PCC4azal teratocarcinoma stem cell differentiation in culture: I. Biochemical studies

PCC4azal embryonal carcinoma cells were observed to spontaneously differentiate under defined culture conditions to endoderm-like cells and subsequently to giant cells. This differentiation was examined by determining the specific activities of several enzymes in the stem and endoderm-like cell populations. With differentiation, the level of alkaline and acid phosphatase activities remained unchanged, plasminogen activator specific activity increased fivefold, and lactate dehydrogenase (LDH) specific activity decreased to 40% of its original level. Isozyme analysis revealed a shift of the LDH isozymes toward LDH₁ with the appearance of LDH₂ for the first time in the endoderm-like cells. The surface antigen SSEA-1 was detected by indirect immunofluorescence on virtually all of the stem cells. However, the SSEA-1 antigen was not present on many of the endoderm-like cells, and it was completely undetectable on giant cells as assayed by immunofluorescence. The expression of H-2 antigen was examined in a similar manner using anti-H-2^b antiserum; this antigen was not detected on the stem, endoderm-like, or giant cells. Thus, there are defined biochemical changes that accompany the differentiation of PCC4azal stem cells in culture.     

Infection of different cell lines of neural origin with subacute sclerosing panencephalitis (SSPE) virus

Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell-free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka-1, Osaka-2, Osaka-3), SSPE virus-infected cell cultures were treated with cytochalasin D to prepare virus-like particles (CD-VLPs). All CD-VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CDVLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD-VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR-32 and SK-N-SH) with varying efficiency. SSPE CD-VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U-251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild-type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain.     

Effects of infection by HIV-1, cytomegalovirus, and human measles virus on cultured human thymic epithelial cells

A tissue culture system for the growth of human fetal and infantile thymic epithelial (TE) cells has been established and characterized. We have investigated the effects of infection of these cells by human cytomegalovirus (CMV), measles virus, and human immunodeficiency virus type-1 (HIV-1). In the case of CMV, morphological changes were apparent by 2-4 days after viral inoculation of infantile TE cells. CMV-related antigens were detected by immunofluorescence after 12 days, and progeny infectious CMV was recovered from culture media after 18 days. Following infection by measles virus, distinctive, multinucleated giant TE cells appeared in both cultures of fetal and infantile TE cells. Measles virus-inoculated TE cells displayed an altered phenotype, as revealed by reaction with monoclonal antibodies with specificity for a variety of TE markers. Finally, infection of TE cells by HIV-1 resulted in cellular disarrangement, increased numbers of Hassall's corpuscles, and multinucleated giant cells. An increase in the number of cells reactive with monoclonal antibodies, specific for Hassall's corpuscles, was observed in the case of cells infected by either measles virus or HIV-1. These findings suggest that a variety of different viruses can successfully infect thymic epithelial tissue. Because of the important role of the thymus in development of the immune system, it is reasonable to conclude that viral infection of thymic tissue might play an important role in virus-mediated suppression of immune responsiveness.     

Immunofluorescence and Cytochemical Studies of Visna Virus in Cell Culture

Sequential morphological changes occurring in sheep choroid plexus cells infected with visna virus were studied by direct immunofluorescence, acridine orange, and hematoxylin and eosin staining methods. Specific immunofluorescence was first detected in the perinuclear cytoplasm of solitary cells 24 hr after infection. As the infection progressed, viral antigen appeared in an increasing number of cells, and rounded globular cells with long slender processes harboring intense fluorescence were seen. Nuclear fluorescence was not observed in infected monolayers. Polykaryocytes formed within 6 hr after inoculation due to the direct cell-fusing effect of the virus inoculum did not show specific fluorescence. Viral antigen was found, however, in the cytoplasm of multinucleated giant cells in cover slips harvested after new infective virus had been released, and later in the course of infection circular fluorescent inclusions were seen in the cytoplasm of polykaryocytes. Comparable eosinophilic inclusions were observed in hematoxylin and eosin preparations, and acridine orange staining of infected monolayers demonstrated similar inclusions which fluoresced with the color characteristic of single-stranded nucleic acid and were susceptible to digestion with ribonuclease. Visna virus appears to be a ribonucleic acid virus which replicates in the cytoplasm.     

Isolation and Biological Characterization of a Measles Virus-Like Agent from the Brain of an Autopsied Case of Subacute Sclerosing Panencephalitis (SSPE)

Isolation of a cytopathic agent causing formation of syncytial giant cells in co-cultivated Vero cells from the brain of an autopsied case of subacute sclerosing panencephalitis (SSPE) is reported. The syncytia usually autolyzed from the center after growing to 1 to 2 mm in diameter and then detached from the culture vessels, and finally made macroscopically recognizable round plaques on the monolayer under liquid overlay. The agent was identified serologically as an agent related to measles virus, by both immunofluorescent tests and plaque reduction tests using anti-measles sera. However, the infected cells did not produce either virions or hemagglutinin, and failed to show hemadsorption and hemolysis of African green monkey red cells even after the 55th passage through Vero cells. Newborn mice, adult mice and hamsters showed neurologic signs after intracerebral inoculations of the infected cells, and most of them died from acute encephalitis. Guinea pigs were unsusceptible. From the brain of the animals with neurologic signs, a similar agent to the inoculated one was recovered. The new isolate appears to be a strain closely related to measles virus on the basis of serology, and was designated as SSPE-“Kitaken-1” strain.     

Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein.

Six monoclonal antibodies directed against respiratory syncytial virus proteins were produced. Each was characterized by immunoprecipitation and indirect immunofluorescence. One was directed against the nucleocapsid protein. NP 44, two were directed against a 37,000-dalton protein, two were directed against the major envelope glycoprotein, GP 90, and one was directed against the 70,000-dalton envelope protein, VP 70. Indirect immunofluorescence stain patterns of infected HEp-2 cells defined GP 90 and VP 70 as viral proteins expressed on the cell surface, whereas NP 44 and the 37,000-dalton protein were detected as intracytoplasmic inclusions. One of the anti-GP 90 antibodies neutralized virus only in the presence of complement but did not inhibit cell-cell fusion. The anti-VP 70 antibody neutralized virus without complement and inhibited cell-cell fusion of previously infected HEp-2 cells, thus identifying VP 70 as the fusion protein.     

Ultrastructure of Measles Virus in Cultures of Hamster Cerebellum

Replication of Edmonston strain of measles virus in cultures of hamster central nervous system tissue was studied by electron microscopy of ultrathin sections. Infected cultures were fixed from 3 hr to 39 days postinoculation (PI). Measles nucleocapsid was first seen within the cytoplasm of giant cells, the latter appearing 5 to 6 days PI. Measles virus particles were most abundant at 10 days PI and appeared to bud off from areas of the cell membrane along which nucleocapsid was aligned. Intranuclear nucleocapsid was more abundant at later stages, and by 39 days PI entire nuclei were seen to be occupied. By this time, the cytoplasmic formations, which had been sequestered by membranes, appeared to lose their regular structure. Budding viral particles at 39 days PI were of a much simplified structure and did not involve the alignment of nucleocapsid about their periphery.     

Giant cell-forming variants of the Miyama strain of type 1 herpes simplex virus which differ in fusion activity

During serial passages of a non-giant cell-forming variant (-GCr) of the Miyama strain of type 1 herpes simplex virus, a new giant cell-forming variant named +GC(81) was isolated. CPE induced by this isolate was compared with that by -GCr and also by +GC(LPV), a derivative strain of the +GC variant of the Miyama strain. Rounding of single cells was observed after infection with -GCr. Remarkable syncytial formation was induced by +GC(LPV), the syncytia containing hundreds of nuclei, while small giant cells were formed by +GC(81). The reason for the appearance of +GC variants that differ in fusion capacity is discussed.     

Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells

Abstract Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.     

Identification of an endosomal antigen specific to absorptive cells of suckling rat ileum

A membrane fraction enriched in apical endosomal tubules was isolated from absorptive cells of suckling rat ileum and used as an immunogen to generate anti-endosome monoclonal antibodies. By immunofluorescence, one of these antibodies bound exclusively to the region of the apical endocytic complex in ileal absorptive cells, but not to other cell types. Immunoblot analysis showed the antigen as a diffuse 55-61-kD band which was highly enriched in the endosome fraction over whole-cell homogenate. The antigen appears to be an intramembrane glycoprotein: it partitioned primarily in the detergent phase after TX-114 extraction, and shifted to 44 kD after chemical deglycosylation. EM immunocytochemistry showed that the antibody bound to the luminal side of endosomal tubule membranes, a portion of endosomal vesicle membranes, and in endocytic pits of apical plasma membranes. However, it did not bind to multivesicular bodies, the giant lysosome, or other organelles. Immunocytochemistry after uptake with adsorbed or soluble tracer proteins showed that the antigen labeled portions of both prelysosomal pathways previously described in these cells (Gonnella, P.A., and M. R. Neutra, 1984, J. Cell Biol., 99:909-917). The function of this glycoprotein is not known, but inasmuch as it has been detected only in absorptive cells of suckling rat ileum, it may serve a function specific to these cells. Nevertheless, this endosomal antigen, designated glycoprotein (gp) 55-61, will serve as a useful marker for exploring membrane dynamics in early stages of the endocytic pathway.     

Viral Infections of Toronto Children During 1965: II. Measles Encephalitis and Other Complications

Among 16 cases of measles encephalitis observed in Toronto during 1964 and 1965, including six who were stuporous or comatose on admission and five who presented with convulsions, measles virus was isolated from CSF of one patient, rising titres of measles antihemagglutinin were detected in another patient, and 14 showed high antibody titres in sera collected as early as two to five days after onset of the measles rash. Increasing levels of measles antibody were detected in paired sera from three of seven patients with uncomplicated measles, and elevated antihemagglutinin titres were found in 16 cases of measles without neural involvement. Measles virus was isolated from lung tissue of a fatal case of giant cell pneumonia. Administration of pooled human gamma globulin to one leukemic patient, and of leukocytes from a convalescent donor to another leukemic child, may have assisted their recovery from measles.     

An ultrastructural study of the response of Blatella germanica (Orthoptera: Blattidae) to the nematode Abbreviata caucasica (Spirurida: Physalopteridae)

An ultrastructural study of the response of Blatella germanica (Orthoptera: Blattidae) to the nematode Abbreviata caucasica (Spirurida: Physalopteridea). International Journal for Parasitology4: 133–138. This study investigates the response of the roach, Blatella germanica L. to the invading spirurid nematode, Abbreviata caucasica v. Linstow. Soon after the first stage nematodes entered the epithelial cells of the colon wall, the surrounding host cells broke down into syncytial giant cells. Large polychromatic epithelial cell nuclei occurred throughout the giant cells and the nematodes moved freely within the cytoplasmic matrix. These giant cells were in turn surrounded by blood cells responding to the disruption. The nematodes developed to the infective third stage juveniles within the giant cells and ingested the syncytial cytoplasm. After reaching the third stage, the parasites remained in a quiescent state within the vacuolated cell which was surrounded by a double tissue layer.Evidence indicated that successful development of the parasite was dependent on the disrupted epithelial cells forming a giant syncytial cell which protected and supplied nourishment to the parasite.     

Isolation of a cytomegalovirus from salivary glands of white-lipped marmosets (Saguinus fuscicollis).

Minced salivary glands from seven white-lipped marmosets (Saguinus fuscicollis and Saguinus nigricollis) and one cotton-topped marmoset (Saguinus oedipus) were cocultivated with marmoset cell cultures. A viral agent, designated SSG, was isolated from two Saguinus fuscicollis. Slowly progressing foci of rounded, vacuolated, refractile cells were first observed at 40-43 days incubation. Electron microscopy revealed intranuclear herpesvirus nucleocapsids and intracytoplasmic and extracellular enveloped particles. Infected cells stained with hematoxylin and eosin contained eosinophilic intranuclear and cytoplasmic inclusion bodies. SSG could be passaged in cell cultures only using viable whole cells; infectious cell-free virus was not detected in either culture supernatants or cell lysates. SSG replicated in marmoset fibroblastic but not in marmoset epithelioid or human fibroblastic cell cultures. Plasma antibodies to SSG were detected by indirect immunofluorescence assays in 16 of 56 (28.6%) adult wild-caught marmosets but were absent in 40 colony-born, hand-reared marmosets. Antigenic cross-reactivity of SSG with a rhesus monkey (Macaca mulatta) cytomegalovirus (bidirectional) and with a human cytomegalovirus (unidirectional) was also demonstrated by indirect immunofluorescence assays. SSG was identified as a herpesvirus by morphology and was classified as a cytomegalovirus by its site of isolation, biologic properties in vitro, and antigenic characteristics.     

Respiratory syncytial virus ts mutants and nuclear immunofluorescence.

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and one spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37 degrees C as well as at the restrictive temperature of 39 degrees C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 mug/ml) for optimal growth, and was not significantly inhibited by mitomycin C (10 mug/ml). It was sensitive to actinomycin D (2.5 mug/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in BS-C-1 cells. Staining of infected monolayers by an indirect immunofluorescence procedure revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning electron microscopy. Filament production was inhibited when certain ts mutants were incubated at 39 degrees C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into three groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear, as well as cytoplasmic, immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.     

Infection of primary human fetal astrocytes by human herpesvirus 6.

Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus which productively infects human CD4+ T cells and monocytes. HHV-6 is the etiologic agent for exanthem subitum (roseola), and it is well-known that central nervous system complications occur frequently during the course of HHV-6-associated disease. In addition, HHV-6 has been associated with encephalitis or encephalopathy. However, very little is known about its tropism for neural cells. There are reports that HHV-6 may infect some glial cell lines, but whether it can infect any primary neural cells is not known. Our studies show that both HHV-6A (GS) and HHV-6B (Z-29) can infect highly purified primary fetal astrocytes in vitro. Infected cells showed cytopathic effects, forming giant syncytia. In dual immunofluorescence assays, the infected cells were detected by antibodies against the HHV-6 p41 nuclear antigen and glial fibrillary acidic protein, indicating that the infected cells are indeed astrocytes. PCR and Northern (RNA) blot analyses also confirmed that the astrocytes are infected by HHV-6. The progeny virus did not alter its host range and could reinfect T cells as well as primary astrocytes. These findings suggest that infection of primary human astrocytes may play a role in the neuropathogenesis of HHV-6.     

Expression of an unidentified immunoglobulin isotype on rabbit Ig-bearing lymphocytes.

A non-IgM immunoglobulin molecule was found on most rabbit Ig-bearing lymphocytes isolated from mesenteric lymph nodes. Membrane bound immunoglobulin light chains and heavy chains were detected by immunofluorescence and by rosetting with antibody-coated erythrocytes on mesenteric lymph node cells stripped of IgM by anti-IgM allotype antibodies. The percentage of cells bearing these residual immunoglobulin molecules was similar to the percentage of cells bearing immunoglobulin before "stripping" with anti-IgM antibody. These residual immunoglobulin molecules were not IgA nor IgG and are believed to be the rabbit analogue of human IgD.     

Isolation of giant mitochondrial nucleoids from the yeastSaccharomyces cerevisiae

Summary The yeast cellsSaccharomyces cerevisiae grown up to stationary phase under either anaerobic conditions, or aerobic conditions in the presence of a respiratory inhibitor, antimycin A, had distinctive giant mitochondrial nucleoids (mt-nucleoids) (apparent diameter 0.6–0.9 m) in contrast with the small mt-nucleoids (apparent diameter 0.2–0.4 m) in respiratory-sufficient cells grown aerobically, as revealed by DAPI-fluorescence microscopy. The cytoplasmic respiratory-deficient cells (rho^– cells), which were induced by treatment of wild-type cells with ethidium bromide, showed both giant and small mt-nucleoids of irregular size. In order to examine the structural and functional differences between giant and small mt-nucleoids, the former were successfully isolated from spheroplasts of three different cells by differential centrifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were free of significant contamination by nuclear chromatin. The number of protein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grown cells, though a few noticeable differences were also recognized. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mt-nucleoids from aerobically grown cells as detected by chromatography on native DNA-cellulose. In contrast, the 67 kDa and 52 kDa proteins were hardly detected in corresponding fractions of giant mt-nucleoids from anaerobically grown cells and from rho^– cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of antimycin A seemed to lack the 67 kDa protein but to have a small amount of the 52 kDa protein. This is the first demonstration of the variance of protein species involved in yeast mt-nucleoids according to the respiratory activity of mitochondria.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Novel peripheral blood-derived human cell lines with properties of megakaryocytes

For 18 mo, we derived 18 cell lines from 11 donors with various clinical profiles ranging from normal to leukemic. Suspension cultures were initiated with 1 X 10(6) mononuclear blood cells/ml of nutrient medium containing 10% human serum and 10% lectin-stimulated human lymphocyte conditioned medium. The cultures were monitored weekly by morphological analyses of Wright-Giemsa-stained cell preparations. All successful cultures showed a significant decline in viability during the first 3-4 wk with rate "lymphoid" cells observed in mitosis. Within the next 2 wk, the proliferating cells gave rise to a rapidly expanding population of mononuclear cells. As the cultures expanded, cell morphology became heterogeneous with respect to cell size and nuclear ploidy, with an accumulation of giant multinuclear cells that were suggestive of megakarocytes. Even though the cells did not have the classical morphology of mature platelet-forming megakaryocytes, 90% of the cells within a cell line were positive by direct or indirect immunofluorescence for the platelet membrane glycoproteins IIb and IIIa; for surface markers HLA-Dr and B2-microglobulin; for intracellular platelet-derived growth factor and platelet factor IV; and for membrane affinity or binding with serum platelet-derived growth factor and platelet factor IV. These results suggest that a blood precursor cell, most likely a primitive megakaryoblast, was isolated from the peripheral blood and was provided with an optimal culture environment for sustained growth. These cells did not mature to a more differentiated stage, perhaps owing to regulatory factor deficiencies in this in vitro system. The remarkable frequency of obtaining cell lines with megakaryocyte properties from normal peripheral blood and the capacity of some normal donors to repeatedly yield these cell lines make this cell culture system indeed unique by being selective for putative megakaryocyte precursors.     

Trophoblast giant cell release of placental lactogens: temporal and regional characteristics

Placental lactogen (PL) production by rat trophoblast giant cells was studied using in vitro methods. The influence of trophoblast giant cell location within the conceptus and day of trophoblast giant cell isolation on the type of PL released in vitro were investigated. The effect of trophoblast giant cell location on the amount of PL, progesterone, and testosterone released in vitro was also evaluated. Trophoblast giant cells release two types of PLs in vitro; a high-molecular-weight lactogen, PL-1, and a low-molecular-weight lactogen, PL-2. The type of PL released by trophoblast giant cells was not influenced by their location within the conceptus at the time of dissection. Location did influence the amount of hormone produced by trophoblast giant cells. Mural trophoblast giant cells were more active in the production of PL, progesterone, and testosterone. The type of PL released by trophoblast giant cells is highly dependent upon the day of gestation the cells are removed for study. Trophoblast giant cells isolated on Day 10 of gestation release predominantly PL-1, while those cells isolated 24 hr later (Day 11 of gestation) release predominantly PL-2. The switch from PL-1 to PL-2 production that occurs in vivo does not occur under the in vitro conditions employed in this report.