ABSTRACT
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.
Subject(s)
Hyaluronic Acid/biosynthesis , Kidney Neoplasms/metabolism , Wilms Tumor/metabolism , Animals , Cattle , Child , Child, Preschool , Female , Fetus/metabolism , Fibrosarcoma/metabolism , Humans , In Vitro Techniques , Kidney/embryology , Kidney/metabolism , Kidney Neoplasms/urine , Male , Rats , Wilms Tumor/urineABSTRACT
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.
Subject(s)
Breast Neoplasms/blood , Fetal Blood/physiology , Hyaluronic Acid/biosynthesis , Animals , Cattle , Cells, Cultured , Humans , In Vitro Techniques , Neoplasm Metastasis , Rats , Sarcoma, Experimental/metabolismABSTRACT
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.
Subject(s)
Blood , Cell Division , Leukocytes, Mononuclear/cytology , Lymphocytes, Tumor-Infiltrating/cytology , Animals , Cattle , Cell Survival , Cells, Cultured , Concanavalin A/pharmacology , Fetal Blood , Humans , Lymphocyte Activation , Mice , Platelet-Derived Growth Factor/pharmacology , Pokeweed Mitogens/pharmacology , Transforming Growth Factor beta/pharmacologyABSTRACT
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.
Subject(s)
Extracellular Matrix/metabolism , Fetal Blood/metabolism , Fetus/physiology , Hyaluronic Acid/metabolism , Wound Healing , Animals , Cells, Cultured , Embryonic and Fetal Development , Female , Fetus/metabolism , Fibroblasts/metabolism , SheepABSTRACT
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.
Subject(s)
Cell Movement , Fetus/physiology , Fibroblasts/physiology , Wound Healing , Animals , Cattle , In Vitro TechniquesABSTRACT
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.
Subject(s)
Blood Preservation/veterinary , Cattle/blood , Leukocytes , Animals , B-Lymphocytes/immunology , Blood Preservation/methods , Cell Separation , Cell Survival , Freezing , Lymphocyte Activation , Monocytes/physiology , T-Lymphocytes/immunologyABSTRACT
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.
Subject(s)
Culture Media , Fetal Blood , Animals , Cattle , Quality ControlABSTRACT
McClain, Mary E. (California State Department of Public Health, Berkeley), and Rex S. Spendlove. Multiplicity reactivation of reovirus particles after exposure to ultraviolet light. J. Bacteriol. 92:1422-1429. 1966.-Exposure of reovirus suspensions to moderate doses of ultraviolet light results in essentially exponential inactivation of infectivity to survivals of 10(-2) to 10(-3). With suspensions of sufficiently high particle concentration, larger doses of ultraviolet light (6 to 12 min) are associated with multiplicity reactivation (MR) which is demonstrable both by immunofluorescent-cell count and by plaque assay in FL human amnion cells. Similar effects are produced by photodynamic inactivation in the presence of proflavine, but not by thermal inactivation at 50 C. All three reovirus types exhibit MR under appropriate conditions, and all three interact in mixed ultraviolet suspensions with high efficiency. Progeny from FL cells infected under conditions of MR were as infectious as those of unirradiated inocula, with yields per cell ranging from 10(4) to 4 x 10(4) infective units.
Subject(s)
Radiation Effects , Reoviridae/growth & development , Reoviridae/radiation effects , Ultraviolet Rays , Amnion , Flavins , Fluorescence , Humans , Immunochemistry , In Vitro Techniques , Virus CultivationABSTRACT
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.
Subject(s)
RNA Viruses/growth & development , Rotavirus/growth & development , Virion/growth & development , Animals , Cattle , Cell Line , Kinetics , Morphogenesis , Virion/analysisABSTRACT
An immunofluorescent cell (IFC) assay technique was developed for the quantification of infectious pancreatic necrosis (IPN) virus of salmonid fishes. Cover slip cultures of rainbow trout gonad (RTG-2) cells were infected with diluted virus preparations. After incubation to permit antigen development, the cells were stained with antiviral fluorescent antibody, and the number of fluorescing (infected) cells was counted. Optimal conditions for the IFC assay procedure are: (i) the use of RTG-2 cells cultured for at least 3 days at 20 C; (ii) 1-h absorption of IPN virus to RTG-2 cells at 20 C or alternatively, 4 h at 4 C; (iii) staining the infected cell cultures at 10 to 12 h postinfection. A linear relationship between the relative concentration of virus in the inoculum and the number of fluorescent cells in the first cycle of infection was observed. The IFC assay method is more sensitive than the plaque method for the assay of IPN virus.
Subject(s)
Fish Diseases/diagnosis , Fluorescent Antibody Technique , Reoviridae/isolation & purification , Adsorption , Animals , Cell Line , Centrifugation, Density Gradient , Evaluation Studies as Topic , Gonads , Methods , Microscopy, Fluorescence , Reoviridae/growth & development , Salmonidae , Temperature , Time Factors , Viral Plaque Assay , Virus CultivationABSTRACT
Infectious pancreatic necrosis virus was stable for 10 days at 4 C in stream and well water, after which the virus had a half-life of 7.5 days. At 15 C, the virus was stable for 5 days, and then had a half-life between 5 and 6 days. Viral antigen in infected cells developed much more slowly at 4 C than at 20 C. Infected cells released infectious viral particles at temperatures as low as 4 C. Nutrition had a greater effect on the production of infectious virus at 4 C than at 20 C.
Subject(s)
Temperature , Viruses/growth & development , Animals , Antigens, Viral , Culture Techniques , Cytopathogenic Effect, Viral , Fluorescent Antibody Technique , Gonads , Necrosis , Pancreas/pathology , Trout , Virus Cultivation , Viruses/pathogenicityABSTRACT
A fluorescent virus precipitin test (FVPT) for the serologic identification of small particulate antigens such as viruses has been described. The test has several advantageous characteristics: (a) It is probably as sensitive as any serologic test (i.e., aggregates with dimensions of 0.2 mum are detectable; therefore, complexes containing as few as three large viruses would give a positive test). (b) Cultivation of the virus is not required. (c) Since an indirect test can be used, only a single fluorescent conjugate is needed to permit the detection of a number of viruses. (d) The indirect test can be used to detect antiviral antibody. (e) The FVPT is rapid and reliable. (f) Its simplicity should enhance its general acceptance and application.
Subject(s)
Antigens, Viral/analysis , Fluorescent Antibody Technique/methods , Precipitin Tests/methods , Animals , Antibody Specificity , Cattle , Diarrhea Viruses, Bovine Viral/immunology , Feces/microbiologyABSTRACT
Thirty-four calf and five infant fecal specimens were tested for the neonatal calf diarrhea virus (NCDV) and for the reovirus-like infantile diarrhea agent; respectively. The procedures used were the fluorescent virus precipitin test and immune electron microscopy. Fourteen of the calf stools contained detectable NCDV, and four of the five infant stools contained the reovirus-like human agent. Infectious NCDV was detected in four of the 34 calf fecal specimens when Madin-Darby bovine kidney cell cultures that had been inoculated with supernatant fluids from stool suspensions were stained with fluorescent antibody. The 20 calf stools that did not have detectable virus were examined for the bovine corona diarrhea virus. Coronavirus was found in two of these specimens.
Subject(s)
Coronaviridae/isolation & purification , Diarrhea Viruses, Bovine Viral/isolation & purification , Diarrhea, Infantile/microbiology , Feces/microbiology , Precipitin Tests , RNA Viruses/isolation & purification , Reoviridae/isolation & purification , Animals , Animals, Newborn , Cattle , Cross Reactions , Fluorescent Antibody Technique , Humans , Infant , Microscopy, ElectronABSTRACT
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.
Subject(s)
Glycoside Hydrolases/pharmacology , Peptide Hydrolases/pharmacology , RNA Viruses/drug effects , Rotavirus/drug effects , Virus Replication/drug effects , Animals , Cattle , Cell Line , Chymotrypsin/pharmacology , Edetic Acid/pharmacology , Kidney , Rotavirus/growth & development , Rotavirus/pathogenicity , Trypsin/pharmacology , Virulence , beta-Galactosidase/pharmacologyABSTRACT
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.
Subject(s)
RNA Viruses/drug effects , Rotavirus/drug effects , Trypsin/pharmacology , Virus Replication/drug effects , Animals , Cattle , Cell Line , Fluorescent Antibody Technique , Haplorhini , HeLa Cells , Humans , Rotavirus/growth & development , Virus CultivationABSTRACT
The nucleic acids of neonatal calf diarrhea virus were characterized by isopycnic centrifugation in Cs2SO4, electron microscopy, ultraviolet absorbance temperature profiles and polyacrylamide gel electrophoresis. These studies indicated that the neonatal calf diarrhea virus genome consists of 11 segments of double stranded RNA with a total molecular weight of 10.75 million daltons.
Subject(s)
RNA Viruses/analysis , RNA, Viral/analysis , Rotavirus/analysis , Centrifugation, Isopycnic , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Molecular Weight , RNA, Viral/isolation & purification , Rotavirus/ultrastructureABSTRACT
Spendlove, Rex S. (California State Department of Public Health, Berkeley), Edwin H. Lennette, Charles O. Knight, and Jean N. Chin. Production in FL cells of infectious and potentially infectious reovirus. J. Bacteriol. 92:1036-1040. 1966.-A comparative study was made of the development in, and release from, FL cells of infectious and potentially infectious (chymotrypsin-activatable) reovirus (Lang strain). The latent period was shorter, the rate of synthesis was more rapid, and the total yield was more than 10-fold greater in potentially infectious virus as compared with infectious virus. Almost all of the potentially infectious virus, but only approximately one-third of the infectious virus, was released from the infected cells.
Subject(s)
Reoviridae/growth & development , Chymotrypsin/pharmacology , Culture Techniques , Cytopathogenic Effect, Viral , Virus CultivationABSTRACT
Lesions of hamster fetal neuraxial tissues, characterized by multifocal and coalescent zones of hemorrhage, edema, and necrosis in the cerebral mantle, brainstem, and spinal cord, were observed in experiments designed to test the teratogenicity of potato preparations. Retrospective and prospective data indicated, however, that the potato preparations were not responsible but that the disease occurred spontaneously in the colony and was associated with direct breeding contact of virgin females with certain males. Observations suggest that an infectious agent may be responsible, but no agent was recovered. Immunofluorescence assay of inoculated cultures indicated that reovirus was not present in affected fetal tissues.
Subject(s)
Central Nervous System Diseases/etiology , Congenital Abnormalities/etiology , Hemorrhage/etiology , Necrosis/etiology , Vegetables/adverse effects , Animals , Axons/ultrastructure , Brain/pathology , Brain Stem/pathology , Breeding , Central Nervous System Diseases/pathology , Congenital Abnormalities/epidemiology , Cricetinae , Female , Freeze Drying , Hemorrhage/pathology , Male , Pregnancy , Reoviridae/isolation & purification , Retrospective Studies , Spinal Cord/pathologyABSTRACT
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.