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PubMed1977

Inactivation of Clostridium haemolyticum toxic fluids and their antigenicity.

One hundred fifty-one isolates of Clostridium haemolyticum were examined for consistent toxin production following repeated serial transfers in laboratory media. Most of these isolates produced only small amounts of toxic materials and serial transfers appeared to reduce toxigenic characteristics. Eleven of the isolates consistenly produced measurable amounts of toxic materials. One of these isolates was used for production of toxic fluids that were concentrated by lyophilization and reconstitution to a smaller volume or by precipitation with ammonium sulphate followed by dialysis against water and glycerol. Known amounts of these substances were inactivated with formalin, heat, beta-propiolactone, ultra-violet irradiation and glutathione. The resulting toxoids were inoculated into guinea pigs and most were judged to be nonimmunogenic because the animals were unable to resist dermal challenge. Toxic materials with added glycine were inactivated with formaldehyde as readily as those without the amino acid but the resulting toxoids were immunogenic while those prepared without the amino acid were not.

Animals↗

PubMedDate not supplied

The assay of influenza virus structural antigens in vaccines by rocket immunoelectrophoresis.

The internally situated influenza virus structural antigens (matrix and nucleoprotein) and the glycoprotein hemagglutinin were assayed using rocket immunoelectrophoresis or single radial diffusion following disruption of purified virus with 1% w/v sodium sarcosyl detergent. Electrophoresis was carried out for 1-4 h in 0.05 M barbitone buffer at pH 8.6. The potency estimations of HA and M activity were similar for the two methods for bivalent beta-propiolactone inactivated whole virus vaccines.

Antigens, Viral↗

PubMed1983

Rabies vaccine prepared from the virus grown in Japanese quail embryo cell cultures.

Fixed rabies virus strain MNIIVP-74 was grown in Japanese quail embryo cell cultures, concentrated by ultrafiltration and inactivated with beta-propiolactone. The resulting vaccine was markedly antigenic and immunogenic for laboratory animals. Human volunteers injected with 2.0 ml vaccine on days 0, 3, 7, 14, 30 and 90 exhibited more intensive and longer antibody production than those injected daily for 14 days.

Animals↗

PubMed1982

Dog response to inactivated canine parvovirus and feline panleukopenia virus vaccines.

Inactivated canine parvovirus (CPV) and inactivated feline panleukopenia virus (FPV) vaccines were evaluated in dogs. Maximal serologic response occurred within 1-2 weeks after vaccination. Antibody titers then declined rapidly to low levels that persisted at least 20 weeks. Immunity to CPV, defined as complete resistance to infection, was correlated with serum antibody titer and did not persist longer than 6 weeks after vaccination with inactivated virus. However, protection against generalized infection was demonstrated 20 weeks after vaccination. In unvaccinated dogs, viremia and generalized infection occurred after oronasal challenge with virulent CPV. In contrast, viral replication was restricted to the intestinal tract and gut-associated lymphoid tissue of vaccinated dogs. Canine parvovirus was inactivated by formalin, beta-propiolactone (BPL), and binary ethylenimine (BEI) in serum-free media; inactivation kinetics were determined. Formalin resulted in a greater loss of viral HA than either BEI of BPL, and antigenicity was correspondingly reduced.

Animals↗

PubMed1983

Effects of p-methoxyphenol and diet on carcinogen-induced neoplasia of the mouse forestomach.

Previously, p-methoxyphenol fed in the diet was found to be the most potent inhibitor of benzo(a)pyrene-induced neoplasia of the mouse forestomach of 18 phenols investigated. In the present study, the effects of p-methoxyphenol on the direct-acting carcinogen, beta-propiolactone (BPL), were determined. p-Methoxyphenol administered at 1 or 4 hr prior to BPL or fed in the diet markedly inhibited BPL-induced neoplasia of the mouse forestomach. Of 10 phenols tested by p.o. intubation, it was the only one that exerted a significant inhibitory activity. Thus far, p-methoxyphenol appears to be an effective inhibitor only when given prior to carcinogen administration. During these studies, it was found that the nature of the diet markedly altered the neoplastic response of the mouse forestomach to BPL but not to benzo(a)pyrene.

Animals↗

PubMed1982

Chemical cocarcinogenesis with the use of a subclone derived from Balb/3T3 cells with catechol as cocarcinogen.

This study was performed for the detection of cocarcinogens by transformation in vitro with the use of a subclone of a Balb/3T3 cell line. Dose response, cytotoxicity, and transformation studies were done with the use of an indirect-acting carcinogen, benzo[a]pyrene (B[a]P), a direct-acting alkylating carcinogen, beta-propiolactone (BPL), and the mouse skin cocarcinogen catechol. The rate of transformation was notably higher in groups treated with B[a]P and catechol or BPL and catechol than in groups treated with either B[a]P or BPL. Catechol alone did not induce any transformation. All the cells isolated from the transformed foci showed characteristics of malignantly transformed cells, such as anchorage-independent growth. Thus chemical cocarcinogenesis was accomplished in vitro similar to that accomplished in in vivo studies reported earlier on mouse skin.

Animals↗

PubMed1980

Evaluation of the effect of betapropiolactone/ultraviolet irradiation (BPL/UV) treatment of source plasma on hepatitis transmission by factor IX complex in chimpanzees.

To evaluate the safety of a beta-Propiolactone/Ultraviolet (BPL/UV), irradiated Factor IX complex preparation we inoculated 8 chimpanzees with 25 units Factor IX/Kilo from a pool of 5 production lots which had been treated in this manner. These lots were derived from approximately 1,000 donors. Animals were followed with weekly tests for hepatitis B serologic markers and transaminases, and biweekly liver biopsies, for 6 months. No evidence of transmission of hepatitis B, or non-A, non-B viruses was observed. To further evaluate the BPL/UV procedure a plasma pool was intentionally contaminated with hepatitis B virus and one half of the pool treated with BPL/UV. Factor IX complex was isolated from the treated and untreated pools and each was inoculated into 4 chimpanzees. The Factor IX derived from untreated plasma infected all four animals with an average incubation period of 10.5 weeks, whereas that prepared from PBL/UV treated plasma infected only one of four animals with an incubation period of 21 weeks. These results were interpreted as suggesting that BPL/UV can inactivate approximately 99.9% of hepatitis B virus infectivity.

Animals↗

PubMed2007

Harvesting and concentration of human influenza A virus produced in serum-free mammalian cell culture for the production of vaccines.

A process scheme for the harvesting and concentration of cell culture-derived human influenza A virus is presented. The scheme comprises two static filtration steps, chemical inactivation by beta-propiolactone and cross-flow ultrafiltration. Human influenza A virus A/PR/8/34 (H1N1) was produced in roller bottles with serum-free medium using MDCK cells as a host. Cultivations resulted in specific hemagglutination (HA) activities of 393 HAU (100 microL)(-1) and turbidities of 0.479 OD measured as the extinction of light at 700 nm (mean values are presented). The concentrations of soluble protein and DNA in the harvests were 72 microg/mL and 5.73 microg/mL, respectively. An average product yield of 79% based on HA activity was achieved after clarification by depth (85%) and microfiltration (93%). The turbidities of cell culture supernatants were reduced to 2% of their initial value. Concentration with 750 kDa hollow-fiber modules by a factor of 20 resulted in 97% recovery of the product when operated at a constant flux of 28 L/(m(2) h) and a wall shear rate of 9,500 s(-1). The amount of protein and DNA could be reduced to 16% and 33% of their initial amount, respectively. An overall product yield of 77% was achieved. Clarified supernatants and concentrates were further analyzed by non-reducing SDS-PAGE and agarose gel electrophoresis. Particle volume distributions of concentrates were obtained by dynamic light scattering analysis. From the results it can be concluded that the suggested process scheme is well suited for the harvesting and concentration of cell culture-derived influenza A virus.

Animals↗

PubMed1994

Induction of protective cytotoxic T cells with viral proteins.

Induction of CD8+, class I-restricted T cells by non-infectious, exogenous antigens has been documented for model protein antigens such as ovalbumin and for major histocompatibility complex restricted short peptides in viral and tumor systems. However, the protective capacity of cytotoxic T cells induced by conventional proteins has not been tested in vivo so far. We, therefore, evaluated the induction of protective cytotoxic T cells against three different full-length recombinant viral proteins derived from a baculovirus expression system, i.e. the glycoprotein and nucleoprotein of lymphocytic choriomeningitis virus (LCMV) and the nucleoprotein of vesicular stomatitis virus (VSV). These viral proteins induced cytotoxic T cells in a T helper cell-independent fashion which lysed infected target cells in vitro and protected mice from viral replication, immunopathological disease and growth of a tumor expressing the same antigen as a tumor antigen. These results are surprising, since it had been shown earlier for completely inactivated nonreplicating viral vaccines and again here for beta-propiolactone-inactivated VSV or UV-light inactivated LCMV that nonreplicating viral vaccines were incapable of inducing protective cytotoxic T cells. Our data show that immunization of mice with as little as 10 micrograms of non-infectious viral proteins triggered long-lasting CD8+ T cell-mediated antiviral immunity. It was found that the protein alone was only weakly able to induce cytotoxic T cells, and that association with cellular debris functioned as an adjuvant. These findings may be relevant for our understanding of the phenomenon of cross-priming and have obvious implications for vaccine strategies.

Animals↗

PubMed1993

A study of the advantages and limitations of immunoblotting procedures for the detection of antibodies against influenza virus.

An immunoblotting procedure was used to determine the specificity and examine some of the properties of antibodies produced following infection of mice with influenza virus or inoculation with noninfectious material with Alhydrogel or complete Freund's adjuvant. The noninfectious material used was beta-propiolactone-inactivated influenza virus and a preparation (HANA) enriched for the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). When influenza viral proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, each of the anti-viral antisera tested exhibited strong binding. Under reducing conditions, however, much weaker binding was observed especially towards the HA1 subunit of HA. This was particularly apparent with antisera raised to virus or HANA in the absence of adjuvant. A panel of monoclonal antibodies directed to HA also bound well to viral HA separated by SDS-PAGE under nonreducing conditions but failed to recognize epitopes on HA1 separated under reducing conditions. These results suggest that when HA is reduced and immobilized on a solid support, it does not display the conformational features essential for the integrity of all epitopes. The immunoblotting procedure was also used to determine the isotype of anti-viral antibody directed against individual viral proteins and to detect matrix protein 2 (M2) in purified influenza virions and influenza-infected cells using antisera raised to a synthetic peptide representing a sequence within the M2 protein.

Amino Acid Sequence↗

PubMed1984

Somatic mutation and recombination test in Drosophila melanogaster.

A novel test system for the detection of mutagenic and recombinogenic activity of chemicals is described in detail. Drosophila melanogaster larvae trans-heterozygous for the mutations multiple wing hairs (mwh) and flare (flr) are exposed to the test compounds for various periods of time ranging from 96 hr to 1 hr. Induced mutations are detected as single mosaic spots on the wing blade of surviving adults that show either the multiple wing hairs or flare phenotype. Induced recombination leads to mwh and flr twin spots and also to a certain extent, to mwh single spots. Recording of the frequency and the size of the different spots allows for a quantitative determination of the mutagenic and recombinogenic effects. This and earlier studies with a small set of well-known mutagens indicate that the test detects monofunctional and polyfunctional alkylating agents (ethyl methanesulfonate, diepoxybutane, mitomycin C, Trenimon), mutagens forming large adducts (aflatoxin B1), DNA breaking agents (bleomycin), intercalating agents (5-aminoacridine, ICR-170), spindle poisons (vinblastine), and antimetabolites (methotrexate). In addition, the test detects mutagens unstable in aqueous solution (beta-propiolactone), gaseous mutagens (1,2-dibromoethane), as well as promutagens needing various pathways of metabolic activation (aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, mitomycin C, and procarbazine). The rapidity and ease of performance as well as the low costs of the test necessitate a high priority for validation of this promising Drosophila short-term test.

Animals↗

PubMed1985

Effects of some DNA-ligands and of some mutagenic compounds on the induction of meiotic disomic or diploid yeast products.

Data were collected following the administration of various compounds to meiotic yeast cultures to investigate their effects on disomic or diploid induction. Epi chlorohydrin, actinomycin D, and 9-aminoacridine were unable to induce either diploidy or disomy. Ethyl methanesulphonate was active in inducing diploids, while hycanthone induced disomics. beta-Propiolactone, N-methyl-N'-nitro-N-nitro-soguanidine, and beta-naphtylamine were able to increase both disomy and diploidy.

DNA↗

PubMed1987

Immunogenicity of a hepatitis A virus vaccine.

Hepatitis A virus (HAV) strain HAV/HFS/GBM was adapted to and grown in human diploid fibroblast cells. The HAV was concentrated by ammonium sulphate precipitation and high-speed centrifugation. The virus was inactivated by beta-propiolacton and purified by sedimentation through a 20% solution of sucrose and by CsCl gradient centrifugation. The immunogenicity of different preparations was tested in mice, guinea pigs, and goats. The immune response after vaccination was tested by determination of anti-HAV with RIA and of neutralizing antibodies with an appropriate test system. Results showed that anti-HAV titers of 1:1,000 and greater, as well as neutralizing antibody titers of 1:1,000 and greater, were found in sera of animals vaccinated with different preparations. It became evident that good anti-HAV titers persisted over a period of at least 1.5 years in goats and mice after immunization with a semipurified HAV vaccine, and titers up to 1:200 were present in mice 2 years after vaccination with a highly purified HAV vaccine.

Animals↗

PubMed1987

DNA damage in the central nervous system of rats after in vivo exposure to chemical carcinogens: correlation with the induction of brain tumors.

The alkaline elution technique has been used to evaluate DNA damage in brain of rats treated with a single equimolar dose of 14 carcinogens of different chemical structure. A clear-cut increase of DNA elution rate, which is considered indicative of DNA fragmentation, was produced by 10 compounds known to induce the development of tumors in the rat central nervous system: N-nitroso-N-methylurea, N-nitroso-N-ethylurea, N-nitroso-N-butylurea, N-nitroso-N-methylurethane, methyl methanesulfonate, ethyl methanesulfonate, dimethyl sulfate, diethyl sulfate, 1,3-propansultone, and procarbazine. Similar amounts of DNA fragmentation were produced by both potent and weak brain carcinogens. In contrast, any significant increase of DNA elution rate was absent in rats treated with N-methyl-N'-nitro-N-nitrosoguanidine, N-nitrosodimethylamine, N-nitrosodiethylamine, and beta-propiolactone, all of which are devoid of carcinogenic activity for the rat central nervous system. These results suggest that the described in vivo brain DNA damage/alkaline elution assay deserves further studies on a wide number of carcinogens and noncarcinogens aimed to establish its possible usefulness for a qualitative preliminary assessment of the ability of a compound to induce neurogenic tumors.

Alkylating Agents↗

PubMed1995

Sendai virus and herpes virus type 1 induce apoptosis in human peripheral blood mononuclear cells.

Recent reports suggest that several viruses, besides human immunodeficiency virus, induce apoptosis in infected cells. We report here that Sendai virus or Herpes simplex virus type 1 (HSV-1), two potent inducers of interferon-alpha, caused cell death in a consistent number of human peripheral blood mononuclear cells. A careful analysis of infected cells by different techniques, such as optical and electron microscopy, DNA agarose gel electrophoresis, and cytofluorimetric analysis of DNA content, showed that cell death was of apoptotic type. Sendai virus was more apoptogenic than HSV-1, and it was further studied to understand the mechanism(s) by which it induced apoptosis. Physical (uv and heat) and chemical (beta-propiolactone) inactivation reduced or abolished the apoptogenic power of Sendai virus. The use of a novel technique, which allows the study of mitochondrial membrane potential (MMP) in intact cells by flow cytometry, showed that a decrease of MMP is concomitant with the appearance of the hypodiploid peak. These results suggest that Sendai virus and HSV-1 can be added to the list of viruses causing apoptosis, which appears to be a general mechanism occurring during viral infection.

Apoptosis↗

PubMed1986

The capacity of various types of immunoglobulin for intravenous use to interact with Fc receptors of human monocytes and macrophages.

The capacity of immunoglobulin for intravenous application (IgG-IV) to interact with Fc receptors of human monocytes and macrophages was tested by quantifying the inhibition of phagocytosis of IgG-sensitized erythrocytes. To this end a spectrometric phagocytosis test has been used. When compared with IgG for i.m. use (IgG-IM), all IgG-IV had reduced activity. This reduction was related, in part, to the reduced amount of IgG dimers and polymers in IgG-IV. On a weight basis dimeric IgG and polymeric IgG exerted 6-fold and 14-fold higher activity, respectively, than monomeric IgG. When this difference was corrected for, chemically modified IgG-IV still had significantly reduced inhibitory activity; DEAE-Sephadex-treated IgG and acid-treated IgG had an activity similar to IgG-IM, and PEG-treated IgG showed a slightly reduced activity. Pepsin-treated IgG was greater than 100-fold less active than IgG-IM. The reactivity of IgG-IV with monocyte and macrophage Fc receptors was closely correlated. The most conspicuous differences found were related to the concentration at which IgG was used. Thus, beta-propiolactone-treated IgG and plasmin-treated IgG were found to have significantly reduced activity at concentrations greater than 20 micrograms/ml, but almost normal activity when used at lower concentrations.

Cells, Cultured↗

PubMed1985

Hepatitis safety of a new prothrombin complex preparation, sterilized according to the method of LoGrippo. Results of a prospective clinical trial.

In a controlled, prospective clinical trial with almost 300 patients undergoing cardiovascular surgery, the hepatitis risk was examined of a new prothrombin complex (PPSB) preparation produced from pooled plasma, which was cold sterilized according to the method of LoGrippo with beta-propiolactone and UV-light. Of 268 evaluable patients, 102 belonged to a group receiving PPSB and 166 belonged to a control group without PPSB. 25 batches of PPSB in dosages of up to 4,500 units per patient, in total, were used. The mean requirement for transfusion of blood or plasma was 10 units per patient in the group receiving PPSB and slightly more than 2 units per patient in the control group. In both of the groups, 3 hepatitis infections occurred (5 cases of non-A, non-B, 1 case of type B), corresponding to hepatitis incidences of 2.9 and 1.8%, respectively. The difference in hepatitis incidence between both groups was statistically not significant. This demonstrates, on the one hand, the hepatitis safety of the sterilized PPSB and, on the other, that multiple transfusion is no longer associated with a high risk of hepatitis when the guidelines of modern donor screening are followed.

Adult↗

PubMed1988

Physicochemical properties of transmissible gastroenteritis virus hemagglutinin.

Transmissible gastroenteritis virus was readily adsorbed onto chicken erythrocytes at 4 degrees C. The hemagglutinin thus adsorbed could be eluted from the erythrocytes by incubating in phosphate buffered saline at 37 degrees C. The receptor on chicken erythrocytes for the hemagglutinin was inactivated by neuraminidase and potassium periodate, but not by trypsin, 2-mercaptoethanol and formalin. The hemagglutinin was inactivated by trypsin, papain, pepsin, alpha-amylase, phospholipase C, neuraminidase, formalin, 2-mercaptoethanol, potassium periodate, ethyl ether, chloroform, Tween-80 and beta-propiolactone, but not by sodium deoxycholate and trichlorotrifluoroethane, suggesting that the active component of the hemagglutinin involved glycoproteins. The hemagglutinin was stable at 37 degrees C or lower temperatures but not at 60 degrees C or higher temperatures. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 45,000 x g for 60 minutes. In rate zonal centrifugation of the hemagglutinin preparation on a sucrose density gradient, the hemagglutinin activity showed a sharp peak at 1.19 g/ml coinciding with the peak of infectivity. The activity in the peak fraction seemed to be structurally associated with virus particles.

Chemical Phenomena↗