Cytostatic effect of immune system cells on tumor cells

The cytostatic effect of different lymphoid cells on tumour cells was studied in mice. It was shown that the cells differed in their ability to manifest cytostatic and natural cytotoxic activity. The interstrain variations of cytostatic activity levels in murine splenocytes were revealed. The degree of cytostatic action of effector cells on tumour cells depended both on effector-target cells ratio and on the incubation time of effector and target cells. The cytostatic effect of splenocytes and macrophages was demonstrated to be unrestricted by H-2 complex and independent of the tumour type. The data suggest that the cytostatic effector cells are heterogeneous and may be distinct from natural killer cells.     

Substrate-dependent differences in growth and biological properties of fibroblasts and epithelial cells grown in microcarrier culture

Normal diploid human fibroblasts and first passage monkey kidney epithelial cells were examined for growth and metabolic activity on microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells cm2 of surface area) on the glass microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells/cm2 of surface area) on the glass microcarriers than they did on the DEAE-dextran microcarriers and morphological differences were observed between the cells growing on the two substrates. On the DEAE-dextran microcarriers, the cells were much more resistant to protease-mediated detachment than were the cells on the glass microcarriers. In these respects, the cells grown on the glass microcarriers were similar to cells grown in conventional monolayer culture. Interestingly, the cells grown on the DEAE-dextran microcarriers expressed higher levels of proteolytic enzyme activity than the cells grown on the glass microcarriers. Substrate-dependent differences in prostaglandin production also occurred--both in unstimulated cells and in cells stimulated with 12-0-tetradecanoyl phorbol acetate. The unstimulated cells on the glass microcarriers produced slightly higher levels of three different prostaglandins than did the cells on the DEAE-dextran microcarriers. However, after stimulation the levels were much higher in the DEAE-dextran microcarrier cultures than in the glass microcarrier cultures. In contrast to these results, there was no significant, substrate-dependent difference in the production of infectious herpes simplex virus. Taken together, these findings suggest that when commercially-useful cells such as normal fibroblasts and epithelial cells are grown in large quantities on microcarriers, the nature of the substrate may have a profound effect on the growth and physiology of the cells. They also suggest that when microcarriers are used, unexpected results based on preliminary work in conventional monolayer culture may be obtained.     

Cadmium injury of cultured human vascular endothelial cells.

The effect of cadmium chloride (CdCl2) on cultured human vascular endothelial (HVE) cells and cultured human fibroblasts (HAIN-55 cells) was investigated. Umbilical vein-derived HVE cells were collected by enzymatic digestion with collagenase. At the concentration of 0-10 microM, Cd had hardly any effect on the cell viability of either cells. The viability of HVE cells decreased markedly at 100 microM, but not that of HAIN-55 cells. Morphologic examination by phase contrast microscopy revealed a more damaging effect of Cd on HVE cells than on HAIN-55 cells. These results suggest that Cd is more cytotoxic to HVE cells than HAIN-55 cells.     

Immunohistochemical localization of the transferrin receptor in the seminiferous epithelium of the rat

The transferrin receptor has been immunohistochemically localized in the seminiferous epithelium of the rat with a monoclonal antibody, MRC OX26, which recognizes the transferrin receptor glycoprotein. The receptor was detectable on mitotically and meiotically dividing germ cells and, less abundantly, on round spermatids. It was lost from germ cells during spermatid elongation and was undetectable on immature spermatozoa. The transferrin receptor was also present on Sertoli cells in the testes of immature animals and on Sertoli cells in the testes of aspermatogenic animals that had been irradiated in utero. It was not detectable on Sertoli cells in the testes of cryptorchid animals. These studies demonstrate that the transferrin receptor is abundant on dividing germ cells as well as dividing somatic cells.     

Binding of diphtheria toxin to CHO-K1 and Vero cells is dependent on cell density

We studied the binding of 125I-labeled diphtheria toxin (DTX) to receptors on monolayer cultures of Chinese hamster ovary cells (CHO-K1) and Vero cells. The number of DTX receptors detected on the cell surface was shown to be dependent on the cell density (number of cells per unit area). Cells at low density (less than 23,000 cells per cm2 for CHO-K1 cells; less than 80,000 cells per cm2 for Vero cells) had more receptors for DTX than cells at higher densities. The difference in receptor number between low- and high-density cells was 33-fold for CHO-K1 cells and 19-fold for Vero cells. We estimated the maximum number of DTX receptors on low-density CHO-K1 and Vero cells to be 50,000 and 370,000 per cell, respectively. The cell density at which the binding of DTX was reduced to 50% of maximum was considerably lower for CHO-K1 cells than for Vero cells (33,000 vs. 220,000 cells per cm2, respectively). Vero cells grown on a surface that had been conditioned by high-density cells bound less DTX, suggesting that interaction of these cells with the underlying extracellular matrix might regulate the number of cell surface receptors for DTX. Low-density cells were more sensitive to DTX than high-density cells, suggesting that low-density cells possessed an increased number of functional receptors that actively transported DTX to the cytosol. CHO-K1 and Vero cells were equally protected by SITS (4-Acetamido-4'-Isothiocyano-Stilbene-2,2'-disulfonic Acid), a compound that has been shown to inhibit the binding and entry of DTX in Vero cells, suggesting that intoxication of CHO-K1 and Vero cells is mediated by a similar mechanism. The data illustrate the importance of taking into account the cell density when measuring the number of DTX receptors on adherent cells.     

Cell growth on microcarriers: comparison of proliferation on and recovery from various substrates

Three commercially-important types of cell were grown on four different microcarrier substrates. The cells, which included normal human diploid fibroblasts (MRC-5), primary chick embryo cells and Madin-Darby bovine kidney cells (MDBK), were compared with regard to proliferation on the substrates and with regard to recovery of viable cells from the same substrates. The substrates used included glass-coated microcarriers (Biosil), collagen microcarriers (Ventregel), DEAE-dextran microcarriers (Cytodex I) and collagen-linked DEAE-dextran microcarriers (Cytodex III). The established cell line (MDBK) grew well on all of the substrates and a high percentage of viable cells could be harvested from each substrate. The MRC-5 cells also grew well on all four substrates but high recovery rates were achieved only with cells grown on the glass-coated microcarriers or collagen microcarriers. In contrast, the primary chick embryo cells grew well only on the glass microcarriers and the recovery rate of cells harvested from this substrate was high. In some industrial operations, the re-utilization of cells after removal from the substrate is necessary. In these situations the appropriate choice of microcarriers for the cultivation of the cells may be critical.     

Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells

To elucidate the biological activities of coral-prostanoids, clavulones, discovered from the Japanese stolonifer Clavularia viridis, we examined the effect of clavulone on the cell growth of human cancer (human promyelocytic leukemia (HL-60) cells and HeLa cells) and normal (Chang liver cells and lung fibroblasts) cells in vitro. Clavulone showed strong antiproliferative and cytotoxic activities in the human cells and it had some selectivity to leukemic (HL-60) cells over other HeLa cells or normal cells on the basis of the IC50 values and cytotoxic effect of the cells. The IC50 value of clavulone in the HL-60 cells was about 0.4 microM (0.2 micrograms/ml). Over 1.0 microM (0.5 micrograms/ml), clavulone showed a significant cytotoxic activity on the HL-60 cells. The data on DNA synthesis and flow cytometric analysis revealed that clavulone arrests the cells in the G1-phase and inhibits the cell growth of HL-60 cells by inhibiting S-phase DNA synthesis. These results suggest that clavulone has a potent antileukemic effect on HL-60 cells.     

The exocrine glands of the male of Eldana saccharina walker (Lepidoptera,galleriinae): Intervention in precopulatory behavior

The scent apparatus of male Eldana saccharina is a glandular complex on the costal area of the forewing. It consists of two parts; glandular complex 1 is composed of five kinds of cells (epidermal cells, scale cells, glandular cells, supporting cells, duct cells); glandular complex 2 also shows five types of cells (epidermal cells, scale cells, glandular cells, duct cells, trichogen cells). The secretory products of the two parts are discharged into separate ducts which converge before opening onto the lower side of the wing. The male also has two prominent hair-pencils borne on the coremata and large secretory trichogen cells on the genital valves. Each of these exocrine gland components plays an important part in formation of the chemically complex pheromones utilized in the precopulatory behavior of the male.     

Interferon gamma modulates the ability of autologous non-T cells to stimulate T cells to produce and respond to interleukin 2

The interactions of T-cell receptor with self-Ia antigen on non-T cells induced IL-2 production and IL-2 receptors on the cell surface and thus responsiveness to IL-2 of T cells in autologous mixed-lymphocyte reaction (AMLR). Four-day-cultured autologous non-T cells lost their ability to stimulate T cells to produce and respond to IL-2 with concurrent decrease of HLA-DR and HLA-DQ antigen expressed on the cell surface. Culturing of non-T cells with 500 U/ml of recombinant interferon gamma (IFN-gamma) maintained their stimulating ability which was otherwise lost. Treatment of non-T cells with monoclonal anti-HLA-DR or anti-HLA-DQ antibody before mixture with T cells abrogated their ability to induce IL-2 production and IL-2 responsiveness of T cells. The combined data suggested that Ia antigen expressed on non-T cells is modulated by IFN-gamma, which increases the ability of non-T cells to stimulate autologous T cells to produce and respond to IL-2.     

差速贴壁分离法:获得小鼠骨髓内皮祖细胞诱导分化培养的最佳贴壁时间

本文旨在比较差速贴壁方法分离的不同时间点贴壁的小鼠骨髓单个核细胞诱导分化为内皮祖细胞的生物学特性,探讨最适宜贴壁时间.Ficoll密度梯度离心分离小鼠骨髓单个核细胞,接种于预先铺有纤维连接蛋白的培养板上,定义为1d贴壁细胞组,取1d非贴壁细胞再接种为3d贴壁细胞组,继续培养2d取非贴壁细胞再接种为3d非贴壁细胞组,继续...     

Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.     

肿瘤干细胞标记分子CD133在胶质瘤细胞U87中的稳定表达

目的：在体外胶质瘤U87细胞中稳定表达肿瘤干细胞标记分子CD133。方法：通过脂质体介导将表达载体质粒CD133-1／pCR3．1-Uni转染U87细胞,G418筛选稳定表达抗性的细胞株;用细胞免疫荧光染色鉴定表达CD133分子的U87细胞。结果：转染CD133表达载体的U87细胞可以被CD133单抗识别,而转染空载体的U87细胞免疫染色结果为阴性,表明CD133分子在U87细胞中稳定表达。结论：U87细胞稳定表达CD133分子,为体内外分析CD133阳性U87细胞特性奠定了基础：U87CD133阳性细胞可以作为免疫组化或流式细胞术等检测其他肿瘤干细胞CD133表达的阳性对照细胞。     

Selective and efficient culturing of retinal pigment epithelial cells using a feeder layer

BACKGROUND: The techniques to isolate and purify retinal pigment epithelial (RPE) cells from small piece of autologous tissues are extremely difficult, and it is important to develop an efficient cell culture technique for RPE cells. The purpose of this study was to investigate the effect of 3T3-J2 cells and conditioned medium from 3T3-J2 cells on the proliferation of cultured RPE cells. METHODS: RPE cells from pigmented rabbits and a human RPE-derived cell line, ARPE-19, were used. First, the effects of co-culturing RPE cells with 3T3-J2 cells on the growth of the cells were analyzed. Second, the effects of the conditioned medium from 3T3-J2 cells on the proliferation of both types of cells were investigated. And third, the effects of the conditioned medium on RPE cell culture from a surgically removed choroidal neovascular (CNV) membrane were investigated. RESULTS: The 3T3-J2 cells increased the proliferation of both rabbit RPE cells and ARPE-19 cells. The number of rabbit RPE cells cultured in a mixture of the conditioned medium from 3T3-J2 cells was significantly higher than that in the reported optimal condition, and a similar tendency was observed for ARPE-19 cells. The results from enzyme-linked immunosorbent assay showed the presence of PDGF-AB, VEGF and IGF-I in the conditioned medium. The conditioned medium also promoted selective growth of human RPE cells from CNV. DISCUSSION: The results from this study present the conditions for efficient and selective culture of primary RPE cells.     

Isolation of a variant of human adenovirus serotype 2 that multiplies efficiently on monkey cells.

A variant of adenovirus serotype 2 (Ad2) that overcomes the block to multiplication of the wild-type Ad2 on monkey cells is described. The variant was selected, after nitrous acid mutagenesis, by sequential passage on monkey cells. This variant forms plaques with similar efficiency on human and monkey cells cells. The kinetics of its growth and its burst size on human and monkey cells are similar. These growth properties are similar to those found for wild-type Ad2 on monkey cells when the block is overcome by coinfection with simian virus 40.     

Nanomechanical analysis of pigmented human melanoma cells

Based on hitherto measurements of elasticity of various cells in vitro and ex vivo, cancer cells are generally believed to be much softer than their normal counterparts. In spite of significant research efforts on the elasticity of cancer cells, only few studies were undertaken with melanoma cells. However, there are no reports concerning pigmented melanoma cells. Here, we report for the first time on the elasticity of pigmented human melanoma cells. The obtained data show that melanin significantly increases the stiffness of pigmented melanoma cells and that the effect depends on the amount of melanin inside the cells. The dramatic impact of melanin on the nanomechanical properties of cells puts into question widely accepted paradigm about all cancer cells being softer than their normal counterparts. Our findings reveal significant limitations of the nanodiagnosis approach for melanoma and contribute to better understanding of cell elasticity.     

两种传代细胞系对轮状病毒D36株(P[4]G2)敏感性的比较

目的探讨轮状病毒D36株在MDCK细胞和Vero细胞上培养的适应性,确定其培养的最佳细胞基质及培养条件。方法将D36株以MOI 1.0按不同培养瓶分组接种MDCK细胞和Vero细胞,补充含有不同浓度胰酶的维持液,于不同时间观察两种细胞病变的情况,同时抽样检测病毒滴度,分析两种细胞对D36株的敏感性。结果D36株病毒感染MDCK细胞后第6天病毒滴度达到最高,为(5.00～5.50)lgCCID50/mL;而D36株病毒感染Vero细胞后病毒滴度于第8天达高峰,为(4.50～4.75)lgCCID50/mL。另外,在两种细胞维持液中加入约0.8μg/mL的胰酶均可提高病毒滴度。结论两种细胞系在同等条件下感染D36株病毒后,MDCK细胞比Vero细胞出现病变的时间早,每一批MDCK细胞培养物病毒滴度高于同批次试验的Vero细胞培养物。     

Effects of environmental temperature on thymus and spleen weights and lymphocytes in mice

Thymus and spleen weights and lymphocytes in the blood were examined in mice transferred from 22 degrees C to 12 degrees C or 32 degrees C environments. After the exposure to either environment, organ weights tended to decrease. In mice exposed to 12 degrees C, the number of WBCs and mononuclear cells and the ratio and the number of T cells decreased on day 1 and/or 3 after the exposure. The number of B cells also declined on day 3, but the ratio of B cells increased on days 3 and 7. In mice exposed to 32 degrees C, the number of WBCs and mononuclear cells and the number of T cells decreased on day 1 and increased on day 7 and/or 14. The ratio of T cells declined on days 1, 3 and 7, while that of B cells increased on day 3, and the number of B cells increased on day 7. These results show that wide variations in environmental temperature affect the weights of organs and the number of cells which act on immune responses in mice.     

CD34+ cells homing: quantitative expression of adhesion molecules and adhesion of CD34+ cells to endothelial cells exposed to shear stress

To investigate the function of the main adhesion receptors (CD62L, CD49d, CD49e, CD11b and CD18) on CD34+ cells during homing, their expression was quantified by flow cytometry using calibration beads. CD34+ cells were isolated from bone-marrow (BM), cord blood (CB) or peripheral blood (PB) from patients with myeloma. As this process might mimic the mature leukocyte migration, we also observed the effect of exposing endothelial cells to shear stress (7 dyn/cm(2)) on the adhesion of CB CD34+ cells.The proportion of CD34+/CD62L+ cells was greater in PB than in BM (p<0.05). Likewise, we found a significantly greater expression of CD62L receptor on PB cells compared to BM cells (p<0.05) and on BM cells compared to CB cells (p<0.05). The proportions of CD34+/CD49d+ cells and CD34+/CD49e+ cells were significantly higher in the BM and CB than in PB. However, no significant difference in CD49d or CD49e antigen densities was observed. The beta_2 integrins (CD11b and CD18) receptors are also implicated in CD34+ cells homing to BM. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. However quantitative analysis revealed that CD18 was more strongly expressed on BM cells than on PB and CB cells.The adhesion assay showed that fluid flow may favour a firm adhesion of CB CD34+ cells to endothelial cells whereas static conditions just allowed CD34+ cells sedimentation.In conclusion, quantitative expression of the main receptors on CD34+ cells indicates that the three main sources of CD34+ cells currently used for transplantation have neither the same phenotype nor the same number of antigenic sites for a receptor. So, we hypothesize that migrational capacity of these cells might be different. Moreover, it seems that shear stress could favor adhesion of CD34+ cells to endothelial cells.     

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.     

In vitro immune response of human peripheral lymphocytes. I. The mechanism(s) involved in T cell helper functions in the pokeweed mitogen-induced differentiation and proliferation of B cells.

Human peripheral lymphocytes (PBL) upon stimulation with PWM proliferate and differentiate to IgM- and IgG-producing cells. The PWM-induced Ig production in B cells was dependent on T cells, and cell-free supernatant (CFS) obtained from PWM-stimulated PBL or T cell-rich fraction replaced T cell helper functions. The active substance(s) in CFS were most likely derived from T cells. The kinetic studies showed that the proliferation of B cells took place in advance of the final differentiation to Ig-producing cells and that T cells or T cell product(s) had to exist at the initiation of cultures in order to give the maximum helper effect. However, the final differentiation of B cells to Ig-producing cells was not dependent on T cells. The helper effect of T cells or T cell product(s) on PWM-induced proliferation and differentiation of B cells was exerted across the MHC barrier. This may make it possible to apply this experimental system to the assessment of quantitative and/or qualitative changes in human helper T cells in several immunologic diseases.