In vitro tests for the measurement of clostridial toxins, toxoids and antisera. II. Titration of Clostridium perfringens toxins and antitoxins in cell culture

The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated. Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical. However, the cytopathic effects of the same preparations are caused by other entities. Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not. Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It follows that all three activities can be valid indicators for toxin neutralization tests. Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test. This test has, in turn, been shown to reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test. It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines.     

In vitro stimulation of human lymphocytes by alpha, beta and delta toxins and toxoids of Staphylococcus aureus.

Alpha, beta and delta toxins of Staphylococcus aureus stimulate human peripheral blood lymphocytes to blastic transformation and formation of IgM, IgG and IgA. The toxins are efficient at concentrations that are not toxic for the cells in culture. A dose of a toxin suitable for stimulation is 100 ng/ml but a stimulation can be observed also at 10 ng/ml, in the case of Ig formation even at a concentration of 1 ng/ml. Toxoids are approximately as effective to elicit blastic transformation as the toxins themselves, their efficiency to stimulate Ig formation being somewhat lower but significant. Alpha and delta toxins and toxoids at the appropriate concentration appear to act as medium-strength polyclonal activators of lymphocytes. Beta toxin and its toxoid are weak polyclonal activators.     

Growth of Clostridium tertium and Clostridium septicum in chemically defined media.

Defined media for the growth of Clostridium tertium and Clostridium septicum are described. The requirements for growth of these two species are compared with each other and with those of Clostridium perfringens.     

Detection of neutralizing antibodies against α-toxin of different Clostridium septicum strains in cell culture

Clostridium septicum, a ubiquitious organism, is the pathogen which causes the classical malignant edema after injuries. Because of its strong cytotoxic alpha-toxin, infections are often lethal. To prevent losses in animals, vaccination with alpha-toxoid vaccines is carried out. Quality control of the vaccines is done by a neutralization test in mice. A cytotoxin test and as an alternative method to detect neutralizing antibodies, a cytotoxin inhibition test was standardized. In the studies, alpha-toxin of the C. septicum reference strain (NC 547) from the National Collection of Type Cultures was compared with alpha-toxin of a field strain from an outbreak in Germany. Sera from five heterologous polyvalent and three monovalent vaccines from eight rabbit groups were available. Each vaccination had been carried out according to the procedure of the German Pharmacopoeia. In three out of the five sera of the groups vaccinated with the heterologous polyvalent vaccine, cytotoxin neutralizing antibodies were detected. High antibody titers were observed in sera of rabbits vaccinated with a vaccine of strain NC 547, lower titers in the sera of rabbits vaccinated with a vaccine of the field strain. No cytotoxin neutralizing antibodies could be found in the sera of rabbits vaccinated with the monovalent C. chauvoei vaccine. The toxins of all strains showed the same ranking of the vaccines. Vaccines which caused high antibody titers in the animals were detected by all toxins as such, as well as vaccines which had medium or low antibody inducing capacity. The results were independent of the C. septicum strain used for the production of alpha-toxin.     

In vitro culture of the hypothalamic supraoptic nucleus. I. General morphological characteristics

Kinetics and morphology of supraoptic nuclei (SON) organ culture of newborn rat hypothalamus have been described. Explants of SON were cultured for 7-90 days. Cell migration, growth of neuron sprouts, formation of nerve bundles and growth zone, and mitotic activity were followed. Neurosecretory cells (NSC) differing in size, shape and degree of activity, were identified in addition to other cell elements of SON. The neurosecret was shown to appear at the beginning of the second week of cultivation in large and middle size NSC. In small and old cultures of NSC no neurosecrete was discovered. It was established that NSC complete the terminal differentiation and maintain for a long time their viable and functional activity in vitro.     

Diversity in Molecular Mass of the Common EDTA-Soluble Antigens of Clostridium chauvoei and Clostridium septicum

Common EDTA-soluble antigens of Clostridium chauvoei and C. septicum were examined by indirect-immunofluorescence (IFA) and immunoblot analysis. The monoclonal antibodies (mAbs) specific for the 35 kDa antigen of C. chauvoei strain ATCC 10092 were used. These mAbs reacted with all 11 strains, 6 of C. chauvoei and 5 of C. septicum, in IFA. In immunoblot analysis with the mAbs, the bands at molecular mass of 35 kDa were found in all C. chauvoei strains, while the bands at 36 kDa were found in 4 of 5 strains of C. septicum. These results indicate that the 35 kDa antigen of C. chauvoei and the 36 kDa antigen of C. septicum possess a similar epitope recognized by the mAb.     

Polypeptides controlling hematopoietic cell development and activation. I. In vitro results

Recombinant DNA technology has been central in answering some of the most relevant questions in the research of regulation of the functional status of hematopoietic progenitor cells and their progeny. This leading article will focus on recent results that have emerged from studies utilizing recombinant molecules that control hematopoietic blood cell development and activation. The following features will be detailed: The molecular and biological characteristics and biochemistry of hematopoietic growth factors, synergizing factors and releasing factors, their role in the regulation of hematopoiesis and activation of normal and leukemic cells, their cellular sources, and regulation of production.     

In vitro cell culture of the Ararat cochineal insect, Porphyrophora hamelii Brandt]

The possibility of cultivating the Ararat cochineal cells in the nutrient media developed for cell cultures of the other insect species was investigated. The initial cochineal cell culture was obtained in suspension. The cell culture on the substrate (glass) dies within 7 days. 2 peaks of proliferative activity was observed in the suspension culture. The increase of cell ploidy and their death were found between these two peaks. The small cells survived and kept their capacity for proliferation. The hemolymph did not stimulate cell proliferation but enabled their better attachment to the substrate. The use of different media did not reveal marked differences in cell behaviour. The pigment granules disappeared during the first week of cultivation.     

Determination of the biological activity of Clostridium difficile toxins in in vivo and in vitro experiments

The biological activity of the filtrates of 29 C. difficile strains was studied in vivo (suckling white mice) and in vitro (cell cultures of different species and origin). The action of the filtrates on the experimental models in vivo was evaluated from the cytotoxic effect index, while in vitro the intensity of the cytotoxic effect was evaluated from the percentage of dead cells in the monolayer. The results of the comparative determination of toxicity characteristics in vivo and in vitro demonstrated that cell cultures were more sensitive experimental models than suckling white mice. The use of cell cultures permitted the quantitative evaluation of the cytotoxic activity of the filtrates under study, as well as the detection of their cell-directed action at minimal concentrations.