A defective nontransmissible recombinant Sendai virus mediates efficient gene transfer to airway epithelium in vivo.

Recombinant Sendai virus (SeV)-mediated gene transfer to differentiated airway epithelial cells has shown to be very efficient, because of its ability to overcome the intra- and extracellular barriers known to limit gene delivery. However, this virus is transmission competent and therefore unlikely to be suitable for use in clinical trials. A nontransmissible, replication-competent recombinant SeV has recently been developed by deleting the envelope Fusion (F) protein gene (SeV/DeltaF). Here we show that SeV/DeltaF is able to mediate beta-galactosidase reporter gene transfer to the respiratory tract of mice in vivo, as well as to human nasal epithelial cells in vitro. Further, in an ex vivo model of differentiated airway epithelium, SeV/DeltaF gene transfer was not importantly inhibited by native mucus. When compared to the transmission-competent SeV in vivo, no difference in gene expression was observed at the time of peak expression. The development of an F-defective nontransmissible SeV, which can still efficiently mediate gene transfer to the airway epithelium, represents the first important step towards the use of a cytoplasmic RNA viral vector in clinical trials of gene therapy.

Necessity of thromboxane A2 for initiation of platelet-mediated contact sensitivity: dual activation of platelets and vascular endothelial cells.

To investigate the crucial role of platelet-derived thromboxane A(2) (TXA(2)) in initiating Ag-specific contact sensitivity (CS), a platelet-dependent CS model using genetically mast cell-deficient W/W(v) mice, was provided. In vivo treatment with BAYu3405, a TXA(2) receptor antagonist, markedly suppressed CS responses in a dose-dependent manner. This inhibitory effect occurred when BAYu3405 was administered before an early initiating phase, suggesting that TXA(2) may be a potent initiator of platelet-mediated CS responses. When platelets were pretreated with BAYu3405 in vitro, platelet aggregation as well as serotonin release, which is able to induce the early phase response allowing local recruitment of CS effector T cells due to direct activation of vascular endothelial cells, was inhibited. The addition of U46619, a TXA(2) agonist, or a mixture of platelets and thrombin-enhanced expression of both ICAM-1 and VCAM-1 on isolated mouse aortic endothelial cells, which was completely abolished by pretreatment with BAYu3405. Furthermore, intradermal injection of U46619 into the ear of platelet-depleted mice led to CS responses with marked expression of ICAM-1 and VCAM-1 on the vascular endothelium. These findings suggest that TXA(2) generated from platelets activated with Ag may mediate initiation of CS responses through inducing serotonin release from platelets and the subsequent aggregation and up-regulated expression of ICAM-1 and VCAM-1 on vascular endothelial cells.

Identification and partial purification of a basic fibroblast growth factor-like growth factor derived from bovine colostrum.

Bovine colostrum that had been collected up to 6 h postpartum was fractionated by ammonium sulfate precipitation, and various fractions were examined for basic fibroblast growth factor activity. Activity that stimulated cell growth was detected in the cream fraction, which was purified by isoelectric focusing and heparin affinity chromatography. Three peaks were eluted from the heparin affinity column at approximately 0.5, 1, and 1.75 M NaCl. Although activity that stimulated cell growth was detected in the second and third peaks, a reaction with antibasic fibroblast growth factor antibody was observed only in the third peak. Fractions in the second and third peaks were examined by SDS-PAGE and Western blot analysis. Activity that stimulated cell growth was detected in the second and third peaks; however, after Western blot analysis using antibasic fibroblast growth factor, only the third peak yielded positive bands at 15 and 28 kDa. These fractions were further subjected to a neutralization test using antibasic fibroblast growth factor antibody. The activity that stimulated cell growth in the second peak was virtually unchanged; however, the activity in the third peak was diminished, showing a relative activity of less than 10% at 1.25 micrograms/ml. Therefore, neutralization of the activity that stimulates cell growth by antibasic fibroblast growth factor antibody suggests that the third peak, which was eluted at approximately 1.5 to 2 M NaCl in heparin affinity chromatography, might be a basic fibroblast growth factor-like growth factor.

Detection of Epstein-Barr virus DNA in virus-infected cells by electron microscopic in situ hybridization.

We describe a procedure for in situ hybridization using a biotinylated Epstein-Barr virus (EBV) sequence with detection at the light and electron microscopic levels. In situ hybridization using an immunogold-silver staining detection system was used to identify biotinylated DNA probes in cell smears and in Lowicryl K4M-embedded EBV-infected and -noninfected cell lines. At the light microscopic level, the reaction product of hybridized EBV DNA sequence seemed to be located mainly in the nuclei. The labelling was dependent on the cell strains. However, at the electron microscopic level, the reaction product was evident as spots or clusters distributed not only in the nuclei of EBV-infected cells but also in the cytoplasm and extracellular particles. These findings suggest that immature particles in the cytoplasm contain EBV DNA. This procedure can be applied to the observation and identification of virus infection.

Hodgkin disease occurring in a patient with extremely high serum antibody titers to Epstein-Barr virus--associated antigens without chronic illness.

PURPOSE: We described for the first time a patient with long-lasting, extremely high serum antibody titer against Epstein-Barr virus (EBV) viral capsid antigen and early antigen without clinical symptoms suggestive of active EBV infection; the patient finally developed Hodgkin disease (HD) after 7 years of follow-up. PATIENT AND METHODS: High serum EBV antibody titers were noted at 2 years of age. Immunological evaluation was performed at the age of 7 years. EBV-specific cytotoxic T-lymphocyte activity was normal. None of the other results showed any significant abnormalities except for the abnormal antibody titers against EBV-associated antigens. RESULTS: The patient developed HD at the age of 9 years. In addition, EBV genomes were found in the nuclei of Hodgkin and Reed-Sternberg cells in the lymph node. CONCLUSIONS: This case suggests that (a) a patient with extremely high serum antibody titers against EBV-associated antigens may develop HD after a prolonged period, even though no clinical symptom suggestive of active EBV infection is observed; (b) EBV may play an important role in the occurrence of HD.

Detection and quantification of virus DNA in plasma of patients with Epstein-Barr virus-associated diseases.

Epstein-Barr virus (EBV) causes various diseases, such as infectious mononucleosis (IM), fatal IM, EBV-associated hemophagocytic syndrome (EBVAHS), and chronic active EBV infection (CAEBV). In the present study, cell-free EBV DNA was detected in the plasma of patients with EBV-associated diseases by PCR assay. The patients included 20 patients with IM, 2 patients with fatal IM, 4 patients with EBVAHS, 4 patients with CAEBV, and 38 healthy children (20 EBV seropositive and 18 EBV seronegative). In patients with IM, plasma samples were positive for EBV DNA in all patients (100%) in the acute phase and in 44% of the patients in the convalescent phase, but plasma samples from the 38 healthy control children were negative (0%) for EBV DNA. Quantitative PCR assay revealed that plasma from patients with IM contained the highest amount of virus DNA within 7 days following the onset of disease (mean, 6 x 10(4) copies per ml). The EBV DNA concentration decreased thereafter as the patients recovered. Plasma from patients with fatal IM contained more than 100 times more copies of EBV DNA (3 x 10(7) copies per ml) than plasma from patients with IM. Plasma from patients with the acute phase of EBVAHS contained 10 times more copies of EBV DNA (5 x 10(5) copies per ml) than plasma from IM, and then patients with the number of copies decreased similarly in both groups of patients in the convalescent phase (2 x 10(4) copies per ml). The amount of virus DNA in patients with CAEBV (6 x 10(4) copies per ml) was similar to that noted in patients with IM; however, it became higher (1 x 10(6) copies per ml) when the patients' clinical status deteriorated. These data suggest that the presence of cell-free EBV DNA in plasma is a common phenomenon in patients with EBV-associated diseases. The concentration of EBV DNA in plasma seems to be higher in patients with the more severe clinical categories of EBV diseases.

An infantile case of cytomegalovirus induced idiopathic thrombocytopenic purpura with predominant proliferation of CD10 positive lymphoblast in bone marrow.

An infant with cytomegalovirus infection (CMV) developed idiopathic thrombocytopenic purpura (ITP) at 4 months of age. A bone marrow (BM) aspiration showed a remarkable increase of immature megakaryocytes and prominent proliferation of lymphoblasts. Flow cytometric analysis of the bone marrow cells showed that the predominant cells in the lymphocyte cluster were of B-lineage (CD19) with CD10 (common acute lymphoblastic leukemia antigen) positive. Virus study showed a higher titer of CMV antibody. Cytomegalovirus DNA was detected by the polymerase chain reaction (PCR) method in urine, peripheral cells and marrow cells. Low-grade fever, diarrhea and petechiae were accompanied by mild liver dysfunction. Complete remission was made with intravenous high-dose immunoglobulin (IVIg) without progression to overt acute leukemia. The percentage of CD10+/CD19+ lymphocytes in bone marrow also diminished. We postulated that the proliferation of immature lymphocytes and megakaryocytes in bone marrow was caused by maturation arrest that might result from CMV infection.

Rapid detection of cytomegalovirus DNA in sera using the polymerase chain reaction: relationship with clinical diagnosis of cytomegalovirus infection after renal transplantation.

A 21-year-old man had chronic renal failure and received a renal transplant. We used the polymerase chain reaction technique to detect cytomegalovirus (CMV) DNA in serum, blood, and urine for the diagnosis of CMV infection after the renal transplantation. CMV DNA in the serum was detected by polymerase chain reaction only during the active stage, while CMV DNA in urine and blood was detected even during the silent stage of CMV infection. It is suggested that the use of the polymerase chain reaction in serum would be useful for the rapid diagnosis of active CMV infection.

Epstein-Barr virus activity in patients on chronic hemodialysis.

Patients with uremia are susceptible to viral infections, especially to Epstein-Barr virus (EBV). Sixty-one patients with end-stage renal diseases on chronic hemodialysis (HD), 14 patients with impaired renal function (CRF), and 27 healthy controls were studied with regard to EBV infection. Uremic patients (HD and CRF) had a significantly higher incidence of EBV infection and higher titers of anti-EBV VCA-IgG antibody than healthy controls. The anti-EBNA-1 titer was significantly higher in patients whose dialysis period was more than 3 months than in whom the dialysis period was 3 months or less. Immunoblotting analysis also showed stronger EBNA-1 signals in hemodialysis patients than EBNA-2, which was strongly detected in the CRF group and in healthy controls. EBV DNA was detected by Southern blot hybridization after PCR amplification of peripheral leukocytes, and occurred at a greater incidence in hemodialysis patients than in the other groups. Taken together, these results demonstrated that hemodialysis patients had persistent EBV infection.

[Rapid diagnosis of cytomegalovirus (CMV) infection in a renal transplant recipient by polymerase chain reaction (PCR)].

We used the PCR method to detect CMV-DNA in serum, urine, and peripheral blood of a patient who had received a renal transplant. A correlation was found between the active stage of the infections and the serum level of CMV-DNA. While no correlation was found in urine or blood. To detect CMV-DNA as early as possible, the serum samples were prepared by glass powder treatment and subjected to PCR followed by nonradioactive DNA hybridization. Moreover, to increase the sensitivity of detection a nested PCR method was employed and we found that the PCR-products were easily detected on the ethidium bromide-stained gel without the following hybridization. Thus, PCR in serum would be useful for rapid diagnosis of the infection and monitoring anti-viral drug therapy.

[A case of Budd-Chiari syndrome treated by radical operation under hepatic perfusion and extracorporeal circulation].

A 41-year-old male patient of Budd-Chiari syndrome associated with membranous obstructions of the retrohepatic inferior vena cava and the left hepatic vein was reported. A radical operation was carried out. The retrohepatic inferior vena cava was reconstructed by a ringed EPTFE patch graft after endovenectomy with the aid of extracorporeal circulation for caval and portal bypasses utilizing cold hepatic perfusion. The patient has been doing well 18 months after the operation.

Detection of human cytomegalovirus DNA in dried newborn blood filter paper.

To detect human cytomegalovirus (HCMV) DNA, filter paper spotted with peripheral blood from newborns 4 to 7 days after birth was dried and subjected to the polymerase chain reaction (PCR) followed by Southern blot hybridization with a nonradioactive oligonucleotide probe. The detection rates were 25.1% in healthy individuals and 33.0% in low body weight neonates weighing not more than 2500 g at birth, most of whom appeared to have been infected transplacentally or by other means within the uterus. HCMV was detected after only heating the dried blood on the filter paper, and may be applied as a screening method for the early diagnosis of HCMV.

Structural study of the sugar chains of porcine factor VIII--tissue- and species-specific glycosylation of factor VIII.

The asparagine-linked sugar chains of blood coagulation factor VIII purified from porcine plasma were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and the sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns. Structural study of each oligosaccharide by sequential exoglycosidase digestion and by methylation analysis revealed that porcine factor VIII contains high mannose-type and bi-, tri-, and tetraantennary complex-type sugar chains. Sixty-seven percent of the complex-type sugar chains contained the Gal alpha 1-->3Gal group, and 23% of the biantennary complex-type sugar chains contained the bisecting N-acetylglucosamine residue. These structures were not detected in the sugar chains of human plasma factor VIII. An in vitro competition study of von Willebrand factor and anti-Gal antibody for binding to factor VIII revealed that von Willebrand factor prevented antibody binding to Gal alpha 1-->3Gal groups in porcine factor VIII sugar chains. This suggests that anti-Gal antibody present in human plasma may not interact with the sugar chains of therapeutic porcine factor VIII. Reverse-transcription polymerase chain reaction was used to identify porcine tissues producing FVIII mRNA. These studies revealed that the kidney is one of the major tissues expressing factor VIII which may contain the sugar chains with the bisecting N-acetylglucosamine residue.

Fatal Epstein-Barr virus-associated hemophagocytic syndrome.

A virus-associated hemophagocytic syndrome is characterized by high fever, liver dysfunction, coagulation abnormalities, pancytopenia, and a benign histiocytic proliferation with prominent hemophagocytosis in bone marrow, lymph node, spleen, and liver. We describe six Japanese children with fatal Epstein-Barr virus (EBV)-associated hemophagocytic syndrome. Five of the six patients had serologic evidence of primary EBV infection at the onset of their diseases. EBV genomes were detected in all the patients by Southern blot hybridization or the polymerase chain reaction. Furthermore, clonality analysis of the EBV genome showed that EBV-infected cells proliferated monoclonally or biclonally in three examined patients. In situ hybridization study using EBV-encoded RNA 1 (EBER1) showed that EBER1 was detected in one of two examined liver tissues, which localized in hepatocytes.

Detection of Epstein-Barr virus transcripts in chemically or immunologically-activated cells and in a null cell-line (HLN-STL-C) by in situ hybridization with alkaline phosphatase-linked oligonucleotide probes.

We report a simple procedure for the detection of Epstein-Barr virus (EBV) by in situ DNA-RNA hybridization with an alkaline phosphatase-linked oligonucleotide probe. EBV-producing cell lines P3HR-1 and Akata were treated with phorbol ester and n-butyrate, and anti-human IgG, respectively. This treatment resulted in highly increased populations of cells with EBV transcripts of the latent membrane protein 1 (LMP1) and envelop glycoprotein gp350/220, but not of EBV-encoded small nuclear RNAs (EBERs). Synthesis of the LMP1 protein, which was encoded by the induced mRNA, was mostly dependent on viral DNA synthesis, as shown by double or single labeling for in situ DNA-DNA hybridization with the oligo-nucleotide probe, and immunoperoxidase staining with a monoclonal antibody against LMP1. In situ hybridization of the null cell line HLN-STL-C established from an adult T-cell leukemia patient showed that 100% of the cells contained both EBERs and LMP1 mRNA and about 0.1% of the cells contained gp350/220 mRNA, indicating that a few of the null cells which carried the EBV genome spontaneously entered the late EBV replication cycle.

Epstein-Barr virus genome-positive tubulointerstitial nephritis associated with Kawasaki disease-like coronary aneurysms.

A patient with recurrent renal failure due to massive interstitial nephritis caused by Leu 3a + 3b-positive T-cell infiltration and associated with multiple thromboembolic attacks is reported. He died of gastrointestinal bleeding after treatment with anticancer agents. At autopsy, diffuse necrosis of the bilateral kidneys was noted as well as giant coronary aneurysms filled with thrombus that resembled those seen in Kawasaki disease and multiple old myocardial infarcts were also present. Among the various Epstein-Barr virus (EBV)-specific antibodies, the titers anti-viral capsid antigen (VCA) and anti-early antigen (EBEA) IgG antibody were always very high in contrast to the relatively low titers of anti-EB nuclear antigen (EBNA) antibodies. DNA extracted from kidney tissue obtained at autopsy was analyzed by Southern blot hybridization after the amplification of EBV-specific DNA by the polymerase chain reaction. In situ hybridization of kidney tissue obtained at biopsy was also performed using an enzyme-linked probe derived from the EBV-encoded RNA 1 (EBER1) gene. As a result, the EBV genome was found both at autopsy and in the biopsy tissue, which clearly revealed EBER1 in the interstitial cells. Taking account of the progressive ST-T changes of the electrocardiograms which were normal early in his course, multiple myocardial infarction associating multiple giant aneurysms probably occurred during this disease process. Thus, it could be concluded that chronic active EBV infection contributed massive interstitial nephritis mediated by the activation of Leu 3a + 3b-positive T cells.

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 by using polymerase chain reaction DNA amplification.

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 (MDV1) was attempted by polymerase chain reaction (PCR) using the primers chosen from the sequence within the long inverted repeats of MDV1 DNA. PCR of the DNAs extracted from oncogenic-strain-infected cells and Marek's disease tumor cell lines produced a major product containing two or three copies of 132-base-pair (bp) repeat units, whereas PCRs of the DNAs extracted from nononcogenic-strain-infected cells yielded amplified products with various sizes corresponding to the number of 132-bp repeat units. The primers chosen from the glycoprotein A genes of MDV1 and herpesvirus of turkeys also were used for determination of their serotype specificity. The PCR procedure was found to be a simple and sensitive procedure for identification of MDV1 and herpesvirus of turkeys and for estimation of oncogenicity of MDV1.

Poly(ADP ribose) polymerase-defective mutant cell clone of mouse L1210 cells.

By a sequential mutation and selection utilizing N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen, we succeeded in separating a poly(ADP ribose) polymerase-defective mutant clone (Cl-3527) from a mouse L1210 cell clone (Cl-3). The enzyme activity per cell in Cl-3527 cells was only 8% of that in wild type L1210 (CCL 219) cells. Immunoblot analysis of the enzyme protein in crude extracts of the mutant and wild type cells revealed that the enzyme defect was manifested as the loss of a 113-kDa wild type enzyme band in Cl-3527. Further analysis of partially purified enzyme from Cl-3527 by immunoblotting revealed that the molecular size of the enzyme in Cl-3527 was 108 kDa and that the amount of the mutant enzyme protein was markedly decreased in Cl-3527. The mutant enzyme was much more heat-labile than the wild type enzyme but the Km for NAD+, requirements for Mg2+ and nicked DNA, and the inhibition by 3-aminobenzamide, a potent inhibitor of the enzyme, however, were not so different from those of wild type enzyme. The mutant cells showed prolonged doubling time, increased temperature-sensitivity, increased percentage of active enzyme on a treatment of cells at high temperature, and increased expression of plasma membrane NADase, compared to wild type cells. Introduction of wild type ADPR pol gene into Cl-3527 cells partially restored the ADPR pol activity and the heat-resistance.