Glomerulonephropathies in Plasmodium inui-infected rhesus monkey: a primate model and possible applications for human quartan malaria.

SUMMARY None of the few animal models proposed for the study of human quartan malaria nephritic syndrome have shown complete pathological findings that are similar to those seen in humans. This study investigated the histopathological changes in kidneys in 10 Plasmodium inui infected Macaca mulatta monkeys by light and electron microscopy in order to develop a suitable animal model for human quartan malaria. Ten healthy adult rhesus monkeys were infected with P. inui and clinical chemistry and haematologic tests were done before and after infection. A renal biopsy sample was collected before infection as a baseline control and another biopsy was collected after infection. Histopathological changes examined by light and transmission electron microscopy (TEM) revealed abnormalities in all infected monkeys to variable degrees. Several electron-dense discrete or diffused mesangial deposits, and increase in mesangial cells and matrix were associated with the morphological changes observed by light microscope. This pattern is consistent with membranoproliferative glomerulonephritis type reported in humans infected with Plasmodium malariae. Results strongly support that the P. inui-infected rhesus monkey develop an immune-complex-mediated glomerulonephritis in the course of the infection. Therefore, this experimental model represents a useful tool to better understand the different parameters and the consequences of quartan malaria infections comparable to situations in humans.

Cellular and clonal aging in the suctorian protozoan Tokophrya infusionum.

Quantitative methods for the study of aging in single organisms of the suctorian protozoan Tokophrya infusionum are described. New cell lines are initiated by metamorphosis of a ciliated embryo to form a sessile adult. The life history of adult cells consists of a sequence of age-related stages, culminating in cessation of reproduction and feeding, and eventual death. Lifespans of single cells were measured and were found to range rather widely about a mean, even when the cells compared were closely related within a single lineage. Variation appears to be inherent in the aging process in Tokophrya. Clones of Tokophrya undergo a gradual deterioration on a scale many times longer than the lifespan of individual cells. Lifespans of individual cells were determined when each of two clones were relatively young and later when their reproductive vigor had begun to decline. In both cases, the lifespan of individual cells were strikingly reduced in the old, as opposed to the young clones. The two types of senescence are thus experimentally separable, but nonetheless coupled phenomena. The similarity of aging in Tokophrya to that of other protozoa, fungi, and tissue culture cells is described and possible mechanisms are discussed.

An alternative secretory pathway in Plasmodium: more questions than answers.

The malaria parasite extensively modifies the host erythrocyte. Many of these modifications are mediated by proteins exported from the parasite and targeted to specific locations within the infected erythrocyte. However, little is known about how the parasite targets proteins to specific locations beyond its own plasma membrane. Treatment of infected erythrocytes with brefeldin A results in the accumulation of many exported Plasmodium proteins into a compartment distinct from the ER. Proteins destined for the host erythrocyte membrane, the parasitophorous vacuole or inclusions within the erythrocyte cytoplasm accumulate in this novel compartment, and co-localization studies indicate that there is a single compartment per parasite. Exported proteins only accumulate in this novel compartment if brefeldin A treatment is concurrent with their synthesis. This novel compartment is probably a membrane-bound organelle located at the parasite periphery, and may be the first step in an alternative secretory pathway that specializes in the export of proteins into the host cell. Such an alternative secretory pathway raises questions about how exported proteins are differentially targeted to this novel organelle versus the ER and the fate of exported proteins after this novel organelle.

A role for peripheral blood fibrocytes in Lyme disease?

It is proposed that peripheral blood fibrocytes will be a new and important player in the pathogenesis of Lyme disease. Peripheral blood fibrocytes are a circulating leukocyte subpopulation that: (a) express collagen; (b) are an abundant source of cytokines, chemoattractants and growth factors; and (c) are able to recruit and activate naive T-cells and memory T-cells. We predict that peripheral blood fibrocytes will represent a new and important antigen-presenting cell which will play an important role in directing the immune response from the pathogenic Th1 to the protective Th2 response cell in Borrelia infections.

Ultrastructure of erythrocytes from Aotus trivirgatus and Saimiri sciureus monkeys infected by Plasmodium vivax.

Erythrocytes from Aotus and Saimiri monkeys parasitized by Plasmodium vivax show dramatic changes starting during the early stages of parasite development. Invaginations of the erythrocyte membrane, caveolae, are found during all parasite development stages. Up to six vesicles can be fused with one caveola, forming a caveola-vesicle complex. As the parasite grows, large accumulations of these vesicles can be seen within the erythrocyte cytoplasm. In addition to these caveolae-vesicle complexes, knob-like structures appear on the erythrocyte surface that are similar to those seen on the host-cell surface of P. falciparum-infected red cells. Extensive membrane-bound clefts spread throughout the erythrocytic cytoplasm, sometimes forming stacks or large whorls. The density of the red cell cytoplasm begins to decrease at an early stage of parasite development. All of these changes may be responsible for an increased fragility of the P. vivax-infected red cell from Aotus or Saimiri monkeys. Moreover, the large amount of parasite material that is released during rupture of the red cell may account for the high fever paroxysms that are characteristic of P. vivax malaria infection.

Effect of the 8-aminoquinoline primaquine on culture-derived gametocytes of the malaria parasite Plasmodium falciparum.

Gametocytes from the Honduras I/CDC clone HB-3 of Plasmodium falciparum were exposed to different concentrations of primaquine diphosphate. It could be shown that the drug acts specifically on the mitochondria of the cells by destroying their internal structure and causing these organelles to swell.

Pronuclei of Heliophrya erhardi Matthes during conjugation and their differential association with coated and uncoated microtubules.

Microtubules surrounding the pronuclei during conjugation of the suctorian ciliate Heliophrya erhardi can be divided into 2 distinct classes by electron microscopy. Microtubules around the stationary nucleus have a conventional appearance and presumably serve as a skeleton, anchoring that nucleus in its cytoplasmic position. Microtubules surrounding the prospective migratory nucleus are coated with electron-dense material and are in some cases associated with 7-nm filaments. These coated microtubules supposedly function to transport the migratory nucleus into the conjugation partner.

Early stages of axonal regeneration in the goldfish optic tract: an electron microscopic study.

Two hours after the goldfish optic tract was cut, the severed axons in the retinal stump of the tract showed ballooning of the axoplasm and myelin sheath in the region of the cut, with accumulation in the swollen axon of various organelles, including dense cored vesicles. By day 1 the myelin sheath had degenerated back to a node of Ranvier and the tip of the severed axon had formed a myelin-free terminal bulb with a well-organized core of 9-10 nm filaments. By 2 days, such terminal bulbs were often seen to be extended on a neck of cytoplasm a few micrometers in length, presumably indicating axonal outgrowth. In addition, occasional small bundles of axon sprouts were first seen at this time. The sprouts had a diameter of about 2 micrometers and contained a central core of 9-10 nm filaments surrounded by a mantle of cell organelles (smooth endoplasmic reticulum, mitochondria and diverse vesicles), with few if any microtubules. Sprouts within a bundle were separated by fairly uniform 10-15 nm spaces. Beginning at 3 days, significant numbers of microtubules appeared in the sprouts, and there was an increasing proportion of small diameter (greater than or equal to 0.3 micrometer) sprouts. Thus it was not until 3 days that the sprouts took on the appearance usually considered to be typical of regenerating axons. By 6 days a dense layer of glial cells or macrophages formed a cap over the cut surface of the tract. Penetrating this layer were bundles containing up to 20-30 axon sprouts and also single axons which may have been serving as 'pioneering' fibres to which later-emerging axons would attach. There was no evidence that the regenerating axons were guided by the glial cells. At 6 days astroglia began to separate individual axons within the bundles but oligodendrocytes were still inactive at this time.

Rapid transport of the acidic phosphoproteins of Plasmodium berghei and P. chabaudi from the intraerythrocytic parasite to the host membrane using a miniaturized fractionation procedure.

A miniaturized procedure for the separation of the host erythrocyte membrane from malarial parasites based on saponin lysis and density-gradient centrifugation with Percoll is described. The procedure requires only 20-35 microliters packed infected erythrocytes, is simple to perform, needs no sophisticated equipment, and can be completed in less than 2 h. Analysis of the isolated erythrocyte membranes and parasites using marker enzymes and electron microscopy revealed that both the purity and the yield of these fractions were relatively high. Erythrocyte membrane proteins, including spectrin, ankyrin, and band 4.1, were not found on the parasitophorous vacuolar membrane, which remained associated with some but not all of the isolated parasites. Application of this method to pulse-chase experiments indicated that the acidic phosphoproteins of Plasmodium berghei and P. chabaudi were rapidly transported from the parasite to the erythrocyte membrane immediately after their synthesis. The rapid export of these acidic phosphoproteins from the parasite distinguishes them from other proteins exported by the malarial parasite.

Comparative infectivity of knobless and knobby clones of Plasmodium falciparum in splenectomized and intact Aotus trivirgatus monkeys.

In two experiments, two knobless (K-) and two knob-producing (K+) clone-cultures of Plasmodium falciparum, FCR-3/Gambia strain, were injected into four Aotus trivirgatus monkeys. The parasitemia in the K(-)-infected splenectomized (S-) monkey rose to a peak of 2.1% on the 16th day, while it reached only 0.7% at the same time in the K+ infected S- animal. Passage from these animals (karyotype VI) into two intact (S+), naive monkeys of karyotype III resulted in very light infections somewhat higher with K+ than with K-. This experiment was repeated with two different clones in two other S- monkeys of karyotype III. Again, the parasitemia of the K+ infected monkey was appreciably below that of the K- monkey. Transfer of parasites into S+ animals of karyotype II resulted in very light infection and, as before, the K+ did somewhat better. About 2 months after its initial infection, the K(+)-infected S- animal from the second experiment came down with a recurrent malaria infection. Electron-microscopic observations on blood from this monkey revealed that the previously K+ parasites had become knobless (K-). Transfer of this material into an S+, naive monkey, again, gave a barely detectable infection. After splenectomy a recrudescence occurred. The results strongly indicate that K- clones of P. falciparum are more infectious to S- Aotus monkeys than K+ clones, whereas in S+ monkeys the situation is reversed.

Intranuclear structures in pyrimethamine-resistant isolates of the malaria parasite Plasmodium falciparum.

The ultrastructure of the malaria parasite Plasmodium falciparum is well known, both from natural infections and from culture material ( Aikawa , 1977, Langreth et al., 1978). It is noteworthy that all of these studies were done with pyrimethamine-sensitive strains, e.g. FCR-3/Gambia. Except for spindle microtubules during schizogony, no intranuclear structures have been described in any of the asexual erythrocytic stages. In the course of isolating clones from the pyrimethamine-resistant strain Honduras I/CDC (V.K. Bhasin and W. Trager , in print) and checking by electron microscopy for the presence or absence of knobs, we noticed intranuclear structures that might be correlated with pyrimethamine resistance. For comparison, we then examined the multi-drug-resistant strain Indochina 1. We present here a first report on these structures as a basis for further studies.

Initial extracellular development in vitro of merozoites of Plasmodium falciparum.

Late schizonts from continuous cultures of P. falciparum were concentrated over Percoll, inoculated to various experimental media at the rate of about 20 X 10(6) per 0.5 ml of medium, and incubated in a candle jar at 37 degrees for 1 day. Controls in standard culture medium showed a heavy invasion with young rings in the previously uninfected red cells introduced with the inoculum of schizonts. In a medium of high potassium content containing a 33% extract of human erythrocytes, this invasion was inhibited and many free merozoites were present. If, however, this same medium was supplemented with both ATP, as the dipotassium salt at 1.6 mM, and sodium pyruvate at 3.6 mM, there appeared large numbers of extracellular forms resembling young rings. Examination of these by electron microscopy shows that they are indeed merozoites that have begun to differentiate extracellularly. This suggests that the trigger for differentiation of merozoites may not depend on the process of entry into a red cell but rather on specific factors within the red cell.

Export of proteins via a novel secretory pathway.

The intraerythrocytic location of the malaria parasite necessitates modification of the host cell. These alterations are mediated either directly or indirectly by parasite proteins exported to specific compartments within the host cell. However, little is known about how the parasite specifically targets proteins to locations beyond its plasma membrane. Mark Wiser, Norbert Lanners and Richard Bafford here propose an alternative secretory pathway for the export of parasite proteins into the host erythrocyte. The first step of this pathway is probably an endoplasmic reticulum (ER)-like organelle that is distinct from the normal ER. Possible mechanisms of protein trafficking in the infected erythrocyte are also discussed. The proposed ER-like organelle and alternative secretory pathway raise many questions about the cell biology of protein export and trafficking in Plasmodium.

Characterization of the parasitophorous vacuole membrane from Plasmodium chabaudi and implications about its role in the export of parasite proteins.

Little is known about how the malaria parasite transports and targets proteins into the host erythrocyte. Parasite proteins exported into the host cell not only have to cross the parasite plasma membrane but also must traverse the parasitophorous vacuolar membrane (PVM) that surrounds the parasite. The PVM of Plasmodium chabaudi-infected erythrocytes was analyzed by immunofluorescence using an antibody against a known PVM protein, a fluorescent lipid probe, and electron microscopy. These analyses reveal qualitatively different membranous projections from the PVM. Some PVM projections are uniformly labeled with the antibody and with lipid probes and probably correspond to the Maurer's clefts. In contrast to this uniform labeling of the PVM and projections, a 93-kDa P. chabaudi erythrocyte membrane-associated protein is occasionally detected in vesicle-like structures adjacent to the parasite. These vesicle-like structures are found only coincident with protein synthesis and are located at discrete sites on the PVM. These observations suggest that the 93-kDa protein does not move along the membranous projections of the PVM toward the erythrocyte membrane. It is proposed that the 93-kDa protein is secreted directly into the erythrocyte cytoplasm at discrete PVM domains and then binds to the cytoplasmic face of the erythrocyte membrane.

Accessibility and distribution of intraerythrocytic antigens of Plasmodium-infected erythrocytes following mild glutaraldehyde fixation and detergent extraction.

Malarial antigens on the surface of infected erythrocytes have been described by many investigators. However, few of these antigens have been unambiguously demonstrated to be exposed on the surface of erythrocytes. This study demonstrates that mild glutaraldehyde fixation results in the cytoplasmic face of the host membrane becoming accessible to antibody under conditions that normally do not expose the cytoplasmic face of uninfected erythrocytes. These results indicate that caution should be used in interpreting data on the membrane disposition of malarial antigens. Detergent extraction of the glutaraldehyde-fixed erythrocytes results in an increased permeabilization such that malarial antigens on the parasite surface and within the cytoplasm of the infected erythrocyte are accessible to antibody. The accessibility of these antigens was demonstrated by both immunofluorescence and two-color flow cytometry. The antigens within the host cytoplasm were not diffuse but associated with patchy aggregates. Analysis of the antigens associated with the cytoplasmic aggregates by immunoelectron microscopy indicated that they were not associated with membrane-bound compartments. The fixation and permeabilization protocol described herein will have useful applications for the characterization and analysis of malarial antigens.

Gametogenesis and sporogony of Hepatozoon mocassini (Apicomplexa: Adeleina: Hepatozoidae) in an experimental mosquito host, Aedes aegypti.

The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti, an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiation were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a parasitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporozoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endoplasmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.

Effect of pyrimethamine resistance on sporogony in a Plasmodium berghei/Anopheles stephensi model.

A pyrimethamine-resistant line of Plasmodium berghei was derived by treating infected mice with high doses of pyrimethamine and selecting for recrudescence. This resistant line was compared with the parental pyrimethamine-sensitive line in order to ascertain whether drug resistance is associated with a biological advantage. Overall, the pyrimethamine-resistant line is quite similar to the sensitive line, except that it proceeds through sporogonic development more slowly than the pyrimethamine-sensitive parental line. However, under pyrimethamine pressure the sensitive line is unable to undergo the sporogonic cycle, whereas the resistant line is unaffected. These results indicate that the transmission of pyrimethamine resistance in this model is favored only under conditions of drug pressure.

A novel alternate secretory pathway for the export of Plasmodium proteins into the host erythrocyte.

The malarial parasite dramatically alters its host cell by exporting and targeting proteins to specific locations within the erythrocyte. Little is known about the mechanisms by which the parasite is able to carry out this extraparasite transport. The fungal metabolite brefeldin A (BFA) has been used to study the secretory pathway in eukaryotes. BFA treatment of infected erythrocytes inhibits protein export and results in the accumulation of exported Plasmodium proteins into a compartment that is at the parasite periphery. Parasite proteins that are normally localized to the erythrocyte membrane, to nonmembrane bound inclusions in the erythrocyte cytoplasm, or to the parasitophorous vacuolar membrane accumulate in this BFA-induced compartment. A single BFA-induced compartment is detected per parasite and the various exported proteins colocalize to this compartment regardless of their final destinations. Parasite membrane proteins do not accumulate in this novel compartment, but accumulate in the endoplasmic reticulum (ER), suggesting that the parasite has two secretory pathways. This alternate secretory pathway is established immediately after merozoite invasion and at least some dense granule proteins also use the alternate pathway. The BFA-induced compartment exhibits properties that are similar to the ER, but it is clearly distinct from the ER. We propose to call this new organelle the secondary ER of apicomplexa. This ER-like organelle is an early, if not the first, step in the export of Plasmodium proteins into the host erythrocyte.