A recombinant PSMA-specific single-chain immunotoxin has potent and selective toxicity against prostate cancer cells

Prostate cancer is the most commonly diagnosed form of cancer and the second leading cancer-related death among men in the Western civilization. Since no effective therapy exists for this tumor after progression beyond resectable boundaries, there is an urgent need for new treatment strategies. Prostate specific membrane antigen (PSMA) represents an excellent target on prostate cancer cells, and therefore specific immunotherapy may be a novel therapeutic option for the management of this tumor. We constructed a fully recombinant immunotoxin (A5-PE40) from a single-chain antibody fragment (scFv) against cell-adherent PSMA and a truncated form of Pseudomonas exotoxin A (PE40) lacking its natural binding domain Ia. The scFv A5 was obtained from a mAb elicited with native PSMA by phage display technology and direct selection on cells carrying the antigen. The bacterially expressed and purified immunotoxin A5-PE40 specifically binds to PSMA-positive prostate cancer cells and induces a 50% reduction of viability (IC50) at a concentration of 20 pM, while PSMA-negative cells remain unaffected. Due to its high and specific toxicity this recombinant immunotoxin is a promising candidate for therapeutic applications in patients with prostate cancer.     

SLE患者血清中SARS-CoV抗体阳性原因分析

为了探讨严重急性呼吸综合征(SARS)冠状病毒(SARS-CoV)抗体测定在系统性红斑狼疮(SLE)患者中的假阳性问题,应用酶联免疫吸附试验(ELISA)和荧光定量RT-PCR技术检测了66例正常对照和31例SLE患者血清中SARS-CoV抗体的阳性率。结果,66例正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3.0%(2/66);31例SLE患者中,IgM抗体和IgG抗体阳性率分别为29%(9/31)和58.1%(18/31),IgG抗体和IgM抗体同时阳性为22.6%(7/31)。经RTPCR检测,上述阳性病例均为阴性。结论:用非纯化抗原制备的ELISA试剂盒测定SLE患者的SARS-CoV抗体,可能出现假阳性,两种抗体同时测定可降低诊断的假阳性率,提高诊断的特异性。在SLE患者中出现假阳性的原因可能与包被的抗原有关。     

SLE患者血清中SARS—CoV抗体阳性原因分析

为了探讨严重急性呼吸综合征(SARS)冠状病毒(SARS—CoV)抗体测定在系统性红斑狼疮(SLE)患者中的假阳性问题,应用酶联免疫吸附试验(ELISA)和荧光定量RT—PCR技术检测了66例正常对照和31例SLE患者血清中SARS—CoV抗体的阳性率。结果,66例正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3．0％(2／66);31例SLE患者中,IgM抗体和IgG抗体阳性率分别为29％(9／31)和58．1％(18／31),IgG抗体和IgM抗体同时阳性为22．6％(7／31)。经RT—PCR检测,上述阳性病例均为阴性。结论：用非纯化抗原制备的ELISA试剂盒测定SLE患者的SARS—COV抗体,可能出现假阳性,两种抗体同时测定可降低诊断的假阳性率,提高诊断的特异性。在SLE患者中出现假阳性的原因可能与包被的抗原有关。     

肿瘤患者血清中SARS-CoV抗体阳性原因分析

探讨SARS冠状病毒(SARS—CoV)抗体在SARS病原学诊断中的特异性及其在肿瘤患血清中的假阳性问题。应用ELISA和荧光定量RT-PCR技术检测了111例正常对照和40例肿瘤患血清中SARS—CoV抗体的阳性率。在111例正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3．6％(4／111);IgG抗体诊断SARS的特异性为96．4％,两种抗体同时阳性诊断SARS的特异性为100％。40例肿瘤患中,IgM抗体均阴性,IgG抗体阳性率17．5％(7／40)。经RT—PCR检测,上述肿瘤患阳性病例均为阴性。结果表明,同时测定SARS—CoV的两种抗体可降低诊断的假阳性率,提高诊断的特异性。用非纯化SARS—CoV抗原制备的ELISA试剂盒测定肿瘤患的SARS—CoV抗体,可能出现假阳性。在肿瘤患中出现假阳性的原因可能与包被的抗原有关。     

Development and Characterization of an Antibody-Labeled Super-Paramagnetic Iron Oxide Contrast Agent Targeting Prostate Cancer Cells for Magnetic Resonance Imaging

In this study we developed, characterized and validated in vitro a functional superparagmagnetic iron-oxide based magnetic resonance contrast agent by conjugating a commercially available iron oxide nanoparticle, Molday ION Rhodamine-B Carboxyl (MIRB), with a deimmunized mouse monoclonal antibody (muJ591) targeting prostate-specific membrane antigen (PSMA). This functional contrast agent is intended for the specific and non-invasive detection of prostate cancer cells that are PSMA positive, a marker implicated in prostate tumor progression and metastasis. The two-step carbodiimide reaction used to conjugate the antibody to the nanoparticle was efficient and we obtained an elemental iron content of 1958±611 per antibody. Immunofluorescence microscopy and flow cytometry showed that the conjugated muJ591:MIRB complex specifically binds to PSMA-positive (LNCaP) cells. The muJ591:MIRB complex reduced cell adhesion and cell proliferation on LNCaP cells and caused apoptosis as tested by Annexin V assay, suggesting anti-tumorigenic characteristics. Measurements of the T2 relaxation time of the muJ591:MIRB complex using a 400 MHz Innova NMR and a multi-echo spin-echo sequence on a 3T MRI (Achieva, Philips) showed a significant T2 relaxation time reduction for the muJ591:MIRB complex, with a reduced T2 relaxation time as a function of the iron concentration. PSMA-positive cells treated with muJ591:MIRB showed a significantly shorter T2 relaxation time as obtained using a 3T MRI scanner. The reduction in T2 relaxation time for muJ591:MIRB, combined with its specificity against PSMA+LNCaP cells, suggest its potential as a biologically-specific MR contrast agent.     

丙肝病毒IgM抗体检测方法的初步研究

选择东燃公司的重组结构区和非结构区抗原建立的抗ＨＣＶ－ＩｇＭ检测方法,简便、快速、特异性强、重复性好、敏感性高。只在丙肝病人组检出而健康献血员均为阴性,与抗ＨＡＶ、ＨＢＶ的ＩｇＭ抗体无交叉反应,且排除了ＲＦ干扰和ＩｇＧ占位引起的假阳性和假阴性,适用于抗ＨＣＶ－ＩｇＭ的临床检测。对２４例丙肝病人的抗ＨＣＶ－ＩｇＭ检测结果显示,急性丙肝病人血清抗ＨＣＶ－ＩｇＭ检出率较高（７５％,６／８）,且随ＡＬＴ正常而消失或滴度下降。慢性病人抗ＨＣＶ－ＩｇＭ检出率为５６．３％（９／１６）,其中７例ＩｇＭ持续阳性者为慢性活动性丙肝,说明慢性病人抗ＨＣＶ－ＩｇＭ与疾病的活动性密切相关。结果提示抗ＨＣＶ－ＩｇＭ的检测在急性肝炎的诊断及慢性丙肝的预后和转归上具有临床意义。     

肠道病毒EV71 IgM抗体参考品的建立

目的建立肠道病毒EV71 IgM抗体检测用参考血清盘。方法通过收集手足口病感染早期患者的咽拭子和血清,对咽拭子病毒进行分离、鉴定、基因分型,通过血清中和试验及酶联免疫吸附试验(捕获法)的验证,确定EV71 IgM抗体阳性样品,组建EV71 IgM抗体参考血清盘,并经3家实验室对该参考血清盘进行协同标定和确认。结果 12名患儿的咽拭子中均分离到EV71病毒,在RD细胞上引起细胞病变。通过套式PCR扩增、序列测定后进化树分析确认均为流行的C4基因亚型。每名患儿的双份血清均可中和EV71病毒,抗体效价为1∶512~1∶2048。通过捕获法进行EV71 IgM抗体检测结果均为阳性。以此12份样品为原料制备EV71 IgM抗体阳性参考品,同时以12份EV71 IgM抗体阴性血清制备阴性参考品,建立了由12份阳性参考品、12份阴性参考品、1份最低检出限参考品和1份精密性参考品组成的肠道病毒EV71 IgM抗体参考血清盘。通过3家实验室协同标定,各实验室间差异均无统计学意义。结论建立了EV71 IgM抗体检测参考血清盘,为相关检测试剂的质量控制和评价提供了参考标准。     

Expression of prostate specific membrane antigen (PSMA) in prostatic adenocarcinoma and prostatic intraepithelial neoplasia

The prostatic membrane antigen (PSMA) is a protein that is expressed in the prostatic epithelium. We studied the expression of PSMA in a series of 55 patients with different stages of prostate cancer and we compared the PSMA staining in prostate cancer cells, in high-grade prostatic intraepithelial neoplasia (PIN) and in histologically benign prostatic epithelium for the same specimen. For this purpose archival paraffin-embedded specimens were studied by immunohistochemistry with a monoclonal antibody 7E11-C5.3 against PSMA using the streptavidin-biotin method. The mean percentage of PSMA immunoreactivity was 56.67% in prostate cancer (CaP) cells, and 48.6% in PIN cells, which was significantly higher than benign-appearing prostatic epithelium (5.72%) (for each pair, p<0.001). PSMA expression was greater in CaP with a higher Gleason score (p=0.01), but no relationship was found with serum PSA value. We conclude that PSMA overexpression is detected in high-grade PIN and is associated with a higher Gleason score of prostate cancer. It is a potential marker for studying carcinogenesis and progression of prostate cancer.     

Expression of Prostate-Specific Membrane Antigen in Lung Cancer Cells and Tumor Neovasculature Endothelial Cells and Its Clinical Significance

BackgroundProstate-specific membrane antigen (PSMA) has been found in tumor neovasculature endothelial cells (NECs) of non-prostate cancers and may become the most promising target for anti-tumor therapy. To study the value of PSMA as a potential new target for lung cancer treatment, PSMA expression in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) tissues and its relationship with clinicopathology were investigated in the current study.MethodsImmunohistochemistry was used to detect PSMA expression in a total of 150 lung specimens of patients with lung cancer. The data were analyzed using univariate and multivariate statistical analyses.ResultsThe percentages of NSCLC patients who had PSMA (+) tumor cells and PSMA (+) NECs were 54.02% and 85.06%, respectively. The percentage of patients younger than 60 years old who had PSMA (+) tumor cells was 69.05%, which was significantly greater than the percentage of patients aged 60 years or older (40.00%, p<0.05). A significant difference was observed in the percentage of NSCLC patients with PMSA (+) NECs and stage I or II cancer (92.98%) and those patients with stage III or IV cancer (76.77%). In the SCLC tissues, NEC PSMA expression (70.00%) did not differ significantly from NSCLC. SCLC tumor cells and normal lung tissues cells were all negative. There was no significant correlation between the presence of PSMA (+) NECs in SCLC patients and the observed clinicopathological parameters.ConclusionsPSMA is expressed not only in NECs of NSCLC and SCLC but also in tumor cells of most NSCLC patients. The presence of PSMA (+) tumor cells and PSMA (+) NECs in NSCLC was negatively correlated with age and the clinicopathological stage of the patients, respectively.     

Expression of prostate specific membrane antigen in androgen-independent prostate cancer cell line PC-3

During the progression of prostate cancer from androgen-dependence or sensitivity to androgen-independence, the overall expression of prostate specific membrane antigen (PSMA) increases with its appearance in plasma membrane. However, surprisingly some androgen-independent metastatic prostate cancer cell lines do not express this protein. Estradiol (E2) and basic fibroblast growth factor (bFGF) due to their recognized and strong involvement in prostate growth, development, and pathology were selected with the aim of restoring the expression of PSMA in markedly dedifferentiated prostate cancer PC-3 cells and in Du 145. E2 (10(-7)-10(-11)M) and bFGF (10ng/ml) stimulated the expression of mRNAs for PSMA (2- to 4-fold increase) that apparently were further translated and processed to its membrane form in LNCaP, PC-3, and Du 145 cells. The values of interaction force between the same anti-PSMA antibodies and all studied cells were almost identical (45-64pN), indicating antigenic similarity of the membrane form of PSMA expressed in LNCaP, PC-3, and Du 145 cells.     

用人工合成的丁型肝炎病毒抗原多肽检测丁肝病毒IgM抗体及其初步应用

用人工合成的丁型肝炎病毒抗原（ＨＤＶ－Ａｇ）肽建立了检测抗ＨＤＶ－ＩｇＭ抗体的ＥＬＩＳＡ方法,本法操作简便、快速,重复性好,特异性强,与抗ＨＡＶ－ＩｇＭ、抗Ｈｋ－ＩｇＭ、抗ＨＢｓ－ＩｇＭ、抗ＨＣＶ－ＩｇＩ、抗ＣＭＶ－ＩｇＭ、抗ＲＶ－ＩｇＭ、类风湿因子（ＲＦ）及抗核抗体（ＡＮＡ）阳性血清均不起反应,且可被２－巯基乙醇阻断而不起反应。经初步临床应用,３１例正常人血清抗ＨＤＶ－ＩｇＭ全部阴性,２８例慢活肝患者检出率为３２．１％（９／２８）,１７例慢迁肝患者血清阳性率为１１．８％（２／１７）１８例肝癌和肝硬化病人血清阳性率为２２．２％（４／１８）这三组病人与正常对照者相比较均有显著性差异（Ｐ＜０．００１）。此外,抗ＨＤＶ－ＩｇＭ阳性血清的ＡＬＴ值均明显高于正常参考范围,提示在ＨＤＶ感染过程中,患者肝细胞进一步受损。实验结果证明,抗ＨＤＶ－ＩｇＭ是诊断ＨＤＶ感染的重要指标,对ＨＤＶ感染早期诊断具有重要价值。     

Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.     

Characterisation of disseminated tumor cells in the bone marrow of breast cancer patients by the Thomsen–Friedenreich tumor antigen

The detection of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has proved prognostic significance in all stages of the disease. Further characterisation of those cells could help to improve the biological understanding of metastases, develop targeted therapies and define surface markers for enrichment techniques. The Thomsen–Friedenreich (TF) antigen has been shown to be a tumor specific antigen in breast cancer. The aim of this study was to investigate the expression of TF on DTC-BM in 25 patients. Bone marrow samples were first double-stained by a Cy3 conjugated cytokeratin (CK) antibody (ab) A45 B/B3 (IgG) and anti-TF ab Nemod 2 (IgM), followed by Cy2 conjugated goat anti-mouse IgM ab. For further characterisation samples were also double-stained with anti-TF ab Nemod 2 (IgM), followed by Cy2 conjugated goat anti-mouse IgM ab, and anti MUC1 ab A76-A/C7 IgG, followed by Cy3 conjugated goat anti-mouse IgG. CK positive DTC-BM showed co-expression of TF antigen in 22/23 patients (96%) and 61 of 62 detected cells (98%). Mononuclear BM cells without CK expression were also negative for TF. All of the TF positive cells showed strong MUC1 expression. This is the first study showing co-expression of CK and TF as markers of DTC-BM. Double staining experiments of TF and MUC1 expression showed that MUC1 is the carrier protein of TF in these cells. As TF is a specific marker of DTC-BM, it could be used as a target for antibody based therapy and immunomagnetic enrichment techniques for the isolation of DTC-BM.     

Activation of human B cells and inhibition of their terminal differentiation by monoclonal anti-mu antibodies

Anti-mu antibody preparations have been found to exert both positive and negative effects on B cell activation and differentiation. To explore these paradoxical influences of IgM cross-linkage on human B cells, three gamma 1 kappa murine monoclonal antibodies specific for human mu-chains (DA4.4, AB6.4, 145.8) were examined for their comparative effects on activation of B cells and inhibition of terminal plasma cell differentiation. All three antibodies appeared equally efficient in immunoprecipitation of surface IgM molecules; however, fluorescence-activated cell sorter analysis revealed that the DA4.4 and AB6.4 antibodies saturated the B cell surface IgM at slightly lower concentrations than did the 145.8 antibody. When the affinity-purified antibodies were added in varying concentrations to cultures of small resting B cells, all three antibodies induced B cell enlargement and DNA synthesis, but with varying degrees of efficiency (DA4.4 greater than AB6.4 much greater than 145.8). In striking contrast, large B cells isolated either by FACS or density gradient separation were unresponsive. The anti-mu-induced proliferative response of small B cells required relatively high B cell densities, but not T cells or the Fc portion of the antibody molecules. The maximal proliferative response was obtained during the third day of culture, and the response curve suggested that anti-mu induced only one round of B cell replication. All three antibodies were capable of completely inhibiting T cell factor-induced differentiation of large B cells into IgM plasma cells; both F(ab')2 fragments and intact anti-mu antibodies were effective in final concentrations as low as 1 microgram/ml. Significant suppression of IgG and IgA plasma cell differentiation was also achieved, but required higher concentrations of the anti-mu antibodies. For each antibody, there was a close correlation between the efficiency of inducing small B cell proliferation and of inhibiting large B cell differentiation into plasma cells. The results show that the B cell response to cross-linkage of cell surface IgM varies according to the differentiation stage. We postulate that the mature resting B cell represents the only stage in the life history of the B cell during which surface Ig cross-linkage leads to a positive signal, negative signals being the rule at other stages in B cell replication and differentiation.     

三种基因转导方法在不同代龄复制性衰老细胞中的比较研究

以增强型绿色荧光蛋白 (EGFP)为报告基因 ,检测腺病毒、逆转录病毒和脂质体对 2 0代和 33代WI 38细胞的转导效率、EGFP的表达强度、对细胞增殖能力和生物学行为影响 ,比较病毒及非病毒方法对不同代龄成纤维细胞 (WI 38)细胞基因转导的特性 .结果显示 :对 2 0代和 33代WI 38细胞,腺病毒转染效率均最高 ,逆转录病毒其次 ;EGFP表达强度腺病毒最高 ,逆转录病毒最低 ;各方法2 0代细胞转导效率及表达强度均高于 33代细胞 (P <0 0 5 ) .逆转录病毒对细胞繁殖和SA β gal染色没有影响 ,腺病毒和脂质体均能导致细胞增殖能力下降 ,SA β gal染色阳性率上升 ,尤以脂质体明显 (P <0 0 5 ) .表明逆转录病毒介导基因对WI 38细胞衰老进程几乎没有影响 ,且转导效率较高 ,适于衰老细胞转基因研究     

IgM antibody against hepatitis B core antigen (IgM anti-HBc): diagnostic and prognostic significance in acute HBsAg positive hepatitis.

IgM antibody against hepatitis B core antigen (IgM anti-HBc), a marker of recent hepatitis B virus infection, was sought by radioimmunoassay in sera diluted 1/4000 from 376 patients presenting to four centres in Italy with acute, apparently type B hepatitis (hepatitis B surface antigen (HBsAg) positive). In 320 patients (85%) a positive IgM anti-HBc test result confirmed that hepatitis was due to primary infection with hepatitis B virus. In the remaining 56 patients absence of the IgM marker indicated that they were previously unrecognised long term carriers of HBsAg. Further serum analysis often showed delta infection and occasionally hepatitis A or cytomegalovirus infection as the true cause of their illness. After six to eight months circulating HBsAg persisted in 38 of 45 patients (84%) without IgM anti-HBc but in only six of 150 patients (4%) with the IgM antibody (p less than 0.0001). A negative IgM anti-HBc test result in patients with acute HBsAg positive hepatitis points to a factor other than hepatitis B virus as the cause of the liver damage and predicts the carriage of HBsAg.     

用间接免疫荧光法检测流行性出血热病人尿中特异性抗体的研究

用间接免疫荧光法检测110例不同病程、病期及病型的流行性出血热病人尿中及血清中特异性抗体。尿中IgM型抗体阳性率为62.7％。尿中IgG型抗体阳性率91.8％与血清IgG型者90.9％相似,而总阳性率(IgG或IgM有一项以上阳性者的总检出率)99.1％则高于血清IgG者。20例其它疾病及10例正常人尿抗体均为阴性。结果表明尿抗体检查法是特异且可靠的,它比血清学方法简便、灵敏、为临床诊断可早期快速得出结果,不用采血有利于病人。IgM型抗体阳性率受病程、病期、病型及尿蛋白量的影响较明显。     

靶向PSMA多价纳米抗体的制备及其生物学活性表征*

目的:构建原核表达系统,制备靶向前列腺特异性膜抗原(prostate-specific membrane antigen,PSMA)多价纳米抗体并初步评价其生物学活性。方法:Bglbrick法构建多价纳米抗体表达载体,转化至大肠杆菌表达并利用亲和层析法纯化。联合蛋白质电泳和Western blot验证纯化产物,BCA法检测表达量。通过免疫荧光和流式细胞术定性评估PSMA特异性亲和能力,细胞ELISA法定量检测PSMA亲和水平,流式细胞术检测内吞效率。结果:成功构建靶向PSMA单价、二价、三价和四价纳米抗体大肠杆菌表达菌株。发酵结果表明四种纳米抗体均能在摇瓶水平实现高效可溶表达,其中二价纳米抗体表达量最高[(259.14±23.56) mg/L],单价纳米抗体表达量最低[(100.58±6.27) mg/L]。亲和实验结果证实四种纳米抗体均能特异性识别并结合PSMA阳性肿瘤细胞,与单价纳米抗体相比,二价、三价和四价纳米抗体对PSMA亲和能力分别提高了3.32倍、2.29倍和2.03倍。最后的内吞实验显示四种纳米抗体均能被PSMA阳性肿瘤细胞高效摄取,30 min内的摄取率均在80%以上。结论:靶向PSMA的多价纳米抗体,尤其是二价纳米抗体,具有比单价纳米抗体更高的产量和亲和水平,且具备不亚于单价纳米抗体的内吞效率,是未来基于PSMA肿瘤诊疗试剂开发的重要候选。