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1.
The phorbolester 12-0-tetradecanoylphorbol 13-acetate (TPA) was used for the induction of differentiation in cells of the human leukemia cell line SPI-802. The cellular morphology, surface marker antigen expression, and isoenzyme profiles of four enzymes (carboxylic esterase, acid phosphatase, hexosaminidase, and lactate dehydrogenase) served as parameters for monitoring the induced phenotypical changes. TPA led to distinct alterations of the morphology and significantly affected the growth rate with cessation of cell proliferation. No major increase in the number of nitro blue tetrazolium-positive cells or aggregation of cells, phagocytosis of latex beads, adherence to plastic surface, or development of pseudopodia were observed. As TPA-treated SPI-802 cells remained negative for these markers of the monocyte-macrophage complex, it can be concluded that the cells did not differentiate into monocytes and macrophages. The immunological marker profile based on testing of 55 monoclonal antibodies, terminal deoxynucleotidyl transferase and two erythrocyte rosette tests indicated a differentiation of SPI-802 cells along the granulocytic cell lineage. This was confirmed by isoenzyme analysis, especially that of carboxylic esterase. An isoenzyme specific for monocytes and macrophages was not detected. In earlier studies it was found that SPI-802 cells produce hemoglobin upon exposure to TPA or hemin. This latter observation and the present results suggested a comparison with the two erythroleukemia cell lines K-562 and HEL. SPI-802 cells appear to have the potential to differentiate along several cell lineages. 相似文献
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Christoph M. Griessinger Andreas Maurer Christian Kesenheimer Rainer Kehlbach Gerald Reischl Walter Ehrlichmann Daniel Bukala Maren Harant Funda Cay Jürgen Brück Renate Nordin Ursula Kohlhofer Hans-Georg Rammensee Leticia Quintanilla-Martinez Martin Schaller Martin R?cken Bernd J. Pichler Manfred Kneilling 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(4):1161-1166
T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific 64Cu-monoclonal antibody (mAb)–TCR complex enables a stable labeling of T cells. The TCR–mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied 64Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.Noninvasive in vivo imaging is an emerging method that enables the examination of T-cell migration kinetics, homing patterns, and the sites of T-cell proliferation and activation. These data are needed for a better understanding of the T-cell response during autoimmune diseases, allergies, infections, and cancer (1). Furthermore, this method reveals the basic mechanisms required for the understanding of T-cell-based immunotherapies and the development and optimization of personalized therapies (2). CD4+ IFN-γ–producing T-helper cells (TH1) and IL-4–producing T-helper cells (TH2) are key players in organ-specific autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, bronchial asthma, and insulin-dependent diabetes mellitus (3). In addition, tumor-associated antigen-specific TH1 cells can inhibit tumor growth (2, 4–6). However, the exact mode of action, as well as the sites of in vivo homing, proliferation, and trafficking kinetics remain unknown for most immune cells; therefore, noninvasive imaging tools are required to visualize their location and trafficking patterns to enable organ-specific temporal quantification.For in vivo investigation of physiological lymphocyte trafficking, it is strictly required to establish a labeling method with little or no impairment to lymphocyte viability and functionality. One frequently used cell-labeling compound for PET is [64Cu]Pyruvaldehyde bis(N4-methylthiosemicarbazone) ([64Cu]PTSM), which has been applied to mouse lymphocytes, primate stem cells, and human dendritic cells (7–9). We recently established a [64Cu]PTSM-labeling protocol that minimizes the harmful effects on T-cell functions (10). Nevertheless, intracellular [64Cu]PTSM labeling continues to impair viability and functionality as well as to induce apoptosis/necrosis and DNA damage in TH1 cells. The use of T-cell receptor (TCR)-specific radiolabeled monoclonal antibodies (mAbs) as a label might minimize the harmful effects of 64Cu on lymphocytes compared with [64Cu]PTSM labeling.Our study aimed to develop a new, highly specific intracellular labeling strategy for T cells that enables whole-body, noninvasive in vivo T-cell tracking. We targeted the antigen-specific TCR using radiolabeled mAbs. We labeled chicken-ovalbumin-TCR-transgenic TH1 cells (cOVA-TCRtg-TH1) with 64Cu-DOTA–modified cOVA-TCR–specific mAbs in vitro and investigated the endocytosis-dependent intracellular accumulation of the mAb–TCR complex. We performed temporal and quantitative PET imaging of systemically transferred and labeled cOVA-TCRtg-TH1 cells in a mouse model of OVA-induced acute airway delayed-type hypersensitivity reaction (DTHR) using chicken, turkey (t), and pheasant (ph) OVA to detect cOVA-specific T-cell homing. This labeling approach was successfully transferred to different mouse TCRs and to the expression marker Thy1.2 to label T cells as well as T-cell–depleted lymphocytes of the spleen. 相似文献
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T Yoshimura T Miyanomae M Tanaka Y Iwai M Mayumi H Mikawa 《European journal of haematology》1987,39(2):136-143
A murine cultured cell line (MKM-O) was established from a tumor of a BALB/C (nu/nu) mouse that had been subcutaneously inoculated with human hepatoma tissue fragments in the same location. The MKM-O cell line was proven to be of endothelial origin by morphological examinations and positive staining with fluorescein-labeled antibody against factor VIII antigen. MKM-O cells grown in vitro produced a colony-stimulating factor (CSF) and a leukemia cell-growth factor (LC-GF). CSF from MKM-O acted on both human and murine bone marrow-derived granulocytes and macrophage colony-forming units (CFU-GM). The activity of LC-GF on human leukemia cell lines (HL-60, HSB-2, CEM, DAUDI and K562) was demonstrated both in conditioned medium (CM) of MKM-O and of rat vascular endothelial cells. The CM of MKM-O also demonstrated a limited growth promoting activity against the mononuclear cells from human cord blood and improved their survival, suggesting that endothelial cells play an important role in the proliferation and differentiation of blood cells. 相似文献
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Nephrin,podocin及α-actinin在小鼠肾小球足细胞系的表达与分布 总被引:3,自引:1,他引:3
目的 :探讨nephrin ,podocin及α actinin在小鼠肾小球足细胞系 (MPC5)的表达与分布 ,为进一步研究上述分子间的相互作用建立稳定的实验平台。 方法 :培养小鼠MPC5,以γ 干扰素诱导在 33℃传代增生 ,继而在37℃培养两周使细胞分化成熟以用于实验研究。相差显微镜观察足细胞形态 ;免疫荧光染色观察nephrin ,podocin ,α actinin及WT1在足细胞的分布 ;RT PCR检测足细胞nephrin ,podocin及α actinin 4的mRNA ;免疫蛋白印迹检测nephrin ,podocin ,α actinin及WT1蛋白。 结果 :成熟足细胞呈星形且有突起形成。免疫荧光及免疫蛋白印迹显示足细胞特异性的表达WT1分子。免疫荧光染色可见nephrin和podocin在足细胞内均呈线状分布于胞膜表面 ,α actinin呈细丝状分布于胞质内及伸出的突起中。在mRNA水平及蛋白水平均检测到nephrin ,podocin和α actinin 4表达 ,其蛋白大小分别为 180KDa,4 5KDa和 10 0KDa。 结论 :首次在mRNA水平及蛋白水平证实了小鼠MPC5能够表达nephrin ,podocin及α actinin ,为进一步研究这些分子间的相互作用奠定了基础。 相似文献
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Ottmar Janssen Barbara Heinze Claus R. Bartram Michael Pawlita Thomas Binder Hermann Heimpel Klaus Diehl Dieter Kabelitz 《Annals of hematology》1990,60(3):206-212
Summary We have established a new cell line from a patient with centrocytic B cell lymphoma. Highly purified peripheral blood B cells from patient DUL (WBC counts 158,000/µl) were infected in vitro with Epstein-Barr virus (EBV), and CD 20+ B cells were cloned into 96 well culture plates with the aid of a cell sorter autoclone device. As shown by GTG-banding and Southern blot analysis, outgrowing EBV-positive clones had the same chromosomal abnormalities and identical monoclonal IgH gene rearrangement as the original EBV-genome-negative leukemic B cell clone. Surface marker analysis with a panel of monoclonal antibodies revealed identical patterns on EBV-negative and -positive clones, with the exception of PCA 1 (reactive with plasma cells) which was negative on freshly explanted leukemic B cells but positive on EBV-converted clones.List of abbreviations
E
sheep erythrocytes
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EBV
Epstein-Barr virus
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FCC-NHL
follicular centrocytic non-Hodgkin's Lymphoma
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FCS
fetal calf serum
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LCL
lymphoblastoid cell line(s)
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mab
monoclonal antibody
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MNC
mononuclear cells
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NHL
non-Hodgkin's lymphoma
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 322/B 2)This work forms part of the Ph. D. thesis of O. Janssen 相似文献
7.
The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones has, in the past, required the isolation of cells from each of the adrenocortical zones. Indeed, the lack of a human adrenocortical cell line retaining the ability to produce any of the major adrenal steroid products has slowed studies on normal and abnormal adrenal function. This obstacle has now been largely overcome with the availability of H295 cells, which represents the first adrenocortical cell line to maintain the ability, under specified conditions, to produce all the adrenocortical steroids (i.e., mineralocorticoids, glucocorticoids, and adrenal androgens). Thus, H295 cells appear to act as pluripotent adrenocortical cells capable of being directed to produce each of the zone-specific steroids. The H295 cell line should prove to be of value in studying the molecular and biochemical mechanisms controlling adrenal Steroidogenesis. 相似文献
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Establishment and characterization of a novel myeloid cell line from the bone marrow of a patient with the myelodysplastic syndrome 总被引:2,自引:0,他引:2
Kaoru Tohyama Hiroshi Tsutani Takanori Ueda Toru Nakamura Yataro Yoshida 《British journal of haematology》1994,87(2):235-242
SUMMARY. A novel long-term cultured myeloid cell line was established from the bone marrow of a patient with myelodysplastic syndrome (MDS). This cell line, designated MDS92. proliferated in the presence of interleukin-3 or granulocyte-macrophage colony-stimulating factor and transiently in the presence of Steel factor, with a tendency for gradual maturation, and formed myeloid colonies in the semi-solid culture condition.
In addition, the MDS92 cell line represented rather complicated karyotypic abnormalities including the deletion of fifth and seventh chromosomes and a point mutation at codon 12 of the N-ras oncogene.
These characteristics of the MDS92 cell line are exclusively compatible with the property of preleukaemia. 相似文献
In addition, the MDS92 cell line represented rather complicated karyotypic abnormalities including the deletion of fifth and seventh chromosomes and a point mutation at codon 12 of the N-ras oncogene.
These characteristics of the MDS92 cell line are exclusively compatible with the property of preleukaemia. 相似文献
10.
In vitro chemically induced differentiation of the JOK-1 cell line, derived from a patient with hairy cell leukemia, has been studied. Non-induced JOK-1 cells expressed sIgM, B-1, CALLA, Ia, and C3 receptors. Following exposure to 2% DMSO, or 100 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the cells underwent a phenotypic change from lymphoblastoid to monocytoid. In the presence of TPA, they became adherent and developed dendritic processes. Associated with these changes was a decrease in sIgM and CALLA and variable expression of C3 receptors. No monocyte/macrophage-associated antigens were detected. These results suggest that the JOK-1 cell line may have a limited ability to differentiate but is not mature enough to complete the differentiation process. 相似文献
11.
Boström H Leuchowius KJ Hallböök H Nordgren A Thörn I Thorselius M Rosenquist R Söderberg O Sundström C 《European journal of haematology》2008,81(3):218-225
B-cell lymphomas/leukemias with simultaneous t(14;18)(q32;q21) and MYC rearrangements have recently been shown to constitute a separate diagnostic entity, presenting with a rapid clinical course and a very poor prognosis. We describe the establishment of an Epstein–Barr virus negative cell line, designated U-2973, from a male patient with a de novo aggressive B-cell lymphoma/leukemia and very high peripheral blast cell count. Flow cytometry of bone marrow cells and U-2973 displayed a mature B-cell phenotype, and immunostaining showed expression of MYC and BCL2. IG gene rearrangement data were consistent with a lymphoid neoplasm of germinal centre derivation. Cytogenetic studies using conventional G-banding, fluorescent in situ hybridization, spectral karyotyping and single nucleotide polymorphism array demonstrated a complex karyotype with both a t(14;18) and double translocations between MYC and a non- IG gene partner located at chromosome 12p12.1. 相似文献
12.
Andrew M. Thomson Nigel A. Roberts & William G. Wood 《British journal of haematology》1998,102(4):976-985
We have characterized a murine hybrid cell line, Bb1-3, generated by the fusion of mouse primary erythroblasts with MEL cells. It proliferated in serum-free medium and displayed a low level of spontaneous erythroid and megakaryocyte differentiation. Terminal erythroid differentiation could be induced with HMBA and DMSO and was enhanced by serum. Treatment with phorbol esters resulted in a high proportion of megakaryocytes and the expression of megakaryocytic specific lineage markers. Bb1-3 cells contain a human β-globin transgene that was expressed at levels of 20–50% of the endogenous mouse globin genes. Initially, expression was largely limited to the β-globin gene but after adaptation to serum free growth, equal expression of both the human γ- and human β-globin genes was observed. This cell line provides further evidence that the differentiation potential of mouse erythroleukaemia cells is not restricted to the erythroid lineage and should be useful to study the mechanisms underlying both developmental globin gene regulation and the terminal differentiation of bipotential erythroid/megakaryocytic progenitor cells. 相似文献
13.
A novel factor-dependent human myelodysplastic cell line, MDS92, contains haemopoietic cells of several lineages 总被引:2,自引:0,他引:2
Kaoru Tohyama Yumi Tohyama Takashi Nakayama Takanorf Ueda Toru Nakamura Yataro Yoshida† 《British journal of haematology》1995,91(4):795-799
Summary. A novel long-term cultured interleukin(IL)-3-dependent human myelodysplastic cell line, MDS92, was shown to contain several myeloid-lineage cells such as neutrophils, macrophages, eosinophils, and a small number of megakaryocyte-lineage cells. Therefore this cell line possesses at least bipotential characteristics of myeloid- and megakaryocyte-lineages. Granulocyte colony-stimulating factor clearly promoted the neutrophil alkaline phosphatase activity of MDS92 cells. To the contrary, the incidence and growth of CD41-positive cells were hardly affected by the addition of IL-6, IL-11, c-mpl ligand (thrombopoietin, TPO) or erythropoietin. TPO slightly supported the growth of CD34-positive cell fraction, but not CD41-positive cell fraction of MDS92 cells in combination with IL-3 or Steel factor. This cell line will be a useful tool for the study of MDS stem cells, but the mechanism of commitment of differentiation in MDS stem cells remains unknown. 相似文献
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B Nielsen P S Madsen A W Jensen P Hokland M Hokland 《European journal of haematology》1992,48(3):146-151
In further studying the mechanism of action of IFN-alpha in HCL, we cultured the HCL cell line JOK-1 and the IFN-sensitive Burkitt cell line Daudi with and without IFN-alpha and investigated the changes in density of a number of surface antigens by use of mAb and flow cytometry analyses. During culture with IFN-alpha, reproducible changes were induced in both cell lines, which were qualitatively similar but differed quantitatively with small and transient changes in JOK-1. Significant decreases in surface antigen expression were observed for CD 19, 23, 37, and for IgM on both cell lines. Moreover, decreases were seen for CD 10, 22, 45, and MHC class II on Daudi, and for CD 20, 21, 27, and 40 on JOK-1. By contrast, only a few antigens increased in density, including CD 39, A96/G8 and SC9, on both cell lines, CD 22 on JOK-1, and CD 21 on Daudi. The increase in CD 39, A96/G8 and SC9 was probably directly related to the mechanism of action of IFN-alpha, whereas the other changes were most consistent with an unspecific inhibition of protein synthesis, possibly due to an accumulation of cells in G0, even though a differentiating effect cannot be ruled out. Thus, the unique in vivo effect of IFN-alpha in HCL was not paralleled by a specific direct effect on JOK-1 in vitro. Our findings therefore do not support the theory that IFN's mechanism of action in vivo is a direct effect on HC, but suggest that indirect effects are involved. 相似文献
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Daniele Perna Florian A. Karreth Alistair G. Rust Pedro A. Perez-Mancera Mamunur Rashid Francesco Iorio Constantine Alifrangis Mark J. Arends Marcus W. Bosenberg Gideon Bollag David A. Tuveson David J. Adams 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(6):E536-E545
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Acute lymphoblastic leukemia with the phenotype of a putative B-cell/T-cell bipotential precursor 总被引:2,自引:0,他引:2
Biphenotypic acute leukemias (BALs) are uncommon. Most are of myeloid-B-cell or myeloid-T-cell lineage. We report herein a 70-year-old man with an unusual acute leukemia where the blasts expressed both B- and T-lymphoid markers. He presented to us with an enlarging cutaneous tumor. The presenting peripheral blood and bone marrow aspirate showed 40% and 90% blasts, respectively, which were negative for the usual cytochemical stains. The flow cytometric analysis revealed that the blasts were positive for CD19, CD20, CD22, cytoplasmic (Cyt) CD79a, CD10, Cyt CD3, CD5, CD7, CD4, HLA-DR, TdT, and were negative for myeloid markers. According to the scoring system from the European Group for the Immunological Characterization of Acute Leukaemias (EGIL), this case was an unequivocal B-cell/T-cell BAL. Conventional cytogenetic analysis revealed 46XY [t(4;11)(q31;q13), add(8)(q24), der(9)del(9)(p21)del(9)(q32q34), -13, +mar] in all 25 metaphases analyzed. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) for 11q23 rearrangements as well as t(9;22) were negative. PCR for both TCR-gamma and IgH gene analyses revealed polyclonal rearrangements. We postulate that this case of BAL might have arisen from the putative common lymphoid progenitor cell. 相似文献
19.
M. Yasunaga R. Ryo Y. Konaka S. Sawada H. Taniguchi H. Sawada M. Goto J. Jikai W. Sugano K. Saigo N. Yamaguchi 《Annals of hematology》1994,68(3):145-151
Summary A 25-year-old man noted swelling of the right cervical lymph nodes in October 1983. Diagnosis of malignant lymphoma was made on the basis of pathological examination of biopsies. Despite both chemotherapy and irradiation treatment, blast cells appeared in the peripheral blood and bone marrow in April 1984. Immunophenotypic analysis demonstrated that the blasts in the patient's peripheral blood expressed CD13, CD33, CD41a, and no markers for T or B lymphocytes, suggesting that he had been suffering from megakaryocytic sarcoma. We established a new cell line derived from the blasts in the peripheral blood, designated KH184. KH184 cells expressed glycoprotein (GP) Ib (CD42b) and GPIIb/IIIa (CD41a), while platelet peroxidase (PPO) activity was negative in an ultrastructural study. Both Northern blot and flow cytometric analysis of surface antigens and DNA content revealed that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) did not induce the maturation of these cells. Various cytokines such as interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) had no effect in promoting the growth of KH184 cells. KH184 cells expressing CD41a seem to possess unusual characteristics. KH184 cells, human GPIIb- and GPIIIa-positive leukemia cells, which lack response to TPA-induced differentiaton, provide a new and unique model for the characterization of factors that are implicated in the terminal differentiation of megakaryocytes, and should aid in studies of the mechanism underlying the occurrence of megakaryocytic sarcoma. 相似文献
20.
Cecilia Gidlf Barbro Carlson Mikael Dohlsten Thomas H. Ttterman 《European journal of haematology》1998,60(4):233-239
Abstract: Bacterial superantigens (SAgs) bound to MHC class II molecules on target cells are efficient activators of cytotoxic T cells expressing certain T cell receptor (TCR) Vβ regions. We described earlier that the specificity of the SAg Staphylococcus enterotoxin A (SEA) can be changed by introducing a D227A point mutation in the major MHC class II binding site and by genetically fusing the SEA mutant (SEAm) to protein A (PA). This SEAm-PA fusion protein can then be used to direct cytotoxic T cells to tumour cells coated with monoclonal antibodies (mAbs). In this communication, we tested the PA-SEAm fusion protein together with mAbs against the myeloid cell surface antigens CD13, CD15 and CD33. A SEA-reactive T cell line was used as effector cells against 10 different myeloid leukaemic cell lines. Optimal lysis of antigen positive leukaemic cells was obtained at a PA-SEAm concentration of 1 ng/ml and effector: target cell ratios of 15 : 1. No correlation between target cell sensitivity and the level of surface antigen expression could be seen. The 6 acute myeloid leukaemia (AML) cell lines tested appeared to be more sensitive than the 4 chronic myeloid leukaemia (CML) cell lines. The sensitivity of the AML cell line HL-60 could be improved further by stimulation with TNFα. This was accompanied by increased surface ICAM-1 expression whereas specific target molecule expression (CD13, CD33) was unchanged. This suggests that sensitivity to lysis is related to the leukaemic subtype and ICAM-1 expression but not to the tumour antigen density. Our results show that it is possible to direct cytotoxic T cells to myeloid leukaemia cells by using SAgs linked to mAbs, and encourage the construction and testing of a recombinant direct SAg-mAb fusion protein as a candidate drug for therapy of myeloid leukaemias. 相似文献