Antigen related to cell proliferation in malignant gliomas recognized by a human monoclonal antibody

A human monoclonal antibody (CLN-IgG) was produced from a human-human hybridoma derived from lymphocytes of a patient with cervical carcinoma. The reactivities of this antibody with various human glioma tissues and cultured glioma cells and the characterization of the antigen recognized by CLN-IgG on malignant glioma cells were analyzed and reported. CLN-IgG reacted with various human glioma cells and glioma tissues, especially glioblastoma, but did not react with normal brain tissues or fetal brain tissues. A large amount of antigen recognized by CLN-IgG was expressed on cell membranes of undifferentiated glioma cells and of glioma cells at the G2/M tumor growth phase in cycling cells. Antigen recognized by CLN-IgG was detected in only one of seven samples of cyst fluid, and was not detected in 27 serum samples or 18 samples of cerebrospinal fluid from glioma patients. CLN-IgG exhibited antibody-dependent cell cytotoxicity against U-25 1 MG glioma cells and primary cultured cells of glioblastomas and anaplastic astrocytomas. These data suggest that the antigen recognized by CLN-IgG might be related to cell proliferation in malignant gliomas. Thus, CLN-IgG might be useful for immunotherapy or immunoimaging of malignant gliomas.     

Monoclonal antibodies with cytotoxic reactivities against human gliomas

Monoclonal antibodies (MAb's) reactive with human malignant glioma cells were derived from mice inoculated with cells from fresh glioma tissue. Seven MAb's were selected for study based on their high-level binding in immunoperoxidase and immunofluorescence assay to most of the glioma tissues derived from various patients and based on the absence of binding to normal bone marrow cells. Four of the seven MAb's did not bind to any of the four normal brain tissues tested, whereas three MAb's bound to one or two of these tissues. Two MAb's bound to the surfaces of cultured glioma cells in radioimmunoassay. One of these MAb's (AS-AY1, immunoglobulin (Ig)(G1) lysed cultured glioma cells with human lymphocytes or murine macrophages as effector cells; the other MAb (AS-AY2, IgM) was reactive in complement-dependent cytotoxicity assay. These two MAb's therefore seem especially promising reagents in approaches to immunotherapy of human malignant glioma.     

A heterophile system in human renal transplantation. V. Relationship of heterophile transplantation antigen and common antigen of Enterobacteriaceae.

Heterophile transplantation antigen and common antigen of Enterobacteriaceae appear serologically to be separate specificities. However, both antigens are common to Enterobacteriaceae, rat erythrocytes, and some human kidneys. Both antigens are obtained from various tissues by the same chemical procedure. Immunity to each antigen is frequently produced by renal transplantation. We suggest that the antigens are either separate molecules which are similar in chemical structure in the region of the antigenic determinant as well as in tissue distribution or separate reactive sites located on the same molecule. The possibility that common antigen may be a human alloantigen raises theoretical possibilities relative to susceptibility to infection and pyelonephritis, as well as to its relationship to histocompatibility.     

Immunohistochemistry of human malignant astrocytoma cells xenografted to rat brain: apolipoprotein E

Fresh xenografted human malignant astrocytoma cells migrate throughout the host rat brain. Cells from three Grade 3 human malignant astrocytomas were prelabeled with Phaseolus vulgaris leucoagglutinin (PHAL) and then xenografted into implantation pockets in rat host cerebral cortex. The human malignant astrocytoma cells in the host brain were immunocytochemically double-labeled for the presence of PHAL, which is used as a marker for graft derived cells, and either glial fibrillary acidic protein (GFAP), a specific marker for astrocytes and astrocytoma cells, or apolipoprotein E (APOE) 7 days, 14 days, 21 days, and 1 month later. Fresh human malignant astrocytoma cells (Grade 3 and 4) contained APOE and GFAP. The xenografted cells preserved APOE and GFAP in the host. PHAL double-labeled human malignant astrocytoma cells were found on the glia limitans along the entire circumference of the brain, in the corpus callosum, internal capsule, entopeduncular nucleus, optic tract, and median eminence. In addition, astrocytoma cells were observed in the cingulum, habenula, arcuate, and supraoptic nucleus. Astrocytoma cells entered the space of Virchow-Robin, migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. APOE was a consistent marker for the migrating human malignant astrocytoma cells, but not an exclusive marker of the xenografted cells, since host rat reactive astrocytes also expressed APOE.     

Selective uptake of hematoporphyrin derivative into human cerebral glioma

The uptake of hematoporphyrin derivative (HpD) into human cerebral glioma was measured using a porphyrin extraction technique. Patients with cerebral glioma were injected with HpD at a dose of 5 mg/kg body weight 24 hours before surgery and photoradiation therapy (PRT). Biopsies of tumor, and where possible, adjacent brain and normal brain were taken for analysis of HpD uptake. HpD was selectively localized into all grades of glioma, and there was a direct correlation between the grade of glioma and HpD level in the tumor. The levels were highest in glioblastoma multiforme (mean uptake of 5.9 micrograms of HpD/g of tumor wet weight) and lower in the intermediate-grade anaplastic astrocytoma (mean uptake of 2.4 micrograms/g of tumor) and the low-grade astrocytoma (1.6 micrograms/g of tumor). Uptake into normal brain tissue taken from HpD-sensitized patients was 0.2 microgram/g. HpD was also localized into the "brain adjacent to tumor" region. The selective uptake into the low-grade glioma suggests that PRT may be of use as an adjuvant therapy in these tumors and the detection of HpD in this region indicates that PRT may control the spread of tumor infiltrating into the adjacent normal brain.     

Immunohistochemical analysis of tumor-infiltrating lymphocytes and major histocompatibility antigens in human gliomas and metastatic brain tumors

A subpopulation of tumor-infiltrating lymphocytes (TILs) and the expression of major histocompatibility complex (MHC) antigens of the tumor cells were examined in 14 glioma and 13 metastatic brain tumor tissues. In both tumors, most of the TILs were T lymphocytes, and both phenotypes of the cytotoxic/suppressor and helper/inducer T lymphocyte were found. On examination of MHC antigens, β₂-microglobulin was shown intensely on tumor cells in all cases, and the monomorphic determinant of the human leukocyte antigen (HLA-DR) was shown in 10 glioma and in 5 metastatic cases. The correlation between the number of TILs and MHC antigen expression on tumor cells was equivocal as a whole in cases of both glioma and metastatic brain tumor.     

Role of estrogen receptor-related antigen in initiating the growth of human glioma cells

OBJECT: The expression of estrogen receptor-related antigen (ER-D5) has been demonstrated in many tumors, including those of the brain, but the actual role of ER-D5 in cell growth is unknown. The authors evaluated the role of ER-D5 in the growth of gliomas in vitro. METHODS: Human glioblastoma multiforme (GBM) cell lines A172, T98G, U87MG, and U118MG; rat C6 glioma and 9L gliosarcoma; AS human astrocytoma; GBM in primary culture and tumor tissues; and normal brain tissues were examined for ER-D5 by using immunohistochemical, Western immunoblot, flow cytometry, and enzyme-linked immunosorbent assays. The ER-D5 was detected in all tumor cell types of human origin, but not in rat cell lines and normal brain; the expression of ER-D5 was not related to cell cycle phase. Kinetic analysis of ER-D5 expression in cultured cell lines revealed that an enhanced and sharp accumulation of ER-D5 occurred during the first 24 hours of culture, followed by a sharp fall in the next 24 hours. Gradual decreases of ER-D5 during the subsequent days were demonstrated in all human cell lines, and in primary cultures of GBM. This accumulation pattern of ER-D5 was confirmed on Western blot analysis. The ER-D5 was also detected in cells cultured in serum-free medium. Culture cells were treated with D5 antibody against ER-D5 for 48 hours and the effects were evaluated using a monotetrazolium colorimetric assay; the result revealed that growth of cultured cells was inhibited in a dose-dependent manner, and that addition of a single median inhibitory concentration dose resulted in complete growth inhibition and arrest of cell growth at the G0/G1 phase at 96 hours posttreatment. CONCLUSIONS: These findings indicated that synthesis and accumulation of ER-D5 is an essential event in the very early phase of in vitro growth of human gliomas.     

Interleukin-10 Inhibited the Expression of Tumor Antigens and Major Histocompatibility Complex Antigen on EJ-ras Oncogene Transformants

Abstract: Interleukin-10 (IL-10), a novel inhibitory cytokine, is one of Th-2 (T helper) cytokine. It inhibits mixed lymphocyte reaction, and the production of inflammatory cytokine and monokine downregulates major histocompatibility complex antigen (MHC) class II antigen expression. However, the effect of IL-10 on tumor cells is not known. Therefore, the mechanism of tumor tolerance induced by IL-10 was investigated. (WKA rat fetus-derived fibroblast) (WFB) and W14 and W31 (EJ-ras oncogene transformants of WFB) were cultured with recombinant human (rh)IL-10 (0, 10, 50, 100 ng/ml). FACS analysis was performed using the following monoclonal antibodies: anti-rat MHC class I monoclonal antibody; and monoclonal antibody 109 (anti-natural killer [NK] target molecule on W14). Monoclonal antibody (mAb) 109-defined antigens were newly expressed during the transforming process by EJ-ras oncogene transfection to WFB. In addition, the effects of rhIL-10 on the ability of proliferation and susceptibility to NK cells were assessed. The cultivation with rhIL-10 resulted in a dose-dependent decrease in the expressions of MHC class I antigen and monoclonal antibody 109-defined antigen. The proliferation and susceptibility to NK cells of W14 were inhibited. These data demonstrated a possibility that IL-10 could induce tumor tolerance to host immunity by inhibiting the expression of tumor-associated antigens and MHC class I.     

Monoclonal antibody-dependent, cell-mediated cytotoxicity against human malignant gliomas.

Two monoclonal antibodies (MAbs), IgG2a MAb ASHG4 and IgG2b MAb ASHE2, were produced in mice immunized with cultured human malignant glioma cells. Both MAbs bound strongly to the surfaces of long-term cultured glioma cells, and MAb ASHE2 also bound strongly to short-term cultured glioma cells. Sections of frozen glioma tissues bound both MAbs strongly, whereas normal brain tissues showed weaker reactivities, and tissues derived from carcinomas of various histological types were completely unreactive. Furthermore, the MAbs did not bind to peripheral blood cells or bone marrow cells. Although both MAbs bound to the same Mr 27,000-29,000 protein, they may detect different or overlapping epitopes on this antigen. Because MAbs ASHE2 and ASHG4 lysed cultured glioma cells with human peripheral blood lymphocytes as effector cells, they are promising reagents for approaches to immunotherapy of human malignant gliomas.     

Murine monoclonal antibodies reactive with a variety of androgen independent Dunning rat prostate adenocarcinoma sublines also reactive with human prostate adenocarcinoma

Murine hybridoma-derived monoclonal antibody (MCA) to Dunning rat prostate adenocarcinoma R-3327HIS (androgen independent type) has been produced by fusing P3x63 Ag8-653 myeloma cells with splenocytes of BALB/c mice which were immunized with R-3327HIS tumor cell membranes. One monoclonal antibody designated MCA-R1 (IgG2a subtype) produced an intense immunostaining of various androgen independent Dunning rat prostate tumor sublines (HIS, HIM, HIF, AT-1, AT-2, AT-3, MAT-Lu and MAY-Ly-Lu), but did not stain other tumors of rat origin or normal rat tissues. Marginal immunostaining was detected in the androgen responsive R-3327H and R-3327G tumor subline. Although MCA-R1 antibody did not react with the regressed prostate tumor of R-3327H or R-3327G, it strongly reacted with the relapsed prostate tumor from either R-3327H or R-3327G tumor derived from rats were treated with diethyl stilbestrol (DES) or castration. MCA-R1 antibody also produced a strong cross-reaction with human prostate adenocarcinoma. Like the Dunning rat tumor, human adenocarcinoma exhibited distinct immunostaining patterns with respect to intracellular localization among well differentiated, moderately differentiated and poorly differentiated tumor. Benign prostatic hyperplasia or other normal tissues did not stain. Immunofluorescence, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioautographic analysis revealed that the Dunning rat prostate tumor antigen recognized (m.wt. 50 and 120 Kd) by MCA-R1 antibody are localized on both cell surface and in the cytoplasm. This MCA may represent a potential reagent for the study of tumor biology and immunotherapy of prostate tumor.     

Dunning rat prostate tumors and cultured cell lines fail to express human prostate carcinoma-associated antigens.

The objective of this study was to determine if human prostate carcinoma-associated tumor markers were expressed by Dunning rat prostate carcinomas. Frozen and formalin-fixed paraffin-embedded tissues from 12 different sublines of Dunning tumors were evaluated for marker expression by immunoperoxidase staining by using a panel of 9 monoclonal antibodies, including antibodies against human PAP and PSA. None of the Dunning tumors were found to express any of the human prostate tumor markers. Both fixed and live immunofluorescent assays were performed on 5 cultured Dunning tumor cell lines, evaluated either as single cells or as monolayers. As with the Dunning tumor tissues, none of the cell cultures expressed any of the 9 human prostate tumor markers. The lack of antigen expression by the Dunning tumor tissues and cell lines suggests that these human prostate tumor markers are quite species specific. These results limit the use of the Dunning prostate tumors as models to explore the preclinical application of these human prostate carcinoma-associated monoclonal antibodies and their target antigens.     

A cell-type specific ganglioside of glomerular podocytes in rat kidney: an O-acetylated GD3.

We recently described a monoclonal antibody (clone 27A) that detected a membrane antigen specific for glomerular podocytes in adult rat kidney. After binding in vivo, the antibodies induced rapid changes in the foot processes. Here we show that in other rat tissues the antigen is detectable only in cells of adrenal medulla, in some cells of neural or neural crest origin, and in 1 to 5% of the cells of a rat pheochromocytoma cell line PC-12. Attempts to isolate the antigen revealed that it is an acidic, sialic acid containing lipid, as shown by thin layer chromatography and immuno-overlay techniques. Further characterization of the gangliosides extracted from rat glomeruli, bovine kidney, rat adrenal glands, or from PC-12 cells by ion exchange, thin layer, and gas liquid chromatography identified the antigenic lipid as a modified disialosyllactosylceramide (GD3). The results of mild alkaline treatment or periodate oxidation of the antigenic ganglioside, as well as chemical O-acetylation studies of standard gangliosides, showed that the modified ganglioside is O-acetylated, most probably at the 9-carbon of its terminal sialic acid residue. To our knowledge this is the first report of cell-type specific expression of gangliosides in the kidney.     

Successful treatment of a malignant rat glioma with cytotoxic T lymphocytes.

Brain tumors are highly resistant to therapy. Their diffuse infiltrative nature and the relative inaccessibility of brain tissue to blood and lymph are barriers to surgical and cytotoxic treatments alike. The purpose of this study was to produce immune cells specifically reactive with an anaplastic rat glioma (RT2) and determine whether those cells could affect tumor progression in the brain. RT2-specific cytotoxic cells were prepared by priming rats in vivo with RT2 tumor cells and Corynebacterium parvum and stimulating the primed lymphocytes in vitro with irradiated RT2 tumor cells and interleukin-2 (IL-2). Cultured cells exhibited a high level of cytotoxicity against RT2, but not C6 (an allogeneic glioma), 3M2N (a syngeneic mammary tumor), or CSE (a syngeneic fibrosarcoma) tumor cells. To generate a model for therapy, rats were injected intracerebrally with RT2, generating progressing brain tumors, which killed untreated rats in approximately 2 weeks. To test the therapeutic potential of the effector cells, tumor-bearing rats were treated by intravenous injection of lymphocytes on Day 5 of tumor growth. Treated rats also received a 5-day course of systemic IL-2 beginning on Day 5. Treatment with IL-2 alone, RT2-primed spleen cells, or RT2-primed spleen cells stimulated in vitro with C6 did not affect rat survival. However, tumor-bearing rats treated with RT2-stimulated lymphocytes exhibited increased survival or were cured. Systemic IL-2 was an essential adjunct, because survival was not affected by treatment with effector cells alone. Therapy initiated on Day 8 of tumor progression lacked effect on survival.(ABSTRACT TRUNCATED AT 250 WORDS)     

血管内皮生长因子在人脑胶质瘤的定位及表达

目的 通过检测人脑胶质瘤及瘤周组织中血管内皮生长因子(VEGF)的基因表达及定位,探讨VEGF促进肿瘤血管生成及瘤周组织水肿的作用机制。方法采用原位杂交方法检测23例人脑胶质瘤及瘤周组织中VEGF基因表达情况,并与7例正常脑组织进行对照研究。不着色者为阴性,细胞胞浆着色呈棕黄(褐)色者为阳性。结果VEGF在胶质瘤组织主要表达于毛细血管、血管内皮细胞及肿瘤细胞,瘤周组织表达于变异的星形胶质细胞,而正常脑组织几乎无表达。胶质瘤组织标本中血管内皮细胞及瘤细胞内VEGF的表达率分别为22.60±2.31和20.18±4.25,而瘤周组织标本中血管内皮细胞及变异的星形胶质细胞内VEGF的表达率分别为8.44±3.17和45.20±6.11,正常脑组织标本中血管内皮细胞及细胞内VEGF的表达率分别为1.76±1.20和1.84±1.51,胶质瘤组织与瘤周组织阳性细胞表达率显著高于正常脑组织(P<0.01);胶质瘤组织中血管内皮细胞VEGF的阳性表达率高于瘤周组织(P<0.01);而瘤周组织星形胶质细胞的阳性表达率高于肿瘤组织(P<0.01)。结论提示VEGF在脑胶质瘤及瘤周组织中的高表达与肿瘤血管生成及瘤周组织水肿的发生密切相关。     

Increased Expression of ABCB6 Enhances Protoporphyrin IX Accumulation and Photodynamic Effect in Human Glioma

Background

Glioma recurrence usually occurs close to the tumor resection margins as a result of residual infiltrating glioma cells. 5-aminolevulinic acid (ALA) fluorescence-guided resection of gliomas has been demonstrated to enhance discrimination of tumor tissue and to improve survival. ALA-based photodynamic therapy is an effective albeit still experimental adjuvant treatment option for gliomas. However, insufficient protoporphyrin IX (PpIX) accumulation may limit the benefits of fluorescence-guided resection and photodynamic therapy.

Methods

We investigated the expression of the ATP-binding cassette transporter ABCB6, which regulates porphyrin synthesis, in surgical specimens from human gliomas and manipulated ABCB6 in human glioma cell lines.

Results

Our findings demonstrated that expression levels of ABCB6 were greatly elevated in human gliomas compared with normal brain tissues and correlated with World Health Organization histologic grade. A previously undescribed finding was that ABCB6 mRNA expression in solidly fluorescing tumor tissues was higher than that in vaguely fluorescing tumors, suggesting that ABCB6 may be at least in part responsible for PpIX accumulation in glioma cells. Accordingly, ABCB6 overexpression in glioma cell lines caused a marked increase in intracellular levels of PpIX, and was more sensitive to ALA-induced photodynamic therapy—events that could be prevented by silencing ABCB6 via siRNA treatment.

Conclusions

Our findings indicate a crucial role of ABCB6 in ALA metabolism and accumulation of PpIX in glioma. ABCB6 overexpression is a potential approach to enhance accumulation of PpIX for optimizing the subjective discrimination of vague fluorescence and improving the efficacy of ALA-based photodynamic therapy.     

Angiogenesis induced by rat glioma cells in vitro]

Angiogenesis induced by rat glioma cells was examined in vitro using a double chamber co-culture system. Cultured microvascular endothelial cells from Fisher 344 rat brain, rat C6 glioma cells and rat T9 gliosarcoma cells were used for this study. Endothelial cells, cultured on type I collagen, formed capillary-like structures. In the co-culture system, C6 glioma cells promoted this formation. On the other hand T9 gliosarcoma cells had no effect on it. The supernatants of C6 glioma cells and T9 gliosarcoma cells suppressed the proliferation of the endothelial cells. C6 glioma cells probably produce and release soluble factors promoting angiogenesis. The proliferation of endothelial cells is thus suppressed while angiogenesis is made more intense. This in-vitro model is useful to elucidate the mechanism of tumor angiogenesis and to evaluate the promoting and inhibiting factors of angiogenesis.     

An autopsy case of multicentric glioma of multiple histopathology

An autopsy case is described of an 66-year-old man with multicentric glioma of multiple histopathology, i.e. protoplasmic astrocytoma and glioblastoma. Enhanced CT scan revealed three separate lesions in the right cerebral hemisphere, pons, and cerebellar vermis. Initial diagnosis by CT included metastatic and primary brain tumor, multiple abscess, fungal infection, parasites, tuberculoma, and so on. Biopsy of the right frontal mass revealed astrocytoma grade-2. An autopsy revealed gelatinous, clear marginal mass in the right frontal, parietooccipital and cerebellar vermis; an opaque marginal mass with necrosis in dorsal pons was found. At microscopic examination, the right frontal tumor exhibited continuity with both the paraventricular and the right parietooccipital tumor. The right cerebral hemisphere and cerebellar vermis tumors showed protoplasmic astrocytoma; the dorsal pons tumor showed glioblastoma. CSF examination revealed no tumor cells. Tumor invasion of the internal capsule and the meninges was also not found. Accordingly, we diagnosed as multicentric astrocytoma of multiple histopathology. Only 11 case reports of multicentric glioma were recorded in Japan; only one of which was of multiple histopathology. Worldwide, only 7 case reports of multicentric glioma of multiple histopathology were recorded; this is the first case of protoplasmic astrocytoma and glioblastoma. Seen in terms of pathogenesis of multicentric glioma, this case is thought to be very interesting.     

一种G422胶质母细胞瘤瘤株标准化动物模型的建立

目的建立标准化小鼠G422胶质母细胞瘤动物模型。方法将接种G422胶质母细胞瘤昆明小鼠的皮下肿瘤，接种于国际标准化小鼠双侧上肢皮下，每侧分别注射0．1、0．2、0．3ml。接种后5、10、15、20d观察小鼠的生存状态及肿瘤的生长特性；用HE染色及胶质纤维酸性蛋白（GFAP）和S-100蛋白免疫组织化学分析。结果标准化小鼠G422胶质母细胞瘤模型生长稳定，成瘤率高，3个实验组均100％成瘤，无1例死亡（P〈0．05）；Gn世及S-100蛋白免疫组织化学表达有棕黄色颗粒呈强阳性，符合胶质源性肿瘤的特点。结论国际标准化小鼠G422胶质母细胞瘤模型，生长特征及病理表现与人脑胶质母细胞瘤相似，是一种较理想的动物模型。