A surface membrane study of human cell lines with different interferon sensitivities]

A comparative study of surface membranes of human cell J-96 and J-48 cultures with different sensitivity to alpha/beta interferon (IF). Reduced sensitivity of J-41 cells to IF-alpha/beta was found to be accompanied by a loss of highly specific receptors for IF-alpha, the lack of changes in the cell surface structures upon treatment with IF-alpha/beta, reduced intensity of cell fusion upon successive treatment with IF and polyethylene glycol. The results are discussed in connection with the observed changes in the activity of superoxide dismutase in J-96 and J-41 cell lines after treatment with IF.     

The human hematopoietic progenitor cell antigen (CD34) in vascular neoplasia.

The human hematopoietic progenitor cell antigen CD34 is synthesized and expressed by early normal hematopoietic progenitor cells and by many acute leukemias. Anti-CD34 antibodies also have been reported to stain blood vessels in tissue sections, and, more recently, CD34 mRNA has been detected in vascular endothelial cells. Therefore, the authors studied the diagnostic utility of immunohistochemical CD34 antigen detection in tumors of endothelial cell derivation and compared the results with stains for von Willebrand (vW) factor. A wide variety of epithelial and mesenchymal neoplasms also were examined to assess the specificity of CD34 for vascular neoplasia. Seven cases of angiosarcoma (seven of seven), five cases of Kaposi's sarcoma (five of five), and eight cases of epithelioid hemangioendothelioma (eight of eight) were moderately to strongly positive for CD34. This reactivity was equally intense in frozen sections, alcohol-fixed tissue, and formalin-fixed specimens. In many cases, the malignant endothelial cells stained more strongly than adjacent benign endothelium. Moreover, in most cases CD34 positivity was quantitatively and qualitatively stronger than staining for vW factor. Two cases of hemangiopericytoma (two of two) were CD34 positive but stained less intensely than the angiosarcomas, Kaposi's sarcomas, or hemangioendotheliomas. Five of six cases of hemangioma also stained positively for CD34; the nonreactive tumor in this group was the only one among 28 vascular neoplasms studied that was not reactive for CD34. In comparison, 9 of the 28 vascular tumors did not stain for vW factor. Three hundred fifty-seven tumors of nonvascular derivation also were examined for CD34 antigen expression. Focal light staining was seen in one pulmonary squamous cell carcinoma; moderate to intense staining was observed in half of the epithelioid sarcomas studied (8 of 16) and in a minority of leiomyosarcomas (3 of 22). These findings indicate that CD34 is a sensitive and relatively specific marker for neoplasms of vascular origin.     

HLA-E在肝癌细胞系中的表达

目的: 研究人类白细胞抗原E(HLA-E)在肝癌细胞系中的表达情况。方法: 通过real-time PCR和Western blotting技术研究5种肝癌细胞系和胎肝细胞系中HLA-E mRNA 和蛋白的表达情况,进行相对定量分析。结果: Real-time PCR 检测显示,HepG2 细胞、Bel7402 细胞、 PLC 细胞、MHCC97细胞和Hep3B2.1-7 细胞这5种肝癌细胞系与L02胎肝细胞系的HLA-E mRNA 水平比较,除Hep3B2.1-7细胞表达几乎接近缺失(P<0.01)外,其余无显著差异(P>0.05)。HepG2细胞、Bel7402 细胞、 PLC 细胞、MHCC97 细胞和Hep3B2.1-7 细胞与L02 细胞的HLA-E 蛋白水平比较,差异显著(P<0.01),其中Hep3B2.1-7细胞表达缺失。结论: 肝癌细胞系的HLA-E mRNA和蛋白表达不同步,可能存在转录后调控。     

Expression of Leishmania antigen on the surface membrane of infected human macrophages in vitro.

Resolution of leishmaniasis is associated with host immunological responsiveness to parasite antigens. In clinical disease, leishmania are found as amastigotes contained with macrophages. We investigated the possibility that Leishmania antigens are expressed on the infected macrophage surface by reacting infected macrophages with antibody to Leishmania. In vitro-infected human monocyte-derived macrophages were labelled with antibody to amastigotes when examined with immunofluorescent or immunoelectron microscopic techniques. Infected macrophages were poorly labelled by antibody to promastigotes (insect forms of Leishmania). Certain antisera that reacted with the surface membranes of amastigotes did not label the infected macrophage surface. These results indicate that human macrophages infected in vitro express Leishmania amastigote antigen(s) on their surface membranes, that such antigen(s) may not be present in large quantities in promastigotes, and that certain antigen(s) on the amastigote surface are not expressed on the surface membranes of infected macrophages.     

Effect of human recombinant tumor necrosis factor on the growth of different human and mouse long-term hematopoietic cell lines

Previous studies have demonstrated that one of the most salient features of tumor necrosis factor (TNF) is its ability to induce tumor necrosis in vivo, and the specificity of its cytotoxic/cytostatic activity for tumor cells has been demonstrated in in vitro studies in which this lymphokine has been shown to kill cultured cells of malignant lines and to have no effect on cells of normal diploid lines. Studies described herein defined the effect of highly purified human recombinant TNF on cells of 34 different human and murine hematopoietic cell lines, particularly human leukemic T and B cells of long-term lymphoblastoid cultures. Results of these studies demonstrated that TNF at concentrations of 3,600 U/ml had no significant effect on the growth of these cells as defined by cytotoxicity, measured with the use of the trypan blue dye-exclusion assay and as defined by cytostasis, assayed by the enumeration of cells and the uptake of [3H]-thymidine and -uridine. In contrast, positive control cultures of TNF-sensitive cells from a murine tumor (L-M/clone L-929, connective tissue) displayed at 50% (LD50) reduction in growth by TNF at approximately 5 U/ml. Likewise, human tumors (MCF-7, breast, and HT-29, colon) were also highly sensitive (LD50 less than 100 U/ml). These studies demonstrate that T and B cells of lymphoblastoid lines as well as cells of other hematopoietic lines display little or no sensitivity to TNF.     

Identification by monoclonal antibody of a major (28 kDa) surface membrane antigen of Schistosoma mansoni

Monoclonal antibody M.1 was generated from mice immunized with membrane enriched extracts of mechanically transformed schistosomula. M.1 bound to the surface membranes of cercariae and young (0-24 h post-transformation) schistosomula but did not bind to older schistosomula or cultured worms. M.1 immunoprecipitated an antigen of approximate molecular weight 27-28 kDa from schistosomula. Experiments using metabolic labeling showed that the antigen was actively synthesized by developing schistosomula. Further M.1 immunoprecipitated a similar 27-28 kDa antigen from membrane-enriched extracts of miracidia, lung and adult worms as well as from schistosomula.     

Role of CD34 antigen in myeloid differentiation of human hematopoietic progenitor cells

CD34 is a transmembrane protein that is strongly expressed on hematopoietic stem/progenitor cells (HSCs); despite its importance as a marker of HSCs, its function is still poorly understood, although a role in cell adhesion has been demonstrated. To characterize the function of CD34 antigen on human HSCs, we examined, by both inhibition and overexpression, the role of CD34 in the regulation of HSC lineage differentiation. Our results demonstrate that CD34 silencing enhances HSC granulocyte and megakaryocyte differentiation and reduces erythroid maturation. In agreement with these results, the gene expression profile of these cells reveals the upregulation of genes involved in granulocyte and megakaryocyte differentiation and the downregulation of erythroid genes. Consistently, retroviral-mediated CD34 overexpression leads to a remarkable increase in erythroid progenitors and a dramatic decrease in granulocyte progenitors, as evaluated by clonogenic assay. Together, these data indicate that the CD34 molecule promotes the differentiation of CD34+ hematopoietic progenitors toward the erythroid lineage, which is achieved, at least in part, at the expense of granulocyte and megakaryocyte lineages.     

Restricted expression of a human T cell lineage membrane antigen

An antiserum raised against human fetal and childhood thymocytes (anti-Thy) and absorbed with peripheral lymphocytes (tonsil) detected an antigen(s) shared by thymocytes, T cell-acute leukemias, activated peripheral T cells and a subset of peripheral T cells. The antigen was expressed by the negative circulating T cell subset after mitogen activation of that separated population. The antigen was shown to be separate from the E rosette receptor, another anti-T cell serum detected antigen, and β₂-microglobulin; its expression was not related to a particular phase of the cell cycle. The results suggest that the antigen is expressed by T cells only under certain maturational and proliferative conditions.     

Expression of CD34 on human B cell precursors.

CD34 is a 110-kD glycoprotein previously shown by a variety of monoclonal antibodies (MoAbs) to be expressed selectively on immature hematopoietic cells. However, more detailed characterization of CD34+ cells has been hampered by lack of anti-CD34 MoAbs that can be labelled directly with fluorochromes to facilitate subpopulation analysis by multi-parameter flow cytometry. We have recently isolated a murine anti-CD34 MoAb, designated as 8G12, that can be directly labelled with fluorochromes such as FITC. In this study, we have exploited this property of 8G12 to compare the reactivity of 8G12 and My10 with normal and leukaemic human marrow cells and to characterize normal early human B cell precursors by two- and three-colour immunofluorescence analysis. Comparison of three-colour staining profiles of normal bone marrow cells incubated with both 8G12 and MY10, and either anti-CD10 or anti-CD19 MoAb revealed the reactivity patterns of 8G12 and MY10 to be indistinguishable. This conclusion was confirmed by a similar comparative analysis of 8G12 and MY10 staining of blood and bone marrow cells from 4 patients with B lineage acute lymphoblastic leukaemia (ALL). Of interest, both 8G12 and MY10 detected a CD34+CD10+CD19- population in normal adult bone marrow. To determine whether a CD34+CD10+CD19- precursor population previously reported by others to exist in fetal liver could also be identified, CD10+CD16- marrow cells were first isolated by FACS and the sorted cells then re-analysed for expression of CD19 and CD34. These studies showed that all of the sorted CD10+ cells that expressed CD34 appeared to coexpress CD19. No CD34+CD10+CD19- cells were detected (at a sensitivity of less than or equal to 0.1%). Further studies will be required to determine whether a very minor population of CD34+CD10+CD19- cells may still be generated in the normal development of B cells in adult human marrow.     

Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines

Background

We have devised a simple and efficient fluorescence-based method to track antigen uptake and processing in human B lymphoblastoid cells (B-LCL). Fluorescein labelled subtilisin was used to optimize antigen uptake conditions and identify processed peptides from human cell lines.

Results

Fluorescein labelled subtilisin conjugates had 0.06 to 2 moles of fluorescein per subtilisin molecule. High performance liquid chromatography and mass spectrometry (NanoESI-LC/MS/MS) analysis identified fluorescein conjugated to K141, K256, and the N terminus. Conjugates retained antigenic specificity to subtilisin specific antibodies and could be processed by whole cell extracts into low molecular weight fragments at pH 5.2. Maximal antigen uptake and processing occurred when PMSF (phenylmethylsulfonyl fluoride) inhibited subtilisin conjugate was incubated with cells at 100–200 μg/ml for 16 to 24 hr. Once optimal uptake conditions were established, processed subtilisin peptides were isolated and identified from human cell lines.

Conclusion

Our studies show that FITC-conjugation provides an efficient tool to track the uptake and processing of this protease antigen and to facilitate identification of processed antigenic peptides from human cell lines.

     

The surface membrane antigen phenotype of human blood basophils

Basophils are effector cells of allergic reactions and express a unique profile of cellular antigens (Ag). Using a combined toluidine-blue/immunofluorescence staining method, we were able to study the cell membrane Ag phenotype of normal human blood basophils with monoclonal antibodies (mAbs) against established and novel CD antigens. According to previous findings, basophils express CD9 (p24), CDlla (LFA-1 alpha-chain), CDllb (C3biR), CDllc (CR4), CD13 (aminopeptidase N), CDwl7 (lactosylceramide), CD18 (beta-chain of β₂), CD25 (IL-2R alpha-chain), CD26 (dipeptidylpeptidase), CD31 (PECAM), CD35 (CR1), CD38 (T10), CD43 (leukosialin), CD44 (Pgp-1), CD45 (pan-leukocyte Ag), and CD63 (basophil activation Ag). Various novel CD Ags were detected on basophils, including membrane cofactor protein (MCP) (CD46), the N-linked glycan CD47, decay-accelerating factor (DAF) (CD55), membrane attack complex inhibitory factor (MACIF) (CD59), LFA-3 (CD58), ICAM-2 (CD 102), ICAM-3 (CD50), C5a receptor (CD88), MIC-2/E2 (CD99), and the interleukin-1 (IL-1) R type II (CD121b). These data provide further evidence that basophils express a unique profile of surface membrane receptors for cytokines and immunomodulating compounds, as well as adhesion molecules and surface glycolipids.     

EB病毒膜蛋白在CHO细胞中的表达

目的研制ＥＢ（Ｅｐｓｔｅｉｎ－Ｂａｒ）病毒基因工程疫苗。方法构建了含有Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）膜蛋白（ＭＡ）基因的重组表达质粒ｐＣＭＶ／ＭＡ，转染ＣＨＯ细胞，研究表达产物的生物学性状，及培养液中不同血清含量、冻存、Ｇ４１８对ＣＨＯ细胞分泌ＭＡ的影响。结果获得两个稳定表达ＭＡ的细胞株。免疫印迹检测表达产物的分子量约为３４０ｋＤ和２２０ｋＤ，间接免疫荧光和免疫斑点确定表达产物能与抗ＭＡ的单克隆抗体特异性结合，用薄层扫描和Ｌｏｗｒｙ法计算ＭＡ的表达量为每天１９μｇ／ｍｌ。经纯化的ＭＡ免疫小鼠，在血清中检测到抗ＭＡ的特异性抗体。结论为开发有效的表达系统用于ＥＢ病毒基因工程疫苗的生产提供有利的实验基础。     

Expression of leukocyte common antigen (CD45) on various human leukemia/lymphoma cell lines

CD45 antigen (leukocyte common antigen), a unique and ubiquitous membrane glycoprotein with a molecular mass of about 200 kDa, is expressed on almost all hematopoietic cells except for mature erythrocytes. However, the biological function of this glycoprotein still remains to be resolved. In order to clarify the role of CD45 antigen in hematopoietic cell differentiation and function, its expression on human leukemia/lymphoma cell lines was studied by membrane immunofluorescence. Thirty-eight established cell lines were analyzed using T29/33, a monoclonal antibody (MoAb) that recognizes the common epitopes of this glycoprotein molecule. Conventional cell marker studies were also carried out on these cell lines to compare their CD45 expression. It was shown that CD45 expression varies among B-lineage cells depending on cell differentiation, in contrast to its stable expression on leukemic T cell (6/6, positive) and myeloid (5/5, positive) lineage cell lines. On the other hand, only two out of six histiomonocytoid lineage cell lines were positive. Human T cell leukemia/lymphoma virus type I (HTLV-I)-associated T cell lines derived from peripheral blood leukocytes of patients with adult T cell leukemia/lymphoma (ALT/L) in Japan did not express CD45 on their cell surface. Taken together, these observations suggest that CD45 has a functional role in hematopoietic cell activation and differentiation.     

Expression of CD34 antigen in testicular mixed germ cell tumor

The expression of CD34 was investigated in 14 cases of testicular mixed germ cell tumor to elucidate the relationship between its expression and histological patterns. Seven of 12 yolk sac tumor components were focally immunoreactive for anti-CD34 antibody. Of these, five showed focal but intense CD34 staining, while the remaining two tumors showed weak staining in small clusters or isolated tumor cells. Positive immunostaining for CD34 was seen predominantly in solid and reticular patterns of the yolk sac tumor components. Seminoma, embryonal carcinoma, and choriocarcinoma components were invariably negative for CD34. If present, immunoreactivity for CD34 in yolk sac tumors is focal and variable, but useful for the distinction from other germ cell tumors.     

Expression of vaccinia virion surface tubule protein as a virus specific cell surface antigen

Summary The sequential expression of a vaccinia virus specific antigen on the surface of infected cells has been followed by¹²⁵I-labelled anti-vaccinia IgG. After an initial drop (during the first 30 minutes of infection) the amount of viral antigen at the cell surface increased steadily for the 12 hours tested. The expression of the antigen was found to depend on protein and RNA synthesis from the start, but dependent on DNA synthesis only after 4 hours. The sensitivity of the phenomenon to ultraviolet light irradiation of the virus suggests that the genetic information needed for the expression of the antigen resides in the viral genome. The antigen has been identified as the virion surface tubule, a tubule-like structure on the surface of the intact virion. It is known that vaccinia virus infection of cells starts with the fusion of the virion envelope with the host plasma membrane.It is here proposed that initially tubule from input virus is detected as viral antigen on the cell surface. Subsequently, virus tubule protein synthesisedde novo migrates and is detectable as the virus specific cell surface antigen.With 3 Figures     

Expression and activation of pro-gelatinase A by human melanoma cell lines with different tumorigenic potential

The production of various proteolytic enzymes by tumor cells facilitate the invasion of solid tumors into surrounding tissues. We examined three cell lines (M1Dor, M4Be and M3Da) derived from malignant melanoma which exhibited different abilities to grow in nude mice following subcutaneous grafting. By in vitro invasion assay using Boyden-chambers technique, we found that none of those cell lines were able to invade the Matrigel. Several studies have substantiated the role of matrix metalloproteinases (MMP), mainly gelatinases MMP-9 and MMP-2, in melanoma cell invasion. Each cell line constitutively produced MMP-2 (but not MMP-9) in its latent form only, with stronger production for the most tumorigenic cell line in vivo (M3Da). Integrity of the MMP-2 activation process was studied since MMP-2 was also recovered as zymogen at the cell plasma membrane. All cell lines secreted TIMP-1 and TIMP-2 in a constitutive manner and again, but TIMP-2 production as well as MT1-MMP expression were found inversely related to their tumorigenic potential. Plating cells onto type I or type IV collagen did not trigger pro-MMP-2 activation; on the contrary, conversion of pro-MMP-2 to its active form could be evidenced when melanoma cell lines were seeded in a three dimensional type I collagen lattice. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flow cytometric measurements of proliferation-associated nuclear antigen p105 and DNA content in non-Hodgkin's lymphomas

Non-Hodgkin's lymphomas are characterized by heterogeneity in cell kinetics, which may be related to differential expression of proliferation-associated nuclear antigens. We have performed two-color flow cytometric quantitation of nuclear antigen p105 and DNA content in nuclear suspensions from 80 paraffin-embedded lymphomas. High-grade lymphomas with diploid DNA histograms tended to express high levels of p105 in all phases of the cell cycle. Aneuploid populations, observed in 14 of 80 cases, showed high p105 expression compared with diploid cells of the same case, with the highest aneuploid:diploid p105 ratios in the high-grade group. We conclude that high-grade or DNA aneuploid lymphomas tend to have increased expression of p105; but considerable variations within each histologic subtype were observed, and the differences between histologic grades were not statistically significant. Further studies that correlate p105 expression and clinical outcome will help to determine the prognostic value of this marker.     

P21,p53蛋白和增殖细胞核抗原在膀胱癌中的表达及意义

目的研究同一膀胱癌中癌基因蛋白产物和抗癌基因蛋白产物的表达。方法应用免疫组织化学ＡＢＣ方法检测５例正常膀胱组织和５８例膀胱癌组织中增殖细胞核抗原（ＰＣＮＡ）,Ｐ２１和ｐ５３蛋白的表达水平。结果５例正常膀胱组织中未发现Ｐ２１,ｐ５３和ＰＣＮＡ的表达,５８例膀胱癌组织中Ｐ２１,ｐ５３和ＰＣＮＡ阳性检出率分别为６２．１％、５５．２％和５８．６％。表达阳性的ＰＣＮＡ和ｐ５３均定位于肿瘤细胞核内,Ｐ２１蛋白定位于细胞膜上。Ｐ２１,ｐ５３和ＰＣＮＡ阳性表达率与膀胱癌的病理分级,临床分期和患者术后生存率关系密切。结论检测ｐ２１,ｐ５３和ＰＣＮＡ有助于评估膀胱癌的预后。     

Reversible acquisition of a host cell surface membrane antigen by Trypanosoma cruzi.

The ability of Trypanosoma cruzi, the etiological agent of Chagas' disease to acquire host cell surface antigen was tested. Parasites emerging after intracellular replication in WOS sarcoma monolayers expressed a sarcoma-associated surface antigen. This antigen was deleted from these parasites after replication in the MNS control monolayer, which does not express WOS sarcoma-associated surface antigen, or by replication in cell-free medium. This type of reversible acquisition of host surface antigen has not been previously described in T. cruzi or other intracellular protozoan parasites.