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.

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.

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.

Mechanism of antiviral action of quercetin against cardiovirus infection in mice.

Oral treatment with quercetin protected ABD2F1/Jena mice significantly against intraperitoneal encephalomyocarditis, Col, SK, MM, Mengo M,L and MengoM virus infections, but not against intracerebral challenge with MengoM virus. Enhanced resistance to MengoM virus were induced in the genetically different DBA 2/Jena, C57BL/Jena, C57BL/Lati and ABC2F1/Jena mice, C57BL6/Jena nu/nu mice were also protected, indicating that the thymus was non-essential to the protective effects of quercetin. In AB/Jena and Lati:CFLP mice the drug failed to be effective. Quercetin was not virucidal and did not interfere with Mengo virus replication in L cells. Interferon was not detected (less than 1 : 8) in sera of ABD2F1/Jena mice 1-48 h after oral administration of the drug. The virus spread from the site of injection to the lymph nodes and other target organs were impaired. Silica treatment, to suppress macrophage function, did not evidently increase the susceptibility of ABC2F1/Jena mice to Mengo virus. However, this treatment abolished the antiviral activity of quercetin, indicating the requirement for macrophages for quercetin to be effective. Virus replication could not be demonstrated in cultures of adherent peritoneal macrophages from either untreated or quercetin-treated ABD2F1/Jena mice.

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.

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.

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.

Sensitivity of formation of adenovirus specific tumour antigen to leukocyte interferon.

The effect of various concentrations of chicken leukocyte interferon was examined on the expression of adenovirus tumour (T) antigen in chick embryo cells. The inhibition of T antigen formation was dependent on the multiplicity of infection. The reduction of T antigen-producing cells was greater when interferon was applied before and after virus infection.

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.

Physico-chemical characteristics of interferons induced by human adenovirus in chick fibroblasts and leucocytes.

The physico-chemical characteristics of interferons with different anti-viral activities produced in chick fibroblast cells and leucocytes in response to the human adenovirus type 12 were investigated. The molecular weights of interferons produced by chick fibroblasts and leucocytes were similar: the main anti-viral activity was found at 27000 and 32000 and a low-titered shoulder at 28000 and 23000 respectively. Under our experimental conditions no oligomeric structure of interferons could be detected. The isoelectric pattern of leucocyte interferon was slightly different from that of the fibroblast interferon.

T antigen synthesis and resistance to interferon in human adenovirus type 12 infected chick cells.

Tye specific T antigen formation has been demonstrated in primary and secondary chick embryo cells (CEC) infected with adenovirus type 12. The frequency of cells synthesizing T antigen was closely dependent on the multiplicity of infection (MOI). At a MOI of 2.85 TCD50 per cell, T antigen was formed in 50% of cells. Cycloheximide inhibited T antigen formation while cytosine arabinoside had no such effect. CEC infected with adenovirus 12 produced interferon and T antigen, both appearing early and at about the same time of infection Exogenous chick interferon had no inhibitory effect on the formation of T antigen. In adenovirus 12-infected CEC, virus specific structural antigen could not be detected.

Effect of quercetin on the course of mengo virus infection in immunodeficient and normal mice. A histologic study.

Quercetin protects mice from lethal Mengo M virus infection when given orally 12 and 1 hr before and 8, 24, 36, 48 and 56 hr after inoculation. No differences in the course of infection have been found between normal splenectomized or congenitally athymic mice. Likewise the effect of drug treatment was similar in all three models. Necrotic lesions in the main target organs (central nervous system, salivary and lacrimal glands, thymus, pancreas, kidneys and spleen) from both normal and immunodeficient animals were less severe and developed later in quercetin-treated mice than in placebo-treated ones. In a few quercetin-treated virus-infected survivors a slight and persistent encephalitis was seen. Quercetin enhanced the graft-versus-host reaction, but failed to affect the humoral antibody response of mice to sheep red blood cells. It is concluded that T- and B-lymphocytes seem involved neither in the pathogenesis of acute Mengo virus infection nor in the antiviral effect of quercetin.

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.

[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.

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.