Biomedical subjects
Publications and source records attributed to T M Seed.
Trypanosoma brucei gambiense has been observed growing in extravascular sites throughout a solid Ehrlich ascites tumor (EAT) in Microtus montanus. These trypanosomes appear in clusters in the tumor. The possible importance of this observation to the host-parasite relationship is discussed.
Aplastic anemia and myelogenous leukemia are prominent pathologic effects in beagles exposed to continuous, daily, low-dose gamma irradiation. In the present work, granulocyte reserves and related mobilization functions have been sequentially assessed by the endotoxin stress assay during the preclinical and clinical phases of these hemopoietic disorders. Characteristic patterns of granulocyte reserve mobilization are described that reflect given stages of pathologic progression. For radiation-induced leukemia, a five stage pattern has been proposed. In contrast, a simple pattern of progressive, time-dependent contraction of granulocyte reserves and mobilization capacity was noted in the development of terminal aplastic anemia. Early preclinical phases of radiation-induced leukemia appear to involve an extensive depletion of the granulocyte reserves ((phase I) during the first approximately 200 days of exposure followed by a partial renewal of the reserves and associated mobilization functions approximately 200 and 400 days (phase II). Sustained, subnormal granulocyte mobilizations (phase III) following endotoxin stress typify the responses of dogs during the intermediate phase, whereas late preclinical, preleukemic stages (phase IV) are characterized by a further expansion of the reserves and in the mobilization capacities, particularly of the less mature granulocytes. Such late alterations in the pattern of granulocyte mobilization, together with other noted cellular aberrancies in the peripheral blood and marrow, appear to indicate leukemia (phase V) onset.
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A purebred female Beagle dog that had received 2,000 R of protracted wholebody gamma-irradiation from 60Co when 14 months old had hematologic changes consistent with a myeloproliferative disorder 3 years after the termination of radiation exposure. Peripheral blood and bone marrow findings during the 7-month period before death showed progressive anemia with increased numbers of platelets; immature granulocytes, monocytes and promonocytes. A period of partial remission occurred during which time the peripheral blood was aleukemic, although there was marked thrombocytosis and abnormal erythropoiesis which was evidenced by bizarre circulating nucleated red cells, anisocytosis, poikilocytosis and Howell-Jolly bodies. The dog had a terminal crisis with marked leukocytosis, most cells in the peripheral blood being bizarre monocytes and promonocytes. Tissues obtained at necropsy showed diffuse as well as focal infiltration of the spleen, liver, lymph nodes, heart, kidney and gastrointestinal wall with immature neoplastic cells resembling monocytes and monocytic precursors. The monocytic differentiation of the invasive cell population was confirmed by morphological, cytochemical, histological, ultrastructural and in vitro cell culture studies.
A method is described to prepare clones of hemopoietic cells grown in soft agar for scanning electron microscopy (SEM). A critical modification of the otherwise quite standard SEM processing procedure for biological samples involved the use of silver micropore disks as an adherent substrate to support the highly labile, deformable agar slabs. This support allows maintenance of the normal flat pancake shape of the specimen through the thiocarbohydrazide osmium ligand binding steps, dehydration, and critical point drying. With this support and careful dissection of the surface agar with a fine steel needle using a stereomicroscope, selected areas and depths within the colony can be exposed and examined by SEM. Surface topography of cloned cells can be correlated with intracellular cytological features by excising areas of interest and directly embedding them in plastic for thin-section preparation and viewing by transmission electron microscopy (TEM). The dried-specimen-teasing method appears useful, because of the ease of preparation of the specimens, its reproducibility, and the degree of visibility and preservation of cell surface structures and intraclonal relationships. Our initial observations, using combined EM techniques, indicate that clonal cell topography is highly variable and that this variability appears to be related both to the relative age and proliferative status of the colony. Based on work to date, we suggest that topographical and spatial analysis, in vitro of cloned, agar-embedded hemopoietic stem cells is possible with simple modifications of conventional SEM preparative techniques.
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A method has been developed for the rapid large scale isolation of plasma membranes and intact nuclei from RAJI lymphoid cells utilizing hypotonic lysis of cells after intracellular loading with glycerol followed by combined flotation-sedimentation within a discontinuous sucrose gradient. Nuclei may be isolated in about 1 h and plasma membranes in about 6 h from 1 to 20 g of cells. Intact nuclei, obtained in 90 to 95% yield based on lysed cells, was isolated by differential centrifugation and contained 16% DNA and about 30% of total cell sialic acid. A crude plasma membrane fraction was isolated by centrifugation onto a cushion of 38% sucrose (d 1.1683) and subsequently resolved into two subfractions. The less dense vesicles had an average d 1.127 and showed a 7-fold increase in specific activity for thymidine phosphodiesterase while the more dense (d 1.151) had a 20-fold concentration of enzyme activity. Activity of enzymes indicative of contamination with lysosomes, microsomes, mitochondria, and cytoplasm was negligible in these plasma membrane fractions. The less dense vesicles had a cholesterol:phospholipid ratio of 0.97 which was higher than that of the more dense vesicles (0.69). Otherwise, the analytical values for the two types of membrane vesicles were similar as both fractions contained like percentages of protein (approximately 30%), lipid (approximately 30%), and carbohydrate (approximately 15%) with trace amounts of RNA and DNA. Twenty-five per cent of the total cell sialic acid was in the plasma membrane fractions.
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The surface charge and lectin-binding capacity of isolated malaria parasites and host erythrocytes were analyzed and compared by chromatographic, electrophoretic, and cytochemical methods. Results indicated that at physiological pH values both freshly prepared and glutaraldehyde-fixed parasites and erythrocytes possess a net negative surface charge. Both cell types were strongly bound to cation-exchange resins and underwent cathode-directed electrophoretic migration. The isoelectric points for erythrocyte-free parasites and uninfected erythrocytes were approximately 3.0 and 4.0, respectively. The different effects of selective enzymatic digestion and solvent extraction on the electrophoretic mobilities of free parasites and erythrocytes suggested that the chemical constituents responsibile for the net negative surface charges on each type of cell are different. The surface charge of the free parasites seemed mainly to be a function of ionized phospholipids rather than of the ionogenic sialic acid moieties, which are the major contributors to the negative charge on erythrocytes. Results of lectin-binding studies indicated that specific glycosidimoieties (i.e., glucose, galactose, mannose, and n-acetyglucosamine), common to the erythrocyte surface, were either absent or in low concentration at the parasite's surface. These observations suggest that the normally intracellular malaria parasites have surface characteristics, differing from those of the host cell, characterized by a scarcity of lectin-binding receptors and sialic acid residues and by the major contribution of lipids to their surface charge.
The characteristics of a selected population of small blood stage parasites obtained by differential centrifugation of a population of P. berghei parasites freed by continuous flow sonication are described. About 10% of these free parasites are merozoites, many others are transitional forms having some merozoite characteristics. The parasite preparations are infectious and sufficiently resistant to incubation at 37 degrees C to be useful experimentally. Disc gel electrophoresis analysis indicates that these small parasites differ in composition from an unselected intraerythrocytic P. berghei population.
The African trypanosome (Trypanosoma brucei brucei) in its bloodstream phase has been found to possess rather unique surface properties. Results of lectin and iron colloid binding studies have indicated that the parasite's surface coat is devoid of many types of exposed carbohydrate residues which commonly are expressed on a variety of mammalian cell types. These include: glucose, mannose, galactose, n-acetylglucosamine, n-acetylgalactosamine, and neuraminic acid. A number of these carbohydrates (E.G., glucose, mannose, galactose and n-acetylgalactosamine) are, however, buried within the surface coat as evidenced by lectin binding to trypsinized parasites. The surface of well preserved parasites also lack exposed sialic acid and phospholipid moieties. Moribund parasites (or trypsinzed organisms) with altered surface coats were found to possess well defined phospholipid regions, but not negatively charged sialyl residues. Results presented tend to suggest that carbohydrates and phospholipids are not present in the outermost portion of the glycocalyx, thus exerting little or no influence on surface-mediated biological activities. However, the presence of such components on the plasmalemma is presumed to be structurally significant; especially during those developmental phases where the glycocalyx is absent, i.e., fly midgut phase.
The relationship of stage of development to structure in Babesia rodhaini and B. microti was studied by freeze-etching, carbon replication, and thin sectioning. The trophozoites of these Babesia are surrounded by a single membrane over most of their surfaces, but in some regions of the cytoplasm, membranous structures may parallel the plasmalemma, providing a double membrane. Merozoites are produced from trophozoites by budding. An early external indication that a bud will form is the organization of a pellicle complex at the site of the bud. The trophozoite nucleus divides before bud formation starts. The pellicle forms by development of a thick subplasmalemmal layer just under the plasmalemma in a bulging area that contains one of the daughter nuclei. The subplasmalemma layer of the pellicle of babesial merozoites is similar to that of plasmodial merozoites as it is divided into a series of roughly hexagonal plates by a reticular network which resembles coarse chicken wire. Thus, not only are the merozoites of these two species of Babesia budded in a fashion similar to that by which plasmodial merozoites are budded, but they also have similar pellicles.
The nature of erythrocyte membrane alterations in Plasmodium simium infections was determined employing light microscopy, carbon replication and transmission electron microscopy. Light microscopy of Giemsa stained preparations shows that infected cells initially acquire a faint stippling (schuffnerization) which becomes pronouced with subsequent parasite development. Enlargement of the host cell usually accompanied stippling. Both phenomena appear to depend on host cell age since infected mature erythrocytes were neither stippled nor enlarged. Carbon replicas show numerous indentations over the outer membrane surface of most infected cells. Their distribution suggests that they account for Schuffner's granules. The surface indentations are manifest as small infundibular which open to the infected cell's surface. Cytoplasmic microvesciles in the infected cell's stroma frequently are observed adjacent or catenated to the surface infundibula. Images suggest their funsion with the surface infundibula thus adding membrane to the cell's surface and accounting for host cell enlargement.
Morphological and cytochemical surface characteristics of isolated malaria parasites (Plasmodium berghei) and host erythrocytes were compared by electron microscopy by using thin section and carbon replica techniques. Erythrocytes were uniform in shape and had fine, granular surfaces. In contrast, free parasites exhibited a variety of sizes, shapes, and surface textures. Fine surface stippling was a common topographical feature of isolated parasites. Small, infective forms often had patterned surfaces resulting from the protuberance of an underlying thick intermediate layer. Results of cytochemical analysis using a sialophilic colloidal iron stain indicated that the malaria parasite's surface lacked exposed sialic acid groups which would normally give rise to a net negative surface charge common to erythrocytes. Biochemical assay demonstrated that malaria parasites contained about one-half the amount of sialic acid per unit weight as did control red cell extracts. Similarly, external acidic mucopolysaccharide coats of free parasites, as revealed by ruthenium red staining were extremely thin as compared with the thick glycocalyx layer of red cells. Lipid plaques at the surface of parasites and red cells were localized by lipophilic iron colloid staining. Although the gross patchwork distribution of plaques was somewhat similar for the two cell types, the parasites were stained more intensely and had a closer-knit patchwork pattern than those exhibited by the erythrocytes. Such findings indicate that there are slight differences in the arrangement of phospholipids at the surfaces of limiting membranes of host cells and parasites. The significance of the above cytochemical surface properties of the malaria parasite (which are seemingly akin to those of intracellular organelles is discussed in relation to certain host-parasite interactions, such as parasite adhesion to target cells and enhanced clearance of extracellular parasites.
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