Design of stable immunoglobulin erythrocyte diagnostica. 1. Erythocyte fixation and their sensitization by specific immunoglobulins]

The work presents the results of developing the method of fixation of erythrocyte constituting the cellular base of immunoglobulin erythrocytic diagnostic preparations and the sensitization of erythrocytes with immunoglobulin preparations of various specificity. Based on Ingraham's method, modified method of erythrocyte stabilization has been developed; it consists in the treatment of 50% cell suspension with 4% formaldehyde solution in the presence of 0.5% sucrose (erythrocyte suspension and formaldehyde solution being in the ratio 1 : 2.5). An economic and highly productive technique of sensitizing erythrocytes with immunoglobulin preparations has been developed. The essence of this technique lies in the interaction between 6% suspension of erythrocytes treated with formalin and tannin and the equal volume of sensitin taken in a working dose. The work also presents the method of synthesizing the bifunctional compound fluoro-borate bis-daizonium complex (obtained from benzidine) and discusses the comparative possibilities of the methods of developing immunoglobulin erythrocytic diagnostic preparations by sensitization of tannin-treated erythrocytes and by chemical conjugation.     

Nonspecific means of increasing the sensitivity of the passive hemagglutination reaction]

The effect of some methods of preliminary treatment of erythrocytes on the PHAT depended on the sensitin náture and the method of erythrocyte load. In case of erythrocyte load with nonprotein and immunoglobulin sensitins without any conjugating agents the simulating effect of heating and periodate treatment was caused not by increase of stable sensitin binding, but by the reduction of physico-chemical resistance of erythrocytes. This effect of erythrocyte treatment permitted to increase the sensitivity of the antibodies and antigens determination. In loading the erythrocytes with the aid of conjugating agents and in sensitization with protein antigens after Boyden no stimuating effect of the treatment was noted.     

Study of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> antigenic structure to obtain a sensitin for design of species-specific diagnostic kits

A method for isolation of outer membrane proteins from Yersinia pseudotuberculosis, which are perspective for further application as sensitin for design of species-specific pseudotuberculosis antigenic diagnostic kits, has been modified. Common species-specific antigens of nine Y. pseudotuberculosis serovars (with molecular weight from 80.62 to 12.2 kDa) were detected by SDS-PAAGE electrophoresis and immunoblotting of the outer-membrane protein preparations. These antigens react with neither the rabbit experimental antiplague antiserum nor antiserum to 39 Y. enterocolitica serovars and normal rabbit serum.     

Purified Protoplasmic Peptides of Mycobacteria: Isolation of Species-Specific Peptides from Protoplasm of Mycobacteria

Infections with mycobacteria other than tubercle bacilli are responsible for a variable percentage of cross-reactions to tuberculin. Two major suggestions for circumventing this problem have been made: the first, development of a quantitative tuberculin test, is based on the fact that most cross-reactions are smaller than those caused by true tuberculous infections; the second, preparation of purified skin test antigens from other mycobacteria, is based on the hope that greater specificity will be displayed by homologous sensitin. Effort so far has been focused on the culture filtrates as the source of antigen. This article describes the preparation of low molecular weight purified protoplasmic peptides (PPP) of specificity and sensitivity superior to purified protein derivatives.     

The protective activity of preparations made from the tick-borne encephalitis virus grown using different cell cultures]

The preparations of tick-borne encephalitis (TBE) virus grown in swine embryo kidney cell culture have been shown to possess pronounced protective activity per unit of virion protein E in comparison with TBE virus preparations derived from cell culture 4647 and chick embryo cell culture. The antigenic activity of all virus preparations under study has proved to be practically the same. The role of post-translation modifications of TBE virus protein E in the manifestation of some of its biological properties is discussed.     

Molecular aspects of the interaction of albumin and tannined erythrocytes in the process of hemosensitization. II. The effect of temperature]

A study was made of a combined influence of the temperature of the medium and of sensitin concentration on the process of interaction of tannin-treated sheep erythrocytes and human serum albumin. On the basis of determination of the number of molecules bound by a single erythrocyte during hemosensitization,, depending on the mentioned conditions, it was revealed that the relative total albumin binding increased with the elevation of temperature. Elevation of temperature also led to the absolute and the relative increase in the stable and a reduction in the loose albumin binding by a single erythrocyte and the growth of sensitivity of the passive hemagglutination test. The role of chemical mechanisms in the erythrocyte albumin loading was demonstrated; this permitted to carry out erythrocyte albumin sensitization at a comparatively high temperature for the purpose of increasing the efficacy of passive hemagglutination test.     

Comparative study on the immunogenic properties of Clostridium perfringens type A toxoid

The paper presents the results of a study on the immunogenic properties of toxoid preparations from Cl. perfringens type A obtained using the routine method of detoxifying alpha = toxin in the culture medium (commercial preparations) and by means of detoxifying a previously purified alpha = toxin (experimental preparations). When tested in immunized guinea pigs, the immunogenicity of experimental preparations was found to be 4.5 to 6 times that of commercial preparations. In mice, there was no difference in the immunogenic properties of the two types of preparations as determined by the ED30 of the antigen and the serum levels of Cl. perfringens antitoxin. The possibility is discussed of using the guinea pig as a laboratory animal model due to its ability to reflect most clearly the differences in the immunogenicity of Cl. perfringens type A toxoid preparations.     

How the carotid body works: different strategies and preparations to solve different problems

This is a review of the different experimental approaches developed to solve the problems in our progress towards a comprehensive understanding of how arterial chemoreceptors operate. An analysis is performed oi the bases, advantages and limits of the following preparations: studies of ventilatory reflexes originated from carotid bodies (CBs) in the entire animal; recordings of CB chemosensory discharges in situ; CB preparations perfused in situ; CB explants in oculo; CB explants in ovo; CB preparations incubated in vitro; CB preparations superfused in vitro; CB preparations perfused and superfused in vitro: CB tissue slices in vitro; cells acutely dissociated from CBs; CB cells in tissue culture; petrosal ganglia superfused in vitro; petrosal ganglion cells in tissue culture; and co-cultures of CB and sensory ganglion cells. A brief historical account is given of the passage from one preparation to the next one. Emphasis is placed on personal experience with the different preparations whenever possible. Examples are given of the importance of selecting the appropriate experimental preparation for solving each particular theoretical problem. In fact, brilliant ideas on how the CB works have been unproductive until finding the adequate experimental approach to explore the validity of such ideas.     

Degradation of agarophytic red algal cell wall components by new crude enzyme preparations

Several new crude enzyme preparations were isolated from a marine association of the agarolytic bacterium Cytophaga diffluens and the infusorium Uronema marinum, an axenic culture of Cytophaga diffluens, some species of land micro- and macromycetes adapted to assimilate red algal biomass and from the marine mollusc Littorina littorea. Fungal and mollusc enzyme preparations were shown to have cellulase, xylanase, protease and agarase activities. Fungal agarase activity was revealed only after 3–4 passages of the culture on the medium containing algal biomass. Enzyme preparations from the association and the pure bacterial culture growing on the medium with bactoagar as the sole carbon source contained only agarase activity. The maximum specific agarase activity was found in a preparation from the marine association. The preparations obtained can be used for isolating protoplasts and single cells from red seaweed thalli. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relation between the mitosis-inhibitory activity of a chalone preparation of Ehrlich ascites tumor and its diurnal secretion

Mitosis inhibitory activity of chalone-containing preparations extracted from cells of Ehrlich's ascites carcinoma (EAC) at varying time of day (9, 13, 17, 21 1, 5 and 9 o'clock) was studied in a temporary suspension culture of EAC. The inhibitory action of the preparations depended on the time of their extraction. This may be indicative of rhythmical alterations of the production or activity of EAC chalone. The maximum activity was demonstrated by the preparations extracted at 5, 9 and 13 o'clock. The activity of the preparations, extracted at 17, 21 and 1 o'clock was considerably less. The degree of mitotic activity inhibition also depends on the dose of duration of action of the chalone-containing preparation.     

Effect of mouse interferons on the development of Ehrlich ascitic carcinoma

Intraperitoneal injection of various preparations of mouse interferons (L cell tissue culture interferons, concentrated or partly purified, and also serum interferon) significantly inhibited the development of Ehrlich's ascites carcinoma in randombred mice. In view of comparatively low activity of serum interferon, the effect of normal mouse serum on the tumour development and its action on L cell tissue culture interferon was investigated. It was shown that normal mouse serum inhibits the action of L cell tissue culture interferon and promotes the development of Ehrlich's ascites carcinoma.     

The development of a process for culturing Bordetella pertussis immobilized on a polyurethane carrier

The processes of the cultivation of Bordetella pertussis, immobilized on polyurethane carrier in a fermenter, were carried out and studied. Acellular pertussis preparations were produced from the culture fluid obtained in the batch and multi-cycle cultivation processes with immobilized cells, as well as in the process with interrupted fermentation (for confirming the possibility of the preservation of cell viability). The content of protein and B. pertussis toxin in these preparations, as well as their leukocytes-stimulating and hemagglutinating activity, did not differ from similar characteristics of preparations obtained from culture fluid in homogeneous cultivation.     

Study of the enterotoxic action of the neurotoxin of the Sonne dysentery microbe in a rabbit small intestine loop model]

The enterotoxic action of neurotoxin from Sonne dysentery microbes (obtained by the method of Mesrobeanu et al.), and also of the culture autolysates and homologous Boiven's endotoxin was studied on a model of the isolated loop of the rabbit small intestine. Neurotoxin preparations obtained from virulent strains as well as autolysates of these cultures possessed enterotoxic activity, whereas purifed endotoxin preparations in doses of 1--10 mg failed to cause any dilatation of the isolated intestinal segment. A significant individual rabbit sensitivity to the enterotoxic action of the neurotoxin preparation was revealed. Lyophilization of neurotoxin preparation did not influence its enterotoxicity. However dialysis against distilled water and boiling of the neurotoxin preparations led to the loss of enterotoxic activity.     

Studies in the Physiology of Parasitism: XIX. On the Killing of Plant Cells by Enzymes from Botrytis cinerea and Bacterium aroideae

1. Enzyme preparations were obtained from culture filtratesof the soft-rot pathogens Botrytis cinerea Pers. and Bacteriumaroideae (Townsend) Stapp grown in simple synthetic nutrientmedia. Crude culture filtrates and preparations purified byacetone-precipitation and dialysis had three characteristicproperties. They (i) decreased viscosity of pectin and pectatesolutions, (ii) macerated parenchymatous tissues of higher plants,and (iii) killed cells of tissues so macerated. A parallelismwas demonstrated between activity estimated by these three criteria. 2. B. cinerea enzyme preparations were active from about pH3.5 to pH 6.0, activity decreasing rapidly from pH 6.o to nearlynil at pH 8.0. Conversely B. aroideae was most active abovepH 8.0, activity decreasing progressively to nearly nil at pH5.5. 3. Both enzymes lost much activity on prolonged dialysis againstdistilled water and this was not recovered on readdition ofdialysed salts. On dialysis against certain salts or salt mixturesreduced or negligible losses occurred. 4. Plasmolysing concentrations of salts or non-electrolytesgreatly retarded the killing action of the enzyme preparations,the effect being out of all proportion to that on macerationor on rate of pectin degradation. 5. Protoplasts were isolated in the plasmolysed condition fromcertain tissues. These were resistant to toxicity in similarmanner to those inside the tissue.     

An improved technique of preparing bone-marrow specimens for cytogenetic analysis.

Sixty-six bone-marrow specimens, derived from patients with hematological and nonhematological disorders, were processed for cytogenetic analysis. Modifications of various parameters of the standard (direct) culture procedure were investigated and the quality of the preparations determined. As a result of these experiments, an improved culture technique was developed that yielded significantly better quality chromosome preparations. This method is based on a short incubation (25-min) of the bone-marrow specimen, immediately following aspiration, in a solution containing both hypotonic KCl and colcemid and completely omits the use of tissue culture medium.     

Endotoxin of Pseudomonas pseudomallei detected by the body weight-decreasing reaction in mice and comparison of it with those of P. cepacia and P. aeruginosa.

The heat-treated whole cells, culture supernatants, and extracted endotoxin preparations of Pseudomonas pseudomallei were examined for endotoxin by the mouse body weight-decreasing (BWD) test. The experiments were conducted also with those of P. cepacia and P aeruginosa. Endotoxin was detected in all the samples of P. pseudomallei. Endotoxin of P. cepacia was detected in whole cells, but not in culture supernatant. The BWD activity of P. aeruginosa was 30 times as high as that of P. pseudomallei. This result was confirmed by the experiments with endotoxin preparations. In the limulus amebocyte lysate gelation (LAL) test, however, the endotoxin preparations of the two species showed the same level of activity.     

Activation of Clostridium botulinum type E toxin purified by two different procedures

Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.     

On the possible mechanism of the protective effect of Escherichia.

A model on a HEp-2 cell culture was elaborated, permitting the study of the ability of microbes to be adsorbed an to proliferate on the surface of cells and of the mechanism of their protective effect. The ability of E. coli strains to be adsorbed and to proliferate on the surface of a cell culture was found to differ. It has been demonstrated that the protection of the cell culture from subsequent infection with virulent Shigella can be explained not only by the antagonistic activity of E. coli strains, but also by their ability to be adsorbed and to proliferate on the surface of cells. A similar mechanism of protective effect is supposed in preparations of the Colibacterin type.     

Visualisation of specific binding sites for atrial natriuretic peptide on non-neuronal cells of cultured rat sympathetic ganglia

Summary The distribution of atrial natriuretic peptide binding sites on cells in dissociated culture preparations of neonatal rat superior cervical ganglia and in explant cultures of rat thoracic sympathetic chain ganglia has been studied. The autoradiographic visualisation of atrial natriuretic peptide binding sites has been combined with the use of specific immunocytochemical markers for glial cells (antiserum to S-100 protein), fibroblasts (antiserum to fibronectin) and neurones (antiserum to protein gene product 9.5) in order to achieve unambiguous identification of the cell types in culture. Specific binding sites for rat¹²⁵I-atrial natriuretic peptide_(1–28) were observed over subpopulations of fibronectin-like-immunoreactive fibroblasts and S-100-like-immunoreactive glia in the dissociated superior cervical ganglion cultures. However, only a subpopulation of fibronectin-like-immunoreactive fibroblasts possessed atrial natriuretic peptide binding sites in the explant culture preparations. No atrial natriuretic peptide-like-immunoreactive cells were present in either culture. The distribution of autoradiographic grains over individual cell surfaces in culture was uniform, but there were distinct differences in the density of labelling of single cells of the same type. This apparent variation in the number of binding sites on glial cells and fibroblasts in culture did not seem to be related to the morphology of the cells or the surrounding cell types. No sympathetic neurones were labelled with autoradiographic grains in either the dissociated or explant culture preparations. However, the presence of atrial natriuretic peptide binding sites on non-neuronal cells of sympathetic ganglia in culture may be linked to the relationship between atrial natriuretic peptide and the sympathetic nervous system.     

Study of the action of phytohemagglutinin on continuous human cells]

A study was made of the agglutinating and growth-stimulating properties of phitohemaagglutinin (PHA) preparations in experiments on continuous human cells (clone line HeLa k-41 and CaVe). PHA and PHA gamma-globulin fraction possessed poor hemagglutination properties against human erythrocytes of A, B and O groups, but expressed marked cytoagglutination properties against HeLa k-41 and CaVe cells. With high PHA doses (100 and 500 microng/ml) proliferation and mitotic indx of the culture cells was lower and the percentage of dead cells and the agglutinin titre in the preparations was greater (1 : 256). With lower PHA doses (5 and 25 microng/ml) the growth was much stronger and the percentage of dead cells lower. The agglutinin titre in the preparations decreased to 1 : 16-1 : 32. PHA gamma-globulin fraction produced the strongest growth-stimulating action with the least amount of dead cells. However, the agglutinin titre in the preparations remained high (1 : 128). A conclusion was drawn that the action of PHA preparations stimulating and inhibiting the growth of continuous human cells proved to be directly connected with the agglutinin content in the preparations, since gamma-globulin PHA fraction expressed the greatest cytoagglutinating and growth-stimulating action.