Victor G. Edy

ADMINISTRATION OF HUMAN FIBROBLAST INTERFERON IN CHRONIC HEPATITIS-B INFECTION

One patient with hepatitis-B surface antigen (HBsAg)-positive chronic aggressive hepatitis, and two chimpanzee carriers of HBsAg, were each given seven doses of 10(7) I.U. of human fibroblast interferon over two weeks. The main differnce observed after treatment was a depression of the nucleocapsid hepatitis-0 core antigen in the liver, indicating that hepatitis-B virus infection is sensitive to interferon. Except for a short febrile reaction, no undesirable effects were seen after the administration of human fibroblast interferon which has not been previously given to man.

Human Fibroblast Interferon for Clinical Trials: Pharmacokinetics and Tolerability in Experimental Animals and Humans

Human fibroblast interferon (F-interferon) purified by adsorption on controlled-pore glass was given intramuscularly to patients at daily dosages of up to 20 × 106 units. Serum levels of antiviral activity were low or undetectable. In contrast, reasonably high serum titers were found in patients receiving interferon prepared from leukocytes (L-interferon). Similarly, in rabbits lower serum titers were seen with F-interferon than with L-interferon. These results are at variance with those obtained earlier (V. G. Edy, A. Billiau, and P. De Somer, J. Infect. Dis. 133:A18–A21, 1976). Possible explanations for this discrepancy are discussed. The F-interferon evoked febrile reactions, delayed skin reactivity, and transitory lymphopenia in humans. Some patients developed an allergic state of the reaginic type as evidenced by a weal and flare reaction after intradermal challenge. However, these patients did not show allergic symptoms after intramuscular injections. None of the side effects was severe enough to prohibit continuation of the treatment; most of them seemed to be due to contaminants not removed by the purification method. The possibility is considered that some of the side effects, e.g., delayed skin reactivity, are sufficiently specific to justify identification of the active principals. Images

Purification of interferon by adsorption chromatography on controlled pore glass.

Human fibroblast and mouse L929 cell interferons can be purified by adsoprtion to and subsequent elution from Controlled Pore Glass. Purification of 40 to 90-fold to specific activities of 1 to 5 times 10(6) units/mg of protein can be achieved in a single step, with good recovery of activity. Human leukocyte interferon does not bind to the glass and cannot be purified in this way.

Human fibroblast interferon for clinical trials: production, partial purification, and characterization.

The production and partial purification of human fibroblast interferon for performing clinical trials is described. The interferon was produced by superinduction (exposure to riboinosinic-ribocytidylic acid, cycloheximide, and actinomycin D) of large numbers of human diploid fibroblast cultures. The yield averaged 750 units per cm2 of culture area. The interferon was concentrated and purified by a two-step procedure involving acid desorption from controlled-pore glass beads and dialysis against polyethylene glycol. Human plasma protein was added as a stabilizer. The lyophilized end product had a specific activity of 0.5 × 106 to 1 × 106 units/mg of protein; it could be reconstituted for injection at a concentration of 2 × 106 units/ml. The composition of this interferon was characterized by crossed immunoelectrophoresis with polyspecific antibodies prepared against the principal sources of potential contaminants: human serum, calf serum, and normal human fibroblasts. Several components of each source were detected. Although the major component of calf serum, bovine serum albumin, was absent, other minor components were retained by the production and purification sequence. One of the main contaminants of fibroblast origin was found to be fibronectin. Images

Distinct Molecular Species of Human Interferons: requirements for Stabilization and Reactivation of Human Leukocyte and Fibroblast Interferons

Human fibroblast interferon preparations were completely stabilized to 100 degrees C by sodium dodecyl sulphate (SDS) in the presence of mercaptoethanol, but only a minor fraction of their activities were stabilized by SDS without mercaptoethanol. On the contarary, human leukocyte interferon preparations were completely stabilized to 100 degrees C by SDS in the absence of mercaptoethanol, but only a minor fraction of their activities were stabilized by SDS in the presence of mercaptoethanol. Furthermore, human fibroblast interferon preparations whose activities had been destroyed by boiling at 100 degrees C were completely reactivated by SDS under reducing conditions, but only a minor part of their activities were restored by SDS in the absence of reduction. On the contrary, human leukocyte interferon preparations whose activities had been destroyed by boiling at 100 degrees C were completely reactivated by SDS in the absence of reduction, but only a minor part of their activities were restored by SDS under reducing conditions. These data suggest that there are distinct molecular species of human interferons.

Human fibroblast and leukocyte interferons show different dose-response curves in assay of cell protection.

Preparations of human fibroblast and leukocyte interferons of similar potency show markedly different dose-response curves in an assay which measures the degree of protection of tissue cultures against virus c.p.e. The effect is observed with both vesicular stomatitis virus (VSV) and Mengovirus, and is not altered by purification of the interferons.

Stabilisation of mouse and human interferons by acid pH against inactivation due to shaking and guanidine hydrochloride.

Summary The inactivation of poly(I)·poly(C)-induced and virus-induced human fibroblast interferon, virus-induced human leukocyte interferon, poly(I)·poly (C)-induced and virus-induced mouse tissue culture interferon and virus-induced mouse serum interferon by shaking and by guani-dine hydrochloride was studied. Only human leukocyte interferon was completely resistant to these treatments. All other interferons examined were largely inactivated, but a small amount of each preparation was resistant to inactivation. The residual activity from either treatment was completely resistant to the other. Inactivation by both shaking and guani-dine hydrochloride was prevented in acid (pH 2 and 3.5) conditions. Possible explanations for these observations are considered.

Effect of human and mouse interferon and of polyriboinosinic acid·polyribocytidylic acid on the growth of human fibrosarcoma and melanoma tumors in nude mice☆

Abstract Human leukocyte interferon, human fibroblast interferon, mouse interferon and an interferon inducer, polyriboinosinic acid·polyribocytidylic acid (poly(I)·poly(C)), were examined for their effect on the growth of two human tumor cell lines in athymic nude mice. The human tumor cell lines were derived from a fibrosarcoma (designated HT- 1080 ) and a nonmelanotic melanoma (designated A- 375 ). Repeated doses of 5 mg/kg of poly(I)·poly(C), administered every other day starting 1 day after tumor cell inoculation, caused a significant inhibition of tumor growth. On the contrary, human (leukocyte or fibroblast) interferon and mouse interferon, at doses up to 5 × 10 6 units/kg , did not affect the growth of either of the two human tumor cell lines tested.

Antimetabolic activities of 2-fluoro-L-histidine

Abstract 2-Fluoro-L-Histidine inhibits protein synthesis in various cell cultures, as measured by 3 H-leucine incorporation. This histidine analog also inhibits the cytopathogenicity of a number of RNA and DNA viruses in primary and continuous cell cultures; it blocks the transformation of normal mouse (MO) cells by murine sarcoma virus, and partially suppresses the release of murine leukemia virus by a continuously infected mouse cell line (JLSV5). In human skin fibroblasts, it reduces the interferon-inducing capacity of poly(I)·poly(C). Inhibition of cell protein synthesis may be the common cause of the various effects. 4-Fluoro-L-histidine is essentially inert in all of the test systems examined.

Neutralization of Interferon Activity in Homologous and Heterologous Cells with Homologous and Heterologous Antibody

Abstract Human fibroblast interferon (HFIF) and human leukocyte interferon (HLIF) can protect mouse L-929 cells against the cytopathic effects of vesicular stomatitis virus (VSV), although HLIF is about 60 times and HFIF about 1200 times less active than mouse interferon (MIF). This relationship suggests that, with repsect to the mouse L-929 receptor, there exists a greater similarity between the active sites of MIF and HLIF than between MIF and HFIF. Employing antibodies directed against MIF, HFIF, and HLIF as a probe, we found interferon-cell interactions to be stronger for homologous than for heterologous combinations. A variable-fit model is proposed to explain the differences that interferons display in cross-species protection. Thus, the closer the fit between interferon and receptor, the greater the antiviral response.