Inhibition of protein synthesis in noninfected L cells by partially purified interferon preparations.

Partially purified interferon preparations, obtained from L-cell monolayers infected with Newcastle disease virus (NDV), were shown to inhibit protein synthesis in noninfected L cells. The incorporation of several amino acids-(14)C was equally sensitive to the pretreatment of the cells with the interferon preparation. Treatment of L-cell monolayers for 24 hr with 800 units of interferon resulted in a 50% decrease in amino acid incorporation. The degree of inhibition was found to be a function of the interferon concentration and the time of exposure of the cells to the partially purified preparations. No inhibitory effect was detected in medium obtained from noninfected cells and purified in an identical manner. The inhibitory effect was shown to be cell specific in that the partially purified interferon from L cells did not reduce amino acid incorporation in heterospecific cell lines. Heating the interferon preparations at 60 degrees C destroyed their antiviral activity and their ability to inhibit valine-(14)C incorporation in L cells.

Encapsulation of anticancer drug and magnetic particles in biodegradable polymer nanospheres.

In this study, we have prepared PLGA (poly-D,L-lactide-co-glycolide) nanospheres loaded with biocompatible magnetic fluid and anticancer drug taxol by a modified nanoprecipitation technique and investigated their magnetic properties. A magnetic fluid, MF-PEG, with a biocompatible layer of polyethylene glycol (PEG), was chosen as a magnetic carrier. The PLGA, whose copolymer ratio of D,L-lactide to glycolide is 85:15, was utilized as a capsulation material. Taxol, as an important anticancer drug, was chosen for its significant role against a wide range of tumours. The morphology and particle size distributions of the prepared nanospheres were investigated by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and showed a spherical shape of prepared nanospheres with size 250 nm. Infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and thermogravimetry (TGA) analysis confirmed incorporation of magnetic particles and taxol into the PLGA polymer. The results showed good encapsulation with magnetite content 21.5 wt% and taxol 0.5 wt%. Magnetic properties of magnetic fluids and taxol within the PLGA polymer matrix were investigated by SQUID magnetometry from 4.2 to 300 K. The SQUID measurements showed superparamagnetism of prepared nanospheres with a blocking temperature of 160 K and saturation magnetization 1.4 mT.

Suppression of human macrophage function in vitro by delta 9-tetrahydrocannabinol.

The ability of macrophages to function in the presence of delta 9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, was evaluated. THC added to macrophage cultures prepared from human peripheral blood inhibited macrophage spreading and phagocytosis of yeast. The effects of THC were concentration dependent, with inhibitory effects observed from 10 to 1 micrograms/ml or lower. These results suggest that macrophages are more sensitive to THC than are lymphocytes because macrophage functions were inhibited by THC at concentrations that did not affect lymphocyte function. Thus, inhibition of lymphocyte function(s) by THC could be attributed to a direct effect of the drug on macrophages which indirectly results in lowered lymphoid cell activity.

Physical integrity of herpes simplex virus following thermal inactivation.

The effect of thermal inactivation on the gross physical integrity of herpes simplex virus (HSV) was investigated. Purified preparations of HSV containing 3H-thymidine were subjected to thermal inactivation at 36 degrees C, ph 6.3 and pH 7.8 and then were analysed by sucrose gradient centrifugation. The data indicated that although HSV inactivation at 36 degrees C was significantly greater at pH 7.8 than at 6.3, the gross physical integrity of most of the HSV in both of these inactivated virus populations was maintained. In addition, heat inactivated HSV adsorbed to mammalian cells as readily as non-inactivated HSV. Thus, thermal inactivation of HSV does not result in the physical disassembly of the virion particles or in the destruction of virus receptor activity.

Thermal-pH inactivation of herpes simplex virus: interdependence of the medium composition and the pH on the rate of virus inactivation. Brief report.

The composition and pH of the suspending medium were shown to be interdependent effectors of the in vitro thermal inactivation of herpes simplex virus (HSV). In addition, enhanced thermal sensitivity of HSV at alkaline pH was a medium dependent effect and, therefore, is not an inherent property of the virus.

pH mediated inhibition of the cell to cell spread of herpes simplex virus infection.

The relationships between the environmental pH and the replication and spread of herps simplex virus (HSV) infection in rabbit skin (RS) cell cultures was studies. Relative to the plaque formation at pH7.0, incubation of RS cells with medium adjusted to pH 6.6 resulted in a 50 to 70 per cent decrement in plaque number. The addition of overlay media adjusted to pH 6.0 or 6.3 precluded HSV plaque formation. Select HSV-1 (type 1) and HSV (type 2) strains readily survived a 3 day incubation with medium adjusted to pH 6.3 as demonstrated by plaque production following a medium shift to pH 7.0. Rs cells incubated with medium at pH 6.3 did not replicate. The survival of RS cells incubated for 3 days with medium adjusted to pH 6.3 was demonstrated by renewed cell proliferation following a medium change from pH 6.3 to 7.0. The progeny virus yields of two HSV strains replicating in RS cells incubated with medium adjusted to pH 6.3 or 7.0 were equivalent at 24 or 48 hours post infection and were indicative of a productive infection. The inhibition of HSV plaque formation at pH 6.3 was not due to an alteration of cell receptors but was the result of an inhibition in the cell to cell spread of the virus. These results are discussed with regard to the possibility that the decline in pH associated with the inflammatory response may serve as a host defense against HSV infections.

Marijuana and immunity: tetrahydrocannabinol mediated inhibition of lymphocyte blastogenesis.

The effects of delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-OH delta-9-tetrahydrocannabinol (11-OH THC) on mitogen driven lymphocyte blastogenic transformation (LBT) were studied. THC inhibited LBT responses to the T lymphocyte mitogens phytohemagglutinin and concanavalin A but not the B lymphocyte stimulant pokeweed mitogen. The metabolite 11-OH THC caused a comparable, but less significant, inhibition of LBT responses to the T cell mitogens. These inhibitions were dependent upon the drug doses and the time of incubation with the lymphocytes. There was no significant inhibitory activity of THC to the LBT when it was added 24 or 48 h after mitogen. In addition, exposure of lymphocytes to THC for 3 h, followed by removal of the drug prior to addition of mitogen had no effect on the cells' ability to respond to the mitogen. Thus, there appears to be a specified temporal period during which exposure of lymphocytes to THC results in an inhibition of blastogenesis. Interleukin 2 (30 units/ml) could not preclude the THC induced inhibition of lymphocyte blastogenesis. We conclude that THC and 11-OH THC inhibit T lymphocyte blastogenesis. However, unlike the THC mediated inhibition of natural killer cell activity (as shown previously), the process is not responsive to the cytokine interleukin 2.

Mapping of genes in BamHI fragment M of Epstein-Barr virus DNA that may determine the fate of viral infection.

We used nuclease digestion to map RNA transcripts encoded in the BamHI M fragment of the Epstein-Barr virus (EBV) genome (strain B95-8). Of the five RNAs, three are rightwardly transcribed, have different cap sites but common 3' termini, and are unspliced. The two remaining RNAs are leftwardly transcribed and are 5' and 3' coterminal. One of these transcripts is spliced, resulting in the removal of a small intron from the 5' region of this RNA. We have previously published data which indicated that the BamHI M region is the first actively transcribed region of the viral genome during the replicative cycle, suggesting that one or more genes in this region is important in the initiation of EBV replication. We have now mapped two large EcoRI restriction fragments which span approximately 75% of the P3HR-1 defective genome and which contain DNA from the BamHI M region of the standard genome. The data indicate that only the coding and 5' flanking sequences for the leftwardly transcribed RNAs are intact within the defective genome. Fewer than 500 bases coding for the 3'-most regions of the rightwardly transcribed RNAs are intact, and it is unlikely that these encode functional native polypeptides. Therefore, it seems that transcriptional activation of the BamHI M-region genes is not mediated directly by the rearrangement of M genes in defective P3HR-1 EBV.

Abortive infection of neural cells by herpes simplex virus type 2.

The nature of the refractoriness of C6 rat glioma cells to herpes simplex virus type 2 (HSV-2) was examined. Infection of C6 cells with HSV-2 results in low virus yields, not exceeding the input virus. Although virus growth studies suggested a restricted cycle of virus replication, synthesis of HSV-2 DNA and HSV-2-specific antigens could not be detected. In addition, HSV-2 yields in C6 cells were unaffected by interferon, cycloheximide, tunicamycin, actinomycin D and cytosine arabinoside. However, trypsin, but not EDTA, treatment of infected C6 cells at 4 hours postinfection (p.i.) reduced maximal HSV-2 yields at 24 hours p.i. by 61 percent. These data: 1) indicate that HSV-2 fails to replicate in C6 cells and is prohibited from directing the synthesis of virus macromolecules; and 2) suggests that the increment of HSV-2 yields observed during the synthesis phase of the virus growth cycle represents re-envelopment and egress of a portion of the input virus.

A simple and rapid test for the identification of clinical herpes simplex virus isolates.

A simple procedure is described which permits the rapid identification of clinical herpes simplex virus isolates. The test utilizes Staphylococcus aureus to which anti-viral immunoglobulins had been adsorbed. Adherence of the antibody-coated bacteria to virus-infected cells is readily seen by light microscopy. Indirect immuno-fluorescence and the S aureus adherence reaction were found to be approximately of equal sensitivity for the detection of virus antigens.

Loss of Marek's disease virus tumorigenicity is associated with truncation of RNAs transcribed within BamHI-H.

The attenuation of Marek's disease virus (MDV) is associated with loss of pathogenicity and tumorigenicity. Previous studies have demonstrated a strong correlation between attenuation and amplification of a specific sequence located within the MDV terminal and internal repeats. We recently reported that the regions containing the amplified sequences, the BamHI D and H fragments, were transcriptionally active. However, differential transcription activity was observed to exist between attenuated and pathogenic MDV strains. Specifically, a major transcript of 1.8 kilobases was found to be produced by pathogenic MDV and not by attenuated MDV. We now report that the disappearance of this transcript is concomitant with the production of a 0.4-kilobase RNA, an RNA resulting from the truncation of the tumorigenicity-related transcript.

Suppressive effect of delta-9-tetrahydrocannabinol on herpes simplex virus infectivity in vitro.

Delta-9-Tetrahydrocannabinol (THC) was found to reduce the infectivity of herpes simplex virus and was without effect against adenovirus type 2 or poliovirus. The effective THC concentration resulting in an 80% decrement in virus viability was dependent upon the presence or absence of serum in the incubation mixture, as a 5% serum concentration decreased the drug activity by approximately 50-fold. THC-mediated inactivation of herpes simplex virus was both time and dose dependent and did not result in virion disassembly or clumping. The THC-related effect was not influenced by the pH of the suspending medium, suggesting that the mechanism of inactivation differed from that associated with the thermal inactivation of the virus. Thus, the data suggest that THC preferentially reduces the infectivity of the enveloped herpes simplex virus, and that this activity is modulated by the presence of serum proteins.

Delta-9-tetrahydrocannabinol augments murine retroviral induced immunosuppression and infection.

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, and the murine retrovirus, Friend leukemia virus (FLV), have been demonstrated to depress cellular immune function, including lymphocyte blastogenic transformation and natural killer cell activity. The present study demonstrates tht the two agents can work in concert to depress these immune activities more severely than either agent administered by itself. When 7.5-10 micrograms/ml THC was added in vitro to spleen cells from mice infected 2-4 weeks earlier with FLV there was a noticeable decrease, beyond that seen with the drug or virus alone, for both lymphocyte blastogenesis and natural killer cell cytotoxicity. In addition, when both FLV and THC were administered to mice concurrently with infection by herpes simplex virus (HSV), mortality attributed to the retrovirus infection occurred significantly more rapidly than in the absence of the drug and HSV. The data indicate that THC acted in the presence of a HSV infection to enhance the FLV induced mortality. By extrapolation to the human condition, these results suggest that marijuana could serve as a cofactor, possibly in conjunction with opportunistic pathogens, in the progression of infection due to the human immunodeficiency virus from latency to overt acquired immunodeficiency syndrome.

Identification of Epstein-Barr virus genes expressed during the early phase of virus replication and during lymphocyte immortalization.

Transcription of the Epstein-Barr virus (EBV) genome in Raji cells superinfected with P3HR-1 EBV in the presence of cycloheximide was compared to transcription in human lymphocytes infected with transforming EBV (B95-8). This was done to identify regions of the EBV genome which contain genes that may mediate initiation of virus replication. Hybridization of 32P-labeled cDNA to cloned fragments of EBV DNA (dot blot hybridization) was employed to identify transcriptionally active regions of the viral genome in these cells. DNA in the BamHI A, F, H, and M restriction fragments was found to encode poly(A) RNA during the early phase of EBV replication. In the absence of cycloheximide the earliest detectable transcripts were transcribed from the BamHI M region. The most transcriptionally active region of the EBV genome in lymphocytes following infection with EBV (B95-8) was the BamHI W-Y-H-F region and, to a lesser extent, the K region. Transcription of the BamHI M region was not detected in these cells. The data suggest that expression of a gene or genes located in the BamHI M region of the EBV genome is an important event in the initiation of EBV replication, whereas expression of the genes in the BamHI W-Y-H-F and K regions may be important in the establishment of latency and cellular immortalization.

Delta-9-tetrahydrocannabinol-(THC)-mediated inhibition of macrophage macromolecular metabolism is antagonized by human serum proteins and by cell surface proteins.

Our previous study demonstrated THC-inhibited DNA synthesis and the phagocytic activity of P388D1 cells [Tang, Lancz, Specter & Bullock (1992) Int. J. Immunopharmac., 14, 253-262]. The ability of proteins in human and bovine sera and of constitutive cellular proteins to modulate the biologic activity of THC was investigated. Both human and fetal bovine sera antagonized a THC-mediated inhibition of P388D1 cell DNA synthesis in a dose-dependent manner. This antagonism was proportional to the protein concentration present in the medium. Both albumin and gamma-globulins influenced THC's inhibitory effects, although they were less potent alpha/beta serum lipoproteins. Exclusion of fatty acid moieties from the albumin did not diminish its ability to antagonize THC. Tritium-labeled THC was acid precipitable only after incubation with bovine or human serum albumin but not DNA, suggesting a physical interaction between the cannabinoid and the protein. Further studies showed that pre-treating cells with trypsin to remove surface proteins significantly enhanced the inhibitory activity of sub-toxic concentrations of THC. Thus, the data indicate that the magnitude of THC's biological effects is determined by the presence and concentration of soluble proteins in the microenvironment and by constitutive proteins present on the cell surface.

Bovine serum albumin preparations enhance in vitro production of tumor necrosis factor alpha by murine macrophages.

Tumor necrosis factor alpha (TNF-alpha) is most commonly produced by macrophages stimulated by lipopolysaccharide (LPS). The present study shows that BSA in place of FBS in RPMI 1640 medium accelerated the rate of LPS-induced TNF-alpha production by resident peritoneal macrophages from BALB/c mice when compared to LPS in serum free medium. Using 10 or 100 ng LPS/ml and 100 U IFN-gamma/ml in RPMI 1640 medium plus 0.5% BSA, both cytoplasmic TNF-alpha mRNA and TNF-alpha precursor and extracellular TNF-alpha production by mouse macrophages were increased when compared to stimulation by LPS plus IFN-gamma in medium without BSA and FBS. The level of TNF-alpha produced was shown to be related to the BSA concentration. Medium containing BSA but no LPS also stimulated macrophages to produce TNF-alpha, but BSA's TNF-alpha inducing activity varied among different lots and was not blocked by polyclonal antibody to BSA. This effect appeared to be associated with the presence of immunoglobulin in BSA products. Confirmation that BSA activity was not due to LPS contamination was achieved by testing macrophages from LPS-nonresponder C3H/HeJ mice, as well as testing TNF-alpha induction in the presence of polymyxin B (10 micrograms/ml), an LPS inhibitor.