A role of HLA-DQ molecules of stimulator-adherent cells in the regulation of human autologous mixed lymphocyte reaction

In this study, we have found that treatment of stimulator autologous adherent cells with anti-HLA-DQ mAb resulted in markedly enhanced proliferative response of T cells in human autologous mixed lymphocyte reaction system wherein T cells were cultured with autologous adherent cells at near ratio of adherent cells to T cells in peripheral blood, in which T cells minimally proliferate. However, treatment of stimulator-adherent cells with anti-HLA class I, anti-DR and anti-DP mAb had no effect on the proliferative response of T cells under the condition. It was further observed that CD4-enriched cells could significantly proliferate in the presence of autologous adherent cells either untreated or treated with anti-DQ mAb, although treatment of adherent cells with anti-DR mAb blocked proliferative response of CD4-enriched cells. Moreover, the proliferative response of CD4-enriched cells was suppressed by addition of CD8-enriched cells in the presence of untreated adherent cells, whereas the proliferative response of CD4-enriched cells could not be suppressed by CD8-enriched cells when the adherent cells in the culture were treated with anti-DQ mAb. These observations suggest that CD8 T cells are required for suppression of proliferative response of CD4 T cells to HLA-DR molecules on autologous stimulator-adherent cells, and that HLA-DQ molecules on the surface of adherent cells play an important role in the induction of suppression by CD8 T cells.     

Induction of mammotroph development by a combination of epidermal growth factor,insulin, and estradiol-17beta in rat pituitary tumor GH3 cells

Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E (2)) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E(2) on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E(2), while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.     

四种生态型芦苇叶中离子分布对生境的生理适应

采用X射线微区分析技术 ,测定了 4种生态型芦苇 (Phragmitesaustralis (CaV .)Trin .exSteud .)叶的表皮泡状细胞、叶肉细胞和叶脉维管束鞘细胞离子的含量。结果表明 :沼泽芦苇的鞘细胞内 ,K 、Na 、Ca2 、Mg2 和Cl-分布均较叶肉细胞和泡状细胞高。沙丘芦苇的泡状细胞中Ca2 分布较叶肉细胞和鞘细胞高 ,而Mg2 在其叶肉细胞 ,以及K 、Na 和Cl- 在其鞘细胞内分布均较高。在轻度盐化草甸芦苇的叶肉细胞内分布较多的Na 和Mg2 ,而在鞘细胞内K 、Ca2 和Cl- 的分布均较叶肉细胞和泡状细胞为高。重度盐化草甸芦苇的泡状细胞内Na 和Mg2 的分布较多 ;同样 ,在叶肉细胞中K 、Ca2 和Cl- 的分布也较多。最后 ,讨论了上述各种离子在不同生态型芦苇叶内分布的状况 ,以及与其环境适应的生理意义。     

Localization of cathepsin D in rat liver. Immunocytochemical study using post-embedding immunoenzyme and protein A-gold techniques

Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Transcriptome atlas of glutamine family amino acid metabolism‐related genes in eight regenerating liver cell types

To explore glutamine family amino acid metabolism of eight liver cell types in rat liver regeneration, eight kinds of rat regenerating liver cells were isolated by using the combination of Percoll density gradient centrifugation and immunomagnetic bead methods, then Rat Genome 230 2.0 Array was used to detect the expression profiles of the genes associated with metabolism of glutamine family amino acid in rat liver regeneration and finally how these genes involved in activities of eight regenerating liver cell types were analysed by the methods of bioinformatics and systems biology. The results showed that in the priming stage of liver regeneration, hepatic stellate cells and sinusoidal endothelial cells transformed proline and glutamine into glutamate; hepatocytes, hepatic stellate cells, sinusoidal endothelial cells and dendritic cells catabolized glutamate to 2‐oxoglutarate or succinate; hepatic stellate cells and sinusoidal endothelial cells catalysed glutamate into glutamyl‐tRNA for protein synthesis; urea cycle, which degraded from arginine, was enhanced in biliary epithelia cells, sinusoidal endothelial cells and dendritic cells; synthesis of polyamines from arginine was enhanced in biliary epithelia cells, sinusoidal endothelial cells, Kupffer cells and dendritic cells; the content of NO was increased in sinusoidal endothelial cells and dendritic cells; degradation of proline was enhanced in hepatocytes and biliary epithelia cells. In the progress stage, biliary epithelia cells converted glutamine into GMP and glucosamine 6‐phosphate; oval cells converted glutamine into glucosamine 6‐phosphate; hepatic stellate cells converted glutamine into NAD; the content of NO, which degraded from arginine, was increased in biliary epithelia cells, oval cells, pit cells and dendritic cells. In the termination stage, oval cells converted proline into glutamate; glutamate degradation, which degraded from arginine, was enhanced in hepatocytes and dendritic cells; the content of NO was increased in oval cells, sinusoidal endothelial cells, pit cells and dendritic cells. The synthesis of creatine phosphate was enhanced in hepatocytes, biliary epithelia cells, pit cells and dendritic cells in both progress and termination stages. In summary, glutamine family amino acid metabolism has some differences in liver regeneration in different liver cells.     

Ultrastructure of distal nephron cells in rat renal cortex

Distal nephron segments in the rat renal cortex contain distal convoluted tubule cells (DCT cells), connecting tubule cells (CNT cells), intercalated cells (I cells), and principal cells (P cells). The present study was carried out to expand present knowledge on the ultrastructure of these cells. The cells were sampled from superficial cortex and analyzed by electron microscopy. Several morphometric parameters were determined and statistical comparison between cell types was performed. Significant structural differences between the cell types were demonstrated. DCT cells showed the highest volume density of mitochondria whereas the amplification of basolateral membranes was higher in CNT cells than in I and P cells. The surface density of the membrane that bounds intermediate vesicles in the apical cytoplasm was twofold higher in I cells than in the other cell types. The morphological differentiation found in the present study adds to available evidence indicating a functional differentiation between the cell types and provides a reference for structure-function correlations in these cells.     

Basal cells in the mouse olfactory epithelium after axotomy: immunohistochemical and electron-microscopic studies

Summary The olfactory epithelium of mice after axotomy was investigated to clarify the stem cells of olfactory cells by double immunostaining using antikeratin (MA903) and anti-bromodeoxyuridine (BrdU) antibodies and by conventional electron microscopy. When a single dose of BrdU was given to mice 9 days after axotomy, immunostaining for BrdU was found in the globose basal cells which were negative for MA903, but not in the basal cells proper which were positive for MA903. The BrdU-immunoreactive cells increased 3-to 6-fold over the number of these cells in the controls, indicating active cell proliferation. At other postoperative days (4 and 14 days), fewer BrdU-immunoreactive cells were found. Furthermore, three pulses of BrdU resulted in numerous BrdU-immunolabelings in the globose basal cells and a few in the basal cells proper. There was no detectable difference in the number of labeled basal cells proper in operated and unoperated mice. In the electron micrographs 9 days after axotomy, the basal cells proper, flat-shaped in unoperated mice, appeared cylindrical or pyramidal in shape and the globose basal cells often lay between the basal cells proper. In unoperated controls, the globose basal cells were located above the flat-shaped basal cells proper. The results suggest that the stem cells of the olfactory cells are globose basal cells and not basal cells proper, and that the shape of basal cells proper changes in relation to the active proliferation of stem cells.     

胆囊收缩素和胰岛素在发育中小鼠胰腺内的表达

目的观察昆明小鼠发育过程中胰腺内胆囊收缩素免疫反应阳性（CCK—IR）细胞和胰岛素免疫反应阳性（Ins—IR）细胞的发生、分布和形态。方法用免疫组织化学和免疫荧光双标法观察小鼠胚胎11d至出生后45d胰腺内的CCK—IR细胞和Ins—IR细胞。结果小鼠胚胎第11d,早期分化的胰腺中即出现CCK—IR细胞和Ins—IR细胞。自胚胎期至生后发育成熟,小鼠胰腺中均可见到Ins—IR细胞。CCK—IR细胞的形态不规则,与Ins—IR细胞相邻分布。在胚胎期免疫反应较强,生后胰腺内依然可见到发弱荧光的CCK—IR细胞。胰岛内还可见CCK—Ins—IR细胞。CCK—IR细胞和Ins—IR细胞主要分布于胰岛内,外分泌部的腺泡和导管处也偶可见到。结论发育中小鼠胰腺内的CCK—IR细胞具有旁分泌细胞的形态特征;免疫荧光双标法可见CCK与胰岛素共存于同一细胞内的现象。     

Paracrine role of Sertoli cells

Data from several experimental approaches strongly suggest that Sertoli cells exert a paracrine control of the two main testicular functions, androgen secretion and spermatogenesis. Further evidence supporting this role of Sertoli cells was obtained by coculture of Sertoli cells with other testicular cells. Coculture of pig or rat Sertoli cells with pig Leydig cells produces an increase in the hCG receptor number and an increase in the steroidogenic activity of Leydig cells. Pretreatment with FSH further increases the values of these two parameters. These biochemical changes were associated with ultrastructural changes in Leydig cells. The effects of Sertoli cells on Leydig cells depend upon the ratio of the two cells and on the substrate in which the cells are cultured. Moreover, Leydig cells produce an increase in the FSH receptor number and in the FSH stimulation of plasminogen activator production by Sertoli cells. Coculture of rat or pig Sertoli cells with rat germ cells, induces an increase in the RNA and DNA biosynthetic activities of germ cells. Most of the stimulatory effects seemed to be mediated by diffusible factors, secreted by Sertoli cells, but full expression of the stimulatory action was observed when germ cells were in contact with other cells. In this coculture system, a fraction of rat germ cells containing mainly mature forms of spermatocytes inhibited rat Sertoli cell RNA and DNA synthesis, but had no effect on pig Sertoli cells. On the contrary, a fraction of rat germ cells richer in spermatogonias and preleptotene spermatocytes, stimulated rat Sertoli cell DNA synthesis but was without effect on pig Sertoli cells. These results clearly show that the stimulatory effects of Sertoli cells on Leydig and on germ cells which are not species specific are mediated mainly by diffusible factors, the secretion of which is regulates by FSH.     

Enhancement of mucus accumulation in a human gastric scirrhous carcinoma cell line (KATO-III) by fibroblasttumor cell interaction

Human fibroblasts (WI-38 cells) were found to enhance mucus accumulation by human scirrhous carcinoma cells (KATO-III cells). Coculture of KATO-III with WI-38 cells resulted in enlargement of the KATO-III cells and increases in the proportions of PAS- and colloidal iron-positive KATO-III cells. These morphological alterations were reversed when the KATO-III cells were again cultured without WI-38 cells. Conditioned media from cultures of WI-38 cells or cocultures of KATO-III and WI-38 cells induced the same morphological alterations in KATO-III cells, suggesting that WI-38 cells produce a factor or factors that enhance mucus accumulation in KATO-III cells. This factor seemed to be a protein with a molecular weight of more than 10,000 daltons.     

Ultrastructural aspects of cat submandibular glands

Submandibular glands of five adult female cats were examined by conventional electron microscopic techniques. All gland acini are mucous secreting and each acinus is capped with mucous secreting demilunar cells. Secretory product of demilunar cells is more electron lucent than that of acinar cells. The demilunes show intercellular tissue spaces and intercellular canaliculi whereas similar specializations are absent between acinar cells. Mitochondria and arrays of granular endoplasmic reticulum are more numerous in demilunar cells than in acinar cells. In acinar and demilunar cells secretory droplets first appear as enlarged Golgi saccules which subsequently become closely related to cisternae of the granular endoplasmic reticulum. Filamentous structures, interpreted as mucin molecules, are present in secretory droplets of acinar cells. Intercalated ducts are short, consisting of several junctional cells between acini and striated ducts. Striated ducts are long and tortuous and contain light cells, dark cells and basal cells. Light cells contain numerous membrane bound granules in their distal ends whereas dark cells show electron lucent vesicles in the same position. Basal cells contain a paucity of organelles and membrane plications but exhibit hemidesmosomes along their basal plasma membranes. Myoepithelial cells are abundant in relation to acinar and demilunar cells. Nerve terminals are present in some instances between acinar cells or between acinar and myoepithelial cells.     

Fluctuation of the SP/non-SP phenotype in the C6 glioma cell line

Using the C6 glioma cell as a paradigm, we found that (i) the clonogenicity of C6 cells is several orders of magnitude higher than the percentage of SP cells; (ii) non-SP cells are able to generate SP cells, and conversely SP cells generate non-SP cells; (iii) non-SP sorted cells behave as tumorigenic cells. Hence, in C6 cells cultured in serum-containing medium, SP cells can be generated from non-SP cells. This dynamic equilibrium explains in C6 cells the maintenance of the SP phenotype with cell passaging and demonstrates the existence of tumorigenic non-SP cells.     

Ultrastructure of the primary gill lamellae of Scomber australasicus

The ultrastructure of the primary epithelium, and the efferent half of the subepithelium, of the primary gill lamellae of slimy mackerel ( Scomber australasicus ) is described. The following cells are identified and described: light nucleated epithelial cells (surface and basal), dark nucleated cells, mucous cells, acidophilic cells, type 1 cells, type 2 cells, type 3 cells and chloride cells in the epithelial region, and subepithelial cells A and B, fibroblasts, chondrocytes, cells of the wall of efferent blood vessel and some blood cells in the subepithelium.     

Suppression of the in vitro secondary response to syngeneic tumor and of in vivo tumor therapy with immune cells by culture-induced suppressor cells.

Mice with advanced disseminated syngeneic tumor can be successfully treated with a combination of chemotherapy and adoptively transferred syngeneic immune cells. We have previously demonstrated that in vivo primed cells secondarily sensitized in vitro became more effective in tumor therapy, whereas primed cells cultured for 5 days without tumor stimulation became less effective than an equal number of uncultured fresh primed cells. Therefore, we examined stimulated and unstimulated cultures of tumor-primed cells for the presence of culture-induced suppressor cells, and determined whether in vivo tumor therapy with immune cells could be inhibited by concurrent inoculation of immune effector cells and cultured normal spleen cells, which contain culture-induced suppressor cells but are devoid of additional effector cells. The in vitro primary allogeneic response was suppressed by cultured normal spleen cells, or tumor-primed spleen cells previously cultured for 5 days with or without tumor stimulation. In vitro secondary sensitization to syngeneic tumor was suppressed by normal or tumor-primed cells that had previously been cultured for 5 days without stimulation. The majority of this suppression was mediated by T cells in the cultured populations. The efficacy of fresh tumor-primed cells, as well as primed cells secondarily sensitized in vitro, in adoptive chemoimmunotherapy of advanced tumor was diminished by concurrent inoculation of cultured normal cells. The cells mediating suppression of in vivo therapy required previous in vitro culture for induction, and were radiation sensitive.     

三角帆蚌外套膜细胞的超微结构

用透射电镜观察了三角帆蚌(Hyriopsis cumingii Lea)外套膜细胞的超徽结构。外套膜内表皮细胞中,有具纤毛的柱状细胞,顶端具小分泌泡的杯状细胞,胞质中含大分泌泡以及线粒体聚集成群的柱状细胞四类。各类表皮细胞的细胞质电子密度和内含物均有差异。细胞核呈椭圆或棒形。线粒体和高尔基体多分布在细胞核上方细胞的游离端。各类细胞游离表面都密生微绒毛,其中内表皮的柱状细胞还着生有纤毛,但无分泌泡。其余几种表皮细胞在细胞顶部都具有大小不等,电子密度不同的分泌泡。在外表皮中只观察到一般的柱状表皮细胞和胞质中具有成群线粒体的细胞两类。另外,外表皮细胞中具有较多的溶酶体。从三角帆蚌外套膜表皮细胞超微结构来看,内、外表皮都具有分泌和吸收机能。内表皮的纤毛柱状细胞有运动功能,而外表皮细胞有较强的消化吸收功能。     

Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo

Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate into the retina in vivo.     

Extrusion of nuclei of murine suspension culture cells with microtubule poisons

Microtubule poisons such as colchicine, colcemid and vinblastine caused extrusion of nuclei of murine suspension culture cells (mastocytoma p-815 cells, myeloma PU-3 cells, leukemia M1 cells). Enucleation did not follow spontaneously in most cells, but it did occur when the treated cells were centrifuged in Separate-L gradient. These poisons did not induce nuclear extrusion in cells growing in monolayer (L cells, BALB/c 3T3 cells, SV40-transformed BALB/c 3T3 cells, histiocytoma HC-11 cells). Cytochalasin B (CB) that had been reported to cause nuclear extrusion in the cells cultured in monolayer [1] did not induce the extrusion in the suspension culture cells but inhibited the colchicine-induced nuclear extrusion.     

Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells

The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components.     

Possible role of neuraminidase in activated T cells in the recognition of allogeneic Ia

In a primary MLR, predominant stimulators in spleen cells are adherent cells and not B cells, although B cells are one of the cell types expressing a large amount of Ia molecules. Our previous experiments showed that T cells treated with neuraminidase (Nase) responded to an allogeneic Ia on B cells. In our experiments, the relationship between the responsiveness to the allogeneic Ia molecules on B cells and Nase activity of T cells was examined. The results showed that T cells increased in Nase activity with the acquisition of the reactivity to Ia on B cells. T cells from normal mice increased in Nase activity after the incubation for 3 days or more in MLR, and these T cells responded to allogeneic Ia on B cells. However, T cells from mice genetically deficient in Nase responded poorly to the Ia on allogeneic B cells even after the incubation in MLR for 3 days. T cells incubated for 3 days in MLR decreased in electrophoretic mobility, indicating the decrease of net negative charge of the cells, and increased in their binding of peanut agglutinin which has been reported to bind to galactosyl residues exposed on T cell surface by removing sialic acids. These results suggest that Nase in T cells was activated by the cultivation in MLR for 3 days, and sialic acids of some molecules on T cell surface were removed by the enzyme and, in turn, T cells acquired the responsiveness to allogeneic B cells in a secondary MLR. Thus, Nase was suggested to play a regulatory role in the recognition of Ia molecules in T cells.