Interferon Preparations Research Articles

Enhancement of human monocyte cytotoxicity by both interferon-γ and -β and comparison to other stimuli

Recombinant interferon preparations caused a dose-dependent increase of human monocyte cytotoxicity to the K562 and Daudi cell lines. Both rIFN-γ and rIFN-β enhanced this function to a similar extent, while rIFN- α c had less effect when compared on the basis of their anti-viral effects. Endotoxin and concanavalin A increased basal monocyte cytotoxicity while phagocytosis of latex particles had no effect. The increased monocyte cytotoxic effect of rIFN-β was completely abrogated by monoclonal antibody to IFN-β, while monoclonal antibody to IFN-γ had no effect. However, monoclonal antibody to IFN-γ only reduced the increased cytotoxic effect caused by rIFN-γ by 25%. Catalase inhibited both basal monocyte cytotoxicity and the increase in cytotoxicity following addition of rIFN-γ only slightly, suggesting that mechanisms other than the oxidative burst were active and could be induced by rIFN-γ.

Interferon-induced organic mental disorders associated with unsuspected pre-existing neurologic abnormalities

Eleven patients ranging in age from 52 to 80 years, undergoing treatment for cancer with various preparations of interferon received neurobehavioral evaluations after experiencing unexpectedly severe organic mental disorders. The reactions ranged from delirium to extrapyramidal symptoms, mania, and neurasthenia with catatonic episodes. Computed tomographic (CT) scans of the brain disclosed unsuspected pre-existing neurologic abnormalities in all patients, including cerebral atrophy (6/11), brain metastases (4/11), and evidence of head injury incurred 40 years earlier (1/11). These findings suggest that cancer patients with pre-existing neurologic dysfunctions are at increased risk for severe interferon neurotoxicity.

Establishment and characterization of a human cell strain, KT, with high sensitivity to UV-killing and to cell proliferation inhibition by interferon.

We have established a human cell line, designated KT, with high susceptibility to both cell proliferation inhibition by interferon and UV‐killing, from a metastatic breast carcinoma. A tumor marker, a pregnancy‐specific glycoprotein (Schwangerschaftsprotein 1; SP1), and carcinoma characteristics compatible with ductal carcinoma of the breast were seen in KT cells by electron microscopic observation. KT cells were slightly more resistant to X‐ray‐induced toxicity than fibroblastic cells, termed KS, from the scalp of the patient. But, KT cells had lower cloning efficiency after UV irradiation than did KS cells: D0 values of 1.5 J/m2 and 7.2 J/m2, respectively. KT cells also appeared more susceptible to human interferon (HuIFN) preparations (α, β, γ and natural or recombinant) than did KS cells, as measured by cell colony formation ability, proliferation rates, and [3H]deoxythymidine incorporation levels into acid‐insoluble cell materials. The sensitivity of KT cells to UV and HuIFN was greater than that of human RSa cells, a cell line with high sensitivity to both agents. KT cells had more capacity for UV‐induced DNA‐repair replication synthesis than did RSa cells, the capacity being much the same as that of KS cells. There was no significant difference in levels of antiviral activity induced by HuIFN and binding capacity for 125I‐labeled IFN‐αA between KT and KS cells. KT cells appeared refractory to cell proliferation inhibition by tumor necrosis factor (TNF) preparations.

Opposite effects of murine interferons on erythroid differentiation of friend cells

Interferons (IFNs), in addition to inducing an antiviral state in uninfected cells, are able to affect cell physiology, including cell differentiation. In this respect hematopoiesis is certainly the area in which most data have accumulated. In general IFN-α or-β inhibit cell growth of normal progenitors of hematopoietic lineages. In leukemia cell cultures IFNs may either stimulate or inhibit cell growth and differentiation. We report here different biological effects of murine (mu) IFN- α 1, -β and -γ species on the erythroid differentiation of dimethyl sulfoxide (DMSO)-induced Friend leukemia cells. Treatment with mu recombinant IFN-β enhances DMSO-induced FLC differentiation, whereas treatment with IFN- α 1 species as well as with natural and recombinant mu IFN-γ preparations only inhibits it. All these observed effects are neutralized by monoclonal antibodies against IFN-α, -β and -γ species. When mu fibroblast IFN (a mixture of α and β species) was used, the inhibitory effect attributable to IFN-α was partly overshadowed by the simultaneous presence of a majority of IFN-β molecules exerting the opposite effect. This is in agreement with data obtained neutralizing fibroblast IFN preparations with excess amounts of monoclonal antibodies against IFN-β (G. B. Rossi etal., 1988, “The Status of Differentiation Therapy of Cancer,” Raven Press, New York) and with our previous reports indicating that mu fibroblast IFN can either enhance or inhibit DMSO-induced differentiation when administered at low (<500 U/ml) or high (>5000 U/ml) doses, respectively. The inhibitory effect of IFN- α 1 on cell differentiation is not linked to any inhibitory effect on cell growth. Results obtained analyzing the effect of IFN- α 1 and -β on various IFN-resistant FLC clones indicate that different mechanisms underlie the stimulatory effect of IFN-β and the inhibitory effect of IFN- α 1. These results shed light on possibly distinct physiological roles of the various species of IFNs.

Effects of Different Interferons on the Replication of Herpes Simplex Virus in Human T Lymphocytes

Human T cells were activated with PHA and after 48 h they were treated with preparations of different human interferons (IFNs). After a further incubation period of 24 h, the cells were washed and infected with herpes simplex virus type 1 (HSV-1). Reduced virus titers were observed in T cells pretreated with IFN-alpha or IFN-beta, whereas IFN-gamma showed no antiviral effects. These findings indicate that IFN-gamma does not exert protection against viral infection in this system. We investigated the synthesis of HSV-coded early and late proteins in T cells pretreated with IFN-alpha and IFN-beta. Immunofluorescence studies revealed inhibition of expression of the immediate early alpha-protein ICP 4. Induction of DNA-polymerase, a viral beta-protein, was inhibited both by IFN-alpha and IFN-beta in a dose-dependent manner. As suggested by SDS-polyacrylamide gel electrophoresis, other viral beta- and gamma-proteins of HSV were inhibited by IFN-alpha and IFN-beta as well.

Effect of Sendai inducing virus in cytopathic effect inhibition assay of bovine leukocyte interferon

Various methods for removal or inactivation of Sendai inducing virus from natural bovine leukocyte interferon (IFN) preparations were compared and the effects of Sendai virus in microtiter cytopathic effect (CPE) inhibition assay were investigated. Ultraviolet light and ultracentrifugation at either 108 000× g or 145 000× g for 2 h proved to be inadequate methods for complete removal of inducing virus from IFN preparations, as judged by the effects of residual virus in CPE inhibition assay. Optimal methods for inducer virus removal/inactivation were ultracentrifugation at 175 000× g or dialysis against pH 2.0 buffer. Sendai virus itself was capable of inducing IFN production in assay cells at concentrations commonly found in virus-induced IFN preparations.

Effects of human alpha-interferon on granulocyte-macrophage progenitor cells (CFU-GM) in vitro.

Two preparations of human interferon (IFN)-alpha were assessed for their influence on granulocyte-macrophage progenitor cells (CFU-GM) in vitro. Both highly purified human IFN-alpha Ly and recombinant IFN-alpha 2a suppressed CFU-GM colony formation in a dose-dependent manner using low-density bone-marrow target cells. Suppression of CFU-GM colony formation was accompanied by an increase in clusters. However, depletion of monocytes, T lymphocytes and B lymphocytes from low-density bone-marrow cells resulted in insensitivity of progenitor cells to IFN-alpha. These results demonstrate that the effects of human IFN-alpha on myeloid progenitor cells (CFU-GM) are mediated by accessory cells within the bone marrow.

Effects of human interferon on cellular response to UV in UV-sensitive human cell strains

Cells of human RSA cell line, with high senstivity to UV killing and low capacity for DNA repair, when pretreated with 1–100 units/ml of human interferon (HuIFN) preparations for more than 12 h before irradiation, acquired an enhancement of UV-induced DNA-repair relications synthesis in association with recovery from inhibition of total cellular DNA synthesis and UV survival. Prompt and transient induction of plasminogen activator activities was also found within 5 min after UV irradiation in the cells pretreated with HuIFN but not in the cells non-pretreated with HuIFN. The enhancement and induction effects of HuIFN were observed, irrespective of the kind of HuIFN preparation used (α, β or γ, and natural or recombinant) and in other UV-sensitive fibroblast cells which were derived from Cockayne syndrome and xeroderma pigmentosum fibroblasts (XP1KY). However, all of the enhancement of DNA-repair synthesis and the induction of plasminogen activator activities by HuIFN was suppressed by treatment with cycloheximide immediately after UV irradiation.

Antibacterial action of interferon preparations

Native leukocytic interferon preparations were shown to inhibit the growth of the bacterium Staphylococcus epidermidis strain 33 as well as to activate cell respiration, to induce the dissipation of transmembrane potential and disturb the initial structure of the cytoplasmic membrane.

Antibacterial action of interferon preparations

Native leukocytic interferon preparations were shown to inhibit the growth of the bacterium Staphylococcus epidermidis strain 33 as well as to activate cell respiration, to induce the dissipation of transmembrane potential and disturb the initial structure of the cytoplasmic membrane.

Interferon treatment markedly inhibits the development of tumor metastases in the liver and spleen and increases survival time of mice after intravenous inoculation of Friend erythroleukemia cells.

To investigate the effect of interferon treatment on the development of tumor metastases, DBA/2 mice were injected i.v. with 2 X 10(6) Friend erythroleukemia cells (FLC) (equivalent to about 5 X 10(5) LD50). FLC multiplied rapidly in the liver and spleen and all untreated or control treated mice died between 7 and 12 days. Daily treatment of mice with potent preparations of mouse interferon alpha/beta was initiated 3 to 72 hr after i.v. inoculation of tumor cells, at times when FLC were already present in the liver and spleen. Interferon treatment resulted in a 100 to 1,000-fold inhibition of the multiplication of FLC in the liver and spleen and a marked increase in mean survival time. Small numbers of tumor cells persisted in the liver and spleen in some interferon-treated mice and could be recovered by bioassay several weeks after tumor inoculation. Most interferon-treated mice died with tumor in the ensuing months. Three of 34 interferon-treated mice were considered cured as they were alive at 386, 325 and 284 days after tumor inoculation. Daily treatment of tumor-inoculated mice with human recombinant interferons alpha D and alpha BDDD, which had antiviral activity on mouse cells in culture, also increased the survival time of mice injected i.v. with FLC. The use of the interferon-resistant 3C18 line of FLC suggests that the marked inhibition of development of established liver and spleen metastases was not due to a direct effect of interferon on the tumor cells, but was host-mediated.

Mast Cell Differentiation in Cultures of T Cell-Depleted Mesenteric Lymph Node Cells from &lt;i&gt;Nippostrongylus brasiliensi&lt;/i&gt;&lt;i&gt;s&lt;/i&gt;-lnfected Mice

Lymphoid and bone marrow cells from normal and horse serum-immunized mice and lymphoid cells from Nippostrongylus brasiliensis-infected mice were cultured on monolayers of embryonic skin fibroblasts to analyze the factors which regulate the differentiation and proliferation of mast cells in vitro. Our results indicate that T cells can regulate the development of mast cells in vitro by either enhancement or suppression. In cultures of horse serum-immune spleen cells, inducer T cells are required for mast cells to develop. However, in cultures of mesenteric lymph node cells from N. brasiliensis-infected mice, inducer T cells are not required for mast cell development. This suggests that the development of mast cells may occur at discrete interleukin-3 (IL-3)-dependent and IL-3-independent stages. Mast cell precursors in the mesenteric lymph nodes of N. brasiliensis-infected mice may have already been acted on by inducer cells in vivo to become mast cell committed. While IL-3 does not appear to be required for mast cell development, these precursors do require the presence of a connective tissue microenvironment such as embryonic skin. The precursors can be inhibited from development by interferon preparations.

Antiproliferative Effects of Interferons against Human Bladder Carcinoma Cell Lines in vitro

In order to evaluate the antiproliferative effects of recombinant human interferon-gamma 2c (rHu IFN-gamma 2c), recombinant human interferon-gamma (rHu IFN-gamma), natural interferon-beta (IFN-beta), and their combination with cytotoxic agents, 17 different human bladder carcinoma cell lines were tested in vitro. The antiproliferative effects were compared in evaluating the tumor cell inhibiting potency of the different interferon (IFN) classes. It could be demonstrated that interferons have inhibiting effects on the bladder cancer cell multiplication rate, yet there are significant differences in the susceptibility of different IFN preparations on different cell lines. The cell lines BT1, RT4, EJ, 468P, 253J, SD, TCCSUP, and SW1738 can be defined as sensitive, T24, 647V, VM-CUB2, and J82 as semisensitive, HT1376, 5637, VM-CUB1, 639V and SW1710 as resistant upon treatment with IFN. The combination of rHu IFN-alpha 2c, IFN-beta, and rHu IFN-gamma seems to be more effective than treatment with rHu IFN-alpha 2c alone. The cytotoxic effect of doxorubicin on bladder cancer cells can be intensified by combining it with IFN.

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

From Old Results to New Perspectives: A Look at Interferon's Fate in the Body

In a previous report, we presented preliminary data on interferon-alpha A (IFN-alpha A) pharmacokinetics through the simultaneous study of distribution of the radiolabeled protein by numerical dynamic scintigraphy and the trichloroacetic acid (TCA)-precipitable radioactivity in whole blood and serum, as well as the radioactivity linked to blood mononuclear cells. The interferon preparation then used was contaminated with free iodine which gave parasite images and counts. Here we present further analysis of those data, enlightened by additional information collected in new studies with interferon preparations without free iodine. Interferon uptake by the kidneys, liver, and heart was a consistent finding. On the contrary, images in stomach and duodenum seem mostly related to their well-known iodine uptake. At present, whether interferon is present in saliva and nasal secretion is unclear. The extent to which blood radioactivity corresponded to blood cells or plasma was analyzed. TCA-precipitable radioactivity measured after administration of filtered radiolabeled interferon was mainly confined to serum. The marginal amount bound to cells seemed to be in leukocytes. Current research points to compare the pharmacokinetic profiles of IFN-alpha and IFN-gamma.

Interferon alpha/beta induces changes in the metabolism of polyenoic phospholipids and diacylglycerols in the livers of suckling mice.

Suckling mice were injected daily from birth for 10 days with potent preparations of mouse interferon alpha/beta. Interferon treatment resulted in a markedly lower concentration of polyunsaturated fatty acids (20:4 omega 6 and 22:6 omega 3) in the two principal liver phospholipids, phosphatidylcholine and phosphatidylethanolamine, than in livers of control-treated mice. This effect appeared to correlate with a low level of synthesis of polyunsaturated phospholipids in the livers of interferon-treated mice. Thus, in control mice, synthesis of species of polyunsaturated phospholipids increased markedly in the first 10 days of life, whereas in 10-day-old interferon-treated mice, the level of synthesis of species of polyunsaturated phospholipids was comparable to that in newborn mice. In parallel, a marked increase in the diacylglycerol content without change of its renewal was observed in the livers of interferon-treated mice. We suggest that interferon treatment results in an inhibition of one of the processes that leads to activation of the enzymatic systems responsible for the synthesis of species of polyunsaturated phosphatidylcholine and phosphatidylethanolamine in the liver of suckling mice. It seems likely that these results are related to the inhibition of liver cell maturation and the marked cell necrosis that are observed in interferon-treated suckling mice.

Effect of recombinant interferons alpha and gamma on human bone marrow- derived megakaryocytic progenitor cells

Interferons (IFNs) have been shown to suppress the proliferation of human pluripotent hematopoietic progenitor cells, CFU-GEMM, and committed erythroid (BFU-E, CFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. However, no information is yet available concerning the effect of IFNs on human megakaryocytic progenitor cells CFU-Mk. Furthermore the mechanisms underlying the inhibitory activity of IFNs are still controversial. Therefore highly purified recombinant IFN preparations, rIFN-alpha and rIFN-gamma, were assessed for their influence on in vitro growth of human bone marrow-derived CFU-Mk as well as CFU-GEMM. In addition, the role of hematopoietic accessory cells, that is, adherent cells and T lymphocytes, in the mediation of the suppressive effect of rIFNs was examined. When added to unseparated bone marrow cells, both rIFN preparations significantly inhibited colony formation with 50% inhibition of CFU-Mk occurring at 22 U/mL for rIFN-alpha and 59 U/mL for rIFN-gamma, while 50% inhibition of CFU-GEMM occurred at 59 U/mL for rIFN-alpha and 101 U/mL for rIFN-gamma. The suppressive effect of rIFN-alpha and rIFN-gamma was selectively abolished by monoclonal antibodies (MoAbs) against rIFN-alpha and rIFN- gamma, thus confirming that the inhibitory activity was due to the rIFN preparations used. The antiproliferative effect of rIFN-alpha and rIFN- gamma on CFU-GEMM growth was not associated with a decrease in the percentage of mixed colonies containing megakaryocytic cells as assessed by use of the MoAb C17.28 against platelet glycoprotein IIIa. Removal of adherent cells and T lymphocytes from the target bone marrow cells had no influence on the suppressive effect of rIFN-alpha, whereas it significantly reduced the inhibitory effect of rIFN-gamma on the growth of megakaryocytic colonies and the other hematopoietic progenitors. The data indicate that (1) human megakaryocytopoiesis is markedly inhibited by rIFN-alpha and rIFN-gamma, and (2) the inhibitory effect of rIFN-alpha is due to a direct action on hematopoietic progenitor cells, whereas the effect of rIFN-gamma is mediated to a significant degree through accessory cell populations.

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.

A phase I clinical trial of recombinant DNA gamma interferon.

Recombinant gamma interferon (r-GIFN) demonstrates in vitro and in vivo characteristics that contrast with those of alpha and beta interferons. It has relatively weak antiviral properties, yet relatively potent immunomodulatory effects. A phase I trial was performed with r-GIFN (specific activity 2.6 X 10(6) IU/mg protein), administered as a continuous intravenous (IV) infusion over 24 hours for five days (Cl X 5) and repeated every 28 days. This schedule was chosen based on the short half-life of r-GIFN in animal systems and the in vitro augmentation of biologic effects with continuous exposure to interferons. Twenty-one patients with refractory solid tumors received 46 evaluable courses of therapy. The dose-limiting toxicities included fever, flu-like symptoms, cardiovascular toxicity, and neurotoxicity. The cardiovascular toxicity included hypotension and one episode of cardiac ischemia with chest pain. Neurotoxicity consisted of lethargy and confusion. These toxicities were reversible, and although dose-limiting, occurred sporadically throughout all dosage levels. Mild to moderately severe non-dose-limiting toxicities included nausea and vomiting, leukopenia, and liver function abnormalities. Other infrequent toxicities included hypocalcemia, diarrhea, constipation, and alopecia. The maximally tolerated dose of r-GIFN on this schedule is 0.5 X 10(6) IU/m2/d. Partial responses were seen in one patient with metastatic melanoma and in one patient with renal cell carcinoma. Toxicity and antitumor activity were seen at doses where interferon serum levels could not be detected by radioimmunoassay. In addition, the toxicity and antitumor activity seen were at much lower doses than previously described for shorter infusion schedules of other recombinant gamma interferon preparations. Differences in biologic activity of interferon preparations and/or differences in scheduling may account for this variability. Although this study defines a recommended phase II dose of r-GIFN based on the maximally tolerated dose, the optimal therapeutic index may exist at a lower dosage level.

Interferons and Their Clinical Value

Interferons were discovered from their antiviral effects. It is now known that they are the products of families of genes found in many vertebrate species and that they bind to cell surfaces and produce a range of effects, varying from an antiviral state to increased expression of tissue transplantation antigens, decreased cell division, and modified immune responses, among others. Interferons are induced by exposure to viruses and double-stranded RNA, and one class, gamma interferon, is a lymphokine produced after a mitogenic or antigenic stimulus. The genes for human alpha, beta, and gamma interferons have been cloned and sequenced, and some have been expressed. When given parenterally to humans, interferons cause signs and symptoms such as fever, tachycardia, and malaise, and they may well be responsible for symptoms of this sort in viral infections. Preparations of natural and recombinant alpha interferon have been shown to be active in prophylaxis and treatment of certain viral infections.