Levamisole and bovine immunity: in vitro and in vivo effects on immune responses to herpesvirus immunization

Levamisole was shown to enhance in vitro blastogenic responses of bovine lymphocytes to nonspecific mitogens (phytohemagglutinin and pokeweed mitogen) as well as to infectious bovine rhinotracheitis virus and purified protein derivative. Greatest enhancement was observed at suboptimal concentrations of viral antigen. In addition to enhancing lymphocyte reactivity levamisole also affected macrophage activity as determined by increased Fc receptor activity and [3H]glucosamine incorporation. Levamisole (5-50 micrograms/mL) enhanced type II immune (or gamma) interferon production by macrophage-lymphocyte cultures. Administration of levamisole and attenuated infectious bovine rhinotracheitis vaccine virus in vivo did not elevate cellular or humoral responses.     

Identification and initial characterization of concanavalin A- and interferon-induced human suppressor factors: evidence for a human equivalent of murine soluble immune response suppressor (SIRS)

Human suppressor T cells activated by leukocyte interferon have properties similar to murine suppressor cells activated by interferon or by concanavalin A. Murine suppressor cells release a soluble mediator, soluble immune response suppressor (SIRS), which accounts, at least in part, for suppressive activity in murine systems. To compare and contrast murine and human suppressor pathways, we evaluated the suppression of human polyclonal plaque-forming cell responses by concanavalin A, by leukocyte interferon, and by immune interferon, or by suppressor cells activated by these agents. In each instance, suppressive activity was prevented by levamisole, ascorbic acid, catalase, or 2-mercaptoethanol, agents known to interfere with murine SIRS activity. Furthermore, concanavalin A, immune interferon, and leukocyte interferon induced T lymphocytes to release 110,000 to 150,000 m.w. proteins which suppressed responses only when added early in the culture period. As with murine SIRS, suppression by each of these human factors was inhibited by 2-mercaptoethanol, ascorbic acid, catalase, or levamisole. The reaction of human suppressor factors with H2O2 (10(-6) M) activated suppressor factors so that they suppress responses when added late in the culture period. Human suppressor factors were protease- and acid (pH 2)-sensitive. The similarities between these human suppressor factors and murine SIRS show the existence of a human SIRS pathway.     

The induction of interferon by levamisole in mice.

Viral inhibitor(s) with the properties of interferon (IF) was found in the sera of DDI mice injected intraperitoneally with 5 to 10 mg/kg of levamisole. A significant level of IF activity appeared by 20 hr and reached a peak by 24 hr after the injection. The induction was abrogated when the mice were pretreated with either whole-body X irradiation of more than 500 R or 2.5 mg of hydrocortisone acetate but was not affected by macrophage-specific depressors such as carrageenan and trypan blue. Also, no induction was detected in thymus-defective nude mice. These results suggest that thymus-derived lymphocytes in the mouse may be required for IF induction by levamisole.     

Activated macrophages mediate interferon-independent inhibition of herpes simplex virus

Activated macrophages exhibit extrinsic antiviral activity (inhibition of virus replication in other cells) which may involve mechanisms similar to macrophage antitumor activity or macrophage-mediated immunosuppression. Peritoneal macrophages elicited in mice by Corynebacterium parvum vaccine suppressed the growth of herpes simplex virus (HSV) in infected cells by an interferon-independent mechanism. This was demonstrated by expression of activity against HSV-infected xenogeneic (Vero) cells. Culture supernatant fluids also did not mediate antiviral activity, and did not contain detectable levels of interferon (< 3 IU/ml). Moreover, antiviral activity was not affected by the presence of anti-mouse interferon IgG. Antiviral activity was expressed at 12–16 hr after infection, at the end of the first cycle of virus replication. Cell contact was required for optimal activity. No enhanced adsorption or phagocytosis of HSV by C. parvum macrophages could be detected nor was macrophage cytotoxicity responsible for the activity. Cytotoxicity (⁵¹Cr release) by macrophages for virus infected cells was low (< 6% specific cytotoxicity), and was not significantly higher with C. parvum macrophages than with resident macrophage controls. Although C. parvum macrophages were not cytotoxic at the macrophage-host cell ratio employed, they did significantly inhibit uptake of [³H]leucine by the host Vero cells. This suggests that inhibition of host cell metabolism by the macrophage, similar to macrophage immunosuppression, may be responsible for the antiviral activity in this system.     

Staphylococcal Hyaluronate Lyase: Purification and Characterization Studies

Staphylococcal hyaluronate lyase (hyaluronidase) derived from a pathogenic strain of staphylococcus was purified by means of salt fractionation with ammonium sulfate and gel filtration through Sephadex G-100. Most of the enzyme activity from concentrated culture supernatant fluids of staphylococci was obtained in a fraction precipitated by 90 to 100% saturation with ammonium sulfate. A small amount of enzyme was also precipitated by 80 to 90% saturation with the salt. The hyaluronidase-rich fractions did not contain other staphylococcal enzymes, such as coagulase, protease, lipase, and staphylokinase. These enzymes were present in the original concentrates. Molecular sieving chromatography of the partially purified enzyme by filtration through Sephadex G-100 resulted in a further increase in specific enzyme activity. However, more than one active peak was obtained after gel filtration, thus suggesting that there may be more than one molecular form of the enzyme. Immunodiffusion in agar gel of the chromatographically purified enzyme fraction, with immune serum from rabbits injected with concentrated staphylococcal culture supernatant fluids, indicated that there was one major antigen. A similar antigen, giving reactions of identity with the purified material, was present in the original culture supernatant fluid.     

Stabilization of the colchicine-binding activity of tubulin by glutathione and by dietary selenium-deficiency

The lability of tubulin in supernatant fluids from rat brain was studied by measuring the loss of colchicine-binding activity. During incubation at 37 degrees C for 1 hr approximately 50% of the binding activity of rat brain supernatant fluids was lost. This loss was reduced by mercaptoethanol or reduced glutathione. Anaerobic conditions did not affect loss of binding, while oxygen markedly reduced it, especially in the presence of reduced glutathione. Supernatant fluids prepared from brains of animals deficient in selenium showed a lower loss of binding than those from selenium-supplemented controls.     

复合干扰素突变体在毕赤酵母中的表达、纯化及活性分析

根据毕赤酵母密码子偏性合成了复合干扰素突变体基因 ,克隆至分泌型酵母表达载体pMEX9K ,将重组载体pMEX CIFNm用SacⅠ线性化后 ,转化毕赤酵母GS115 .转化子经诱导后 ,培养上清有抗病毒活性的蛋白产生 .经过离子交换 ,疏水层析 ,凝胶过滤三步层析纯化 ,得到了纯度大于95 %的重组复合干扰素突变体 ,经N端氨基酸序列分析表明 ,该蛋白N端序列与理论值一致 ,质谱测定分子量为 19 3kD ,与理论值一致 .用细胞病变抑制法测定其活性 ,并结合Lowry法蛋白定量计算其比活性为 6× 10 8IU mg ,与复合干扰素的比活相当 .     

In vitro stimulation of natural killer (NK) cells by soluble factor(s) generated in antigen-stimulated rhesus monkey peripheral blood lymphocyte cultures.

Peripheral blood lymphocytes from rhesus monkeys (Macaca mulatta) sensitized to keyhole limpet hemocyanin (KLH), when stimulated in vitro with KLH, developed natural killer (NK) cell activity that was assayed with Rous Sarcoma virus-transformed marmoset fibroblasts as targets in a 4-hr 51Cr-release assay. The supernatant fluids from 24- to 25-hr KLH-activated cultures were capable of stimulating NK development in nonsensitive lymphocyte cultures. The effector cells were neither macrophages nor B cells (plastic and nylon-wool nonadherent) and did not form E-rosettes with neuraminidase-treated sheep red blood cells. Cultures depleted of EA-rosetting cells, i.e., Fc-receptor-bearing lymphocytes, were incapable of generating NK activity when stimulated in vitro. Kinetic studies showed that peak DNA synthesis, as measured by 3H-T incorporation, preceded maximum cytotoxicity. Elimination of dividing cells by 5-bromo-2'deoxyuridine (BrdU) and light treatment during the interval from day 1 to day 4 inhibited the development of cytotoxicity on day 7. Cell replication was required for the induction of NK cells with KLH as well as with antigen-activated culture supernatant fluids. When cultures were left unstimulated for 4 days, NK activity could not be developed subsequently either by adding antigen, mitogen (PHA), or supernatant fluids from activated cultures.     

Levamisole regulates the proliferation of murine liver T cells through Kupffer-cell-derived cytokines

 We have previously shown that levamisole increases the cytotoxic, cytostatic, and proliferative activity of murine nonparenchymal liver cells (NPC) in vitro. We have also shown that the nonadherent subpopulation of NPC, which are composed predominantly of T lymphocytes, is very responsive to this agent when administered to mice. Kupffer cells or immigrant macrophages are also responsive to levamisole but to a lesser extent. These findings prompted us to investigate changes in cytokine production by NPC following-treatment of mice with levamisole (25 mg/kg, i.p.), which may help explain the observed alterations in the immune functions of these cells. We found that levamisole treatment of mice causes a threefold increase in production of interferon (IFN) α/β by adherent NPC (more than 80% – 90% Kupffer cells) in vitro. When IFN α/β was added to cultured cells, it decreased the proliferative capacity of liver T cells in a dose-dependent manner. In contrast, the addition of anti-IFNα/β was shown to augment levamisole-induced proliferation of unfractionated NPC and Kupffer cells. NPC production of interleukin 1 (IL-1) and interleukin-6 (IL-6) in vitro was also increased threefold following treatment of mice with levamisole. IL-6 added in vitro to cells significantly augmented levamisole-induced proliferation of liver T cells while anti-IL-6 reduced proliferative activity to control levels. These findings suggested that IFNα/β, IL-6, and IL-1 play important regulatory roles in controlling the proliferative response of murine liver-associated T lymphocytes to levamisole. Finally, the proliferation of bone marrow cells was increased in mice given 5-fluorouracil (5FU). On the other hand, the proliferation of NPC was dramatically suppressed when 5FU was administered. However, the proliferation of these cells was restored when levamisole was given after 5FU. Received: 27 November 1995 / Accepted: 16 October 1996     

Properties and subcellular localization of adenosine diphosphatase in arterial smooth muscle cells in culture

The properties and subcellular localization of adenosine diphosphatase (ADPase) activity in smooth muscle cells cultured from pig aortas have been investigated. The pH optimum of ADPase activity was 7.3 and the apparent K_m for ADP was 10.3 μM. ADPase activity was inhibited completely by EDTA and was restored by the addition of divalent cations. The enzyme activity was not inhibited by 2-glycerophosphate, a substrate for non-specific phosphatases, nor by levamisole, a specific inhibitor of alkaline phosphatase. Smooth muscle cells were homogenized and a post-nuclear supernatant was applied to a sucrose density gradient in a Beaufay automatic zonal rotor. The distribution of ADPase activity in the density gradient was similar to that of 5′-nucleotidase activity, a marker enzyme for the plasma membrane, and distinct from the distributions of the marker enzymes for the other organelles. When the cells were homogenized in the presence of digitonin, an agent which binds to cholesterol and increases the equilibrium density of the plasma membrane, the modal equilibrium densities of ADPase activity and of 5′-nucleotidase activity were increased to similar extents, thus confirming the plasma membrane localization of ADPase activity.     

Production of monoclonal antibodies to Shigella. Enzyme-linked immunosorbent assay for screening hybridoma antibodies with intact bacteria

A simple and rapid enzyme-linked immunosorbent assay (ELISA)-type assay has been developed to screen hybridoma supernatant fluids with whole viable or killed bacteria as the antigen. The optimum concentration of acetone-killed and dried cell antigen for coating was 25–100 μg/ml. Screening of hybridoma supernatant fluids against whole cells, both with and without fixation, was assessed and both were equally sensitive. The data indicate that bacteria] fixation is detrimental in ELISA probably because of loss of antigenic structure. A highly specific monoclonal antibody (laM3) was produced against Shigella flexneri la and was employed to optimize the assay procedure.     

Production of soluble suppressor factors by herpes simplex virus-stimulated splenocytes from herpes simplex virus-immune mice.

Indirect evidence indicates that herpes simplex virus (HSV)-specific cytotoxic-T-lymphocyte induction is regulated by suppressor cells. To search for such suppressor effects, supernatant fluids from splenocyte cultures from normal and HSV-immune mice cultured either with or without viral stimulation were tested for their ability to inhibit HSV-specific cytotoxic-T-lymphocyte induction. Only the supernatant fluid from the HSV-stimulated, HSV-immune cultures contained a suppressor activity (HSV-SF). HSV-SF was produced by nylon-wool-purified Thy 1+ cells. HSV-SF was detectable after 3 days of culture and would only suppress cytotoxic-T-lymphocyte induction if HSV-SF was added within 24 h of initiation of the test cultures. HSV-SF was neither dialyzable nor heat stable. Molecular sieve chromatography of HSV-SF yielded multiple peaks of suppressor activity. Although most of these peaks exhibited nonspecific suppressor activity, the suppression mediated by the 90,000 to 150,000-molecular-weight fractions was antigen specific and genetically restricted. These results provide direct evidence for the regulation of HSV-cytotoxic-T-lymphocyte induction by a novel suppressor factor.     

Bleomycin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity

We determined whether human lung fibroblasts might release chemotactic activity for neutrophils (NCA) and monocytes (MCA) in response to bleomycin. The human lung fibroblasts supernatant fluids were evaluated for chemotactic activity by a blind well chamber technique. Human lung fibroblasts released NCA and MCA in a dose- and time-dependent manner in response to bleomycin. Checkerboard analysis of supernatant fluids revealed that both NCA and MCA were chemotactic. Partial characterization revealed that NCA was partly heat labile, trypsin sensitive, and predominantly ethyl acetate extractable. In contrast, MCA was partly trypsin sensitive and ethyl acetate extractable. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by leukotriene B4 receptor antagonist and anti-IL-8 and G-CSF Abs. MCA was attenuated by leukotriene B4 receptor antagonist, and monocyte chemoattractant protein-1, GM-CSF, and TGF-beta Abs. Leukotriene B4 receptor antagonist and these Abs inhibited the corresponding m.w. chemotactic activity separated by column chromatography. The concentrations of IL-8, G-CSF, monocyte chemoattractant protein-1, GM-CSF, and TGF-beta in the supernatant fluids significantly increased in response to bleomycin. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to bleomycin.     

PHA激活的脐血T细胞条件培养液对人骨髓CFU—C的抑制

Cord blood T cells were enriched by nylon wool colomn, and effects of PHA-stimulated T cell supernatant collected from 18 h to 7 days on the proliferation of CFU-c were studied. The results showed that the supernatant collected at 18 h (PHA-TCM) could significantly inhibit the growth of CFU-c and the inhibition was PHA-TCM dose dependent, suggesting there is a CFU-c inhibitory activity in PHA-TCM. Kinetic studies demonstrated that the activity was decreased in the supernatant collected at 48 h and disappeared at 7 days. On the other hand, unstimulated T cell supernatant and PHA alone had no inhibitory effect on CFU-c growth. Indomethacin did not affect the production of the inhibitory activity and no interferon activity could be detected in PHA-TCM. These suggested that the inhibition was mediated by a non-interferon, non-prostaglandin suppressor. Further studies revealed that the suppressor was a protein stable at 56 degrees C and lost in pH 2 and pH 11 for 3 h, its molecular weight was large than 10,000 dolton.     

Inhibition of aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus

Six isolates of Bacillus pumilus were tested for their ability to inhibit aflatoxin production of Aspergillus parasiticus NRRL 2999 in yeast extract sucrose (YES) broth. Aflatoxin production was inhibited in both simultaneous and deferred antagonism assays, suggesting that the inhibitory activity was due to extracellular metabolite(s) produced in cell-free supernatant fluids of cultured broth. The inhibition was not due to organic acids or hydrogen peroxide produced by B. pumilus since the inhibitory activity was not lost after pH adjustment or treatment of supernatant fluids with catalase. A range of media tested for the production of inhibitory metabolite(s) in supernatant fluids showed that all media supported bacterial growth and production of the metabolite(s). The metabolite(s) were produced over a wide range of temperature (25 to 37°C) and pH (4 to 9) of growth of B. pumilus. They were stable over a wide range of pH (4 to 10) and were not inactivated after autoclaving at 121°C for 30 minutes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.     

Biochemical relationship between murine immune interferon and a killer cell helper factor.

Concanavalin A and alloantigen-stimulated mouse spleen cells release into the supernatant at least two distinct antigen-nonspecific factors that enhance the generation of cytotoxic cells in vitro. As previously reported, analysis of the supernatant by ammonium sulfate fractionation, gel filtration, hydroxylapatite chromatography, and hydrophobic chromatographic showed one of the two killer cell helper factors (KHF) to be associated with thymocyte mitogenic factor. This report demonstrates the second KHF to be separable from thymocyte mitogenic factor but inseparable from type II (immune) interferon. In addition, this KHF exhibits the same sensitivity to exposure to pH-2 buffer as does immune interferon. The KHF activity of an unfractionated supernatant, which is greater than of either the TMF-associated or interferon-associated KHF alone, is the result of the additive activities of the two independently acting helper factors.