Inhibition of DNA synthesis and alteration of cyclic adenosine 3',5'-monophosphate levels in RSa cells by human leukocyte interferon.

Human leukocyte interferon inhibits the proliferation of the virus-transformed human embryonic cells RSa. Incorporation of [3H]thymidine (TdR) into an intracellular pool and the activity of TdR kinase were reduced in the interferon-treated RSa cell culture. High-degree (90%) inhibition of [3H]TdR incorporation was associated with concentrations of added interferon that produced more than a twofold increase in the intracellular cyclic AMP (cAMP) level, and low-degree inhibition was associated with smaller increases in cAMP. In the IFr cell culture, which is relatively resistant to the anticellular action of interferon, considerably less inhibition of TdR incorporation and a slight increase in cAMP were observed. Extracellularly added dibutyryl-cAMP inhibited the proliferation of both RSa and IFr cells to almost the same degree. A decrease in cAMP level and the initiation of DNA synthesis of G1-phase-arrested RSa cells by serum addition were prevented when cells were pretreated with interferon. These results indicated that intracellular cAMP may mediate the inhibitory effect of interferon on DNA synthesis and cell growth.

Inhibition of DNA synthesis of synchronized RSa cells by human leukocyte interferon.

A human leukocyte interferon preparation suppressed DNA synthesis and subsequent mitosis of synchronously growing virus-transformed human RSa cells. DNA synthesis was inhibited completely when interferon was added to cell cultures in late G1 phase, but was not so inhibited when interferon was added in G1/S boundary phase. Overall protein synthesis was reduced only slightly by interferon treatment. The findings suggested that critical events leading to the inhibition of DNA synthesis and subsequent mitosis by interferon action take place in late G1 phase or S phase.

Mechanism of stimulation by human interferon of prostaglandin synthesis in human cell lines.

Human interferon beta (IFN-beta) stimulated the synthesis of prostaglandin E (PGE) and prostaglandin F2 alpha in IFN-sensitive RSa and GM258 cell lines, but not in IFN-resistant HEC-1 cells. IFN-beta at a concentration of 1000 units/ml elicited 2- to 3-fold increases in PGE production in these cell lines. In the presence of exogenous arachidonic acid (1 microgram/ml), IFN-pretreated cells produced 5-fold more PGE compared to the cell cultures in the absence of arachidonic acid. Prednisolone, an inhibitor of phospholipase A2, at a concentration of 2 micrograms/ml inhibited the enhanced synthesis of PGE by IFN-pretreated cells. Indomethacin (4 micrograms/ml), a potent fatty acid cyclooxygenase inhibitor, also inhibited the increased synthesis of PGE. IFN stimulated the release of [14C]arachidonic acid from phospholipids but did not stimulate the activity of fatty acid cyclooxygenase. These data suggest that IFN stimulates prostaglandin synthesis by promoting the release of arachidonic acid from phospholipids. Since cycloheximide and actinomycin D inhibited the stimulation of PGE synthesis, the stimulation of prostaglandin synthesis by IFN seemed to be due to de novo enzyme synthesis which catalyzes the release of fatty acid. Addition of exogenous PGE suppressed the growth of RSa and GM258 cells. Prednisolone and iodomethacin partially inhibited anti-cell growth activity of IFN, suggesting a possibility that IFN-inhibited cell growth was partly mediated by prostaglandin.

Expression of human papillomavirus type 6b E2 gene product with DNA-binding activity in insect (Bombyx mori) cells using a baculovirus expression vector.

Human papillomavirus type 6b (HPV6b) has been shown to be a major etiologic agent of genital condylomas. The E2 gene, one of the early genes, has been shown to activate the enhancer element in trans in cells transformed with bovine papillomavirus type 1a (BPV1a) but the E2 gene product of any HPV has not been identified. The E2 gene of HPV6b was inserted into the polyhedrin gene of a Baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV), 156 bp downstream from the translational start codon, and transferred into BmNPV by allelic replacement in a cotransfected Bombyx mori cell line, Bm-N. The predicted 46-kDa protein was produced by a recombinant virus in the infected Bm-N cells at a high level under the control of the polyhedrin promoter. We obtained the antibody against the putative E2-polyhedrin fusion protein by immunizing a rabbit with this protein. This protein reacted with the antibodies against polyhedrin and the fusion protein. This protein did specifically bind to the upstream regulatory region of the HPV6b and BPV1a genomes. This DNA binding activity was blocked by the antibody against this protein.

Expression of the E2 open reading frame of papillomaviruses BPV1 and HPV6b in silkworm by a baculovirus vector.

To produce and characterize the nature of the E2 protein of papillomaviruses, we have developed a system to express the DNA sequence containing an open reading frame (ORF) as a fusion protein in cultured insect cells and silkworms. The DNA fragments of the E2 ORF predicted from the DNA sequence of bovine papillomavirus type 1 and human papillomavirus type 6b were linked to the N-terminal part of polyhedrin gene of the Baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) vector. Hybrid proteins composed of polyhedrin protein (52 amino acids) and the E2 proteins of BPV1 (410 amino acids) or HPV6b (346 amino acids) were efficiently produced in B. mori cells and silkworm larvae infected with recombinant viruses. The amount of E2 fusion proteins produced by recombinant viruses was comparable to that of polyhedrin produced by wild type BmNPV. The hybrid proteins were immunologically reactive to antiserum against polyhedrin.

The effect of methylprednisolone on myocardial beta-adrenergic receptors and cardiovascular function in shock patients.

The effect of methylprednisolone on the myocardial beta-adrenergic receptors after long term ( > 72 h) catecholamine infusion was studied. In 56 patients with pulmonary arterial catheter, 10 mg/kg of methylprednisolone was given as an intravenous bolus. Significant increases could be seen in cardiac output and blood pressure in patients who were simultaneously treated with vasopressors like dopamine and/or dobutamine. In patients who were on dopamine infusion higher than 10 micrograms/kg/min methylprednisolone there was an increase in the systemic vascular resistance. Patients who were not on vasopressors but received methylprednisolone, had no similar changes in hemodynamic parameters. In an in vitro analysis of tissue from the myocardium in 12/56 patients who succumbed and in four additional patients who expired after multiple trauma, a beta-adrenergic receptor assay was performed. It was found that the long term infusion of catecholamines decreased the receptor number and the methylprednisolone abolished or caused the decrease to be less pronounced. In this study we could not control the selection of the patients; a randomized study needs to be conducted in the future.

Effect of glycolipids and glycophorin on the activity of human interferon-beta and -gamma.

We have studied neutralization of the antiviral activity of human fibroblast (IFN-beta) and immune interferon (IFN-gamma) by incubation with glycolipids (including the gangliosides GM1, GM2 and GM3, as well as various other glycolipids) and with the sialoglycoprotein, glycophorin. When 100 units/ml of IFN-beta were preincubated with 30-80 microM of the gangliosides, all antiviral activity was abolished. Similarly, 120 microM of glycophorin completely reversed the antiviral activity of 100 units/ml of IFN-beta. Glycolipids containing more than two sugars also showed moderate inhibitory effects. GM2 at a concentration of 200 microM almost completely inhibited the antiviral activity of 100 units/ml of IFN-gamma, but GM1, GM3 and glycophorin had only a moderate inhibitory effect. These results suggest that the terminal N-acetylneuraminic acid (NANA) residues of gangliosides and of glycophorin play an important role in the inhibition of IFN-beta, and that they may be similarly involved in the inhibition of IFN-gamma.

Expression of the anti-retrovirus action of interferon in human cells which exhibit a reduced number of receptors for interferon.

The anti-viral and anti-cell fusion actions of human gamma interferon (IFN) were examined on human rhabdomyosarcoma cells and compared with the actions of IFN-alpha. Treatment of A204 and RD114-C1 cells with IFN-gamma resulted in significant inhibition of retrovirus production and cell fusions which were induced by Sendai virus, but IFN-gamma did not induce 2'-5' oligoadenylate (2-5A) synthetase or dsRNA-dependent protein kinase, and failed to inhibit EMC virus replication in RD114-C1 cells as previously observed on IFN-alpha treatment (Tomita, Y. et al. (1982) Virology 120, 258-263). Although IFN-gamma induced 56K protein more strongly than IFN-alpha in human transformed HEp-2, HeLa, RSa, IFr, and A204 cells, no significant induction of this protein was observed in RD114-C1 cells after IFN-alpha or IFN-gamma treatment. Specific bindings of 125I-labeled human IFN-alpha A to HeLa, A204 and RD114-C1 cell surfaces showed that the numbers of the binding sites on RD114-C1 cells were reduced to less than 22% of those on A204 cells. These results suggest that RD114-C1 cells exhibit a reduced number of receptors for IFN on the cell surface and that the receptors are functional for the expression of the anti-retrovirus and anti-cell fusion actions of IFN, but are not enough in number for expression of the anti-EMC virus action of IFN.

Effects of concanavalin A on the antiviral and cell growth inhibitory action of human interferons.

Sensitivities of human transformed cell line RSa and its variant cell line IFr to the cytotoxicity of Con A were compared. IFr cells were more resistant than RSa to Con A. Con A-resistant cell lines, Con Ar-1 and Con Ar-3, were isolated from RSa, and they were slightly more sensitive than RSa cells to the cell growth-inhibitory actions of interferons. Agglutinability of RSa, IFr, and Con Ar cells by Con A was compared and found to be almost equal. The combined effects of Con A and interferon upon growth and viability of these cell lines were tested. When RSa and IFr cells were treated simultaneously with Con A and Le-IF, growth of the cells was suppressed more markedly than when treatment was with Con A or Le-IF alone. To clarify the mechanism of this phenomenon, binding of 125I-labeled Con A was examined. Though ther wee some differences, both leukocyte and fibroblast interferon enhanced the binding of Con A to RSa cells and also in Con Ar cells but, in interferon-resistant IFr and HEC cells, enhancement of Con A binding was low or not observed. Therefore, the combined effect of Con A and interferon on the inhibition of cell growth is not considered to be merely due to the enhanced binding of Con A by interferon action. Successive treatment of RSa or Con Ar cells with Con A and interferon did not enhance the antiviral action of interferon at all. On the contrary, simultaneous treatment with Con A and interferon suppressed the antiviral action of interferon, depending on the concentration of Con A used. Thus, the effect of Con A on the antiviral and cell growth-inhibitory actin of interferon seems rather different.

Corticosteroid effect on early beta-adrenergic down-regulation during circulatory shock: hemodynamic study and beta-adrenergic receptor assay.

OBJECTIVES: The steroid effect on critically ill patients remains controversial. The aim of this study is to characterize the effect of methylprednisolone on the heart in a beta-adrenergically down-regulated condition. DESIGN: A prospective hemodynamic study and retrospective receptor assay. SETTING: Multidisciplinary ICU in a university hospital. SUBJECTS: 42 patients who required pulmonary arterial catheters and an additional 4 corpses who were available for study within 3 h of their deaths. INTERVENTION: Intravenous methylprednisolone (10 mg/kg). MEASUREMENTS AND RESULTS: We pursued a hemodynamic study following a glucocorticoid administration. In patients who had undergone a long term (> 72 h) catecholamine treatment, the cardiac index increased. In patients who had undergone a short term (1-72 h) catecholamine treatment and in patients with no record of catecholamine administration, the cardiac index showed no remarkable change. Among the corpses, who died soon after their arrival, and the patients, who later died in the ward and were available for further study, we measured beta-adrenergic receptor density in the left ventricular myocardium. It was found that receptor density was decreased after long term catecholamine treatment. Methylprednisolone, on the other hand increased the receptor density. CONCLUSION: Methylprednisolone improved the cardiac index, intriguingly, in patients with long term catecholamine treatment in circulatory shock. Myocardial beta-adrenergic receptor also increased in number after the administration of methylprednisolone. However, the hemodynamic improvement caused by methylprednisolone was not observed in patients without beta-adrenergic down-regulation.

Measurement of nitric oxide in the rat cerebral cortex during hypercapnoea.

Changes in nitric oxide (NO) concentration and cerebral blood flow (CBF) in the parietal cortex during hypercapnoea were investigated in anaesthetized rats, using a NO-selective electrode and laser Doppler flowmetry. When hypercapnoea was induced by inhalation of 5% CO2 for 10 min, both the NO concentration and CBF increased. After administration of 7-nitroindazole, a neuronal NO synthase (nNOS) inhibitor, both the basal NO and CBF decreased, and responses to hypercapnoea were also significantly suppressed by 70.1% and 73.2%, respectively, compared with the control state. These results suggest that NO derived from nNOS is involved not only in maintaining resting cerebral circulation but also in regulating CBF response during hypercapnoea.

Induction of polyploidization in the human erythroleukemia cell line (HEL) by protein kinase inhibitor (K252a) and the phorbol-ester TPA.

Endomitosis (polyploidization) is a distinctive feature of megakaryocyte differentiation. We examined this mechanism in an erythromegakaryocytic cell line, HEL, using a protein kinase inhibitor K252a or a phorbol-ester TPA. HEL cells treated with K252a showed a marked increase in the proportion of CD41 positive cells and polyploid cells as well as in cellular size and nuclear size. TPA showed similar results but induced multi-nucleation instead of enlargement of nuclear size. K252a added at the G1/S boundary phase did not inhibit the first and second round DNA synthesis, but inhibited cell division. K252a did not inhibit the expression of genes involved in mitosis such as cyclin B, cdc25B and cdc2, in the first round S phase. However, the cyclin B associated Cdc2 kinase activity needed for mitosis during the G2/M phase was reduced by K252a. TPA delayed DNA synthesis and expression of these genes, and suppressed Cdc2 kinase activity in the second round G2/M phase. These results suggest that the polyploidization induced by K252a results from inhibiting mitosis possibly caused by suppression of Cdc2 kinase activity. TPA may induce the multi-nucleation through a different mechanism.

HIV infection of megakaryocytic cell lines.

Thrombocytopenia is a common hematologic disorder in HIV infection and occurs in both asymptomatic and AIDS patients. An autoimmune mechanism has been postulated for the platelet destruction associated with some forms of thrombocytopenia. However, recent studies revealed that megakaryocytes are susceptible to HIV infection and suggested the possibility that HIV can directly impair the platelet production from megakaryocytes. This study was designed to characterize the HIV receptor expression in megakaryocytic cells and the responsiveness to HIV infection. Four different megakaryocytic cell lines at different stages of differentiation were established from the peripheral blood of different individuals with hematologic malignancies. CMK and CMY cells (differentiated cell lines) expressed CD4, but CMS and CTS cells (poorly differentiated cell lines) did not. The HIV coreceptor CXCR4 was also expressed in CMY and CMK cells. HIV-1 (HTLV-IIIB) replicated in CMY cells persistently but not in other three cell lines. CMY cells as well as CMK cells were also susceptible to the lytic infection of HIV-2 (LAV2). Pretreatment of the CMY cells with anti-CD4 antibody inhibited the infection by both HIV-1 and HIV-2. Our results indicate that mature megakaryocytic cells express CD4 along with HIV coreceptors and are susceptible to HIV infection.

Expression of multidrug resistant gene (mdr-1/P-glycoprotein) in a megakaryoblastic cell line, CMK, and its enhancement during megakaryocytic differentiation.

Multidrug resistance is a severe clinical problem in the chemotherapy of malignant disease. Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often resistant to many anti-cancer chemotherapeutic drugs. Here we report the expression of the mdr-1/P-glycoprotein in a cell line, CMK, established from a patient with AMKL. Expression of mdr-1 mRNA in CMK11-5 cells, a well differentiated subline, was higher than in CMK6 cells, a poorly differentiated subline. The level of P-glycoprotein was also higher in CMK11-5 cells. The cytokines interferon-gamma (IFN-gamma), GM-CSF and IL-3, which were shown to induce megakaryocytic differentiation of CMK cells, enhanced the expression of the mdr-1 mRNA and levels of P-glycoprotein. These results imply that differentiated megakaryocytic cells may have higher levels of the P-glycoprotein expression, suggesting a possible normal physiological function of P-glycoprotein in mature megakaryocytes.

Establishment of a new human megakaryoblastic cell line, CMY, with chromosome 17p abnormalities.

A new megakaryoblastic cell line CMY was established from a Down's syndrome patient suffering from acute megakaryoblastic leukemia. The karyotypes of CMY showed deletion of chromosome 17 or the translocation of 17p, whereas the blasts of the patient did not reveal these abnormalities of chromosome 17 by conventional karyotype analysis. Blasts of the patient failed to respond to chemotherapy and complete remission could not be attained. The abnormalities of 17p became progressively predominant in the patient. These results suggest that the blasts of a minor clone which had the abnormalities of chromosome 17p might have existed in the patient from the beginning and CMY was established from the minor clone. Investigation of p53 gene by PCR-SSCP analysis revealed that blasts of the patient showed normal patterns, while CMY showed an abnormally migrating band in exon 5 alone. This result suggests that another novel oncogenic factor(s) besides p53 might be present on chromosome 17p and other tumor suppresser genes need to be studied.

A UV-sensitive human clonal cell line, RSa, which has low repair activity.

The repair activity of a human transformed cell line, RSa, which was found to be highly sensitive to the lethal effects of 254 nm far-ultraviolet radiation, was compared with that of HeLa cells by evaluating the range of UV-induced incorporation of [methyl-3H]thymidine ([3H]dThd) or 5-[6-3H]bromodeoxyuridine ([3H]BrdUrd) into deoxyribonucleic acid. Direct scintillation counting was used for measuring the extent of unscheduled DNA synthesis (UDS) in UV-irradiated cells, which were treated with hydroxyurea or with arginine deprivation. More quantitative measurements were made by using the density labeling and equilibrium centrifugation method for assaying repair replication. All the amounts of UDS and repair replication in RSa cells were markedly below those in HeLa cells. The possible relationships of the low repair activity to abnormally high UV sensitivity in RSa cells are discussed.

Current status of bacterial contamination of autologous blood for transfusion.

Autologous transfusion, although not without risk, does decrease the risk of transmitted diseases via homologous transfusion. However, strict quality control is required for autologous transfusion. In Japan, a recent enactment requires that written informed consent be obtained prior to blood transfusion, which therefore requires that clinicians provide sufficient explanation of the risks involved with this procedure. To the best of our knowledge, this is the first study to comprehensively evaluate the manner in which the safety of autologous blood transfusion can be compromised by bacterial contamination. For a 24-month period, between April 1996 and March 1998, bacterial contamination of all kinds of autologous blood samples was tested by sampling the culture immediately prior to transfusion. Subculturing, identification and susceptibility testing of the isolates were performed. From the 287 units of all kinds of autologous blood transfused, 18 were culture positive (6.3%). Positive blood cultures were obtained in two of the 59 units (3.4%) of autologous transfusion donated preoperatively (ATDP) that was infused intraoperatively, in three of the 117 units (2.6%) of hemodilution/autologous transfusion (HAT) and in three of the 81 (3.7%) of ATDP infused postoperatively. There was a high percentage (33.3%) of positive blood cultures in the cases of intraoperative blood salvage (IOBS). The total rate of positive blood cultures was 6.3% including IOBS and 3.1% excluding IOBS. The most common microorganism isolated from autologous blood was coagulase-negative Staphylococci in 12 of 18 culture-positive units (66.7%). Alpha Streptococcus uiridans was isolated in 2 units (11%) and Staphylococcus aureus was isolated in 1 unit (5.5%). However, none of the patients who received the culture-positive autotransfusion blood showed clinical signs or laboratory findings of bacteremia. Safe ATDP is threatened by bacterial contamination that can be introduced by numerous sources, such as the donors' blood, the skin at the site of venipuncture, the environment and the phlebotomist's finger. In the cases of IOBS, protection against bacterial contamination at the surgical site is crucial. Here we discuss the relevance of our findings to the efforts to minimize the risks of contamination associated with autologous blood transfusion; risks that must be communicated to the patient in the process of informed consent. Continued research is required to identify the safest method of autologous blood transfusion.

In vitro and in vivo evaluation of BO-2727 against imipenem- and/or meropenem-resistant Pseudomonas aeruginosa.

The in vitro and in vivo activity of BO-2727, a carbapenem antibiotic, against resistant clinical isolates of Pseudomonas aeruginosa was studied. The geometric mean MICs against three groups of clinical isolates resistant to imipenem, meropenem and both carbapenems were 4.28, 4.08 and 5.44 micrograms/ml, respectively. BO-2727 also inhibited multiply antibiotic resistant isolates and laboratory mutants including a nalB-type mutant, which showed resistance to antibiotics such as imipenem, meropenem, ceftazidime, and/or ciprofloxacin, at less than 1.56 micrograms/ml. Overall, BO-2727 was 4-fold more active than biapenem, meropenem, panipenem and imipenem with an MIC90 of less than 6.25 micrograms/ml. The presence of basic amino acids in minimal medium less affected the antipseudomonal activity to a minimal extent, suggesting that BO-2727 has diverse penetration routes through the outer membrane other than OprD channel, which facilitates the diffusion of basic amino acids and carbapenems. The in vitro activity of BO-2727 reflected well in its therapeutic efficacy in experimental systemic infection in mice. These results suggest a possibility for the development of antipseudomonal carbapenems having activity against imipenem- and/or meropenem-resistant P. aeruginosa as well as a broad spectrum encompassing Gram-positive and -negative bacteria.

Drainage for acute pancreatitis.

In severe acute pancreatitis, the drainage of activated pancreatic enzymes, infectious substances and necrotic tissue from the abdominal cavity is critical, especially after the operation. We use Faycon drainage tubes together with multi-use feeding tubes. Just after the operation, normal saline is added through the feeding tube and the Faycon drainage tube is suctioned continuously. Irrigation is necessary for more than two weeks.

Effect of gravity change on the production of thrombopoietic growth factors.

It is reported that the stay in the space develops anemia, thrombocytopenia, and altered function and structure of red blood cell. The mechanism of these abnormalities was not clarified yet. The cloning of the thrombopoietin (TPO), followed by the analysis of TPO and c-mpl (its cellular receptor) knockout mice confirmed its role as the primary regulator of thrombopoiesis. TPO has been shown to stimulate both megakaryocyte colony growth from marrow progenitor cells and the maturation of immature megakaryocyte to form functional platelet. This process includes the massive cytoskeletal rearrangement, such as proplatelet formation and fragmentation of proplatelet. In this study we have focused on the production of thrombopoietic growth factors in mice those were exposed to gravity change by parabolic flight (PF).