Swine blood leukocytes--new source of production of inferferon active in human cells

Swine blood leukocytes have first been found capable of producing interferon the antiviral activity in human cells of which is comparable to that of native human leukocyte interferon. The influence of various factors on production of porcine leukocyte interferon active in human cells were studied. Thus, when serum was substituted with MAF in the medium used for leukocyte cultivation the titer of the produced interferon increased 4--16-fold and the content of protein impurities in the preparation decreased 10-fold or more. Marked individual interferon-producing capacity of blood leukocytes from different swine and seasonal variations in the level of this capacity were established. The optimal conditions for porcine leukocyte interferon production are: the use of leukocytes in the first few hours after bleeding of the animals; leukocyte concentration in the medium 10 million cells per 1 ml; medium 199 with 10% MAF; New-castle disease virus as interferon inducer in a dose of 10--100 CPD50 per cell. Priming of leukocytes with interferon enhanced their interferon-synthesizing capacity 2--8-fold. This new source of interferon production is readily available, and interferon is useful for public health and veterinary needs.     

Stimulation by calcium chloride of virus-induced interferon formation by blood, haemopoietic organ and continuous human cells

Calcium chloride stimulated virus-induced production of leukocyte interferon by human and animal blood and haemopoietic organ cells. CaCl2 treatment of surviving cells (leukocytes, human and mouse bone marrow) and Namalva cells was the most effective when carried out simultaneously with adsorption of the virus-inducer or when CaCl2 pretreatment was combines with its subsequent addition together with the virus-inducer. Optimal CaCl2 concentrations were 5 mM for human bone marrow cells and 10 mM for human leukocytes and mouse bone marrow cells. CaCl2 treatment was equivalent to priming in case of interferon induction in human leukocytes and, as distinct from priming, it considerably increased virus-induced interferon production by Namalva cells.     

Rhinovirus induces natural killer-like cytotoxic cells and interferon alpha in mononuclear leukocytes.

Natural killer-like cellular cytotoxicity was augmented by incubation of human rhinovirus serotype 2 with peripheral blood mononuclear leukocytes collected from healthy donors. The production of alpha interferon but not gamma interferon was identified in the same cell cultures. A specific interaction of conformationally intact rhinovirus with peripheral blood mononuclear leukocytes was required for induction of the response, since the response was extinguished at reduced quantities of infectious rhinovirus, and acid inactivated rhinovirus did not augment cellular cytotoxicity. Productive replication of rhinovirus was not observed in cultures of peripheral blood mononuclear leukocytes. The replicative failure was not related merely to interferon production, since the rate of disappearance of rhinovirus was similar to that observed in cell free medium. The findings suggest that natural killer cells should be considered as a potential component of the local nasopharyngeal pathophysiology of rhinovirus infection.     

Treatment of human peripheral blood leukocytes with proteases does not affect Sendai virus-induced interferon production.

Trypsinization of human peripheral blood leukocytes was found to have no effect on Sendai virus adsorption or on interferon induction by the virus. alpha-Chymotrypsin and papain also did not affect interferon induction, although the three proteases did remove part of the leukocyte surface material. In contrast, treatment of leukocytes with neuraminidase reduced virus adsorption and thoroughly abolished interferon induction. We conclude that protease-resistant structures on leukocyte surfaces serve as the receptor of Sendai virus for the induction of interferon.     

Feasibility of using human spleen cells to produce interferon

Spleen cells of subjects who died suddenly are capable of producing large amounts of interferon in response to induction with Newcastle disease virus (NDV) and Sendai virus (1-2.5 IU per 1000 cells). The optimal conditions for interferon production by these cells include using NDV, the H strain, as an inducer in a dose of 10 ID50/cell and fresh spleen cells stored no more than 24 hours at 4-6 degrees C in a concentration of 0.5-1.0 x 10(7)/ml, with the incubation time of 20-24 hours. It is recommended that spleen cells from subjects who died suddenly be used for human interferon production.     

Lymphoid cell blastogenesis as an in vitro indicator of cellular immunity to Legionella pneumophila antigens.

The lymphocyte blastogenic transformation assay was adapted to study responsiveness of lymphoid cells from animals and humans to Legionella pneumophila antigens in vitro. Spleen cells from guinea pigs after active immunization with Legionella vaccine, but not from normal animals, responded by blast cell transformation when stimulated in vitro with killed Legionella whole-cell vaccine, sonic extracts thereof, or a purified somatic antigen. The response was dose dependent. Similar lymphocyte blastogenesis occurred with spleen cells from mice sensitized to Legionella by sublethal infection with the bacteria. In addition, blastogenesis occurred with peripheral blood leukocytes from human volunteers tested in vitro with whole-cell vaccine, sonic extracts, or purified somatic antigen. Maximum responsiveness generally occurred 4 to 5 days after stimulation of human peripheral blood leukocytes, but a day or two earlier with spleen cells from normal or sensitized mice. Guinea pig spleen cells generally showed peak responses at the same time as human peripheral blood leukocytes after stimulation in vitro. Blastogenic responses with purified antigen were comparable to those with the whole-cell vaccine or sonic extract. Such antigens from Legionella provide a useful material for inducing responses in vitro as a correlate of cellular immunity to these bacteria.     

The influence of temperature on interferon in cell cultures of homoiothermic and heterothermic mammals

Subnormal temperature was found to depress the production of interferon by cultures of fibroblasts of homoiotherms and heterotherms after virus or poly I.poly C induction. However, in comparison to human (HEF) and mouse (MEF) fibroblasts (homoiotherms) induced with NDV-R or poly I.poly C, interferon production in fibroblasts of spotted sousliks (SL) (heterotherm) and in the aneuploid line of mouse origin (L929) exhibits a greater cold resistance. In contrast to HEF and MEF cells in SL and L929 cells interferon was produced even at 21 degrees C after induction with NDV-R and at 26 degrees C after induction with poly I.poly C. A comparison of alpha and gamma interferon production by mouse and spotted souslik leukocytes did not reveal such distinct differences. At a low temperature (26 degrees C) the production of NDV-R induced interferon was depressed, but it was higher after induction with LPS in both types of leukocytes. PHA and Con A induced interferon was produced in both types of leukocytes only at 37 degrees C. These data confirm that alpha, beta and gamma interferon induction is triggered by different mechanisms and suggest a resistance to cold of beta interferon production in heterothermic animals.     

The effect of temperature on production and function of bovine interferons

Summary Subnormal temperatures were found to depress the production of interferon by bovid herpesvirus 2 (BHV-2)-infected bovine testicular cells, bovine peripheral blood leukocytes, and bovine monocytes, as well as by BHV-2 antigen-stimulated immune peripheral blood leukocytes. Interferon titers generated at 30° C were approximately 10 percent of those at 40° C. Also, subnormal temperatures depressed interferon function. Bovine testicular cells treated at 40° C for 24 hours with high concentrations of BHV-2-induced bovine monocyte interferon or BHV-2 antigen-stimulated immune peripheral blood leukocyte interferon, and then infected with BHV-2 and retreated with interferon at 40° C, had little or no viral growth or cytopathic effect after 72 hours. Cultures without interferon, or those treated with the same amount of interferon at 30° C, had significantly more cytopathic effect and had viral titiers up to 10⁷ TCID₅₀ higher than cultures at 40° C. Earlierin vitro studies done without exogenous interferon showed that BHV-2 replicated to high titers at 30° but not at 40° C. Thus, at low temperatures (30° C)in vitro, BHV-2 induced little interferon, was not highly suppressed by interferon, and replicated to high titers. At higher temperatures (40° C), BHV-2 replication induced high interferon levels, was strongly suppressed by interferon, and replicated poorly. This may help explain the restriction of BHV-2 lesions to skin despite the presence of virus in both skin and internal organs in infected cattle.With 5 Figures     

Use of filter-adsorbed enterotoxins for isolating human immune interferon

The use of A and B enterotoxins of St. aureus adsorbed on Millipore nitrocellulose filters for induction of donor blood leukocytes resulted in production of immune interferon free of the inducer. The interferon produced by this method is acid-labile and inactivated by antiserum to human immune interferon but not to human leukocyte interferon. The advantage of this type of induction consists in the possibility of multiple use of the filters with adsorbed enterotoxins.     

Induction of interferon in mice and cell cultures by Mycoplasma pneumoniae.

Inoculation of mice and L and human embryonic lung (HEL) cell cultures with Mycoplasma pneumoniae failed to induce the production of interferon. M. pneumoniae multiplied in these cell cultures without a marked cytopathic effect. M. pneumoniae induced interferon in human peripheral blood leukocytes with maximum titres of 32 units/ml at 48 hours after infection.     

Bone-marrow interferon

Nucleated bone-marrow cells of mice, rats, guinea pigs, hens, cows, and man are shown to be capable of producing interferon on induction with Newcastle disease virus in vitro. Interferon production by these cells was characterized by high stability. Bone-marrow interferon is not inferior in its activity to interferon obtained by the use of blood leukocytes and spleen cells.     

Dipyridamole is an interferon inducer

2,6-Bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]-pyrimidine (dipyridamole) induced interferon production in vitro in explanted mouse peritoneal leukocytes and to a lower degree in non-lymphoidal cell cultures of mouse (L cells primary embryo fibroblasts) and human (diploid embryo lung fibroblasts) origin. Dipyridamole induced interferon also in mice after intravenous administration. Peak interferon levels in the blood (128 IU/ml) were attained at 49 hr after injection of 0.1 mg dipyridamole per kg body weight and at 24 and 12 hr after injection of 0.6-1.8 and 16.7 mg/kg respectively. By its pH stability, thermostability and antigenic properties the interferon induced in mice, mouse peritoneal leukocytes and L cells corresponded to IFN-alpha and IFN-beta. This interferon-inducing capacity of dipyridamole may account for its broad-spectrum antiviral effect.     

Rat interferons. III. Antiviral activity and effect of "priming" on interferon production in homologous and heterologous cell lines.

Studies on interferon from rat serum sensitive to pH 2-0 showed the following: 1) Its action is stronger in cultures of heterologous L cells than in rat fibroblast cultures. 2) Resistance acquired under the influence of interferon lasts longer in a heterologous than in a homologous system. 3) Actinomycin D added to cultures after 4 hours of contact with interferon inhibits its activity in L cells, and potentiates its activity in RE cells. 4) In rat embryonic cells treated with interferon, interferon production following induction with ND virus is blocked. Similar blocking was observed in cultures of L cells after 24-hour priming; after 6 hours of exposure of L cells to rat interferon, on the other hand, production of interferon was enhanced.     

Interferon-producing capacity of human tonsil cells and properties of interferon produced by these cells.

Tonsils excised from human beings for chronic tonsillitis have been shown to be an accessible and sufficiently rich source of human lymphocytes. Tonsil cells produced interferon as intensively as blood leukocytes and the properties of this interferon were similar to those of leukocyte interferon. The optimal conditions for interferon production by tonsil cells were established.     

Human immune interferon: production and action

The most marked production of immune interferon by human peripheral blood leukocytes and splenocytes stimulated with phytohemagglutinin (PHA) and staphylococcal enterotoxin A (SEA) was shown to be achieved when lymphoid cells are propagated under conditions of constant sparing mixing on roller apparatus at a temperature of 37 degrees +/- 0.5 degrees C. The resulting interferon was sensitive to low pH, thermolabile, inactivated by treatment with trypsin, and not neutralised by antisera to human alpha- and beta-interferons. The antiviral properties with regard to vesicular stomatitis and Semliki Forest viruses were practically similar in PHA- and SEA-induced interferon and human alpha- and beta-interferons. The capacity to inhibit colony formation by HeLa cells was 30 times higher in gamma-interferon than the antiproliferative activity of alpha- and beta-interferons.     

The role of immunocytes in interferon production in mice in response to inosiplex induction

Studies in CBA mice showed that after intraperitoneal injection of inosiplex in a dose of 25 mg/mouse interferon could be detected in the blood serum, peritoneal exudate, and spleen of the animals, the peak of its production being observed in 3-4 days. Interferon was produced by T- and B-lymphocytes. The former were characterized by delayed interferon synthesis while the latter released interferon into the medium as early as 24 hours after induction. The regulatory role of the cells of the monocyte-macrophage system is discussed.     

The E receptor regulates interferon-gamma production: four-receptor model for human lymphocyte activation

The E receptor (binds sheep erythrocytes) is found on virtually all human T cells. Here we show that a monoclonal antibody 9.6, which recognizes and binds the E receptor, inhibited interferon-γ production by human peripheral blood mononuclear leukocytes induced with the mitogens phytohemagglutinin, concanavalin A, Staphylococcal enterotoxin A and the monoclonal antibody OKT3. Metabolic activation (RNA and DNA synthesis) in human peripheral blood mononuclear leukocytes in response to mitogens was also sharply inhibited by 9.6. This inhibitory effect occurred early during the induction phase since 9.6 had much diminished inhibitory effects when added 15-24 h after induction; peak IFN-γ production and DNA synthesis occurred 3-4 days post induction. An early event inhibited by 9.6 appeared to be interleukin 2 (IL2) receptor formation since: (a) the ability of mitogen-stimulated peripheral blood mononuclear leukocytes to absorb IL2 was inhibited by 9.6, and (b) lines of T lymphocytes which already expressed IL2 receptors were largely resistant to the inhibitory effects of 9.6 on IFN-γ production and DNA synthesis. The tumor promoters 12-O-tetradecanoyl phorbol-13-acetate and teleocidin largely reversed the inhibition by 9.6 of IFN-γ production and metabolic activation induced by mitogens. A model for the control of IFN-γ induction involving four receptors, those for mitogens, tumor promoter, IL2 and erythrocyte, is proposed.     

Interferon-inducing and protective action of polyguacyl in an experimental influenza infection

A comparative study of the interferon-inducing and anti-influenza activity of polyguacyl, an interferon inducer, given to human volunteers intranasally and as an aerosol, was carried out. The induction of endogenous interferon in the blood to 100-127 units/ml was observed only after intranasal administration of 5 mg polyguacyl. By both routes of administration the inducer did not diminish the implantation of the virus and development of postvaccination reactions in volunteers to intranasal administration of live influenza A (H1N1) vaccine.     

Physico-chemical properties of interferon produced by a mixed leukocyte suspension.

Physico-chemical properties of partially purified interferon produced by a mixed culture of human peripheral blood leukocytes following induction with double-stranded RNA extracted from f2 phage infected Escherichia coli were studied. Molecular heterogeneity of the interferon preparation was demonstrated by gel chromatography on a Sephadex G-100 column, disc electrophoresis in polyacrylamide gel and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The first two methods revealed 5 peaks, the latter 7 peaks of interferon activity. There was no difference in the molecular nature of interferon produced by cultures exposed to the inducer for the whole period of incubation and that produced by cultures from which the inducer was removed after a short induction time.     

Lack of transmission to polymorphonuclear leukocytes and human umbilical vein endothelial cells as a marker of attenuation of human cytomegalovirus.

The development of a human cytomegalovirus (HCMV) vaccine for the prevention of perinatal disease is urgently needed. However, markers of HCMV attenuation are not defined at present. An in vitro model for the study of interactions of HCMV-infected human fibroblasts and peripheral blood polymorphonuclear leukocytes showed that (1) known laboratory-adapted HCMV strains as well as cell culture-adapted HCMV isolates were not transmitted to polymorphonuclear leukocytes; (2) each of the 80 HCMV recent isolates was consistently transmitted to polymorphonuclear leukocytes as both infectious virus and viral components (nucleic acid and proteins); and (3) all 15 polymorphonuclear leukocyte-tropic strains tested thus far were also endothelial cell-tropic. The in vitro transmissibility to polymorphonuclear leukocytes (and endothelial cells) is proposed as a surrogate marker of pathogenicity of HCMV strains. It seems reasonable to assume that a HCMV strain candidate for a vaccine be verified as deprived of the transfer property to polymorphonuclear leukocytes (and endothelial cells) before involvement in clinical trials assessing safety and immunogenicity.