Biomedical subjects
Publications and source records attributed to N Fuchsberger.
Elevated synthesis of 23 K protein by human hepatoma PLC/PRF/5 cells was observed after their treatment with conditioned medium from concanavalin A stimulated peripheral-blood monocytes. Increased amount of this protein was first determined 4 hr after the treatment and its maximal level was reached 48 hr later. The role of the 23 K protein remains so far unknown.
Recombinant interferon alpha enhanced the MHC class I antigen density on human leukaemia/lymphoma cell lines REH, U-937 and HL-60, as measured by immunocytofluorometry using specific monoclonal antibodies. A similar effect was induced (as demonstrated in REH cells), also by human leukocyte interferon-alpha. The latter, however, caused no major alterations in the expression of leukocyte common antigen (ICA; CD45) and transferrin receptor (CD71) in the cell lines examined. In REH cells, there was no interferon-induced alteration of CD10 antigen (CALLA), which in this cell line is markedly down-regulated by 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A decrease of CD4 antigen density on the cell membrane was induced by interferon-alpha in monoblastoid U-937 cells. No induction of MHC class I and II antigens by interferon-alpha was found in K-562 cell subline.
Human leukocyte interferon (HLIF) can be contaminated by several antigens which may include also potentially pathogenic agents. For this reason, gel filtration as a separation method was included into the routine procedure of preparation and purification of HLIF for clinical use. Since production of HLIF requires the cultivation of leukocytes in the presence of serum (or albumin), serum proteins represent then the majority of proteins contaminating IF preparations. Measuring the total protein content, as a control of various antigens present in HLIF preparations during purification, becomes ineffective because, essentially, it indicates only the decrease of the amount of proteins derived from cultivation medium. For a better visualization of dissociation of different antigens from molecules with IF activity during the purification procedure, the method of quantitative immunoelectrophoresis was applied utilizing a sheep antiinterferon serum in assay. Our results indicate that this method represents a valuable control test.
No changes in sensitivity of human embryo cells (HEC) to the antiviral action of interferon early (i.e. up to 17 days) after infection with simian virus 40 (SV40) could be detected. At the same time the sensitivity of cells to the cell-growth inhibitory effect of interferon decreased considerably. The changes in sensitivity of HEC to interferon action occurred during 5 successive passages following the infection with SV40 and remained on a similar level thereafter. During this period, no morphological alterations of cells were observed.
The electroimmunoassay (quantitative "rocket" immunoelectrophoresis) method was adopted for analysis and characterization of interferon preparations. Ammonium sulphate-precipitated anti-interferon globulin also containing unknown antibodies against antigens that contaminate interferon preparations was used in the tests. By comparing the results of the electroimmunoassay of fractions obtained by polyacrylamide gel electrophoresis of human leukocyte interferon with the antiviral activity of the fractions, an excellent dissociation of molecules with interferon activity from the bulk of contaminating antigens was achieved. The method is extremely sensitive and requires very small volumes for assay.
Studies performed in the CSV subline of mouse L-cells suggest that activated endogenous and/or exogenous viral infection might be the factor that modifies the cell surface and, in consequence, the sensitivity of the cell to the cell-growth inhibitory action of interferon. In contrast with the parental L-cells, the CSV subline obtained by prolonged passage of L-cells in the presence of interferon shows an altered electron microscopic morphology, absence of C type particles and a decreased agglutinability with Concanavalin A. It is resistant to the cell-growth inhibitory effect of interferon but retains the sensitivity toward its antiviral effect. However, the sensitivity of the CSV subline toward the cell-growth inhibitory effect of interferon increased significantly after treatment with 5-iododexyuridine, infection with the Harvey strain of mouse sarcoma virus and/or prolonged passages in vitro. In this respect, the CSV subline resembles the primary mouse embryonic cells. Since sensitivity of CSV cells increased also after treatment with cyclic 3'-5' adenosine monophosphate and/or prostaglandin E2, it is possible that the cell-growth inhibitory effect of interferon is mediated through the "second messenger" system.
A one-step purification procedure using affinity chromatography on purified anti-interferon antibodies makes possible to obtain highly purified interferon preparations. Antibodies against purified mouse fibroblast interferon were covalently bound to activated CNBr-Sepharose 4B. This immunoadsorbent had a binding capacity of 125,800 mouse fibroblast interferon units per 4.8 ml of gel. The binding capacity for mouse leukocytes was similar while for serum interferon it was lower. When analyzed in polyacrylamide gel, the electrophoretic profiles of purified IF had a similar shape to that of unpurified IF. The results of this purification procedure are not influenced by volume, biological activity and/or purity of the starting material.
After prolonged immunization of rabbits with a semipurified mouse interferon preparation in Freund-incomplete and/or Al-Span-Oil-adjuvant a specific interferon neutralizing immunoglobulin was obtained from antiserum. The specific activity of the antiserum and immunoglobulin was confirmed in tests in which the interaction of antibodies with the cell-surface was ruled out. The antiserum (and the immunoglobulin) neutralized both the anti-viral and the cell-growth inhibitory activity of interferon. The "slow" and the "fast" fractions of purified interferon preparations were equally sensitive toward the neutralizing effect of antibodies. On the other side, the reaction of heat-inactivated interferon with the antiserum did not diminish its neutralizing activity suggesting a destruction of interferon-antigenic sites.
After prolonged immunization of rabbits with a semipurified mouse interferon preparation in Freund's incomplete or Al-Span-Oil adjuvant, a specific interferon-neutralizing immunoglobulin was obtained from antiserum with a capacity of neutralizing about 49000 mouse interferon units per ml. The specific activity of the antiserum and immunoglobulin was confirmed in tests in which the interaction of antibodies with the cell surface was ruled out. The antiserum (and the immunoglobulin) neutralized both the antiviral and the cell-growth inhibitory activities of interferon. The "slow" and the "fast" fractions of purified interferon preparations were equally sensitive to the neutralizing effect of antibodies. On the other hand, the reaction of heat-inactivated interferon with the antiserum did not diminish the neutralizing activity of the latter, suggesting a destruction of interferon antigenic sites.
Interferon preparations of a high degree of purity were obtained by a one-step procedure using affinity chromatography on specifically purified immunoadsorbent. The procedure consisted of binding interferon harvested from serum-free medium and purified by Zn-acetate precipitation and SP-Sephadex chromatography to CNBr-activated Sepharose 4B (Column No. 1). In the next step, antiinterferon globulin was purified by affinity chromatography on Column No. 1 with the bound interferon. In this way, antibodies against purified interferon, which were free from non-antibody components, were obtained. The purified antibodies were then coupled to CNBr-activated Sepharose 4B forming Column No. 2. The latter had a binding capacity of 125 800 mouse interferon units per 4.8 ml of gel. This capacity was not altered during an 8-month period of use. The gel was capable to bind interferons obtained from fibroblasts and leukocytes and, partially, from serum. The resulting purified, products were similar, i.e. they were not influenced by volume, interferon activity, or purity of the starting material. The electrophoretic profiles of the products had a similar shape irrespective of the origin of the starting material.
Purified mouse L-cell interferon induced with Newcastle disease virus was subjected to polyacrylamide gel electrophoresis. A profile characterized by two components was obtained. The faster peak represented about 90-99 per cent of antiviral activity. Both components exerted a cell-growth inhibitory effect on L cells. A study of the quantitative relations between antiviral and cell-growth inhibitory activites of the two peaks revealed a discrepancy between these two activities in the fast peak. An analysis of this phenomenon suggests that the binding capacity of the fast moving molecular species of interferon is lower in young cells than in aged cells, i.e. those incubated for 3 days. Such differences were not observed when the slow molecular species was examined. It was concluded that both inherent properties of molecular components of interferon and the age (and species) of cells used in tests influence the outcome of interferon action on the cell.