In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03

The sulfated polysaccharide, p-KG03, purified from the marine microalga, Gyrodinium impudium, is a unique compound comprising homogenous galactose units conjugated to uronic acid and sulfated groups. Although previous studies showed that p-KG03 suppresses tumor cell growth and infection by encephalomyocarditis virus, its effect against enveloped virus infection and the biological mechanism of action have not been elucidated. In this report, the inhibitory activity of p-KG03 against influenza virus was examined and compared with that of other sulfated polysaccharides (fucoidan and pentosan polysulfate) and antiviral agents (oseltamivir phosphate, oseltamivir carboxylate, amantadine, and ribavirin). The results of a cytopathic effect reduction assay using MDCK cells demonstrated that p-KG03 exhibited the 50% effective concentration (EC(50)) values of 0.19-0.48 μg/ml against influenza type A virus infection (selectivity index >200) but not all influenza type B viruses. Mechanism studies showed that inhibition of influenza virus replication was maximized when p-KG03 was added during or within 6 h after viral infection, suggesting that mainly the viral adsorption and internalization steps are targeted by this compound. The results of influenza virus binding assay to p-KG03 and fluorescence microscopy indicate that the antiviral activity of p-KG03 is directly associated with its interaction with viral particles. The sulfated polysaccharide p-KG03 is a potent and specific influenza A viral entry inhibitor and may be a candidate for antiviral drug development.     

Inhibition of histamine-stimulated inositol phospholipid hydrolysis by agents which increase cyclic AMP levels in bovine tracheal smooth muscle.

1. The effect on histamine-stimulated [3H]-inositol phosphate accumulation of a range of agents which increase the accumulation, or mimic the actions, of cyclic AMP has been investigated in bovine tracheal smooth muscle. 2. Salbutamol (1 microM), forskolin (1 microM) and vasoactive intestinal peptide (VIP, 1 microM) inhibited the inositol phosphate response to 0.1 mM histamine and increased the accumulation of [3H]-cyclic AMP in [3H]-adenine-labelled slices of bovine tracheal smooth muscle. The effect on inositol phospholipid hydrolysis was mimicked by the membrane permeant analogues of cyclic AMP, dibutrylcyclic AMP (1 mM) and 8-bromo-cyclic AMP (1 mM). 3. In contrast to salbutamol, which was equally effective at producing the two effects, forskolin produced large increases in [3H]-cyclic AMP accumulation (EC50 = 1.2 microM) at much higher concentrations than those required for inhibition of histamine-stimulated [3H]-inositol phosphate accumulation (EC50 = 0.09 microM). However, significant increases in [3H]-cyclic AMP accumulation, of similar magnitude to those obtained with salbutamol and VIP, were observed over the concentration range appropriate for inhibition of the inositol phosphate response to histamine. 4. In the presence of histamine (0.1 mM), isobutylmethylxanthine (IBMX, 1 mM) and rolipram (0.1 mM) both significantly (P less than 0.05) elevated tissue [3H]-cyclic AMP levels. IBMX, rolipram and (to a lesser extent) SKF 94120 significantly (P less than 0.05) reduced histamine-stimulated [3H]-inositol phosphate accumulation by 81%, 68% and 20%, respectively. M&B 22948 was without a significant effect on either [3H]-cyclic AMP or histamine-induced [3H]-inositol phosphate accumulation. 5. Both rolipram and forskolin reduced the increase in incorporation of [3H]-inositol into membrane phospholipids which followed stimulation with histamine. However, a significant inhibition of [3H]-inositol phosphate accumulation could be demonstrated under conditions in which there was no change in the level of [3H]-inositol incorporation.     

In vitro uptake of gelatin nanoparticles by murine dendritic cells and their intracellular localisation.

The long term goal of this study is to develop an efficient nanoscopic vaccine delivery system, based on the biodegradable and natural polymer gelatin, to deliver therapeutic protein antigens along with adjuvants into dendritic cells (DCs). In this study, gelatin nanoparticles were tested for qualitative and quantitative uptake in murine DCs in vitro. A second aim of this study was to prove that the carrier system is able to deliver tetramethylrhodamine conjugated dextran (TMR-dextran), as a model drug into the DCs. The TMR-dextran was incorporated during the preparation of the gelatin nanoparticles. DCs were generated from murine bone marrow cells by an established ex vivo technique. Flow cytometry showed that 88% of the cells positive for the specific murine DC marker CD11c took up TMR-dextran loaded gelatin nanoparticles, whereas only 4% of the soluble form of TMR-dextran was taken up. Double color confocal laser scanning microscopy (CLSM) showed that gelatin nanoparticles were phagocytosed by DCs and the triple color CLSM showed that the TMR-dextran was localized mainly in lysosomes as expected, but partly also outside the lysosomes, presumably in the cytoplasm. An in vitro release study of TMR-dextran from gelatin nanoparticles demonstrated that there was hardly any release in phosphate buffered saline (PBS), but by trypsin-assisted degradation of gelatin nanoparticles resulted in the release of about 80% of the TMR-dextran from the particles. These results suggest that gelatin nanoparticles hold promise as a new biocompatible tool for vaccine delivery to DCs, with applications in cancer immunotherapy.     

In vitro effect of inosiplex on T lymphocytes. I. Influence on T cells with receptors for IgG (T gamma)

Inosiplex, a complex of inosine and 2-hydroxypropyldimethyl ammonium-4-(acetylamino)benzoate, 1:3 molar ratio, originally developed for antiviral use, is now under wider investigation because of its immunopotentiating properties. This compound can have some actions on T cells at various stages of differentiation, thus promoting an enhancement of their blastogenic responses to varied mitogenic agents (PHA, Con A, PWM, MLC, tetanus toxoid, and viral antigens). Our studies demonstrated that under the influence of inosiplex human peripheral blood T lymphocytes bearing Fc IgG receptors have an augmented receptor avidity for SRBC which result in an increased E active rosette formation, and that T cells preincubated with the drug at the appropriate concentrations express more Fc IgG receptors. Even though T gamma cells exert "in vitro" immunoregulatory properties, the increase in percentage of T gamma lymphocytes do not correlate with a potentiation of the Con A-induced suppressor activity of T cells. Moreover, the lymphocytes treated with the substance in the absence of Con A exert helper functions, increasing the mitogenic responses of the second culture PHA--treated lymphocytes. These data appear to suggest a pro-proliferative inosiplex-induced effect which could mask a concomitant suppressor cell induction.     

In vitro inhibition of human influenza A virus infection by fruit-juice concentrate of Japanese plum (Prunus mume SIEB. et ZUCC)

Using a plaque reduction assay, treatment of human influenza A viruses with the fruit-juice concentrate of Japanese plum (Prunus mume SIEB. et ZUCC) showed strong in vitro anti-influenza activity against human influenza A viruses before viral adsorption, but not after viral adsorption, with 50% inhibitory concentration (IC50) values against A/PR/8/34 (H1N1) virus, A/Aichi/2/68 (H3N2) virus and A/Memphis/1/71 (H3N2) virus of 6.35+/-0.17, 2.84+/-1.98 and 0.53+/-0.10 microg/ml, respectively. The plum-juice concentrate exhibited hemagglutination activity toward guinea pig erythrocytes. Its hemagglutination activity was inhibited by the monosaccharide N-acetylneuraminic acid and a sialoglycoprotein (fetuin), but not by the other tested monosaccharides (mannose, galactose, glucose and N-acetylglucosamine), suggesting the presence of a lectin-like molecule(s) in the Japanese plum-juice concentrate. Our findings suggest that the fruit-juice concentrate of Japanese plum may prevent and reduce infection with human influenza A virus, possibly via inhibition of viral hemagglutinin attachment to host cell surfaces by its lectin-like activity.     

Marijuana components suppress induction and cytolytic function of murine cytotoxic T cells in vitro and in vivo.

Killer lymphocytes play a major role in host defense against tumors and infectious diseases. Previously, we reported that delta-9-tetrahydrocannabinol (THC) and II-hydroxy-delta-9-tetrahydrocannabinol (II-hydroxy-THC) suppressed the cytolytic activity of cultured natural killer (NK) cells. Also, we showed that the drugs appeared to be affecting a stage in the killing process subsequent to the binding of the killer cell to the target cell. In the present report, we have extended these studies to an examination of the effect of cannabinoids on the activity of cytotoxic T lymphocytes (CTLs). The cytolytic activity of CTLs generated by cocultivation with either allospecific stimulators or TNP-modified-self stimulators were suppressed by both THC and II-hydroxy-THC treatment. Allospecific CTLs generated in vivo were also inhibited by an in vitro exposure to either THC or II-hydroxy-THC, and the sensitivity of these cells to drug effects appeared to be greater than the sensitivity of the in vitro generated CTLs. Suppression of cytolytic function by THC and II-hydroxy-THC was maximal after a 4-h drug treatment, suggesting that the drug effects were inducible and therefore required a finite period of time to develop maximally. As seen in previous studies involving NK cells, drug treatment of mature CTLs appears to have little effect on the binding capacity of these cells for the target. However, the maximal killing capacity of the cells and the frequency of CTLs were significantly reduced by drug treatment. In addition to suppressing the cytolytic activity of mature effector CTLs, we also show that drug treatment inhibits both the proliferation of lymphocytes responding to an allogeneic stimulus and the maturation of these lymphocytes to mature CTLs. Similarly, CTL activity developing in vivo could be inhibited by THC injection. These results suggest that CTLs are inhibited by cannabinoids by at least two mechanisms. First, the cytolytic activity of mature killers is suppressed at some point beyond the binding to the target cell. Second, the cannabinoids appear to suppress the normal development of these mature effector cells from less mature precursor cells.     

Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP.

1. In bovine aortic endothelial cells (BAEC), thrombin (1 mu ml-1), bradykinin (1-10 nM) and adenosine triphosphate (ATP) (0.3 microM-100 microM) each induced a biphasic elevation of cytosolic calcium ([Ca2+]i), consisting of an initial transient followed by a sustained plateau phase. 2. Pretreatment of BAEC with 4 beta-phorbol 12-myristate 13-acetate (PMA; 100 nM) reduced the magnitude of the initial transient elevation of [Ca2+]i, induced by thrombin (1 mu ml-1), low concentrations of bradykinin (1 nM) or ATP (0.3 microM, 3 microM), but not by higher concentrations of the latter two agonists. Addition of PMA (100 nM) during the plateau phase of the increase in [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (10 nM) or ATP (30 microM) resulted in a fall in [Ca2+]i. 3. The inhibitory effects of PMA (100 nM) were inhibited by staurosporine (100 nM) but not mimicked by the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD; 100 nM). Furthermore, staurosporine (100 nM) increased [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by thrombin or bradykinin. In contrast, staurosporine (100 nM) reduced [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by ATP (30 microM). 4. Pretreatment with forskolin (10 microM) had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (1 nM and 10 nM) or ATP (30 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of platelet cyclic GMP but not cyclic AMP suppression in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro.

1. Incubation of endothelial cells with platelets in the absence or the presence of PAF (10 nM) markedly increased platelet cyclic AMP levels, which were significantly decreased by indomethacin (3 microM). Co-incubation of endothelial cells and platelets with polymorphonuclear leukocytes (PMNs) did not change the platelet cyclic AMP levels. 2. Incubation of endothelial cells with platelets in the absence of PAF increased platelet cyclic GMP levels, which were increased 3.5 fold by PAF. These cyclic GMP levels were significantly decreased by NG-nitro-L-arginine (100 microM), and completely by methylene blue (10 microM). When endothelial cells and platelets were co-incubated with PMNs, the cyclic GMP level in the cell mixture was 42.5 and 65.3% lower than that in endothelial cells and platelets without and with PAF stimulation, respectively. 3. PAF induced platelet adhesion to endothelial cells only when PMNs were present. Methylene blue dose-dependently potentiated the PMN-dependent platelet adhesion induced by PAF, although it had no effect in the absence of PMNs. 4. Sodium nitroprusside and 8-bromo cyclic GMP but not dibutyryl cyclic AMP significantly, although partially, inhibited the platelet adhesion. Inhibition of cyclic GMP-specific phosphodiesterase by zaprinast slightly inhibited the PMN-induced platelet adhesion and potentiated the inhibitory effect of 8-bromo cyclic GMP, while these drugs markedly inhibited the adhesion of platelet aggregates induced by PMN sonicates. 5. These results suggest that the impairment by activated PMNs of EDRF-induced platelet cyclic GMP formation is involved in part in the mechanism of PMN-dependent platelet adhesion to endothelial cells induced by PAF in vitro. The precise mechanism still remains to be clarified.     

In vitro immunosuppressive potency of deflazacort, a new bone-sparing corticosteroid on T lymphocytes, NK and K cells.

The in vitro immunosuppressive effect of deflazacort, a new bone-sparing glucocorticoid, and its biologically active metabolite, 21-deacetyl-deflazacort, was examined on phytohaemagglutinin (PHA) stimulated human peripheral blood lymphocytes (PBL) as well as on natural killer (NK) and killer (K) cell activity. Deflazacort and the 21-deacetyl metabolite were as potent as prednisolone and hydrocortisone in suppressing PHA stimulated lymphocytes in a dose dependent way, but all were less potent than methylprednisolone. The physiological metabolites of hydrocortisone, dihydrocortisol and tetrahydrocortisol were without any immunosuppressive effects in vitro. Deflazacort, 21-deacetyl-deflazacort, and methylprednisolone suppressed NK cell activity, while hydrocortisone and aldosterone had no effect on NK cells. K cell activity was resistent to all tested glucocorticoids except methylprednisolone at high concentrations. The present results indicate that deflazacort and 21-deacetyl-deflazacort are potent immunosuppressive drugs in vitro and, on a molar basis, equally as potent as prednisolone.     

Effects of lidocaine on the function of immunocompetent cells. I. In vitro exposure of mouse spleen lymphocytes and peritoneal macrophages

In vitro exposure of mouse lymphocytes and macrophages for 24 h to noncytotoxic doses of lidocaine (10(-4) to 10(-6)M) resulted in inhibition of random macrophage motility and in an interference with the production of macrophage migration inhibitory factor or with its interaction with the cell surface. The effects of lidocaine, membrane-stabilizing local anesthetic, were related to its concentration in the medium and to its ability to interact with the cell surface and cause changes in the ionic configuration of the plasma membrane. The drug conferred permanent changes on the surface of lymphocytes at all concentrations tested, but changes in the surface of macrophages induced by 10(-5) and 10(-6)M lidocaine were reversible. The presence of noncytotoxic doses of lidocaine in the cellular environment resulted in significant changes in cellular functions that appeared to be related to the ability of the drug to interact with cell membranes in a manner determined by the specific properties of the cell.     

Enhancement by beraprost sodium, a stable analogue of prostacyclin, in thrombomodulin expression on membrane surface of cultured vascular endothelial cells via increase in cyclic AMP level.

Prostacyclin and beraprost sodium (beraprost), a stable analogue of prostacyclin, increased cyclic AMP (cAMP) levels of cultured human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. The elevation of cAMP by beraprost was sustained longer than that by prostacyclin. The expression of thrombomodulin (TM) on membrane surface of HUVEC was enhanced by beraprost and prostacyclin, and the persistence of the increase in TM expression by beraprost was greater than prostacyclin. Dibutyryl cAMP (db-cAMP) mimicked the effects of beraprost and 3-isobutyl-1-methylxanthine enhanced the effects. Beraprost, prostacyclin and db-cAMP also effectively blocked the interleukin-1- and tumor necrosis factor-induced depression of TM expression substantially. These results suggest that TM expression is positively regulated by cAMP in HUVEC, and that beraprost may be potentially effective for reducing thrombotic events through the mechanism which initiates the stimulation of cAMP/TM system in vascular endothelial cells.     

Alteration of DNA topoisomerase II activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: a target for potential therapeutic agents

Infection of H9 cells with human immunodeficiency virus type 1 (HIV-1) was found to decrease the phosphorylation of DNA topoisomerase II during the initial phase of infection. Simultaneously, with a later overshoot of phosphorylation and the subsequent activation of DNA topoisomerase II, the production of HIV-1 started. Applying three new protein kinase C inhibitors from the class of O-alkylglycerophospholipids we demonstrated that inhibition of protein kinase C-mediated phosphorylation of DNA topoisomerase II resulted in an inhibition of HIV-1 production. Based on the differential effect of the two protein kinase C activators, phorbol ester and bryostatin, we conclude that phosphorylation of DNA topoisomerase II is mediated by the form alpha and gamma of protein kinase C. These data suggest that agents which inhibit these two forms of protein kinase C are also potential candidates for an anti-HIV therapy.     

Bupleurum kaoi inhibits Coxsackie B virus type 1 infection of CCFS-1 cells by induction of type I interferons expression.

Coxsackie B virus type 1 (CVB1) infection is known to cause high morbidity and mortality in children, however, there is no effective drug for treating this disease. The present study aimed to examine the antiviral activity of Bupleurum kaoi (BK), a popular herbal drug for treating viral and bacterial infections, against CVB1 infection and its mechanisms of action. Our data showed that BK neutralized the CVB1-induced cytopathic effect in human neonatal foreskin fibroblast cell line (CCFS-1/KMC), with IC50 and EC50 values around 12.38 microg/ml and 50.93 microg/ml, respectively. Its CC50 and SI values were 883.56 microg/ml and 17.34, respectively. These results suggest that BK possessed anti-CVB1 activity, and showed no effect on CCFS-1 cell viability and growth at concentration 250 microg/ml. The time-of-addition studies showed that BK (50, 100 and 200 microg/ml) added at various time of preinfection (-1 to -3 h), coinfection (0 h) and postinfection (1-3 h) could inhibit CVB1 infection. Interestingly, BK also showed an inhibition on viral replication through the induction of IFN-alpha/beta expression. In conclusion, BK possessed antiviral activity against CVB1 infection. It interfered the early stage of viral replication and viral replication after infection through the induction of type I interferon expression.     

In vitro inhibition of enterobacteria-reactive CD4+CD25- T cells and suppression of immunoinflammatory colitis in mice by the novel immunomodulatory agent VGX-1027

VGX-1027 is an isozaxoline compound that has recently been found to primarily target the function of murine macrophages but not of T cells, inhibiting secretion of tumor necrosis factor (TNF)-alpha in response to different Toll-like receptor agonists in vitro and in vivo. The well-defined role of innate immunity in inflammatory bowel diseases prompted us to consider the use of VGX-1027 in these diseases leading us to in vitro and in vivo test the drug in related experimental conditions. These consist, respectively, of the proliferation assay of CD4+CD25- T cells to enterobacteria, and the acute inflammatory colitis induced in mice by intracolonic challenge with dinitrobenzene sulfonic acid. The data from the two sets of experiments revealed that VGX-1027 inhibited both proliferation of enterobacterial antigen-reactive CD4+CD25- T cells in vitro and the development of clinical and histological signs of colitis in vivo. The beneficial effect in this model was associated with reduced colonic production of proinflammatory cytokines such as interleukin (IL)-1beta, TNF-alpha, IL-12p70 and interferon (IFN)-gamma and lower content of nuclear factor (NF)-kappaB (p65). These findings seem to warrant investigations of VGX-1027 for use in human.     

Modulation by cyclosporin A of murine natural resistance against herpes simplex virus infection. II. Influence on the HSV-induced natural killer cell responses, macrophage activities and interferon levels

Cyclosporin A (CyA) interfered locally at the site of injection with several resistance functions which are of potential importance in experimental herpes simplex virus (HSV) infections of mice. HSV-induced stimulation of macrophage phagocytosis was reduced by CyA when the mice were infected 5 days before the assay. The in vitro replication of the virus in macrophages, however, was enhanced. Natural killer (NK) cell response were severely impaired. To some extent this could be attribute to the induction of suppressive macrophages by the drug treatment. Interferon levels induced by HSV were not diminished but rather enhanced in some experiments. Inhibitory effects ceased after termination of CyA treatment and could be prevented by presensitization of the mice with attenuated HSV type 2.     

Enhancement of transformed foci and induction of prostaglandins in Balb/c 3T3 cells by palytoxin: in vitro model reproduces carcinogenic responses in animal models regarding the inhibitory effect of indomethacin and reversal of indomethacin's effect by exogenous prostaglandins.

Cell transforming activity of palytoxin, a non-TPA type tumor-promoter, was investigated with the two-stage transformation assay using Balb/c 3T3 cells. Palytoxin showed potent promoting activity; treatment at 1.9 pM or more increased the number of transformed foci after initiation by 3-methylcholanthrene (MCA). Determination of prostaglandin (PG) E2 and PGF(2alpha) concentrations in the culture medium revealed that palytoxin (1.9-3.7 pM for 24 h) stimulated the production of PG in Balb/c 3T3 cells (the concentration reached 3-4 microM), and treatment with PGE2 or PGF(2alpha) itself increased the number of transformed foci of Balb/c 3T3 cells after initiation by MCA. Neither palytoxin nor PGs showed initiating activity. Indomethacin suppressed the promoting activity of palytoxin, but not that of PGE2 and PGF(2alpha). Interestingly, concomitant treatment with PGE2 or PGF(2alpha) in addition to indomethacin markedly reversed the suppressive effect of indomethacin. These findings indicated that the in vitro transformation model could reproduce experiments that have been performed in animal models regarding the inhibitory effect of indomethacin on carcinogenic responses and reversal of indomethacin's effect by exogenous prostaglandin and, therefore, may provide insight into molecular modes of action of palytoxin. In the present study, palytoxin also induced prostaglandin synthesis, and therefore, the Balb/c 3T3 cell model should provide insight into the molecular mechanism by which palytoxin regulates prostaglandin biosynthesis.