-60, and slightly with other bands. The data suggest that interactions among similar molecules in the sporozoites and host cells may play a role in cellular invasion.     

Intravital observation of Plasmodium berghei sporozoite infection of the liver

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.     

Intravital Observation of Plasmodium berghei Sporozoite Infection of the Liver

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.     

Development of Eimeria crandallis Honess from Sheep in Cultured Cells*

SYNOPSIS. Cell lines of embryonic lamb trachea (LETr), lamb thyroid (LETh), and bovine liver (BEL) as well as an established cell line of Madin-Darby bovine kidney (MDBK) were used in a study of the in vitro development of Eimeria crandallis from sheep. Excysted sporozoites were inoculated into Leighton tubes containing coverslips with monolayers of the different cell types. Coverslips were examined with phase-contrast and interference-contrast at various intervals up to 20 days after inoculation; thereafter the monolayers were fixed and stained in various ways. Freshly excysted sporozoites, with 2–10 spheroidal refractile bodies, entered all of the cell types in relatively small numbers; intracellular sporozoites were first seen 2 min after inoculation. After 24 hr, most intracellular sporozoites had only 1 or 2 refractile bodies. Before and during transformation of sporozoites, the nucleus and peripheral nucleolus increased markedly in size. Transformation resulted in usually spheroid but sometimes ellipsoid trophozoites. Trophozoites were seen first 3–4 days, and binucleate schizonts at 4–5 days after inoculation. Immature schizonts increased considerably in size and eventually had large numbers of nuclei. Some of the parasites became lobulated and the lobules often separated to form individual schizonts. In BEL, LETr and LETh cells, mature schizonts, up to 150 μm in diameter, were seen first 11–14 days after inoculation. The BEL cells were the most favorable for development. Merozoites were formed by a budding process from the surface of the schizonts as well as from blastophores. Some merozoites were seen leaving mature schizonts, but no further development was observed. Merozoites frequently were motile and had a sharply bent posterior end. Marked nuclear and cytoplasmic changes were observed in parasitized cells.     

钐在小鼠肝脏细胞中的动态观察

It is generally considered that the rare earth compounds are plasma membrane-impermeable, thus affecting the cells only on their surface. Recently, we found that after repeated injections to mice of large dose of samarium trichloride, a soluble compound of rare earth, samarium aggregates appeared in Kupffer cells and hepatocytes of liver. In this study, we aimed at observing the route by which samarium enters the liver cells and the process of the formation of samarium aggregates. Samarium trichloride was given to Swiss mice at one dose of 70 mg/kg intravenously. Thereafter, at different intervals from 15 min to 48 h after the injection, the samarium in liver was traced dynamically by electron microscopy and X ray microanalysis. From 15 min to 2 h both Kupffer cells and hepatocytes endocytosed samarium-containing particles and formed phagosomes, in which the ingested particles were progressively concentrated. Besides, the small phagosomes fused with each other. Phagocytosis was especially active in Kupffer cells. During the 4 h to 24 h many Kupffer cells were degenerated and broken. In hepatocytes the phagosomes gathered mostly around the bile canaliculi. Groups of highly electron-dense particles were found in the lumen of bile canaliculi, implying the excretion of samarium by bile. At the 48 h, the samarium-containing phagosomies were found still in both kinds of cells in the liver.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Ultrastructural Observations on the Infection of Rat Liver by Plasmodium berghei Sporozoites In Vivo1

The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.     

Ultrastructural observations on the infection of rat liver by Plasmodium berghei sporozoites in vivo

The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.     

A histochemical study about the involvement of rat liver cells in the uptake of heterologous immune comples from the circulation

Summary Intravenously injected immune complexes (ICx) composed of bovine serum albumin (BSA) and rabbit anti-BSA were taken up by the liver. Insoluble complexes, made in antibody excess, were rapidly taken up by Kupffer cells and were metabolized within 24 h. Soluble complexes, made in antigen excess, were only partly taken up by Kupffer cells. In addition these complexes were found, taken up and metabolized by endothelial cells. Until 2 h after injection soluble complexes could also be observed along the microvilli of hepatocytes. No signs of endocytosis in hepatocytes could be observed. It is concluded, that ICx can be taken up by Kupffer cells as well as by endothelial cells. The physical state of the complexes, soluble or insoluble, determines the cell type in which uptake occurs. To Prof. Dr. H.G. Goslar on the occasion of his 65th birthday