Enhanced proliferation of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes in a plasma-derived cell culture fluid.

The proliferation and activation of murine and human T-lymphocytes in a high-protein lymphocyte culture fluid (LCF) is compared to that of cells cultured in 10% fetal bovine serum (FBS). Tumor-infiltrating lymphocytes (TIL) proliferate exponentially in the LCF for up to 46 days and generate cell numbers that are nearly 100,000-fold greater than cells cultured in FBS. This rapid growth of T cells in LCF could have an important impact in adoptive immunotherapy and gene therapy since cell growth is a limiting factor in these technologies.

A control for fetal bovine serum.

The FBS Control is designed to assist cell culture scientists who have been frustrated with lack of consistency in FBS lots. Continued use will reduce the time and expense needed in classical lot selection methods.

Hyaluronic acid-stimulating activity in the pathophysiology of Wilms' tumors.

Markedly elevated levels of hyaluronic acid occur in the serum and urine of some patients with Wilms' tumor. We have recently described a glycoprotein factor in fetal serum that stimulates deposition of hyaluronic acid. In a survey of bovine fetal tissue extracts, we have identified the fetal kidney as the source of this circulating activity. Wilms' tumors arise from transformed "rests" of fetal kidney. We demonstrate here that such tumors continue to produce this fetal factor and that the hyaluronic acid-stimulating activity is found in the urine of children with Wilms' tumors. In the three patients with Wilms' tumor who were followed, elevated levels of hyaluronic acid-stimulating activity were found in their urine before treatment. By 2 months after surgical removal of their tumors, these levels had returned to baseline. We propose that hyaluronic acid-stimulating activity is the mechanism for the elevated levels of hyaluronic acid in the sera and urine of these patients. The activity is an oncofetal protein and the first for which a function has been identified. It also is a marker for this common childhood solid tumor and has the potential for identifying children at increased risk.

Studies in fetal wound healing: I. A factor in fetal serum that stimulates deposition of hyaluronic acid.

Fetal wound healing without scar formations, fibrosis, or contracture might be accompanied by major differences in the wound extracellular matrix. The matrix of fetal wounds is rich in hyaluronic acid, a glycosaminoglycan found in high concentrations whenever there is tissue proliferation, regeneration, and repair. Although hyaluronic acid is a critical molecule for both embryonic development and wound healing, no factor has yet been identified that modulates hyaluronic acid in a consistent manner. We describe here a substance present in fetal sheep serum that stimulates hyaluronic acid synthesis by cultured fibroblasts. This glycoprotein factor appears to be ubiquitous, present in fetal sheep and bovine serum, reaching a peak in each at 40% of the way through gestation. This factor is also present in amniotic fluid. It might control hyaluronic acid deposition. In turn, hyaluronic acid, by creating an extracellular environment permissive for cell motility and proliferation, might be critical for fetal development. We suggest that the same sequence of events underlie the unique properties observed in fetal wound healing.

Studies in fetal wound healing: II. A fetal environment accelerates fibroblast migration in vitro.

We have used an in vitro model of wound healing using scratches made in a confluent monolayer of fibroblasts. The effects of fetal calf and postnatal calf serum on the migration of fibroblasts were compared. Differences between fetal and calf serum-incubated fibroblasts grown on coverslips were observed within 15 minutes of exposure. Cells in fetal serum began to change both shape and orientation and to move into the trough created by the scratch. The fibroblasts incubated in fetal calf serum completely filled in the trough within 16 hours while those incubated in calf serum did not do so even after 24 hours. We estimate that, at any point, there was a 50% lag time in the migration of the fibroblasts in the presence of postnatal calf serum. This difference in migration and filling was not a function of mitogenesis; the mitogenicity of the two sera were comparable. The results suggest that fibroblast migration in vitro is accelerated by the fetal serum. A similar mechanism may occur in vivo and may underlie the ability of the fetal wound to heal more rapidly.

Hyaluronic acid-stimulating activity in sera from the bovine fetus and from breast cancer patients.

The sine qua non of malignancy is the ability of tumor cells to migrate and invade surrounding tissue. There are many substances that have been described that enhance cell motility and hyaluronic acid is prominent among these. Hyaluronic acid is a high molecular weight alternating disaccharide polymer found in abundance in extracellular matrices whenever rapid cell proliferation or tissue regeneration and repair occur. It creates a permissive environment for cell motility during embryogenesis, and high levels of hyaluronic acid also correlate with increased tumor cell invasion and aggressiveness. Little is known about the regulation of hyaluronic acid production, either in normal tissue or in malignancy. In this study, we characterize a hyaluronic acid-stimulating activity in fetal calf serum and describe a similar activity in the sera of breast cancer patients. The stimulating activity was measured by placing aliquots of test substance on fibrosarcoma cells. These indicator cells, which synthesize copious quantities of hyaluronic acid, respond to stimulation in a time- and dose-dependent fashion. The fetal calf serum hyaluronic acid-stimulating activity is maximum early in gestation and then falls rapidly to essentially no activity at term. This activity was partially purified from 120-day fetal calf serum by concanavalin A-Sepharose affinity and ion exchange chromatography and is accounted for by a glycoprotein with a molecular weight of 150,000 on gel filtration under native conditions. The sera of breast cancer patients with measurable burden of disease also contained hyaluronic acid-stimulating activity, which was not present in normal serum donors or in breast cancer patients without evidence of disease. The production of this stimulating activity may contribute to the development of the malignant phenotype by inducing hyaluronic acid-rich microenvironments that are permissive to tumor cell invasion and metastases.

Influence of malnutrition and alterations in dietary protein on murine rotaviral disease.

The possible correlation between malnutrition and degree of severity of rotavirus-associated infantile diarrhea which appears to occur in human populations was studied using a mouse model. To determine the effects of general malnutrition or altered levels of dietary protein, female mice were fed throughout pregnancy and infection periods with diets diluted with 0, 300, or 600 g glucose/kg, designated as normal nutrient to calorie ratio (N/C) diet, 70% N/C diet, or 40% N/C diet or with diets containing 75, 150, or 300 g casein/kg, as low-, normal-, or high-protein diets. Murine rotavirus was given by gavage to the 2-day-old offspring of these dams, and the extent of infection determined. Marked increases in severity of diarrheal disease were seen in the infants from dams receiving the 40 and 70% N/C diets and the low-protein diet. Severity of infection was seen as increased deaths, reduced weight gain, and increased passage of diarrheic feces. Intestinal viral levels and intestinal diarrhea scores did not vary appreciably. Serum interferon remained below detectable limits throughout the studies, but serum antibody was determined in dams 30 days post-virus exposure. The latter titers were lower in the infected mice from dams fed the 40 and 70% N/C diets, but were essentially the same in all the protein diet groups. Cross-fostering was done using the 40 and 100% N/C diets, wherein mice from dams fed either diet were placed on mothers fed the opposite diet. Increased severity of infection was again seen when the virus was given 2 days after the exchange, although the greatest infection occurred in animals from dams fed 40% N/C diet which were then fostered by other similarly fed dams. The increased host sensitivity to the rotaviral infection appeared to be a result of both pre- and postnatal dietary effects.

Aerosol stability of infectious and potentially infectious reovirus particles.

The aerosol stability of two particle forms, infectious and potentially infectious, of reovirus were examined under static conditions for a range of relative humidities at 21 and 24 degrees C. Virus aerosolization efficiency was determined for two methods of dissemination: Collison nebulizer and Chicago atomizer. Suspensions of Bacillus subtilis var. niger spores were added to reovirus preparations that included both particle forms and disseminated into a dynamic aerosol toroid to estimate the physical decay of the aerosols. At 90 to 100% relative humidity, both reovirus particle forms showed less than 10-fold loss of infectivity after 12 h of aging. At lower relative humidities the aerosol decay curve showed rapid initial decay followed by a markedly lower decay rate. Our findings reveal that reovirus particles are relatively stable in the airborne state.

Protamine precipitation of two reovirus particle types from polluted waters.

Two forms of virus particle are released from reovirus-infected cell cultures, infectious reovirus and potentially infectious reovirus (PIV). PIV particle forms have a complete outer coat and are not infectious until the outer coat is altered or removed. The PIV concentration in polluted waters, however, has not been determined. Protamine sulfate precipitation, using 0.25% fetal bovine serum and 0.005% protamine sulfate for the first precipitation of the sample and 0.0025% for the second, was employed to concentrate infectious reovirus and PIV from water and sewage. Infectious reovirus and PIV particles were concentrated over 500-fold from river water inoculated with virus, and virus recoveries of between 80 and 100% were achieved. Virus precipitates stored at -20 degrees C as a protamine-virus concentrate showed a 5% loss of PIV after 14 days. Virus preparations were assayed, before and after treatment, with 200 micrograms of chymotrypsin per ml, using a fluorescent-antibody procedure. Protamine sulfate precipitation and fluorescent-antibody detection are effective ways to recover and assay reoviruses present in raw sewage.

Evaluation of cell lines and immunofluorescence and plaque assay procedures for quantifying reoviruses in sewage.

Twelve continuous cell lines were tested to determine their sensitivity to reovirus types 1, 2, and 3 isolated from sewage. Madin-Darby bovine kidney (MDBK), rhesus monkey kidney (LLC-MK2), and human embryonic intestinal (intestinal 407) cells were most sensitive, respectively. In a similar study, MDBK cells were more sensitive than LLC-MK2 and Buffalo green monkey kidney (BGM) cells to sewage-isolated, protamine-precipitated reoviruses which had not been serotyped and had no previous cell contact. Sewage-isolated, protamine-precipitated reoviruses were also used in conjunction with MDBK cells in a comparative evaluation of immunofluorescent cell count and plaque assay procedures. The immunofluorescence assay is more sensitive and more rapid than the plaque assay. Reoviruses in excess of 10(4)/liter of raw sewage were detected by the immunofluorescent cell count assay.

Inhibition of rotaviruses by selected antiviral substances: mechanisms of viral inhibition and in vivo activity.

Several RNA virus inhibitors were evaluated against simian (SA11) rotavirus infections in vitro and murine rotavirus gastroenteritis in vivo. Test compounds included 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 3-deazaguanine (3-DG), 3-deazauridine, and 9-(S)-(2,3-dihydroxypropyl)adenine [(S)-DHPA]. All drugs inhibited total infectious SA11 virus yields in MA-104 cells. Ribavirin, 3-DG, and (S)-DHPA affected [3H]uridine uptake into uninfected MA-104 cells in both the acid-soluble and -insoluble fractions. All drugs reduced the levels of dense (precursor) and light (complete) SA11 particle yields compared with control but did not alter the relative amounts of dense compared with light particles, suggesting that the agents did not interfere with virus assembly. Ribavirin and 3-DG inhibited SA11 polypeptide synthesis, as determined by polyacrylamide gel electrophoresis studies. None of the agents or mono- and triphosphate derivatives of ribavirin inhibited SA11 RNA polymerase activity. In murine rotavirus studies, oral therapy with ribavirin-2',3',5'-triacetate and (S)-DHPA increased mean survival time, but no increase in survivor rate was observed. 3-DG- and (S)-DHPA-treated mice had a more rapid weight gain than controls, suggesting a probable lessening of the severity of the disease.

Inhibition of bluetongue and Colorado tick fever orbiviruses by selected antiviral substances.

The effects of four ribonucleic acid virus inhibitors were evaluated in cell cultures and in mice to determine inhibitory effects against bluetongue virus and Colorado tick fever virus (CTFV). Test compounds included 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 3-deazaguanine, 3-deazauridine, and 9-(S)-(2,3-dihydroxypropyl)adenine. Ribavirin-2',3',5'-triacetate (ribavirin triacetate) was evaluated in vivo against CTFV. Inhibition of cytopathic effect and plaque reduction were used to evaluate antiviral activity. In cytopathic effect inhibition studies, bluetongue virus was markedly inhibited by 3-deazaguanine and 3-deazauridine in Vero cells with moderate inhibition by the other agents. Ribavirin and 3-deazaguanine markedly inhibited CTFV in MA-104 cells, 3-deazauridine was slightly less active, and 9-(S)-(2,3-dihydroxypropyl)adenine was negative. Ribavirin was less effective in Vero cells against CTFV. When mice were inoculated intracerebrally with CTFV and treated by a single intracerebral injection with drug, ribavirin triacetate increased the number of survivors, 3-deazaguanine increased mean survival time, and ribavirin was negative. Intraperitoneal treatment of infected mice with ribavirin triacetate for 1 week significantly increased the number of survivors and mean survival time, providing strong evidence that the agent is active across the blood-brain barrier.

Trypsin enhancement of rotavirus infectivity: mechanism of enhancement.

The infectivity of most rotaviruses is enhanced by treatment with trypsin. We studied the mechanism of enhancement of examining the effect of trypsin on rotavirus infectivity, aggregation, early interactions with host cells, and structure. The results indicated that trypsin does not increase levels of infectious virus by dispersion of aggregates or affect the efficiency or rate of attachment of virus to cells. A fraction of virus that was not infections without trypsin treatment was found to attach to cells, but did not initiate antigen synthesis. When cells were infected with labeled, purified virus, increased levels of uncoated particles were found in cells infected with trypsin-treated virus. Infection of cells with trypsin-treated virus also led to greater levels of RNA synthesis early in the infection. The results suggest that trypsin converts a noninfectious fraction of virus into infectious virus by allowing this fraction to uncoat in the infected cell. Trypsin was found to cleave an 88,000-dalton structural polypeptide of bovine rotavirus generating 67,000- and 20,000-dalton cleavage products.

Development of antiviral levels of ribavirin in serum and urine of orally treated rats.

The levels of ribavirin or its antivirally active metabolic products were determined in the serum and urine of rats treated with single oral doses of 1,000 or 100 mg/kg of the compound, using a newly developed micromethod in which measles virus inhibition was assayed in BS-C-1 cells. At the high dosage level, maximum ribavirin serum levels of 24 microgram/ml were observed 2 h postribavirin administration. Approximately 10-fold less active material was seen in the rats receiving the lower ribavirin dosage; this peak effect was seen 1 h after treatment. Urine excretion was maximal between 4 and 20 h after treatment in both dosage groups.

Bioassay system for determining ribavirin levels in human serum and urine.

A simple, sensitive, micromethod was developed to determine levels of ribavirin, or its active metabolic products, in human serum or urine. The procedure utilized the inhibition of measles virus cytopathic effect in BS-C-1 cells. Based upon maximum dilutions of human serum or urine containing ribavirin which inhibited the measles virus, the bioassay detected ribavirin in concentrations as low as 0.006 microgram/ml in serum or 0.03 microgram/ml in urine. Herpesvirus 1, parainfluenza virus 3 and reovirus 1 were also tested for sensitivity to ribavirin. Minimum inhibitory concentrations of ribavirin in serum or urine against these other viruses were no lower 0.32 microgram/ml.

Role of two particle types in bovine rotavirus morphogenesis.

The involvement of light (L) and dense (D) bovine rotavirus particle types during virus replication has been studied. It was found that infectious parental L virions are uncoated in vivo to a particle similar to native D particles. Differences in the rate of synthesis and relative yields of L and D particles in MDBK and MA-104 cells have been detected. Results from pulse-chase labeling experiments indicate that D particles serve as morphogenic precursors to the complete L virion.

Cryopreservation of bovine mononuclear leukocytes.

A technique of cryopreservation of bovine mononuclear leukocytes for use in lymphocyte stimulation tests and cell identification studies has been developed. Dimethyl sulfoxide was used as the cryopreservation agent. Cells were frozen to --40 C at a controlled rate of --1 C/minute and then to --80 C at a rate of --4 C/minute, and were subsequently stored in liquid nitrogen (--109 C). The cells were than recovered by rapid thawing in a 37-C water bath, diluted with tissue culture media, washed, and used for lymphocyte stimulation and cell identification tests. The magnitude of the lymphocyte blastogenic responses of frozen and thawed mononuclear leukocytes was similar to that seen with freshly collected cells. Additionally, percentages of T cells, B cells, and monocytes were similar between frozen and thawed cells and freshly collected cells.

Effect of enzymes on rotavirus infectivity.

The infectivity of a bovine rotavirus was enhanced 140-, 8-, and 3-fold, respectively, by trypsin, protease, and lactase. Ficin, carboxypeptidases A and B, lysozyme, and beta-galactosidase had little effect on the infectivity. Chymotrypsin caused a threefold decrease in the infectivity. Trypsin acts directly on the rotavirus and not on the host cell.

Production of high-titer bovine rotavirus with trypsin.

Titers of bovine rotavirus in excess of 10(9) immunofluorescent infectious units per ml of culture fluids have been produced, using trypsin treatment of the virus. Infectivity of preparations of the virus can be increased with as little as 1 ng of trypsin per ml, with maximum increases of 1 to 2 log10 with 1 microgram of trypsin per ml. The virus grows to titers in excess of 10(5) immunofluorescent units per ml in MDBK, LLC-MK2, MA-104, and HeLa cells. When MDBK cells are infected with a multiplicity of infection of 20, maximum yields of cell-associated, trypsin-enhanceable virus are obtained 4 to 8 h postinfection. Maximum yields of cell-free, trypsin-enhanceable virus are produced 16 to 20 h postinfection. The results presented here indicate that trypsin can be used to produce high-titer stocks of bovine rotavirus.