Epstein-Barr virus: detection of genome in somatic cell hybrids of Burkitt lymphoblastoid cells.

Somatic cell hybrids of Burkitt lymphoblastoid cells, from which Epstein-Barr virus can be recovered, were examined for the presence of virus DNA by DNA-RNA hybridization. Four clones of hybrid cells, each negative for virus antigens by immunofluorescence, contained virus DNA in varying genomic equivalents. The number of virus genome equivalents increased in the hybrid cells after induction of virus with iododeoxyuridine.

Oxygen therapy during exercise training in chronic obstructive pulmonary disease.

BACKGROUND: Exercise training within the context of pulmonary rehabilitation improves outcomes of exercise capacity, dyspnea and health-related quality of life in individuals with chronic obstructive pulmonary disease (COPD). Supplemental oxygen in comparison to placebo increases exercise capacity in patients performing single-assessment exercise tests. The addition of supplemental oxygen during exercise training may enable individuals with COPD to tolerate higher levels of activity with less exertional symptoms, ultimately improving quality of life. OBJECTIVES: To determine how supplemental oxygen in comparison to control (compressed air or room air) during the exercise-training component of a pulmonary rehabilitation program affects exercise capacity, dyspnea and health-related quality of life in individuals with COPD. SEARCH STRATEGY: All records in the Cochrane Airways Group Specialized Register of trials coded as 'COPD' were searched using the following terms: (oxygen* or O2*) AND (exercis* or train* or rehabilitat* or fitness* or physical* or activ* or endur* or exert* or walk* or cycle*). Searching the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library), MEDLINE, EMBASE and CINAHL databases identified studies. The last search was carried out in June 2006. SELECTION CRITERIA: Only randomized controlled trials (RCTs) comparing oxygen-supplemented exercise training to non-supplemented exercise training (control group) were considered for inclusion. Participants were 18 years or older, diagnosed with COPD and did not meet criteria for long-term oxygen therapy. No studies with mixed populations (pulmonary fibrosis, cystic fibrosis, etc) were included. Exercise training was greater than or equal to three weeks in duration and included a minimum of two sessions a week. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials for inclusion in the review and extracted data. Weighted mean differences (WMD) with 95% confidence intervals (CI) were calculated using a random-effects model. Missing data were requested from authors of primary studies. MAIN RESULTS: Five RCTs met the inclusion criteria. The maximum number of studies compared in the meta-analysis was three (31 on oxygen versus 32 control participants), because all included studies did not measure the same outcomes. When two studies were pooled, statistically significant improvements of oxygen-supplemented exercise training were found in constant power exercise time, WMD 2.68 minutes (95% CI 0.07 to 5.28 minutes). Supplemental oxygen increased the average exercise time from 6 to 14 minutes; the control intervention increased average exercise time from 6 to 12 minutes. Constant power exercise end-of-test Borg score (on a scale from 1 to 10) also showed statistically significant improvements with oxygen-supplemented exercise training, WMD -1.22 units (95% CI -2.39 to -0.06). One study showed a significant improvement in the change of Borg score after the shuttle walk test, by -1.46 units (95% CI -2.72 to -0.19). There were no significant differences in maximal exercise outcomes, functional exercise outcomes (six-minute walk test), shuttle walk distance, health-related quality of life or oxygenation status. According to the GRADE system most outcomes were rated as low quality because they were limited by study quality. AUTHORS' CONCLUSIONS: This review provides little support for oxygen supplementation during exercise training for individuals with COPD, but the evidence is very limited. Studies with larger number of participants and strong design are required to permit strong conclusions, especially for functional outcomes such as symptom alleviation, health-related quality of life and ambulation.

[Open stent implantation for ascending aorta aneurysm with severe calcification].

A 77-year-old woman had an ascending aortic aneurysm and aortic regurgitation due to aortitis syndrome. Computed tomography showed that ascending aorta was 55 mm in diameter and had severe calcification between the ascending aorta and distal aortic arch involves neck vessels. We performed open stent implantation and aortic valve replacement with bioprostheses valve. No adverse event occurred after the operation and the patient was discharged on the 28th postoperative day. The open stent implantation was useful for the treatment of the aneurysm in high risk patients.

Human nasopharyngeal carcinomas positive for Epstein-Barr virus DNA in North America.

Nasopharyngeal carcinomas (NPC) from 2 black patients and 1 Caucasian patient were positive for Epstein-Barr virus (EBV) DNA. Of the tumors, 2 were lymphoepitheliomas (undifferentiated NPC) and 1 was a moderately differentiated NPC. All 3 patients had high IgG titers against EBV early antigen and high IgG and IgA titers against virus capsid antigen (VCA). In one patient, the levels of anti-VCA IgA were different than those of anti-VCA IgG over the course of the disease. Our data support the association of EBV and NPC in North America.

Athymic nude mice: induction of tumors containing Epstein-Barr virus using Burkitt's-related cell lines.

We studied tumor induction in athymic nude mice by D98/HR-1 cells, an epithelial somatic cell hybrid containing the Epstein-Barr virus (EBV) genome, and by the parental D98 and HR-1 cells. Groups of animals were inoculated with cells grown in culture, with cells from tumors induced by the cell lines, or with cells from lines derived from tumors. The tumors induced by D98/HR-1 cells were undifferentiated carcinomas; those induced by D98 cells were carcinomas and those induced by HR-1 cells were poorly differentiated lymphomas. Preliminary data suggest that the number of EBV genome equivalents was sharply reduced in cells from both D98/HR-1 and HR-1 tumors. Subsequent passage of tumor cells in vitro resulted in a partial recovery in the number of EBV genome equivalents in HR-1 cells and a complete recovery in D98/HR-1 cells. The reduction in the number of EBV genomes in the tumor cells suggests that in vitro passage can influence the number of EBV genomes in these cells.

Partial restriction map of Marek's disease virus DNA.

A partial restriction map of Marek's disease virus (MDV) DNA was constructed by digestion with endonucleases BamHI, Bg/I and SmaI and by blotting hybridization. The data suggest that there is a terminal heterogeneous sequence at least on one end of the MDV DNA molecule. The data did not reveal four different orientations of the terminal fragments of MDV DNA molecules despite the observation that MDV DNA contains inverted repeat sequences as also present in Herpes simplex virus (HSV) DNA molecules (Cebrian et al., 1981). Terminal deletion of MDV DNA, SalI-H and I, was found in high passage number preparations.

Cloning of Epstein-Barr virus (EBV) DNA fragments in pBR32 and Charon3A.

Restriction fragments of Epstein-Barr virus (EBV; B95-8) DNA were cloned in the Tc gene of pBR322 (HindIII-F, -G, -I, -J, -K, -L, and -M) and in Charon3A (EcoRI-GI and -G2). Altogether these cloned fragments covered 39% of the entire viral genome. The cloned EcoRI-G2 fragment of EBV (B95-8) DNA was shown to contain, in addition to HindIII-J, two more HindIII-fragments : HindIII-M, which had not been located on the linkage map of the viral genome (Given and Kieff, 1978) and HindIII-N, which had been unrecognized up to now. The utility of this cloning method is discussed in regard to the detailed mapping of a viral genome and large-scale production of the viral DNA.

Common antigenic epitopes are present on heat-labile oligomers of MDV glycoprotein B and on HSV glycoprotein B.

The antigenic cross-reactivity between the Marek's disease virus glycoprotein B (MDV gB) and glycoprotein B (gB) of herpes simplex virus type 1 and 2 (HSV1 and HSV2) was analysed by the immunoblotting method. We studied cell lysates in both denatured and in undenatured form (i.e., unheated) and reacted them with convalescent sera from chickens infected with the RBIB MDV strain and with human anti-HSV1 gB. Both sera detected the heat-labile MDV gB and the HSV gB oligomers. In addition, monospecific antibodies to the MDV gB 230 kDa oligomer (strain CVI988) were immunoaffinity purified from both the chicken and the human sera. The chicken and human monospecific antibodies detected the homologous and the heterologous gB oligomers in native MDV- and HSV1-infected cell lysates. 15 human sera were tested by immunoblotting and by immunofluorescence on HSV1-, CVI988-and herpes virus of turkeys (HVT)-infected cells. By both assays about half of the human sera reacted with MDV-infected cells. This study demonstrates that the MDV gB heat-labile oligomers possess conformational epitopes shared with the human alpha-herpes virus HSV1 and HSV2 gB heat-labile oligomers.

Inhibition of human immunodeficiency virus forward and reverse transcription by PC6, a natural product from cones of pine trees.

We have previously shown that PC6, a natural product extracted from cones of Pinus parviflora Sieb et Zucc, can inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in CD4+ T cells and in monocyte/macrophage cell lines. Here, we show by immunoprecipitation of HIV-1 proteins with a specific pooled serum that PC6 inhibited the expression of all HIV-1 proteins in CEM cells. PC6 did not affect the posttranslational processing of the HIV-1 proteins. Northern, Southern, and kinetics analyses revealed that PC6 inhibited HIV-1 reverse and forward transcription in CEM cells. Transient transfection of CEM cells with HIV-1 long terminal repeat (LTR) linked CAT DNA also showed that PC6 inhibited LTR-driven gene expression at the transcriptional level.

Modification of human immunodeficiency viral replication by pine cone extracts.

We have shown previously that two fractions (PC6 and PC7) extracted from cones of the Japanese white pine Pinus parvifloria Sieb. et Zucc have potent immunopotentiating effects. Here, we show that PC6 and PC7 inhibited HIV-1 replication (greater than 95%), in a dose-dependent manner, in chronically infected CR10/HIV-1 cells and in acute cytolytic HIV-1 infection of CEM cells. Treatment of CEM cells, prior to or after acute infection with HIV-1, reduced subsequent viral production, but the best inhibitory effect was obtained with treatment before and after infection: an 80% inhibition was achieved with as little as 3 micrograms/ml of PC6. Comparable results were also obtained when PC6 was used to inhibit HIV-1 replication in the U937 human histiocytic lymphoma cell line. Both PC6 and PC7 were relatively nontoxic to cells. The anti-HIV-1 effect of PC6 and PC7 we observed in this report, coupled with earlier reports of their immunopotentiating properties suggest their potential as ideal therapeutic agents for the treatment of AIDS.

Superinfection of a defective human immunodeficiency virus type 1 provirus-carrying T cell clone with vif or vpu mutants gives cytopathic virus particles by homologous recombination.

The partially CD4-expressing T cell clone, Vpr-1, which carries a latent vpr-defective HIV-1 genome and expresses HIV-1 Nef protein only, was permissive to superinfection by HIV-1. Superinfection of Vpr-1 with vif- or vpu-defective mutants, which were noncytopathic, reactivated the vpr-defective virus and led to homologous recombination and cytopathogenesis. The data provide an experimental model for homologous recombination being an important mechanism whereby HIV-1 acquires genetic heterogeneity, and when occurring among defective virus in vivo bestows novel biological activities and virulence.

2 gene families of mareks-disease virus (mdv) with a potential role in tumor-induction in chicken (review).

Marek's disease virus (MDV) is one of the most oncogenic herpesviruses and the only cancer virus that has a successful vaccine. Based on the short-latency and polyclonal nature of the tumors, it is likely that MDV encodes its own oncogenes. Recent studies have provided insights into possible MDV genes involved in oncogenesis. We describe the characterization of two gene families encoded by the BamHI-H and I-2 fragments. The BamHI-H and I-2 fragments, located contiguously in the IR(L) region, are unique to oncogenic (serotype) MDV and consistently expressed in MDV-transformed tumors and T-cell lines. The structural alteration of the BamHI-H fragment, specifically the expansion of a 132 bp repeat, has been correlated with the attenuation of the oncogenicity of MDV. Several open-reading frames, some resulting from alternative splicing, can be identified. One of the open-reading frames, BHa, encodes a 7 Kd polypeptide. BHa shares limited homology with a T-cell lymphoma oncogene TLM and stimulates extended growth of chicken embryo fibroblasts, when transfected into the cell. The other gene implicated in latency or oncogenesis is Meg whose coding sequences span portion of the BamHI-I-2 and the Q(2) fragment. Meq has an interesting structure, resembling a chimera between Jun/Fos oncoproteins and WT-1 tumor suppressor protein. It contains a bZIP (basic leucine zipper) domain and a long proline-rich domain. The proline domain has trans-activating function, whereas the bZIP domain allows Meq to dimerize with a variety of transcriptional factors and expands the enhancer repertoire it acts on. As neoplastic transformation usually requires the complementation of multiple oncogenes, BHa and Meq could be two of the proteins participating in this process.

A 7-kda protein encoded by the bamhi-h gene family of mareks-disease virus is produced in lytically and latently infected-cells.

Marek's disease virus (MDV) BamHI-H gene family was transcribed specifically in cells infected with oncogenic MDV, and the transcripts were prematurely terminated in cells infected with non-oncogenic attenuated strains of MDV due to the amplification of a 132 bp repeat located downstream of their promoter. There are six small open reading frames in the two differently spliced and two unspliced transcripts. This report describes expression of BHa gene open reading frame A, present in 1.7 kb unspliced transcript. Antibody was raised against BHa C-terminal polypeptide. The antibody showed specific reaction to the GST fusion protein derived from BHa protein. Immunoprecipitation with the lysates of infected cells was carried out. The results indicated that 7 kDa BHa protein was immunoprecipitated from the lysates of oncogenic MDV infected chicken embryo fibroblasts (CEF) and a MSB-1 lymphoblastoid cell line derived from Marek's disease tumor but not from non-oncogenic MDV infected CEF or uninfected CEF. 36 kDa protein was co-immunoprecipitated with 7 kDa BHa protein in oncogenic MDV infected CEF but was not detected in MSB-1 cells. This is the first report to show that a small open reading frame of the BamHI-H gene family was in fact expressed in MDV infected cells.

Prolonged proliferation of primary chicken-embryo fibroblasts transfected with cdnas from the bamhi-h gene family of mareks-disease virus.

Oncogenic virus-specific 1.69 kb and 1.5 kb cDNAs, derived from the tumorigenicity-associated BamHI-H gene family of Marek's disease virus (MDV), were cloned into the eukaryotic expression vector (PRC/CMV) and designated as PRC/CMV-1.69 and PRC/CMV-1.5, respectively. Chicken embryo fibroblasts (CEFs) transfected with PRC/CMV-1.69 or PRC/CMV-1.5 plasmid DNA showed reduced serum-dependence for growth compared with PRC/CMV vector-transfected CEFs. Also, PRC/CMV-1.69 or PRC/CMV-1.5 DNA transfected CEFs proliferated for a longer period of time (20-22 passages) compared with the CEFs transfected with PRC/CMV plasmid DNA (10 passages). Reduced serum-dependence for growth and prolonged proliferation of CEFs after in vitro transfection with PRC/CMV-1.69 or PRC/CMV-1.5 indicated that these two cDNAs stimulate cell growth. These data indicated that one of the functions of the BamHI-H gene family of MDV is cell-growth control, which may play an important role in the establishment and maintenance of uncontrolled growth of tumor cells induced by MDV.

Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts: method and efficacy of adenovirus-mediated transduction.

OBJECTIVE: Allograft-targeted immunosuppressive gene therapy may inhibit recipient immune activation and provide an alternative to systemic immunosuppression. We studied the optimal technique and efficacy of intracoronary gene transfer of viral interleukin-10 and human transforming growth factor-beta 1 in a rabbit model of heterotopic heart transplantation. METHODS: Replication-defective adenoviral vectors were constructed, expressing viral interleukin-10 (AdSvIL10) or transforming growth factor-beta 1 (AdCMVTGF-beta 1). Intracoronary delivery of vectors was accomplished ex vivo by either bolus injection or slow infusion. The allografts were implanted heterotopically in recipient rabbits and collected 4 days after the operation. Vector dose was 4 x 10(9) to 6 x 10(10) pfu/gm of donor heart. Transfer was confirmed by DNA amplification for both genes. Gene product expression in tissue was quantified by immunoassay and visualized by immunohistochemical staining. RESULTS: Allograft viral uptake was only 9.9% +/- 2.4% with bolus injection, but increased to 80.5% +/- 6.8% at 1 ml/min infusion rate (p = 5 x 10(-14)). Uptake ratio was not affected by vector quantity or slower infusion rates. Transforming growth factor-beta 1 was consistently detected in allografts infected with AdCMVTGF-beta 1, but not with control adenovirus or AdSvIL10. Expression was proportional to infused vector quantity and reached 10 ng/gm of allograft at infused 10(10) pfu/gm. Transforming growth factor-beta 1 was also detected in recipient's serum at less than 1 ng/ml. Viral interleukin-10 was detected in minor amounts only (< 1 ng/gm) in allografts infected with AdvIL10 up to 5 x 10(10) pfu/gm. Nevertheless, it was detected in recipient serum at concentrations up to 0.4 ng/ml. CONCLUSIONS: Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts during cold preservation is feasible. Slow infusion is superior to bolus injection. In vivo effects on allograft rejection remain to be determined.

Intracoronary adenovirus-mediated transfer of immunosuppressive cytokine genes prolongs allograft survival.

BACKGROUND: Intracoronary transfer and expression of recombinant genes in the intact heart is now feasible. In the transplant setting, local modulation of host immune responses by a genetically modified allograft may offer an attractive alternative to systemic immunosuppression. METHODS: We tested the efficacy and in vivo effect of intracoronary transfer of two immunosuppressive cytokine genes. First-generation E1-deleted adenoviral vectors expressing the Epstein-Barr virus interleukin-10 (AdSvIL10) or human transforming growth factor--beta 1 (AdCMVTGF-beta) were used. Rabbit cardiac allografts were transduced during cold preservation by slow (1 ml/min) intracoronary infusion of 10(10) pfu/gm diluted viral vectors and then implanted heterotopically. Controls included E1-deleted adenovirus (Ad5dI434) and AdCMVLacZ. Beating allografts were collected on day 4 for analysis of gene transfer efficacy and distribution. Additional grafts were used for evaluation of alloreactivity (n = 34). RESULTS: Mean allograft viral uptake was 81% (up to 91%). Polymerase chain reactions and reverse transcription-polymerase chain reactions confirmed the presence and expression of both genes in the grafts. beta-Galactosidase staining in AdCMVLacZ-infected grafts demonstrated efficient gene expression, which was highest (100%) in subepicardial regions. More homogeneous transmyocardial distribution of the transgene (in 25% to 40% of cells) could be achieved by pulsatile slow delivery. Allograft survival was 6.9 +/- 0.9 days in controls (n = 12), 11.1 +/- 1.7 days in AdCMVTGF-beta-infected grafts (n = 11, p < 10(-4)), and 11.2 +/- 3 days in AdSvIL10-infected grafts (n = 11, p < 10(-4)). Histologic scores (blinded) showed significantly (p < 0.005) higher regression coefficients for rejection in controls compared with both cytokine-transduced groups. Perioperative administration of cyclosporine A (INN: ciclosporin) to recipients had no effect on survival of AdCMVTGF-beta-infected grafts but reduced survival of AdSvIL10-infected grafts. CONCLUSIONS: Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts is efficient and effectively prolongs graft survival. Vectors that would induce long-term expression of such genes may make this approach clinically applicable.

Inhibition of influenza virus infection by pine cone antitumor substances.

The anti-influenza virus activity of polysaccharides and other high molecular weight fractions from pine cone extract (PCE) of Pinus parviflora Sieb. et Zucc. was investigated. None of the fractions affected the growth of MDCK cells. The acidic PCE substances markedly suppressed the growth of the influenza virus in MDCK cells. Significant inhibition of both the viral protein synthesis in infected cells and virion-associated RNA-dependent RNA polymerase activity was observed with these acidic fractions. Although amantadine inhibited virus plaque formation as effectively as PCE fractions, it was less effective in inhibiting the RNA polymerase activity. These results suggest that PCE, which has been shown to contain antitumor substance(s), also contains anti-influenza virus substance(s).

Inhibition of herpes simplex virus infection by tannins and related compounds.

Several chemically defined plant extracts were investigated for their antiviral action on herpes simplex virus (HSV-1, HSV-2)-infected African green monkey kidney cells and human adenocarcinoma cells, using a plaque formation assay. Among them, the monomeric hydrolyzable tannins, oligomeric ellagitannins and condensed tannins, having galloyl groups or hexahydroxydiphenoyl groups, had the most potent anti-HSV activity. Their 50% effective doses (0.03-0.1 microgram/ml) were by two-three orders of magnitude lower than their 50% cytotoxic doses (greater than 10 micrograms/ml). On the other hand, gallic acid, neutral polysaccharides, chemically modified (N,N-dimethylaminoethyl-, carboxymethyl-, and sulfated-) glucans, sialic acid-rich glycoproteins, and uronic acid-rich pine cone polysaccharide showed little or no activity. Using radiolabeled virus particles, we demonstrated that the anti-HSV effect of the tannins is due to inhibition of virus adsorption to the cells.

Clinical assessment of prolonged myocardial preservation for patients with a severely dilated heart.

BACKGROUND: The purpose of this study was to compare the myocardial protective effect of histidine-tryptophan-potassium and glucose-insulin-potassium cardioplegic solutions in patients with a dilated heart (left ventricular diastolic diameter > 55 mm, left ventricular systolic diameter > 45 mm) associated with prolonged cross-clamp time (longer than 200 minutes). METHODS: We selected 20 patients with dilated hearts due to severe aortic regurgitation. Glucose-insulin-potassium cardioplegia was used in 11 patients and histidine-tryptophan-potassium cardioplegia was used in 9 patients. RESULTS: After operation, the cardiac index was significantly increased in the histidine-tryptophan-potassium group (p < 0.05). Postoperative percent fractional shortening was 13.4% +/- 3.1% in the glucose-insulin-potassium group and 23.6% +/- 2.6% in the histidine-tryptophan-potassium group (p < 0.05). Creatine kinase levels were significantly lower in the histidine-tryptophan-potassium group than that in the glucose-insulin-potassium group (p < 0.05). The incidence of ventricular arrhythmia (higher than Lown's grade 2) was lower in the histidine-tryptophan-potassium group. CONCLUSIONS: These data support the superiority of the histidine-tryptophan-potassium method over the glucose-insulin-potassium method for protection of the dilated heart during prolonged ischemia in open heart operations.

A novel EBNA-1 titration method and putative anti-EBNA-1 protein.

A novel and rapid EBNA-1 titration method has been developed which uses immunoprecipitation of specific DNA-protein complexes with EBNA-1-positive serum. The method is more sensitive than the conventional immunofluorescence method and has potential value as a diagnostic reagent for clinical laboratories. TPA induction of putative anti-EBNA-1 protein of cellular origin is discussed, which may play a key role for the shift from latent to lytic replication of EBV.