Production of proinflammatory cytokines by syncytiotrophoblasts infected with human cytomegalovirus isolates.

Forty per cent of women with primary cytomegalovirus (CMV) infection during gestation transmit the infection to their fetuses, which may result in abnormalities for the newborn, varying in degree from mild to severe. The factors whereby CMV in the placenta develops into a fulminating infection and spreads to the fetus are not known. In this study the production of proinflammatory cytokines was investigated in syncytiotrophoblast (ST) cultures infected with CMV strains. The interrelationships between the cytokines produced in the ST cultures and the number of nuclei of ST expressing the CMV immediate-early (IE) gene were examined. To resemble a natural infection, clinical CMV isolates and a low multiplicity of infection were used. TNF-alpha and IL-1 beta were not detected in the supernatants of any ST cultures. Similar or increased amounts of IL-6 were found in the CMV-infected cultures. The IL-8-inducing capacities of the CMV strains differed in the ST cultures. The IE gene expression of the virus provided was dependent on the amount of IL-8 produced in the STs. Our observations indicate that certain CMV strains induce high amounts of IL-8, which in turn enhances CMV replication in the placenta, while others can replicate if the IL-8 is provided by a co-infecting agent.

Human cytomegalovirus gB genotype 1 is dominant in congenital infections in South Hungary.

On the basis of the sequence variation of the glycoprotein B (gB) gene, human cytomegalovirus (HCMV) can be classified into four gB genotypes. Genotyping of HCMV from congenital infections was carried out on the assumption that the envelope gB may influence the outcome of prenatal infection. Sixty-three pregnant women were included in the study: 40 pregnant women whose fetuses were strongly suspected of having viral infection, and 23 women with normal pregnancies, from whom amniotic fluid was taken for fetal karyotype assessment. The amniotic fluid, fetal blood, blood, and urine of the newborns were examined for HCMV DNA by a nested polymerase chain reaction, and the gB genotype was determined by restriction fragment length polymorphism. HCMV DNA was detected in 12 cases in which the fetuses were suspected of having a viral infection and in 3 of the normal pregnancies. All the HCMV DNA had identical genotype, gB1. These data clearly indicate the dominance of the gB1 genotype in congenital HCMV infections. The clinical outcome of these pregnancies, however, cannot be predicted on the basis of the involvement of this genotype.

Membrane associated antitumor effects of crocine-, ginsenoside- and cannabinoid derivates.

In the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions. Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells. Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL. Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells. The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells. Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression. However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL. The crocin had no antiviral effect [on HSV-2 infected vero cells] up to 25 micrograms/mL concentration. Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump. Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells. It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities.

Antitumor activity of benzo[a]phenothiazines.

We have previously reported on the diverse biological activities of benzo[a]phenothiazines, such as the induction of antitumor and antimutagenic activity in vivo, and differentiation and apoptosis in vitro. The relationship of radical generation and pi-spin density or dipole moment was investigated, using quantum-chemical calculation with UHF/PM3. These data suggest that the origin of radical generation by active benzo[a]phenothiazines, which affect such biological activities might be N-atom at position 12.

Relationship between tumor (T) antigen expression and substituent effects on benzo [a] phenothiazines.

Human adenovirus, oncogene-type 12 infected HEp-2 cells were exposed to six benzo[a]phenothiazines. 5-Oxo-5H-benzo[a]phenothiazine (4) and 6-hydroxy-5-oxo-5H-benzo[a]phenothiazine (5) were moderately toxic. 9-Methyl-12H-benzo[a]phenothiazine (2), 10-methyl-12H-benzo[a]phenothiazine (3), 6-methyl-5-oxo-5H-benzo[a]phenothiazine (6), and 12H-benzo[a]phenothiazine (1) were not toxic in the system tested. 6-Methyl-5-oxo-5H-benzo[a]phenothiazine (6) enhanced the expression of viral oncogene product (tumor antigen) in the adenovirus infected cells. 5-Oxo-5H-benzo[a]phenothiazine (4) and 6-hydroxy-5-oxo-5H-benzo[a]phenothiazine (5) reduced this effect. 6-Methyl-5-oxo-5H-benzo[a]phenothiazine (6), with hyperconjugation due to the methyl group, increased the T antigen activity at higher dose concentrations, whereas 6-hydroxy-5-oxo-5H-benzo[a]phenothiazine (5) with a hydroxy substituent had the opposite effect on T antigen expression. The methyl substitution at positions C9 or C10 increased the T antigen expression of adenovirus infected cells.

Transcriptional repression by human adenovirus E1A N terminus/conserved domain 1 polypeptides in vivo and in vitro in the absence of protein synthesis.

The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40-80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors.

Effects of two benzo[a]phenothiazines on multi-drug resistance (mdr) and tumor antigen expression.

Two benzo[a]phenothiazines 5H-benzo[a]phenothiazin-5-one (1), and its derivative 6-methyl-5H-benzo[a]phenothiazin-5-one (2) inhibited the proliferation of human and mouse tumor cell lines. The multi-drug resistant (mdr) subline was more sensitive than its parent cell line to 5H-benzo[a]phenothiazin-5-one (1), 6-methyl-5H-benzo[a]phenothiazin-5-one (2) was equally antiproliferative against the three cell lines tested. Rhodamine 123 efflux of mdr cells was more efficiently inhibited by 5H-benzo[a]phenothiazin-5-one (1) than by 6-methyl-5H-benzo[a]phenothiazin-5-one (2). The exposure of adenovirus infected cells to 5H-benzo[a]phenothiazin-5-one (1) resulted in a reduction of tumor-antigen expression, whereas 6-methyl-5H-benzo[a]phenothiazin-5-one (2) enhanced the T-antigen expression.

Study on the glycoprotein nature of chicken IFNs by chemical cleavage and inhibition of glycosylation.

The considerable molecular heterogeneity of chicken IFNs suggested the possible glycoprotein nature of these IFNs. The carbohydrate-specific oxidation and cleavage by neuraminidase indicated that fibroblast IFN may contain sugar moieties. Among the antimetabolites preventing glycosylation, monensin diminished IFN production. However, since it had the same effect on unglycosylated human leukocyte IFN, the drug very probably blocks intracellular transport. It rather proved that chicken IFNs are also secretory proteins. Tunicamycin diminished the formation of leukocyte IFN, but the decrease in titre of this IFN is due to the inhibition of protein synthesis. It is suggested that chicken fibroblast IFN is a glycoprotein, while leukocyte IFN lacks a sugar moiety. The IFN produced on induction with mitogen is closely related to leukocyte IFN, and therefore it seems that the chicken IFN system does not involve the conventional gamma type.

Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: autonomous protein domains for induction of cellular DNA synthesis and for trans activation.

The human papillomavirus type 16 E7 protein belongs to a family of nuclear oncoproteins that share amino acid sequences and functional homology. To localize biochemical activities associated with E7, we chemically synthesized the full-length 98-amino-acid polypeptide and several deletion mutant peptides. We show that the E7 polypeptide is biologically active and possesses at least two functional domains; the first induces cellular DNA synthesis in quiescent rodent cells, and the second trans activates the adenovirus E1A-inducible early E2 promoter and binds zinc. Further, each domain is autonomous and can function on separate peptides. DNA synthesis induction activity maps within the N-terminal portion of the molecule, which contains sequences related to adenovirus E1A conserved domains 1 and 2 required for cell transformation and binding of the retinoblastoma gene product. trans-Activation and Zn-binding activities map within the C-terminal portion of the molecule, a region which contains Cys-X-X-Cys motifs. trans Activation does not require protein synthesis, implying a mechanism that involves interaction with a preexisting cellular factor(s). E7 trans activates the adenovirus E2 promoter but not other E1A-inducible viral promoters, suggesting the possibility that E7 trans activation involves interaction, directly or indirectly, with cellular transcription factor E2F.

Mutational analysis of autonomously functioning trans-activating peptides encoded by adenovirus E1A: evidence for two subdomains.

We have shown previously that a chemically synthesized adenovirus E1A region 3 peptide of 49 amino acids, protein domain 3 (PD3; residues 140 to 188 of the 289-amino-acid protein), trans activates viral genes in vitro and in vivo. To study structure-function relationships, we synthesized N-terminal deletion and cysteine substitution mutant peptides and tested their activities in a cell microinjection assay. Peptides lacking 1 to 12 N-terminal residues exhibited 5- to 50-fold-reduced molar specific activities, whereas those lacking 16 or 18 residues were inactive. Substitution of each of five PD3 cysteine residues with alanine resulted in substantial losses of activity: mutants in the PD3 N-terminal portion showed 40 to 55% of wild-type activity but required a 20-fold-higher concentration than PD3, whereas those in the C-terminal half were as much less active. These peptide mutant studies suggest the existence of two PD3 functional regions: one, localized in the C-terminal 70 to 75% of the molecule, is essential for trans activation; the other, localized in the N-terminal 25 to 30%, can be overridden to a significant extent at high peptide concentrations.

An adenovirus E1A protein domain activates transcription in vivo and in vitro in the absence of protein synthesis.

We have shown previously that a synthetic peptide of 49 amino acids, encoding mainly adenovirus E1A protein domain 3 (PD3), functions as an autonomous transcriptional activator. Here we provide two lines of evidence showing that E1A transactivation does not require the induction of cellular protein synthesis. First, PD3 rapidly transactivates E1A-inducible early viral genes in the presence of inhibitors of protein synthesis, as demonstrated by microinjection-in situ hybridization experiments. Second, PD3 greatly stimulates transcription of E1A-inducible genes in vitro. Mutant PD3 peptides with single amino acid substitutions in conserved cysteine residues are defective in transactivation both in vivo and in vitro. Our findings provide compelling evidence that protein synthesis is not required for E1A transactivation, and support a model in which E1A modifies the activity of a preexisting cellular protein(s) involved in the regulation of transcription.

Interferon induction by adenovirus type 12: stimulatory function of early region 1A.

Adenoviruses are generally weak interferon inducers, triggering chicken embryo fibroblast cells by a UV-resistant viral component, probably the capsid or capsid elements, to produce 50 to 100 IU of interferon per ml. Adenovirus types 12, 18, and 31, however, can induce by a UV-sensitive mechanism 10 to 20 times more interferon than other types do. By using mutant and recombinant adenoviruses, we demonstrated that early region 1A was responsible for the enhanced interferon production of chicken cells infected with adenovirus type 12.

The role of interferon in the adenovirus-induced augmentation of granulocyte-mediated cytotoxicity in chicken.

The effects of human adenoviruses on the granulocyte-mediated natural cytotoxicity of chicken leukocytes were investigated. A significant, but transient augmentation of granulocyte cytotoxicity was observed 24 h after virus injection, followed by a relatively long period of its suppression. A good correlation was found between the augmented cytotoxicity and interferon induction. The interferon-inducing capacity of adenovirus type 6 and type 12 in vitro similarly ran parallel with their ability to stimulate granulocyte-mediated cytotoxicity. An adenovirus-induced elevation of cytotoxicity was not observed when IFN production was inhibited by pretreatment of the leukocytes with monoclonal antibody specific for bursal cells and monocytes. In addition, anti-IFN antibody abrogated the stimulation of cytotoxicity as well. During the in vitro experiments in which granulocyte-specific monoclonal antibody was applied, evidence was found that the effector cell activity is associated with the granulocytes. These results suggest that both the in vitro and the in vivo adenovirus-induced augmentation of granulocyte-mediated cytotoxicity is due to the IFN-inducing capacity of the virus. In chickens, the rapid augmentation of the granulocyte cytotoxicity may be important in the acute stage of infection, increasing the resistance to the virus in question and also to bacterial infections.

Production and characterization of interferon induced in chicken leukocytes by concanavalin A.

Interferon (IFN) was induced in chicken peripheral blood leukocytes by exposure to Concanavalin A (Con A). It has reached a maximum level 96 hr after stimulation with the optimum dose of 10 micrograms/ml. The IFN was partially purified by chromatography on controlled-pore glass adsorbent and was characterized as a fairly acid-stable and heat-resistant, trypsin-sensitive and species-specific substance with Mr of 20,500 Da. Antiviral response by this type of IFN in chick fibroblast culture developed within several hours. This study provides first evidence of the presence of IFN in supernatants of mitogen-stimulated chicken peripheral leukocytes.

Are granulocytes the main effector cells of natural cytotoxicity in chickens?

Purified peripheral blood granulocytes from chicken were tested for cytotoxic activity against two types of virus-transformed chicken cell line, LSCC-H32 and LSCC-RP9. Strong cytotoxicity could be demonstrated, as measured in a 4-hr 51Cr-release assay, especially against the fibroblastoid LSCC-H32 cells. The degree of cytotoxicity was dependent on the E:T ratio. Normal CEF cells were completely resistant to the cytotoxicity. No cytotoxicity of human granulocytes could be observed against a variety of adherent and non-adherent target cells, as measured by the same microcytotoxicity technique. The priority of granulocytes in the natural cytotoxicity in the avian system is, therefore, suggested.

Effect of human adenovirus on natural killer cell activity in mice.

The influence of human adenovirus type 6 on the natural killer cell activity in mice has been investigated. The cytotoxic effect of splenic mononuclear cells of different mouse strains (Balb/c, CBA and BlO) against YAC-1 cells was estimated using a 51Cr release system assay. The effector cells responsible for cytotoxicity were identified by Percoll density gradient fractionation as LGL cells. A single intraperitoneal injection of the virus enhanced the natural killer activity in all three strains of mice. The cytotoxicity was enhanced 24 to 48 h after the virus injection, depending on the host strains, but it then decreased and the preinjection level was reached after 72 h. A good correlation was found between the augmented cytotoxicity and interferon induction.

Effect of arginine-butyrate on interferon induction by adenovirus.

Treatment of human adenovirus type 12 infected chick cells with a low concentration of arginine-butyrate strongly inhibited interferon formation. At this concentration, as evaluated by the rate of protein synthesis the drug exerted no significant toxic effect on the cells. The early virus gene expression, not being affected in the butyrate-treated chick cells, it is autonomous and is not influenced by the effect of butyrate on chick cells. This is at variance with what has been observed in other semipermissive systems infected with adenovirus.

[Molecular aspects of interferon induction by adenoviruses]. / Molekuliarnye aspekty induktsii interferona adenovirusami.

The capacity of adenovirus to induce interferon in the infected cells was studied. The examined adenovirus strains of early types were grouped in 2 groups according to their capacity to induce interferon and to the sensitivity of the infecting and interferon-inducing activity to UV-irradiation. A common property of adenoviruses, potent interferon inducers, is their high sensitivity to UV-irradiation.

Different interferon-inducing ability of human adenovirus types in chick embryo cells.

Human adenovirus (Ad) types differ in their ability to induce interferon (IFN) in chick cells. Of 12 types investigated, Ad8, Ad12, Ad 18 and Ad 31 proved to be more effective IFN inducers than Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad15 and Ad19. Ultraviolet (UV) irradiation decreased the IFN-inducing ability of the more effective inducers only, indicating that transcription of viral DNA might play a role in IFN induction by these types. DNAs isolated from Ad2, Ad5 and Ad12 alike induced low amounts of IFN in chick cells. The IFN-inducing capacity of phage DNA was similar to that of adenovirus DNA, but induction by non-viral DNA (prokaryotic and eukaryotic) did not result in detectable IFN production. It is assumed that viral DNA and virus particles promote IFN production in different ways. Probably the viral component responsible for IFN induction by the effective Ad types differs from those having lower IFN-inducing ability.