Consistent sequence variation of Epstein-Barr virus nuclear antigen 1 in primary tumor and peripheral blood cells of patients with nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) has been proven as a cancer associated with Epstein-Barr virus (EBV). This study was performed to examine sequence variations of the EBV nuclear antigen 1 gene (EBNA-1) in primary tumor and peripheral-blood cells of NPC patients from Taiwan. EXPERIMENTAL DESIGN: DNA extracted from freshly frozen tumor tissues and corresponding peripheral-blood cells of 13 previously untreated NPC patients were subjected to PCR and direct sequencing using EBNA-1-specific primers. We compared the sequence data and analyzed the clinical outcomes. RESULTS: We obtained a 100% positive-detection rate of EBV DNA in the primary tumors of all patients irrespective of the degree of differentiation. The EBNA-1 gene of all tumor samples was the "V-val" strain, showing the same clustered point mutations. They included 21 nucleotide exchanges, leading to 14 amino-acid mutations and 6 silent exchanges, relative to B95-8 cell line. Two of 13 tumors exhibited an additional point mutation at codon 585. EBV DNA was also detected in peripheral-blood cells of 9 of 13 patients under our experimental conditions. Direct-sequencing data showed match alterations of EBNA-1 gene between the primary tumor and peripheral-blood cells. Tumor relapse was observed in four of nine patients with detectable EBNA-1 DNA in their peripheral-blood cells, whereas none of the four patients without detectable EBNA-1 DNA in their peripheral-blood cells developed tumor relapse. CONCLUSIONS: Results of the current study represents the first demonstration of consistent sequence variation of EBNA-1 in primary tumors and peripheral-blood cells. Clinical observations support that the presence of EBV DNA in the peripheral-blood cells may arise from disseminated cancer cells, resulting in a higher relapse rate and poor prognosis.     

家族遗传性结肠息肉病恶变过程中PCNA变化

目的　为研究家族遗传性大肠息肉病 (FPC)恶变演变过程。方法　采用LAB免疫组化染色对 2 9例家族遗传息肉病 ,2 4例大肠癌和 2 8例腺瘤性息肉增殖细胞核抗原 (PCNA)进行对比研究。结果　在恶变FPC和大肠癌组织中PCNA表达较高 ,明显高于恶变FPC组未恶变息肉 ,未恶变FPC组息肉和腺瘤性息肉 (P <0 .0 1) ;恶变FPC组织PC NA表达明显高于大肠癌组织 ,有显著差异 (P <0 .0 5 ) ;恶变FPC组中未恶变的息肉明显高于未恶变FPC组息肉和腺瘤性息肉 ,有显著差异 (P <0 .0 5 ) ;未恶变FPC组息肉PCNA表达与腺瘤性息肉比较无显著差异 (P <0 .0 5 )。结论　FPC恶变演变过程是逐渐发生的 ,应进行连续监测 ;FPC一但恶变 ,恶性程度较高 ,易发生转移 ,预后较差 ;FPC恶变后另一些未癌变息肉尽管没有癌变但已属于高危癌前病变 ,手术中应将所有息肉一并切除。     

Feline oncornavirus-associated cell membrane antigen. V. Humoral immune response to virus and cell membrane antigens in cats inoculated with Gardner-Arnstein feline sarcoma virus.

Tumor growth responses in 5- to 6-week-old kittens inoculated with the Gardner-Arnstein strain of feline sarcoma virus exhibited three distinct pattern: 1) complete tumor regression or no detectable tumor growth in approximately one-third of 43 inoculated kittens, 2) rapid tumor progression which led to debilitation and death within 16.2 +/- 4.2 weeks following infection in an additional one-third, and 3) slow tumor growth or temporary regressions in the remaining third. The feline oncornavirus-associated cell membrane antigen (FOCMA) antibody response was closely correlated with tumor progression; rapid progressors had the lowest antibody titers, whereas those in the "no tumor or permanent regression" categories had the highest titers. These results agreed with those previously observed with another virus strain, the Snyder-Theilen feline sarcoma virus. Cats in the intermediate categories of tumor growth also had intermediate levels of FOCMA antibody. The presence of virus-neutralizing (VN) activity was not always correlated with anti-FOCMA activity. Animals in the rapid-progressor category, compared to the regressors or slow progressors, were more likely to have detectable VN antibody during early periods. Conversely, animals in the regressor group or group with no tumors were more likely to show an early rise in detectable anti-FOCMA activity than animals in either of the progressor groups.     

EBV-determined nuclear antigen (EBNA)-positive cells in the peripheral blood of infectious mononucleosis patients.

After removal of SRBC rosette-forming T-cells from the peripheral blood, the residual, largely B-lymphocyte fraction of five infectious mononucleosis patients was found to contain 0.5-2% blast cells, positive for the EBV-determined nuclear antigen (EBNA). There was a rough parallelism between the presence of large lymphoblasts in the hematological smear, EBNS-positive large blasts in the B-cell fraction and the ability of the T-cell fraction to exert an EBV-specific lymphocytotoxicity on established cell lines in vitro. EBNA-positive B-cells and EBV-specific killer T-cells disappeared after the acute phase of the disease.     

Inhibition of gastric cancer cell proliferation by antisense oligonucleotides targeting the messenger RNA encoding proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is a nuclear protein that regulates DNA synthesis by DNA polymerase delta, and is essential for DNA replication. PCNA expression level is related to the malignancy of gastric cancer cells. Seven different gastric cancer cell lines and two kinds of control cell lines were treated with antisense oligonucleotides complementary to the messenger RNA of PCNA. Treatment of each gastric cancer cell line with antisense oligonucleotides at concentration of 10-40 microM inhibited the cell growth, colony formation and PCNA protein production in a dose-dependent manner, but only affected normal cells slightly. A random sequence oligomer showed no effect. These results show that PCNA is essential for gastric cancer cell proliferation and that the use of synthetic oligonucleotides is an effective way of producing antisense-mediated changes in the behaviour of human gastric cancers.     

Persistence of Epstein-Barr viral nuclear antigen (EBNA) in cells entering the EB viral cycle.

It was shown by double immunofluorescence studies that Epstein-Barr viral nuclear antigen (EBNA) was preserved in EBV-infected cells after they had entered the productive viral cycle, as signalled by the appearance of the early antigen (EA)complex. A nuclear component of the EA comples could be clearly distinguished from EBNA with regard to antigenic specificity.     

Pilot experiments with EB virus in owl monkeys (Aotus trivirgatus). II. EB virus in a cell line from an animal with reticuloproliferative disease

A continuous cell line has been established in culture from a pathological lymph node of an owl monkey with reticuloproliferative disease after inoculation with EB virus. The cells grow in suspension in clumps or as single individuals and show the characteristic features of lymphoblasts with some differentiation to reticulum cells; their karyotype is typical of one sub-species of owl monkey. A herpes virus has been found in the cells and has been identified as EB virus in two types of immunofluorescence test using two different specific antisera; this identification was confirmed by the ability of the virus to transform normal human cord-blood lymphocytes and make them grow as a continuous cell line. Infection by a carcinogenic simian virus, herpesvirus saimiri, has been excluded by tests for neutralizing antibodies and virus growth in susceptible cells. The continuous growth of the monkey cells, the presence in them of EB virus, and the possible relationship of the virus to the original reticuloproliferative disease of the inoculated monkey, are considered and discussed.     

The expression of proliferating cell nuclear antigen in paraffin sections of peripheral, node-negative non-small cell lung cancer.

Cell proliferation of 40 peripheral, node-negative non-small cell lung cancers (NSCLC) treated with surgery alone was investigated by immunohistochemical analysis with the monoclonal antibody (MoAb) PC10, which recognizes a proliferating cell nuclear antigen (PCNA) in formalin-fixed and paraffin-embedded material. Results were correlated with DNA ploidy and S-phase fraction (SPF) analyzed by DNA flow cytometric study. Mitotic count (MC) was analyzed by light microscopic study and histopathologic features. PCNA immunoreactivity was seen in all samples and confined to the nuclei of cancer, but not to the surrounding, tumor-negative cells; its frequency ranged from 0-70% (median, 15%), and tumors expressed either a low (0-25%, n = 25) or intermediate (26-75%, n = 15) proliferative activity. There was no relationship between PCNA immunoreactivity and tumor stage or among size, histologic type, and mitotic count (MC). Tumors with intratumoral blood vessel invasion (BVI) showed a significantly higher (P less than 0.005) PCNA immunoreactivity than BVI-negative tumors. PCNA scores were significantly higher (P less than 0.005) in DNA aneuploid (n = 22) than in DNA diploid (n = 18) tumors and correlated significantly with the SPF of DNA aneuploid tumors (r = 0.825, P less than 0.0001), but not with diploid tumors (r = 0.002, P = 0.9). Intermediate proliferating tumors had a significantly higher (P less than 0.01) MC than their counterparts. In univariate analysis, significant predictors of survival were tumor classification (T1 versus T2), tumor size (less than or equal to 2.6 cm versus more than 2.6 cm), BVI (BVI-negative versus BVI-positive), MC (less than or equal to 8 versus more than 8), and PCNA immunoreactivity (low versus intermediate). DNA ploidy and SPF did not influence survival significantly. Only PCNA immunoreactivity retained its independent level of significance (P = 0.02) by multivariate analysis. It was concluded that PCNA immunostaining is a simple and clinically useful method for estimating cell proliferation in formalin-fixed, paraffin-embedded tissue of resected peripheral, node-negative NSCLC.     

Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation

The production of a mouse monoclonal antibody, Ki-67, is described. The Ki-67 antibody recognized a nuclear antigen present in proliferating cells, but absent in resting cells. Immunostainings with Ki-67 revealed nuclear reactivity in cells of germinal centres of cortical follicles, cortical thymocytes, neck cells of gastrointestinal mucosa, undifferentiated spermatogonia and cells of a number of human cell lines. The Ki-67 antibody did not react with cells known to be in a resting stage, such as lymphocytes, monocytes, parietal cells and Paneth's cells of gastrointestinal mucosa, hepatocytes, renal cells, mature sperm cells, brain cells, etc. Expression of the antigen recognized by Ki-67 could be induced in peripheral blood lymphocytes after stimulation with phytohaemagglutinin, whereas it disappeared from HL-60 cells stimulated with phorbol esters to differentiate into mature macrophages in a resting stage. These findings suggest that Ki-67 is directed against a nuclear antigen associated with cell proliferation. A first series of immunostainings of tumour biopsies indicated that Ki-67 may be a potent tool for easy and quick evaluation of the proportion of proliferating cells in a tumour.     

外周血和鼻咽组织EB病毒DNA检测对鼻咽癌诊断和防控的临床观察

目的探讨外周血和鼻咽组织EB病毒DNA检测对鼻咽癌诊断和防控的临床意义以及鼻咽腔冲洗对防控鼻咽癌的价值。方法选取2010年12月至2012年4月55例鼻咽癌患者为观察组,抽取同期55例健康者为对照组,采用EB病毒DNA PCR荧光定量检测技术检测外周血和鼻咽组织EB病毒DNA,比较鼻咽癌患者与健康者外周血和鼻咽组织EB病毒DNA检测结果。对另外1组67例EB病毒抗体阳性而鼻咽活检结果为阴性者进行鼻咽腔冲洗,比较治疗前、后EB病毒抗体检测结果。结果观察组患者外周血、鼻咽组织中EB病毒DNA阳性检出率均高于对照组健康者,差异均有统计学意义(均P<0.01)。经鼻咽腔冲洗治疗后,EB病毒抗体阳性者检测转阴率为19.4%,患者临床症状明显改善。结论鼻咽组织EB病毒DNA检测有助于鼻咽癌的临床诊断,对EB病毒抗体阳性者进行鼻咽腔冲洗治疗对于鼻咽癌的防控具有重要意义。     

基因工程膜抗原检测EB病毒MA—IgA抗体研究及与VCA—IgA...

With gene engineering EB virus membrane antigen as the diagnostic antigen, indirect immunofluorescence (IF) assay was used to detect IgA antibody against EB virus membrane antigen (MA-IgA) in sera from 202 nasopharyngeal carcinoma (NPC) patients and 315 controls (normal and patients with other tumors). MA-IgA antibody was positive in 96.8% of the pre-treatment NPC patients with a GMT of 1:36.3. MA-IgA detection by this method was more sensitive than EA-IgA detection by IE. In contrast, patients with tumors other than NPC were negative for MA-IgA antibody. 9.1% of VCA-IgA positive persons were MA-IgA positive with a GMT of less than 1:5. No MA-IgA positive was found in VCA-IgA negatives. The results indicated that this method was relatively specific. In the treatment group, the positive rate and GMT of MA-IgA antibody declined with increase in survival time and the decline was faster than VCA-IgA. When recurrence or distant metastasis developed, similar to VCA-IgA and EA-IgA antibodies, the positive rate and GMT of MA-IgA antibody increased to its pretreatment level. Therefore, MA-IgA detection might be valuable in the early diagnosis and monitor of NPC.     

Prognostic value of cell proliferation in breast cancer as determined by proliferating cell nuclear antigen (PCNA) immunostaining.

A series of 175 biopsies from primary tumours were subjected to immunostaining for proliferation cell nuclear antigen (PCNA) and histopathological analysis for prognostic factors in 175 women with breast cancer followed up for nine years. In normal breast epithelium, only occasional nuclei were positive for PCNA. In breast carcinomas, the fraction of nuclei positive for PCNA showed considerable intratumoural variation, usually being highest at the invasive tumour margins. The fraction of positive nuclei was significantly related to histological grade (p less than 0.001), histological type (p = 0.049) and tumor recurrence (p = 0.01). The fraction of positive nuclei predicted recurrence-free survival (p = 0.007) and overall survival (p = 0.0158) in univariate analysis. In the multivariate analysis including also the standard prognostic factors, the PCNA positivity predicted recurrence-free survival (p = 0.004) and overall survival (p = 0.030) independently. The results show that the light microscopic assessment of the growth fraction as determined by PCNA immunolabeling has independent prognostic value in breast carcinomas like some other (e.g. S phase fraction, mitotic index, Ki-67) previously characterized proliferation indices.     

Cell proliferation in rat colon measured with bromodeoxyuridine, proliferating cell nuclear antigen, and [3H]thymidine.

Epithelial cell proliferation was studied in the normal colonic mucosa of 5-week-old Sprague-Dawley rats, comparing [3H]thymidine incorporation (group 1) with two newer proliferation markers, bromodeoxyuridine (group 2) and proliferating cell nuclear antigen (group 3). Microautoradiography (group 1) or immunoperoxidase assays (groups 2 and 3) were carried out. Cells were counted for positive reaction and position along 50 colonic crypt columns/animal. No significant differences were found in number or distribution of labeled epithelial cells in proliferative compartments in crypt columns of normal colonic mucosa; labeled cells were mainly in the lower 60% of colonic crypts. Thus, in this model, bromodeoxyuridine and proliferating cell nuclear antigens were comparable to [3H]thymidine as reliable markers of proliferating epithelial cells in rat colon.     

Antibodies to EB virus capsid antigen and to soluble antigen of lymphoblastoid cells in infectious mononucleosis patients

Sera from infectious mononucleosis patients aged 2 to 35 years and from normal subjects aged 11 to 35 years were examined for the presence of antibody to the EB virus capsid antigen (VCA) in the indirect immunofluorescence test, and for the presence of antibody reactive with the soluble (S) antigen of lymphoblastoid cell lines in the complement-fixation test. Sera from all infectious mononucleosis patients taken either in the acute stage of the disease or up to 9 years after its onset contained VCA antibody. The sera taken in the acute stage of the disease were free of S antibody; out of 97 sera investigated only one was weakly reactive. Only one of the seven sera taken 4 to 5 months after the onset of the disease contained S antibody. After the 7th month following onset, the majority of the subjects possessed S antibody. These data indicate that the S antibody appears much later in the course of EB virus infection than the VCA antibody. The predominant majority of sera from subjects with no history of the disease possessed VCA antibody; most of these also possessed S antibody. The incidence of S antibody in these subjects and those who had been suffering from infectious mononucleosis 1 to 9 years previously were comparable.     

Changes in the immune cell population and cell proliferation in peripheral blood after gemcitabine-based chemotherapy for pancreatic cancer

Purpose

Regulatory T cells (Tregs) play a role in the immunosuppressive state in pancreatic cancer patients. We aimed to evaluate the changes of immune cells population including Tregs caused by gemcitabine (GEM)-based chemotherapy.

Methods

Fifty-three patients with pancreatic cancer were enrolled in this study, of which 32 received GEM- based chemotherapy. Blood samples were collected before and at least 2 weeks after the last dose of chemotherapy. The peripheral blood mononuclear cells (PBMCs) were subjected to flow cytometry analysis after labeling with anti-CD4, anti-CD25, and anti-Foxp3 antibodies. Other lymphocytes and NK cell markers were also measured. The proliferative capacity of PBMCs stimulated with anti-CD3 was analyzed using H³ thymidine.

Results

The percentage and number of Tregs were significantly decreased after chemotherapy (p = 0.032, p = 0.003, respectively). The other immune cells and the proliferative capacity did not change.

Conclusion

This study showed that GEM-based chemotherapy produced an immunomodulatory effect via the depletion of Tregs.     

Lymphoblastoid cell lines and burkitt-lymphoma-derivee cell lines differ in the expression of a second epstein-barr virus encoded nuclear antigen

Twenty-seven Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and 27 EBV-carrying Bur-kitt-lymphoma-derived lines were analyzed for expression of the second EBV-encoded nuclear antigen (EBNA-2) by immunoblotting and anticomple-ment immunofluorescence with EBNA-2-specific sera. While all lymphoblastoid cell lines expressed EBNA-2, only 10 of the 27 BL lines were EBNA-2-positive. Comparison of the EBNA-2 coding Bam HI W-Y- and H-fragments of EBV-DNA in the different cell lines by restriction enzyme analysis suggests that EBNA-2 negativity is due either to sequence diversity or to a deletion in the BamHI WYH region.