Marc De Ley

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

Metallothioneins in Human Kidneys and Associated Tumors

Human kidneys and their associated tumors (nonneoplastic kidney tissues from patients with a transitional cell carcinoma or an adenocarcinoma and the adenocarcinomas themselves) were evaluated for their Zn, Cd, and Cu contents as well as for their metallothionein (MT) level. The total Cd content was correlated with the MT content, and both values were significantly decreased in the adenocarcinomas in comparison with the other tissues. After extraction and separation by anion-exchange chromatography, MT-0 was identified in the nonneoplastic tissues from both the adenocarcinomas as well as the transitional cell carcinomas. Since until now MT-0 protein was only found in human fetal liver and in Zn-stimulated human monocytes, a possible role for this isoform as an oncofetal marker is hypothesized. Separation of the isoforms of MT by reversed-phase high-performance liquid chromatography and sequence analysis showed besides MT-1e and MT-1l the isoform-MT-1g, which is not expressed in the healthy kidney, and MT-1k, an isoform which is not yet demonstrated in renal tissues. We conclude that the expression profile of the MT isoforms in the kidney changes due to the presence of a tumor.

Messenger RNA of human immune interferon: Isolation and partial characterization

Abstract Suspension cultures of total and lymphocyte-enriched peripheral white blood cells were used as a source for the preparation of mRNA for human γ-interferon. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose and further purified on a preparative sucrose gradient. The RNA preparations were translated by oocytes into (a) protein(s) that showed biological activity in the interferon-assay. On sucrose gradient centrifugation the translatable fractions of the RNA migrated with a sedimentation coefficient of approximately 15 S, a value compatible with the molecular weight of human γ-interferon. The translation product was further characterized by serological cross reactivity with purified γ-interferon in neutralization reactions.