Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody

Chondroitin-sulfate containing proteoglycan (CSPG) of the extracellular matrix (ECM) was visualized in chick tissues and cell cultures with a monoclonal antibody, CS-56. Cultured cells of various origins contained dense punctate layers of CSPG on both the substrate and the cell surface, as determined by immunofluorescent and immunogold staining. Under culture conditions the CSPG-containing matrix was usually excluded from stable cell-to-substrate focal contacts. The substrate-attached CSPG exhibited remarkable chemical stability but could be successfully removed by pronase or chondroitinases ABC and AC. Incubation of living cells with CS-56 antibodies resulted in the clustering of surface CSPG into patches, indicating that the surface-bound CSPG is free to move laterally along the plasma membrane. The unique properties of the CSPG-containing ECM revealed by CS-56 antibodies and their relationships to specific types of cell contacts are discussed.     

The influence of paraquat on cell cycle--analysis of cell kinetics using DNA/BrdU staining]

We examined the in vitro and in vivo effects of paraquat on the cell cycle. After we exposed paraquat to the cultured cell (MDCK), we examined cell kinetics by flow cytometry with BrdU-PI double staining. And we administered paraquat intravenously to the rats, we collected and separated rat's liver and pulmonary cells, and we examined the cell kinetics by same method. Consequently our study found that the cells in the S-phase were noted to be abundant, and we suggested that paraquat apparently arrested the cell cycle at S time, and that DNA damage was importance in the mechanism of paraquat toxicity.     

Proliferation kinetics of NK/Ly tumor cells after single and fractionated irradiation with fast electrons]

With single irradiation, the inhibition of tumor cell division and DNA synthesis was more pronounced than with fractionation irradiation. In fractionated schedules, the dose increase per fraction, and the interval prolongation between fractions, with decrease of the number of fractions (within the same time of irradiation), enhanced the effect of radiation. Yield of pahologic mitoses and extent of morphologic injury of cells was less expressed with fractionated irradiation and did not depend on the schedule of fractionation of the total dose.     

Analysis of cell survival after multiple fractions per day and low-dose-rate irradiation of two in vitro cultured rat tumor cell lines

Two rat tumor cell lines which differ significantly in radiosensitivity, a rhabdomyosarcoma (R-1) and a ureter carcinoma (RUC-2), were treated with multiple fractions per day and low-dose-rate gamma radiation. The purpose of these experiments was to investigate (i) the influence of fraction size and interfraction interval on repair of sublethal damage (SD) and (ii) whether low-dose-rate irradiation can be simulated by giving multiple fractions per day which might be applied in clinical treatments. In both cell lines, multiple doses were given at 1- to 4-hr intervals. SD repair was at a maximum in 2 hr but did not reach the theoretically expected level. For both cell lines, survival at higher total doses was different from that theoretically expected if repair of SD was assumed to be completed and at the maximum level. To account for the observation that less than complete repair of SD occurred, theoretical survival curves were calculated with the assumption of a constant but less than 100% level of SD repair. Experimental data correlated well with these calculated curves. There were only very small differences in survival after the different multiple fractions per day regimens. Survival after irradiation at a dose rate of 1.00 Gy/hr was found to be similar to that after multiple fractions per day.     

A monoclonal antibody that recognizes Golgi-associated protein of cultured fibroblast cells

We have obtained a hybridoma clone, JLJ5a, which secretes monospecific antibody directed against a 110-kdalton protein of gerbil fibroma cells, Rat-1 fibroblasts, and L6 myoblasts. It appears to be localized in the Golgi apparatus by the following criteria: (a) In double- staining experiments the localization of the 110-kdalton protein by the JLJ5a monoclonal antibody was coincident with the reaction products of thiamine pyrophosphatase (one of the enzyme markers of the Golgi apparatus; Novikoff and Goldfischer, 1961, Proc. Natl. Acad. Sci. U.S.A. 47:802-810) in the same cells. (b) The staining pattern of the JLJ5a monoclonal antibody became fragmented and dispersed into vacuoles after pretreatment of the cells with Colcemid or monensin.     

Localization of betav tubulin in the cochlea and cultured cells with a novel monoclonal antibody

Tubulin, the dimeric structural protein of microtubules, is a heterodimer of alpha and beta subunits; both alpha and beta exist as numerous isotypes encoded by different genes. In vertebrates the sequence differences among the beta(I), beta(II), beta(III), beta(IV) and beta(V) isotypes are highly conserved in evolution, implying that the isotypes may have functional significance. Isotype-specific monoclonal antibodies have been useful in determining the cellular and sub-cellular distributions and possible functions of the beta(I), beta(II), beta(III), and beta(IV) isotypes; however, little is known about the beta(V) isotype. We here report the creation and purification of a monoclonal antibody (SHM.12G11) specific for beta(V). The antibody was designed to be specific for the C-terminal sequence EEEINE, which is unique to rodent and chicken beta(V). The antibody was found to bind specifically to the C-terminal peptide EEEINE, and does not cross-react with the carboxy-termini of either alpha-tubulin or the other beta-tubulin isotypes. However, the antibody also binds to the peptide EEEVNE, but not to the peptide EEEIDG, corresponding respectively to the C-terminal peptides of bovine and human beta(V). Immunofluorescence analysis indicates that beta(V) is found in microtubules of both the interphase network and the mitotic spindle. In gerbils, beta(V) also occurs in the cochlea where it is found largely in the specialized cells that are unique in containing bundled microtubules with 15 protofilaments.     

Inhibition of human tumor growth by IgG2A monoclonal antibodies correlates with antibody density on tumor cells

Eight monoclonal antibodies (MAb) of IgG2a isotype that were produced against human melanomas were tested for tumor growth-inhibiting properties in nude mice injected with human melanoma cells of various origins. Four of the eight MAb inhibited growth of these tumors, and all four of these antibodies reacted in antibody-dependent macrophage-mediated cytotoxicity (ADMC) assays in vitro. The MAb that were inactive in vivo also did not react in these assays in vitro. The number of antibody-binding sites per cell on the tumor cell surface was significantly higher for tumoricidal MAb as compared to unreactive MAb. On the other hand, the percentage of tumor cells binding the MAb and the binding affinity to these cells were the same for the two groups of MAb. Also, tumoricidal and nontumoricidal MAb bound with similar affinity and antibody density to Fc receptors on macrophages. The importance of the number of antibody sites on the tumor cell surface for tumor destruction by MAb was confirmed by the demonstration of tumoricidal effects of mixtures of MAb that were by themselves not tumoricidal. MAb binding to different molecules on melanoma cells were complementary in ADMC, whereas MAb directed to the same molecule but to different epitopes were not.     

Plasminogen activating enzyme in cultured glioblastoma cells. An immunofluorescence study with monoclonal antibody

A monoclonal antibody against a 52,000 dalton human plasminogen activating enzyme (HPA52) was used for immunofluorescence staining of cultured glioblastoma cells. The fluorescence was located in the cytoplasm of the cells. A pronounced variation in the staining intensity was observed between the individual cells. The specificity of the fluorescent stain was supported by the findings that 1) no staining was obtained with a monoclonal antibody of the same subclass, but with irrelevant specificity (anti-2,4,6-trinitrophenyl); 2) adsorption with HPA52 purified to homogeneity removed the ability of anti-HPA52 to mediate staining; 3) the glioblastoma cells contained HPA52, as measured by enzymatic assay, while melanoma cells that were not stained did not contain HPA52 activity; 4) dexamethasone reduced both the enzymatically determined HPA52 content and the immunofluorescence in parallel, while progesterone affected none of these parameters; 5) we have previously found that culture fluid conditioned by the glioblastoma cells apart from HPA52 does not contain detectable amounts of any protein that binds to anti-HPA52. Several advantages of immunohistochemical detection of plasminogen activators compared with enzyme histochemical methods are discussed, among these that the immunohistochemical method distinguishes between plasminogen activators of different types.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Inhibition of attachment and growth of tumor cells on collagen by a monoclonal antibody

Summary A murine monoclonal antibody, VM-1, which binds to basal cells of normal human epidermis, reduces the ability of human squamous cell carcinoma cells (SCL-1) derived from the skin to attach and spread on collagen by about 50% and causes cell rounding. Similar effects have been previously shown using normal human keratinocytes. The attachment of cell lines derived from human lung squamous cell carcinomas (SW1271 and SW900), melanoma A375, glioblastoma 126, and fibrosarcoma HT1080 is also inhibited by this antibody. VM-1 antibody does not bind to normal human fibroblasts, benign nevus cells, or the human B-cell-derived line 8866. VM-1 antibody inhibits the growth of SCL-1 cells in vitro as measured by cell numbers and [³H]thymidine ([³H]TdR) incorporation. It is not cytolytic in the presence of complement as measured by⁵¹Cr release. Repeated treatment of SCL-1 cells with VM-1 antibody significantly reduces the proportion of SCL-1 cells that attach to collagen. In addition, after treatment of SCL-1 cells with VM-1 antibody, several proteins can no longer be demonstrated by gel electrophoresis of the cell-free supernatant. The VM-1 antibody effect on attachment and spreading is partially reversed by pretreatment of the collagen surface with laminin and fibronectin, but not with the carbohydrates chondroitin-6-sulfate or hyaluronic acid or with the protein lysozyme. By fluorescence staining, the antigen recognized by VM-1 antibody is membrane-bound and Triton X-100 extractable. The VM-1 antigen is excluded from Bio-Sil TSK-400 and sediments at about 10.5 S. It has a covalent molecular weight on the order of 10⁶. Proteinase K digestion produces VM-1 antibody reactive fragments, assumed to be polysaccharides, with a polydisperse molecular weight distribution in the range 5000 to 30 000. The VM-1 antigen is partially lost from solution on boiling and is no longer detectable in the aqueous or organic phase after chloroform-methanol extraction. The properties of the VM-1 antigen are consistent with those of a proteoglycan involved in attachment and spreading of kerationcytes and certain tumor cells on collagen. This research was supported by a grant from the Elsa U. Pardee Foundation, a Training Grant from the National Institutes of Health, Bethesda, MD, and the Psoriasis Research Institute. Part of this work has appeared as an abstract in Fed. Proc. 43:1929, Abst. #2994, 1984.     

BrdU related direct revelation of typical dynamic R- and G-banding with the use of monoclonal anti-BrdU antibody.

Pulse 5-bromodeoxyuridine (5-BrdU) incorporation during the last S-phase is known to produce R- or G-banded chromosomes after photolysis-plus-Giemsa (FPG) staining. The authors applied an immunological staining with monoclonal anti-BrdU antibody instead of the FPG protocol. The results offered banded chromosomes with an immunological typical R-banding (RBI) on the GBG cultivated cells (early pulse incorporation), and an immunological G-banding (GBI) on the RBG cultivated ones (late pulse incorporation). After a further FPG protocol following an immunological treatment, an inverted banding pattern became evident whereas a faint immunological staining remained. Thus the method superimposed a GBG-banding on the RBI-staining or a RBG on the GBI one. This allows a rapid and easy R and G double chromosomal identification on the same metaphase cell, using first the immunological banding then the classical FPG staining. The method allows a reproducible dynamic G-banding with an easy monitored late 5-BrdU pulse incorporation specially attractive in spontaneous dividing cells from bone marrow. This dynamic G-banding protocol should be extended to chorionic villi and malignant cells. Our data are in agreement with a connection between dynamic banding and chromosomal portions containing or not BrdU. The lack of an immunological staining after the FPG protocol has been noticed and assume the photolysis degradation-elution of the DNA in BrdU-substituted areas.     

Copper adsorption kinetics of cultured algal cells and freshwater phytoplankton with emphasis on cell surface characteristics

Comparative studies were carried out on the adsorption of copper by a range of laboratory-cultured algae and freshwater phytoplankton samples. The level of surface mucilage associated with the cultured algae ranged from high (Anabaena spiroides, Eudorina elegans) to moderate (Anabaena cylindrica, Microcystis aeruginosa) to complete absence (Chlorella vulgaris, Asterionella formosa, Aulacoseira varians, Ceratium hirundinella). With laboratory cultures, the rapid uptake, EDTA release and quantitative similarity between living and dead (glutaraldehyde-fixed) algae were consistent with physical binding of Cu at the cell surface. The higher Cu adsorption per unit surface area and longer adsorption time of mucilaginous algae in the time-course study, and the relatively high level of Cu bound to mucilage found by X-ray microanalysis suggest that mucilage played an important role in metal binding. For all species examined, Cu adsorption kinetics (external Cu concentrations 1 to 1000 mg L⁻¹) showed a clear fit to the Freundlich, but not the Langmuir isotherm, indicating a monolayer adsorption model with heterogenous binding sites. The Freundlich adsorption capacity constant (K_f) was higher in mucilaginous (3.96–12.62) compared to nonmucilaginous (0.36–3.63) species, but binding intensity (Freundlich constant 1/n) did not differ between the two cell types. The results suggest that mucilaginous algal species may have potential as biosorbents for treatment of industrial effluents containing heavy metals. Investigation of the Cu adsorption behavior of four mixed phytoplankton samples also revealed a good fit to the Freundlich, but not the Langmuir, isotherm. Freundlich constants were in the range 2.3–3.2 for samples dominated by Chlorophyta, Bacillariophyta and Cyanophyta, but recorded a value of 7.4 in the sample dominated by Dinophyta. Comparison with data from laboratory monocultures suggested that the adsorption kinetics of mixed environmental phytoplankton samples cannot be predicted simply in terms of the major algal species.     

T cell immunity to lymphoma following treatment with anti-CD40 monoclonal antibody

In this study we demonstrate that treatment with anti-CD40 mAb eradicates a range of mouse lymphomas (BCL(1), A31, A20, and EL4), but only when used against i.v. tumor doses in excess of 10(7) cells. Only partial protection was seen against smaller tumor loads. We saw no evidence that anti-CD40 mAb changed the phenotype of the lymphomas or inhibited their growth in the initial period following treatment, but it did result in a rapid expansion of cytotoxic CD8(+) cells that was able to clear the neoplastic disease and provide long-term protection against tumor rechallenge. The CTL responses were blocked by mAb against a range of coreceptors and cytokines, including CD8, B7-1, B7-2, LFA-1, and IFN-gamma, but not CD4 or CTLA-4, indicating the presence of a conventional cellular Th1 response. Furthermore, we found evidence of cross-recognition between lymphomas (BCL(1) and A20) as measured by cytotoxicity and IFN-gamma responses in vitro and using tumor rechallenge experiments, suggesting common target Ags. Finally, although anti-CD40 was shown to stimulate NK cell killing, we could find no role for these cells in controlling tumor growth. These data underline the ability of anti-CD40 mAb to potentiate CTL responses and the potency of cellular immunity in eradicating large quantities of syngeneic tumor.     

Studies on BrdU labeling of hematopoietic cells: stem cells and cell lines

Studies using chronic in vivo BrdU exposure, isolating primitive stem cells, and determining BrdU labeling, indicate that stem cells cycle. BrdU is also incorporated into DNA during damage/repair. DNA, which has incorporated BrdU due to cycle transit is heavier than normal, while the density of DNA with damage/repair incorporation is intermediate. DNA density of purified lineage-rhodamine low (rho(low)) Hoechst low (Ho(low)) stem cells or FDC-P1 cell line cells-was assessed in vitro, after exposure to cytokines and BrdU (cycling model) or cytokines and BrdU with bleomycin to induce strand breaks and hydroxyurea to halt cycle progression (damage/repair model). We determined DNA density using cesium chloride (CsCl) gradients and either fluorometry or dot blot chemiluminesence. DNA from BrdU labeled cycling Lin-rho(lo)Ho(lo) or FDC-P1 cells was heavier than normal DNA, while damage repair DNA had an intermediate density. We then assessed BrdU labeling of Lin-rho(lo)Ho(lo) cells in vivo. We found that 70.9% of lin-rho(lo)Ho(lo) cells labeled at 5 weeks. DNA density of these cells was low, in the damage/repair range, but similar results were obtained with stem cells, which had proliferated in vivo. Dilution of BrdU in in vitro culture of proliferating FDC-P1 cells also resulted in damage/repair density. We conclude that in vitro BrdU labeling models can distinguish between proliferation and damage/repair, but that we cannot obtain high enough in vivo levels to address this issue. All together, while we cannot absolutely exclude damage/repair as contributing to stem cell BrdU labeling, the data indicate that primitive bone marrow stem cells are probably a cycling population.     

Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody

Summary Six patients with progressive B cell non-Hodgkin's lymphoma have been treated with an IgG2a mouse monoclonal antibody (mAb) against the B cell differentiation antigen CD19, with total doses varying from 225 mg to 1000 mg. Free mAb was detected in the serum after doses of 15–30 mg. After the mAb infusions the number of circulating tumour cells was temporarily reduced, but in some cases antibody-coated cells remained in the circulation for several days. mAb penetrated to extravascular tumour sites; in general higher doses were required to saturate cells in the lymph nodes than to sensitize tumour cells in the bone marrow. mAb doses of up to 250 mg were given i.v. over 4 h without major toxicity. One patient twice achieved a partial remission after two periods of mAb treatment with an 8-month interval; the second remission lasted for 9 months. One patient showed a minor response. None of the patients made antibodies against the mouse immunoglobulin. Serum immunoglobulin levels were followed as a measure of the function of the normal B cell compartment; no significant changes were seen up to 6 months after mAb treatment.Supported by the Dutch Cancer Society (grant NKI 84-14)     

重组单克隆抗体药物的工程细胞培养工艺研究

单克隆抗体(monoclonal antibody, mAb)是一类重要的基因工程生物技术衍生药物,其药物适应证包括自身免疫性疾病、移植后并发症、心血管疾病、感染性疾病和各种类型的癌症。此类适应证药物通常使用时间长、使用剂量大,因此需具备大批量生产的能力。由于mAb药物的生产成本以及产品质量控制的复杂性,要求有效且经济地提供批间一致性较好的高质量药物。中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞是生物技术产业中用于生产治疗性糖蛋白最常用的真核表达宿主,也是生产治疗性抗体药物最常使用的细胞。在细胞培养过程中,工程细胞株、培养基和工艺条件是mAb药物生产中影响其质量的三个重要因素。现分别从培养基、培养参数和培养模式等对生产mAb药物的细胞培养技术作一概述。