NS-7 (nervous system antigen-7): a cell surface antigen of mature brain, kidney, and spermatozoa shared by embryonal tissues and transformed cells

Xenogeneic immunization of rabbits with cells of a mouse undifferentiated neural tumor, MUNTAD, produces antisera which after appropriate absorption bind to the cell surfaces of MUNTAD cells and to three normal adult mouse tissues: brain, kidney, and spermatozoa. Any one of these tissues quantitatively absorbs all of the activity for MUNTAD cells from the antisera, indicating that a single antigen or antigens is shared. This is designated NS-7. The adult tissues positive for NS-7 are the same as those that carry NS-6, but NS-7 is distinguished serologically from NS-6 by being present on many embryonic tissues, tumors, and cell lines which completely lack NS-6. Blocking and capping studies show that on the MUNTAD cell surface, some NS-7 antigenic sites and all NS-6 sites are physically related.     

A new lymphocyte alloantigen (Ly-10) controlled by a gene linked to the Lyt-1 locus

Spleen cells from a (BALB/c x C57BL/6)F1 mouse immunized with CBA/J spleen cells were fused with the myeloma cell line NS-1. One of the six established hybrid cell lines continuously secreted antibody that recognized a new antigenic specificity, tentatively called "Ly-10.1". This newly found antigen is expressed on thymocytes, on splenic T and B cells, on bone-marrow cells, and on the cells derived from brain, kidney and liver. It is also expressed on a continuous cell line, 416B, with stem-cell characteristics. The unique tissue distribution and, furthermore, a distinct strain distribution pattern distinguishes Ly-10.1 from any known murine lymphocyte alloantigen. On the basis of reactivity with cells of the C57BL/6-Lyt-1a congenic strain, one gene governing Ly-10 expression is assigned to the Lyt-1 region of chromosome 19.     

Central nervous system antigen (NS-5) and its presence during murine ontogenesis

An antiserum raised by immunization of C3H.SW/Sn mice with cerebellum from 4-day-old C57BL/6J mice recognizes a cell surface component(s) [NS-5] present in different degrees on various parts of the mouse central nervous system. When analyzed by an antiserum-and complement-mediated cell cytotoxicity test and by the ability of various tissues to absorb anti-NS-5 antiserum activity, the antigen(s) was detectable on cerebellum, retina, olfactory bulb, cortex, basal ganglia, and medulla, but not on nonneural tissues with the exception of mature spermatozoa and 4-day-old kidney. The antigen(s) detected by the anti-NS-5 antiserum was found in similar quantities on young and adult rat and mouse cerebellum; however, it was not detectable on any of 16 clonal cell lines derived from the rat central nervous system. During preimplantation stages of murine development, the antigen could be detected on all cells of (2–4)-cell and (8–16)-cell stages and on the trophoblastic cells of blastocysts by indirect immunoflourescence. Embryos on day 9 of gestation, the earliest stage tested after implantation, expressed the antigen(s), but expression was restricted to the nervous system.     

Serological characterization of a nervous system/sperm antigen (NS-52): demonstration of its presence on murine preimplantation embryos.

Anti-NS-5 antiserum raised in C3H.SW/Sn mice against cerebellum of 4-day-old C57BL/6J mice could be shown to recognize two cell surface antigens on cerebellar cells, NS-5₁ and NS-5₂, the latter antigen being shared with mouse and rat but not rabbit sperm. An antigen operationally identical to NS-5₂ was detected using indirect immunofluorescence staining on mouse preimplantation stages of development. While the unfertilized ova did not express detectable antigen on the cell surface, the fertilized egg expressed antigen shortly before the first cleavage division. From that stage onward, the anti-NS-5 antiserum stained the blastomeres of all stages, including the trophoblast cells and inner cell mass cells of the blastocyst. No difference in staining activity was observed for preimplantation embryos of various mouse strains analyzed: C57BL/6J, BALB/c, 129/J, C3H/DiSn, CKB × BALB.K, C3H.SW/Sn, and Swiss Webster mice. The staining activity was removed when the antiserum was preabsorbed with cerebellum or sperm from any of these mouse strains or with cerebellum and sperm of rats. Lymphocytes, thymocytes, liver, kidney, and skeletal muscle from early postnatal and adult mice and heart from early postnatal mice did not absorb the staining activity and neither did rabbit sperm nor cerebellum.     

Expression of nervous system antigen-3 by lines of mouse fibroblasts and kidney cells and continued expression in hybrid cells

In a survey of the expression on cultured mouse cells of the cell surface antigen known as nervous system antigen-3 (NS-3), it was found that RAG, a renal adenocarcinoma line, expressed that antigen. It was also observed that 3T3, a fibroblast line of unknown tissue origin, expressed NS-3. Cells of these two lines were hybridized with cells of two mouse L cell lines that did not express NS-3. Four hybrid clones were tested for both the 3T3 × L cell cross and the RAG × L cell cross, and all the hybrids were found to be NS-3 positive. All the hybrids had at least 40% as much activity as the NS-3 positive parent. Of the four parental mouse cell lines used, only 3T3 expressed Thy-1.2 antigen on the cell surface. In contrast to the continued expression of NS-3 on hybrid cells, Thy-1.2 antigen was not detectable on two clones of 3T3 × L cell hybrids that were tested.     

Studies of the mouse Ly-6 alloantigen system

Three monoclonal antibodies were produced by fusing mouse myeloma cell line NS-1 with spleen cells from C3H/An mice hyperimmunized with B6-H-2^k spleen cells. These antibodies recognized an alloantigen displaying a similar strain distribution pattern to the Ly-6.2 and Ala-1.2 alloantigens. Analysis of C×B and B×H recombinant inbred mice revealed close linkage of genes controlling Ly-m6 and Ly-6. The monoclonal antibodies lysed 70 percent of cells in lymph nodes and 60 percent in spleen in direct cytotoxicity assays, but did not lyse significant numbers of cells of thymus and bone marrow. Separated T and B cells were reactive with the antibodies, but T cells were more sensitive to the antibody and complement than B cells. Virtually all cells in cultures of cells activated in the mixed lymphocyte reaction or by Concanavalin A were reactive with the monoclonal antibodies. Direct plaque-forming cells were completely eliminated by the monoclonal antibody and complement. By absorption tests, cells from all organs tested so far (thymus, lymph node, spleen, bone marrow, brain, kidney and liver) were shown to express the Ly-m6 determinant. Tumor cell lines with T, B or stem cell characteristics were reactive with the monoclonal antibody by direct cytotoxicity and absorption assays.     

A cell surface antigen, TER, expressed by embryos and germ cells.

An antiserum prepared in rabbits against the C3HeB/FeJ mouse ovarian teratocarcinoma E6496 was absorbed in vivo in C3HeB/FeJ mice. This absored antiserum identified an antigen, denoted TER, that is present on sperm, ova, embryonic germ cells, and cells of the early mouse embryo. TER was absent from all adult somatic cells tested, but found on several murine tumors.     

Detection of a cell surface antigen common to a mouse myeloma and a population of mouse thymocytes.

A new antigen termed PTA (plasmocytoma thymus antigen) is described which occurs on 70--80% of mouse thymocytes of all mouse strains tested and on an IgG 2b producing BALB/c plasma cell tumor. PTA is detectable on mouse spleen and lymph node cells. It is absent from liver, brain, bone marrow, and antibody plaque forming cells. PTA is not related to theta antigen and is not detectable on rat thymocytes.     

A new lymphocyte alloantigen (Ly-10) controlled by a gene linked to theLyt-1 locus

Spleen cells from a (BALB/cxC57BL/6)F₁ mouse immunized with CBA/J spleen cells were fused with the myeloma cell line NS-1. One of the six established hybrid cell lines continuously secreted antibody that recognized a new antigenic specificity, tentatively called Ly-10.1. This newly found antigen is expressed on thymocytes, on splenic T and B cells, on bone-marrow cells, and on the cells derived from brain, kidney and liver. It is also expressed on a continuous cell line, 416B, with stem-cell characteristics. The unique tissue distribution and, furthermore, a distinct strain distribution pattern distinguishes Ly-10.1 from any known murine lymphocyte alloantigen. On the basis of reactivity with cells of the C57BL/6-Lyt-1^a congenic strain, one gene governing Ly-10 expression is assigned to the Lyt-1 region of chromosome 19.We chose the notation Ly-10 rather than Ly-9 to allow for a future decision that Lgp 100 (Ledbetter et al. 1979) should be renamed Ly-9.     

The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions

The human AC133 antigen and mouse prominin are structurally related plasma membrane proteins. However, their tissue distribution is distinct, with the AC133 antigen being found on hematopoietic stem and progenitor cells and prominin on various epithelial cells. To determine whether the human AC133 antigen and mouse prominin are orthologues or distinct members of a protein family, we examined the human epithelial cell line Caco-2 for the possible expression of the AC133 antigen. By both immunofluorescence and immunoprecipitation, the AC133 antigen was found to be expressed on the surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133 antigen, but not its mRNA level, was down-regulated upon differentiation of Caco-2 cells. The AC133 antigen was specifically located at the apical rather than basolateral plasma membrane. An apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney. Electron microscopy revealed that, within the apical plasma membrane of Caco-2 cells, the AC133 antigen was confined to microvilli and absent from the planar, intermicrovillar regions. This specific subcellular localization did not depend on an epithelial phenotype, because the AC133 antigen on hematopoietic stem cells, as well as that ectopically expressed in fibroblasts, was selectively found in plasma membrane protrusions. Hence, the human AC133 antigen shows the features characteristic of mouse prominin in epithelial and transfected non-epithelial cells, i.e. a selective association with apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34(+) bone marrow progenitors revealed the cell surface expression of prominin. Taken together, the data strongly suggest that the AC133 antigen is the human orthologue of prominin.     

6C6鼠-人嵌合抗体的纯化及鉴定

采用基因工程技术 ,将小鼠 6C6单克隆抗体可变区基因与人抗体恒定区基因连接 ,构建了鼠 人 6C6嵌合抗体基因 ,并在CHO细胞中高效表达 .利用ProteinA亲和层析柱从细胞培养上清中分离纯化 6C6嵌合抗体 ,得到电泳纯度大于 98%的 6C6嵌合抗体 ,其重链 (5 5kD)和轻链 (2 4kD)符合IgG相对分子质量的理论值 .Western印迹、细胞免疫荧光和免疫组织化学实验结果均呈阳性 .表明6C6嵌合抗体可识别人乳腺癌细胞表面上的肿瘤相关抗原 ,保持了 6C6单克隆抗体的特性 ,为后续的研究工作奠定了基础     

Expression of the human MHC, HLA-DQW6 genes alters the immune response in C57BL/6 mice

In an attempt to obtain direct evidence for the critical role of HLA class II molecules in regulating the immune response, genomic genes for alpha- and beta-chains of HLA-DQw6 from HLA-Dw12 haplotype were introduced into the C57BL/6 (B6) strain of mouse and a line of HLA-DQw6 transgenic mouse was obtained. Tissue specificity of the expression of the transgenes was much the same as that of murine I-Ab genes. DQw6 molecules were expressed on B cells and macrophages in spleen cells and about 30 to 40% of the I-Ab+ spleen cells were positive for DQw6. The HLA-DQw6 transgenic B6 mouse became tolerant to the DQw6 molecules, as evidenced by the MLR and antibody production specific to the DQw6 molecules. The HLA-DQw6 transgenic B6 mouse showed a strong immune response to streptococcal cell wall antigen (SCW), whereas the B6 mouse was a low responder to SCW. The SCW-specific T cell line was established from the transgenic mouse and this T cell line recognized SCW in the context of HLA-DQw6 molecules expressed on the mouse L cell transfectant or on human monocytes. The proliferative response to SCW of primed lymph node T cells and the SCW-specific T cell line derived from the transgenic mice was inhibited by anti-HLA-DQ mAb. Thus, it is clear that the HLA-DQw6 genes acted as major histocompatibility genes in these transgenic mice.     

Inhibition of protein kinase C arrests proliferation of human tumors

We have shown that inhibition of protein kinase C by 1-5-isoquinolinylsulfonyl-2-methylpiperazine, H7, induces differentiation and inhibits proliferation of Neuro 2a cells. We have now tested if H7 is able to inhibit proliferation of: 1) human tumor cell lines from tissues other than brain; and 2) primary cultured cells from several human brain tumors. H7 inhibits, in a dose-dependent manner, proliferation of all human tumor cell lines tested and of primary cultured cells from human brain tumors. These results indicate that inhibition of protein kinase C inhibits proliferation of tumoral cells, therefore, H7, and likely other inhibitors of protein kinase C, could be useful in the clinical treatment of brain (and probably other) tumors.     

富亮氨酸重复超家族新成员LRRC4的克隆与在脑瘤中的表达分析

在染色体7q31-32多种肿瘤杂合性丢失（loss of heterozygosity,LOH）高频区,采用表达序列标签（expressed sequence tag,EST）介导的定位候选克隆策略获得了一个定位于人染色体7q31-32的新基因（GenBank 登录号: AF196976）.该基因编码653个氨基酸,蛋白质理论pI/m:6.58/72.7 ku.它包含七个典型的LRR、一个IgC2样结构域.此外,它还包含一个N端信号肽、一个C端跨膜区.其结构特征表明它是富亮氨酸重复（leucine-rich repeat,LRR）超家族的新成员.经过人类基因组命名委员会的同意,将该基因命名为LRRC4.此外,通过序列相似性匹配还获得了定位于小鼠6号染色体的LRRC4的同源基因（GenBank 登录号: AF290542）.RNA印迹和RT-PCR检测发现LRRC4在正常人脑组织相对特异表达,而在多种原发性脑瘤表达明显下调或缺失.综合考虑LRRC4基因的序列特征及表达谱,提示LRRC4基因可能在神经系统中发挥重要作用.     

Influence of Host Cell Infiltration on the Glycolipid Content of Mouse Brain Tumors

Abstract: Previous studies showed that levels of some glycosphingolipids (GSLs) expressed in solid brain tumors grown in vivo were reduced or undetectable in cultured cells prepared from the tumors. This phenomenon has been attributed either to suppressed glycolipid synthesis from unknown forces of the tissue culture environment or to the absence of host cells that normally infiltrate the solid tumors growing in vivo. To test further the host cell hypothesis, we examined host cell markers in two experimental mouse brain tumors, the ependymoblastoma and the CT-2A, that were grown as subcutaneous solid tumors in the flank of C57BL/6J (B6) mice or as cultured cells in vitro. The markers included ganglioside N -glycolylneuraminic acid (NeuGc), GA1 (asialo-GM1), and Fc receptor-bearing cells. NeuGc-containing gangliosides, GA1, and Fc receptors are expressed by macrophages and lymphoid-type cells of the mouse host immune system but are not normally expressed by mouse neural cells. Differences in the relative content of Fc receptor-bearing cells in ependymoblastoma and CT-2A tumors grown in vivo (8.3 and 16.8%, respectively) were proportional to differences in the relative content of NeuGc-containing gangliosides (25.5 and 45.1%) and GA1 (8.5 and 13.8%), respectively. Neither cultured tumor cell line expressed Fc receptors, GA1, or NeuGc-containing gangliosides. These findings suggest that non-neoplastic host infiltrating cells (macrophages) contribute significantly to the GSL composition of solid tumors growing in vivo.     

Monoclonal M6 antibody interferes with neurite extension of cultured neurons.

Monoclonal M6 antibody binds to the surface of murine central nervous system neurons as well as to apical surfaces of epithelial cells in the choroid plexus and proximal tubules of the kidney. M6 antigen is expressed in the central nervous system as early as embryonic day 10, most strongly in the marginal zone of the neural tube, and remains detectable in adulthood. IgG or Fab fragments of M6 antibody interfere with the extension of neurites by cultured cerebellar neurons. Effects of the antibody on neurite extension are readily detectable after 24 h. No reduction of cell viability is detected during the first 3 days of antibody treatment. Cultures maintained in the presence of antibody for longer than 5 days exhibit reduced viability of neurons. This reduction in long-term viability in the presence of M6 antibody is largely avoided when 25 mM KCl is included in the culture medium. The antibody-mediated perturbation of neurite outgrowth is not blocked by the presence of elevated KCl. The unusually short and flattened appearance of neurites in these cultures suggests that the M6 antibody selectively affects neurite extension. Time-lapse cinematography of anti-M6-treated neurons reveals no apparent effect on movement of lamellipodia and filopodia of growth cones. Only the overall extension of the neurite appears to be inhibited. M6 antigen is a 35 kD glycoprotein that can be isolated from a deoxycholate- (DOC) solubilized membrane fraction from adult mouse brain.     

Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells

The blood brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) and tightly regulates the transport of molecules from blood to neural tissues. In vitro BBB models from human pluripotent stem cell (PSCs)-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs) were differentiated into endothelial cells (ECs), and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM), in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs) to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.     

Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells

The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.     

Hemagglutinin prepared from cells of Aedes albopictus, clone C6/36, infected with type 1 dengue virus

A high titer of hemagglutinin (HAnin) was found in culture fluids of cells of Aedes albopictus, clone C6/36, infected with type 1 dengue virus. The HAnin (TC antigen) was associated with complete virions and no appreciable small-sized HAnin was produced, in contrast to the case in an infected suckling mouse brain (SMB) homogenate, in which most of the HAnin is smaller than complete virions. Extraction of the TC antigen with Tween 80-ether disrupted the HAnin into smaller-sized particles, resulting in complete loss of infectivity (TE-TC antigen), but it did not affect the titer or reactivity of the HAnin. Comparative hemagglutination-inhibition (HI) assay of human sera using TC antigen, TE-TC antigen or standard antigen extracted from infected SMB (SMB antigen) showed that TC or TE-TC antigen could be used for routine diagnostic or epidemiological HI tests instead of SMB antigen, which is rather hard to prepare.