Involvement of a p53-dependent pathway in rubella virus-induced apoptosis.

In light of the important role of apoptotic cell death in the pathogenesis of several viral infections, we asked whether the cytopathogenicity evoked by rubella virus (RV) might also involve apoptotic mechanisms. The To-336 strain of RV induced apoptosis in Vero and RK-13 cells, but not in fibroblast cell lines. UV-inactivated RV virions did not elicit the apoptotic response, indicating that productive infection is required for the induction of cell death. Both p53 and p21 protein levels were highly elevated in RV-infected Vero cells. The level of p21 mRNA was increased, while expression of the p53 gene was unaffected by RV infection. A dominant-negative p53 mutant (p53(W248)) conferred partial protection from RV-induced apoptosis. These data implicate a p53-dependent apoptotic pathway in the cytopathogenicity of RV, thereby suggesting a mechanism by which RV exerts its teratogenic effects.

Stimulation of interferon and cytokine gene expression by imiquimod and stimulation by Sendai virus utilize similar signal transduction pathways.

The imidazoquinolineamine derivative 1-(2-methyl propyl)-1H-imidazole [4,5-c]quinoline-4-amine (imiquimod) has been shown to induce alpha interferon (IFN-alpha) synthesis both in vivo and in peripheral blood mononuclear cells in vitro. In this study, we show that, in these cells, imiquimod induces expression of several IFNA genes (IFNA1, IFNA2, IFNA5, IFNA6, and IFNA8) as well as the IFNB gene. Imiquimod also induced the expression of interleukin (IL)-6, IL-8, and tumor necrosis factor alpha genes. Expression of all these genes was transient, independent of cellular protein synthesis, and inhibited in the presence of tyrosine kinase and protein kinase C inhibitors. Infection with Sendai virus led to expression of a similar set of cytokine genes and several of the IFNA genes. Imiquimod stimulates binding of several induction-specific nuclear complexes: (i) the NF-kappa B-specific complexes binding to the kappa B enhancer present in the promoters of all cytokine genes, but not in IFNA genes, and (ii) the complex(es) binding to the A4F1 site, 5'-GTAAAGAAAGT-3', conserved in the inducible element of IFNA genes. These results indicate that imiquimod, similar to viral infection, stimulates expression of a large number of cytokine genes, including IFN-alpha/beta, and that the signal transduction pathway induced by both of these stimuli requires tyrosine kinase and protein kinase activity.

Lipofectin-aided cell delivery of ribozyme targeted to human urokinase receptor mRNA.

A 37-mer hammerhead ribozyme has been designed to efficiently cleave the 1.4 kb mRNA of the urokinase plasminogen activator receptor (uPAR). Under in vitro conditions, the chemically synthesized ribozyme cleaved uPAR mRNA and inhibited its translation in a concentration-dependent fashion. The ribozymes were 5'-[35S]thiophosphorylated and used as a model to analyze conditions for RNA delivery in a cultured human osteosarcoma cell system. Ribozymes degraded immediately in cell-conditioned medium but ribozymes complexed with lipofectin were protected from RNases for up to 22 h. Lipofectin rapidly transported ribozyme into the cell, where it accumulated almost exclusively in the cytoplasm. Thus, lipofectin dramatically enhances stability and cytoplasmic delivery of ribozymes, potentially enabling targeting of mRNA in vivo.

Interferon pretreatment regulates interferon and interleukin-6 production in L929 cells in a coordinated manner.

Two sublines of L929 cells with different interferon (IFN)-producing capacities synthesized IFN and interleukin-6 (IL-6) simultaneously in response to Sendai virus infection. IFN pretreatment primed the production of both cytokines. However, the difference in IL-6 production between the "high and low producer" L-cell sublines was about one magnitude of order larger than in the case of their IFN production. The determination of the corresponding mRNA levels also reflected this difference.

Association between chemiluminescence stimulating and IL-2 inducing activities of Staphylococcus aureus strains in human and mouse mononuclear cells.

The plastic adherent fraction of human mononuclear cells (MNC) responded with maximal chemiluminescence (CL) upon stimulation with greater than or equal to 1000 bacteria per cell of heat killed preparations of Staphylococcus aureus strains. Different strains had different CL stimulating activities and their sequences were similar on MNC from different blood donors. IL-2 inducing and CL stimulating activities seem to be parallel features of S. aureus strains, since their sequence set ups established on the basis of these two properties were almost identical. The same phenomenon could also be observed in a mouse system, in which activated peritoneal cells (PC) were the most active CL exhibiting population. The IL-2 inducing activity of staphylococci in mouse spleen cells and their CL stimulating activity in activated PC followed a similar pattern too. The sequences of CL inducing activities of different staphylococcal strains were in good agreement in human and mouse cells. Representative strains with high, moderate and low CL inducing activities followed the same sequence of virulence for mice.

Effects of phorbol myristate acetate on interleukin-2 and accompanying interferon production of human leukocytes induced by heat-inactivated Staphylococcus aureus.

Interleukin-2 (IL-2) production induced by heat--inactivated Staphylococcus aureus (SAU) was enhanced by simultaneous addition of phorbol myristate acetate (PMA). The effect was optimal at a concentration of 10 ng/ml SAU; in the presence of 10 ng/ml PMA, the amount of SAU required for maximal IL-2 production was lower. The kinetics of SAU and of SAU plus PMA-induced IL-2 production were similar. Stimulated mononuclear cells produced interferon (IFN) in addition to IL-2. The titre of accompanying IFN was decreased in cultures stimulated with the SAU plus PMA combination. Plastic nonadherent sheep erythrocyte-positive cells were the most active in the SAU-induced IL-2 production. In contrast, the bulk of the IFN activity was produced by the nonadherent E rosette-nonforming cells. Neutralization of IFN with specific antibodies and pH 2 treatment indicated that SAU-induced IFN consisted mainly of alpha-IFN.

Interferon production by normal mouse tissues in organ cultures.

Freshly removed tissues of normal untreated mice produced relatively high amounts of interferon (IFN) in organ cultures. Lymph nodes, subcutaneous tissue, and the capsule of the kidney were the most active IFN producers. The abdominal wall and the thigh muscle were less active, whereas the lungs and spleen, similarly to the peritoneal exudate and bone marrow cells, produced only threshold amounts of IFN. Liver cultures did not produce IFN under these experimental conditions. Cultures prepared from IFN-pretreated animals produced three- to fourfold more IFN. Homogenates of tissue prepared immediately after their removal did not contain a detectable amount of IFN. The bulk of the IFN activity was produced during the first 6 h of incubation at 37 degrees C. Omission of serum from the culture medium, and the presence of 50 micrograms/ml of polymyxin B, did not inhibit IFN production. Cultures incubated at 0 degrees did not release any IFN. The IFN activity produced by all types of tissue was pH 2 resistant and it was neutralized by an antiserum to murine (Mu) IFN-beta. Different strains of mice produced comparable amounts of IFN under the present experimental conditions.

Essentially pure murine interferon-alpha/beta primes poly rI:rC and Sendai virus-induced interferon production in mice.

Intramuscular (i.m.) injection of mice with 2000 IU/g of essentially pure murine interferon-alpha/beta (MuIFN-alpha/beta) 3 h before the induction of IFN by an intraperitoneal (i.p.) inoculation of 10 haemagglutinating units (HAU) per g Sendai virus or 3 micrograms/g polyriboinosinic and polyribocytidylic acid complex (poly rI:rC) elicited a primed IFN response in both cases. Antiserum to MuIFN-alpha/beta neutralized both the priming and antiviral activities of the IFN preparation used. Comparison of the kinetics of primed and unprimed IFN production by Sendai virus indicated that the early (2-4 h) period of IFN production was affected.

Different interferon-producing capacities of L929 cell sublines and the enhancement of interferon production by priming are controlled pretranslationally.

Two sublines of mouse L929 cells designated L929B and L929M were studied. The L929B cells, which displayed a 2-3-fold higher IFN production in response to Sendai virus than that of the L929M cells, had a higher sensitivity to the antiviral and priming effects of IFN and were more resistant to VSV. In good accord with the amount of IFN produced, more translatable IFN mRNA was isolated from the L929B cells. IFN production and IFN mRNA activities were proportionally increased in the IFN-primed cultures of both sublines. Results indicate that both inherent and priming-induced increased-IFN production are based on pretranslational control mechanisms.