The African swine fever virus lectin EP153R modulates the surface membrane expression of MHC class I antigens

We have modeled a 3D structure for the C-type lectin domain of the African swine fever virus protein EP153R, based on the structure of CD69, CD94 and Ly49A cell receptors, and this model predicts that a dimer of EP153R may establish an asymmetric interaction with one MHC-I molecule. A functional consequence of this interaction could be the modulation of MHC-I expression. By using both transfection and virus infection experiments, we demonstrate here that EP153R inhibits MHC-I membrane expression, most probably by impairing the exocytosis process, without affecting the synthesis or glycosylation of MHC antigens. Interestingly, the EP153-mediated control of MHC requires the intact configuration of the lectin domain of the viral protein, and specifically the R133 residue. Interference of EP153R gene expression during virus infection and studies using virus recombinants with the EP153R gene deleted further support the inhibitory role of the viral lectin on the expression of MHC-I antigens.     

Mapping and sequence of the gene encoding protein p37, A major structural protein of African swine fever virus

The gene encoding protein p37, one of the major structural proteins of African swine fever (ASF) virus has been mapped and sequenced. Protein p37 was obtained from purified virions and the first 27 amino acids from its NH₂-terminal end were identified by automatic Edman degradation. To map the gene encoding protein p37, a mixture of 20-mer deoxyoligonucleotides based upon a part of this amino acid sequence was hybridized to cloned ASF virus restriction fragments. This allowed localization of the gene in fragmentKpnIF/HindIII G₁ of the African swine fever virus genome. An analysis of the DNA sequence from this region revealed an open reading frame encoding 418 amino acids. In this sequence, the 27 NH₂-terminal amino acids determined by sequence analysis of protein p37 are preceded by a stretch of 132 amino acids residues, indicating that protein p37 is synthesized as a polypeptide of higher molecular weight and then posttranslationally processed by cleavage of a Gly-Ala bond. This processing event accounts for the antigenic relationship of protein p37 to a virus-induced, nonstructural protein with a relative molecular weight of 60 kD.     

Enhanced discrimination of African swine fever virus isolates through nucleotide sequencing of the p54, p72, and pB602L (CVR) genes

Complete sequencing of p54-gene from 67 European, American, and West and East African Swine Fever virus (ASFV) isolates revealed that West African and European ASFV isolates classified within the predominant Genotype I according to partial sequencing of p72 were discriminated into four major sub-types on the basis of their p54 sequences. This highlighted the value of p54 gene sequencing as an additional, intermediate-resolution, molecular epidemiological tool for typing of ASFV viruses. We further evaluated p54-based genotyping, in combination with partial sequences of two other genes, for determining the genetic relationships and origin of viruses responsible for disease outbreaks in Kenya. Animals from Western and central Kenya were confirmed as being infected with ASFV using a p72 gene-based PCR assay, following outbreaks of severe hemorrhagic disease in domestic pigs in 2006 and 2007. Eleven hemadsorbing viruses were isolated in macrophage culture and genotyped using a combination of full-length p54-gene sequencing, partial p72-gene sequencing, and analysis of tetrameric amino acid repeat regions within the variable region of the B602L gene (CVR). The data revealed that these isolates were identical in their p72 and p54 sequence to viruses responsible for ASF outbreaks in Uganda in 2003. There was a minor difference in the number of tetrameric repeats within the B602L sequence of the Kenyan isolates that caused the second Kenyan outbreak in 2007. A practical implication of the genetic similarity of the Kenyan and Ugandan viral isolates is that ASF control requires a regional approach.     

野生型p53基因诱导凋亡相关基因ANNEXIN A2的表达研究

目的探讨胡基因过表达介导不同转移潜能肿瘤细胞的凋亡分子机理，寻找与细胞凋亡和肿瘤转移的相关基因。方法应用mRNA差异显示方法分析腺病毒介导的野生型,053基因感染不同转移潜能的肺癌细胞系前后存在的表达差异基因，并用逆转录一聚合酶链反应、Northern印迹和Western印迹方法加以验证。结果分析发现，在柳基因诱导后ANNEXIN A2基因的表达显著差异有统计学意义，经加基因诱导后ANNEXIN A2基因的表达有明显下调。并且以Arfip 973细胞的表达缺失最为显著。经逆转录一聚合酶链反应、Northern印迹和Western印迹方法也验证了这种差异的存在。结论表明ANNEXIN A2基因的表达与p53基因诱导的肿瘤细胞凋亡存在密切的相关性。     

Minute virus of mice (MVMp) infection and NS1 expression induce p53 independent apoptosis in transformed rat fibroblast cells

Parvoviruses infect and kill tumor cells in vivo and in vitro more efficiently than normal cells. Infection of transformed cells by the parvovirus minute virus of mice (MVM) results in high expression of the major non-structural cytolytic viral protein NS1, which induces a cell death modulated by cellular factors. In this work, we show that MVMp infection and/or NS1 protein expression in permissive transformed rat fibroblast cells leads to apoptosis in wild type and p53^−/− cells. Apoptotic cell morphology correlates with mitochondrial membrane permeabilization and activation of caspases 9 and 3 but not caspase 8. Thus, further characterization of the antitumor activity of MVMp and its NS1 protein may contribute to the eradication of tumors, including those lacking p53.     

Evolution of T lymphocytes and cytokine expression in classical swine fever (CSF) virus infection

This study characterized the cell-mediated immune response in pigs inoculated with the Alfort 187 isolate of classical swine fever (CSF) virus. Quantitative changes in the T-lymphocyte population (CD3(+), CD4(+) and CD8(+)) and qualitative changes in cytokine expression (IL-2, IL-4 and IFNgamma) by these cells in serum, thymus and spleen were demonstrated. These changes coincided spatially and temporally with previously described quantitative and qualitative changes in monocyte-macrophage populations, thus demonstrating the contribution of the two cell populations to lymphoid depletion. Moreover, examination of cytokine expression in thymus and spleen samples revealed a type 1 cell-mediated immune response in the early and middle stages of the experiment, giving way to a type 2 immune response towards the end of the experiment; these findings, which accorded with the serological results and lymphopenia, may influence the delayed humoral response characteristic of CSF.     

促红细胞生成素对大鼠脊髓损伤后caspase-3表达及神经细胞凋亡的影响

目的通过观察重组人红细胞生成素（recombinant human erythropoietin，rHu—EPO）对大鼠急性脊髓损伤后Caspase-3表达及凋亡的影响，探讨其脊髓保护的作用机制。方法60只SD大鼠随机分成3组：假手术组，对照组和治疗组，每组20只，采用Allen’s打击法制成急性脊髓损伤模型。观察药物治疗前后大鼠的神经功能行为，用免疫组化染色检测caspase-3表达，原位脱氧糖核苷酸末端转移酶介导的缺口末端标记法（Tunel法）标记凋亡细胞；比较各组间差别。结果HE，免疫组化染色示EPO治疗组脊髓损伤程度小，神经元细胞破坏少；caspase-3在各时相点的表达明显降低，8h和7dTunel标记凋亡阳性细胞显著减少；大鼠神经功能恢复显著优于对照组（P〈0．01）。结论EPO能下调Caspase-3表达，抑制脊髓神经细胞凋亡，对继发性脊髓损伤有保护作用。     

Bcl-xl和p53在人卵巢癌SKOV3细胞中表达及中药干预作用

目的探讨Bcl-xl、p53基因在白毛藤诱导人卵巢癌SKOV3细胞凋亡中的作用。方法不同浓度白毛藤作用于细胞,荧光显微镜观察人卵巢癌SKOV3细胞凋亡作用,RT-PCR法检测Bcl-xl、p53基因表达量的变化。结果白毛藤能诱导人卵巢癌细胞凋亡,并且上调p53和降低Bcl-xl表达。结论白毛藤促进凋亡,其机制可能与激活p53、抑制Bcl-xl表达有关。     

大肠腺癌p53基因cDNA突变与突变型p53基因蛋白表达的相互关系和意义

目的：探讨大肠腺癌突变型ｐ５３基因蛋白表达与ｐ５３基因ｃＤＮＡ突变间的相互关系和意义。方法：对１００例新鲜大肠腺癌组织采用ＰＡｂ２４０单克隆抗体免疫组织化学染色（ＬＳＡＢ法）检测突变型ｐ５３基因蛋白表达、ＲＴ－ＰＣＲ－ＳＳＣＰ检测ｐ５３基因ｃＤＮＡ突变，并比较它们间相互关系。结果：１００例大肠腺癌中，７６例ＰＡｂ２４０单抗阳性（７６％），５１例ｐ５３基因ｃＤＮＡ突变阳性（５１％）；ＰＡｂ２４０单抗反应与ｐ５３基因ｃＤＮＡ突变比较，两者皆阳性４９％，两者中一者阳性２９％，两者皆阴性２２％（Ｐ＜０．０００１）。结论：ｐ５３基因ｃＤＮＡ突变与其基因蛋白产物结构改变高度吻合，大肠腺癌ｐ５３基因ｍＲＮＡ（ｃＤＮＡ）突变是参与和影响突变型ｐ５３基因蛋白结构改变和生物学行为的主要因素     

野生型p53基因导入对U937细胞分化、凋亡和CD36受体表达的影响

目的： 研究野生型p53基因重组腺病毒载体（AdCMV-p53）导入对U937细胞分化、凋亡和清道夫受体CD36表达的影响。 方法： AdCMV-p53导入U937细胞后，用细胞计数、细胞周期分析、台盼蓝染色排除法计数细胞悬液中的活细胞数目和NBT还原反应观察其对U937细胞生长、分化的影响；RT-PCR、免疫荧光和流式细胞分析检测AdCMV-p53导入对CD36表达的影响。 结果： AdCMV-p53可以高效导入U937细胞，野生型p53基因导入促进U937细胞向巨噬细胞分化，台盼蓝染色发现实验组阳性细胞数（64.6±9.2）%较对照组（14.2±5.5）%明显增多，吞噬能力增强；NBT还原反应实验组（49.7±12.6）%较对照组（6.3±1.8）%升高。RT-PCR和流式细胞分析检测，野生型p53基因导入使得CD36 mRNA转录增强，CD36蛋白表达增加。 结论： 野生型p53基因能影响细胞分化和凋亡，并上调清道夫受体CD36的表达，对于动脉粥样硬化的预防和基因治疗具有潜在意义。     

异常黑胆质成熟剂与清除剂对人Hela细胞凋亡基因表达的影响

目的：研究异常黑胆质成熟剂与清除剂两种复方作用前后,Hela细胞中凋亡基因p53,Fas及细胞增殖基因bcl-2的表达及它们之间的差异。方法应用体外细胞培养及RT－PCR对上述3中基因的表达及它们间的差异性进行分析,结果：用1g/L异常黑胆质成熟剂与清除剂处理24h,有大量p53基因表达,而Fas及bcl-2基因未见表达。结论,异常黑胆质成熟剂与清除剂可诱导野生型p53基因表达,这可能是它们清除体内恶变细胞及增强机体抗病功能的分生子生物学机制之一。     

p53基因第7内含子多态性与非小细胞肺癌及其组织p53基因突变的关系

目的研究p53基因第7内含子多态性与非小细胞肺癌（non-small cell lung cancer,NSCLC）及其组织p53基因突变的关系。方法采用病例对照方法选择105例NSCLC患者和100名对照,以聚合酶链反应和ApaⅠ酶切鉴定p53基因第7内含子基因型。然后收集64例NSCLC活检标本及40例NSCLC石蜡包埋组织,鉴定基因型后测序检测第5～8外显子突变。结果在NSCLC中p53基因第7内含子ApaⅠ^＋/＋占23.8%,ApaⅠ^-/-占12.4%,ApaⅠ^＋/-占63.8%;在对照组中ApaⅠ^＋/＋、ApaⅠ^-/-和ApaⅠ^＋/-分别为44.0%、11.0%和45.0%。两组间基因型构成比差异有统计学意义（P〈0.01）。序列分析显示基因型ApaⅠ^＋/＋、ApaⅠ^-/-和ApaⅠ^＋/-的NSCLC组织p53基因第5～8外显子突变率分别为20.0%、50.0%和52.9%,不同基因型之间的突变率差异有统计学意义（P〈0.05）。结论p53基因第7内含子多态性与NSCLC有关。     

喉癌发生中人乳头瘤病毒感染与Langerhans细胞、p53表达的关系

目的：探讨喉癌的发生机理。方法：应用免疫组化ＬＳＡＢ法对３０例喉鳞状细胞癌人乳头瘤病毒（ＨＰＶ）感染、Ｌａｎｇｅｒｈａｎｓ细胞（ＬＣ）及ｐ５３蛋白表达进行了研究。结果：２６．７％的病例可以检测到ＨＰＶ抗原成分。ＨＰＶ感染的癌旁粘膜内ＬＣ数量明显少于无感染者，且形态也发生改变。ｐ５３蛋白表达阳性率在ＨＰＶ感染组（３７．５％）明显低于ＨＰＶ检测阴性组（８３．３３％）。结论：提示ＨＰＶ、ＬＣ、ｐ５３在喉癌发生发展过程中起一定作用，且相互影响，ＨＰＶ感染引起ＬＣ数量减少，局部免疫功能降低，ＨＰＶ感染还可能通过表达的肿瘤蛋白或其他机制使抑癌基因ｐ５３失活，进而导致肿瘤的发生。     

循环癌细胞p53表达在大肠癌患者检测中的临床意义

目的探讨从大肠癌患者外周血检测循环癌细胞胡表达的临床意义。方法用流式细胞术，对术前128例大肠癌患者外周血循环癌细胞胡异常表达进行检测，实验数据用SPSS统计软件处理。结果大肠癌外周血循环癌细胞胡异常表达与患者淋巴结转移显著相关（P〈0．01）；p53异常表达与患者肿瘤分化程度密切并呈正相关（r=0．8476，P〈0．05）；p53异常表达与淋巴结转移在左右结肠癌病例中差异具有统计学意义（P〈0．05）。结论外周血中可检出大肠肿瘤细胞生物学标志物胡突变表达，这可为大肠痛预测复发及远端微转移提供了一个有效的手段。     

The significance of IgG subclasses and mannan-binding lectin (MBL) for susceptibility to infection in apparently healthy adults with IgA deficiency.

The aim of this study was to investigate the significance of IgG subclasses and MBL for susceptibility to infection in association with IgA deficiency. The study population consisted of 139 apparently healthy adult blood donors with IgA deficiency and normal serum levels of IgG and IgM, and an increased susceptibility to infection demonstrated at a population level. Additionally, 216 controls matched for age and sex were investigated. IgG4 deficiency was more common and the mean level of IgG4 lower in persons with IgA deficiency than in the controls. No significant associations could be demonstrated between overt IgG subclass deficiencies and increased susceptibility to infection. However, when the mean concentrations of IgG subclasses were analysed with regard to medical history, that of IgG1 was lower in persons who reported recurrent viral respiratory infections, that of IgG3 in persons who had episodes of severe infection in their history, and that of IgG4 in persons who had recurrent mild respiratory infections, compared with those who had no particular history of infections. In contrast, MBL deficiency-alone or combined with that of the IgG subclass-was not associated with increased susceptibility to infection in persons with IgA deficiency. The results indicate that the proneness to infections observed in a population of otherwise healthy persons with IgA deficiency can only for a small part be accounted for by concomitant deficiencies of IgG subclasses. Contrary to expectations, no synergism between the deficiencies of IgA and MBL could be demonstrated.     

Frequent alteration of p16(INK4a)/p14(ARF) and p53 pathways in the round cell component of myxoid/round cell liposarcoma: p53 gene alterations and reduced p14(ARF) expression both correlate with poor prognosis

In myxoid/round cell liposarcoma (MLS/RCLS), the presence of a round cell (RC) component has been reported to correlate with a worse prognosis for the patients. However, little is known about the molecular genetic differences between conventional myxoid (MX) components and RC components in this tumour. The aim of this study was to investigate the possible implications of molecular alterations of G1 to S-phase check-point genes, especially in the RC component. We evaluated the immunohistochemical expression of p53, MDM2, p14 and p16 protein and assessed proliferative activities using MIB-1 in 29 RC components and 81 MX components from 90 cases. Mutation of the p53 gene, amplification of the MDM2 gene, homozygous deletion, methylation status and mutation of the p16(INK4a)/p14(ARF) genes were also investigated, using concordant paraffin-embedded and frozen material. The data were analysed together with clinicopathological factors to assess their prognostic implications in MLS/RCLS. Immunohistochemically, the over-expression of p53 protein (p = 0.01366) and the reduced expression of p14 (p < 0.0001) and p16 (p < 0.0001) proteins were significantly more frequently observed in RC components than in MX components. Reduced expression of p14 protein correlated significantly with hypermethylation of the p14(ARF) gene promoter (p = 0.0176) and over-expression of p53 protein (p = 0.00837). By univariate analysis, reduced expression of p14 and p53 missense mutation were found to reduce the rate of survival significantly (p < 0.05). Multivariate analysis, including clinicopathological factors, revealed that tumour site (p = 0.0251), the presence of an RC component (p = 0.0113), high MIB-1 labelling index (p = 0.0005) and p53 missense mutation (p = 0.0036) were adverse prognostic factors. In MLS/RCLS, reduction of p14 protein expression and p53 mutation were related to poor prognosis. Accordingly, the p14(ARF)/p53 pathway may contribute to the presence of an RC component and malignant progression in this tumour.     

The relationship between the gene mutation of p53 and the protein expression of p53 and Ki-67 in non-Hodgkin's lymphomas

The relationship between the mutation of the p53 gene and the expression of the p53 protein and the Ki-67 antigen has been investigated in 115 cases with non-Hodgkin's lymphoma, using the immunohistochemical double staining technique, single-strand conformational polymorphism and DNA sequencing methods. Eighteen cases showed more than 10% of p53+ cells and the others showed a few p53+ cells presented sporadically. Alterations in the p53 gene were detected in six cases with B cell type, consisting of five cases with point mutation and one case with point mutation and 15 base pairs deletion. These six cases showed a high percentage of p53+ cells and five cases revealed that the percentage of p53+ cells was higher than that of Ki-67+ cells (p53+ cells > Ki-67+ cells). Excluding the six cases with mutation of the p53 gene, all cases revealed that the percentage of p53+ cells was less than that of Ki-67+ cells (p53+ cells < Ki-67+ cells). Moreover, there was a positive correlation between expression of the p53 protein and of the Ki-67 antigen in histologic types of B cell lymphomas and of T cell lymphomas, respectively, except in small non-cleaved (Burkitt's) and lymphoblastic types. Therefore, sporadic cases showing p53+ cells > Ki-67+ cells revealed alteration of the p53 gene, and expressed abnormal p53 protein (mutant form). Most cases showing p53+ cells < Ki-67+ cells expressed normal p53 protein (wild type), and may reflect the rapid proliferation rate.     

Human immunodeficiency virus (HIV-1) infection selectively downregulates PD-1 expression in infected cells and protects the cells from early apoptosis in vitro and in vivo

Programmed Death-1 (PD-1), a member of T cell costimulatory molecules is expressed in high levels on antigen specific T cells during chronic viral infection, whereas PD-1 expression in the context of HIV-1 infected CD4+ T cells is not known. Here we report that productively infected CD4+ T cells lose PD-1, whereas bystander cells were unaffected. Additionally, p24+/PD-1 negative cells are less susceptible to apoptosis compared to bystander cells in the same infected milieu. Similar results were observed in vivo, as infected T cells isolated from HIV-1+ individuals have significantly low level of PD-1 and the observed loss of PD-1 in vivo is independent of viral load, CD4 count, and/or antiviral treatment. Together these results indicate that productively infected cells are resistant to early apoptosis by downregulating PD-1, whereas PD-1 enhances the susceptibility of effector T cells to apoptosis suggesting a dual role for PD-1 during HIV-1 infection.