Rebeca Falcoff

Treatment of Visceral Leishmaniasis with Pentavalent Antimony and Interferon Gamma

Acute visceral leishmaniasis is associated with an antigen-specific immunosuppression of mononuclear cells as evidenced by defective in vitro production of interferon gamma. We evaluated treatment with recombinant human interferon gamma in combination with conventional pentavalent antimony therapy in patients with visceral leishmaniasis. Six of eight patients with visceral leishmaniasis (mean duration, 17 months) that had been unresponsive to multiple courses of pentavalent antimony responded to treatment with recombinant human interferon gamma (100 to 400 micrograms per square meter of body-surface area per day) in addition to pentavalent antimony (20 mg per kilogram of body weight per day) for 10 to 40 days. The other two patients improved initially but then relapsed and required treatment with amphotericin B. Eight of nine additional patients with previously untreated severe visceral leishmaniasis were also successfully treated with the combination of interferon gamma and pentavalent antimony. The 14 patients who responded to this regimen had marked improvement in symptoms and in measures of anemia and leukopenia, as well as weight gain, a decrease in spleen size, and an absence or reduction of leishmanias in splenic aspirates. These patients had no recurrence of illness after a mean (+/- SE) follow-up of 8 +/- 1 months. Fever was the only major side effect of interferon gamma. We conclude that the combination of interferon gamma and pentavalent antimony is effective in treating seriously ill patients with refractory or previously untreated visceral leishmaniasis.

Synergistic Effect of Interferon‐γ and Tumor Necrosis Factor‐α on Antiviral Activity and (2′–5′) Oligo (A) Synthetase Induction in a Myelomonocytic Cell Line

TNF was not observed to have an antiviral effect on either HL60 or U937 cells. However, it did significantly enhance interferon (IFN)‐γ‐mediated antiviral activity in U937 cells. Treatment of U937 cells with IFN‐γ enhanced (2′–5′) oligo (A) synthetase activity (2.5‐fold) but treatment with TNF did not Combined treatment with TNF‐α + IFN‐γ increased this activity dramatically (20–40‐fold). This increase correlated with the very large increase in the (‘2–5’) oligo (A) synthetase mRNA level in U937 cells. No such effects were observed in HL60 cells. Furthermore, binding studies and cross‐linking analysis showed that IFN‐γ modulated TNF‐α receptors in U937 cells without altering their properties, but did not do so in HL60 cells.

Induction de la synthèse d'interféron par des RNA bicaténaires. II. Etude de la forme intermédiaire de réplication du virus Mengo.

Abstract Interferon-inducing capacity of double-stranded RNA. II. Studies on Mengo virus replicative intermediate Total RNA from Mengo virus infected L cells (RNA-IC) induced interferon in mice when inoculated intraperitoneally and intravenously. When RNA-IC was treated by 2-M LiCl, the multistranded replicative intermediate and the single-stranded viral 37-S RNA were precipitated while the double-stranded replicative form remainded in solution. Both, insoluble and soluble fractions were interferon inducers. When insoluble fraction (replicative intermediate+37-S RNA) was treated in vitro with ribonuclease, the enzyme degraded the viral 37-S RNA and the single-stranded portion of the replicative intermediate, transforming it in replicative-form molecules. The total amount of interferon produced was the same before and after ribonuclease treatment. Chromatography of insoluble fraction was performed on Sepharose 2B column. The front eluting material was the replicative intermediate containing 50% of ribonuclease-resistant radioactivity. The second radioactive peak, 37-S RNA, was completely digested by the enzyme. Only the fractions from the replicative intermediate zone induced interferon. The amount of interferon induced by each fraction of the chromatography was proportional to the nuclease-resistant RNA present. From these results it appears that the interferon-inducing capacity of the replicative intermediate is exclusively associated with its double-stranded core.

Induction de la synthèse d'interféron par des RNA bicaténaires: I. Application à l'étude du cycle de multiplication du virus Mengo (I)

Abstract Interferon inducing capacity of double-stranded RNA. I. Studies on Mengo virus replication Total RNA from Mengo infected L cells (RNA-IC) induces interferon in mice when inoculated in microgram amounts intraperitoneally and intravenously (Fig. 2). The inducer is resistant to ribonuclease (100 μg, 22°, 30 min in 0.3 M NaCl-0.03 M trisodium citrate and formaldehyde (1.5%), respectively. The kinetics of synthesis of the inducer during Mengo virus multiplication is shown in Fig. 3: after exponential growth, maximal amounts of inducer are observed at 6 h. The data presented in Fig. 4 illustrate a sucrose density gradient profile of RNA-IC; 2 mice were inoculated with each fraction and the interferon in the serum was titrated. A peak inducing activity was found at 16 S. Heavy labelled RNA-IC with [5-3H]uridine was fractionated by density gradient (Fig. 5A, 5B). Acid-insoluble radioactivity of each fraction was measured before and after ribonuclease treatment (10 μg/fraction). The inducing interferon activity was also determined. Both peaks were superposable. When 3H-labelled RNA-IC was treated with ribonuclease before sedimentation, acid-insoluble radioactivity and interferon induction peaks were still coincident but displaced to the lighter side of the gradient. Sedimentation rates slower than viral single-stranded RNA and ribonuclease resistance are characteristics of double-stranded replicative form. Interferon inducing capacity appears to be an added property which might be of value in the search for other replicative forms.

Location of enhanced ribonuclease activity and of a phosphoprotein kinase in interferon-treated mengovirus-infected cells

Abstract Infection of interferon-treated L cells by mengovirus increased a ribonuclease activity in a sedimentable fraction known to contain the viral replication complex. The enhanced in vitro phosphorylation of a 67,000 M r protein was also observed in the same fraction.

Recombinant human interferon-gamma inhibits adenovirus multiplication in vitro.

The susceptibility of adenovirus to the inhibitory effect of human interferons in vitro was investigated. We tested recombinant human interferons-alpha 2, -beta 1 and -gamma against adenovirus serotypes 1 and 5 (group C), 3 and 7a (group B), and a wild strain isolated from an acutely ill child who later died. Pretreatment of WISH cells with interferon-gamma for 24 h induced a dose-dependent inhibition of multiplication of all adenovirus strains tested, the TCID50 varying from 25 to 90 IU/ml. Human interferon-alpha 2 was unable to decrease adenovirus multiplication, while interferon-beta 1 at 2000 IU/ml slightly lowered the yield of adenovirus. Similar results were obtained in HEp-2 and FS-4 cells, while A-549 and peripheral blood mononuclear cells were insensitive to interferon-gamma. The difference between the effects of interferon-gamma and interferon-alpha and -beta on adenovirus multiplication in vitro suggests that its mechanism of antiviral action is different.

Recombinant human interferon-gamma inhibits adenovirus multiplication without modifying viral penetration.

We have recently reported that adenovirus replication is inhibited by human recombinant interferon-gamma, but not by recombinant interferon-alpha, in a dose-dependent manner. The aim of this study was to determine whether the antiviral effect of recombinant interferon-gamma could be linked to interferon-induced alteration at the membrane level, inhibiting either adenovirus penetration of or release from WISH cells. Adsorption and penetration were investigated with an 125I-labelled adenovirus binding assay. To test defective virus release, the presence of newly synthesized virus proteins in the cytoplasmic and nuclear compartments was investigated. Binding studies showed that interferons-gamma and -alpha did not modify adenovirus attachment and penetration. Interferon-gamma but not interferon-alpha inhibited hexon protein synthesis in the cytosol as well as its accumulation in the nuclear compartment. The synthesis of polypeptides III, IV and VI was also inhibited. In cells infected before interferon-gamma treatment, its addition could be delayed up to 2 h after the infection to produce an inhibition of virus yield greater than 1 log10 unit (90% inhibition). We conclude that interferon-gamma acts on an intracellular step before or at adenovirus protein synthesis, probably through a mechanism not shared with interferon-alpha.

Variations in susceptibility to Leishmania amazonensis infection in lines of mice selected for high or low immunoresponsiveness

Summary The degree of resistance to a local Leishmania amazonensis challenge has been compared in lines of mice obtained by selective breeding for high or low immunoresponsheness: High and Low antibody responder mice of Selections I and II (H1, H11 and L1 L11 lines) and high and low responder mice to T mitogen PHA (Hi/PHA and Lo/PHA). The aim of this preliminary study was to focus attention on genetic differences related with well defined immune characteristics. Clear‐cut results were obtained, both H1 and H11 mice developed large and disseminating lesions, the rale of symptom aggravation being faster in H11, while L1 and LM proved resistant to parasites, only small and transient lesions being observed for them during a 150 days follow up. The outcome of infection also differs in Hi/PHA and Lo/PHA mice, Hi/PHA having a resistant and Lo/PHA a susceptible phenotype.

Expression of extracellular and intracellular human IFN-γ in mouse L cells transformed with the human IFN-γ cDNA gene

Abstract Cotransformation with a plasmid containing a thymidine kinase gene (pTK2) and a plasmid encoding human IFN-γ (pTG11) has been used to establish murine L cell lines expressing human IFN-γ. The HuIFN-γ gene was present in 30% of the tk+ cell lines and some of these secreted low levels of IFN into the culture medium. Two of the clones obtained after transformation were selected for detailed analysis. Clone 1–12 constitutively secreted very low levels of HuIFN-γ in the culture medium. This antiviral activity was characterized by its species specificity and antigenicity as authentic human IFN-γ In contrast, clone 3–47 produced a HuIFN-γ activity which could only be detected intracellularly. This clone was resistant to infection both by Vesicular stomatitis (VSV) and Mengo viruses and contained increased levels of enzymes known to be induced by interferon. Dur results suggest that clone 3–47 produces a non-secreted HuIFN-γ like molecule which is able to trigger an antiviral state in the murine cell independent of the interaction with a specific IFN-γ surface receptor.

RECOVERY BY INTERFERON OF MENGOVIRUS-INDUCED SHUTOFF OF HOST PROTEIN SYNTHESIS*

In preincubated cell-free extracts prepared from interferon-treated cells the translation of exogenous viral or cellular mRNAs is inhibited equally.’,2 It is also known that the addition to such extracts of dsRNA activates at least two specific enzymes, the consequence being the inhibition of translation. (Reviewed by C. Bagli~ni.~] Since dsRNA seems to play a major role in stimulating interferon-dependent enzymes. it was interesting to study a functional system in which the challenge virus would provide dsRNA molecules. We have chosen as model L cells infected with mengovirus. It is known that soon after infection by a picornavirus there is an inhibition of cellular RNA and protein synthesis. Later in infection, the synthesis of DNA is also d e ~ r e a s e d . ~ The inhibition of the synthesis of macromolecules is often referred to as “shutoff.“ We decided to study first, the possible action of interferon (IF] on the establishment of the shutoff. Since the level of shutoff varies with the cell lines and with the multiplicity of infection we established our experimental conditions. (FIGURE 1). Confluent monolayers of L cells were infected with different multiplicities of infection (MOI] of mengovirus; after 4 h r cells were pulse-labeled for 30 min with methionine, lysed with detergents (NP40 and DOC] and the proteins analyzed by slab gel electrophoresis. The first lane to the left shows the pattern of host proteins; in the second lane, with 5 pfu/cell viral proteins are very easily recognized, although cellular proteins are also present. It should be remembered that the viron RNA acts as messenger and it is translated from one single initiation site. The unique translation product is a “polyprotein” of about 270,000 MW. Primary and secondary cleavages of this protein give rise to precursors, and finally capsid and noncapsid stable proteins. With 20 and 50 pfu/cell the shutoff of host protein synthesis is markedly established. The counts of this experiment are shown in FIGURE 2, represented by the filled circles. With 5 pfu/cell, although viral proteins are actively synthesized, the total amount of methionine is already lower than in the control noninfected cells. Both, host and viral proteins contributed to the total counts. With 20 pfu cellular proteins are almost absent, the counts correspond principally to viral proteins. With 50 pfu/cell more viral proteins are synthesized, which explains the higher incorporation of the label. When we repeated the experiment with cells pretreated with 200 units IF/ml. we were unable to detect viral proteins.