Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity

Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.3pM, and thus were not greatly different in potency from the parent drug PT523. By comparison, the K(i) values of aminopterin (AMT), methotrexate (MTX), and 10-ethyl-10-deazaaminopterin (EDX) were 3.7, 4.8, and 11pM. In assays of competitive inhibition of [3H]MTX influx into CCRF-CEM cells, the K(i) values ranged from 0.21 to 7.3 micro M, as compared with 0.71, 5.4, and 1.1 micro M for PT523, AMT, and EDX. The K(t) for MTX was also re-analyzed and found to be 4.7 micro M, in better agreement with the literature than our previously reported value of 7.1 micro M. The IC(50) values of these compounds as inhibitors of the growth of CCRF-CEM cells after 72hr of drug exposure ranged from 0.53 to 55nM, and were qualitatively consistent with the other results.     

含新城疫病毒血凝素-神经氨酸酶基因重组腺病素的构建及鉴定

目的:构建表达新城疫病毒血凝素-神经氨酸酶(Hemagglutinin-neuramidinase,HN)基因的重组腺病毒,为进一步研究HN基因对消化道肿瘤的抑制作用及分子机制建立基础.方法:用BamHⅠ和XbaⅠ双酶切质粒pVHN,将获得的HN基因片段连接入穿梭质粒pacAd5 CMV K-N pA,构建含HN基因的穿梭质粒pacAd5-HN.利用PacⅠ单酶切对pacAd5-HN和腺病毒基因组质粒(pAd5)进行线性化处理后应用脂质体介导法共转染AAV-293细胞,分别利用蚀斑纯化和RT-PCR、Western blot等方法对重组病毒进行筛选和鉴定,并测定所获得重组病毒的滴度.结果:所构建重组病毒可有效表达HN基因,表达产物分子量约为63 kD,重组病毒滴度为108～1010 PFU/ml.结论:成功构建含HN基因的重组腺病毒,所获得重组病毒的滴度可以满足体内外实验要求.     

鸡胚成纤维细胞培养抗肿瘤NDV疫苗的实验研究

目的用细胞培养法研制高效价人用抗肿瘤NDV。方法采用鸡胚成纤维细胞低血清培养法,在接种NDV之前的细胞培养中,少量使用优质小牛血清（2%）,让单层细胞基本铺满培养瓶底时,用无血清培养液多次洗涤单层细胞,以尽量降低残余牛血清含量。接种NDV之后,以人白蛋白代替小牛血清继续滋养细胞,培养病毒。结果NDV收获液LogPFU≥5.5,血凝效价1：640,经反向间接血凝试验检测,残余牛血清蛋白含量≤50ng/ml。结论NDV效价和活性符合人用疫苗要求。     

Glycoproteins of representative paramyxoviruses: isolation and antigenic analysis using a zwitterionic surfactant.

A new method for the isolation of biologically active envelope antigens from paramyxo- and myxoviruses was developed using a zwitterionic detergent Empigen BB. Essentially complete recovery of hemagglutinin and sialidase from representative paramyxoviruses (PMY, NDV, and Sendai virus) and influenza virus X7 was achieved. The glycoproteins (HN and F) of PMY were purified in a DEAE-Bio-Gel A column equilibrated with bicarbonate buffer containing Empigen, Polyacrylamide gel electrophoresis analysis of pooled HN fractions revealed one band, indicating that it is a common glycoprotein. Gels of pooled F-enriched fractions revealed three bands corresponding to LGP, HN, and F. Gels of PMY virus revealed six polypeptides, and they have been tentatively designated L, LGP, HN, F, NP, and M. LGP, a large glycoprotein, was not detected in gels of NDV and Sendai virus. It has been proposed that LGP may consist of a complex of F. Antiserum was prepared against purified HN and it was found to be monospecific. The antiserum inhibited both hemagglutination and enzyme activities of PMY, which is in support of the hemagglutinin and sialidase of PMY being associated with a common glycoprotein. By enzyme inhibition analysis of PMY, NDV, and Sendai virus, it was shown that the enzymes of these viruses are antigenically distinct. The methods described for the isolation and purification of PMY glycoproteins may be useful for the preparation of myxo- and paramyxovirus subunit vaccines.     

双特异性重组腺病毒Ad-HT对肝癌细胞的体内、外抑制效应

目的:探讨双特异性抗肿瘤重组腺病毒Ad-HT对肝癌细胞的体内、外抑制作用和作用方式。方法:运用噻唑兰法检测重组腺病毒Ad-HT对BEL-7402细胞的抑制作用;应用AO/EB染色法、DAPI染色法、Annexin V检测、Caspase检测、线粒体膜电位检测和活性氧检测等多种方法分析Ad-HT对BEL-7402细胞抑制作用的方式和途径;构建C57BL/6小鼠荷H22肿瘤模型,瘤内注射重组腺病毒,通过非放射性乳酸脱氢酶细胞毒性检测方法,检测NK活性、CTL活性,利用酶联免疫吸附方法检测IL-2、IL-4、IL-10和IFN-γ等细胞因子水平,探讨Ad-HT对体内实体肿瘤的抑制作用和对免疫系统的影响。结果:Ad-HT能够抑制BEL-7402细胞生长,且其作用具有一定时效和剂效关系趋势;Ad-HT感染导致BEL-7402细胞磷脂膜外翻、细胞核皱缩、细胞膜通透性增加、Caspase酶活性增强、线粒体膜电位下降和活性氧水平升高;Ad-HT实验组模型动物平均期在30天以上,显著高于对照组;另外,Ad-HT具有增强NK和CTL活性,上调IL-2和IFN-γ等细胞因子水平的功能。结论:Ad-HT能够通过诱导细胞凋亡,抑制BEL-7402肿瘤细胞增殖,而这种抑制作用可能通过线粒体途径实现。另外,Ad-HT能够有效延长模型动物平均生存期,活化免疫功能细胞,并使免疫趋向Th1优势。     

Recombinant Newcastle disease virus expressing H9 HA protects chickens against heterologous avian influenza H9N2 virus challenge

In order to produce an efficient poultry H9 avian influenza vaccine that provides cross-protection against multiple H9 lineages, two Newcastle disease virus (NDV) LaSota vaccine strain recombinant viruses were generated using reverse genetics. The recombinant NDV-H9Con virus expresses a consensus-H9 hemagglutinin (HA) that is designed based on available H9N2 sequences from Chinese and Middle Eastern isolates. The recombinant NDV-H9Chi virus expresses a chimeric-H9 HA in which the H9 ectodomain of A/Guinea Fowl/Hong Kong/WF10/99 was fused with the cytoplasmic and transmembrane domain of the fusion protein (F) of NDV. Both recombinant viruses expressed the inserted HA stably and grew to high titers. An efficacy study in chickens showed that both recombinant viruses were able to provide protection against challenge with a heterologous H9N2 virus. In contrast to the NDV-H9Chi virus, the NDV-H9Con virus induced a higher hemagglutination inhibition titer against both NDV and H9 viruses in immunized birds, and efficiently inhibited virus shedding through the respiratory route. Moreover, sera collected from birds immunized with either NDV-H9Con or NDV-H9Chi were able to cross-neutralize two different lineages of H9N2 viruses, indicating that NDV-H9Con and NDV-H9Chi are promising vaccine candidates that could provide cross-protection among different H9N2 lineage viruses.     

Protection of chickens against hepatitis-hydropericardium syndrome and Newcastle disease with a recombinant Newcastle disease virus vaccine expressing the fowl adenovirus serotype 4 fiber-2 protein

Newcastle disease (ND) is one of the most important and devastating avian diseases with considerable threat to the global poultry industry. Hepatitis-hydropericardium syndrome (HHS), caused by virulent fowl adenovirus serotype 4 (FAdV-4), is another highly infectious disease in chickens with severe economic impact. The effective way to combat ND and HHS is by vaccinating the poultry. In the present study, a recombinant NDV LaSota vaccine strain expressing full length fiber-2 gene of FAdV-4 (rLaSota-fiber2) was generated using reverse genetics. The FAdV-4 fiber-2 protein was expressed as a soluble form rather than NDV membrane-anchored form. The rLaSota-fiber2 was genetically stable, and it showed growth patterns in embryonated eggs comparable to that of parental rLaSota virus. Since our unpublished data demonstrated that delivery of live rLaSota-fiber2 in drinking water or ocular delivery of the vaccine didn’t produce protection against hypervirulent FAdV-4 challenge, even though the vaccine provide full protection against NDV challenge, the efficacy of the rLaSota-fiber2 was evaluated by delivering the vaccine intramuscularly in this study. Single-dose intramuscular vaccination of 2-week-old SPF White Leghorn chicks with the live or inactivated rLaSota-fiber2 provided complete protection against virulent NDV challenge. However, single-dose intramuscular vaccination with the live rLaSota-fiber2 vaccine provided better protection against virulent FAdV-4 challenge and significantly reduced faecal viral shedding comparing to the inactivated vaccine. These results indicate that the NDV-vectored FAdV-4 vaccine is a promising bivalent vaccine candidate to control both HHS and ND.     

HLA control of interferon production in the human mixed lymphocyte culture

This report describes studies of interferon production in the human mixed lymphocyte culture. Similar levels of interferon were detectable in two-way, unidirectional, and double-irradiated mixed lymphocyte culture combinations. Following an initial lag period of 1-2 days interferon production showed a peak response on days 3-4 and, in most combinations, was detectable through day 7 of the mixed lymphocyte culture. Interferon-gamma was the predominant type of interferon produced, along with a minor component of interferon activity detectable in some culture combinations which appeared similar to interferon-alpha. Interferon was not produced in HLA identical sibling combinations except for one brother-sister pair who were the donor and recipient of a kidney allograft. Interferon was produced in sibling and unrelated individual combinations that expressed HLA-A, HLA-B, or HLA-DR region incompatibility, with higher concentrations of interferon detectable in the HLA-DR region imcompatible combinations.     

人血单核细胞和U937细胞产生IFN-α的比较

一般认为人体单核-巨噬细胞(Mo-Mφ)系在体外诱生IFN-α较为困难,本文试图找到适合的培养条件,使Mo-M(?)系能在体外诱生IFN-α.结果显示:经M-CSF长期预处理和IFN-γ的短期预处理或单用IFN-γ短期预处理后,NDV刺激均能使人外周血Mo-M(?)产生较高水平的IFN-α;U937细胞只能产生低水平的IFN-α.     

A double recombinant herpes virus of turkeys for the protection of chickens against Newcastle,infectious laryngotracheitis and Marek’s diseases

A double recombinant strain of herpes virus of turkeys (HVT) was constructed that contains the fusion (F) gene from Newcastle disease virus (NDV) and the gD plus gI genes from infectious laryngotracheitis virus (ILTV) inserted into a non-essential region of the HVT genome. Expression of the F protein was controlled by a human cytomegalovirus promoter, whereas expression of gD plus gI was driven by an ILTV promoter. The double recombinant vaccine virus (HVT-NDV-ILT) was fully stable genetically and phenotypically following extended passage in cell culture and infection of chickens. Safety of the vaccine virus was confirmed by overdose and backpassage studies in specific-pathogen-free chickens. Chickens vaccinated with a single dose of HVT-NDV-ILT administered by the in ovo route were highly protected from challenge with the velogenic NDV (GB Texas), ILTV (LT 96-3) and Marek’s disease virus (GA 5) strains (97%, 94% and 97%, respectively). Similarly, chickens vaccinated with a single dose by subcutaneous (SC) route at 1 day of age were highly protected from challenge with the same three viruses (100%, 100%, and 88%, respectively). The protection level of a single dose given by in ovo or SC route against challenge with a virulent Marek’s disease virus strain demonstrates that insertion of multiple genes from two different pathogens within the HVT genome had no adverse effect on the capacity of HVT to protect against Marek’s disease. These results demonstrate that HVT-NDV-ILT is a safe and efficacious vaccine for simultaneous control of NDV, ILTV and Marek’s diseases.