Antiviral activity of recombinant interferon-alpha-2b in combination with certain antioxidant

In vitro activity of interferon-alpha-2b in combination with various antioxidants against the influenza virus and Herpes simplex was studied. The standard strains and a clinical strain of Herpes simplex isolated from a patient with resistance to acyclovir were used. The in vitro studie showed that antioxidants, such as alpho-tocoferol acetate (vitamin E), Unithiol and ascorbic acid had a significant antiinfluenzae and antiherpetic action on the influenza virus A/H5N1 and Herpes simplex variants. They protected up to 100% of the cell monolayer from the virus cytopathic effect. The taurin solutions had no antiviral activity irrespective of the infection dose. Combinations of interferon-alpha-2b with alpha-tocopherol acetate (vitamin E), Unithiol or ascorbic acid showed a significant synergistic effect: the antiviral activity of interferon increased several times. The antiinfluenza activity of interferon-a-2b in the presence of various concentrations of taurin did not change.     

Mitogen induction of murine C-type viruses. I. Analysis of lymphoid cell subpopulations.

We reported previously in vitro induction of endogenous C-type viruses from normal mouse spleen cells by lipopolysaccharide (LPS) as well as by combination treatment with concanabalin A and 5-bromo-2'-deoxyuridine (Con A/BrdU). To identify the cell types sensitive to virus induction and to study the relationship of mitogenicity to virus induction we have compared T cell populations (BALB/c thymus cells and cortisone-resistant thymus cells), B cell populations (nu/nu spleen cells and lymph node cells), adherent BALB/c peritoneal cells and mixed populations (BALB/c spleen cells, macrophage-depleted BALB/c spleen cells, and lymph node cells). LPS-induction occurred only in B cell-containing populations. In contrast, induction by Con A/BrdU depended on the presence of both T and B cells. In both instances, neither macrophages nor hemopoietic cells appeared to be a major source of virus. Treatment with anti-Ig serum and complement reduced virus induction by LPS/BrdU but not by Con A/BrdU suggesting that different cell populations produce virus after stimulation with these two different mitogens.     

The effect of the antiherpesvirus drug 5-methoxymethyl-2'-deoxyuridine on the humoral immune response in vivo.

5-Methoxymethyl-2'-deoxyuridine (MMUdR), a drug with potent antiviral activity in vitro against Herpes simplex virus, was investigated for its immunosuppressive effects. Doses as high as 2000mg/kg given daily for 9 days were not immunosupporessive as judged by the fact that treated animals produced normal immune responses to sheep erythrocytes, Brucella bacteria, and Herpes simplex virus.     

Asynchronous depression of responses to T- and B-cell mitogens during acute infection with cytomegalovirus in the guinea pig

The nonspecific functional capacity of spleen cells, taken from female guinea pigs with primary acute cytomegalovirus (CMV) infection, was assessed using lipopolysaccharide (LPS), a B-cell mitogen, and concanavalin A (Con A), a T-cell mitogen. Proliferative responses to the two mitogens were found to be significantly depressed in animals inoculated with CMV as compared to control animals. The defect in Con A responsiveness occurred earlier during the course of the infection than the defect in LPS responses. Although responses to the mitogens were depressed at the time of peak virus activity in the spleen, the possibility of lytic destruction of the spleen cells by the virus during in vitro culture was excluded. In addition, the depression in Con A responsiveness was noted with a wide range of Con A concentrations, and preculture studies failed to result in enhanced reactivity of the cells from infected animals. We conclude that reductions of both B- and T-cell functions, which differ in their timing during the course of acute CMV infection, occur concurrently with an enhanced viral specific immune response in guinea pigs acutely infected with CMV.     

Interaction of reovirus with cell surface receptors. II. Generation of suppressor T cells by the hemagglutinin of reovirus type 3

In studying reovirus interactions with lymphocytes, we have found that reovirus type 3, but not type 1, inhibits the in vitro proliferative response of murine splenic lymphocytes to concanavalin A (Con A). By analyzing recombinant clones containing genes from both reovirus types 1 and 3, we found that the S1 gene, the gene that encodes the viral hemagglutinin, is responsible for the inhibitory effect. In addition we found that type 3, but not type 1, generates suppressor T cells in vitro capable of suppressing Con A proliferation. By analyzing recombinant clones, we also found that the viral hemagglutinin is responsible for the generation of suppressor T cells by reovirus type 3. These effects were observed whether UV-inactivated or live virus was used. Reovirus type 3 inhibition of the proliferative response of murine splenic lymphocytes to Con A was blocked by anti-reovirus type 3 antibody but not by anti-reovirus type 1 antibody. Antiviral antibody had no effect on the ability of reovirus type 3 induced suppressor cells to inhibit Con A proliferation. We have previously demonstrated a receptor on murine lymphocytes for the hemagglutinin of reovirus type 3, and our results suggest that the in vitro suppression of Con A proliferation of murine lymphocytes by reovirus type 3 is secondary to the interaction of the viral hemagglutinin with a receptor on the surface of murine lymphocytes, which results in the generation of functionally active suppressor T cells.     

Target cell heterogeneity in murine leukemia virus infection. III. Identification of susceptible Lyt 1+ and resistant Lyt 2+ T-cell subsets following in vitro infection with Friend murine leukemia virus

The susceptibility of splenic T-cell subpopulations to productive infection with Friend murine leukemia virus was determined after in vitro infection and stimulation with Con A. Con A enhanced the number of productively infected cells in unseparated spleen cells as well as in T-cell-enriched spleen cell fractions. Splenic T cells were fractionated into Lyt 1+ and Lyt 2+ subpopulations using both positive and negative selection techniques; susceptible splenic T cells were recovered in the Lyt 1+ fraction and specific cytotoxic treatment with anti-Lyt 1 antibody and complement reduced the number of infectious center-producing cells by greater than 87%. In marked contrast, Lyt 2+ splenic T cells were resistant to productive infection by Friend murine leukemia virus in vitro.     

Changes in the cell surface architecture in normal and polyoma virus transformed hamster cells after infection with influenza virus

Test of Con A induced cell agglutination, method of binding cells to Con A coated nylon fibres and modified procedure of cell-to-cell binding were used in the investigation of architectural surface changes in normal and polyoma virus transformed hamster cells infected with influenza virus. In both cell types influenza virus infection caused 1) increase in fixation resistant Con A agglutination, 2) decrease in the level of surface membrane fluidity and cell plasticity. It has postulated that influenza virus infection results in stabilization of the cell surface architecture. These changes are amplified by polyoma virus transformation. Con A acts in this system, as an indicator rather than as a modifier of architectural changes.     

Stimulation of secondary anti-MSV cytolytic T lymphocytes with MBL-2 reconstituted membranes

Membranes and solubilized, reconstituted membranes from Moloney-infected tumors MBL-2 have been used to stimulate in vitro secondary cytolytic T lymphocytes (CTL) in C57BL/6 mice primed and Moloney murine sarcoma virus. Membranes are shown to stimulate the generation of Moloney-specific and H-2 restricted CTL. Stimulation with solubilized, reconstituted membranes required the presence of rat lymphocyte Con A supernatant (containing interleukin 2) during the culture. Reconstituted membranes made in the presence of the detergent-insoluble fraction from the plasma membrane were able to stimulate a response in the absence of Con A supernatant.     

Cell-mediated immune responses in patients with recurrent Herpes Simplex infections. II. Infection-associated deficiency of lymphokine production in patients with recurrent herpes labialis or herpes progenitalis.

Herpes simplex virus antigen-induced lymphocyte proliferation and production of leukocyte migration inhibitory factor (LMIF) and lymphocyte-derived interferon were studied in normal individuals and patients with recurrent Herpes labialis and Herpes progenitalis. Virus-specific lymphoproliferative responses were regularly detected in patients with recurrent infection irrespective of the clinical stage of infection. In contrast, transient deficiencies in herpes-specific lymphoid production of both LMIF and interferon were regularly documented at the time of and immediately before herpes simplex-induced vesicular eruptions. During the convalescence, pronounced production of these mediators in response to antigenic stimulation with inactivated virus antigen preparations were regularly detected. The biology of these fluctuations in lymphokine production is evaluated and discussed.     

Concanavalin A-inducible suppressor cells in pleural effusions and peripheral blood of cancer patients

Summary Carcinomatous pleural effusions of 18 of 20 patients with lung cancer contained suppressor cell precursors that could be activated by concanavalin A (Con A) to suppress the proliferative responses of autologous and allogeneic lymphocytes to phytohemagglutinin and Con A. However, pleural effusion cells showed no suppressor function without prior activation by Con A. In contrast, the peripheral blood of the cancer patients exhibiting impaired mitogenic response contained nonspecific spontaneous suppressor cells capable of inhibiting the lymphoproliferative response to mitogens without prior activation by Con A, but these cells were not able to show further suppressor function even after activation by Con A. The maximum suppression was observed after 48-h treatment of lymphocytes with optimally mitogenic doses of Con A. The Con A-inducible suppressor cells of the pleural effusion and spontaneous suppressor cells of the peripheral blood of cancer patients had the same characteristics with regard to the capacity to suppress the mitogenic responses of autologous and allogeneic lymphocytes, belonging to the group of nylon wool-nonadherent T cells and being sensitive to in vitro culture and resistant to treatment with mitomycin C.     

Herpes virus proteins ICP0 and BICP0 can activate NF-kappaB by catalyzing IkappaBalpha ubiquitination

The immediate early proteins ICP0 and BICP0 from Herpes virus are promiscuous activators of both viral and cellular genes and play a critical role in virus life cycle. Here we report that ICP0 and BICP0 could induce NF-kappaB translocation from cytoplasm into nucleus and strongly activate NF-kappaB responsive genes specifically. This process was dependent on the RING domain of both proteins. In addition, ICP0 interacted specifically with IkappaBalpha and its activating effect was attenuated by Ubch5A(C85A) and MG132, but not by IkappaBalpha(S32A/S36A). Remarkably, IkappaBalpha was poly-ubiquitinated by both ICP0 and BICP0, in vitro and in vivo. These data indicate that ICP0 and BICP0, functioning as ubiquitin ligases, are bona fide activators of NF-kappaB signaling pathway. Our study identifies a new way ICP0 and BICP0 explore to regulate gene expression.     

Glycosylation of four acute-phase glycoproteins secreted by rat liver cells in vivo and in vitro. Effects of inflammation and dexamethasone

We have studied the role of the liver in the relative increase of Concanavalin A (Con A)-reactive molecular forms of various positive rat acute-phase glycoproteins (APGPs) occurring in serum during inflammation. Secretion media of hepatocytes isolated from inflamed rats showed a 2 to 5-fold increase of the total amounts of four APGPs studied in comparison to secretion media of control hepatocytes. These changes were in analogy with those observed for corresponding sera, except for alpha 1-antitrypsin. All the different Con A-reactive molecular forms were present in the media, with exception of the most reactive form of ceruloplasmin. In vitro and in vivo, dexamethasone augmented the secretion of three APGPs, and especially of the Con A-most reactive forms. The in vitro effect of dexamethasone--augmented secretion of Con A-reactive molecular forms of alpha 1-acid glycoprotein and haptoglobin--was comparable with the results obtained for hepatocytes isolated from inflamed rats. In vivo, dexamethasone treatment resulted in an even higher increase of the serum concentration of the Con A-most reactive forms of both APGPs than experimental inflammation did. Although an extrahepatic contribution cannot be excluded, these results suggest that alterations in the Con A reactivity of APGPs as observed during the acute-phase of inflammation have their origin in the liver. A change in the Con A reactivity of glycoprotein indicates a modulation of its glycosylation. Since dexamethasone can affect these changes in vivo and in vitro, glucocorticoids most probably are involved in the regulation of the glycosylation of the APGPs during biosynthesis in the liver.     

Relation between hyaluronan and sulphated glycosaminoglycan synthesis and degradation in cultured embryonic fibroblasts. Effect of concanavalin A and ammonium chloride administration.

In order to evaluate the relationship between glycosaminoglycan (GAG) synthesis and degradation, the effect of NH4Cl, which inhibits lysosomal degradation, on GAG production was analysed in vitro in concanavalin A (Con A) stimulated fibroblasts from 7 and 14-day-old chick embryos. 35SO4 incorporation into total proteoglycan (PG), 3H incorporation into individual GAG classes, beta-N-acetyl-D-glucosaminidase and beta-D-glucuronidase activity were determined. The results indicate a correlation between Con A and NH4Cl effects: NH4Cl induced a reduction principally in the GAG classes most stimulated by Con A. Thus HA and DS are much more stimulated by Con A and inhibited by NH4Cl than are CS and HS.     

Reactivity of avian RNA tumor viruses with lectins.

The infectivity of avian RNA tumor viruses was inactivated to varying degrees by treatment with either concanavalin A (Con A) or phytohemagglutinin but not by treatment with wheat germ agglutinin. In general, leukosis viruses reacted preferentially with Con A, whereas sarcoma viruses showed more affinity for phytohemagglutinin. In a more extensive study with subgroup A of Prague Rous sarcoma virus (PR-A), the effect of inactivation by Con A could be specifically prevented by the addition of alpha-methyl-D-mannoside, alpha-methyl-D-glucoside, and N-acetyl-D-glucosamine. These sugars were also capable of eluting [3H]glucosamine-labeled material from disrupted PR-A virus, which was bound to a Con A-sepharose affinity column. A major viral glycoprotein recovered from the column had the same mobility as gp85 in polyacrylamide gel electrophoresis and could be immunoprecipitated with anti-gp85 antiserum. These results suggest that the material reacting with Con A is present on the gp85 component of the viral glycoprotein. The diversity in the reactivity of the glycoproteins of transforming and nontransforming viruses with plant lectins is discussed.     

HSV neutralization by the microbicidal candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium.     

Impaired interleukin 1 and interleukin 2 production following in vitro abortive infection of murine spleen mononuclear cells by Semliki Forest virus

The effect of Semliki Forest virus (SFV) infection of murine spleen mononuclear cells was investigated in vitro. A small percentage of spleen macrophages expressed viral antigens, but no infectious virus particles were released, indicating an abortive-type infection. Wild-type SFV infected a higher percentage of macrophages than the attenuated, demyelinating mutant A7. The proliferation of spleen mononuclear cells under Con A stimulation was inhibited by the viral infection. The supernatant (SN) harvested from infected and Con A-stimulated spleen adherent cells could not stimulate thymocytes in an interleukin 1 (IL-1) assay and indomethacin treatment of infected cultures had no effect. The stimulatory effect of SN from noninfected cultures in the IL-1 assay was reduced when SN from infected cultures was added, suggesting the presence of an IL-1 inhibitor. Interleukin 2 (IL-2) production by splenocytes also decreased after viral infection, but exogenous IL-2 restored the response to Con A stimulation of infected spleen cells. This study demonstrates that abortive SFV infection of spleen macrophages has an immunosuppressive effect which may lead to an aberrant immune regulation.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

The in vitro suppression of spontaneous erythrocyte autoimmune responses with lymphocytes activated with concanavalin A.

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to Br-MRBC. Mitogenic concentrations of Con A prevented the development of both the PFC and TDTH responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and TDTH response against BrMRBC.     

Concanavalin A increases glyoxalase enzyme activities in polymorphonuclear leukocytes and lymphocytes

Glyoxalase I converts methylglyoxal and glutathione to S-lactoylglutathione and glyoxalase II converts this compound to D-lactic acid, regenerating glutathione in the process. A recent study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro whereas concanavalin A (Con A) has been shown to increase microtubule occurrence in polymorphonuclear leukocytes (PMN). The present report describes the dose-dependent activation by Con A of both glyoxalase I and II in PMN and lymphocytes. In nine experiments with PMN, Con A (100 microgram/ml) increased glyoxalase I and II activities by 19 +/- 8% and 12 +/- 10% (mean +/- S.D.). In 17 experiments with lymphocytes, activation of the two enzymes by 10 microgram/ml Con A was 30 +/- 14% and 28 +/- 8%. Changes occurred after a 1-min incubation with Con A and persisted for at least 60 min. Since both enzyme activities are increased it is not clear if S-lactoylglutathione levels are increased or decreased but presumably they change. The present findings are compatible with the hypothesis that Con A increases microtubule occurrence in PMN by affecting the glyoxalase enzymes. They also represent a newly described early biochemical change caused by Con A in lymphocytes.