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.

Antiviral activity of Win 41258-3, a pyrazole compound, against herpes simplex virus in mouse genital infection and in guinea pig skin infection.

Win 41258-3 (4-[6-(2-chloro-4-methoxyphenoxy)hexyl]-3,5-diethyl-1H-pyrazole methane sulfonate) has in vitro and in vivo activity against herpes simplex virus types 1 and 2. In cell culture, a concentration of 2 microgram/ml produced a greater than 50% inhibition of plaque formation of herpes simplex virus type 2, and 3 microgram/ml produced a 100% reduction of herpes simplex virus type 1. Win 41258-3 was effective against herpes simplex virus types 1 and 2 in mouse genital infection after intravaginal administration. Win 41258-3 was administered to mice at 4 h postinfection with solutions containing 1.25, 2.5, 5, or 10% of the compound in saturated tampons. Therapy resulted in a high survival rate (80 to 100%) of treated animals versus 20 to 30% of placebo-treated controls. Win 41258-3 was also effective in guinea pig skin infection produced by herpes simplex virus type 1. Solutions of 2.5, 5, and 10% Win 41258-3, applied to the skin starting 24 h postinfection, resulted in rapid suppression of development of herpetic vesicles and significant reduction of the virus titers in the lesion sites.

In vitro antimicrobial activity of rosoxacin against Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum.

The antimicrobial activity of rosoxacin, a new quinoline antibacterial compound, was determined against the causative organisms of three sexually transmitted diseases. Rosoxacin demonstrated a high degree of activity against Neisseria gonorrhoeae clinical isolates, with the minimal inhibitory concentrations for 50% of these being 0.03 microgram/ml. The corresponding minimal inhibitory concentrations for penicillin, ampicillin, tetracycline, and spectinomycin were 0.25 U/ml, 0.125 microgram/ml, 0.25 microgram/ml, and 16 microgram/ml, respectively. Eleven strains of Chlamydia trachomatis were inhibited by 5 microgram of rosoxacin per ml, and each of seven Ureaplasma urealyticum strains was inhibited by 2 to 8 microgram of rosoxacin per ml. The results of these susceptibility studies, coupled with those of an earlier evaluation of the pharmacokinetics of rosoxacin, provide support for extending or undertaking clinical evaluations of this compound against infections with N. gonorrhoeae, C. trachomatis, and U. urealyticum.

Role of infectious secretions in the transmission of rhinovirus.

In a series of studies aimed at investigating the role of environmental surfaces in the transmission of certain respiratory virus infections, it was shown that small amounts of nasal mucus containing rhinovirus (infectious mucus) can spread from fingertips to door knobs, faucet handles, or other environmental surfaces and remain infectious for many hours. These surfaces can serve as a reservoir of virus and may provide sufficient infectious material to contaminate hands. Recent studies have shown that once virus is on the fingers, it may be transferred to the nasal and conjunctival mucosa by means of autoinoculation. It has been estimated that as little as 1.0 plaque-forming unit can produce an infection in a susceptible human. In the present experiments, the amount of rhinovirus transmitted from fingers contaminated with infectious mucus to environmental surfaces and from there onto the fingers of a volunteer who touched the contaminated objects was quantitated, and the efficiency of transfer was studied. From 3 to 1,800 plaque-forming units of rhinovirus were recovered from the fingertips of volunteers (recipients) who handled either a door knob or a faucet that had previously been manipulated by another volunteer (donor) whose fingers were contaminated with infectious mucus. The average amount of rhinovirus recovered from the fingers of the recipients was approximately 13.5% of the amount recoverable from the fingers of the donor. In experiments in which there was direct hand-to-hand contact between donor and recipient, about 6.7% of the virus present on the fingertips of donors was recoverable from the recipients.

Antibacterial activities, nephrotoxicity, and ototoxicity of a new aminoglycoside, Win 42122-2.

Win 42122-2 is a new aminoglycoside antibiotic obtained from a mutant strain of Micromonospora purpurea. In vitro and in vivo comparisons of Win 42122-2 with gentamicin and amikacin revealed that Win 42122-2 generally was less active than gentamicin against Pseudomonas and many Enterobacteriacae, especially Klebsiella and indole-negative Proteus. Against most gentamicin-susceptible isolates, Win 42122-2 was more active than amikacin. Gentamicin-resistant clinical isolates were usually resistant to Win 42122-2, although it was active against certain gentamicin-resistant organisms, depending upon the aminoglycoside-modifying enzymes harbored by the organism. However, Win 42122-2 was markedly less toxic than gentamicin in subacute nephrotoxicity studies in rats, ototoxicity experiments in guinea pigs, and ataxia determinations in cats. This series of antibacterial determinations and toxicity evaluations indicated that the reduced toxicity of the antibiotic may be sufficient to provide an improved therapeutic ratio over gentamicin and other aminoglycosides, even though Win 42122-2 is less potent than gentamicin against some bacteria.

Preliminary studies of the mode of action of arildone, a novel antiviral agent.

Arildone (also known as Win 38020), a novel aryl diketone, inhibited replication of herpes simplex virus type 2 in tissue culture by interfering with an event that occurs prior to 6 h postinfection. The inhibition could be partially reversed by washing. Although the exact mechanism of action is unknown, neither viral deoxyribonucleic acid nor viral proteins were synthesized in the presence of arildone.

Cutaneous herpes simplex virus infection of guinea pigs as a model for antiviral chemotherapy.

The kinetics of Herpesvirus hominis (Type 1) replication and lesion development in guinea pig skin were determined. The data indicate that lesion scores did not always reflect virus content. Virus replication was detected prior to appearance of lesions and maximum virus content preceded maximum lesion score. Lesions resolved slowly while virus content declined rapidly to an undetectable level. The utility of the guinea pig skin-herpesvirus model for the evaluation of ara-A and kethoxal was studied. Ara-A treatment at three dose levels suppressed lesion development and at the two highest dose levels lesion development was delayed. Lesion virus content, when determined during the period of maximum virus replication, was not affected by treatment. Kethoxal markedly suppressed lesion development and virus growth. Aspects of this experimental model typify cutaneous herpes simplex disease of man. Studies designed to further assess its utility for evaluating antiviral drugs seem warranted.

Sensitivity of rhinoviruses to human leukocyte and fibroblast interferons.

Rhinoviruses were tested for sensitivity to human interferon from two sources: human leukosytes induced with Sendai virus and human diploid fibroblasts induced with polyriboinosinic-polyribocytidylic acid. Twenty-five serotypes of rhinovirus were tested against fibroblast interferon in HeLa cells, and five serotypes were evaluated in fibroblast cultures against fibroblast and leukocyte interferons. Sensitivity varied widely in the HeLa cell assay; rhinovirus type 10 was inhibited by approximately 20-40 units of interferon, whereas rhinovirus type 15 was not inhibited by 5,120 units; the significance of this observation is unclear. In contrast, when fibroblast cultures were used in the assay system, five selected serotypes, including rhinovirus types 10 and 15, had similar levels of sensitivity (0.5-5.0 units) to fibroblast or leukocyte interferon.

Biohazard potential: recovery of infectious virus from the liquid nitrogen of a virus repository.

Flame-sealed glass ampules containing vesicular stomatitis (VSV) were submerged in a liquid nitrogen (LN2) refrigerator for storage. Onseveral occasions cracked or shattered ampules were discovered upon removal from the refrigerator. Subsequently, VSV was recovered from the LN2 of the virus repository indicating a source of potential danger to those employing glass ampules submerged in LN2 for preservation of pathogenic organisms.

Interferon administered orally: protection of neonatal mice from lethal virus challenge.

Interferon was identified in the milk of mice injected with an interferon inducer. The kinetics of interferon appearance in serum and in milk were similar, but maximum concentrations in milk were 10 to 20 percent of those in serum. Interferon administered orally to neonatal mice was detected in their serums. Significantly more newborns survived an oral challenge with vesicular stomatitis virus when interferon had been induced in the lactating mothers.

Evaluation of gentamicin for use in virology and tissue culture.

Data are presented comparing gentamicin to penicillin and streptomycin (Pen-Strep) in tissue culture medium with respect to a number of parameters associated with virology and tissue culture. Unlike Pen-Strep, gentamicin was stable at pH 2 to 10 for 15 days at 37 C in tissue culture medium, and its activity was unaffected by the presence of serum. Moreover, it was stable to autoclaving. Twenty cell types replicated normally at the suggested concentration of 50 mug/ml, and all cells were unaffected by 20 times this concentration. Evidence for its practical use in virus studies was demonstrated in that (i) it was not viricidal to ribonucleic acid or deoxyribonucleic acid viruses at 40 times the suggested concentration at 37 C, (ii) the size and number of plaques were not affected by 20 times the suggested concentration, (iii) interferon assays and production were unaffected by 20 times the suggested concentrations. Gentamicin may be uniquely useful for shipment of clinical specimens and long-term tissue culture and virus studies.

Effect of antiviral agents in equine abortion virus-infected hamsters.

Equine abortion virus, a member of the herpesvirus group, produces a lethal infection in hamsters. With this system, the protective effect of certain inhibitors of deoxyribonucleic acid viruses, inducers of interferon and exogenous interferon, was evaluated. Of the various agents studied, 9-beta-d-arabinofuranosyladenine markedly suppressed mortality, and 5-iodo-2'-deoxyuridine, distamycin A, and N-ethylisatin beta-thiosemicarbazone were inactive. Of the inducers tested, statolon, ultraviolet-irradiated Newcastle disease virus, and polyriboinosinic:polyribocytidylic acid (poly I:C) were protective, and endotoxin, polyacrylic acid, and polymethacrylic acid did not protect. Administration of exogenous interferon did not afford protection. Statolon and ultraviolet-irradiated Newcastle disease virus induced circulating interferon in hamsters, whereas poly I:C, endotoxin, and polyacrylic acid did not produce interferon. Because of the severity of the disease produced in hamsters by equine abortion virus, lack of protective activity by an agent in this system should not preclude possible efficacy against other members of the herpesvirus group.