发育心肌和心肌干细胞分化后心肌特异蛋白表达的比较研究

为探讨MCSC移植修复缺血性心肌的可能性,观察了大鼠发育心肌和MCSC分化为心肌细胞的cTnT和Cx-43表达特点。结果表明从胚胎发育至成年,心肌cTnT mRNA表达逐渐上调;用BMP-2诱导1周的MCSC可检测到cTnT mRNA表达,3～4周表达显著升高,诱导2周能观察到cTnT蛋白,3～4周可见cTnT呈现横纹样结构。胚胎第11 d心肌可检测到Cx-43 mRNA表达,生后7 d达高峰,以后逐渐降低,17 d趋于稳定。胚胎期Cx-43蛋白多分布于肌膜下,生后10 d位于细胞连接处。诱导的MCSC中Cx-43 mRNA表达特征与cTnT mRNA相似,2周可观察到Cx-43蛋白,3～4周可见Cx-43位于相邻细胞连接处。本研究结果提示,在BMP-2诱导下MCSC可分化为心肌细胞,表现为成熟心肌细胞的结构特征。MCSC有望成为治疗缺血性心脏病的理想种子细胞。     

全反式维甲酸体外诱导P19细胞向心肌方向分化

目的探讨不同浓度全反式维甲酸(all-trans retinoic acid,atRA)诱导P19细胞向心肌分化的效力。方法细胞分成P19细胞组,2nm/L atRA诱导组,5nm/L atRA诱导组,8nm/L atRA诱导组。各组细胞经过诱导、聚集培养、聚集体贴壁培养10天后,RT-PCR检测GATA-4,α-肌球蛋白重链(α-myosin heavychain,α-MHC)的mRNA表达,免疫荧光双标检测α-sarcomeric actin和cTnT蛋白共表达,Western blot检测cTnT的蛋白表达。结果 atRA可诱导聚集P19细胞表达GA-TA-4、a-MHC mRNA;α-sarcomeric actin和cTnT的表达和共表达增加;5nm/L atRA组,8nm/L atRA组GATA-4、a-MHCmRNA的表达量显著高于P19细胞组;5nm/L atRA组,8nm/L atRA组两种蛋白的表达和共表达量显著高于P19细胞组,以5nm/L atRA组最高,显著高于其它组。结论 atRA可诱导聚集P19细胞向心肌分化,其中5nm/L atRA组效果最好。     

抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。     

牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定

为制备牛病毒性腹泻病毒(BVDV)糖蛋白E2单克隆抗体(MAb),利用原核表达并且纯化的重组糖蛋白E2(rE2)免疫BALB/c小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合.采用以BVDV为检测抗原的间接ELISA筛选阳性细胞克隆,经3次克隆纯化后获得2株稳定分泌抗E2特异性MAb的杂交瘤细胞株,分别命名为4E3与1G11.用4E3与1G11杂交瘤细胞株接种BALB/c小鼠制备腹水,采用rE2及BVDV包被的ELISA测得的效价分别是6.21×106和6.83×105及6.83×105和7.5×104.间接ELISA、Western blot、IFA试验表明两株杂交瘤细胞所分泌的MAb具有良好的反应性和特异性.经抗体亚类鉴定4E3与1G11均为IgM/K.特异性试验表明4E3与1G11这2株MAb均不与牛传染性鼻气管炎病毒、牛副流感病毒3型、牛腺病毒3型反应;其中4E3不与猪瘟病毒反应,而1G11则可与猪瘟病毒发生交叉反应,这种反应特性可试用于BVDV与猪瘟病毒的鉴别诊断.所制备的4E3与1G11 MAb可以用于BVDV抗原的检测,为建立检测BVDV E2蛋白血清抗体的ELISA奠定了基础.     

关岭牛MyoD玉基因启动子报告质粒的构建及活性验证

为了克隆关岭牛MyoD玉基因启动子并验证其启动活性,根据GenBank中牛的MyoD玉基因序列设计PCR引物,用PCR技术扩增牛MyoD玉基因的启动子,构建重组克隆载体pUCmT-MyoD玉;并通过PCR扩增、限制性酶切、测序及生物信息学分析对阳性克隆进行鉴定;构建报告质粒pGL3-MyoD玉,并将其转染小鼠C2C12、3T3-L1细胞系,检测其24 h后的双荧光素酶活性。实验结果获得了关岭牛MyoD玉基因启动子,其序列长度为993 bp,并成功构建了MyoD玉启动子报告质粒;双荧光素酶活性检测表明pGL3-MyoD玉在小鼠C2C12细胞中的表达是pGL3空载体40.65倍,在小鼠3T3-L1细胞中的表达是空载体的1.13倍,MyoD玉启动子在小鼠C2C12细胞中的表达高于3T3-L1细胞(**p<0.01)。结果表明关岭牛MyoD玉基因启动子具有启动活性,在小鼠骨骼肌细胞中特异性表达。     

牛血清在百日咳毒素CHO细胞簇聚试验中的影响

目的观察不同胎牛血清对百日咳毒素在中华仓鼠卵巢细胞(Chinese hamster ovary cell,CHO)细胞簇聚试验中的影响。方法分别用两个厂家共6批次的牛血清培养CHO细胞,连续传3代后进行细胞簇聚试验,观察添加PT阳性对照、纯化PT、脱毒PT后其细胞的簇聚效果。结果 1、2号牛血清培养的CHO细胞生长缓慢,3~6号牛血清培养的CHO细胞生长正常。质量浓度16 ng/m L的PT阳性对照和纯化PT均未引起1~2号CHO细胞簇聚;3~6号牛血清培养的CHO细胞PT阳性对照判定终点分别为2、1、16、4 ng/m L,其纯化PT判定终点分别为1、0.5、8、2 ng/m L;脱毒PT质量浓度为40μg/m L时,1、2、5号牛血清培养的CHO不簇聚,而3、4、6号牛血清培养的CHO细胞脱毒PT判定终点分别为10、5、20μg/m L。结论 6种牛血清培养的CHO细胞簇聚程度存在差异,其中以4号牛血清培养的CHO细胞对PT阳性对照、纯化PT和脱毒PT最为敏感。需筛选对簇聚试验敏感度高的牛血清用于百日咳毒素CHO细胞簇集试验。     

心肌梗死大鼠钙敏感受体表达与心肌细胞凋亡的变化

目的:观察大鼠急性心肌梗死后不同时间心肌钙敏感受体(CaSR)的表达和心肌细胞凋亡的变化情况。方法:健康Wistar大鼠随机分为假手术组(Sham)和心肌梗死(AMI)组,通过结扎左侧冠状动脉前降支的方法,建立大鼠心肌梗死模型,分别在手术后1、2、4周(每组成功存活n=5)检测心脏形态学和血流动力学的改变,检测心肌组织中CaSRmRNA和蛋白的表达,以及Bax、Bcl-2、caspase-3和caspase-9蛋白的表达,检测血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)活性和肌钙蛋白(cTnT)水平,观察心肌细胞凋亡情况。结果:和Sham组相比,随着心肌梗死的发展,AMI组大鼠心肌组织CaSR的mRNA和蛋白的表达、细胞凋亡指数均明显增加(P<0.05),心肌细胞超微结构损伤严重;左心室收缩压(LVSP)、左心室内压最大上升速率(+dp/dtmax)(mmHg/s)和最大下降速率(-dp/dtmax)(mmHg/s)减少,左心室舒张末期压(LVEDP)明显增大(P<0.05);AMI组血清cTnT水平、CK和LDH活性均升高(P<0.05),随着心肌梗死的发展,cTnT水平和CK活性逐渐降低,LDH变化不明显。心肌组织中促凋亡相关蛋白Bax、caspase-3、caspase-9表达增多,抑制凋亡的相关蛋白(或因子)Bcl-2表达减少(P<0.05)。结论:随着AMI的发展,AMI组大鼠心肌组织中CaSR的mRNA和蛋白的表达增多,细胞凋亡数增加,表明CaSR参与了心肌梗死的发展,其机制可能与促进细胞凋亡有关。     

牛成纤维细胞的分离与体外培养

研究了牛胎儿和成年牛皮肤组织成纤维细胞的分离、培养、纯化方法和生长特征。通过组织块贴壁培养和分离单细胞接种培养均能获得原代牛皮肤细胞。用2.5 g/L胰蛋白酶+1mmol/L EDTA和5 g/L胶原酶I联合消化牛皮肤组织较2.5 g/L胰蛋白酶+1 mmol/L EDTA消化,得到更多的单个细胞,两者之间差异极显著(P<0.01),但其死细胞比率却有较大升高;2.5 g/L胰蛋白酶+1 mmol/L EDTA消化牛胎儿组织得到的单细胞数显著高于皮肤组织消化后得到的细胞数(P<0.01),死细胞比率也高于同种酶消化的皮肤组织。分离纯化的胎儿和皮肤成纤维细胞的生长曲线都正常且相似。2.5 g/L胰蛋白酶+1 mmol/L EDTA消化贴壁细胞后死细胞率明显高于用0.5g/L胰蛋白酶+0.53 mmol/L EDTA消化的细胞(P<0.05);培养24 h后细胞贴壁率前者要明显低于后者(P<0.05)。用0.5 g/L胰蛋白酶轻度消化混杂生长的成纤维细胞和上皮样细胞,经过反复贴壁传代2～3代,可得到较纯的成纤维细胞。     

牛轮状病毒单克隆抗体的制备及其应用

以纯化浓缩的HN-7株牛轮状病毒(牛RV,从河南腹泻犊黄牛粪样中分离)作为免疫原,免疫Balb/c小鼠,取其脾细胞与同系小鼠骨髓瘤细胞SP2/0融合,经克隆化筛选出2株(2C_7和3C_9)具群特异性的单克隆杂交瘤细胞株。2C_7与3C_9的小鼠腹水单克隆抗体(McAb)与牛HN-7、BRV007、BRV014及NCDV,猪Na86,猴SA-11和人Wa株RV细胞培养物的间接ELISA反应效价均达1∶10~5,但它们的HI和NT滴度分别≤1∶8和≤1∶100。McAb的Ig类别和IgC亚类检测结果2C_7为IgG1,3C_9为IgG2b。分别用3C_9 McAb和多克隆抗血清(PcAb)包被微量反应板进行粪样中RV抗原检测,共检测犊黄牛腹泻粪样36份、婴幼儿腹泻粪样40份,McAb和PcAb两系统检测结果的符合率分别达到97.2％(35/36)和100％(40/40)。     

牛结核γ干扰素ELISPOT检测方法的建立

本研究旨在建立一种牛结核γ干扰素ELISPOT检测方法,并评价该方法用于牛结核病检测的价值。通过筛选与天然牛γ干扰素特异性结合的单克隆抗体分别作为包被抗体和检测抗体,并探索不同的实验条件确定最佳包被抗体浓度、最佳细胞数量和最佳检测抗体浓度等,建立牛γ干扰素ELISPOT检测方法。采集30头奶牛尾静脉血并分离外周血单个核细胞,以结核菌素作为刺激原,使用建立的ELISPOT检测方法进行牛结核病检测,并与BOVIGAMTM ELISA试剂盒检测结果进行比较。筛选到两株与天然牛γ干扰素特异性结合的单抗2G5和5E11,确定ELISPOT检测方法的最佳实验条件为:包被抗体2G5浓度2.5μg/m L,每孔细胞数量2.5×105个,检测抗体Bio-5E11浓度1μg/mL。使用建立的ELISPOT检测方法与BOVIGAMTM ELISA试剂盒对30头奶牛进行同步检测,结果显示,以BOVIGAMTM ELISA试剂盒检测结果作为参考标准,14头BOVIGAMTM ELISA试剂盒检测阳性牛中,ELISPOT方法检出的阳性牛为11头,敏感性为78.6%(11/14);16头BOVIGAMTM ELISA试剂盒检测阴性牛中,ELISPOT方法检出的阴性牛为12头,特异性为75%(12/16)。应用结核菌素作为刺激原的牛结核γ干扰素ELISPOT检测方法可用于牛结核病辅助检测,具有潜在的临床应用价值。     

Differential immunomodulation by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell activation: dependence upon the individual anti-CD3 monoclonal antibody used for activation

Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent upon which anti-CD3 mAb was used as the stimulus, being more pronounced when the anti-CD3 mAb 454 was used as the stimulus than when either anti-CD3 mAb 147 or 446 was the stimulus. The effects of neuraminidase-treated sheep erythrocytes (which bind to CD2) were also more pronounced on mAb 454-induced proliferation than on mAb 147- or 446-induced proliferation. Furthermore, the effects of preincubation with anti-CD2 mAb depended upon the responder status of the donor to IgG1 anti-CD3 mAb. Preincubation of high-responder cells with anti-CD2 mAb had little effect on subsequent IgG1 anti-CD3-induced proliferation. In contrast, preincubation of low-responder cells with anti-CD2 mAb usually augmented the otherwise small proliferative response to IgG1 anti-CD3 mAb. Taken together, these observations suggest that interaction of surface CD2 with ligand alters the response of T cells to anti-CD3 mAb, but these effects depend upon the individual anti-CD3 mAb used for stimulation. These studies raise the possibility that perturbation of different parts of the CD3-T cell antigen receptor complex may lead to different sequelae, and, as a result, the T cell may respond to a given immunomodulator in different ways.     

Modulation by antiidiotypic monoclonal antibodies of immune lysis mediated by anti-HLA monoclonal antibodies

The mAb R18-9 recognizes a cross-reacting idiotope outside the Ag-combining site of the syngeneic anti HLA-DQw3 mAb KS13, whereas the mAb R1-38, KO3-34, KO3-256, and KO3-335 recognize spatially close private idiotopes within the Ag-combining site of mAb KS13. All the analyzed Id require the association of the H and L chain of mAb KS13 for their expression. The mAb R1-38 and R18-9 were shown to markedly differ in their ability to modulate immune lysis of target cells mediated by mAb KS13. mAb R18-9 did not affect C-dependent lysis of cultured B lymphoid cells WALK mediated by mAb KS13, but enhanced cell-dependent mAb KS13-mediated lysis. mAb R1-38 inhibited both C and cell-dependent lysis mediated by mAb KS13. The effect was influenced by the incubation conditions. mAb R1-38 completely inhibited lysis when it was preincubated with mAb KS13 before being added to target cells, inhibited it partially when it was added simultaneously with mAb KS13 to target cells and did not affect it when added to target cells which had been preincubated with mAb KS13. Neither mAb R1-38 nor R18-9 in combination with mAb KS13 modulated T cell proliferation induced by allogeneic HLA mismatched lymphocytes. The system we have described may represent a useful in vitro model to investigate the mechanism(s) by which antiidiotypic antibodies may influence the outcome of organs transplanted in recipients with a history of humoral presensitization to donor's HLA Ag.     

Fine specificity and idiotype diversity of a murine anti-HLA-DQw3 monoclonal antibody elicited with a syngeneic antiidiotypic monoclonal antibody

A total of 469 hybridomas was generated from a BALB/c mouse immunized with the syngeneic antiidiotypic mAb KO3-34 that recognizes an idiotope within the Ag-combining site of the immunizing syngeneic anti HLA-DQw3 mAb KS13. One of the hybridomas, named S2B154, was shown with a number of serologic and immunochemical assays to mimic the specificity of anti HLA-DQw3 mAb KS13. This result was unexpected, because the antiidiotypic mAb had been classified as gamma because of the lack of detection of anti-HLA-DQw3 antibodies in sera from BALB/c mice immunized with the antiidiotypic mAb KO3-34. The antiidiotypic mAb S2B154, an IgG1, displays a lower affinity constant to HLA-DQw3 Ag-bearing lymphoid cells than mAb KS13, an IgG2b, but a higher one to antiidiotypic mAb KO3-34. Testing with a panel of antiidiotypic mAb elicited with the mAb KS13 showed that both antiidiotypic mAb S2B154 and mAb KS13 express in their Ag-combining site the idiotopes recognized by the antiidiotypic mAb KO3-256, KO3-335, and R1-38. However, the antiidiotypic mAb S2B154 does not react with the mAb R18-9 that recognizes an idiotope outside the Ag-combining site of mAb KS13. The results we have presented question the validity of the classification of antiidiotypic antibodies based on their ability to inhibit the binding of the corresponding antibodies to Ag and on the specificity of antisera elicited with antiidiotypic antibodies. Furthermore, the hybridomas we have developed in this Id cascade provide us the opportunity to analyze the structural basis of idiotypic Ag mimicry and to evaluate the regulatory properties of antiidiotypic antibodies in the immune response to HLA class II Ag.     

Cytotoxic activity and production of toxic nitrogen oxides by macrophages treated with IFN-gamma and monoclonal antibodies against the 73-kDa lipopolysaccharide receptor.

The hamster IgM mAb 5D3 is specific for an 73-kDa LPS receptor on murine leukocytes. This mAb inhibits binding of radiolabeled LPS to splenocytes and acts as an agonist for induction of LPS-mediated changes in macrophage function. Resident peritoneal macrophages treated with IFN-gamma and mAb 5D3 developed potent cytotoxic activity against tumor cells. Cells treated with IFN-gamma or mAb 5D3 alone were inactive. Macrophage cytotoxic activity induced by IFN-gamma and mAb 5D3 was inhibited by NGMMLA and coincident with high levels of NO2-released into culture fluids. These data show that mAb 5D3 serves as an effective trigger signal for induction of cytotoxic activity with IFN-gamma-primed macrophages. Indeed, mAb 5D3 exactly mimicked the effects of LPS in these same systems. Unlike LPS, effects of mAb 5D3 on induction of macrophage cytotoxic activity and production of nitrogen oxides was abrogated after boiling, and not affected by addition of polymyxin B. The effects of LPS and mAb 5D3 as a trigger signal for IFN-gamma-primed macrophages were associated with production of TNF activity in culture fluids and inhibited by mAb against rTNF-alpha. Expression of class II MHC on macrophages induced by IFN-gamma treatment was suppressed by both LPS and mAb 5D3. These suppressive effects of LPS and mAb 5D3 were not affected by NGMMLA or mAb against rTNF-alpha. Finally, macrophages treated with LPS or mAb 5D3 before exposure to IFN-gamma and LPS or mAb 5D3 did not develop cytotoxic activity or high levels of NO2- in the culture fluids. These same cells developed both effector activities after addition of rTNF-alpha. These results in toto identify the 73-kDa protein as a receptor that mediates LPS-induced changes in macrophage effector function. The mAb 5D3 serves as a specific and defined reagent agonist for analysis of LPS receptor-linked change.     

Induction of macrophage-mediated tumor cytotoxicity by a hamster monoclonal antibody with specificity for lipopolysaccharide receptor.

Experiments have been carried out to assess the immunostimulatory activity of a hamster IgM mAb (mAb5D3) with specificity for an 80-kDa LPS-binding protein expressed on murine macrophages and monocytes. The addition of mAb5D3 to cultures of murine bone marrow-derived macrophages activated these cells to become tumoricidal for mastocytoma cells in vitro. The activity of mAb5D3 was enhanced in the presence of IFN-gamma. Neither mAb5D3 nor LPS were able to activate macrophages from the LPS-hyporesponsive C3H/HeJ mouse, although these cells responded normally to heat-killed Listeria monocytogenes. The results of several experiments establish that the observed LPS-like activity of mAb5D3 was not due to contaminating endotoxin: 1) the activity of mAb5D3 but not LPS was heat labile at 100 degrees C; 2) the activity of LPS but not mAb5D3, was inhibited by addition of polymyxin B; and 3) quantitative estimates of endotoxin contamination by Limulus amoebocyte lysate reactivity. These experiments thus demonstrate that mAb5D3 can serve as an agonist for LPS-dependent macrophage responses and, when considered with those of our companion paper showing specificity of mAb5D3 for the 80-kDa LPS-binding protein, provide strong support for the concept that the 80-kDa LPS-binding protein previously identified serves as a functional receptor for LPS on murine macrophages.     

Inhibition of cytotoxic T cell lysis by anti-CD8 monoclonal antibodies: studies with CD3-targeted cytolysis of nominal antigen-negative targets

The function of the CD8 molecule in lympholysis mediated by cytotoxic T cells was investigated by examining possible contributions of ligands on the target cell to the inhibition of lysis observed with CD8-specific mAb. In order to evaluate a variety of target cells, including those not expressing the nominal Ag (NA) for which the CTL was specific, lysis was effected by cross-linking the CTL and the target cells with anti-CD3 mAb. Such CD3 redirected cytotoxicity was demonstrated to be inhibited by anti-CD8 mAb when low anti-CD3 mAb concentrations were used. The possibility that inhibition by anti-CD8 mAb resulted for competition for the FcR between the anti-CD3 mAb and anti-CD8 mAb was eliminated by targeting TNP-modified cells with an antibody heteroconjugate prepared from Fab fragments of anti-CD3 and anti-DNP antibodies. Inhibition of the lysis of target cells not expressing NA including those deficient in class I expression, demonstrated that neither NA nor class I expression was required for anti-CD8 mAb inhibition. Whether the anti-CD8 mAb inhibition required CD8 Ag interaction with any ligand on the target cell was further investigated by measuring exocytosis of enzyme granule from CTL activated with CD3-coated poly-styrene beads. CD8-specific mAb inhibited such CTL activation in this target cell-free system. A CD8(+), MHC class II-specific CTL clone, was used to show differential inhibition by anti-CD8 mAb, depending on the target cell, therefore providing evidence that anti-CD8 mAb binding does not generate an absolute off signal. These data are consistent with the hypothesis that anti-CD8 mAb affect the lytic process independent of the recognition of a ligand on the target cell by CD8.     

Murine cryoglobulinemia: pathogenic and protective IgG3 self-associating antibodies

A murine IgG3 mAb, 6-19, derived from autoimmune MRL-lpr/lpr mice, is a rheumatoid factor (RF) specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intra-peritoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces skin leukocytoclastic vasculitis and acute glomerulonephritis associated with cryoglobulinemia. Because IgG3 interact with each other, we have determined whether noncryoprecipitating IgG3 mAb were able to inhibit the cryoprecipitation of 6-19 mAb and the development of related tissue lesions. In vitro, the cryoprecipitation of 6-19 mAb was almost completely inhibited by a fourfold excess of a noncryoprecipitating non-RF IgG3 (9-106) mAb derived from MRL-lpr/lpr mice. Cryoprecipitation of five other IgG3 mAb was similarly inhibited by the 9-106 mAb, and two other noncryoprecipitating IgG3 mAb, including the 2-6D antinuclear autoantibody, inhibited the cryoprecipitation of 6-19 mAb. In vivo, pretreatment of BALB/c mice with 9-106 or 2-6D mAb prevented the development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was greatly diminished in 9-106 or 2-6D mAb-treated mice, although their serum levels of 6-19 mAb and RF activity were comparable to those of control mice. This indicated that pretreatment with non-cryoglobulin IgG3 inhibited the cryoglobulin generation and cryoglobulin-associated tissue lesions induced by an IgG3 RF cryoglobulin-generating mAb. These results suggest that the balance of formation of IgG3 autoantibodies with or without the cryoglobulin activity may be critical for the development of IgG3 cryoglobulin-mediated tissue lesions in murine lupus, particularly in MRL-lpr/lpr mice.     

Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line

We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.     

抗GPC3 C端抗体辅助杀伤肝癌细胞HepG2的活性检测及抗原表位鉴定

目的：检测制备的7株抗磷脂酰肌醇蛋白聚糖3（GPC3）蛋白C端单克隆抗体是否具有辅助杀伤肝癌细胞的活性,并研究其识别的抗原表位。方法：用细胞增殖法检测制备的抗体是否具有抗体依赖细胞介导的细胞毒性（ADCC）活性;用生物信息软件分析GPC3蛋白C端（359～580残基）的结构及抗原特征,并据此将其分为4个截短片段,将克隆的各基因片段分别连接到原核表达载体pGEX-4T-1中,进行蛋白表达和纯化,用间接ELISA和Western印迹分析GPC3C端单克隆抗体的表位识别情况。结果与结论：制备的7株单克隆抗体对肝癌细胞HepG2均具有不同程度的辅助杀伤作用,其中5号单克隆抗体的辅助杀伤效果最好;表达并纯化了GPC3C端4个截短片段的重组蛋白;间接ELISA和Western印迹检测结果表明,7株抗体均特异性结合GPC3蛋白的473～525残基区段。     

Expression of a 33-kDa antigen Tm 1 on lymphocyte surface after modulation of cell surface antigens by IgM monoclonal antibody

The mAb Tm 1 was obtained from a fusion of SP2/O tumor cells with spleen cells from CF1 mouse immunized with T cells modulated by an IgM anti-CD3 mAb.mAb Tm 1 reacted with IgM anti-CD3 modulated T cells (66.6%) but not with unmodulated T cells (4.4%). Tm 1 was not expressed on T cells modulated with either IgG2a or IgG1 anti-CD3 mAb. Immunoprecipitation from 125I-labeled CD3-modulated T cells showed that Tm 1 Ag is a single polypeptide of 33 kDa under reducing and nonreducing conditions. Kinetic studies revealed that Tm 1 was detectable on T cells 10 min after incubation and maximally expressed after 4 h of incubation with IgM anti-CD3 mAb. CD3 expression was markedly modulated by this anti-CD3 mAb after the same period of incubation. Studies with cycloheximide revealed that Tm 1 expression on T cells does not require new protein synthesis. Tm 1 expression persisted long after CD3-reexpression 24 h later. Tm 1 was present on a small fraction of circulating T cells, B cells, and monocytes and absent from granulocytes, platelets, E, and thymocytes. Tm 1 was not expressed on T cells after various activation stimuli but was expressed on B cells upon activation. Additional studies indicate that IgM mAb against other T cell differentiation Ag and IgM mAb against B cell Ag also lead to the expression of Tm 1 on these cells. Thus, modulation of surface Ag by IgM mAb externalizes this cytoplasmic Ag. However, one exception has been noted. Purified mAb Tm 1 was not mitogenic and was unable to block either the T cell proliferation induced by 12-O-tetradecanoyl phorbol-13-acetate plus anti-CD3 mAb and other T cell stimuli, or the B cell proliferation induced by B cell mitogens. The role of Tm 1 on lymphocyte function remains to be determined.