Giovanni B. Rossi

Isolation of interferon-resistant variants of friend erythroleukemia cells: Effects of interferon and ouabain

Interferon (IFN)-resistant clones of Friend leukemia cells (FLC) have been isolated by continuous growth in IFN-rich medium followed by plating in semisolid medium in the presence of 1500 U/ml of IFN. FLC variants are fully resistant to the IFN-induced antiviral state against lytic viruses (Mengo and vesicular stomatitis virus) and retrovirus (Friend-MuLV) production at least up to IFN doses 1000-fold higher than those needed to protect parental FLC. With respect to erythroid differentiation in DMSO-induced FLC it is emphasized that: (1) the FLC variant tested was also resistant to the enhancement of erythroid differentiation by low doses (15–480 U/ml) of IFN; (2) IFN doses (5000–20,000 U/ml) practically unable to reduce Friend-MuLV release and VSV yields did inhibit, instead, the erythroid differentiation of the DMSO-induced FLC variant tested almost at the same extent as with susceptible FLC. The striking dichotomy of IFN-induced antiviral versus differentiation effects suggests that different mechanisms underlie different IFN effects, the specificity of which was fully ascertained by employing pure IFN preparations. In the FLC system, treatment with ouabain does not compete IFN-induced antiviral state. Yet, two out of three variants tested were partially resistant to ouabain-induced inhibition of Friend-MuLV release while being fully susceptible to other ouabain-induced effects on FLC. In addition, an ouabain-resistant FLC variant was not resistant to IFN.

2–5A synthetase activity does not increase in interferon-resistant friend leukemia cell variants treated with α/β interferon despite the presence of high-affinity interferon receptor sites

The presence of interferon (IFN) receptors on mouse Friend leukemia cells (FLC) has been investigated in binding experiments with highly purified 125I-labeled mouse alpha/beta IFN. Both IFN-resistant clones and wild-type IFN-sensitive FLC showed a specific saturable binding site for mouse IFN with a similar affinity constant. In contrast to IFN-sensitive FLC, IFN-resistant FLC variants were not inducible by IFN for double-stranded RNA-dependent 2-5A synthetase activity.

Interferons in cell growth and development

Interferons (IFNs), besides inducing an antiviral state in uninfected cells, are also natural regulatory molecules. They play a key role in the regulation both of cell growth and differentiation, and of development. Up- or down-regulation of oncogenes by IFNs may be one of the mechanisms by which these molecules affect cell physiology. The list of IFN-inducible proteins continues to grow rapidly and future research should identify among these the mediators of the biological effects of IFNs.

Inhibition of animal virus production by means of translation inhibitors unable to penetrate normal cells

Abstract The correlation between virus and host protein synthesis, membrane leakiness, and virus production has been studied in vesicular stomatitis virus-infected L cells, herpes simplex virus (type 1)- and Sendai virus-infected 37RC cells. In all three systems, membrane leakiness, as measured by an altered permeability to low-molecular-weight translation inhibitors (e.g., hygromycin B), is detectable at a time when the cells are very actively engaged in virus protein synthesis. The alteration of the membrane increases as the virus life cycle goes on so that an almost total and specific inhibition of viral translation by hygromycin B is achieved late in infection. Although the overall protein synthesis is not shut off in Sendai virus-infected cells, a gradual replacement of host protein synthesis by viral translation parallels an increasing plasma membrane permeability to hygromycin B, which is also correlated with the ever increasing fraction of infected cells. These results indicate that cells actively engaged in viral protein synthesis have lost, at least partially, the permeability barrier that plasma membrane maintains in uninfected cells. The presence of hygromycin B in the culture medium significantly reduces the production of mature virus in the three systems studied suggesting that this approach may prove useful in the search for antiviral agents.

Protection of macaques against simian immunodeficiency virus infection with inactivated vaccines: comparison of adjuvants, doses and challenge viruses

Nine European laboratories contributed a total of 98 macaques towards a collaborative trial to study the ability of formaldehyde-inactivated or subunit SIV vaccines to protect immunized animals against live virus challenges. Four adjuvants, three dose levels and two immunization schedules were compared. Fifty-two of 61 (85%) immunized animals were protected against infection after challenge with either homologous or heterologous virus strains grown in human cells. Optimum protection required a high dose of antigen and a prolonged immunization schedule. On the day of challenge the titres of antibodies to SIV and to host cell components, as well as the titres of neutralizing antibodies, were significantly higher in the protected animals than in the non-protected. Forty-four vaccinated macaques (of which 36 were protected against previous challenges grown in human cells) and 28 naive animals were then challenged with extracellular or cell-associated SIV grown in simian cells. All naive animals and all vaccinees challenged with extracellular SIV became infected. Four of the eight animals challenged with cell-associated viruses were protected. These results clearly indicate that vaccines which potently protect against SIV grown in human cells, do not protect against SIV grown in simian cells. The cell substrate on which challenge viruses are grown is clearly significant in interpreting the results of vaccine trials. This trial has demonstrated that SIV vaccines using different adjuvants can protect macaques against SIV grown in human cells but not against extracellular SIV grown in simian cells. These results have important relevance to the development of HIV vaccines for humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon effects on Friend leukaemia cells. I. EXpression of virus and erythroid markers in untreated and dimethyl sulphoxide-treated cells.

The effects of low doses (40 to 1000 units/ml) of mouse interferon (IF) on the expression of Friend leukaemia virus (FLV) and globin genes in Friend leukaemia cells (FLC) have been examined. IF blocks production of extracellular virus, but virus antigens accumulate in the cytoplasm. In cells treated with IF at the time of seeding, there is a reduction in the amount of RNA specified by the lymphatic leukaemia virus (LLV) component of FLV; with the same IF dose there is a small but definite stimulation of haemoglobin and globin mRNA synthesis. The effects of IF on LLV gene expression are even more pronounced in dimethyl sulphoxide (DMSO)-stimulated LFC. No correlation was found between LLV gene expression and the appearance of erythroid markers.

TGF β induces a sustained c-fos expression associated with stimulation or inhibition of cell growth in EL2 or NIH 3T3 fibroblasts

Abstract We have previously indicated that epidermal growth factor (EGF) plays a fundamental role in the proliferation control of EL2 rat fibroblast line. It is shown here that transforming growth factor β (TGF β) stimulates both DNA synthesis and proliferation of EL2 cells, while exerting an inhibitory effect on the growth of murine NIH-3T3 fibroblasts. We also report the effect of TGF β and EGF on c-fos expression in EL2 cells, as compared to that of TGF β in NIH-3T3 fibroblasts. In EL2 cells EGF induces a transient c-fos expression at both mRNA and protein level, as previously observed in NIH-3T3 fibroblasts treated with platelet-derived or fibroblast growth factor (PDGF, FGF). Conversely, TGF β induces in EL2 cells a sustained expression of fos mRNA and protein, which are still detectable at least 24 and 7 hr after treatment respectively. In NIH-3T3 fibroblasts TGF β causes a sustained fos RNA expression, which is not associated, however, with detectable fos protein. We conclude that in fibroblasts stimulated by mitogens c-fos expression may be differentially modulated, depending of the growth factor and the cell line. This is seemingly due to differential regulation of fos gene expression, not only at the transcriptional and/or post-transcriptional level (transient or sustained fos RNA induction by EGF or TGF β in EL2 cells), but also at the translational level (fos protein(s) induction by TGF β in EL2 but not NIH-3T3 fibroblasts, possibly related to the stimulatory vs inhibitory effect of this factor on the growth of the former vs the latter line).

SEX AS A RISK FACTOR FOR HTLV-I SPREAD AMONG INTRAVENOUS DRUG ABUSERS

289 intravenous drug abusers attending public assistance centers in Rome were studied to evaluate the correlation between HTLV-1 and specific behaviors such as needle sharing and sexual promiscuity. HTLV-1 is a virus which is widespread in certain areas and endemic in southeastern Italy. High rates of seropositivity have been found in intravenous drug addicts and is probably transmitted by routes similar to those for HIV the AIDS virus. HTLV-1 seropositivity was significantly more common among anti-HIV-positive individuals (p<0.001) suggesting similar ways of transmission. Needle sharing was more frequent in seropositives than in seronegatives but the difference was not significant. A closer association was found between HTLV-1 seropositivity and sexual promiscuity. Logistic regression analysis confirmed an association between sexual promiscuity and seropositivity for HTLV-1 and revealed an even stronger association when interaction between sexual promiscuity and needle sharing was also taken into account. These findings emphasize the importance of sexual contact besides needle sharing in determining HTLV-1 spread among intravenous drug abusers.

Renal metabolism of homologous serum interferon

The metabolic behaviour of homologous native and desialylated serum and urinary interferons has been investigated by using an isolated and perfused rabbit kidney, the performance of which is comparable to that in vivo. Serum native and urinary interferons disappear from the plasma perfusate with a fractional turnover rate of 1.8 and 2% and half-lives of 3 and 35 min, respectively. Serum desialylated interferon disappears much more rapidly in keeping with the finding that the glomerular sieve poses less steric hindrance and electrophysical repulsion to the passage of less anionic proteins. These results confirm and extend our previous findings, which indicated that the kidneys have a predominant catabolic role and can explain to some extent the rapid disappearance of interferon from plasma.

Interferon-induced antiviral actions in Friend leukemia cells: Role of membrane gangliosides

Abstract The interactions between interferon (IFN) and membrane gangliosides have been analyzed in Friend leukemia cells (FLC). The G M1 -cholera toxin (CT) model has been selected as a reference system for a high-affinity ganglioside-ligand binding. Two gangliosides have been detected in FLC extracts: a disialoganglioside (∼ 95%) and a monosialoganglioside (∼ 5%) which migrate respectively as G D1a and G M1 . Unilammellar liposomes containing either mixed commercial gangliosides or G D1a or G M1 bind three to eight fold more IFN (both partially purified and pure ) than gangliosides-free liposomes. Only 3–4% of the input IFN was bound, whereas G M1 -containing liposomes bound almost 50% of [ 3 H]acetyl-CT. Neuraminidase treatment of FLC, which converts G D1a -like into the G M1 -like ganglioside, does not significantly modify the IFN-induced antiviral effects. CT binding to FLC as well as CT-induced elevation of endocellular cyclic AMP levels in FLC were instead markedly increased by the same neuraminidase treatment. Further, pretreatment of FLC with either G D1a or G M1 or gangliosides extracted from FLC did not change the IFN-induced establishment of the antiviral state. These data provide convincing evidence that FLC gangliosides do not represent the IFN high-affinity receptors nor specifically mediate IFN effects, even though they bind IFN to some extent.