Effects of nitrosocarbaryl on BALB/3T3 cells.

Carbaryl(N-methyl-1-naphthylcarbamate) and its nitrosated product, N-nitrosocarbaryl, were tested for their effects of BALB/3T3 (clone A31) cells in culture. Nitrosocarbaryl, but not carbaryl, caused transformation of the BALB/3T3 fibroblasts, but neither chemical induced the complete expression of endogenous murine leukemia virus. Transformed cells differed from the parental control cells by loss of contact inhibition, change in morphology, growth in soft agar, growth to higher saturation densities, and tumorigenicity in normal newborn and irradiated weanling mice and athymic (nude) mice. Transformed clones were found to be negative for expression of RNA tumor virus antigens, viral reverse transcriptase, and infectious virus. Thus, it appears that nitrosocarbaryl can transform BALB/3T3 cells to tumorigenic cells with altered biological properties but without complete activation of RNA tumor viruses in the transformed cells. Expression of viral antigen in the transformed cells was inducible by iododeoxyuridine, indicating that the endogenous viral genome was retained in an unexpressed state.

Comparison of amantadine and rimantadine for prevention of type A (Russian) influenza.

The efficacies of 200 mg of daily doses of amantadine and of rimantadine for prevention of infection and illness due to influenza A/USSR/77 (H1N1) virus were compared in a double-blind, placebo-controlled study on a college campus. Frequencies of symptoms that might have been side effects of the drugs were not significantly different from those in placebo recipients. Analyses indicated that the trial was initiated late in the epidemic and that an age-related protective effect against A/USSR virus existed; seroconversion frequencies were 52/139 (37%) among 18-19-year-olds, 33/130 (25%) among 20-21-year-olds, and 5/39 (12.8%) among 22-24-year-olds. Among initially antibody-negative (less than 1 : 4 in complement fixing and neutralizing tests and less than 1 : 8 in hemagglutination inhibition tests) 18-19-year-old students, amantadine was associated with significantly fewer seroconversions (P = 0.01) and both less infection and milder illness than occurred in placebo recipients (P less than 0.05). Although rimantadine was not accompanied by reduction in frequency of seroconversions in the same age group, illness frequency and severity among seroconverters were significantly reduced when compared to placebo recipients (P less than 0.01). Amantadine and rimantadine appear suitable for use in young adults. Although other studies have suggested greater effectiveness of rimantadine than of amantadine against influenza, no evidence for this was seen in the present study which used both drugs at the same dose.

Flow cytometric identification of microorganisms by dual staining with FITC and PI.

The identification of microorganisms by flow cytometry was evaluated by using a double staining technique with propidium iodide and fluorescein isothiocyanate and a two dimensional analysis. A diverse group of 19 different species and strains of microorganisms was tested to determine if they could be differentiated by flow cytometry. The organisms tested displayed characteristic and distinct two dimensional fluorescent patterns which allowed ready grouping and differentiation into subsets of organisms. The slopes and correlation coefficients of the histograms and the ratio of red to green signals expressed these differences quantitatively and allowed organisms to be placed into one of three groups based on these values. In some instances, as with Streptococcus pneumoniae and pyogenes and Staphylococcus aureus and epidermidis, it was possible to distinguish between species of bacteria from the same genus. The use of dual dye labeling and flow cytometry provided a rapid method of identifying selected microorganisms and may be broadly applicable for the detection and identification of many bacteria and fungi.

Observations of cell function and morphology in the presence of silicone gel: an in vitro study.

The project described in this article used in vitro tissue culture techniques and flow cytometry to determine if there are alterations in cell morphology in those cells that have been placed in contact with a commercially available medical-grade silicone gel used in mammary implants. We were unable to demonstrate significant changes in cell cycle characteristics following in vitro exposure for 1 to 12 days using human fibroblasts, mouse fibroblasts, and Chinese hamster ovary cells.

Characterization of bacteria by mixed-dye fluorometry.

We describe an indirect detection method for bacterial identification and differentiation, based on selective adsorption of several fluorescent dyes. The lipid, protein, and nucleic acid components of fixed whole cells are stained with two mixtures, each containing two fluorochromes. The unadsorbed dyes were measured simultaneously with a video fluorometer [Clin Chem 22: 1483, 1976]. A dye-absorption matrix of the response can be generated, and we did so for each of nine bacteria. These responses were compared to a control or "complete" response matrix, and the response ratios of each bacterial species for each of the four dyes were calculated and plotted to obtain a characteristic pattern. From the response-ratio plots, plus simple pattern-recognition techniques, we could differentiate among all the bacteria. This rapid, sensitive technique is potentially applicable to a wide variety of bacteria.

Carcinoembryonic antigen production by human colorectal adenocarcinoma cells in matrix-perfusion culture.

Four human colorectal adenocarcinoma tumor cell lines, previously established and characterized in monolayer culture were grown in a matrix-perfusion culture system to determine the suitability of this technique for synthesis of carcinoembryonic antigen (CEA). Production of CEA in excess of 100,000 ng was attained from one cell line, SW 403, during 15-day growth trials. In growth trials and cell-free diffusion studies, CEA passed through membranes of 100,000-dalton molecular weight porosity but not 10,000 porosity. Using cell cultures of high, moderate, or low producers, CEA synthesis tended to reach a plateau after several days of culture and remained nearly constant as the cells attained a maintenance condition. Basic biologic characteristics of the cell lines, expressed as growth rates and CEA produced per 10(6) cells, were comparable in monolayer and perfusion culture. The high cell densities, (10(8) to 10(9) cells per ml) achieved in matrix perfusion made it possible to routinely obtain continuous high yields of CEA over an extended time period.

Identification of fluorescent Pseudomonas species.

Fluorescent pseudomonads may be identified through examination of the fluorescence "profiles" of diffusible pigments released into the growth medium. The profiles are obtained in the form of an emission-excitation matrix, the elements of which correspond to fluorescence intensity as a function of multiple exciting and emitting wavelengths. Use of a rapid scanning fluorometer to acquire these data provides a relatively fast method for identification of the microorganisms after incubation. This procedure can potentially be expanded for the identification of other microorganisms.

Multiparameter appraoch to the "fingerprinting" of fluorescent Pseudomonads.

A recent study has shown that an emission-excitation matrix could be used to provide a unique fingerprint for the selective identification and characterization of certain species of Pseudomonas. This paper describes the results of a systematic study of the variables that contribute to the uniqueness of the fingerprint. Additional insight concerning the nature of the fluorescent pigments of these species is gained by analysis with a high-performance liquid chromatograph/video fluorometer combination. While providing information about the analytical variables contributing to the analysis, this combination also added the variable of chromatographic retention time for unambiguous fingerprinting.

Ex vivo hemodialysis culture system for assessing the activity of cancer chemotherapeutic agents.

An ex vivo culture system was developed for assessing the activity of cancer chemotherapeutic agents against tumor cells. The system utilizes artificial capillary culture units and the technique of hemodialysis to expose tumor cells to a chemotherapeutic drug and its metabolites following injection of the drug into an experimental animal. This ex vivo culture system was used to test the activity of 5-fluorouracil (5-FU) against four human colorectal adenocarcinoma cell lines (SW 403, SW 480, SW 620, and SW 707). Cell killing by 5-FU or its metabolites in blood dialysate following intravenous injection was measured by determining colony formation of cells attached to plastic and suspended in 0.3% agar after short-term exposures of 1 to 2 h. The technique was shown to discriminate between the sensitivities of these cell lines and the respective sensitivities to the drug were reproducible. Kinetics of drug clearance from the host animal's blood were shown to be similar to that in humans. The results suggest the system may be useful for testing diverse drugs, including those requiring metabolic activation, against a variety of types of tumor cells.

A modified matrix perfusion-microcarrier bead cell culture system. I. Adaptation of the matrix perfusion system for growth of human foreskin fibroblasts.

Two strains of human foreskin fibroblast cells were incapable of sustained growth in a matrix perfusion culture system, possibly because of their inability to attach to the fiber surfaces. Addition of microcarrier beads to the extracapillary space allowed attaining high cell densities in excess of 10(7) cells per culture unit. Microcarrier beads were tested in hollow fiber culture devices containing membranes of 10(4) or 10(5) D nominal porosities. Best results were obtained when initial cell densities of at least (2-3) x 10(6) cells were used in units with 10(5) D pore size membranes and DEAE-Sephadex or polyacryl-amide microcarrier beads in the extracapillary space. This extension of the matrix perfusion system should be useful for growing other anchorage dependent cells while retaining the advantages of perfusion culture.

A modified matrix perfusion-microcarrier bead cell culture system. II. Production of human (beta) interferon in a matrix perfusion system.

Addition of microcarrier beads to a matrix perfusion cell culture system allowed growth of anchorage dependent human foreskin fibroblasts which would not grow in the culture units alone. The utility of the system for collection of cellular products was demonstrated by the induction and harvesting of human (beta) interferon. Interferon production was highest in perfusion cultures when medium was circulated throughout the induction and when inducer containing 100 mug/mL polyriboinosinic: polyribocytidylic acid was placed directly in contact with cells in the extracapillary space. These conditions provided 4-to-10-fold greater interferon yields per cell, and approximately 12-fold increases per vessel, than monolayer cultures. Perfusion grown cells produced interferon at a maximal level for 20 h postinduction compared to approximately 2 h for monolayer grown cells, thus giving a higher total yield of interferon. Other procedures increasing the efficiency of the system included priming with 50 U/mL interferon standard, reinduction of cells, use of antibiotic free medium, reduced serum concentrations, and in vitro aging of the cells.

Rapid screening for chemical carcinogens: transforming activity of selected nitroso compounds detected in a transplacental host-mediated culture system.

The transplacental host-mediated hamster cell culture system was used to test a variety of solvents and chemicals of unknown and known (positive and negative) activity for their ability to induce morphologic transformation of cells and growth in agar. Examination of approximately 13,000 colonies of cells from untreated animals yielded no transformants, thus demonstrating no spontaneous transformation in the system. Similar negative results were obtained after animals were treated with the solvents acetone, ethanol, dimethyl sulfoxide, dimethylformamide, and trioctanoin oil. Several known carcinogens, including benzo[a]pyrene, methylnitrosourethane, urethan, and diethylnitrosamine, were positive for transforming activity. Three pesticides, carbaryl, methomyl, and landrin, and their N-nitroso derivatives were tested. All the nitrosated forms had transforming activity, but only one of the pesticides, landrin, was positive. In all experiments conducted, results of the agar-growth test correlated well with tests for morphologic transformation. The transplacental hamster embryol cell culture system therefore detected transforming activity of N-nitroso compounds and some known carcinogens.

Exteriorized carotid-jugular shunt for hemodialysis of the goat.

A surgical technique was developed for the establishment of a permanent exteriorized artificial shunt between a carotid artery and a jugular vein of a goat. The shunt allowed continuous hemodialysis with an artificial kidney for hours or a prosthetic unit for days and intermittent experimental use of the animal for months.

Hemodialysis culture of Serratia marcescens in a goat-artificial kidney-fermentor system.

Hemodialysis was employed to simulate in vivo conditions for growth in mammalian blood, but without phagocytosis, by using the goat and Serratia marcescens as a host-parasite model. The blood stream was shunted surgically via prosthetic tubing from a carotid artery through the hollow-fiber membranes in an artificial kidney hemodialyzer and back into a jugular vein. The dialysate solution concurrently was pumped from a modular fermentor through the hemodialyzer jacket outside of the membranes and back into the fermentor. Hemodialysis between the two circuits was maintained continuously. When equilibrium was attained, bacteria inoculated into the dialysate circuit multiplied first exponentially at the maximal rate and then arithmetically at a lesser rate equally well under aerobic or anaerobic conditions. When a population of about 10(9) viable bacteria/ml was exceeded, the goat reacted acutely with signs of general toxemia, pyrexia, and leukopenia, apparently because of dialyzable toxic material produced by the culture. The maximal molecular size of the toxic material was defined relative to a rigid globular protein of 15,000 in molecular weight and 1.9 nm in hydrodynamic radius or to a flexible fibrous polyglycol of 5,500 in molecular weight and 2.6 nm in hydrodynamic radius, based on determinations of the membrane porosity threshold for dialysis.

Ex vivo hemodialysis culture of microbial and mammalian cells.

A small hemodialysis culture unit was developed which can be attached to an arterial-venous shunt and worn by an animal for days. The unit consists of a blood channel separated by a membrane from a dialysate chamber in which microbial or mammalian cells can be cultured. Bacterial multiplication proceeded first exponentially at the maximal rate and then arithmetically at a lesser, dialysis-limited rate. In a survey of 16 pathogenic microorganisms and five types of mammalian cell, results indicated that most of the aerobes grew well, but none of the obligate anaerobes grew at all. The separation of the culture from cellular and macromolecular host defense mechanisms allowed the cultivation of parasitic cells on an animal that was not naturally susceptible.

Severity of fever in influenza: studies on the relation between viral surface antigens, pyrexia, level of nasal virus and inflammatory response in the ferret.

Previous work has shown that fever in influenza of ferrets occurs following release of endogenous pyrogen from virus-phagocyte interaction in the upper respiratory tract (URT), and suggested that the poor inflammatory response and correspondingly low fever elicited by A/Puerto Rico/8/34 (H1N1), compared with H3N2 reassortant clones of A/Puerto Rico/8/34-A/England/939/69, were related to its H1 and N1 surface antigens. Nasal virus levels, inflammatory and pyrexial responses produced in ferrets by clones 31 (H3N1) and 64b (H1N2) of the same reassortant system suggested a connection between the H1 antigen and low inflammatory response, but results were not conclusive. Unlike A/Puerto Rico/8/34, two recent H1N1 isolates, A/USSR/90/77 and A/Fiji/15899/83, produced a high inflammatory response yet low fever despite large amounts of virus in the URT, suggesting that either no connection exists between the acquisition of the H1 antigen and production of a low inflammatory response, or the H1 antigen of recent isolates, whilst antigenically related to that of A/Puerto Rico/8/34, is biologically different.

Trivalent attenuated cold-adapted influenza virus vaccine: reduced viral shedding and serum antibody responses in susceptible adults.

Trivalent cold-adapted recombinant (CR) influenza virus vaccines containing types A (H1N1 and H3N2) and B viruses were evaluated in two double-blind, placebo-controlled trials. Susceptible adults were randomly assigned to receive the following vaccines by intranasal drops 1 month apart: two doses of trivalent vaccine, bivalent CR influenza A (Bi A) vaccine followed by monovalent B (Mono B) vaccine or vice versa, or two doses of placebo. All vaccines were well tolerated. Shedding of each of the three vaccine viruses was reduced after the first dose of trivalent vaccine compared with primary vaccination with Bi A or Mono B. Shedding was also reduced after second vaccinations, whether homologous (trivalent-trivalent) or heterologous (Bi A/Mono B or Mono B/Bi A). Reduced viral shedding was associated with reduced serum antibody responses. Thus, both simultaneous and sequential inoculations of susceptible adults with CR influenza vaccine viruses result in reduced viral shedding and serum antibody responses.

Comparison of trivalent cold-adapted recombinant (CR) influenza virus vaccine with monovalent CR vaccines in healthy unselected adults.

A trivalent cold-adapted recombinant (CR) influenza virus vaccine containing types A and B viruses was compared with monovalent vaccines of each virus in a double-blind, placebo-controlled trial. Adults with a wide range of preexisting antibody titers received one 0.5-mL dose intranasally of trivalent vaccine; monovalent A/H1N1, A/H3N2, or B vaccine; or placebo. All vaccines were well tolerated. Serum antibody response frequencies and postvaccination geometric mean antibody titers were similar for recipients of trivalent or the corresponding monovalent vaccine for each of the vaccine components. Stepwise logistic regression analysis of the antibody responses of trivalent vaccine recipients demonstrated that response to one vaccine virus did not adversely affect the likelihood of response to the other viruses. This study failed to find serologic evidence of interference between vaccine viruses, suggesting that trivalent CR influenza virus vaccine may be useful for preventing influenza in adult populations.