Human non-malignant and malignant brain tumor derived cell cultures: proliferation and sensitivity to natural human fibroblast (beta) interferon.

Twenty-one human brain tumor biopsies were processed by mechanical and enzymatic methods to produce mixed cell suspensions. Cultures were prepared in small plastic flasks, and primary outgrowth occurred in 16/21 cultures. The period required for primary outgrowth ranged from 3 days to 14 days. We established serial propagation with 15/16 of the primary cultures. Sensitivity to HuIFN-beta was determined between passages 3 to 12, using a microassay based on cell viability (uptake of a supravital stain, neutral red). Extracted dye was quantified in acidic-methanol using the MR580 Microelisa Autoreader (Dynatech). We observed a broad range of responsiveness to the drug among the 12 cell-strains tested. Thus, 4 cell strains were relatively sensitive; 4 were resistant to 10(4) IRU/ml of purified HuIFN-beta. Four cell strains exhibited a level of responsiveness that was intermediate to that of these two groups. During propagation of these biopsies, cytopathology suggestive of paramyxovirus-infection appeared in 4 of the cell-strains. This characteristic was not uniformly associated with high sensitivity to human beta interferon which is a very potent, naturally occurring antiviral substance. Our results support the concept that information concerning sensitivity to HuIFN-beta and other cytostatic agents may be rapidly obtained using microcultures of brain tumor cultures in conjunction with supravital stain uptake studies. Additionally, these results suggest that further clinical studies with beta interferon should be undertaken to define the parameters which determine successful in vivo application.

Serum protein masking of the thermal sensitivity of the antiviral activity of purified human beta interferon: implications for clinical studies.

Human beta-interferon (HuIFN-beta) exhibits antiproliferative and antiviral properties. Successful clinical application of this drug depends on knowledge of the thermal stability of these activities under physiological conditions. In the present study, both the antiproliferative and antiviral activities were stabilized by the addition of very small quantities of serum proteins. This supplement was sufficient to mask the slightly higher thermosensitivity of the antiviral activity. In the absence of serum proteins, the values of both the half-life and the energy of activation were higher for the antiproliferative activity than for the antiviral function. Each had a half-life of at least 24 h and identical values for the energy of activation in the presence of proteins furnished by 1% fetal bovine serum. This study provides additional evidence to support a thesis recently advanced by Carter et al. that the antiproliferative domain of glycosylated beta interferon may be separable from the antiviral domain. It is concluded that the efficacy of HuIFN-beta, under clinical conditions, will not be seriously impaired by thermal inactivation. Antiviral assays of serum may be freely substituted for antiproliferative assays during pharmacological studies.

Characterization of a virus isolated from a case of human infectious hepatitis.

A new viral agent, isolated from the serum of an infectious hepatitis patient and designated as Agent II-B, was extensively studied in in vitro and in vivo systems. Agent II-B multiplied well in primary and serial animal cell cultures and in embryonated hen's eggs. Quantal and quantitative infectivity assays were performed in monolayers of African green monkey kidney cells. Effective concentrations of 5-iodo-2-deoxyuridine and guanidine hydrochloride did not inhibit the multiplication of Agent II-B, although 2-hydroxybenzyl-benzimidazole was an effective inhibitor. Essential lipids were not detected. The diameter of the agent is 16-25 nm and its buoyant density in CsCl equilibrium density gradients was 1.35 gm/ml. Neutralization test results did not reveal antigenic relatedness between Agent II-B and known human picornaviruses. Apparently, this new viral agent is a picornavirus which possesses the capacity to multiply in unexpectedly diverse cell types.

Inhibition of the multiplication of herpes simplex virus by aliphatic nitrosamines.

Non-toxic doses of dimethylnitrosamine and diethylnitrosamine inhibit the multiplication of herpes simplex virus type 1 and type 2 in HEp-2, Wi-38 and primary HEK cells. These results are somewhat unexpected, since both HEp-2 and Wi-38 cells lack detectable microsomal enzyme activity. The possible significance of the preliminary results is discussed.

Cellular compartmentalization of herpesvirus antigens during viral replication.

HEp-2 cells infected with herpes simplex virus develop five distinct immunofluorescent elements. Three (small nuclear granules, large nuclear granules, and an amorphous mass filling the nucleus) contain antigens which react with a rabbit serum prepared against boiled infected cell debris. A labeled pool of human antibody revealed antigens making up cytoplasmic granules and those responsible for a diffuse cytoplasmic fluorescence. All five immunofluorescent elements are demonstrable with a rabbit serum prepared against unheated infected cell debris. Viral antigens are segregated in the nucleus or in the cytoplasm; within the limits of detection, each antigen accumulates in one compartment only. The antigens responsible for the diffuse cytoplasmic fluorescence and for the amorphous nuclear mass are synthesized early in infection; they are formed in arginine-deprived cells and exist in a form which does not sediment on centrifugation at 79,000 x g for 2 hr. The antigens comprising the nuclear and cytoplasmic granules arise relatively late in infection; they are not formed in arginine-deprived cells, and they are readily sedimented on centrifugation at 79,000 x g for 2 hr. Heating (60 C for 2 hr) confers on one or more cytoplasmic viral antigens a new specificity; the altered antigens are demonstrable with labeled rabbit anti-boiled infected cell serum which normally does not combine with cytoplasmic antigens.