Recovery of Cell-Bound Interferon

Interferon could be recovered from homologous cells to which it was applied but could not be recovered from heterologous cells. The amount of interferon that could be recovered from cells corresponded to the sensitivity of the cells to the antiviral activity of the interferon: mouse embryo fibroblasts, which were 5 to 10 times as sensitive as L-929 cells to interferon, bound 5 to 10 times more interferon than the latter, whereas Lpa cells, which were only one-third as sensitive as L-929 cells to interferon, bound only one-third as much as the latter. The concentration of cell-bound interferon was as much as 150 times the extracellular concentration of interferon applied to the cells. Interferon bound to cells at 4 C with the same efficiency as it did to cells at 37 C, and actinomycin D-treated cells bound interferon as well as normal cells. Even though the total amount of interferon bound to cells was as much as 30% of the amount of interferon applied to them, no loss of antiviral activity was detectable from the medium.     

Protein kinase C and leucine metabolism in intestinal crypt cells

Intestinal cells were separated into fractions rich in villous or crypt cells. Alkaline phosphatase, present in villous cells, but absent from crypt cells, was used as a marker. Crypt cells were 3-6 times as active as villous cells in the metabolism of leucine or mevalonate. The previously reported stimulatory effect of albumin was twice as strong in crypt cells as that in villous cells. Reduced glutathione, spermidine HCl and ethanolamine (0.5-10 mM) did not replace albumin, the effect of which was maximal at 0.02 mM. Protein kinase C was shown to be present mainly in crypt cells.     

Hexaploid H1 (ES) cells established from octaploid H1 cells are as DNA stable as pentaploid H1 cells

Hexaploid H1 (ES) cells (6H1 cells) were established from octaploid H1 cells (8H1 cells), as were pentaploid H1 cells (5H1 cells). 6H1 cells were compared with 5H1 cells. The number of chromosomes of 6H1 cells was 115, 20 more than the 95 of 5H1 cells. The durations of G(1), S, and G(2)/M phases of 6H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 5H1 cells. The cell volume of 6H1 cells was equivalent that of 5H1 cells. The morphology of 6H1 cells was flattened circular cluster, different from the spherical cluster of 5H1 cells. 6H1 cells exhibited alkaline phosphatase activity as well as 5H1 cells. The DNA content of 6H1 cells was stable and maintained for 300 days of culturing, the same as that of 5H1 cells. The DNA stability of 6H1 cells was explained using a hypothesis concerning the DNA structure of polyploid cells because the asymmetric configuration of homologous chromosomes in 6H1 cells inhibited chromosome loss.     

An immunohistochemical study of endocrine cells in the pancreas of the Red-bellied frog (Bombina orientalis)

The regional distribution and frequency of pancreatic endocrine cells in the red-bellied frog, Bombina orientalis, were studied by the immunohistochemical peroxidase anti-peroxidase (PAP) method using five types of specific mammalian antisera to insulin, glucagon, somatostatin, bovine pancreatic polypeptide (PP) and secretin. The frequency was calculated as the mean number of each endocrine cell type/1,000 total cells (including exocrine and endocrine cells) using an automated image analysis process. The percentage of each immunoreactive (IR) cell species to the total IR cell population was also calculated. In the pancreas of the red-bellied frog, all five endocrine cell types were demonstrated. Insulin IR cells were located in the pancreas as single cells or islet-like clusters. The latter were localized in central regions. The insulin-IR cells showed a frequency of 65.40 plus/minus 14.56/1,000 cells. Glucagon IR cells were also detected as single cells or as clusters but in the case of clusters, two distributional patterns were detected - a central core type and a marginally distributed type. They showed an abundance of 32.70 plus/minus 7.32/1,000 cells. Somatostatin-IR cells were dispersed throughout the pancreatic parenchyma as single cells, three to four cells, or clusters. The clusters were located in the marginal regions. The somatostatin-IR cell frequency was 19.40 plus/minus 6.52/1000 cells. PP-IR cells were randomly distributed throughout the pancreatic parenchyma as single cells with a frequency of 14.70 plus/minus 4.92/1,000 cells. Secretin-IR cells were demonstrated as clusters or as single cells, and as clusters they occupied the central regions. They showed a frequency of 39.60 plus/minus 10.36/1,000 cells. This is the first report of the presence of secretin-IR cells in amphibian pancreatic endocrine cells. Overall, there were 37.20 plus/minus 6.84% insulin-, 21.90 plus/minus 5.55% glucagon-, 11.60 plus/minus 4.33% somatostatin-, 8.60 plus/minus 2.72% PP- and 23.40 plus/minus 4.45% secretin-IR cells.     

B cells as antigen-presenting cells: antigen-specific IL-2 production by cloned T cells without expression of IL-2 receptors

A murine T cell clone, 24-2C, responds specifically to human IgG (HGG) in the context of I-Ab. B cells purified from mouse spleen cells were examined for their function as antigen-presenting cells (APC) in the response of 24-2C cells to HGG. B cells functioned as APC for IL-2 production but not for proliferation, whereas spleen cells or spleen-adherent cells functioned as APC for both IL-2 production and proliferation. LPS-activated B cells also failed to induce the proliferative response. The addition of the culture supernatant of 24-2C cells stimulated with HGG presented by irradiated spleen cells to the culture of 24-2C cells, irradiated B cells, and HGG induced the proliferative response of 24-2C cells, whereas IL-1, IL-3, and/or interferon-gamma did not reconstitute the proliferation. The expression of IL-2 receptors (IL-2R) on 24-2C cells was examined using a monoclonal anti-mouse IL-2R antibody AMT 13 or 7D4. 24-2C cells cultured with spleen cells as APC expressed IL-2R. Those cultured alone or with B cells as APC did not express IL-2R. Enlargement of 24-2C cells in response to HGG was also examined, and the relative cell size of those cultured with B cells or spleen cells as APC was larger than that of those cultured alone. These results demonstrate that B cells as APC induce IL-2 production and cell size enlargement in the response of 24-2C cloned T cells to HGG, but not IL-2R expression nor proliferation.     

Prevalence of Heterotypic Tumor/Immune Cell-In-Cell Structure In Vitro and In Vivo Leading to Formation of Aneuploidy

Cell-in-cell structures refer to a unique phenomenon that one living cell enters into another living cell intactly, occurring between homotypic tumor cells or tumor (or other tissue cells) and immune cells (named as heterotypic cell-in-cell structure). In the present study, through a large scale of survey we observed that heterotypic cell-in-cell structure formation occurred commonly in vitro with host cells derived from different human carcinomas as well as xenotypic mouse tumor cell lines. Most of the lineages of human immune cells, including T, B, NK cells, monocytes as well as in vitro activated LAK cells, were able to invade tumor cell lines. Poorly differentiated stem cells were capable of internalizing immune cells as well. More significantly, heterotypic tumor/immune cell-in-cell structures were observed in a higher frequency in tumor-derived tissues than those in adjacent tissues. In mouse hepatitis models, heterotypic immune cell/hepatocyte cell-in-cell structures were also formed in a higher frequency than in normal controls. After in vitro culture, different forms of internalized immune cells in heterotypic cell-in-cell structures were observed, with one or multiple immune cells inside host cells undergoing resting, degradation or mitosis. More strikingly, some internalized immune cells penetrated directly into the nucleus of target cells. Multinuclear cells with aneuploid nucleus were formed in target tumor cells after internalizing immune cells as well as in situ tumor regions. Therefore, with the prevalence of heterotypic cell-in-cell structures observed, we suggest that shielding of immune cells inside tumor or inflammatory tissue cells implies the formation of aneuploidy with the increased multinucleation as well as fine-tuning of microenvironment under pathological status, which may define distinct mechanisms to influence the etiology and progress of tumors.     

An ultrastructural study of the regenerating breast feather of the fowl

The developmental morphology of regenerating male breast feathers of the jungle fowl was studied at the ultrastructural level. The process of keratinization was observed in the three types of cells which form feather barbs: barbule cells, cortical cells, and medulla cells. Keratinization first became evident in the barbule cells and resembled the process of keratinization as observed in hair cortical cells and embryonic down feathers. Eventually the whole cytoplasmic area of the barbule cell was occupied by keratin. The barb cortex cells became keratinized in a similar fashion as the barbule cells but not until they were developmentally twice as old as the barbule cells. When keratinization was complete in these cells, the keratin was in the form of large agglomerates scattered in the cytoplasm. The barb medulla cells showed no obvious signs of keratinization until they were developmentally three times as old as the barbule cells. Keratin filament bundles were first seen near the plasma membranes of the medulla cells. Large empty vacuoles appeared in the cytoplasm which also contained moderate amounts of glycogen.     

Cellular immune response against tumor cells. I. In vitro immunization of allogeneic and syngeneic mouse spleen cell suspensions against DBA mastocytoma cells

Spleen cell populations stimulated in vitro with as few as 1000 tumor cells produce cytotoxic effector cells. Syngeneic as well as allogeneic spleen cells respond to DBA mastocytoma tumor cells. There is a significant cellular immune response to allogeneic tumor cells 72 hr after exposure to antigen. By contrast, the response of DBA spleen cells to DBA mastocytoma tumor cells is first detectable at 120 hr following exposure to antigen. C57BL/6 spleen cells immunized against DBA mastocytoma antigen kill both DBA mastocytoma tumor cells and normal cells from DBA animals. DBA spleen cells immunized against DBA mastocytoma antigen kill only the DBA mastocytoma tumor cells, and not normal cells from DBA animals.     

Glycogen-containing cells in the maternal and embryonic portions of the placenta in the rat and the common vole Microtus subarvalis

Differentiation sequences and further transfiguration of glycogen-rich cells during placenta development were investigated for the rat and field vole Microtus subarvalis (11-20 day gestation). The presence of glycogen is a characteristic feature of decidual cells located in the region of lateral sinusoids, as well as of metrial gland cells, secondary giant trophoblast cells and trophoblast cells in the connective zone of placenta. Glycogen-containing metrial gland cells and trophoblast cells of connective zone of placenta are found to underlie the layer of tertiary giant trophoblast cells that cover the wall of the central arteria. Thus, both maternal and embryo-derived glycogen-containing cells always accompany the tertiary giant trophoblast cells that penetrate deeply into the maternal part of placenta but do not contain glycogen. In the field vole placenta the cells of peripheral trophoblast subpopulation of the connective zone of placenta attaching to the decidua basalis are stained by PAS-reaction more intensely than deeply situated ones. These data, as well as other phenomena revealed here, show that maternal and trophoblastic cells attaching to each other in placenta contain, as a rule glycogen. Glycogen cells in rat placenta and trophoblast cells of peripheral subpopulation of connective zone of placenta are similar in many respects. In this connection, a possible protective role of glycogen-containing cells, that probably favour the co-existence of maternal and embryo-derived cells in placenta, is discussed.     

Ultrastructure and fluorescence histochemistry of endocrine (APUD-type) cells in tracheal mucosa of human and various animal species

This study describes distrinctive cells with ultrastructural and histochemical features of APUD-type endocrine cells within the tracheal epithelium of human fetuses, newborns and children as well as different animal species. These cells referred to as Kultschitzky cells (K cells) were found to be argyrophilic, but not argentaffin, and are considered analogous to the same type of cells in lung and gastro-intestinal tract. Fluorescence histochemistry demonstrated the presence of intracellular amine within tracheal K cells, but only after in-vitro or in-vivo administration of amine precursor (L-DOPA). Ultrastructurally, these cells are characterized by the presence of numerous cytoplasmic granules (dense core vesicles) which show species related morphologic variations. Two different types of K cells were found in trachea of lamb and armadillo, each type possessing morphologically different dense core vesicles. In human and rabbit tracheas, only one type of K cell was identified. K cells in the trachea are distributed as single cells between other epithelial cells; neuroepithelial bodies such as those found in bronchial mucosa were not identified. Well differentiated K cells were found in tracheas of early human fetuses and throughout gestation, infancy, and childhood. Preservation of K cells in human autopsy material and widespread occurence of these cells in various laboratory animals will permit further studies into the nature and function of tracheobronchial endocrine cells.     

Natural killer cells can enhance the proliferative responses of B lymphocytes

In addition to lytic activity against malignant and virally transformed target cells, recent evidence has suggested that natural killer (NK) cells can modulate immune activities such as the suppression of B cell responses through noncytotoxic means. Using human B cells and highly purified autologous NK cells, we have demonstrated that NK cells can substantially augment the proliferative responses of B cells stimulated with the surface immunoglobulin crosslinking agents anti-IgM or Staphylococcus aureus Cowan strain I (SAC). This "enhancer" activity of NK cells was quite potent and was observed at an NK:B cell ratio as low as 0.05. Peak blastogenic responses of B cells cocultured with NK cells in the presence of B cell activators were observed at 2-3 days, similar to the responses of B cells in the absence of NK cells. Using the inhibitor of DNA synthesis mitomycin C, we determined that B cells and not NK cells were proliferating in cocultures of these lymphocytes stimulated with SAC. Activated B cells neither prevented the lysis of the isotope-labeled NK-sensitive target cell line K562 nor formed conjugates with NK cells, suggesting that cell contact was not a prerequisite for the effect. These studies have further expanded the functional repertoire of NK cells to include enhancer as well as suppressor and lytic activities.     

B cells in autoimmunity

B-cell development is tightly regulated, including the induction of B-cell memory and antibody-secreting plasmablasts and plasma cells. In the last decade, we have expanded our understanding of effector functions of B cells as well as their roles in human autoimmune diseases. The current review addresses the role of certain stages of B-cell development as well as plasmablasts/plasma cells in immune regulation under normal and autoimmune conditions with particular emphasis on systemic lupus erythematosus. Based on preclinical and clinical data, B cells have emerged increasingly as both effector cells as well as cells with immunoregulatory potential.     

Cathepsin D and its prognostic value in neuroepithelial brain tumors

Expression of Cathepsin D (Cath D) in some primary neuroepithelial brain tumors and its prognostic value were studied. The research included 65 samples of human primary neuroepithelial brain tumors. There were 50 glial tumors (10 diffuse astrocytomas (DA), 15 anaplastic astrocytomas (AA), 25 glioblastomas (GB), 15 embryonic tumors (15 medulloblastomas (MB) as well as 5 samples of normal brain tissue. Immunohistochemical method was applied to monitor diffuse positive reaction in the cytoplasm of brain tumor cells, endothelial cells and tumor stromal cells and showed diffuse positive reaction for Cath D in the cytoplasm of brain tumor cells, endothelial cells and stromal cells in all analyzed samples of DA, AA, GB and MB as well as in microglial cells, neurons and in endothelial cells in all analyzed samples of normal brain tissue. Qualitative analysis of Cath D expression in the cytoplasm of brain tumor cells and endothelial cells as well as the percentage of brain tumor cells, endothelial cells and stromal cells immunopositive for Cath D showed that there was difference between analyzed brain tumor groups, but according to statistical tests the difference was not statistically significant. Survival correlated with the percentage of stromal cells immunopositive for Cath D. Survival prognosis was influenced by the percentage of stromal cells immunopositive for Cath D and tumor grade. The obtained results singled out the percentage of stromal cells immunopositive for Cath D as an independent parameter. The results of this research on the prognostic value of Cath D in some primary brain tumors of neuroepithelial origin indicate that there is real possibility to use Cath D as an independent prognostic factor in human glioma progression and thus open up possibilities for further scientific research.     

Characterization of an SV40-transformed 3T3 cell line expressing an unusual phenotype.

A transformed variant derived as a clone from normal 3T3 cells infected with simian virus 40 (SV40) has been found to possess a phenotype intermediate between that of normal cells and that characteristic of the transformed state, yet cells of the variant still test positively for the SV40-specific nuclear T-antigen. The variant exercises growth control, although not as stringently as do normal cells. Its cell size more closely resembles that of normal cells than of transformed cells. The variant also exhibits levels of spontaneous agglutination that are in line with those characteristic of the normal cells from which it was derived, and far higher than corresponding values for cells exhibiting the fully transformed phenotype. Plasma membranes of variant cells more closely resemble those of transformed cells than of normal cells as estimated by polyacrylamide gel electrophoresis. Perhaps the most distinguishing characteristic of the transformed variant is its complete immunity to agglutination by concanavalin A (Con A), even at concentrations of the lectin as high as 500 mug/ml. Moreover, trypsinization does not render variant cells as agglutinable in the presence of Con A as are untreated fully transformed cells. By contrast the variant displays a low tolerance of Con A toxicity, as monitored by ability to grow after treatment with the lectin, and on this count resembles transformed cells. Moreover a survey of several normal cell lines has revealed that even they do not consistently show resistance to Con A toxicity. These observations indicate that Con A-mediated agglutination and inability to grow after treatment with Con A are quite independent and do not bear a cause and effect relationship.     

极北鲵消化道嗜银细胞的胚后发生

为了探讨极北鲵(Salamandrella keyserlingii)消化道嗜银细胞胚后发生的形态学特征及分布规律,采用Grimelius银染法,对30～46期的极北鲵幼体进行了研究。结果显示,嗜银细胞在食管、胃、小肠各部分的发生时间不同。胃中的嗜银细胞在第41期出现,小肠中的在第42期出现,食管中的在第43期出现。嗜银细胞形态多样,有圆形、椭圆形、梭形和锥体形,大多分布在消化道黏膜上皮之间。根据嗜银细胞的形态判定其可能具有内分泌和外分泌两重功能。极北鲵胚后发育消化道各部位嗜银细胞出现顺序的不同可能与其消化生理活动及个体生理活动的变化相适应。     

Induction of differentiation of undifferentiated cells into pancreatic beta cells in vertebrates

The beta cells of the pancreatic islets, which maintain glucose homeostasis by secreting insulin, are important cells for sustaining life. In recent years, islet transplantation has been performed as a treatment for type I diabetes. Since there are not enough donors for patients awaiting transplantation, beta cells grown in vitro are expected to be utilized as a substitute for islets. To obtain the cells with properties of human beta cells, it is necessary to understand the process by which human pancreatic islets are formed, as well as their structural characteristics. By using undifferentiated cells, such as Xenopus laevis animal caps and mouse ES cells, pancreatic tissue has shown to be able to be induced in vitro. Various attempts have been made to obtain human beta cells from human ES/iPS cells. Versatile methods have been developed and improved efficiency has been achieved by the use of low molecular weight compounds, but the challenge remains to prevent tumor formation and achieve functional maturation. Inducing the differentiation of somatic stem cells into insulin-producing cells has also brought us closer to clinical application. There are still many challenges related to the practical use of beta cells derived from undifferentiated cells, such as the development of methods to substitute these cells for host beta cells, standardization of the treatment protocol, quality control, and confirmation of safety. Research on the methods of inducing undifferentiated cells to differentiate into beta cells has shown definite progress, suggesting that cell therapy for diabetes may become a preferred therapeutic option over islet transplantation.     

Stimulation of prostaglandin synthesis in cultured rat synovial cells by a factor derived from polymorphonuclear leukocytes

Stimulation of synovial cell prostaglandin production by a factor obtained from casein-induced peritoneal polymorphonuclear (PMN) cells has been investigated. Both the extract and short time cultured medium of rat peritoneal PMN cells stimulate prostaglandin (PG)E2 production as well as collagenase production in the culture of rat synovial cells. PGE2 production by the cells in the presence of the PMN factor is much faster (5 to 24 hr) than collagenase production (24 hr or later, Biomedical Res. 3, 506-516, 1982). This stimulating factor is confirmed to be derived from PMN cells, based on the purification of the cells from peritoneal exudate cells by the Ficoll-Urographin method. Elution profile of the factor on gel filtration has indicated that both PGE2 and collagenase productions by synovial cells are stimulated by the same effluent fractions corresponding to molecular weights of 15,000 - 20,000 daltons and 30,000 - 40,000 daltons. These results suggest that PMN cells are involved in PG production as well as collagenase production in the inflamed tissue by stimulating connective tissue cells such as synovial cells.     

Rous sarcoma virus-transformed avian cells express four different cell surface antigens that are distinguishable by a cell-mediated cytotoxicity-blocking test.

Japanese quails bearing avian sarcoma virus-induced tumors develop immune spleen cells that are cytotoxic in vitro against virally and chemically transformed cells, as well as against embryonic cells. The cell-mediated cytotoxicity can be blocked by soluble antigens extracted from in vitro cultured cells. The existence of partial as well as total blocking effects in tests with extracts from various transformed and untransformed virus-producing cells makes it possible to distinguish up to four different kinds of antigens expressed on sarcoma virus transformed cells: a) a subgroup-specific determinant of the virus-envelope glycoprotein gp85 (s-gp85) is expressed at the surface of productively infected, tranformed as well as untransformed cells; b) a group-specific determinant of gp85 (g-gp85) that is only expressed on the surface of virus-transformed cells; c) embryonic antigens, also detectable on chemically transformed as well as on primary normal embryonic cells, and finally; d) a sarcoma virus transformation-specific antigen (TSSA) that is not a structural constituent of the virus.     

Regulatory responses of hepatocytes,macrophages and vascular endothelial cells to magnesium deficiency

The liver is the organ that responds to nutritional disturbances including magnesium deficiency. The present study evaluated cellular responses to magnesium deficiency using model cells of the liver, namely, HepG2 cells as hepatocytes, RAW264.7 cells as Kupffer cells and human umbilical vein endothelial cells (HUVECs) as vascular endothelial cells; we examined effects of culture with magnesium deficient medium on cell responses in individual types of cells as well as interactive responses among cells. Metabolomic analyses indicated that magnesium deficiency differentially affected the cellular content of metabolites among HepG2 cells, RAW264.7 cells and HUVECs. The cellular content of the metabolites in HepG2 cells and HUVECs was also affected by the conditioned medium from RAW264.7 cells cultured with the magnesium-deficient media. The changes in HUVECs partly resembled those of the livers of magnesium-deficient rats previously described. RNA-seq analyses indicated that magnesium deficiency modulated the expression levels of molecules related to the ubiquitin-proteasome pathway and oxidative stress/antioxidant response in HepG2 cells and RAW264.7 cells, respectively. Furthermore, when HUVECs were co-cultured with RAW264.7 cells, lipopolysaccharide-induced expression of interleukin (IL)-1β and IL-6 was enhanced by magnesium deficiency, depending on the presence of RAW264.7 cells. The present study reveals that magnesium deficiency affects cellular metabolism in HepG2 liver cells, RAW264.7 macrophages and HUVECs, and that the modulation of cellular responses to extracellular magnesium deficiency in HUVECs depends on the presence of RAW264.7 cells. The complex responses in individual cells and through cell interactions partly explain the regulatory reaction to magnesium deficiency in the liver.