Interferon Antibody Research Articles

Relationship between circulating interferon and anti‐interferon antibodies and impaired natural killer cell activity in systemic lupus erythematosus

Interferon (IFN) production and response are impaired in a high percentage of systemic lupus erythematosus (SLE) patients. In addition, elevated serum levels of alpha-IFN or anti-alpha-IFN antibodies are present in some SLE patients. This study examined the relationship of circulating IFN and anti-IFN antibodies to the impairment of natural killer (NK) cell function in SLE. All 15 SLE patients studied had measurable circulating alpha-IFN, while the normal controls had minimal serum IFN. Neither patient nor control sera contained any detectable anti-alpha-IFN activity. However, most of the SLE patients demonstrated defects in NK cell function. Because these defects in NK cell function appeared to be associated with circulating IFN, but not anti-IFN, antibodies, the effect of prolonged in vitro IFN exposure on NK cell function of peripheral blood mononuclear cells was determined. It was found that prolonged exposure to IFN induced both an apparent defect in IFN response and a definite impairment of baseline NK cell function. These results suggest that prolonged elevation of circulating alpha-IFN levels could be responsible, in part, for the defects in natural cytotoxicity present in SLE.

Implications of new technology for pharmacy education and practice

Specific knowledge and skills needed by pharmacists in an era of advancing health-care technology are presented, and the most important modern technologic developments in health care are described briefly. Implementation of new technology in health care will be affected by cost considerations and by social trends such as shifting population demographics, an increase in diseases of lifestyle, consumerism, and self-care. Technologic advances in the following areas are described: computerized information networks, bioelectronics, biotechnology, computer graphics, diagnostic imaging, and drug development in space and in the oceans. Diseases are noted for which diagnosis and treatment using new technology is promising. The roles of interferon and monoclonal antibodies are described, and trends in development of new drugs and drug delivery systems are traced. A new philosophical basis for pharmacy education that is consistent with the needs of a technologically oriented society is needed. Practitioners will look to their professional societies for assistance in identifying and implementing the new technology.

Induction of ppp(A2'p)nA-dependent RNase in murine JLS-V9R cells during growth inhibition.

We recently reported that interferon induces the synthesis of ppp(A2'p)nA(n = 2 to greater than or equal to 4) (2-5A)-dependent RNase in the murine cell line JLS-V9R. These cells normally contain very low levels of the nuclease; after interferon treatment, however, they develop levels approaching those found in murine L or Ehrlich ascites tumor cells. Here, we report a similar increase in the nuclease levels in JLS-V9R cells during the transition from the subconfluent actively growing state to the confluent stationary phase. Levels of 2-5A synthetase increased in parallel with the nuclease. The induced levels of both the nuclease and synthetase returned to low basal amounts after trypsinization, dilution, and culturing of the cells at subconfluent densities. The addition of anti-murine interferon (alpha + beta) antibodies to the medium did not affect the induction of the nuclease nor could any interferon be detected in the culture supernatants as determined by the lack of antiviral activity. The increase in the enzymes was not, therefore, due to the spontaneous production of interferon. The induction of the nuclease during confluency preceded an inhibition of [3H]-thymidine incorporation by the cells into DNA. The regulation of the 2-5A-dependent RNase in JLS-V9R cells may, therefore, be related to the control of cell growth.

A colorimetric assay for quantification of defective interfering particles of respiratory syncytial virus.

A colorimetric assay for defective interfering (DI) particles of respiratory syncytial (RS) virus was developed. This quantitative biological assay is based on neutral red dye uptake by DI particle-protected cells that survive standard virus challenge. This assay was more sensitive than the reduction of infectious yield (RIY) assay and was capable of detecting 1 X 10(4) to 2 X 10(4) DI particles/ml. The coefficient of variation for parallel, simultaneous replicates (n = 10) was 23%. Cell-protecting activity in the colorimetric assay appeared simultaneously with activity in the RIY assay on undiluted passage of plaque-purified virus. Both activities were particulate, were inactivated by RS virus antiserum and exhibited similar ultraviolet-inactivation kinetics. The absolute values of the slopes of dilution curves for both assays were similar, and using regression analysis both assays enabled estimation of similar numbers of active particles. These results suggest that both activities are mediated by the same DI particle. The mechanism of cell protection does not appear to involve extracellular interferon because the inclusion of interferon antibody in the assay did not diminish DI particle cell protection. Finally, the colorimetric assay was used to reveal alternating cycles of infectious and DI virus production on serial undiluted passage.

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.

Evidence for a protective role of interferon in resistance to murine cytomegalovirus and its control by non-H-2-linked genes.

Murine cytomegalovirus (MCMV) induces rapid production of a partially pH 2-stable type 1 interferon, the serum level of which is controlled by non-H-2-linked host genes. The production of high, intermediate, and low levels of interferon was found in C3H/He, C57BL/10, and BALB/c mice, respectively, and the use of H-2 congenic mice on the BALB/c or C57BL/10 background showed that H-2-associated genes were not involved. Administration of large (up to 200,000 U) daily doses of partially purified type 1 (alpha plus beta) interferon failed to protect low-producer BALB/c or BALB.K strains from lethal infection. Treatment of the higher (C3H/He) or intermediate (C57BL/10) producer strains with anti-type 1 interferon antibody significantly reduced their resistance to the virus; however, such treatment had no effect on the low-producer BALB/c strain. The decreased resistance of anti-interferon-treated C3H/He mice was accompanied by a transient reduction in serum interferon titers, decreased activation of natural killer cells, a markedly enhanced viremia, and increased viral titers in the liver. These data strongly support a protective role of interferon in defense against MCMV in certain strains of mice. Furthermore, these data suggest that previous observations of a correlation of non-H-2-linked, genetically determined resistance to MCMV with activation of natural killer cells may have its basis in the genetic control of interferon induction by MCMV.

Cellular origin and interactions involved in gamma-interferon production induced by OKt3 monoclonal antibody.

OKT3 monoclonal antibody, a human T cell mitogen, induced interferon production by cultured mononuclear cells at 10(-11) M concentrations. Interferon was secreted only under conditions wherein OKT3 was mitogenic, and production was correlated with cell proliferation. Thus, like mitogenesis, interferon secretion reached a peak 3 days after OKT3 stimulation, was inhibited by a factor(s) in human serum, and required 1000 times higher concentrations of Fab and F(ab')2 fragments of OKT3 for induction. The interferon was most likely of "gamma" (immune) type, because pH 2 and 56 degrees C treatments denatured it, whereas anti-alpha or -beta interferon antibodies did not. Mononuclear cells were fractionated into subpopulations that contained OKT4+ cells (helper/inducer T cells), OKT8+ cells (cytotoxic/suppressor T cells), and OKM1+ cells (monocytes) by combining sheep red blood cell rosetting and complement-mediated lysis using monoclonal antibodies against specific cell types. Both OKT4+ and OKT8+ cells proliferated upon OKT3 stimulation with the absolute requirement of OKM1+ cells. However, OKT4+ cells plus OKM1+ cells were necessary for the secretion of interferon. Studies with selective pretreatments with mitomycin C suggested that gamma-interferon was secreted by the OKT4+ cells and that the OKM1+ population subserved an accessory function.

Interferon: Wesentlich vereinfachte, enzym-immunologische Bestimmung mit zwei monoklonalen Antikörpern

Human leukocyte interferon (HuIFN-alpha) can be determined by a substantially simplified enzyme-immunological method, based on the solid phase sandwich principle. In a single step, the interferon in the sample or the standard solution (tests and standards are run concomitantly) is bound to a polystyrene bead coated with monoclonal (mouse) anti-interferon, and at the same time it is labelled with a second monoclonal (mouse) interferon antibody, which is coupled to horse radish peroxidase. After this immunological incubation, the non-bound material is removed by washing, and the quantity of peroxidase bound to the bead is measured enzymically. The resulting colour intensity is determined photometrically, and it is directly proportional to the interferon concentration in the sample. The detection limit of the test can be as low as 100 I.U./l interferon, depending on the assay conditions. The variation coefficient within series was 3-6%. Serum samples can be used directly in the test without pretreatment. Depending on the required sensitivity, the incubation may be performed for periods between 30 minutes and 24 hours.

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.

Marrow transplantation for leukemia

Marrow transplantation for selected patients with leukemia, as for patients with severe combined immunologic deficiency or severe aplastic anemia, has now become an accepted clinical procedure. For patients with acute leukemia who have relapsed after achieving a remission of chemotherapy, marrow grafting from an identical twin or an HLA-identical sibling has now been demonstrated to produce median remissions as long as or longer than any reported for combination chemotherapy. In contrast to chemotherapy, marrow transplantation offers the possibility of cure for a small but significant fraction of these patients. Marrow transplantation for patients with ANL in first remission has now resulted in median survivals much longer than any reported with chemotherapy. Although it now appears that more than 50% of these patients can be cured with marrow transplantation, a much longer follow-up is indicated since some patients who achieve a complete remission with combination chemotherapy are now living for a long time, and some of these patients (less than 20%) may also be cured. Current intensive research with new modalities such as interferon, Acyclovir, Cyclosporin A, and monoclonal antibodies can reasonably be expected to improve the overall results of marrow transplantation.

Comparisons of several biological and physicochemical properties of human leukocyte interferons produced by human leukocytes and by E. coli

Human interferon (IFN) prepared from virus-induced human leukocyte suspensions (leukocyte-derived interferon) was compared to the IFN extracted from Escherichia coli harboring a human interferon-α cDNA hybrid plasmid (Hif-SN35-AH-L6). E. coli-derived IFN was 20 to 50 times more active than leukocyte-derived IFN on heterologous bovine, feline, murine and guinea pig cells, relative to the activity on human cells. After partial purification by affinity chromatography on an anti-human lymphoblastoid IFN antibody column, the IFN was analyzed by SDS-polyacrylamide gel electrophoresis. While leukocyte-derived IFN gave a heterogeneous pattern with major peaks of activity at 24000 and 19 000 daltons, E. coli-derived IFN gave a homogeneous peak of activity at about 17–18 000 daltons. The leading edge of leukocyte-derived IFN in SDS-polyacrylamide gels was significantly more active on bovine cells than on human cells and coincided in mobility with E. coli-derived IFN, which was also much more active on bovine than on human cells. After reduction with mercaptoethanol in SDS, the E. coli-derived IFN lost no activity, whereas the leukocyte-derived IFN lost about 90% of its activity. After reduction, E. coli-derived IFN migrated in SDS-polyacrylamide gels as a single peak at 24000 daltons, as did the residual activity of reduced leukocyte-derived interferon. Our data suggest that the interferon produced by the E. coli harboring the clone Hif-SN35-AH-L6 is analogous in size and cross-species activity to one of the molecular species of leukocyte-derived interferon.

Characterization of a subspecies of mouse interferon cross-reactive on human cells and antigenically related to human leukocyte interferon

Mouse interferon preparations from various virus-induced cell cultures exhibited some antiviral activity (about 0.1% of homologous cell titers) on human cells. When analyzed by SDS-polyacrylamide gel electrophoresis the activity of mouse interferon on mouse cells migrated as two bands with activity peaks at 38,000 and 22,000 daltons, while the activity on human cells was detectable only in the faster migrating portion of the lower molecular weight band, at about 21,000 daltons. When the mouse interferon preparation was chromatographed on a column of immobilized anti-human leukocyte interferon antibodies, the specifically retained interferon was found to migrate in SDS-polyacrylamide gels as a 21,000-dalton species which exhibited similar degrees of activity on both mouse and human cells. Thus, mouse interferon preparations contain a low molecular weight interferon species which is both active on mouse cells and is responsible for all the heterologous antiviral activity on human cells and is antigenically related to human leukocyte interferon. These data suggest significant structural similarities between the active cores of certain interferons from phylogenetically diverse animal species.

Mechanisms of Interferon Induced Transfer of Viral Resistance Between Animal Cells

The sequence of events initiated by interferon and leading to the antiviral state were studied as possible sites for the cell-to-cell transfer of interferon induced viral resistance. The possible role of interferon produced by recipient cells was negated by the demonstration of transfer of resistance in the presence of anti-human interferon antibody and under conditions of a single cycle of VSV growth. Transfer of sensitivity of WISH cells to mouse interferon, possibly through transfer of a membrane receptor, seems unlikely since resistance was transferred in the absence of mouse interferon. From kinetic data and the fact that actinomycin D blocked resistance in human cells for 3 h longer than in mouse cells, it seems unlikely that the mouse antiviral protein itself or its mRNA alone is a likely candidate for the transfer of resistance. Thus, by a process of elimination, we suggest that secondary messenger molecules which transmit the interferon signal from the membrane to the nucleus are the effector substance(s) for the transfer process.

The immunosuppressive effect of type II mouse interferon preparations on antibody production

Preparations containing Type II (immune induced) interferon suppressed the immune response to sheep erythrocytes (SRBC) both in vitro and in vivo. Type II interferon preparations were 250 times more active in immunosuppression than Type I (L cell) interferon preparations in parallel experiments. The antiviral and immunosuppressive activities shared several unique physical-chemical activities including pH 2 lability, 56 °C stability, and resistance to inactivation by anti-L-cell interferon antibody. Both activities were denatured by boiling at 100 °C for 2.5 min, but were not renatured by boiling for 1 min with 1% SDS, 1% β-mercaptoethanol and 5 M urea. The bulk of both the immunosuppressive and antiviral activities were recovered from a 41–60% saturated ammonium sulfate precipitate of Type II interferon. Sephadex G-100 column chromatography of Type II interferon preparations yielded two major peaks of anti-viral activity of molecular weights of approximately 40,000 and 90,000, both of which together contained the total immunomodulating activity observed in the proteins of the Chromatographic effluent. The 90,000-dalton species was also detected by its anti-viral and immunosuppressive activity on polyacrylamide gel electrophoresis.

Production and Properties of Migration Inhibitory Factor and Interferon in the Circulation of Mice with Delayed Hypersensitivity

Abstract Further evidence is presented of the coordinate production and similar properties of two mediators, migration inhibitory factor (MIF) and interferon, in the circulation of BCG-infected mice inoculated with specific antigen (Old Tuberculin, OT). In addition, the interferon elicited by OT in BCG-infected mice is shown to be a distinct molecular species of viral inhibitor and is designated “Type II interferon.” The designation “Type I interferon” is used to describe the viral inhibitor produced in BCG-infected or control mice given nonspecific stimuli such as bacterial lipopolysaccharide (LPS) or Newcastle disease virus (NDV). Although Type II interferon possesses the biologic attributes required to classify a viral inhibitor as “interferon,” it, as well as MIF, differs markedly from Type I interferon in stability at pH 2 and at 56°C, range of species specificity, and antigenicity. Antibodies against highly purified L cell interferon induced by NDV (anti-Type I interferon antibodies) neutralize the activity of interferons elicited in mice by nonspecific stimuli such as LPS or NDV. In contrast, the antiviral activity of Type II interferon and the macrophage-inhibitory activity of MIF, elicited by specific antigen in mice with delayed hypersensitivity, are not affected by high concentrations of antibody against Type I interferon. Evidence is presented which emphasizes the striking similarities in properties of MIF and Type II interferon, although it is not possible to determine whether the two mediators are the same or different protein molecules.