Interference by Trypsin in the Interaction of Staphylococcal Enterotoxin B and Cell Cultures of Human Embryonic Intestine

The inhibitory effect of trypsin on the cytotoxicity of staphylococcal enterotoxin B acting with human embryonic intestine cell cultures was examined. Trypsin treatment of the cells rendered them resistant to enterotoxin for a period of 48 hr. The resistance increased proportionally with increased time of exposure of the cells to trypsin. Neither ethylenediaminetetraacetic acid nor scraping, which were used as alternate means of cell suspension, caused any resistance to the toxin. The effect is enzymatic and appears to be similar to the inhibitory action of trypsin and chymotrypsin on the attachment of polioviruses and coxsackieviruses to HeLa cells.     

Enterotoxin B Synthesis by Replicating and Nonreplicating Cells of Staphylococcus aureus

Although 95% of the enterotoxin B produced by Staphylococcus aureus appears during the latter part of the exponential phase of growth, growth per se is not necessary for toxin synthesis. A procedure is described whereby a concentrated suspension (at least 6 x 10(10) cells per ml) of a 16-hr culture of S. aureus was found to be capable of producing toxin, without replication, when air and glucose were present. This technique allows the growth requirement to be separated from toxin formation. Although higher (100 mug/ml) concentrations of toxin appeared in the medium when nitrogen was present, lower levels (30 mug/ml) were produced in the absence of N-Z-amine A. Toxin production proceeded without any net increase in deoxyribonucleic acid, ribonucleic acid, or protein. Chloramphenicol did not inhibit toxin formation in a nitrogen-free medium. The optimal pH for toxin production in a nitrogen-free medium was 8.0 to 8.5; for synthesis in a medium where nitrogen was available, the optimal pH was 7.0 to 7.5. Increasing the rate of aeration increased toxin release during growth, but decreased the amount of toxin subsequently produced when the bacteria were resuspended. These results suggest the presence of a precursor pool in the cells collected after 16 hr of growth.     

Production of Staphylococcal Enterotoxin A on Cellophane-Over-Agar

Up to approximately 50 mug of staphylococcal enterotoxin A per plate was produced by cellophane-over-agar cultures. Enterotoxin was determined by a latex agglutination technique.     

In Vitro Production of Clostridium perfringens Enterotoxin and Its Detection by Reversed Passive Hemagglutination

The reversed passive hemagglutination (RPHA) test yielded a positive reaction in 2 h with as little as 0.5 ng of purified Clostridium perfringens enterotoxin (CPE) per ml as well as with cultures of some C. perfringens grown in Duncan-Strong (DS) medium. This method is the most sensitive, the simplest, and the fastest among all reported. The time course of CPE production of Clostridium perfringens NCTC 8798 in DS was investigated by RPHA. CPE in culture was detectable at 4 h, increased gradually, reached a maximum at 12 to 14 h, and remained at a high level of 20 mug/ml through 48 h of incubation. CPE synthesized within cells is released easily by sonic disruption of young cultures and by aging the cultures 20 h or more. Heat shock of the cell inoculum was essential for CPE production by C. perfringens in DS.     

Mutagenesis in cultured human diploid cells. IV. Induction of 8-azaguanine resistant mutations by phloxine, a mutagenic red dye.

Disodium 9-(3',4',5',6'-tetrachloro-o-carboxyphenyl)-6-hydroxy-2,4,5,7-tetrabromo-3-isoxanthone (phloxine), a red dye used as a food additive, was tested for its activity to induce 8-azaguanine (8AG) resistant mutations in cultured human embryonic cells. Phloxine had a severe cytotoxic effect on the cells at concentrations of 1 to 10 mug/ml. At concentrations of more than 30 mug/ml of phloxine no further decrease in cell survival was found. This cytotoxic effect of phloxine was not dependent on the duration of treatment. After treatment with phloxine for 2 h division of cells in normal medium was inhibited for 120 h. When cells were treated with phloxine at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected with 30 mug/ml of 8AG, an increase in the induced mutation frequency was found. This increase in mutation frequency was dependent on the concentration of phloxine used as a mutagen and treatment with 100 mug/ml of phloxine increased the frequency to six times that in untreated cultures.     

Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay

An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described. The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells. Psychrotrophic B. cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic. Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic. There were pronounced strain differences in the amount of toxin produced by the B. cereus isolates. Some isolates of B. circulans, B. laterosporus/cereus, B. lentus, B. licheniformis, B. mycoides, B. subtilis and B. thuringiensis were also toxigenic.     

Interaction of Staphylococcal Alpha Toxin and the Estrogenic Hormone Diethylstilbestrol

Previous work in this laboratory showed that diethylstilbestrol was capable of suppressing induced furunculosis in rabbits. The present study indicates that the synthetic estrogenic hormone diethylstilbestrol which is used for acne, estrogen deficiency, cancer, and other disorders, can reduce the cytolytic action of staphylococcal alpha toxin. The cytotoxic action of purified alpha toxin for tissue cultures was evaluated by use of such parameters as total and viable cell counts, glucose, and protein determination, and cytopathic effects (CPE) in the presence and absence of steroids. To 3-day-old primary rabbit baby kidney tissue cultures, 1 to 5 μg of diethylstilbestrol per ml was added; growth of tissue cultures in Eagles medium was continued till the 6th day, and then one tissue cytopathic dose per milliliter of alpha toxin was added, and the subsequent fate of tissue cultures was assayed. Such cultures yielded higher total and viable cell counts, utilized more glucose, and contained more protein than the control cultures. In control cultures, CPE was observed on the 3rd hr after the addition of alpha toxin, and it was complete in 24 hr, whereas in tissue cultures treated with diethylstilbestrol, the CPE was significantly reduced. The data presented in this study made possible the availability of a suppressor of the cytolytic action of alpha toxin and might be useful in assaying the action of alpha toxin in an in vitro inexpensive test system.     

Production of Escherichia coli Heat-labile Enterotoxin in Fermenter Dialysis Culture

Production of Escherichia coli heat-labile enterotoxin was investigated with one porcine and one human strain in three different media under different cultivation conditions. Cultivation in aerated fermenters at pH 7·0 yielded 10–20 times more enterotoxin/ml of culture fluid than cultivation in shake flasks. A trypton-yeast extract medium was optimal in fermenter cultures. Comparatively good yields of enterotoxin in fermenters were also obtained in a glucose-salts medium. Continuous feeding of glucose and salts during fermenter cultivation resulted in a lower production of enterotoxin/mg of bacterial cells. Since this decrease in specific yield could be reversed by using dialysis culture, it was concluded that inhibition of toxin formation was due to the accumulation of extracellular low molecular weight metabolites. The highest yield of enterotoxin in dialysis culture was 80 ED₅₀ ml⁻¹ (rabbit jejunal loop test) which is at least eight times more toxin than in ordinary fermenter culture and 80 times more toxin than in shake flask cultures.     

Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP

The regulation of staphylococcal enterotoxin A (SEA) synthesis in a defined medium was studied using continuous culture techniques. SEA production was repressed by glucose and repression could be overcome by addition of exogenous cyclic AMP. As well as this classical catabolite repression control, addition of glucose to de-repressed steady-state cultures resulted in rapid disappearance of toxin from the medium (also mediated by loss of cyclic AMP). When the toxin dissappeared from the medium, it was taken up again by the bacteria without apparent modification.     

Influence of pH on the Heat Inactivation of Staphylococcal Enterotoxin A as Determined by Monkey Feeding and Serological Assay

The effect of pH on the thermal inactivation of staphylococcal enterotoxin A was investigated. Analysis of heated toxin by immunodiffusion in gel indicated that enterotoxin A in beef bouillon was inactivated faster at pH 5.3 than at pH 6.2. The z values (slopes) for the heat inactivation curves at pH 6.2 and 5.3 were 49.5 and 55 F (about 27 and 30 C), respectively. Enterotoxin produced and heated in dialyzed Casamino Acids medium and assayed by monkey feeding was more easily inactivated by heat at pH 5.3 than at pH 7.8. Thermal inactivation curves for enterotoxin A in beef bouillon (5 mug/ml, pH 5.3) were determined by two methods, monkey feeding and serological assay. The z values for the curves obtained by these two methods were both 55 F, although loss of biological or toxic activity of the enterotoxin occurred before loss of serological activity.     

Growth of the IB-RS-2 Pig Kidney Cell Line in Suspension Culture and Its Susceptibility to Foot-and-Mouth Disease Virus

The adaptation of the pig kidney cell line IB-RS-2, clone 60, to growth in suspension culture is described. When fully adapted, an approximate threefold increase in viable cells was obtained within 72 hr from initial cell concentrations of 5 x 10(5) per ml in culture volumes up to 1,500 ml. The monolayer cells (99th passage level) used to initiate the suspension cultures and the fully adapted suspension cells were shown to have an aneuploid chromosome karyotype, whereas earlier monolayer cultures (32nd passage level) had a pseudodiploid karyotype. Replicate virus titrations in monolayers prepared from suspension-adapted cells, IB-RS-2 monolayer cells, BHK monolayer cells, and in suckling mice showed that the suspension cells had retained sensitivity to foot-and-mouth disease virus. The geometric mean peak infectivity of seven strains of foot-and-mouth disease virus grown in IB-RS-2 suspension cells was 10(8.2) plaque-forming units per ml, with a mean complement-fixing activity of approximately 135 complement-fixing units per ml. These preliminary results indicate that submerged cultures of these cells on an industrial scale may be useful for commercial foot-and-mouth disease vaccine production.     

Follicle stimulating hormone stimulation of 125-I-human chorionic gonadotropin binding in porcine granulosa cell cultures.

In order to see if FSH acts directly upon the granulosa cell to stimulate hCG binding, granulosa cells harvested from small 1-2 mm porcine follicles were grown in 250 ml flasks in chemically defined media containing 0.05 mug/ml highly purified human FSH for 2, 4, and 6 days. The defined medium consisted of culture medium 199 plus 0.4% bovine serum albumin, 0.2% lactalbumin hydrolysate and 10 munit/ml insulin. The cultures were harvested by scraping with a rubber policeman and incubated with 0.1 mug/ml 131-I- or 125-I-hCG. Binding expressed as cpm/culture or per mg protein yielded similar results. In five separate experiments addition of FSH stimulated hCG binding two- to fourfold above control cultures. In a typical experiment after 2 days of culture, the specific binding of control cultures to hCG was 962 plus or minus 45 cpm/culture (-x plus or minus SE; n = 3) and the binding in cultures grown in the presence of 0.05 mug/ml FSH was 3933 plus or minus 1787 (n = 3; P less than 0.01). Granulosa cells harvested from large (8-12 mm) follicles grown under similar conditions bound 29,669 plus or minus 948 cpm/culture (n = 4). These data demonstrate that FSH may have a direct stimulatory role upon induction of granulosa cell LH-hCG receptors in vitro.     

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.     

Aflatoxin B1 Induction of Lysogenic Bacteria

A technique for biological verification of aflatoxin B(1) was developed based on toxin-mediated induction of lysis in a lysogenic strain of Bacillus megaterium NNRL B-3695. Reduction of culture turbidity was determined at various concentrations of toxin. Incubation of 1.1 x 10(-4) g (dry weight) of cells/ml of growth medium containing 25 mug of B(1) per ml at 37 C reduced initial turbidity 0.20 absorbance units in 4 hr. If the bacterial lysate of the lysogenic strain, after a 2-hr incubation with 25 mug of B(1) per ml, was plated with a sensitive B. megaterium strain (NRRL B-3694), plaque-forming units increased approximately 150 times relative to the control. Comparable testing of the effects of aflatoxin on the nonlysogenic, sensitive strain demonstrated that 75 mug of B(1) per ml neither induced lysis nor plaque-forming units. Although induction is not an exclusive property of aflatoxin B(1), the differential response of the lysogenic and sensitive Bacillus strains to B(1) offers a unique and rapid technique for biological verification of the toxin.     

Aflatoxin B1 Uptake by Flavobacterium aurantiacum and Resulting Toxic Effects

Removal of aflatoxin B(1) from liquid cultures by resting and growing cells of Flavobacterium aurantiacum NRRL B-184 was studied. Spectrophotometic and thin-layer techniques served as aflatoxin assays. Cells grown in the presence of 5 ppm or higher levels of aflatoxin developed aberrant morphological forms. These toxin concentrations partially inhibited growth, and the nature of the inhibition suggested that aflatoxin interfered with cell wall synthesis. Incubation of 1.0 x 10(11) resting cells per milliliter with 7.0 mug/ml of aflatoxin B(1) during a 4-hr period facilitated complete toxin removal from a buffered aqueous medium. Autoclaved cells and cell wall preparations could remove a fraction of the aflatoxin of a test system. However, the toxin removed by autoclaved cells and cell walls could be extracted by washing with water but the aflatoxin B(1) removed by intact cells could not be extracted into the liquid phase. The uptake of aflatoxin B(1) by resting cells was sensitive to temperature and pH. Ruptured preparations of F. aurantiacum were not able to remove or modify the aflatoxin in an aqueous solution.     

Changes in protein content per cell during growth of mouse L cells

The relationship between cell density and protein content per cell was examined in monolayer and suspension cultures of mouse L cells. In monolayer cultures, the protein content per cell reached a maximum at 6 h after plating and retained this level for 18 h. Thereafter, the protein content per cell declined gradually during the exponential growth phase and finally returned to the initial level at the stationary phase. The changes were neither due to the effect of trypsinization nor to the exhaustion of the medium. The protein content per cell in a sparse culture was always greater than that in a dense culture for monolayer culture of L cells. In suspension culture the increase of protein content per cell during the lag phase was similar to that found in monolayer cultures. However, the gradual decline of protein content per cell observed during the exponential phase of monolayer cultures was not detected during that of a suspension culture. The results suggest that the decrease of protein content per cell in monolayer cultures may be related to some function of cell plasma membrane which could be inhibited by a cell-to-cell contact.     

Relationship Between Staphylococcal Antiserum Titer and Zone Development on Immune Serum Plates

A workable relationship was established between the standard serum titers of staphylococcal immune antisera and the development of precipitin zones on serum agar around colonies of staphylococcal strains producing homologous antigens (enterotoxins). The standard titer of a serum is defined as the reciprocal of that serum dilution which, with 10 mug of pure enterotoxin per ml, will give a precipitin zone 10 mm in length in single gel-diffusion tubes after 7 days of incubation at 25 C. A numerical scale was set up for determining the intensity of precipitin zones on serum agar. A reading of 3 was considered optimum. This correlated well with a standard serum titer of 25, when 1 ml of such a serum was used per 20 ml of medium per serum plate. From this relationship, the minimum volume of serum required to give optimum precipitin zone development can be calculated.     

Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.     

Tumorsphere assay provides more accurate prediction of in vivo responses to chemotherapeutics

Although the sphere culture system has been widely used in stem cell biology, its application for drug screening is limited due to lack of standardized, rapid analytical tools. To optimize sphere cultures for in vitro screening of drugs, we evaluated the properties of primary tumor cells growing as tumorspheres and compared their chemosensitivity to those of cells growing in monolayer. Most cells in tumorsphere cultures were quiescent whereas cells in monolayer culture had a high mitotic index. Moreover, doxorubicin showed better cytotoxicity than paclitaxel in the sphere cultures, but their efficacy was reversed in the monolayer cultures. Importantly, the response of cytotoxic outcomes for suspension cultures matched the in vivo response better than monolayer cultures, providing support for the use of short term suspension cultures of primary cells as a model for drug testing.     

MITOTIC AND NONMITOTIC MULTIPLE-LAYERED PERFUSION CULTURES

Cell types in addition to those previously described (Kruse et al. 1963. J. Nat. Cancer Inst. 31:109; Kruse and Miedema. 1965. J. Cell Biol. 27:273) were found to form multiple-layered cultures by perfusion-culture technique. Dense populations containing 43 x 10⁶ embryonic rat muscle (NF-ER) cells, 23 x 10⁶ diploid human tonsillar (NF-JAM) cells, 77 x 10⁶ human pleural effusion isolate (RPMI 2650) cells, 35 x 10⁶ embryonic diploid human lung (Flow 2000) cells, 21 x 10⁶ bovine lung (FB4BM) cells, 108 x 10⁶ bat lung (Tb1Lu) cells, and 81 x 10⁶ SV-40 virus-transformed embryonic diploid human lung (WI-38VA13A) cells were obtained in 6–14 days from dilute inocula in T-60 or T-75 flasks; these were equivalent to about 4, 3, 3, 4, 2, 4, and eight monolayers, respectively. Perfusion of an NF-ER culture for 6 wk with medium plus 10% whole calf serum yielded a cell density equivalent to 12 monolayers (140 x 10⁶ cells per T-75 flask). This culture exhibited random labeling of nuclei from bottom to top after pulsing for 90 min with thymidine-³H. Medium plus 0.1% serum maintained NF-JAM cultures at constant viable cell numbers with virtual absence of thymidine-³H labeling. Similar results were obtained with WI-38 cultures, but WI-38VA13A cells continued active DNA synthesis and mitosis in medium with 0.1% serum to form 16–20 layers of cells (191–239 x 10⁶ cells per T-75 flask) in 27 days. WI-38VA13A cells ceased proliferation and became nonviable rapidly in serumless medium.