Production of an antiviral factor by murine spleen cells after treatment with Corynebacterium parvum.

We have previously shown that injection of Corynebacterium parvum (CP) in mice protected them against lethal encephalitis induced by herpes simplex virus, (HSV). It is shown here that spleen cells of CP-injected mice in vitro produce a factor capable of inhibiting the replication of HSV in mouse embryo fibroblasts (MEF). A similar activity was produced after in vitro exposure of spleen cells from untreated mice to CP. CP was only slightly mitogenic in contrast to phytohemagglutinin (PHA), concanavalin A (Con A), and bacterial lipopolysaccharide (LPS) which were strongly mitogenic but did not induce antiviral activity high enough to be detected in the HSV-MEF system. The activity produced by CP-treated spleen cells appeared to be interferon since it was trypsin sensitive and species specific and not virus specific, and since preincubation of the cells was required to demonstrate an antiviral effect. However, the identity of CP-induced interferon with any of the previously described subclasses of interferon remains to be determined.     

Protection of mice against influenza virus infection: Enhancement of nonspecific cellular responses by Corynebacterium parvum

Groups of C57Bl/6J, BALB/c, BALB/c nu⁺/nu⁺ mice, inoculated intranasally with Corynebacterium parvum (350 μg/mouse) were protected from death by an otherwise lethal dose of influenza virus, A/WSN (H1N1) inoculated 3 days later. The lungs of C. parvum-trented, virus-infected C57Bl/6J, BALB/c, or BALB/c nu⁺/nu⁺ mice contained significantly less infectious virus than did controls, and this reduction was apparent as soon as 24 hr after virus inoculation. The maximum protective effect correlated with increased lung interferon levels. C. parvum treatment caused an increase in the lung cell number which was in part due to a large increase (ca. 10-fold) in macrophage content, and the natural killer cell activity was also enhanced, though not as markedly as occurred 3 days after infection. Most (>85%) of the resident macrophages in normal lungs were susceptible to infection by virus (as indicated by hemadsorption), whereas most of those recovered from the lungs of C. parvum-treated mice resisted infection. Despite the increase in macrophage content, the level of specific immune responses to infection, such as cytotoxic T-cell activity, DTH reaction, and antihemagglutinin antibody, remained unchanged by C. parvum treatment so that the major if not only effect of this treatment was on the level of the less-specific components of the immune system.     

Differentiation of the immunosuppressive and antiviral effects of interferon.

Virus-induced (virus-type) interferon suppression of the in vitro antibody response of mouse (C57B1/6) spleen cells to sheep red blood cells was blocked by 5 × 10^?5M 2-mercaptoethanol (2-ME). The blockade was not due to a direct effect on interferon since 2-ME was capable of blocking the suppression when added to cultures up to 48 hr after interferon. 2-ME blockade of virus-type interferon immunosuppression was not due to the immunoenhancing property of 2-ME. Similar protective effects of 2-ME were observed during immunosuppression by virus-type interferon inducers, but not T-cell mitogen inducers of interferon (immune interferon). The data suggest that the immunosuppressive properties of virus-type and immune interferon preparations involve different mechanisms. Virus-type interferon inhibited DNA synthesis in unstimulated spleen cell cultures and in 2-ME stimulated cultures, and the degree of inhibition of DNA synthesis appeared to be related to the immunosuppressive property of interferon in the absence or presence of 2-ME. 2-ME did not affect the antiviral properties of either virus-type or immune interferon in nonlymphoid cells. Further, the induction of virustype interferon in spleen cells was neither inhibited nor enhanced by 2-ME, while the induction of immune interferon was enhanced. This enhancement is consistent with 2-ME enhancement of the immunosuppressive effects of immune interferon inducers.There are two possibilities for 2-ME blockade of the immunosuppressive effect of virus-type interferon, while not affecting the antiviral property. Firstly, the immunosuppressive and antiviral properties of virus-type interferon may involve different mechanisms at the subcellular level. Secondly, the selectivity of the blockade by 2-ME could be due to the fact that spleen cells are the target cells in immunosuppression, while L cells are the target cells in inhibition of virus replication. Thus, virus-type interferon may suppress the immune response at the level of the macrophage and 2-ME may reverse this effect by replacing a blocked macrophage function.     

Comparison of antiviral and antitumor activity of activated macrophages.

The antiviral and antitumor activity in vitro of normal, stimulated, vaccinia virus “immune”, and activated peritoneal macrophages were compared. Activated (pyran or corynebacteria induced) PEC exhibited both antitumor and antiviral activity. Stimulated (thioglyocollate) and vaccinia virus “immune” PEC inhibited virus growth but did not possess antitumor activity. Normal (unstimulated) PEC were relatively ineffective in either activity. The antiviral activity was nonspecific, being expressed against herpes simplex and EMC viruses in addition to vaccinia. Although a possible role for interferon was suggested by the lack of activity of mouse PEC on vaccinia virus growth in heterologous FLK cells, definitive proof was not obtained. The activity was most pronounced against multiple cycles of viral infection initiated at a low multiplicity of infection. Single cycle virus growth was not affected, suggesting that the major inhibition was on subsequent cycles of virus growth.     

Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr, whereupon either the inhibition of [¹²⁵I]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8–10% and 10%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4–6 and 6–8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.     

Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses

The cell-wall sulphated polysaccharide of the red microalga Porphyridium sp. has impressive antiviral activity against Herpessimplex viruses types 1 and 2 (HSV 1, 2) and Varicella zoster virus(VZV). Treatment of cells with 1 g mL^-1 polysaccharideresulted in 50% inhibition of HSV-infection as measured by the plaqueassay. Inhibition of the production of new virus particles was also shownwhen pre-infected cell cultures were treated with the polysaccharide. Inaddition, there was indirect evidence for a strong interaction between thepolysaccharide and HSV and a weak interaction with the cell surface.Depending on the concentration, the polysaccharide completely inhibitedor slowed down the development of the cytopathic effect in HSV or VZVpreinfected cells, but did not show any cytotoxic effects on Vero cells evenwhen a concentration as high as 250 g mL^-1 was used. Itseems therefore that the polysaccharide is able to inhibit viral infection bypreventing adsorption of virus into the host cells and/or by inhibiting theproduction of new viral particles inside the host cells. Thus, this alga seems tobe a good candidate for the development of an antiviral drug.     

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.     

Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon.

Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state.     

The potency of acyclovir can be markedly different in different cell types

Acyclovir is an acyclic guanine analog with a considerable activity against herpes simplex viruses. We studied the antiherpetic activity of acyclovir in macrophages and fibroblast cell lines. Utilising a plaque reduction assay we found that acyclovir potently inhibited the HSV-1 replication in macrophages (EC50) = 0.0025 microM) compared to Vero (EC50 = 8.5 microM) and MRC-5 (EC50 = 3.3 microM) cells. The cytotoxicity of acyclovir was not detected at concentrations < or = 20 microM, thus the selective index in macrophages was >8000. This marked difference in antiherpetic activity between macrophages and fibroblasts was not observed with Foscarnet and PMEA. We suggest that this potent antiviral effect of acyclovir is mainly due to a proficient phosphorylation of the drug and/or a favourable dGTP/acyclovir triphosphate ratio in macrophage cells.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

Effects of carrageenan on spontaneous and antibody-dependent cell-mediated cytotoxicity

We examined the effect of carrageenan on in vitro antibody-dependent cell-mediated cytolysis (ADCC) and spontaneous cell-mediated cytolysis (SCMC) in cultures of human peripheral blood mononuclear cells (PBL). Carrageenan, when present in the assay, nonspecifically reduced ADCC and SCMC against both Chang and chicken erythrocyte (CRBC) target cells. This reduction in cytotoxicity could not be attributed entirely to the macrophage toxic and complement-inhibitory properties of carrageenan because neither removal of complement nor macrophage depletion prevented the dose-dependent inhibition. In contrast, pretreatment of effector PBL, with carrageenan followed by removal of Carrageenan by washing did not alter ADCC or SCMC against Chang cells, which are mediated by nonphagocytic cells, but reduced both ADCC and SCMC activity against CRBC targets, which are mediated in part by macrophages. Thus, Carrageenan, when present in in vitro cell-mediated cytotoxicity assays, causes a nonspecific impairment of cytotoxicity that is independent of its anticomplement or macrophage-toxic properties.     

Effect of interferon on replication of herpes simplex virus types 1 and 2 in human macrophages.

Macrophages derived from human peripheral blood and cultured for 1 week were permissive for the replication of herpes simplex virus (HSV) types 1 and 2. Low titers of interferon (IFN) were produced after virus infection. The yield of infectious virions was reduced by pretreatment of cells with natural and recombinant IFN-alpha and natural IFN-beta. Recombinant and natural IFN-gamma exhibited very low antiviral activity. Treatment of cells with IFN-gamma mixed with IFN-alpha or with IFN-beta did not result in a synergistic inhibition of virus yield. We studied the synthesis of HSV type 1- and HSV type 2-coded proteins in macrophages treated with IFN-beta. Induction of the HSV beta-protein DNA polymerase was strongly inhibited in IFN-treated cells in a dose-dependent manner. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, other beta- and gamma-proteins of HSV were inhibited as well. Immunofluorescence studies revealed a strong inhibition of the expression of immediate early alpha-protein ICP4. The results indicate that IFN acts early during the viral replication cycle to inhibit the synthesis of HSV alpha- and beta-proteins.     

Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells

Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2’-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.     

Direct augmentation of cytolytic activity of tumor-derived macrophages and macrophage cell lines by muramyl dipeptide.

Macrophages derived from MSV-induced tumors and several macrophage cell lines showed direct cytolytic activity in an 18-hr ⁵¹Cr release assay against tumor target cells. The cytolytic activity of these macrophages was augmented by the addition of muramyl dipeptide (MDP) to the cytotoxicity assay, an effect similar to that observed with bacterial lipopolysaccharide. The stimulation of macrophage-mediated cytotoxicity by MDP appeared to be under genetic control since macrophages from BALB/c mice were augmented with MDP while those from C57BL/6 animals were not. MDP appears to act directly on the macrophage without the participation of any other cell type, since MDP increased the activity of the macrophage cell lines.     

Epigallocatechin gallate (EGCG) combined with zinc sulfate inhibits Peste des petits ruminants virus entry and replication

Despite the fact that the Peste des petits ruminants virus (PPRV) leads to high morbidity and mortality (up to 100%), antiviral drugs against PPRV are not available. The aim of this study was to estimate the dose of epigallocatechin gallate (EGCG) co-administered with zinc (II) ions as an antiviral agent against PPRV. Treatment of PPRV-infectedVero cells with EGCG and zinc sulfate (zinc II) was administered, and antiviral activities against PPRV in infected Vero cells was evaluated by determination of virus yields, expressed as logTCID₅₀/mL. Cytotoxicity was determined using the tetrazolium-based MTS test. Zinc sulfate at 1.1 mg/mL and EGCG at 25 μM showed low potentiated and potentiated antiviral activities against PPRV, respectively. These agents caused significant inhibition of PPRV in Vero cells (p < 0.05) with a reduction in logTCID₅₀/mL by up to 3-fold. The combination of EGCG (25 μM) and zinc sulfate (1.1 mg/mL) was observed to have strong antiviral activity (p < 0.01) against PPRV with a reduction in logTCID₅₀/mL of the virus up to 4-times without causing any host cell cytotoxicity. This study is the first one to prove that the zinc II has the capability of stimulating EGCG to inhibit in vitro PPRV entry. Moreover, this combination appears capable of reducing infection resistance by hindering viral adaptation.     

Selective protection of murine thymic helper T cells from glucocorticosteroid inhibition by macrophage-derived mediators

We investigated the in vitro effects of dexamethasone (DEX) on the functional capacities of virgin murine T cells cultured in the absence and presence of adjuvant-stimulated macrophages or factors derived from them. Immunologically mature thymocytes, isolated on the basis of their inability to bind peanut agglutinin (PNA^? thymocytes), were used as virgin T cells. Treatment of PNA^? thymocytes with DEX for 24 hr in vitro eliminated their subsequent capacity to function as helper cells for primary humoral responses, to proliferate when stimulated by plant mitogens or allogeneic cells, or to generate T-cell-mediated cytotoxic responses. However, when PNA^? thymocytes were pretreated with DEX in combination with either adjuvant-activated macrophages or their culture supernatants, which contained Interleukin 1, the capacity of the T cells to subsequently express helper activity was preserved. The macrophage products, however, did not prevent DEX from inhibiting the capacities of PNA^? thymocytes to proliferate in response to plant mitogens or alloantigens or to generate cytotoxic effector cells; thus, protection was selective. The data indicate that, prior to activation, helper T cells are distinguished by their capacity to become steroid resistant in response to macrophage products. Although T-cell proliferative and cytotoxic responses have been reported to be protected from DEX inhibition by Interleukin 2, our results suggest that macrophages prevent steroid effects on virgin helper T cells by Interleukin-1-dependent mechanisms that do not involve Interleukin 2. While we have not delineated the biochemical pathways of protection, we show that the acquisition of DEX resistance by helper T cells cannot be attributed to the polyclonal induction of helper activity by macrophage factors.     

Further studies on the antiviral activity of alloferon and its analogues

The subject of our studies was the synthesis, biological evaluation, and conformational studies of insect tridecapeptide alloferon (H‐His‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH) and its analogues such as: [des‐His¹]‐, [Lys¹]‐, [Arg¹]‐, and [Ala¹]‐alloferon. These peptides were synthesized to check the influence of the His residue at position 1 of the alloferon chain on its antiviral activity. Two aspects of the biological effects of these peptides were determined: (i) the cytotoxicity in vitro in the Vero, LLC‐MK2, and HEp‐2 cell lines, and (ii) the antiviral activity in vitro in respect to DNA and RNA viruses. We found that alloferon inhibited the herpes virus multiplication and failed to affect the coxsackie virus replication, whereas [Lys¹]‐alloferon exhibited a high inhibitory action towards both viruses. Moreover, the peptides did not show any cytotoxic activity against the Vero, LLC‐MK2, and HEp‐2 cells. The preliminary circular dichroism conformational studies showed that the peptides investigated seem to prefer an unordered conformation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.