Differentiation of a clone isolated from the HT29 cell line: polarized distribution of histocompatibility antigens (HLA) and of transferrin receptors

The HT29 cell line, derived from a human colon adenocarcinoma, is able to differentiate if galactose replaces glucose in the culture medium. We have isolated a clone (HT29-18) from this cell line which displays differentiated properties of the parent cell line. HT29-18 cells grown in glucose-containing medium form multiple layers of round cells without specific cell-cell adhesion. In contrast, when grown in galactose-containing medium, they form a monolayer with tight junctions and exhibit a well differentiated brush border at their apical membrane, which faces the culture medium. The polarized properties of HT29-18 cells grown in galactose-containing medium were demonstrated by immunofluorescent techniques with antibodies against 2 plasma membrane proteins. Class I histocompatibility antigens (HLA) and transferrin receptors, 2 well characterized integral membrane proteins, are uniformly distributed on the cell surface of undifferentiated HT29-18 cells, but acquire a polarized distribution during differentiation, localized on the basolateral membranes and absent from the apical surface. Binding of 125I-labeled transferrin was used to determine transferrin receptor distribution on apical and basolateral membranes. Functional tight junctions in the differentiated cultures were demonstrated, as the monolayer was impermeable to a permeation dye (ruthenium red) as well as to antibodies. The sealing of these tight junctions is, as in vivo, Ca++-dependent as they could be opened by a short incubation in Ca++-free medium.     

A labile antigen complex at the lymphocyte surface: the effect of the substratum on its expression

Human T lymphocytes carry a surface antigen, detectable by a monoclonal antifibronectin antibody, which appears to consist of 150 and 55 kd components as revealed by SDS-PAGE. After in vitro culture of the lymphocytes on a plastic substratum for 48 hr comparatively few cells (40 +/- 18% in separate individuals) express the antigen. In contrast, the vast majority of lymphocytes cultured on a collagen matrix for the same time period maintains surface expression of the antigen (76 +/- 14% in separate individuals). Conditioned media from lymphocytes on plastic contain substantial amounts of antigenicity detectable by the same antibody, whereas conditioned media from lymphocytes on collagen are devoid of such antigenicity. The expression at the cell surface of other T lymphocyte antigens (Leu-4, Leu-3, and OKT8) is identical during culture on plastic and collagen for 48 hr. Collagen does not activate the cells to DNA synthesis or expression of IL 2 receptors, and consequently the potentiation of antigen expression by this substratum cannot be attributed to a mitogenic effect. The composition of subsets of T lymphocytes and the viability of the cells are the same on plastic and collagen, which excludes that the substratum-dependent variations in antigenicity reflect selection or loss of antigen-bearing cells. Thus, substratum-dependent regulation of the expression at the cell surface appears to be a unique property of the 150/55 kd T cell surface antigen. Culture on collagen substrata augments the number of lymphocytes showing motile behavior two to four times compared with culture on plastic.     

Immunohistochemical localization of cell surface receptors using a novel method permitting simple, rapid and reliable LM/EM correlation

Summary A method is presented which allows correlative serial section analysis by light and electron microscopy of cell surface antigens in monolayer cultures. Sites of antigenicity are shown by deposition of diaminobenzidine after pre-embedding, immunoperoxidase immunocytochemistry. Osmication is replaced by the use of gold chloride which specifically enhances the electron density of diaminobenzidine. In addition gold chloride bound to diaminobenzidine survives embedding and provides the basis for a post-embedding photochemical amplification method. Immunostained cells are embedded in LR White by a rapid technique which preserves their structure and leaves them available for subsequent post-embedding immunocytochemistry. The method is illustrated by the demonstration of epidermal growth factor (EGF) receptors on the EGF receptor-rich human carcinoma cell line A431 using a well characterized monoclonal antibody raised against EGF receptor.     

An immunocytotoxicity assay for neural cells in monolayer cultures

The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell susptnsions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with 10- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.     

Partial characterization of nervous system-specific cell surface antigen(s) NS-2.

Partial biochemical characterization of several neural tissue specific antigens isolated from a murine glioblastoma cell line was accomplished by means of radioiodination of intact cells followed by immunoprecipitation of the cell lysate with a rabbit serum specific for neural tissue antigens. Polyacrylamide gel electrophoresis of the immunoprecipitate in sodium dodecyl sulfate resolved the labeled antigens into several major components: two proteins (or glycoproteins) having apparent m.w.'s of 84,000 and 120,000 and lipid associated components which may be heterogeneous. The protein and lipid associated components apparently possess independent antigenicity because after chloroformmethanol extraction the protein components can be immunoprecipitated from the aqueous phase and the lipid associated component can be immunoprecipitated from the organic phase. Despite their independent antigenicity it is not known whether the components may be noncovalently associated on the cell surface. Although some of these antigens can be isolated from brain or glioma cells (a related tumor), non can be demonstrated in lymphoid tissues or C1300 neuroblastoma cells using identical methods. Therefore, these studies confirm our previous findings concerning the specificity of the anti-NS-2 antiserum by using cytotoxicity tests.     

Identification of multiple cell surface antigens associated with the sex pheromone response of Streptococcus faecalis.

High-molecular-weight surface antigens, obtained by ammonium sulfate precipitation of culture supernatants and identified in Western blots of sodium dodecyl sulfate-polyacrylamide gels, have been correlated with the sex pheromone response of Streptococcus faecalis donor cells. Pheromone-induced cells carrying the conjugative plasmid pCF10 produced both an antigenic component (C130) composed of at least four bands in the range of 130 kilodaltons and a 73-kilodalton antigen (SA 73). The concentration of the C130 antigen in culture supernatants increased with time after exposure of donor cells to pheromone preparations. Gel filtration studies indicated that this antigen exists in the native state as a very large complex that is more than 180,000 daltons in size. The C130 antigen was susceptible to digestion by proteinase K and was not reactive with either concanavalin A or wheat germ agglutinin. The antigenicity of C130 was not destroyed by treatment of blots with trypsin, chymotrypsin, or papain before development with antibody, whereas the antigenicity of SA73 was susceptible to these treatments.     

Theileria annulata: attenuation of a schizont-infected cell line by prolonged in vitro culture is not caused by the preferential growth of particular host cell types

Flow cytometry and monoclonal antibodies to bovine leucocyte surface antigens were used to identify the types of host cells that the sporozoites of Theileria annulata infect in cattle, to determine whether virulent schizont-infected cell lines (lines) differed phenotypically from avirulent lines, and to establish whether attenuation in vitro was accompanied by the preferential growth of particular host cell types. The surface antigens of four pairs of T. annulata (Ta) (Hisar) lines derived ex vivo and in vitro, including the virulent ex vivo-derived Ta Hisar S45 line, were consistent with a myeloid origin for all lines, irrespective of their derivation. The profiles of lines derived from cattle inoculated with a virulent line showed that the schizonts liberated from inoculated cells had transferred to myeloid cells. A number of other lines infected with different stocks of T. annulata expressed myeloid markers; a single line expressed CD21, a B cell marker. During prolonged in vitro culture, the parasites in the ex vivo (virulent)- and in vitro (avirulent)-derived Ta Hisar S45 myeloid lines became clonal, as defined by glucose phosphate isomerase (GPI) polymorphism, and the virulent line became attenuated. The two lines retained phenotypic profiles indicative of a myeloid origin but coexpressed some lymphoid antigens (CD2, CD4, CD8), although not CD3. Cloned schizont-infected lines, representing the three parasite GPI isotypes which constituted the virulent line, expressed similar patterns of myeloid and lymphoid markers to the virulent parent line. Some schizont-infected clones failed to establish as lines during the early weeks of culture because the cells died as the parasites differentiated into merozoites at 37 degrees C, the temperature at which schizont-infected cells normally grow exponentially. These results provided no evidence that prolonged culture induces preferential growth or loss of particular host cell types. However, a number of the alterations in host cell surface antigens induced by prolonged culture were shown to be linked to permanent changes in the parasite genome.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

Immunoelectron microscopy of cell surface antigens: a quantitative analysis of antibody binding after different fixation protocols

Summary The effect of different fixation solutions on the denaturation of membrane-associated antigens in murine lymphoid cells was determined quantitatively using microfluorometric analysis and a radioimmunoassay. Paraformaldehyde and periodate-lysine-paraformaldehyde solutions preserved the antigenicity of cell surface-associated immunoglobulin (S–Ig) antigens when used in concentrations ranging from 0.01 to 4%. However, glutaraldehyde destroyed the antigenicity of S–Ig and Thy 1.2 molecules at concentrations higher than 0.1%. Electron microscopic analysis of the different fixed cell suspensions, after labelling of the cells with a rabbit anti-mouse immunoglobulin-horseradish peroxidase conjugate (RaM-Ig-HRP) showed that prefixation of the sample with 0.1% glutaraldehyde was optimal for immunoelectron microscopical studies, since this concentration preserved both the antigenicity of membrane-associated antigens as well as the ultrastructure of the cells under study. Prolonged fixation periods affected antibody binding. However, S–Ig molecules denatured at a slower rate than Thy 1.2 molecules. A preparation method for the immunoelectron microscopical localization of lymphoid and non-lymphoid cell types in lymphoid organs is reported.TheHistochemical Journal lecture 1979 given by Dr Van Ewijk to the Histochemistry and Cytochemistry Section of the Royal Microscopical Society on 12 July, 1979.     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

Responsiveness of acetylcholinesterase and butyrylcholinesterase activities in neural cells to age and cell density in culture

Acetylcholinesterase activity in neuroblastoma and C-6 glial cells, maintained in monolayer culture, decreased with increasing age and cell density (cells/mm²). Butyrylcholinesterase activity in C-6 glial cells did not change with age, but increased slowly with increasing cell density. AChE reached peak activity at a lower density in neuroblastoma than in C-6 glial cells. The data suggest either that AChE in both cell types is responsive to cell-cell contact or that different enzymes are involved.     

Responsiveness of actylcholinesterase and butyrylcholinesterase activities in neural cells to age and cell density in culture.

Acetylcholinesterase activity in neuroblastoma and C-6 glial cells, maintained in monolayer culture, decreased with increasing age and cell density (cells/mm²). Butyrylcholinesterase activity in C-6 glial cells did not change with age, but increased slowly with increasing cell density. AChE reached peak activity at a lower density in neuroblastoma than in C-6 glial cells. The data suggest either that AChE in both cell types is responsive to cell-cell contact or that different enzymes are involved.     

The effect of ethanol on cell wall antigens ofSaccharomyces cerevisiae and specific isolation of high ethanol producing strains of this yeast,making use of a serological technique

Generally, natural isolates of high ethanol producingSaccharomyces cerevisiae obtained by screening are used in alcoholic industries. The methods involved in their isolation and identification are elaborate. Antigenic analysis using antibodies raised against wholeSaccharomyces cells indicated species specificity of cell wall surface thermostable antigens. By affinity purification, the specific antibodies could be obtained and used for specific isolation ofS. cerevisiae. Antigenic studies using antibodies raised against isolated cell walls of fermentatively grownS. cerevisiae indicated the occurrence of thermolabile antigens common toSaccharomyces species. Higher concentrations of these antigens could be detected in thoseS. cerevisiae that had the ability for high ethanol production. The concentrations of these cell wall common antigens increased with increasing culture age and ethanol accumulation in culture broths. In younger yeast cells, the concentration could be increased by growing the cells in a medium containing added ethanol. Using dilutions of cross absorbed antibody specific for common antigens and Ouchterlony test, high ethanol producingS. cerevisiae could be identified.     

Generation of a radial-like glial cell line

Rat C6 glioma is a cell line that has been used extensively as a model of astroglia. Although this cell line retains many of the properties of developing glia, it does not resemble morphologically the specialized form of glia found embryonically, the radial glia. In experiments designed to study a mutant form of receptor protein tyrosine phosphatase β, we isolated a subclone of C6 called C6-R which, like radial glia, assumes a highly polarized radial-like morphology in culture. C6-R cells and, to a somewhat lesser extent, C6 cells, express cytoskeletal proteins found in developing astroglia including glial fibrillary acidic protein and RC1. As seen with radial glia, cerebellar granule cell bodies and neurites migrated along radial processes of C6-R cells in culture. Morphological analysis of dye-labeled cells injected into the developing forebrain revealed that a large fraction (∼60%) of the C6-R cells in the cortex assumed a radial orientation and about half of these (∼30%) made contact with the pial surface. In contrast, the parental C6 cells generally formed aggregates and only displayed a radial alignment when associated with blood vessels. These results suggest that we have generated a stable cell line from C6 glioma which has adopted certain key features of radial glia, including the ability to promote neuronal migration in culture and integrate radially in vivo in response to local cues. This cell line may be particularly useful for studying receptors on radial glia that mediate neuronal migration. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 291–304, 1998     

Preparation of enteric-coated microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate: The effect of swine serum on micromeritic properties

Summary Cellulose acetate phthalate was used to prepare the Mycoplasma hyopneumoniae vaccine (MHV) microspheres using a solvent evaporation method. Swine serum was used as an additive in the antigen to form the core materials. The addition of serum had a significant effect on surface topography of the MHV microspheres. By using this modified solvent evaporation method, the recoveries of antigens in the MHV microspheres were generally over 90% of the weight and antigenicity of antigens originally added in the formulation.     

Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells

The aim of this study was to investigate the effect of hyperthermia, 6 MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133⁺/CD44⁺ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133⁺/CD44⁺ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6 Gy) and hyperthermia (60 and 90 min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.     

Pathways of Differentiation in Chick Embryo Neuroretinal Cultures

Markers of neuronal cell differentiation (GABA accumulation, choline acetyltransferase activity) are shown to increase initially and then decline sharply in monolayer cultures of 9 day embryo neuroretinal (NR) cells. A glial marker (glutamine synthetase, GSase) is precociously inducible by hydrocortisone (HC) in dense'monolayer' NR cultures (containing aggregates of neuronal cells overlying the glial sheet) as well as in chick embryo retinal explants. The induced level of GSase activity is not maintained in the continued presence of HC, but rather declines by 20 days in vitro. Choline acetyltransferase (CAT) activity is higher in HC-treated cultures than in controls only during the period when induced GSase activity is detectable. Furthermore, the subsequent transdifferentiation of lens cells (monitored as δ crystallin content) in these cultures is delayed by 10 days and much reduced in extent when HC is present throughout the culture period.
We suggest a simple model to account for these results, on the basis of recent evidence that lens cells are derived mainly from the retinal epithelial cells (immature Müller glia) of 9-day embryonic NR, and that transdifferentiation results from a change in cell determination during the early stages of'monolayer' culture. In outline, our model proposes that early dedetermination of the retinal glia is associated with a decline of neuronal cell markers (dedifferentiation) followed eventually by loss of the neuronal cells. Hydrocortisone, by inducing transient glial cell differentiation (GSase activity), both prolongs the expression of a neuronal marker (CAT) and also reduces later transdifferentiation into lens.     

Monolayer culture of cells originating from apreimplantation bovine embryo

Summary The objective of this study was to establish a method by which trophectodermal cells originating from individual preimplantation bovine embryos could be perpetuated in monolayer culture. A single, Day-11 bovine embryo collected nonsurgically from a mixed-breed beef cow was cultured in Ham's F10 medium supplemented with fetal bovine serum, sodium pyruvate, insulin and epidermal growth factor. After 13 d in culture the embryo had adhered to the surface of the plastic culture vessel and a monolayer covering 0.3 cm² had developed in the manner of a tissue explant. The monolayer was successfully dispersed using trypsin-EDTA and the cells were passaged Expansion to a 25-cm² flask was achieved by the 4th passage. By passaging cultures at a dilution ratio of 1∶2, cells were maintained for 38 passages before growth slowed. Transfers beyond the 44th passage were unsuccessful. The cell line, designated BE-13, was successfully frozen and thawed at the 9th, 12th, 15th, and 20th passages. The cell line contains both mono- and binucleate cells with a prominent rough endoplasmic reticulum characteristic of ruminant trophoblast cells. Susceptibility to eight bovine viruses was demonstrated. Such cell lines may provide inexpensive systems for the study of trophoblast metabolism and for investigation of the role of the trophoblast in the pathogenesis of selected bovine abortifacient diseases. Because of their range of viral susceptibility, these cells might also be useful for diagnostic purposes. Published as publication no. 1891 College of Veterinary Medicine, Auburn University, Alabama 36849. This work was funded in part by an Auburn University Faculty Research Grant-in-aid. Preliminary results of the study were presented in abstract form at the 1987 Annual Conference of the International Embryo Transfer Society.     

The “in situ” expression of Human Leukocyte Antigen Class I antigens is not altered by cryopreservation in human arterial allografts

This study was aimed to establish whether the cryopreservation procedure we currently use in clinics can modify arterial homograft antigenicity. To this purpose, we performed an immunohistochemical study on fresh and cryopreserved human arterial homografts to visualize the expression of HLA class I heavy and light chains "in situ" by using the HC-10 and Namb-1 monoclonal antibodies. Human femoral arteries and thoracic aortas were harvested from 18 heart-beating donors and sampled before and after cryopreservation. Arterial segments were frozen in liquid nitrogen vapors in a controlled rate freezing system. After thawing, samples were processed for routine immunohistochemistry. To standardize immunostaining, flow-cytometry indirect immunofluorescence analysis was performed on HUVEC; immunohistochemistry of human ovarian cortical vessels was performed as an additional positive control. Negative controls were performed by omitting tissue incubation with primary antibodies. HLA-class I antigens were markedly expressed by endothelial cells lining surface intima and adventitial vasa vasorum; a moderate expression was found in medial smooth muscle cells. Except for the surface unreactivity caused by loss of endothelium, results from cryopreserved arterial allografts were strictly comparable to those observed in fresh, unfrozen tissues. These results support the view that cryopreserved arterial allografts are immunogenic as their fresh counterparts; apart from smooth muscle cells which retained a moderate expression of HLA class I antigens following cryopreservation, our study suggests that the highly HC-10 positive endothelial cells we found to line the rich adventitial network of vasa vasorum are expected to be one of the major targets of the serological response in the recipient.