Complement-Dependent Cytotoxicity Against Hepatoma Cells Mediated by IgM Antibodies in Serum from Tumor-Bearing Rats-

Complement-dependent cytotoxic antibodies in syngeneic serum from rats carrying transplanted aminoazo dye-induced hepatomas (D23 and D33) and from rats immunized with irradiated tumor cell or homogenates were studied by a short-term 51Cr release assay. The tumor-bearer sera (TBS) were subjected to chromatography on unsolubilized protein A and Sepharose 4B. The cytolytic activity of D23 TBS was recovered in the IgM molecular weight region, whereas no such activity was obtained in the IgG fraction. As judged from immunodiffusion experiments, the IgM molecular weight fraction did not contain any aggregated or complexed IgG. Moreover, in immunofluorescence tests against viable hepatoma cells, using a specific anti-rat IgG conjugate, the TBS were negative. Cross-testing of D23 and D33 TBS against the two hepatomas and cross-absorption of the sera with tumor plasma membranes revealed no tumor specificity in these cytotoxicity reactions. Furthermore, neither fetal nor early postnatal liver cells could absorb out the activity. Absorptions with adult liver plasma membranes, however, abrogated the cytotoxic activity, and even more effective in this respect were homogenates from kidney and small intestine, thus indicating that the cytotoxic antibodies are directed against 'normal' adult antigen(s) present also in tissues other than liver.     

抗人肝癌导向药物的制备及其分析

本文报道了肝癌单克隆抗体Ａ２４－表阿霉素结合物的制备及其对肝癌细胞ＳＭＭＣ－７７２１的体外杀伤作用。先用高碘酸钠把葡聚糖氧化成多醛基葡聚糖，再以此为中介体连接单抗与表阿霉素。结合物中抗体与药物摩尔比为１：４０。单抗活性保存良好。该结合物对肝癌细胞的毒性显著高于游离药物及无关抗体结合物，而对非靶细胞的毒性则明显低于游离药物。说明结合物对肝癌细胞有较强的选择性杀伤作用，有可能用于肝癌导向治疗。     

肝癌细胞"重叠生长"局部成因初探

探索肝癌细胞"重叠生长"形成过程及其成因.运用显微形态学观察、计算机图像处理技术和统计学分析,以及细胞力学和生化检测等研究手段对肝癌细胞原位生长信息进行量化表征.结果发现:(1)肝癌细胞变形调节能力较正常肝细胞强;(2)对"重叠生长"斑进行负压吸吮时,斑片从基底脱落,其下方未见铺展细胞;(3)肝癌细胞株HepG2的整合素表达明显高于正常肝细胞株L02;(4)纤维连接蛋白(Fibronectin, Fn)对肝癌细胞表达整合素β1有下调作用,Fn裱衬基底后,细胞黏附力和形态稳定性增高,圆细胞产生与聚集减少.由此得出(1)所谓"重叠生长层"实际上是由大量圆细胞聚集排列构成;(2)圆细胞产生与整合素β1表达异常及其对细胞黏附特性的影响相关;(3)肝癌细胞"重叠层"的形成与细胞频发形变诱导的圆细胞聚集相关.     

In Vitro Cytotoxicity of Human Lymphocytes: Reduction of the Lymphocyte Cytotoxicity Induced by Antibodies or Phytohemagglutinin by Removing Immune-Complex-Binding Cells

Immune-complex-binding cells have been excluded from suspensions of purified human lymphocytes by a rosette-formation procedure with antibody-sensitized ox erythrocytes. After such treatment the remaining lymphocytes were tested in a cytotoxicity assay, using ⁵¹Cr-labeled chicken erythrocytes as the target cells. Lymphocyte cytotoxicity for antibody-coated target cells was profoundly reduced after removal of immune-complex-binding cells (more than 10 times reduction within 4 to 6 hours of incubation). Lymphocyte cytotoxicity induced by phytohemagglutinin (PHA) was also reduced by the same procedure. This reduction was less marked, the cytotoxicity of lymphocytes lacking immune-complex-binding cells being 26% to 82% (mean, 47%) of control lymphocytes. The results indicate that a population of immune-complex-binding lymphocytes is needed for lymphocyte cytotoxicity induced by target cell antibodies and also for the full expression of PHA-induced cytotoxicity.     

Dependency of B Cells on the Presence of Adherent Cells, or Factors Derived from Them, for the Production of Autoantibodies in Vitro in the Absence of Cell Division

Peritoneal cells from untreated mice secrete autoantibodies after 3-4 days of in vitro culture, although the cells do not divide. Here, peritoneal cells enriched for B cells to contain 95% surface Ig-bearing cells, did not secrete autoantibodies after 3 days of in vitro culture unless plastic-adherent cells derived from the peritoneal cavity were cultured with the B cells. Cell-free media, taken from peritoneal adherent cells that had been cultured for 3 days in vitro, when added a final concentration of 50% in fresh culture medium to purified B cells, substituted for the presence of accessory cells. In contrast to cultures of unfractionated peritoneal cells, little increase in precursor frequency was detected when enriched B cells were cultured in the presence of LPS/DXS. However, the addition of adherent cells, supernatants derived from adherent cells, or cytokines produced by a T-cell hybrid EL4, resulted in an increased precursor frequency when LPS/DXS was added to the culture medium. Three macrophage cell lines, P388-D1, J774, and PU-5-IR, when added to purified B cells. augmented the autoantibody precursor frequency detected in vitro. This is strong evidence that potentially autoreactive B cells require one or more types of accessory cells in order to differentiate into autoantibody secretors during culture in vitro. Further, the results provide indirect evidence that interleukin 1 may be a crucial molecule in the differentiation of B cells.     

Natural Cytotoxicity of Human Lymphocytes against Equine Target Cells in Vitro

Human lymphocytes displayed a frequent natural cytotoxicity (NK) in vitro against normal equine dermal fibroblasts (ED) and against equine tumour cells of a virus-containing cell line (Mc-1). Similarly, human normal sera contained antibodies that induced antibody-dependent cellular cytotoxicity (ADCC) by normal human lymphocytes against the same target cells. Both NK and ADCC varied for different donors. For individual donors, however, cytotoxicity against the two target cells was significantly correlated both in NK and ADCC. For ED there was also a significant correlation between ADCC and NK activity. Both NK and ADCC showed some selectivity as assessed by cold target cell inhibition. Inhibition studies with Fab fragments of anti-human IgG established the involvement of immunoglobulins in the NK reaction. In this context, a marked and mainly immunoglobulin-dependent increase in both NK and ADCC activity against Mc-1 was observed in a laboratory worker frequently exposed to the target cells. The results indicate that variations of natural cytotoxicity in individual donors may sometimes be an indication of an ongoing spontaneous sensitization.     

体外培养树突状细胞成熟诱导物的研究进展

树突状细胞(DC)是体内功能最强的抗原提呈细胞(APC),参与多种自身免疫疾病、肿瘤、免疫缺陷病及某些炎症反应的病理过程.以成熟DC为基础制备的DC疫苗具有明显的诱发抗肿瘤免疫应答的能力,其显著的抗肿瘤效应已经得到广泛认可.目前体外培养DC的方法尚没有统一的标准,致使培养得到的DC在均一度和功能上有很大的差别,很大程度上限制了DC的基础研究和DC疫苗的临床应用工作的开展.以下综述了体外扩增DC所用成熟诱导物的特点,为相关研究者体外扩增DC提供了参考.     

Comparison of the Effector Cells in Human Spontaneous Cellular Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity: Differential Sensitivity of Effector Cells to in Vivo and in Vitro Corticosteroids

The effector cells mediating antibody dependent cellular cytotoxicity (ADCC) and spontaneous cellular cytotoxicity (SCC) in humans have been reported to possess similar characteristics. Multiple cell separation techniques were employed in an attempt to physically separate and distinguish the effector cells in these two types of cellular cytotoxicity. Subpopulations of mononuclear cells obtained by a variety of fractionation procedures which either enriched or depleted monocytes, lymphocytes bearing a receptor for sheep erythrocytes (SRBC), a receptor for complement (CRL) or an Fc receptor for IgG always had similar effects on both ADCC and SCC. Aggregated gamma globulin blockade of Fc receptors produced similar dose-dependent depressions of ADCC and SCC. Despite our inability to physically separate the effector cells of ADCC and SCC, administration of in vivo dexamethasone caused a relative increase in ADCC but a profound decrease in SCC. Furthermore, in vitro dexamethasone in pharmacologic and suprapharmacologic concentrations caused no change in ADCC but significantly decreased SCC. This study demonstrates that although the effector cells cannot be physically separated, ADCC and SCC are differentially sensitive to corticosteroids and are hence functionally distinct either on the basis of different subsets of effector cells with similar surface markers or different mechanisms of cytotoxicity by the same effector cell.     

Inhibition of in Vitro Generation of Cytotoxicity Against Tumor Cells by Monoclonal Antibody to Thy-1

The Thy-1 molecule on murine T lymphocytes has been suggested to play a role in cellular activation events leading to a variety of immunologic functions. We present evidence that this molecule may be involved in signals leading to the in vitro generation of cytotoxic T cells against several tumor cell lines used as stimulators in mixed tumor-lymphocyte culture. The presence of monoclonal antibody against a polymorphic determinant on the Thy-1 molecule markedly reduced the generation of cytotoxicity after three days of culture of murine splenocytes with stimulator tumor cells bearing low levels of Ia antigen. In contrast, no effect was seen when the stimulators were either allogeneic splenocytes, or a tumor cell line expressing large amounts of Ia. These results suggest that the Thy-1 molecule is critically involved in events leading to the generation of cytotoxic effectors under some, but not all conditions.     

In Vitro Proliferation of Murine Spleen Cells: I. Strain Variation of Response to Medium from Cultures of EL-4 Cells

Proliferative responses of murine lymphoid cells were elicited in vitro with supernatant fluid from cultures of EL-4 thymoma cells stimulated with phorbol ester. It was demonstrated that such responses depend on IL-2 contained in the supernatant fluid, but reflect co-stimulation with IL-2 and phorbol ester. Striking differences in the magnitude of proliferative responses of spleen, splenic T lymphocytes, thymus and bone marrow cells from various strains were observed. Three classes of responders could be identified. The differences in responsiveness, at least in part, reflected differences in the frequency of responsive cells and were genetically controlled by codominant alleles of two independent somatic genes.     

In Vitro Proliferation of Murine Spleen Cells: I. Strain Variation of Response to Medium from Cultures of EL-4 Cells

Proliferative responses of murine lymphoid cells were elicited in vitro with supernatant fluid from cultures of EL-4 thymoma cells stimulated with phorbol ester. It was demonstrated that such responses depend on IL-2 contained in the supernatant fluid, but reflect co-stimulation with IL-2 and phorbol ester. Striking differences in the magnitude of proliferative responses of spleen, splenic T lymphocytes, thymus and bone marrow cells from various strains were observed. Three classes of responders could be identified. The differences in responsiveness, at least in part, reflected differences in the frequency of responsive cells and were genetically controlled by codominant alleles of two independent somatic genes.     

Two- and Three-Dimensional Culture of Keratinocyte Stem and Precursor Cells Derived from Primary Murine Epidermal Cultures

In the skin, multipotent keratinocyte stem cells (KSC) are localised in the hair follicle bulge region. Although, KSC can be cultivated and grown in two-dimensional (2D) culture they rapidly lose stem cell markers when isolated from their niche. Currently, there is no KSC culture method available which recapitulates an environment similar to the KSC niche in the hair follicle. Here we describe the successful establishment of an in vitro 3D stem cell culture model developed from clonally growing keratinocyte lines derived from neonatal mice using culture conditions previously established for human keratinocytes. After 20 passages, keratinocyte lines showed a stable ratio of holoclones (stem cells), meroclones (stem and precursor cells) and paraclones (differentiating cells), with approximately 29% holoclones, 54% meroclones and 17% paraclones, and were thus termed keratinocyte stem and precursor cell (KSPC) cultures. In high calcium medium, KSPC cultures grown at the air-liquid interphase differentiated and formed epidermal equivalents. Notably, and in contrast to primary keratinocytes, keratinocytes from KSPC cultures were able to aggregate and form spherical clusters in hanging drops, a characteristic hallmark shared with other stem cell types. Similar to the in vivo situation in the hair follicle bulge, KSPC aggregates also showed low proliferation, down-regulation of keratin 6, absence of keratin 1, and expression of the KSC markers keratin 15, Sox9, NFATc1 and Zfp145. KSPC aggregates therefore provide an optimal in vitro 3D environment for the further characterisation and study of normal and genetically modified KSPC.     

Blocking Effect of Rheumatoid Factor on the In Vitro Cytotoxicity of Lymphoid Cells from Carcinoma Patients

Human cryo-IgM rheumatoid factor (RF) preparations blocked the tumor-specific in vitro cytotoxicity of ovarian or bladder carcinoma patients' lymphoid cells in microcytotoxicity assays. The effect was mediated by pretreatment of the effector cells. Cryo-IgM RF free of detectable IgG blocked in a dilution-dependent manner, and immunosorbent purification of contaminating IgG from another preparation did not abrogate the blocking effect. Control IgM preparations lacking RF activity did not block the cytotoxicity, and normal human serum preincubation of the RF preparations rendered them inactive, indicating that the blocking effect was due to the anti-IgG activity of the RF.     

Characteristics of cis-Diamminedichloroplatinum-selected In Vitro Passaged Cells Derived from a Transplantable Rat Malignant Fibrous Histiocytoma

A cis -iamminedichloroplatinum (CDDP)-selected cell line (MT-R10) was induced by continuous exposure of an in vitro passaged cell line (MT-P) established from a transplantable rat malignant fibrous histiocytoma (MFH-MT) to CDDP. MT-R10, capable of proliferating in the presence of l.0 μg CDDP/ml, was passaged in CDDP free medium. The doubling time of MT-R10 at passage 10 (MT -R10/10) was almost the same as that of MT -P, being 22.3 and 25.5 h, respectively. The concentration of CDDP required for 50% inhibition of MT-R10/10 proliferation was two-fold higher than that of MT-P. MT-R10 consisted of round, epithelial-type cells arranged in compact sheets. Ultrastructurally, MT-R10 had numerous free ribosomes, some mitochondria, and other poorly developed cytoplasmic or-ganelles suggesting its undifferentiated nature. MT -R10 showed no reaction for acid phosphatase or non-specific esterase. Tumors induced in syngeneic rats by inoculation with MT-R10 consisted of small, round, undifferentiated cells with scanty cytoplasm. They showed organoid and trabecular patterns, and were often arranged in compact sheets. The neoplastic cells showed no reaction for any of the histiocytic lysosomal and antigenic markers tested, but exhibited a strong reaction for alkaline phosphatase. Bone formation was often observed in the tumors. These observations suggest that CDDP selected, undifferentiated cells may have osteogenic potential and may be one of the progenitor cells of MFH-MT.     

Effect of TNF Expression by Stromal Cells on Hemopoietic and Stromal Precursor Cells in Long-Term Bone Marrow Cultures Derived from TNF-Deficient Mice

The effect of TNF expression by wild type stromal sublayer on hemopoiesis was studied in cultures after second inoculation of TNF-deficient bone marrow cells. Long-term maintenance of hemopoiesis was determined solely by the absence of autocrine expression of TNF by hemopoietic cells. The production of hemopoietic precursors of all studied types in TNF-deficient cultures on a wild type sublayer was significantly higher than in cultures without TNF production. Presumably, initial expression of TNF in the sublayer promotes the survival of hemopoietic precursors with high proliferative potential in the culture. It is also obvious that TNF is required for normal functioning of stromal precursor cells. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 11, pp. 561–564, November, 2005     

T Cell-Mediated Cytotoxicity Against p53-Protein Derived Peptides in Bulk and Limiting Dilution Cultures of Healthy Donors

The p53 tumour suppressor gene product plays an important role in the development of most human cancers. Point mutations in the p53 gene are common in malignant states and results in over-expression of wild type and mutant determinants of the p53 protein. This process might generate MHC-I restricted epitopes for T cell recognition and p53-derived peptides have been suggested as targets for tumour-specific cytotoxic T lymphocytes (CTL). Our primary aim was to estimate the frequencies of p53-peptide reactive CTL precursors (CTLp) in peripheral blood from healthy young individuals. We selected wild type and mutated peptides derived from the p53 sequence with a binding motif for HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from healthy HLA-A2 donors were stimulated in vitro in bulk cultures as well as in limiting dilution cultures using autologous cells pulsed with p53 peptides as stimulator cells. T cell reactivity was observed towards both wild type and mutated p53 peptide epitopes with CTL precursor frequencies varying from 1: 2 × 10⁴ to 1: 1.5 × 10⁵. These results might suggest the presence of an ongoing immune response in normal individuals against cells expressing increased levels of p53 protein.     

Anti-Tumor Activity of Four Ayurvedic Herbs in Dalton Lymphoma Ascites Bearing Mice and Their Short-Term In Vitro Cytotoxicity on DLA-Cell-Line

The anti-tumor activity and chemopreventive potential of four Ayurvedic herbs viz. Curcuma longa L., Ocimum sanctum L., Tinospora cordifolia (Wild) Miers ex Hook.f & Thomas and Zizyphus mauritiana Lam. were evaluated using Dalton Lymphoma ascites (DLA) tumor model in Swiss Albino mice. The outcome was assessed using survival time, peritoneal ascitic fluid (Tumor volume) and hematological indices as parameters. Animals were divided into five groups (n = 6) viz. one DLA control and four Herb + DLA treated groups. All the four herb + DLA groups were pre-treated with respective herbs for 7 days and hematological indices were measured for entire five groups. On day-8 animals were inoculated with 1×10⁶ DLA cells i.p., and Herb + DLA groups were continued with oral herbal treatment for 21-days. Hematological parameters and tumor volume were assessed to find the effects of herbs. Short term in vitro cytotoxicity was determined by Trypan Blue exclusion method and LDH leakage assay using different concentrations of herbal extracts and 5-FU as a positive control and IC₅₀ for each herbal extract and 5-FU were determined. Oral administration of crude herb increased the survival time and decreased the peritoneal ascitic fluid content significantly. Hb, RBCs and total WBC which were altered by DLA inoculation were restored significantly by all the herbs except O. sanctum. All the four herbs showed in vitro cytotoxic activity against DLA cell-line. Moreover inter group comparison of all the four herbs for anti-tumor activity showed efficacy in the following order- T. cordifolia > Z. mauritiana ≥ C. longa > O. sanctum respectively.