Effect of poly-L-lysine and neuraminidase on the infectivity of Trypanosoma cruzi in cultured HeLa cells

The percentage of parasitisation and index of adherence of Trypanosoma cruzi has been studied when host HeLa cells or metacyclic forms were pretreated with neuraminidase or with poly-L-lysine. The percentage of parasitisation was significatively reduced (P less than or equal to 0.001) when cells were pretreated with poly-L-lysine while pretreatment with neuraminidase caused no apparent effects. On the other hand, the adherence of the metacyclic forms pretreated with poly-L-lysine or neuraminidase was significantly higher than that of the control group.     

Effect of human recombinant interferon on cytotoxic activity of natural killer (NK) cells and monocytes

Studies have been performed on the in vitro immunologic effects of homogeneous recombinant human leukocyte interferon, IFLrA. Large granular lymphocytes, enriched for natural killer (NK) cell activity, were pretreated wtih IFLrA or natural interferon preparations and then tested for augmentation of NK activity and of antibody-dependent cell-mediated cytoxicity (ADCC). Monocytes were tested for cytolytic and cytostatic activity in 48–72 hr radioisotopic assays performed in the presence or absence of interferon. Treatment with IFLrA caused significant augmentation of NK, ADCC, and monocyte-mediated cytotoxic activities. Even 10 units of IFLrA induced augmentation of NK activity, and 100 units or more boosted monocyte-mediated activity. The effects in each of these assays were species-specific, with no detectable effects on the activity of mouse effector cells. These results indicate that homogeneous recombinant interferon has potent in vitro immunomodulating effects and thus provide a basis for carefully examining the in vivo effects of this protein on host defenses in forthcoming clinical trials with cancer patients.     

Influenza virus-induced interferon production in mouse spleen cell culture: T cells as the main producer.

Interferon production by spleen cells from unimmunized C3H mice challenged in vitro with influenza virus AO/PR8 was investigated. Glass-nonadherent cells (lymphocytes) produced significant levels of interferon, although cocultivation of glass-adherent macrophages was needed for optimal production. Treatment of the cells with antithymocyte serum and complement markedly reduced the interferon production. When glass-nonadherent cells were fractionated on a nylon wool column, the T-cell-enriched fraction consistently produced more interferon than the B-cell-enriched fraction. It is concluded that T cells are an important producer of interferon in spleen cell cultures from normal mice upon challenge with influenza virus, although non-T cells (macrophages and B cells) also may produce interferon under suitable conditions.     

Modulation of the human in vitro antibody response by human leukocyte interferon preparations

The ability of human leukocyte Interferon to modulate the plaque-forming-cell response of human peripheral blood leukocytes to horse red blood cells was examined. Human peripheral blood mononuclear cells were cultured in vitro with the addition of varying doses of human leukocyte interferon 24 hr prior to, simultaneously with, and 24 hr after sensitization of the cultures with horse red blood cells. Plaque-forming-cell responses were measured 5 days after sensitization with antigen using poly-L-lysine-coupled horse red blood cell monolayers. When human leukocyte interferon preparations were added 24 hr prior to sensitization with antigen, a significant enhancement of the plaque-forming-cell response was observed. When the interferon was added simultaneously with antigen, the plaque-forming-cell response was significantly suppressed. Therefore, human leukocyte interferon appears to have a time-dependent immunomodulatory activity. The kinetics of immunomodulation appear to be different from those of previously described mouse models.     

Effect of nucleosides on interferon production and development of antiviral state induced by poly I.poly C.

Effect of nucleosides both on induction of antiviral state in chick embryo cells (CEC) or rabbit kidney cells (RK13) and on interferon production in RK13 or mouse fibroblast cells (L cells) by polyriboinosinic-polyribocytidylic acid (poly I.poly C) was studied. Addition of inosine or a fifty-fifty mixture of inosine and uridine at a final concentration of 0.1 mM to 10 mM to a growth medium enhanced development of antiviral state in CEC. The nucleoside effect was also observed in RK13 at 0.1 mM but not at a concentration higher than 1 mM. Interferon production in RK13 by superinduction (sequential treatment with metabolic inhibitors after exposure to poly I.poly C) was enhanced 1.5- to 4.0-fold by addition of the nucleoside mixture to the growth medium. When RK13 was pretreated with 10 units per ml of interferon and then superinduced by inhibitors, the enhancing effect of nucleosides on interferon production was not observed. Interferon production in L cells was potentiated a little by addition of 1 mM of the nucleoside mixture to the growth medium. The effect of nucleoside was not observed when the nucleosides were added after exposure to poly I.poly C. The nucleoside effect may be applicable for production of high titered interferon.     

Mouse interferon messenger RNA: characterization of the Xenopus laevis oocyte translation product

Mouse interferon messenger RNA was isolated from Newcastle disease virus-induced mouse Lpa cells and then translated in Xenopus laevis oocytes. The resulting oocyte homogenate containing translated interferon activity was unstable to treatment with 5 m urea and to repeated freeze/thaw cycles, and it was 1% cross-reactive on human cells, as was native mouse interferon. Both native mouse interferon and the mouse interferon produced by the translation of mouse interferon mRNA behaved almost similarly on CPG, poly(U)-Sepharose, and anti-mouse interferon antibody columns. When the oocyte-translated product was partially purified and analyzed on sodium dodecyl sulfate-polyacrylamide gels, it migrated as a major single band of activity at 21–22,000 daltons with a trailing edge at 22–30,000 daltons. Only minor activity was detected in the region of 35–40,000 daltons where the vast majority of the native mouse interferon migrated. Thus, the oocyte-translated mouse interferon product comigrated largely with the minor species of native mouse interferon with a little activity which corresponds with the larger molecular weight species of native mouse interferon.     

Interferon blocks interleukin 1-induced prostaglandin release from human peripheral monocytes

We have studied the short-term effects of interleukin 1, lipopolysaccharide, and interferon on prostaglandin release from freshly isolated human peripheral monocytes. When the cells were pretreated for 8 to 9 hr with either E. coli lipopolysaccharide or recombinant interleukin 1 (beta), prostaglandin release increased. Inclusion of recombinant IFN-alpha or IFN-gamma during the pretreatment phase blocked subsequent prostaglandin release. Interferons were effective at concentrations in the range of 1 to 10 antiviral units/ml, and the inhibition was manifested within several hours after exposure to the lymphokine. Similar trends were observed by measuring thromboxane release. These data suggest antagonistic roles for interleukin 1 and interferon in the regulation of eicosanoid release from monocytes.     

Fermentation of Cellulosic Substrates in Batch and Continuous Culture by Clostridium thermocellum

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was compared in batch and continuous cultures. Maximum specific growth rates per hour obtained on cellulosic substrates were 0.1 in batch culture and >0.13 in continuous culture. Cell yields (grams of cells per gram of substrate) in batch culture were 0.17 for pretreated wood and 0.15 for Avicel. Ethanol and acetate were the main products observed under all conditions. Ethanol:acetate ratios (in grams) were approximately 1.8:1 in batch culture and generally slightly less than 1:1 in continuous culture. Utilization of cellulosic substrates was essentially complete in batch culture. A prolonged lag phase was initially observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuous culture with ~5 g of glucose equivalent per liter in the feed, substrate conversion relative to theoretical ranged from 0.86 at a dilution rate (D) of 0.05/h to 0.48 at a D of 0.167/h for Avicel and from 0.75 at a D of 0.05/h to 0.43 at a D of 0.11/h for pretreated wood. At feed concentrations of <4.5 g of glucose equivalent per liter, conversion of pretreated wood was 80 to 90% at D = 0.083/h. Lower conversion was obtained at higher feed substrate concentrations, consistent with a limiting factor other than cellulose. Free Avicelase activities of 12 to 84 mU/ml were observed, with activity increasing in this order: batch cellobiose, batch pretreated wood < batch Avicel, continuous pretreated wood < continuous Avicel. Free cellulase activity was higher at increasing extents of substrate utilization for both pretreated wood and Avicel under all conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity, are essentially the same for pretreated mixed hardwood and Avicel under a variety of conditions. Hydrolysis yields obtained with C. thermocellum cellulase acting either in vitro or in vivo were comparable to those previously reported for Trichoderma reesei on the same substrates.     

The Mode of Production of Endotoxin-Induced Interferon in Rabbit Tissue Cells

In vitro production of endotoxin-induced interferon in rabbit tissue cell cultures could be enhanced by pretreatment with interferon. The enhancible state developed from the first hr of incubation at 37 C and a maximal priming effect was attained at 6 hr of incubation. Yields of interferon from unprimed cultures were usually 20–200 units/ml. In contrast, the primed cultures constantly yielded 1,000–2,500 units/ml of interferon. The pretreatment with interferon seemed to cause an earlier appearance of detectable interferon and the primed cells became more sensitive to endotoxin. It turned out that 10–30 units/ml of rabbit interferon were enough to develop the maximal priming. Even when cells were pretreated with higher doses of rabbit interferon such as 1.0 × 10⁴–1.0 × 10⁵ units/ml, the same level of priming effect was always observed without diminution. Various types of homologous (rabbit) and heterologous (human and mouse) interferon preparations showed similar dose-dependent enhancement of interferon production in proportion to the antiviral titers of these preparations as tested with RK-13 cells of rabbit origin.     

The effect of interferon on lymphocyte-mediated effector cell functions: selective enhancement of natural killer cells.

The effect of human interferons on different types of lymphocyte-mediated killer assays was explored. Killing by T cells generated through mixed lymphocyte cultures as well as antibody-dependent lymphocyte-mediated cytotoxicity was not influenced by the addition of interferon. Enhancement of cytolysis produced by natural killer cells was observed when interferon was added during the assay, but enhancement could also be induced if the effector cells were pretreated with interferon for 2 hr prior to the lytic reaction. Killing of a cell line susceptible to natural killing was increased and a cell line which is normally relatively resistant to this type of killing became a susceptible target.     

Interferon activates DNA repair genes in UV-irradiated human cells]

Unscheduled DNA synthesis in human fibroblasts pretreated with natural and recombinant interferons was studied by scintillated radiometry. UDS was increased in fibroblasts pretreated 7 days before UV-irradiation. Newly synthesized proteins induced in cells after interferon treatment were compared with heat shock proteins.     

Influence of low doses of an oxazaphosphorine on natural killer activity of human lymphocytes

Summary The influence of cis-4-sulfoethylthio-cyclophosphamide (mafosfamide) on natural killer cell activity was examined in vitro in order further to elucidate the possible immunological mechanisms of tumor regressions following low-dose oxazaphosphorine therapy. It was observed that cytotoxicity of human blood lymphoid cells was unchanged or reduced when the lymphocytes were pretreated for 24 h with mafosfamide or when the drug was present during incubation with K562 cells. However, when lymphoid cells were preincubated with human leukocyte interferon plus mafosfamide, natural killer activity was enhanced above the level caused by interferon alone. This enhancement was noted at mafosfamide concentrations of 1 nM-1 M and was only present when the lymphocyte preparation was contaminated with monocytes.     

Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Augmentation of human monocyte-mediated cytolysis by interferon

Human monocytes, separated by either plastic adherence or adherence to microexudatecoated surfaces, from the peripheral blood of most normal donors were shown to have significant cytolytic activity against TU5, a mouse SV40-transformed target cell. Spontaneous cytolysis ranged from 0 to 32% at a 40:1 effector:target (E:T) ratio. Augmentation of cytolysis was usually seen when human fibroblast interferon (IF) (10³–10⁴ units/ml) was cultured with the effector and target cells for the duration of the assay. The mean increase in percentage cytolysis at 40:1 and 20:1 E:T ratios was greater with monocytes obtained by a microexudate method (24.1 and 22.4%) than with monocytes obtained by a plastic adherence method (16.0 and 8.1%). Only a slight augmentation of cytotoxicity was observed when the effector cells were pretreated with IF for 1-hr. The increased levels of cytotoxicity observed when IF was present during the assay did not appear to be due to the toxic effects of IF on the target cells or to a stable increase in the susceptibility of the target cells to lysis.     

An approach for determining the capacity of human cells to repair gamma-induced DNA damage using interferon

The method of ultracentrifugation of a nucleoid in a neutral sucrose gradient in the presence of ethidium bromide was used to detect gamma radiation-induced DNA breaks and their resynthesis in human HEp-2 cells and fibroblasts taken from a skin biopsy of patients with homocystinuria (HCN). In HEp-2 cells pretreated with interferon the nucleoid sedimentation rate after gamma irradiation did not differ from that in intact cells, that is, interferon exerted its protective effect whereas in HCN cells interferon was ineffective. After incubation with interferon, the resynthesis of the induced breaks was enhanced in these cells as well.     

Enhancement by alkylating agents of chemical carcinogen transformation of hamster cells in culture

When Syrian hamster embryo cells were pretreated with a weak chemical carcinogen, methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS), or with a physical agent such as X-irradiation prior to being exposed to a potent cancer-producing chemical, transformation (crisscrossing of cells not seen in control) occurred up to nine times more often than when the cells were not pretreated. The degree of enhancement appears independent of carcinogen dose. The transformation frequency associated with the carcinogens benzo(a)pyrene (BP), dimethylbenz(a)anthracene (DMBA), 3-methylcholanthrene (MCA), N-acetoxy-2-acetylaminofluorene (AcAAF), and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was increased. There are similarities in the enhancement produced by pretreatment of hamster cells with X-irradiation and with alkylating agents: with both, maximum enhancement occurred approx. 48 h after treatment and lethality attributable to the pretreatment was 10–20% relative to control. However, enhancement produced by X-irradiation pretreatment was slightly greater than that obtained with MMS. The exact cause of the enhancement in transformation resulting from the interaction of these agents is not yet known, but the enhancement associated with MMS pretreatment cannot be related to partial cell synchronization or disruption in the cell cycle. Hamster cells pretreated with 250 μM of MMS demonstrated no alteration in normal cel DNA synthesis through 48-h post-treatment. Analysis of unscheduled DNA synthesis by autoradiography or by alkaline sucrose gradients indicated that the damaged DNA was rapidly repaired after treatment. Therefore, repair of DNA damage as it is now understood is probably not involved.     

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation

Background aimsMesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α–pretreated human bone marrow–derived MSCs on resting or activated T cells.MethodsMSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed.ResultsUnprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions.ConclusionsUnprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.     

Heterogeneity of mouse lymphocytes in interferon production upon influenza virus challenge in culture

Heterogeneity of mouse lymphocytes with respect to interferon production upon influenza virus challenge in culture was studied. Spleen cells produced much more interferon than thymocytes or mesenteric lymph node cells. Spleen cells mainly responsible for interferon production belonged to the hydrocortisone-sensitive population. When spleen lymphocytes were separated into seven fractions by centrifugation on a serum albumin density gradient, they were found to differ greatly in interferon producing capacity; a small fraction of intermediate density, representing a few percent or less of the total lymphocytes, produced markedly high levels of interferon.     

Cell-mediated tumor-killing effect studied by using bilayer lipid membranes

The bilayer lipid membrane (BLM) system was used to investigate the tumor killing effect of natural killer (NK) cells under various experimental conditions. It was found that NK cells interact specifically with BLMs made from lipids and proteolipids isolated from target K562 cells inducing an increase of the membrane conductance. This effect was more pronounced when the NK cells were pretreated with interferon. A similar effect was observed when NK cells were pretreated with sodium selenite. The results suggest that changes in membrane conductance and permeability are involved in the mechanism of the tumor-killing effect mediated by NK cells.