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Biomedical subjects

W R Fleischmann

Publications and source records attributed to W R Fleischmann.

At least 55 records · Page 3Linked to original sources
PubMed1985

An inhibitor of interferon action: I. Physical association of the inhibitor with interferon-gamma.

Previously, an inhibitor of interferon action has been identified in mouse lymphokine preparations. The inhibitor was produced following IFN-gamma production and thus may be involved in regulation of IFN-gamma action. We now show that native inhibitor activity as isolated from crude and partially purified IFN-gamma preparations migrates along with interferon activity on molecular sieving gels. IFN-gamma which comigrates with inhibitor migrates at a slightly higher molecular weight than IFN-gamma free of inhibitor. This inhibitor activity correlates with the decreased antiviral activity of crude IFN-gamma produced late in the course of mitogen stimulation. This is apparent for both plaque reduction and yield reduction assays for interferon. This association of IFN-gamma with its inhibitor suggests a mechanism for the local regulation of IFN-gamma activity.

Animals↗
PubMed1985

Evidence that IFN-alph/beta induces two antiviral states active against different viruses.

IFN-alpha/beta has been suggested to require only one round of mRNA and protein synthesis to induce an antiviral state. We have examined the mechanism of induction of the antiviral states shown against three types of viruses: mengovirus (plus strand, sense RNA), vesicular stomatitis virus (VSV, minus strand RNA), and vaccinia virus (DNA). Mouse L cells were treated with IFN-alpha/beta and cycloheximide and then with actinomycin D on a schedule which allowed only one round of mRNA and protein synthesis. The cells were challenged with virus under single cycle growth conditions to determine the amount of antiviral activity against the particular challenge virus employed. These studies confirmed that most of the antiviral effect directed against VSV is achieved with one round of macromolecular synthesis. However, most of the antiviral effect directed against mengovirus and vaccinia virus seemed to require more than one round. These results suggest that IFN-alpha/beta induces two different antiviral states: one requiring one round of synthesis which is primarily responsible for the inhibition observed for VSV; and another requiring more than one round of synthesis which is primarily responsible for the inhibition observed for mengovirus and vaccinia virus.

Animals↗
PubMed1984

Potentiation of interferon action by mixtures of recombinant DNA-derived human interferons.

Natural and essentially pure recombinant DNA-derived HuIFN-alpha and HuIFN-gamma were examined for their relative abilities to potentiate interferon action. Potentiation of human interferon's antiviral and antiproliferative activities were studied. The essentially pure recombinant DNA-derived human interferons were found to be as effective in their potentiating interactions as their natural counterparts. The results demonstrate that it is the human interferons themselves which interact to potentiate human interferon's varied activities.

Antiviral Agents↗
PubMed1984

Potentiating effect of murine interferon-gamma-containing lymphokine preparations on the antiviral and antiproliferative effects of murine interferon-alpha/beta: identification of the potentiation factor as murine interferon-gamma itself.

Mixed preparations of murine interferon-gamma (MuIFN-gamma) and murine interferon-alpha/beta (MuIFN-alpha/beta) have been shown to induce more than additive levels of antiviral protection, when compared to those induced by these interferons given separately. MuIFN-gamma preparations contain many lymphokines and several of these as well as MuIFN-gamma itself may participate in this potentiation. In the present study, natural as well as three recombinant DNA-derived MuIFN-gamma's, in combination with antibody to MuIFN-gamma have been employed to examine the precise role of MuIFN-gamma. The antiviral effect was examined with a single cycle virus yield reduction assay and the antiproliferative effect with a colony formation inhibition assay. Recombinant DNA-derived MuIFN-gamma was as effective as natural MuIFN-gamma at participating in the potentiation of both the antiviral and antiproliferative activities. Antibody to MuIFN-gamma effectively blocked the potentiation of both the antiviral and the antiproliferative activities of natural and recombinant DNA-derived MuIFN-gamma's. Since the recombinant DNA-derived preparations from E. colie can be assumed not to contain mammalian proteins other than MuIFN-gamma, the data conclusively demonstrate that the potentiation factor in MuIFN-gamma preparations is MuIFN-gamma itself.

Animals↗
PubMed1984

Inhibition of anticellular activity of mouse gamma interferon by an inhibitor of interferon action.

An inhibitor of interferon's (IFN) antiviral action was evaluated for its ability to block interferon's anticellular action. Crude and partially purified mouse immune interferon (MuIFN-gamma) preparations with high and low levels of an inhibitor of IFN's antiviral action were prepared. These preparations were examined for their relative antiviral and direct anticellular activities. The inhibitor present in the IFN-gamma preparations was found to be an equally potent inhibitor of both the antiviral and the direct anticellular activities of IFN-gamma. Further, while the inhibitor of IFN-gamma was inherently present in most IFN-gamma preparations, it was not an inherent feature of all IFN-gamma molecules. Inhibitor-free IFN-gamma could be separated from inhibitor-containing IFN-gamma by column chromatography. The results suggest that the inhibitor generally antagonizes interferon action and should be taken into account when evaluating the biological properties of IFN-gamma preparations.

Animals↗
PubMed1984

Requirement for IFN gamma in potentiation of interferon's antiviral and anticellular activities: identity of mouse and human systems.

Potentiation was originally demonstrated as a nonadditive, synergistic enhancement of interferon (IFN) activity in the mouse system for mixed preparations containing MuIFN-alpha/beta and MuIFN-gamma. Potentiation of the antiviral and anticellular activities has now been studied for mouse and human systems, and in both systems IFN-alpha and IFN-beta interacted synergistically with IFN-gamma, but not with each other. Further, the antiviral and anticellular activities of IFN-alpha and IFN-beta were potentiated equally by IFN-gamma. Potentiation was demonstrated for HuIFNs with specific activities of 10(7) U/mg of protein and higher. Naturally produced and recombinant HuIFN-alpha s had the same relative abilities to be potentiated by HuIFN-gamma. It was concluded that IFN-gamma with either IFN-alpha or IFN-beta was essential for potentiation, that potentiation of anticellular and antiviral actions occurred in similar manners, and that a close correlation existed between potentiation in mouse and human systems. These results suggest that potentiation was caused by the interaction of two dissimilar IFN types (immune versus virus-type) and that potentiation studies in the mouse may be directly relevant for humans.

Animals↗
PubMed1984

Potentiation of interferon's antiviral activity by the mutually synergistic interaction of MuIFN-alpha/beta and MuIFN-gamma.

The interaction of the interferons (IFNs) that cooperate to potentiate the antiviral action of IFN was studied. Serial dilutions of MuIFN-gamma and MuIFN-alpha/beta were employed separately and in combination to block virus replication in one-step, virus yield reduction experiments. To calculate the potentiation of IFN activity, protection levels obtained for each combination of MuIFN-gamma and MuIFN-alpha/beta were compared with those obtained for the separate IFNs. Potentiation levels increased with increasing concentrations of each of the IFNs in a dose-dependent manner, suggesting that potentiation of IFN's antiviral activity was the result of the mutually synergistic interaction of the IFNs. Three challenge viruses were employed: Mengo virus (positive-strand RNA virus), vesicular stomatitis virus (negative-strand RNA virus), and vaccinia virus (DNA virus). Identical results were observed with the three different viruses, suggesting that mutual synergism was a basic feature of the potentiation of IFN's antiviral activity by combined preparations of MuIFN-gamma and MuIFN-alpha/beta.

Animals↗
PubMed1984

Discrimination between nonmalignant and malignant cells by combinations of IFN-gamma and IFN-alpha/beta.

Combination treatment with interferon (IFN)-gamma and either IFN-alpha or IFN-beta synergistically enhances (potentiates) the anticellular effects of the IFNs. IFNs have been shown to have anticellular activity against normal as well as malignant cells. To study the relative effectiveness of IFN's anticellular activity against nonmalignant and malignant cells in the mouse system, two paired sets of nonmalignant/malignant cells (JB-1/JB-8; D-1/D-11a) were studied using 3-day kinetics of cell growth experiments. Treatment of the nonmalignant cells with IFN-gamma or IFN-alpha/beta employed separately had little or no effect on the growth of the cells at the IFN concentrations employed. Combination IFN treatment had only a slight effect on the growth of these cells. Treatment of the malignant cells with IFN-gamma or IFN-alpha/beta employed separately had low levels of effect on the growth of the cells. In contrast, treatment of these malignant cells with combined IFN-gamma and IFN-alpha/beta had a very marked anticellular effect, dramatically showing the growth of, or even killing, the malignant cells. These observations appeared to be independent of the growth status of the cells, since stationary-phase and log-phase malignant cells maintained their relatively greater sensitivity to the IFNs. Thus, IFN treatment, particularly combination IFN treatment, had a differential effect on the growth of the paired sets of nonmalignant and malignant cells and appeared to sharply differentiate between nonmalignant and malignant cells.

Animals↗
PubMed1984

Eradication of cultured human melanoma cells by immune interferon and leukocytes.

For the determination of the conditions for the most effective cytolysis of human melanoma cells, leukocyte interferon (IFN-alpha), fibroblast interferon (IFN-beta), and immune interferon (IFN-gamma) were compared for their abilities to kill cultured human melanoma cells in the presence and absence of peripheral blood mononuclear leukocytes (PBL). A microassay was employed in which the viability of melanoma target cells was determined after various times of incubation with interferons alone or with PBL. On 7 human melanoma cell lines (from 6 different patients), IFN-gamma had significantly greater direct anticellular effect than IFN-alpha or IFN-beta. When PBL were added, all target cells were killed after 48 hours with IFN-gamma, but they were not killed with IFN-alpha or IFN-beta. When IFN-gamma was added to either IFN-alpha or IFN-beta, a potentiation of the anticellular effect was observed both with and without PBL. The actions of this "natural" IFN-gamma could be reproduced with recombinant IFN-gamma and could be neutralized by an antibody to a synthetic peptide encoded by the 5'-end of IFN-gamma complementary DNA. It was concluded that IFN-gamma is significantly more active against these human melanoma cell lines than either IFN-alpha or IFN-beta and, most significantly, that eradication can occur in the presence of IFN-gamma and PBL. Furthermore, synergistic anticellular action can be observed when IFN-gamma is added to IFN-alpha or IFN-beta. These findings point to the need for preclinical trials to evaluate eradication in vivo.

Cell Line↗
PubMed1983

Interferon potentiation occurs at a pretranscriptive stage.

Combination of interferon-gamma (IFN-gamma) with a mixture of IFN-alpha and IFN-beta (IFN-alpha/beta) produce a potentiated antiviral state. The onset of induction of the potentiated antiviral state was observed to be more rapid than the onset of induction of either IFN-gamma or IFN-alpha/beta separately. The onset of viral resistance to combined IFN-gamma and IFN-alpha/beta (25 U/ml each) began after 2 h, compared with 4 h for IFN-alpha/beta (25 U/ml) alone and 8 h for IFN-gamma (25 U/ml) alone. By adding actinomycin D at various times after interferon treatment, this more rapid onset of induction of the potentiated antiviral state was shown to be the result of a more rapid onset of cellular transcription. To determine whether increased concentrations of IFN-alpha/beta acted through a similar mechanism, we performed kinetic experiments with several concentrations of IFN-alpha/beta and with combined preparations of IFN-gamma and IFN-alpha/beta. The findings showed that treatment of cells with the combined interferons and with a high concentration of IFN-alpha/beta resulted in more rapid onset of cellular transcription and more rapid development of the antiviral state. Thus, the cellular response to potentiation appeared to involve perception by the cell of the combined interferons as a higher-than-expected effective concentration of interferon, resulting in more rapid onset of cellular transcription of mRNA for antiviral proteins.

Animals↗
PubMed1983

Potentiation of lymphocyte natural killing by mixtures of alpha or beta interferon with recombinant gamma interferon.

Human lymphocytes were treated with human alpha (IFN-alpha), beta (IFN-beta), or recombinant gamma (IFN-gamma) interferons separately or in combination to determine their ability to enhance natural killing against mouse L cell targets. Our results showed that recombinant IFN-gamma was approximately 50 times more active per unit of antiviral activity than either IFN-alpha or IFN-beta. Moreover, the levels of natural killing by lymphocytes treated with combinations of IFN-alpha and IFN-beta were additive, whereas combinations of recombinant IFN-gamma and IFN-alpha or recombinant IFN-gamma and IFN-beta were synergistic. The development of natural killing in lymphocytes treated with recombinant IFN-gamma did not occur more rapidly but reached higher levels (62%) than that observed with lymphocytes treated with IFN-alpha or IFN-beta (15%). The results suggest the importance of IFN-gamma and mixtures of IFN-gamma with IFN-alpha or IFN-beta in the enhancement of natural killing activity against virus infections and neoplasia.

Adjuvants, Immunologic↗
PubMed1982

Direct cytolysis by partially-purified preparations of immune interferon.

Mouse IFN gamma preparations purified 30-fold were found to have direct cytolytic activity against a number of tumor and normal cells. Cell killing was determined using a sensitive, rapid and accurate assay which employed very low numbers of cells and very small quantities of interferon. The cytolytic activity of IFN gamma on 11 murine tumor cell lines was investigated. A 20-fold difference was found between the most-sensitive cell type, P-388 lymphoma, versus the most resistant cell type, C127v leukemia. A number of normal mouse cells was also found to have low to intermediate sensitivity to the cytolytic action of IFN gamma. Human IFN gamma was also shown to have cytolytic activity which, like mouse IFN gamma, was relatively species-specific. Direct cytolysis was not found to be a characteristic of IFN-alpha/beta. Different mechanisms of action for the antiviral and cytolytic activities of IFN gamma are indicated because the cytolytic titer of IFN gamma did not parallel its antiviral titer on most cell types and increasing the cell number produced a decrease in the cytolytic titer and an increase in the anti-viral titer. High concentrations of IFN gamma (i.e., 2,900 units/ml) resulted in complete lysis of cells within 24 h, while lower concentrations (i.e., 700 units/ml) resulted in a reversible inhibition of cell growth during this time period. Evidence that the cytolytic substance in the IFN gamma preparation was IFN gamma include the following: (1) both antiviral and anticellular activities copurified through a 30-fold purification; and both activities were (2) relatively species-specific; (3) sensitive to heat; (4) inactivated by low pH and (5) neutralized by antibodies to IFN gamma. Therefore, we propose the possibility that direct cytolysis is yet another of IFN gamma's distinctive antivities.

Animals↗
PubMed1982

Potentiation of the direct anticellular activity of mouse interferons: mutual synergism and interferon concentration dependence.

Mouse immune (IFN-gamma)2 and virus-type (IFN-alpha/beta) interferons were used separately and in combination in cloning studies with B-16 melanoma cells. IFN-gamma was found to be a more potent mediator of the direct anticellular effect of interferon than was IFN-alpha/beta, as shown not only by a greater sensitivity of B-16 cells to IFN-gamma but also by a steeper slope of the anticellular sensitivity curve of the IFN-gamma. The differences in the slopes of the curves defining their anticellular effect appeared to be inherent in the interferons themselves and not due to an inhibitor of interferon, a stimulator of cell growth, or another factor possessing anticellular activity. The results are consistent with the interpretation that IFN-gamma and IFN-alpha/beta exert their anticellular effects by different mechanisms. The anticellular activity of interferon against B-16 melanoma replication until development was potentiated by mixed preparations of IFN-gamma and IFN-alpha/beta. The potentiation appeared to be an expression of a property of the interferons themselves. Replication units resistant to the potentiated activity of the interferons were not detected. Potentiation levels were dependent on the concentrations of both IFN-gamma and IFN-alpha/beta and continued to increase dramatically as the interferon concentrations increased. Maximum potentiation observed was 214-fold at the highest interferon concentrations used. The data suggested that potentiation is a mutual, synergistic interaction of IFN-gamma and IFN-alpha/beta.

Animals↗
PubMed1982

Gamma interferon (IFN gamma) and IFN alpha/beta suppress murine myeloid colony formation (CFU-C)N: magnitude of suppression is dependent upon level of colony-stimulating factor (CSF).

The effect of different types of interferon (IFN) on macrophage and granulocyte colony formation (CFU-C) in vitro was studied using bone marrow cells from C57BL/6 mice. CFU-C formation was measured at 7 days using a standard methylcellulose culture system with L-cell conditioned medium (LCCM) as a source of colony-stimulating factor activity (CSF), IFN gamma was obtained from staphylococcal enterotoxin A- (SEA) stimulated mouse spleen cells and was partially purified (50-fold) using affinity chromatography. IFN alpha/beta was obtained from Newcastle disease virus-infected mouse L-cells and was partially purified (286-fold) using an AcA 202 column. IFN gamma inhibited CFU-C formation in a dose-dependent manner and was 100 times more inhibitory than IFN alpha/beta. IFN gamma completely suppressed CFU-C formation with as little as 15 units of antiviral activity. In contrast, 1000 to 2000 units of antiviral activity of IFN alpha/beta was required for complete suppression of CFU-C formation. The ability of IFN gamma to inhibit CFU-C formation was completely abolished after pH 2 treatment (24 hr), heat treatment (60 degrees C, 2 hr), or treatment with a specific antiserum against IFN gamma, which indicated that IFN gamma was the mediator of CFU-C inhibition. When IFN gamma and IFN alpha/beta were mixed together and added to bone marrow cells, they had a synergistic effect on the suppression of CFU-C formation. The level of inhibition mediated by both IFN gamma and IFN alpha/beta was totally dependent upon the amount of CSF present in the culture system; increasing the level of CSF could completely overcome the IFN suppressive effect. These results suggest that IFN gamma and IFN alpha/beta may have regulatory roles in hematopoiesis.

Animals↗