Tissue and cellular distribution of an adhesion molecule in the carcinoembryonic antigen family that serves as a receptor for mouse hepatitis virus.

BACKGROUND: The receptor for the murine coronavirus mouse hepatitis virus (MHV)-A59, called MHVR or Bgp1a, is a glycoprotein in the carcinoembryonic antigen family of the Ig superfamily. Biliary glycoprotein (Bgp) isoforms play a role in cell adhesion, bile acid transport, and ecto-ATPase activity. MHV-resistant SJL/J mice express a different allele of Bgp1 called Bgp1b. Analysis of the tissue and cellular distribution of Bgp1 proteins can therefore provide new insight on both cellular functions and MHV-A59--induced pathogenesis. EXPERIMENTAL DESIGN: Bgp1 expression was analyzed in the digestive, respiratory, endocrine, urinary, and central nervous systems of adult BALB/c and SJL/J mice by immunocytochemistry and immunoelectron microscopy using a monoclonal Ab specific for the N-terminal domain of the Bgp1a proteins and polyclonal rabbit anti-Bgp1, which recognizes both the Bgp1a and Bgp1b proteins. The function of Bgp1 proteins as viral receptors was tested on tissue sections by a virus binding assay. MHV-A59 replication was analyzed by immunocytochemistry. RESULTS: Bgp1 expression was observed on membranes of epithelial cells (including hepatocytes, intestinal, endocrine, and respiratory epithelial cells), kidney proximal tubules, and endothelial cells in many tissues. It was usually localized at the apical pole of the cells and, when present, on the brush borders and the cilia. A new direct virus binding assay showed that MHV attachment onto cells correlates with Bgp1 expression. Viral infection was detected in hepatocytes, lymphoid tissue, and the exocrine pancreas but not in endocrine cells, enterocytes, kidney, or respiratory cells. In the central nervous system, no immunolabeling of neurons or glial cells was found with anti-Bgp1 Ab. CONCLUSIONS: Bgp1 proteins are present on BALB/c and SJL/J epithelia and endothelia. These glycoproteins might be involved in cell-to-cell contacts, for example between hepatocytes. On BALB/c mice, Bgp1a expression is consistent with the tropism of MHV-A59 for the liver. However, Bgp1a was also expressed on cells that were not infected by MHV-A59.

Characterization of Pneumocystis carinii preparations developed for lipid analysis.

Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10(8)-10(9) organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80-95%.

Plasmodium falciparum antigens associated with membrane structures in the host erythrocyte cytoplasm.

Hybridomas were made from mice immunized with plasma membranes from erythrocytes infected with Plasmodium falciparum. Among the monoclonal antibodies produced, a series reacted with antigens in the host cell cytoplasm. Immunoelectron microscopy, along with indirect fluorescent antibody double labeling experiments, were used to further localize the antigens to membrane structures (presumably Maurer's clefts) in the erythrocyte cytoplasm. The epitopes thus localized are found on three parasite proteins (20 kDa, 29 kDa, and 45 kDa) and one parasite glycoprotein (45 kDa). They are likely to be part of a transport system for the parasite.

Two Plasmodium falciparum merozoite surface polypeptides share epitopes with a single Mr 185 000 parasite glycoprotein.

The malarial parasite Plasmodium falciparum synthesizes a major glycoprotein (gp) of Mr 185 000 during its asexual blood cycle. Immunoprecipitation of [35S]methionine- or [3H]glucosamine-labeled schizont antigens indicated that two groups of polypeptides were distinguished with anti-gp 185 mouse monoclonal antibodies: group A was composed of glycosylated molecules of Mr 185 000, 120 000, 90 000, 88 000, 46 000, and 40 000 while group B contained, in addition to gp 185, polypeptides of Mr 152 000, 106 000 and 83 000. The latter polypeptides lacked detectable amounts of radiolabeled saccharide. The smaller Mr polypeptides were specifically immunoprecipitated and not merely coprecipitated with gp 185. Our results suggest that gp 185 contains at least two structurally distinct domains which may be processed independently into either the group A or group B polypeptides. Although gp 185 may not be a merozoite surface protein, representative group A and group B-specific monoclonal antibodies bound to surface antigens of the merozoite as demonstrated by immunolabeling followed by electron microscopy. Therefore, at least one group A antigen and one group B antigen appeared to be on the extracellular surface of the merozoite. The proteins found in immunoprecipitates after both (1) sonication in aqueous medium and ultracentrifugation and (2) solubilization and phase separation of parasite molecules with Triton X-114 suggested that the group A and group B polypeptides and glycoproteins are either soluble or peripheral membrane proteins. Some of these, therefore, may be components of the surface coat of the merozoite.

Pathogenicity, stability, and immunogenicity of a knobless clone of Plasmodium falciparum in Colombian owl monkeys.

The pathogenicity, immunogenicity, and morphological stability of a knobless clone of strain FCR-3 of the human malaria parasite Plasmodium falciparum was investigated in Aotus monkeys. An early knob-bearing (K+), wild-type isolate of strain FCR-3 and the D3 knobless (K-) clone were adapted to Aotus monkey erythrocytes in continuous culture, establishing the parasites in Aotus cells without exposure to in vivo cellular or humoral immune responses. All monkeys, intact or splenectomized, which were infected with wild-type FCR-3 adapted to Aotus cells in vitro, developed virulent infections and had to be drug treated. The intact nonsplenectomized animals which received knobless D3 cloned parasites did not develop virulent infections even after multiple infections. The splenectomized monkeys which received the K- D3 clone had virulent infections. Late-stage wild-type K+ parasites sequestered in both intact and splenectomized monkeys, whereas late-stage D3 K- parasites did not sequester in the splenectomized animals. These results suggest that two elements affected the pathogenicity of the malaria parasites in these experiments. Knobs on K+-infected erythrocytes enabled these parasites to sequester, presumably by attachment to capillary endothelium. When present, the spleen eliminated circulating K- late-stage erythrocytes, presumably by selection on the basis of their nondeformability. Although clone D3 K- parasites are nonvirulent in intact monkeys, they induced some immunological protection against challenge with wild-type K+ parasites. The surface morphology of K--infected erythrocytes remains unaltered throughout these experiments, suggesting that loss of knobs is a stable condition.

Long term cultivation of Plasmodium falciparum in Aotus trivirgatus erythrocytes.

The erythrocytes of the Colombian owl monkey Aotus trivirgatus griseimembra can be used for the long-term in vitro cultivation of Plasmodium falciparum employing a modified Trager -Jensen method. Cultures are grown in HEPES-buffered RPMI-1640 using a 4% suspension of monkey erythrocytes and 10% pooled heat-inactivated human AB serum, with initial parasitemias in a range between 0.2 and 0.5%. Adaptation of new strains from human erythrocytes cultures can be performed by simply subculturing from human to owl monkey erythrocytes in a stepwise manner. When 5% human AB serum is included in cultures to support growth, as much as 5% monkey serum can be added in order to investigate serum effects, such as antibody activity against P. falciparum. The Aotus trivirgatus continuous culture system has provided a stable, consistent source of infected erythrocytes for in vitro experimentation, and the techniques developed have been used to further refine and support the animal experiments in progress.

Fine-structural alterations in Leishmania tropica within human macrophages exposed to antileishmanial drugs in vitro.

The mechanism of action of antileishmanial compounds is poorly understood. Ultrastructural changes in Leishmania tropica within human macrophages exposed in vitro to Pentostam, pentamidine, amphotericin B, WR 6026, ketoconazole, and Formycin B were examined in these experiments. In Pentostam-treated cultures, some organisms exhibited diminished definition of mitochondrial and other membranes, while other organisms had completely disintegrated. Pentostam-exposed macrophages demonstrated loss of membrane definition in the absence of further alterations; it is therefore hypothesized that impaired macrophage membrane function may contribute towards the effect of this drug against macrophage-contained organisms. Leishmania parasites in pentamidine-treated cultures initially demonstrated swollen kinetoplasts and fragmentation of the kinetoplast DNA core. The initial observed effect of the other four drugs on the parasites was cytoplasmic condensation. These ultrastructural studies suggest that all five non-antimonial drugs may have different mechanisms of action than antimony (Pentostam) against Leishmania.

Immunization of owl monkeys to Plasmodium falciparum with merozoites from cultures of a knobless clone.

Summary Aotus trivirgatus monkeys of karyotype 2 were treated as follows. Three received two injections of purified merozoites of a knobless (K-) clone of Plasmodium falciparum with muramyl dipeptide as adjuvant; three received similar injections but with merozoites of a wild-type knobby (K+) strain; three controls received MDP with human erythrocytes in the amounts estimated to be present as contaminants in the merozoite preparations. A month after the second injection all nine monkeys were inoculated with parasites of the wild-type knobby strain from another infected Aotus. The monkeys that had received the K- material developed only extremely low infections markedly different from the infections in the controls. Of those that received K+ material, one died early with low parasitaemia, one was protected and one had the same level of infection as the controls. In the combined group of immunized animals, four out of six were protected.

Inefficacy of metronidazole in experimental infections of Leishmania donovani, L. mexicana, and Trypanosoma brucei brucei.

Metronidazole has been claimed in several earlier reports to be active in human cases of leishmaniasis and trypanosomiasis. Its efficacy against the protozoa causing these diseases was tested in hamsters infected with Leishmania mexicana or L. donovani, and in mice infected with Trypanosoma brucei brucei. In separate experiments, hamsters were either inoculated intradermally into the nose with 5 million amastigotes of L. mexicana or intracardially with 10-30 million amastigotes of L. donovani, and mice were infected intraperitoneally with 30 million T. b. brucei. Metronidazole was administered in four oral doses on alternate days for a total of 375 mg/kg to hamsters and 500 mg/kg to mice. Sodium stibogluconate (Pentostam) served as a positive control. In hamsters the extent of infection was assessed by the appearance of flagellates in blood agar cultures of nose and spleen, by counting amastigotes in nose and liver impression smears, and by measuring the size of nose lesions. Ultrastructure of nose lesions before and after treatment with metronidazole or Pentostam was also evaluated. Infection in mice was assessed by the extent of parasitemia and/or survival to 30 days. In no case did metronidazole-treated animals differ from untreated controls. Metronidazole shows no activity against experimental infections of leishmaniasis or trypanosomiasis in these animal models.

Interferon-induced enhancement of macrophage Fc receptor expression: beta-interferon treatment of C3H/HeJ macrophages results in increased numbers and density of Fc receptors.

Previous studies demonstrated that the augmentation of Fc receptor-(FcR) mediated phagocytosis and binding of opsonized sheep erythrocytes is cytokine mediated. Specifically, beta-interferon- (beta-IFN) and gamma-IFN-rich preparations were shown to increase binding and phagocytosis of opsonized erythrocytes by C3H/HeJ macrophages. In this report we extend these studies by examination of the mechanisms by which highly purified beta-IFN increases FcR capacity in C3H/HeJ macrophages. Our findings indicate that beta-IFN augments FcR function for both IgG2a and IgG2b receptor subclasses. Moreover, this increased ability to bind and phagocytose opsonized erythrocytes is associated with a concomitant increase in both the number and surface membrane density of FcR.

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis.

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.

Antigenicity of the infected-erythrocyte and merozoite surfaces in Falciparum malaria.

The antigenicity of altered structures induced by Plasmodium falciparum in the membranes of infected Aotus monkey and human erythrocytes was examined. Antisera were obtained from monkeys made immune to malaria. Bound antibodies were shown to be localized on the knob protrusions of infected erythrocytes of both human and monkey origin and from both in vitro and in vivo infections. Therefore, P. falciparum infection has produced similar antigenic changes in the erythrocyte surfaces of both man and monkey. Uninfected erythrocytes and all knobless-infected erythrocytes bound no antibody from immune sera. Strains of P. falciparum from widely different geographic areas that were cultured in vitro in human erythrocytes induced structures (knobs) which have common antigenicity. Merozoites were agglutinated by cross-linking of their cell coats when incubated with immune sera. The binding of ferritin-labeled antibody was heavy on the coats of both homologous and heterologous strains of the parasite, indicating that the merozoite surfaces of these strains share common antigens.

Isolation of stages of the human parasite Plasmodium falciparum from culture and from animal blood.

Procedures for isolation of various forms of the asexual erythrocytic stages of the human parasite Plasmodium falciparum are outlined. The procedures employ the plasma expander Physiogel, which is composed of a chemically modified, partially hydrolysed gelatin dissolved in Ringer's lactate. Based on the observation that parasitized cells which are easily separable by this technique differ appreciably at the ultrastructural level, a mechanism by which separation occurs is proposed.

Fine structure of human malaria in vitro.

The erythrocytic cycle of the human malaria parasite, Plasmodium, falciparum, was examined by electron microscopy. Three strains of parasites maintained in continuous culture in human erythrocytes were compared with in vivo infections in Aotus monkeys. The ultrastructure of P. falciparum is not altered by continuous cultivation in vitro. Mitochondria contain DNA-like filaments and some cristae at all stages of the erythrocytic life cycle. The Golgi apparatus is prominent at the schizont stage and may be involved in the formation of rhoptries. In culture, knob-like protrusions first appear on the surface of trophozoite-infected erythrocytes. The time of appearance of knobs on cells in vitro correlates with the life cycle stage of parasites which are sequestered from the peripheral circulation in vivo. Knob material of older parasites coalesces and forms extensions from the erythrocyte surface. Some of this material is sloughed from the host cell surface. The parasitophorous vacuole membrane breaks down in erythrocytes containing mature merozoites both in vitro and in vivo. Merozoite structure is similar to that of P. knowlesi. The immature gametocytes in culture have no knobs.