Human Leukocyte Interferon Preparations Research Articles

4696899 Process for the preparation of human leukocyte and human gamma interferon: Miklos Toth, Valeria Endresz, Ilona Beladi, Sandor Toth, Szeged, Hungary assigned to EGYT Gyogyszervegyeszeti Gyar

4696899 Process for the preparation of human leukocyte and human gamma interferon: Miklos Toth, Valeria Endresz, Ilona Beladi, Sandor Toth, Szeged, Hungary assigned to EGYT Gyogyszervegyeszeti Gyar

Characterization of three species of Escherichia coli-derived human leukocyte interferon A separated by reverse-phase high-performance liquid chromatography.

A preparation of recombinant human leukocyte interferon A (rIFN-alpha A) obtained from Escherichia coli cells was found by reverse-phase high-performance liquid chromatography (HPLC) to contain three species. The three species, named Mf-1, Mf-2, and Ms in the order of their elution, were separated and characterized to elucidate their structural differences. The amino acid compositions, the amino-terminal amino acid sequences (Cys1-Asp2-Leu3-), and the carboxy-terminal amino acids (Glu) of the three species agreed with those predicted from the cDNA sequence, although the sequence analysis yields of Mf-2 and Ms were extremely low. They were found to have the same disulfide bonds, Cys1-Cys98 and Cys29-Cys138, by amino acid analysis of the tryptic peptides, but the recovery of the peptides linked by a Cys1-Cys98 disulfide bond for Mf-2 and Ms was extremely low. The results demonstrate that although the primary structures of the three species are almost identical, small difference(s) exists among them especially at the amino-terminal portion.

4534906 Removal of impurities from human leukocyte interferon preparations

4534906 Removal of impurities from human leukocyte interferon preparations

4534906 Removal of impurities from human leukocyte interferon preparations: Paul D Johnston assigned to Genentech Inc

4534906 Removal of impurities from human leukocyte interferon preparations: Paul D Johnston assigned to Genentech Inc

Effects of human interferon preparations on neutrophil function.

We have studied effects of two partially purified human leukocyte (alpha) interferon (IFN) preparations (PIF-A and PIF-B) and a highly purified fibroblast (beta) IFN on the functional activity of normal human neutrophils (PMNs). In vitro, PIF-B conferred a significant and dose-dependent enhancement of chemiluminescence (CL) induced both by phagocytosis and a soluble stimulus, f-Met-Leu-Phe, and decreased killing of Staph. aureus. In contrast, PIF-A caused only a slight inhibition of bactericidal activity and had no effects on CL. beta-IFN had no effects on either bactericidal activity or CL. Migration under agarose was decreased with all of the IFN but phagocytosis and release of enzymes was not affected. PMNs from seven patients treated with PIF-A for multiple myeloma exhibited increased CL responses but no other PMN functions were affected. The findings that human IFN preparations affect PMN functions indicate that high-dose IFN therapy of immunocompromised patients should be carefully evaluated for the possibility of increased infectious complications.

The failure of human leukocyte interferon to influence the growth of human glioma cell populations: in vitro and in vivo studies.

Five high-grade (3 grade III and 2 grade IV) astrocytoma tumour cell populations were treated with a preparation of Human Leukocyte Interferon either in monolayer cell culture or as multicellular spheroids in vitro or as xenografts growing in immune-deprived mice in vivo. A moderate and transient sensitivity was seen in one grade III tumour when tested in both of the in vitro assays, but no inhibition of growth was seen in vivo. Two tumours which were apparently resistant to Interferon treatment responded to orthodox chemotherapy. When used in conjunction with BCNU, Interferon was not effective in prolonging delay in tumour growth. It is concluded that Interferon is unlikely to be an effective agent in the treatment of malignant brain tumours.

Characterization of a UV-resistant strain, UVr-10, established from a human clonal cell line, RSb, with high sensitivity to UV, 4NQO, MNNG and interferon.

Characterization was performed of a UV-resistant variant strain. UVr-10, derived from a human clonal cell line, RSb, with high sensitivity not only to the lethal effect of 254-nm far-ultraviolet (UV) irradiation but also to the effects of 4-nitroquinoline 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and to the cell proliferation inhibition (CPI) effect of human leukocyte interferon (HuIFN-alpha) preparations. Colony-formation assays confirmed the increased resistance of UVr-10 cells to both UV and 4NQO, but no increased resistance to MNNG. The marked recovery from the inhibition of the total cellular DNA synthesis of UVr-10 cells, estimated by [methyl-3H]thymidine ([3H]dThd) uptake into the cellular DNA materials, was seen during 6 h after UV irradiation or 4NQO treatment even under the conditions without the recovery uptake into those of the parent RSb cells, but not during 6 h after MNNG treatment. Comparative studies on the activity of DNA repair synthesis between UVr-10 and RSb cells, by measuring the extent of UV-, 4NQO- or MNNG-induced unscheduled DNA synthesis (UDS) and DNA repair replication, revealed an increased activity of UVr-10 cells to UV and 4NQO but no significant increase of the activity to MNNG. These results suggest that increased DNA repair activities of a UVr-10 cell line may account for its becoming resistant to the lethal effect of UV and 4NQO. Concerning the CPI effect of HuIFN-alpha, UVr-10 cells showed increased resistance. Further, the DNA synthesis activity of UVr-10 cells was not so inhibited by HuIFN-alpha exposure as that of RSb cells. However, HuIFN-alpha-exposed UVr-10 cells showed more enhanced levels of activity of pppA(2'p5'A)n synthetase (2-5A synthetase) than the exposed RSb, thus suggesting that HuIFN-alpha could exert enough intracellular effect even in UVr-10 cells. The implication of the increased resistance of UVr-10 cells to the effects of UV, 4NQO and HuIFN-alpha, but not to those of MNNG, is discussed.

Interferon increases HLA synthesis in melanoma cells: interferon-resistant and -sensitive cell lines.

We report that human leukocyte interferon preparations increase the expression of beta 2-microglobulin by 100-200% on the surface of normal fibroblast and melanoma cell lines sensitive to interferon. This increase in expression can be correlated with an increase in HLA synthesis as measured by incorporation of [35S]methionine in these antigens. This enhanced HLA synthesis, which is 5- to 17-fold, is time dependent and dose related. Synchronized cells in the G0/G1 phase of the cell cycle appear to be more sensitive to this interferon action. Neither an increase in surface expression nor in HLA synthesis is observed in a melanoma cell line resistant to the antiviral and antigrowth effects of interferon. Furthermore, there appears to be a stronger correlation between this increased HLA synthesis and the antiviral function than between it and the antiproliferative action of interferon.

Modulation of the human in vitro antibody response by human leukocyte interferon preparations: Parker MA, Mandel AD, Wallace JH, Sonnenfeld G:Cell immunol 58: 464, 1981

Modulation of the human in vitro antibody response by human leukocyte interferon preparations: Parker MA, Mandel AD, Wallace JH, Sonnenfeld G:Cell immunol 58: 464, 1981

Human lymphocyte production of corticotropin and endorphin-like substances: association with leukocyte interferon.

Human leukocyte interferon (hIFN-alpha) preparations contain immunologically and biologically recognizable endorphin and corticotropin-like (ACTH-like) activities. The ACTH bioactivity was demonstrable only after pepsin or acid treatment. Highly purified hIFN-alpha was composed of two molecular species of interferon (18,500 and 23,000 daltons). Endorphin activity was associated with both of these molecules. Pepsin treatment of the 23,000-dalton but not the 18,500-dalton hIFN-alpha generated ACTH activity. In acid, the 23,000-dalton hIFN-alpha broke down into the 18,500-dalton form and ACTH (4500 daltons). The ACTH derived from hIFN-alpha by pepsin digestion comigrated with a purified ACTH standard in NaDodSO4/polyacrylamide gel electrophoresis. hIFN-alpha-producing lymphocytes showed positive immunofluorescence after staining with highly specific antisera to ACTH alpha-(1-13) and gamma-endorphin. Essentially 100% of the human peripheral lymphocytes were capable of producing both ACTH and gamma-endorphin-related substances, presumably associated with hIFN-alpha. These results strongly suggest a circuit between the immune and neuroendocrine systems which involves neuroendocrine hormone-like substances, some of which are associated with hIFN-alpha

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.

31] Preparation of human leukocyte interferon in the presence of tunicamycin and its characterization

This chapter presents methodology to show that the molecular size heterogeneity of human leukocyte interferon (HLIF) can be eliminated when interferon is produced in the presence of tunicamycin, a specific glycosylation inhibitor. The evidence suggests that the molecular basis of this heterogeneity may be the relative extent of glycosylation of its molecular components. Tunicamycin is used as a specific inhibitor, to probe the glycosylation status of HLIF. Tunicamycin is shown to alter the properties of HLIF molecules that are released into the production media. The use of tunicamycin as a probe, is particularly revealing, because its inhibitory action suggests the N-glycosidic nature of linkage between the polypeptide and its saccharide moiety. HLIF is known to contain two distinct molecular weight components when subjected to sodium dodecyl sulfate-disc gel electrophoresis: 21,000 and 16,000 dalton components in the approximate ratio of 3:7. If HLIF contains sialic acid, then impairment of its glycosylation by tunicamycin should alter its behavior on isoelectric focusing.

64] Measurement of effects of human leukocyte interferon on human granulocyte colony-forming cells in vitro

Publisher Summary This chapter describes the use of in vitro semisolid agar culture system in delineating the human leukocyte interferon (HLIF) effects on human granulopoiesis. The various concentrations of HLIF are incorporated in the upper layers of the agar culture dishes, and the target marrow cells are stimulated by HPCM present in the underlayers. To prepare HPCM, a source of colony-stimulating activity (CSA), a freshly delivered human placenta is washed with sterile saline and then placed on a sterile stainless steel tray inside a laminar vertical-flow hood. Since the HLIF preparations used in experiments did not stimulate any colony or cluster formation in the absence of human placental conditioned medium (HPCM), the increase in clusters noted with increasing HLIF concentrations suggested either an inhibition of proliferation at a latter stage of the granulopoietic maturational process or a prolongation of the generation time of each successive mitosis.

69] Purification of human leukocyte interferon by two-dimensional polyacrylamide gel electrophoresis

The homogeneous human leukocyte interferon can be obtained by devising a purification scheme that includes the elimination of heterogeneity by chemical oxidation with sodium periodate, molecular sieve column chromatography, affinity column chromatography, and a modification of the two-dimensional gel electrophoresis technique. A modified two-dimensional gel electrophoresis system is used to further purify the human leukocyte interferon preparations. The modifications made are (1) the omission of reducing agents, such as β-mercaptoethanol, in all solutions and (2), the increased length of the isoelectric focusing gel from l I cm to 24 cm with the corresponding increase of the second-dimension sodium dodecyl sulfate (SDS) gel slab to 35 cm wide by 30 cm in height. Homogeneous spots of two forms of human leukocyte interferons are obtained with this method, each with a final specific activity of about 10 9 units per milligram of protein. This method of two-dimensional gel electrophoresis of periodatetreated preparations can be used to isolate two distinct species of human leukocyte interferon molecules differing in human to bovine activity ratios.

Role of G0-G1 arrest in the inhibition of tumor cell growth by interferon.

We report here that human leukocyte interferon preparations are capable of influencing the transition of human melanoma cells from the "A" state to the "B" phase. Human melanoma cells that enter a quiescent stage at high cell density are more sensitive to the cytostatic action of interferon than those that continue to proliferate under similar conditions. Cell cycle perturbations caused by interferon in these cells include a decreased transition rate out of G0-G1 ("A" state) into S ("B" phase) and a prolongation of S. These findings support the idea that some metabolic event is required for progress through the G0-G1 phase of the cell cycle and is susceptible to interferon action.

Interferon and spontaneous cytotoxicity in man. I. Enhancement of the spontaneous cytotoxicity of peripheral lymphocytes by human leukocyte interferon

A purified preparation of human leukocyte interferon used at this hospital in the treatment of malignant diseases was tested for its ability to modify the spontaneous cytotoxicity of peripheral lymphocytes from healthy donors. The inhibitory effect of allogeneic lymphocytes on the (3H)thymidine incorporation of a lymphoblastoid cell line, Raji, was augmented by the presence of interferon or by pretreatment of the lymphocytes with interferon. This form of pretreatment also increased lymphocytes' capacity for reducing the number of surface-adherent tumor cells in a microassay. Moreover, lymphocytes treated with interferon exhibited an enhanced cytotoxic capacity for target cells on incubation with such cells labelled with 51CR.

Characterization of the heterogeneous molecules of human interferons: differences in the cross-species antiviral activities of various molecular populations in human leukocyte interferons.

SUMMARY Human leukocyte interferon (HuLeIF) preparations were separated into populations of molecules with different sizes, by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and with different charges, by isoelectric focusing. These populations with different sizes and charges were analysed for their antiviral activity on homologous cells and on heterologous (bovine) cells. The distribution of interferon activity into two broad peaks by SDS-PAGE was similar whether assayed on human or bovine cells. However, within these peaks, the relative ratio of the activity in human cells and bovine cells varied significantly: while most of the size components had similar human/bovine cell activities, the fastest migrating component (apparent mol. wt. ∼ 13500) was more than 100 times more active on bovine cells than on human cells. The peaks of activity in isoelectric focusing were distributed from pH 5.5 to 7.0. There was generally correspondence between human and bovine cell activities, but while the more neutral pH range peaks were consistently slightly more active on human cells than on bovine cells, the more acid range peaks were always slightly more active on bovine cells than on human cells. However, with the most acidic peak, there was more than 100 times greater activity on bovine cells than on human cells. These data show that the heterogeneity of HuLeIFs is greater than merely two size populations, and data confirm that different forms of human leukocyte interferon can vary markedly in biological activity.

Elimination of size and charge heterogeneities of human leukocyte interferons by chemical cleavage

Human leukocyte interferon (HuLeIF) preparations contain distinct molecular forms of interferon exhibiting significant heterogeneties in sizes when analyzed by electrophoresis in sodium dodecyl sulfate (NaDodSO(4))/polyacrylamide gels, migrating in two broad bands of activity with peaks at about 21,000 and 15,000 daltons. HuLeIFs exhibit extensive charge heterogeneities when analyzed by isoelectric focusing, resolving into several major peaks of approximately equal activity distributed from pH 5.7 to 7.0. When HuLeIF preparations are treated with 0.01 M sodium periodate buffer, pH 4.5, at 4 degrees , both the size and charge heterogeneities rapidly disappear: periodate-treated HuLeIF migrates as a single, narrow band at 15,000 daltons in NaDodSO(4)/polyacrylamide gels and focuses as a single, narrow band at pH 5.7. Quantitative considerations suggest that either the larger, heterogeneously charged HuLeIFs are converted to the smaller, size- and charge-homogeneous interferon by extensive chemical deglycosylation, or, alternatively, the smaller, 15,000 dalton, pH 5.7 interferon is much more stable to periodate treatment than are the other interferon forms. However, the activity of each of the variously charged forms of HuLeIF isolated from focusing gels exhibited the same stability as the pH 5.7 component; similarly, the activity of each of the size-forms of HuLeIF isolated from NaDodSO(4)/polyacrylamide gels exhibited the same stability as the 15,000-dalton interferon.

Production and preparation of human leukocyte interferon

Production and preparation of human leukocyte interferon

Effects of Interferon on the Human Clonal Cell Line, RSa: Inhibition of Macromolecular Synthesis

Multiplication of the human clonal cell line, RSa, is completely inhibited by human leukocyte interferon preparations. Synthesis of DNA and protein is markedly reduced in these cells in proportion to the concentration of interferon applied. Interferon treatment leads to accumulation of cells of an epithelial morphology which do not enter the division cycle. It is suggested that the growth inhibitory effects of interferon on RSa cells may result from these effects.