Cell cycle arrest and lytic induction of EBV-transformed B lymphoblastoid cells by a histone deacetylase inhibitor, Trichostatin A.

Latent infection of the Epstein-Barr virus (EBV) is strongly associated with the pathogenesis of several human tumor types. The restricted expression of the latent EBV antigens is critical for EBV-associated tumors to escape from immune surveillance. EBV lytic replication can be triggered by various treatments and the induced lytic genes cause strong cytotoxic T lymphocyte (CTL) responses. Histone acetylation or deacetylation is associated with chromatin remodeling and regulates gene expression. Histone deacetylase (HDAC) inhibitors affect cell cycle progression as well as gene expression in a wide variety of transformed cells. We examined whether an HDAC inhibitor, TSA, can affect cell cycle progression and induce EBV lytic replication in EBV-transformed lymphoblastoid cell lines (LCLs). TSA caused cell cycle arrest at low concentrations and induced apoptosis at higher (>300 nM) concentrations in the LCLs and EBV negative BJAB cells. To clarify the underlying mechanism of TSA-induced cell cycle arrest, expression of cell cycle regulatory factors was examined by RNase protection assay and Western blot analysis. Following TSA treatment, a reduced expression of cyclin D2 and an induction of p21 may have played an essential role for G1 arrest in LCLs, while p21 induction might have arrested BJAB cells in G1 phase. A Cdk inhibitor, p57, was increased by 300 nM TSA in both LCLs and BJAB cells, indicating its role in apoptosis. Moreover, immunofluorescene assay and Western blotting showed that TSA induced EBV lytic replication in LCL cells. These results suggest that TSA may exert an enhanced anti-tumor effect for EBV-associated tumors not only by inducing a cell cycle arrest and apoptosis, but also by triggering an EBV lytic cycle.

Enhancement of Q(m) by co-doping of Li and Cu to potassium sodium niobate lead-free ceramics.

Lead-free piezoelectric ceramics KNN modified by Li-substitution and CuO addition have been synthesized, and the piezoelectric and dielectric properties were measured. A morphotropic phase boundary (MPB) between orthorhombic and tetragonal phases was formed with Li-substitution. The co doping of Li and Cu markedly enhanced the mechanical quality factor (Q(m)) in comparison with the sole doping of Li and Cu. Anomalous anti ferroelectric-like hysteresis curves were observed in 2 mol% CuO-doped ceramics. The anti-ferroelectric-like curves were changed to that of normal ferroelectrics following poling. A model based on the formation of the internal bias field (Ei) due to the movements of space charges was proposed to explain these phenomena. It was considered that the Ei stabilized the spontaneous polarization (Ps) and suppressed the domain wall motion to enhance the Q(m). The highest Qm obtained in this study was 742. The [(Na0(0.5)K0(0.5))(0.96)Li0(0.04) ] NbO(3) + 0.45 mol% CuO ceramics showed a high Q(m) value of 414 with a high piezoelectric constant d(33) of 100 pC/N.

Increased frequency of antigen-specific CD8(+) cytotoxic T lymphocytes infiltrating an Epstein-Barr virus-associated gastric carcinoma.

Gastric adenocarcinomas carrying Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor-infiltrating lymphocytes (TILs), we isolated and cultured such cells from a surgically resected EBV-associated gastric carcinoma. They were found to be positive for CD3, CD8, T-cell receptor beta chain, and cytotoxic molecules. The isolated TILs consisted of human leukocyte antigen (HLA) class I-restricted CD8(+) cytotoxic T lymphocytes (CTLs), which killed autologous EBV-transformed cells (but not phytohemagglutinin blast cells) and recognized HLA-A24 as restriction molecules. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 molecules, nor HLA-A24(+) fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV(+) gastric carcinomas do not express conventional target proteins of EBV-specific CTLs, and the data suggest that some cellular proteins may be involved in the strong T-cell response to EBV-associated gastric carcinoma. In addition, our data suggest that class I-restricted, antigen-specific CD8(+) CTLs are specifically expanded within EBV(+) gastric carcinoma tissue.

Oncolytic virotherapy with an HSV amplicon vector expressing granulocyte-macrophage colony-stimulating factor using the replication-competent HSV type 1 mutant HF10 as a helper virus.

Direct viral infection of solid tumors can cause tumor cell death, but these techniques offer the opportunity to express exogenous factors to enhance the antitumor response. We investigated the antitumor effects of a herpes simplex virus (HSV) amplicon expressing mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) using the replication-competent HSV type 1 mutant HF10 as a helper virus. HF10-packaged mGM-CSF-expressing amplicon (mGM-CSF amplicon) was used to infect subcutaneously inoculated murine colorectal tumor cells (CT26 cells) and the antitumor effects were compared to tumors treated with only HF10. The mGM-CSF amplicon efficiently replicated in CT26 cells with similar oncolytic activity to HF10 in vitro. However, when mice subcutaneously inoculated with CT26 cells were intratumorally injected with HF10 or mGM-CSF amplicon, greater tumor regression was seen in mGM-CSF amplicon-treated animals. Furthermore, mGM-CSF amplicon treatment prolonged mouse survival. Immunohistochemical analysis revealed increased inflammatory cell infiltration in the solid tumor in the mGM-CSF amplicon-treated animals. These results suggest that expression of GM-CSF enhances the antitumor effects of HF10, and HF10-packaged GM-CSF-expressing amplicon is a promising agent for the treatment of subcutaneous tumors.

Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins in insect cells.

The insect-baculovirus expression system has proved particularly useful for producing recombinant proteins that are biologically active. Overexpression of foreign proteins using the recombinant baculovirus system is often accompanied by aggregation of the overexpressed protein, which is thought to be due to a limitation of the translated protein folding in the infected cells. Co-infection of a recombinant baculovirus capable of expressing the human chaperone Hsp70 slightly increased the solubility of the overexpressed Epstein-Barr virus replication protein, BZLF1. Co-expression of Hsp70 and its co-factor, Hsdj or Hsp40, was here found to improve the solubility of the target protein several fold. Thus, a baculovirus expression system producing these molecular chaperones may find application for improved production of target foreign gene products in insect cells.

Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 2 and comparison with the type 1 counterpart.

The complete nucleotide sequence of the DNA polymerase gene of herpes simplex virus (HSV) type 2 strain 186 has been determined. The gene included a 3720-bp major open reading frame capable of encoding 1240 amino acids. The predicted primary translation product had an Mr of 137,354, which was slightly larger than its HSV-1 counterpart. A comparison of the predicted functional amino acid sequences of the HSV-1 and HSV-2 DNA polymerases revealed 95.5% overall amino acid homology, the value of which was the highest among those of the other known polypeptides encoded by HSV-1 and HSV-2. The functional amino acid changes were spread in the N-terminal one-third of the protein, whereas the C-terminal two-third was almost identical between the two types except a particular hydrophilic region. A highly conserved sequence of 6 aa, YGDTDS, which has been observed in DNA polymerases of HSV-1, Epstein-Barr virus, adenovirus, and vaccinia virus, was also present at positions 889 to 894 in the C-terminal region of HSV-2 DNA polymerase.

Method to express and purify nm23-H2 protein from baculovirus-infected cells.

High-throughput protein expression and purification are major bottlenecks in the postgenomic and proteomic era. We show here an automated method to express and purify nm23-H2, a nucleoside diphosphate kinase (NDPK), in a 96-well format, by the use of a robotic workstation, from insect Spodoptera frugiperda (Sf9) baculovirus-infected cells using nickel-nitrilotriacetic acid (Ni-NTA) agarose beads. The automated method is coupled to mass spectrometry for a validation and quality-control analysis. To verify the bona fide of the recombinant protein, several tests have been produced, including NDPK assay, Western blotting, and in vitro phosphorylation experiments, thus confirming the value of the protocol developed. The method has been validated for the expression of several proteins, thus confirming the value of this automated protocol. The research presented here is a useful method both for industrial and academic environments to produce in a high-throughput mode recombinant eukaryotic proteins to be assayed for a specific function in a systematic manner.

Early intervention in post-transplant lymphoproliferative disorders based on Epstein-Barr viral load.

Using a real-time quantitative PCR assay, we identified two patients with EBV-related lymphoproliferative disorders at a very early stage. Both had received an unmanipulated bone marrow transplant with anti-thymocyte globulin for conditioning. To estimate virus-specific immunity, the frequencies of EBV-specific CD8+ T cells were measured using an enzyme-linked immunospot assay. The frequencies of EBV-specific CD8+ T cells of the two were 3.2 and 7.7%, respectively, which had possibly expanded in vivo. After withdrawing the immunosuppressive agents or administering donor lymphocytes transfusion, their symptoms regressed in parallel with the viral load.

Molecular cloning of herpes simplex virus type 2 DNA.

Restriction enzyme HindIII digestion of the whole genome of herpes simplex virus type 2 strain 186 yielded 10 DNA fragments with molecular weights ranging from approximately 22 X 10(6) to 1.2 X 10(6), which were cloned into the HindIII site of bacterial plasmid pACYC 184. The cloned fragments were identified by hybridization to HSV-2 virus DNA and by double digestion with restriction endonucleases. The recombinant plasmids, even if they carried DNA sequences with molecular weights of more than 10(7), were efficiently replicated in E. coli HB101.

Blunt chest trauma with deep pulmonary laceration.

BACKGROUND: Deep pulmonary laceration (DPL) is rare and its survival rate is low. The present study focused on the prognostic factors of DPL. METHODS: The present study concerned 17 DPL patients treated in Tokai University Hospital between 1988 and 1998. The prognostic factors of DPL were compared with systolic blood pressure (SBP), PaO2, and the volume of intrathoracic blood loss. Characteristic findings of initial chest roentgenograms of DPL were investigated. RESULTS: Eleven patients were saved and 6 patients died. An SBP of less than 80 mm Hg on arrival at the hospital and a blood loss of more than 1,000 mL through the chest tube within 2 hours after arrival were poor prognostic factors. Hypoxemia on arrival was not a poor prognostic factor. Chest roentgenograms showed macular infiltrative shadow with moderate lung collapse and deviation of the mediastinal shadow toward the unaffected side. Selective bronchial occlusion with a Univent prevented suffocation by intrabronchial blood. CONCLUSIONS: Two poor prognostic factors of DPL are SBP less than 80 mm Hg on arrival and blood loss of more than 1,000 mL through the chest tube within 2 hours after arrival.

Evaluation of cytology and tumor markers of pure pancreatic juice for the diagnosis of pancreatic cancer at early stages.

To evaluate the value of cytology of pure pancreatic juice (PPJ) and tumor marker determination in PPJ and serum for the diagnosis of early pancreatic cancer (EPC), PPJ was obtained endoscopically from 16 patients with EPC (< 20 mm, confined to pancreas), 16 patients with chronic pancreatitis (CP), and 20 controls. Cutoff levels of CEA, CA19-9, and POA in PPJ were set from ROC curves at 40 ng/ml, 7,500 U/ml, and 1.5 U/ml, respectively. For the differentiation of EPC from CP, the sensitivity of CEA, CA19-9, and POA was 71.4, 42.9, and 50%, respectively; specificity was 93.3, 46.7, and 80%, respectively; and diagnostic accuracy was 82.8, 44.8, and 65.5%, respectively. Determination of serum tumor markers was useless. Sensitivity of cytology was 75%, specificity was 93.8%, and diagnostic accuracy was 84.4%. Combined cytology and CEA determination in PPJ increased the diagnostic accuracy to 93.1%. The combination was useful in supporting and supplementing endoscopic retrograde cholangiopancreatography (ERCP) findings for the correct diagnosis in 11 and 4 patients, respectively, with EPC; in one patient EPC was correctly diagnosed on ERCP findings alone. One of 16 patients with CP showed false-positive results. We conclude that cytology and CEA determination in PPJ with ERCP is a useful combination for the diagnosis of pancreatic cancer even in early stages.

A case of chronic pancreatitis successfully treated by endoscopic removal of protein plugs.

A 56 years old male with chronic pancreatitis complained of intractable abdominal pain, anorexia, emaciation and peripheral edema. Medical treatment initiated only partial improvement in the general condition and hypoproteinemia. Endoscopic retrograde cholangiopancreatography revealed multiple filling defects in the dilated main pancreatic duct. Endoscopic aspiration of pure pancreatic juice yielded numerous protein plugs. The endoscopic removal of protein plugs from the pancreatic duct resulted in remarkable improvement in symptoms, laboratory findings and ERCP findings. We consider this procedure to be an important new treatment of chronic pancreatitis.

Nerve fiber analysis and age-related changes of the human mandibular nerve.

We morphometrically analyzed nerve fibers of the human mandibular nerve with a discriminative staining method that makes it possible to separate nerve fibers into myelinated and unmyelinated fibers. We counted numbers and transverse areas of myelinated axons under the microscope using an on-line image-analyzer. This study revealed the morphometric changes which affect the human mandibular nerve during the aging process.

Saving the cost and energy by an interactive multimedia system with ISDN 128 Kbps for telemedicine.

The telemedicine systems offer many potential advantages for health care delivery. In 1997, we implemented Phoenix, the interactive multimedia system of the Nippon Telegraph and Telephone Corporation (NTT) for clinical application involving teleconsultations over a wide area for delivery of special care in emergency medicine at Tokai University linking with eight hospitals via the Integrated Service Digital Network (ISDN) 128 Kbps. This study was designed to determine the potential saving of the cost and energy through the interactive multimedia network. By using the interactive multimedia system with some modifications, we achieved a satisfactory real-time contact regarding clinical matters. We believe that this network has allowed appropriate transfer of information between medical centers. This system has also significantly reduced the estimated cost for clinical meetings.

Availability of Japanese subsidies for international telemedicine projects.

In this article, the authors report the methods for obtaining subsidies for overseas telemedicine projects from Japanese sources based on their own personal experiences. The Japanese Government is already subsidizing such specialized NGOs (Non-Governmental Organizations) as the Telemedicine Society of Japan and Basic Human Needs and, Japanese trading companies are also hiring telemedicine experts. Prospective methods for obtaining subsidies are outlined as under the following headings: Assistance without compensation, Technology transfer, Grass-roots grant assistance, the Telecommunications Advancement Organization, Postal Savings for International Voluntary Aid, Venture business development funds provided by the Ministry of International Trade and Industry, Mission demonstration satellites by the National Space Development Agency of Japan, the Sasakawa Pacific Island Nations Fund, and International Communications Foundation. Key points of the applications are noted under (1) Degree of contribution to local residents, (2) Significance of project continuation and (3) Novelty and economic impact.

[A case of pulmonary infarction secondary to subacute bacterial endocarditis with subpulmonary VSD].

A case of pulmonary infarction secondary to subacute bacterial endocarditis of pulmonary valve which is associated with subpulmonary VSD is presented. The jet stream of blood through the subpulmonary VSD made damage to the pulmonary valve, which may be one of the reasons why subacute bacterial endocarditis was associated with the subpulmonary VSD. Echocardiography of the right-sided valves will be very useful in order to detect the pulmonary valve endocarditis in congenital heart disease presenting with fever.

[Surgical treatment of bullous emphysema using Gore-Tex sheet].

Surgical treatment for bullous emphysema using Gore-Tex sheet and mechanical stapler is reported. The techniques were followed in a Juetnner's report: Small cut is made at the center of a 10 cm square of a Gore-Tex sheet, through which the area of bullous lung planned for resection is tented out with a lung forceps. A stapling instrument charged with a 4.8 mm clips is placed over the sheet around the suture line, and closed over both sheet and lung parenchyma. The excess sheet is removed after resection of the bullous lung. Five cases of bullous emphysema with a history of pneumothorax were successfully treated by the above method. Air leakage disappeared on the first postoperative day at latest. We assume Gore-Tex sheet, although a non-absorbable material, is in use as well as polydioxanone ribbon for prevention of air leakage in surgical cases of bullous emphysema.

[Operation of pleural dissemination of thymoma].

Nine patients underwent operation for pleural dissemination of thymoma and received radiotherapy postoperatively. In 5 patients dissemination was resected along with the invasive thymoma at the initial operation. In 6 patients resection of a part of the diaphragm was needed. In 4 patients recurrent dissemination of thymoma appeared in the phrenico-costal sinus, where not only the location of dissemination but also postoperative radiotherapy had not been performed properly at the time of the previous operation. Seven are alive 2 months to 7 years 10 months after the first operation for dissemination. Two died respectively 7 years and 3 years 5 months after the first operation for dissemination.

[Experimental and clinical studies of bestatin as an immunomodulator].

The experimental and clinical effects of bestatin were examined and the results obtained were as follows. Bestatin which is an immunomodulator discovered by Umezawa did not increase the bone marrow stem cells examined by the method of the spleen colony assay in the 60Co irradiated mouse. However, it prolonged the survival time slightly when it was administered 10-25mg/kg intraperitoneally. Clinically, bestatin was administered to the patients with gastrointestinal cancer. It did not influence on the PHA-induced lymphocyte blastformation rate, but it increased the peripheral lymphocyte count and PPD skin reaction in the cases of curative resection and increased the peripheral lymphocyte counts in the cases of nonresection after 1 month of the operation.

[Abnormalities of blood coagulation and effect of anticoagulant therapy in postoperative patients with lung cancer].

To evaluate coagulable state, beta-thromboglobulin (beta-TG) levels have been followed sequentially in 70 postoperative patients with lung cancer. Nineteen out of them were treated with warfarin plus ticlopidine at a dosage enough to prolong the thrombo-test time to approximately 20% of normal value. There was a significant rise in beta-TG compared with control subjects and beta-TG was correlated with stages of disease. Serial beta-TG determinations revealed that beta-TG and CEA levels fairly paralleled with each other which suggested beta-TG might be useful in following tumor progression or response to therapy in postoperative period. As to the relation between beta-TG levels and five-year survivals, patients whose beta-TG were under 50 ng/ml showed more favorable prognoses than those who had higher levels. Long term anticoagulation with warfarin plus ticlopidine reduced the beta-TG levels of 19 stage 3 or 4 patients, especially in stage 4 the rate of reduction was marked. Nineteen patients with anticoagulant-treated group demonstrated a more prolonged time from beginning of treatment to first evidence of disease progression than 18 non-treated patients. Also anticoagulant-treated group had a more prolonged clinical course than non-treated group after disease progression. These results might be associated with disease stabilization which achieved with anticoagulant therapy. Survival at 30 months after initiation of treatment was 74% in the treated group and 64% in the nontreated group. Although there was no statistical difference in two groups, survival of treated group exceeded that of the non-treated group throughout the observation period. In stage 4 patients, however, the difference between two groups was statistically significant.