Selectivity and potency of agonists for the three subtypes of cloned human beta-adrenoceptors expressed in Chinese hamster ovary cells

The selectivities, potencies and efficacies of beta3-adrenoceptor (beta3-AR) agonists on human three beta-AR subtypes expressed in Chinese hamster ovary (CHO) cells were investigated using radioligand binding assay and cyclic AMP (cAMP) accumulation assay. The three beta-AR subtypes showed the nature of G protein-coupled receptors with the constitutive activity. BRL37344, CL-316,243 and a newly synthesized beta3-AR agonist N-5984, 6-[2-(R)-[[2-(R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-2,3-dihydro-1,4-benzodioxine-2-(R)-carboxylic acid, were compared for the potency and selectivity for the beta3-AR. In the radioligand binding assay, the affinity of N-5984 for beta3-ARs was 14, 70 and 220 times more potent than those of BRL37344, isoproterenol and CL-316,243, respectively. N-5984 had higher selectivity than BRL37344 for human beta3-ARs compared with either for beta1-ARs or beta2-ARs. N-5984 showed higher potency and intrinsic activity of cAMP production than BRL37344 in CHO cells expressing the beta3-ARs. CL-316,243 had almost no activity of cAMP production in CHO cells expressing any subtype of beta-ARs. These results indicate that N-5984 is the most potent and selective agonist for human beta3-ARs than any other agonists tested.     

Antagonism by antimuscarinic and neuroleptic compounds at the five cloned human muscarinic cholinergic receptors expressed in Chinese hamster ovary cells.

We determined the affinity and selectivity of binding for 24 compounds: nine antimuscarinics (including some antiparkinson drugs) and 15 neuroleptics (including the atypical compounds clozapine, fluperlapine, melperone, rilapine, risperidone, tenilapine, tiosperone and zotepine) at the five human muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Equilibrium dissociation constants (Kd) were obtained from competitive radioligand binding studies with [3H]quinuclidinyl benzilate and membranal preparations of these cells. As expected, QNB had the highest affinity of the compounds studied at the five receptor subtypes and was not selective (Kd ranged from 0.027-0.088 nM). Benztropine had the next highest affinity of the antimuscarinic compounds and thioridazine had the highest affinity of the neuroleptics. Among the antiparkinson drugs, biperiden was the only one selective for the m1 subtype; and among the neuroleptics, the atypical drug clozapine was also selective for the m1 subtype. This selectivity may explain clozapine's unusual efficacy in refractory schizophrenic patients and its low incidence of extrapyramidal side effects. However, because most other atypical neuroleptics studied lacked high affinity and selectivity at muscarinic receptor subtypes, it is likely that other mechanisms are involved as well.     

Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3

BACKGROUND: Alloantibodies against human neutrophil antigen‐3 (HNA‐3) are responsible for the fatalities reported in transfusion‐related acute lung injury. Consequently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA‐3 antibodies. STUDY DESIGN AND METHODS: HEK293T were transfected with HNA‐3a or HNA‐3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA‐3 antibodies in flow cytometry and antibody capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test. RESULTS: In flow cytometry, 12 of 14 HNA‐3a sera reacted specifically with HNA‐3aa cells. One serum sample showed positive reaction with HNA‐3bb cells. All HNA‐3b sera recognized HNA‐3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA‐3a sera (12/12) showed positive reactivity with HNA‐3aa cells with no cross‐reactivity with HNA‐3bb cells. Again, all HNA‐3b sera reacted with HNA‐3bb cells only. Furthermore, genotyping of 249 individuals detected a new HNA‐3 allele caused by a nucleotide substitution C>T at Position 457 leading to L₁₅₃F mutation in choline transporter‐like protein‐2. This mutation impairs polymerase chain reaction with sequence‐specific primers based HNA‐3a typing. However, analysis with cells expressing F₁₅₃ isoform showed that this mutation did not alter the binding of HNA‐3 antibodies. CONCLUSIONS: This study demonstrated that HEK293T cells expressing stable recombinant HNA‐3 are suitable for the detection of HNA‐3 alloantibodies allowing reliable screening of blood products.     

Detection of HNA-3a and -3b antibodies using transfected cell lines and recombinant proteins

BACKGROUND: HNA‐3 is a diallellic system located on choline transporter‐like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA‐3a antibodies are of clinical importance in transfusion‐related acute lung injury but antibody detection requires labor‐intensive granulocyte isolation from HNA‐typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA‐3 antibodies. STUDY DESIGN AND METHODS: HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55‐230) and full‐length membrane bound CTL2 with HNA‐3a and ‐3b epitopes. Soluble fragments were used in enzyme‐linked immunosorbent assays to detect HNA‐3 antibodies. The cell lines expressing full‐length proteins were trypsin treated to remove HLA antigens and frozen at ?80°C. Thawed cells were then used to detect HNA‐3 antibodies by flow cytometry. RESULTS: Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti‐HNA‐3a sera and by both available anti‐HNA‐3b sera. Twenty‐one anti‐HLA sera reacted variably with untreated cell lines expressing full‐length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti‐HNA‐3a and two anti‐HNA‐3b sera bound to the corresponding cell line. CONCLUSION: Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA‐3 antibodies, the HEK293 cells expressing full‐length CTL2 proteins were useful in the detection of HNA‐3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use.     

Neurokinin(3) receptors couple to the activation of neuronal nitric-oxide synthase in stably transfected Chinese hamster ovary cells

Several physiological effects induced by activation of neurokinin(3) (NK(3)) receptors are mediated by the production of nitric oxide (NO). We investigated the intracellular coupling of NK(3) receptors to NO synthase (NOS) using a Chinese hamster ovary cell line that was stably transfected with both the NK(3) receptor and type I (neuronal) NOS. NOS activity in the transfected cell line was assayed directly, by measuring the formation of L-citrulline, another product of NOS, as well as indirectly, by measuring the production of cGMP in cultured rat fetal lung fibroblasts (RFL-6 cells). MePhe(7)-neurokinin B (NKB) stimulation of L-[(3)H]citrulline production was concentration-dependent and yielded a two-site model for the concentration-response relationship. The production of L-citrulline in response to two other tachykinins, substance P or neurokinin A, revealed only a one-site nature of the response. The production of cGMP in response to MePhe(7)-NKB had an EC(50) value that corresponded to the high-potency component of MePhe(7)-NKB-induced production of L-[(3)H]citrulline. Agonist-induced calcium signaling was also concentration-dependent, and the acute increase in the production of cGMP by MePhe(7)-NKB (0.1 nM) was dependent on the release of calcium from intracellular stores. Results of this study provide the first direct evidence that NK(3) receptors couple to the generation of NO within the same cell.     

Use of Chinese hamster ovary cells with altered glycosylation patterns to define the carbohydrate specificity of Entamoeba histolytica adhesion

We compared the adherence of E. histolytica trophozoites with a panel of lectin-resistant CHO mutants with altered glycosylation patterns. Our results coupled with data from saccharide inhibition studies indicate that terminal N-acetyllactosamine units on Asn-linked complex type oligosaccharides provide the major determinants on the cellular receptor for E. histolytica adhesion.     

Highly specific enzyme immunoassay for the beta-subunit of human thyrotropin with use of monoclonal antibodies

A highly specific enzyme-linked "sandwich" immunoassay is described for determining free human thyrotropin (hTSH) beta-subunit in serum by using a anti-hTSH beta-subunit monoclonal antibody conjugated with beta-D-galactosidase (EC 3.2.1.23) and a solid phase consisting of silicone rods coated with another monoclonal antibody. We could detect as little as 0.04 ng of beta-subunit per assay. The measurable range of hTSH beta-subunit concentrations in serum was 0.4 to 50 micrograms/L. The assay demonstrated little or no cross reactivity with intact hTSH, hTSH alpha-subunit, or human choriogonadotropin. The mean CVs were 12.2% within assay, 13.9% between assay. The hTSH beta-subunit was not detectable in sera from healthy subjects, patients with hyperthyroidism, or two patients with pituitary tumors producing TSH. It was measurable (at concentrations of 0.65 to 2.70 micrograms/L) in sera from eight of 23 hypothyroid patients. In five of the hypothyroid patients examined, the concentration of hTSH beta-subunit in serum increased after administration of thyroliberin. This method may be useful in elucidating the physiological and pathological significance of the hTSH beta-subunit and in examining the function of the hypothalamus-pituitary-thyroid axis.     

Use of Epstein-Barr virus-transformed B cell lines for the generation of immunoglobulin-producing human B cell hybridomas

HGPRTase-deficient EBV-transformed B cell lines were shown to be effective fusion partners with mitogen-activated human B cells for the construction of Ig-producing human B cell hybridomas. In a series of experiments using these lines and B cells from several tissue sources, approximatley 20% of the cultures plated were consistently positive for growth after hypoxanthine-aminopterin-thymidine selection and approximatley 30% of these synthesized significant new Ig. A marked increase in Ig secretion was observed after hybridization, which was due to new Ig; Ig from the parental lime was shown to disappear in several instances. Special analyses were carried out on a human hybridoma secreting antibody specific for tetanus toxoid and tetanus toxin and stable subclones were derived. These studies suggest that EBV-transformed lines will prove useful in human hybridization studies, thus making a large library of B cell lines available for the generation of human monoclonal antibodies.     

Choice of immunoglobulin G purification method in assays for antibodies to the thyrotropin receptor

Activity of autoantibodies to the thyrotropin receptor in the serum of patients with active Graves's disease was compared when the patients' IgG was purified by three different procedures: ammonium sulfate precipitation (I), a modified batch diethylaminoethyl cellulose method (II), and affinity chromatography on Protein A-Sepharose CL-4B (III). IgG extracted by I was significantly less potent in inhibiting binding of 125I-labeled thyroid membranes than that prepared by either II or III, and was significantly less effective than II in stimulating adenyl cyclase activity in thyroid membrane. Thyroglobulin, a serum protein whose concentration is increased in patients with various thyroid diseases, was coprecipitated in amounts sufficient to significantly inhibit binding only when method I was used, but not with either of the other two procedures. Evidently method I is inferior to either of the other two when used for purification of autoantibodies to the thyrotropin receptor. Method II used in this study, being faster and more economical than I and of equivalent efficacy, is a feasible alternative method for clinical use.     

不同程序接种重组(CHO)乙型病毒性肝炎疫苗免疫效果观察

目的 评价成人按不同免疫程序接种乙型病毒性肝炎(乙肝)疫苗的免疫效果。方法 在湖州市南浔区6个乡镇街道选择16~49岁成人,对自愿参加的人群采血2 ml,分离血清,使用ARCHITETi2000化学发光免疫分析仪采用化学发光法检测乙肝表面抗原(hepatitis B surface antigen,HBsAg)、乙肝表面抗体(hepatitis B surface antibody,抗-HBs)和乙肝核心抗体(hepatitis B core antibody,抗-HBc),对HBsAg、抗-HBs和抗-HBc均为阴性者按乡镇(街道)分为A、B两组,其中A组按0,1,3月程序接种20 g重组(CHO)乙肝疫苗,B组按0,1,12月程序接种。对完成全程免疫的人群,第三针接种1个月后采血3 ml,分离血清检测抗-HBs。评价成人按不同免疫程序接种20 g重组(CHO)乙肝疫苗后抗-HBs的阳转率和抗体滴度水平。结果 共435人完成全程免疫并完成第三针后1个月采血;B组的抗-HBs阳转率和几何平均浓度(geometric mean concentration, GMC)分别为95.87%和893.53 mIU/ml;A组的抗-HBs阳性率和GMC分别为63.59%和31.99 mIU/ml;两组的抗-HBs阳性率差异有统计学意义(2=70.207,P0.001);两组抗-HBs GMC差异有统计学意义(F=89.609,P0.001)。结论 按0,1,12月程序接种乙肝疫苗可获得较好的免疫效果。     

Use of a sensitive enzyme immunoassay for human thyrotropin in the diagnosis of hyperthyroidism

A sensitive, simultaneous sandwich enzyme immunoassay for TSH was evaluated especially for its ability to distinguish hyperthyroid patients from the euthyroid population. A total of 140 patient samples was analzyed by this assay as well as with a two-step sandwich radioimmunoassay. The diagnostic sensitivity of the thyrotropin assay was 92.5% and the specificity was 88%. False negatives by thyrotropin assay included two patients with Graves' disease who were being treated with propranolol at the time of testing and one patient who was considered hyperthyroid while receiving synthroid. Twelve patients with elevated free thyroxine index levels were considered euthyroid and 50% of these had thyrotropin values that were undetectable; most were elderly patients with nonthyroidal illnesses. Although the thyrotropin enzyme immunoassay had good sensitivity and precision for the detection of hyperthyroidism, our data suggest the limitation of a single thyrotropin determination in establishing the euthyroid state, especially in elderly patients with associated nonthyroidal illnesses and hyperthyroxinemia.     

Lack of effect of high-intensity pulsed ultrasound on sister chromatid exchange and in vitro Chinese hamster ovary cell viability

The frequency of sister chromatid exchanges (SCEs) in in vitro Chinese hamster ovary cells and their viability were not affected by 3-min exposures to 2-4 MHz, focused, pulsed ultrasound with a pulse repetition rate of 200 Hz, pulse duration of 10 mu sec and intensities (SPTP) of 500 W/cm2 and 2500 W/cm2. The viability results are consistent with those reported elsewhere; the SCE response does not verify a specific previously reported positive response.     

人白血病细胞株重组激活基因表达及T细胞受体基因重排的检测

背景：淋巴细胞特异性重组激活基因编码的重组激活基因1与重组激活基因2蛋白是参与V（D）J重排机制的重要的重组酶。除参与V（D）J重排以外，近年的研究结果表明重组激活基因介导的转位作用可能与染色体易位及淋巴性恶性肿瘤的发生有关，但迄今尚未有明确定论。目的：检测重组激活基因、DNA修复因子Ku70／Ku80和末端脱氧核苷转移酶mRNA表达以及T细胞受体基因重排在人白血病和淋巴癌细胞株的发生情况。设计：重复测量实验。单位：南方医科大学生物技术学院分子免疫研究所。材料：T淋巴白血病细胞株Jurkat和6T-CEM购自上海细胞生物研究所；T淋巴白血病细胞株Molt-4，皮肤T细胞淋巴癌细胞株HuT102，Burkitt’s淋巴癌细胞株Raji和Daudi以及原髓细胞白血病细胞株HL-60和慢性髓原白血病细胞株K562均由本实验室保存。细胞用含有体积分数0．1胎牛血清的RPMI1640培养基于37℃，体积分数0．05C02条件下培养。方法：实验于2005-10／2006-01在南方医科大学生物技术学院分子免疫研究所完成。采用反转录聚合酶链反应检测重组激活基因1，重组激活基因2，非同源末端连接装置途径中的DNA修复因子Ku70／Ku80。以及末端脱氧核苷转移酶mRNA表达；采用巢式、半巢式聚合酶链反应、连接介导的聚合酶链反应等方法检测T细胞受体重排删除DNA环和T细胞受体B链重组信号序列两端的断裂点。了解参与V（D）J重排过程的基因表达和T细胞受体基因重排中间体的产生情况。主耍观察指标：重组激活基因、DNA修复因子Ku70／Ku80和末端脱氧核苷转移酶mRNA表达以及T细胞受体基因重排在人白血病和淋巴癌细胞株的发生情况。结果：反转录聚合酶链反应检测结果显示：重组激活基因1mRNA在4种T细胞株中均被检测到，在两种B细胞株和两种髓性白血病细胞株中未检测到；重组激活基因2和末端脱氧核苷转移酶mRNA表达仅在Jurkat，Molt-4和6T-CEM3种T细胞株中检测到，但在6T-CEM表达较弱；除HL-60细胞未检测到Ku80表达外，所有细胞株均检测到Ku70和Ku80表达。对4种T细胞株T细胞受体重排中间体检测结果表明：仅在Jurkat细胞中检测到DB2-J132 sjTRECs与DB25’端和3’Rss断点，表明Jurkat细胞发生T细胞受体基因重排。同时发现Jurkat TCR Dβ2-Jβ2重排删除环结合区具有明显的多样性特征。结论：重组激活基因可能与T细胞白血病具有更为密切的关系。Jurkat细胞有可能成为研究重组激活基因与T细胞淋巴性肿瘤的一个潜在的细胞模型。     

人白血病细胞株重组激活基因表达及T细胞受体基因重排的检测

背景:淋巴细胞特异性重组激活基因编码的重组激活基因1与重组激活基因2蛋白是参与V(D)J重排机制的重要的重组酶。除参与V(D)J重排以外,近年的研究结果表明重组激活基因介导的转位作用可能与染色体易位及淋巴性恶性肿瘤的发生有关,但迄今尚未有明确定论。目的:检测重组激活基因、DNA修复因子Ku70/Ku80和末端脱氧核苷转移酶mRNA表达以及T细胞受体基因重排在人白血病和淋巴瘤细胞株的发生情况。设计:重复测量实验。单位:南方医科大学生物技术学院分子免疫研究所。材料:T淋巴白血病细胞株Jurkat和6T-CEM购自上海细胞生物研究所;T淋巴白血病细胞株Molt-4,皮肤T细胞淋巴瘤细胞株HuT102,Burkitt’s淋巴瘤细胞株Raji和Daudi以及原髓细胞白血病细胞株HL-60和慢性髓原白血病细胞株K562均由本实验室保存。细胞用含有体积分数0.1胎牛血清的RPMI1640培养基于37℃,体积分数0.05CO2条件下培养。方法:实验于2005-10/2006-01在南方医科大学生物技术学院分子免疫研究所完成。采用反转录聚合酶链反应检测重组激活基因1,重组激活基因2,非同源末端连接装置途径中的DNA修复因子Ku70/Ku80,以及末端脱氧核苷转移酶mRNA表达;采用巢式、半巢式聚合酶链反应、连接介导的聚合酶链反应等方法检测T细胞受体重排删除DNA环和T细胞受体β链重组信号序列两端的断裂点。了解参与V(D)J重排过程的基因表达和T细胞受体基因重排中间体的产生情况。主要观察指标:重组激活基因、DNA修复因子Ku70/Ku80和末端脱氧核苷转移酶mRNA表达以及T细胞受体基因重排在人白血病和淋巴瘤细胞株的发生情况。结果:反转录聚合酶链反应检测结果显示:重组激活基因1mRNA在4种T细胞株中均被检测到,在两种B细胞株和两种髓性白血病细胞株中未检测到;重组激活基因2和末端脱氧核苷转移酶mRNA表达仅在Jurkat,Molt-4和6T-CEM3种T细胞株中检测到,但在6T-CEM表达较弱;除HL-60细胞未检测到Ku80表达外,所有细胞株均检测到Ku70和Ku80表达。对4种T细胞株T细胞受体重排中间体检测结果表明:仅在Jurkat细胞中检测到Dβ2-Jβ2sjTRECs与Dβ25’端和3’RSS断点,表明Jurkat细胞发生T细胞受体基因重排。同时发现JurkatTCRDβ2-Jβ2重排删除环结合区具有明显的多样性特征。结论:重组激活基因可能与T细胞白血病具有更为密切的关系。Jurkat细胞有可能成为研究重组激活基因与T细胞淋巴性肿瘤的一个潜在的细胞模型。     

Detection of cell surface and intracellular antigens by human monoclonal antibodies. Hybrid cell lines derived from lymphocytes of patients with malignant melanoma

This study represents an initial attempt to analyze the humoral immune reactions of patients with malignant melanoma by hybridoma methodology. Using lymphocytes from regional lymph nodes, peripheral blood and tumor infiltrates, 158 fusions were performed with SKO-007 (human myeloma line), LICR-LON-HMy2 (LICR-2), GM 4672 (human lymphoblastoid lines), or NS-1 (mouse myeloma line). Fusion of lymph node lymphocytes with NS-1 resulted in a 3-4 times higher frequency of clones than fusion with LICR-2, and a 10 times higher frequency than fusion with SKO-007 or GM 4672. In the case of peripheral blood lymphocytes, fusion with NS-1 gave greater than 25 times higher frequency of clones than fusion with LICR-2 or SKO-007. Production of human mu, gamma, or alpha heavy chains was detected in 50-80% of wells containing growing clones, and the levels of immunoglobulin ranged from 0.3 micrograms to 40 micrograms/ml. NS-1-derived clones could be easily subcultured, while LICR-2 and SKO-007 clones grew more slowly on subculturing. In this study, Ig secretion appeared to be a more stable property of LICR-2- derived clones than NS-1-derived clones. A panel of 20 human cancer cell lines was used to screen 771 Ig-secreting cultures for antibody to cell surface or intracellular antigens. Reactivity with cell surface antigens was found infrequently (6 cultures), whereas reactivity with intracellular antigens was more common (27 cultures). A new cell surface antigen with properties of a glycolipid was defined with an IgM monoclonal antibody secreted by a tetraploid cell derived from a fusion of LICR-2 with lymphocytes from the axillary lymph node of a patient with melanoma. The hybrid cell line has been subcloned four times and secretes 5 micrograms IgM/ml. The antigen detected by this IgM antibody was found on 5 of 23 melanoma cell lines and 12 of 30 epithelial cancer cell lines. No reactions were found with 11 cultures derived from normal cells. Stable cell lines secreting human antibody that detected nuclei, nucleoli, cytoskeletal elements, Golgi complex, or other cytoplasmic components were also isolated in this study. One of these antibodies detected an intracellular antigen that is restricted to cells of neuroectodermal derivation, and a second antibody reacted primarily with cells of epithelial origin. Using these methods to isolate and analyze human monoclonal antibody, it should now be possible to define the repertoire of the humoral immune response to melanoma.     

Detection of antigens specific for B-lymphoid cultured cell lines with human alloantisera

Human sera were tested for cytotoxicity to pairs of long-term tissue- cultured cell lines. Each pair had been derived from the same individual and one of the pairs possessed the characteristics of either "T" or "B" cells. The alloantisera used were HL-A-typing reagents or sera obtained from Amish multiparas. Selected cytotoxicity was found against the B-cell lines by direct testing. Cytotoxicity was abolished by absorption with B-cell line but not by absorption with the T-cell lines. The results suggest that a group of allotypic antigens may be expressed exculsively on human B cells.