Purification and characterization of a novel cytotoxic substance from cell-free extract of Streptococcus pyogenes

A cytotoxic substance designated as streptococcal cytotoxic protein (SCP) was isolated from a cell-free extract of the Su strain of Streptococcus pyogenes possessing cytotoxic and antitumor activity. SCP was purified with a series of column chromatography and preparative PAGE to give a homogeneous single band as revealed by PAGE analysis. The purified SCP has a molecular mass of 165 kDa, composed of four 43 kDa subunits, and its pI is 4.3. SCP was sensitive to proteinases and was labile to heat and at acidic or alkaline pH. SCP showed inhibitory effects on the [3H]thymidine, [3H]uridine and [3H]leucine uptakes and on the growth of cells, and released 51Cr from cells when the protein was added to the cultures of Ehrlich ascites carcinoma (EAC), mouse mammary tumor (MM-2), leukemia (L-1210) and NIH-3T3 mammalian cells in vitro. SCP also showed an antitumor effect on EAC or MM-2 tumor-bearing mice but not on L-1210 tumor-bearing mice in vivo.     

Immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice

Studies on the immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice are summarized in this paper. There was a striking difference in the radiation response of lymphocyte subsets; B cells consist of a fairly radiosensitive homogeneous population, whereas T cells consist of a large percentage (greater than 90 per cent) of radiosensitive and a small percentage (less than 10 per cent) of extremely radioresistant subpopulations. Ly 1+ and Ly 2+ lymphocytes appear equally radiosensitive, although the percentage of radioresistant cells was slightly larger for the former (approximately 5.5 per cent) than the latter (approximately 2.5 per cent). There was a significant strain difference in the radiosensitivity of immune-response potential in mice; immunocompetent cells of C3H mice were more radioresistant than those of BALB/c, C57BL/6, and B10.BR mice. Studies on the long-term effect of radiation on immune system in mice indicated no evidence for accelerated ageing of the immunologic functions when radiation exposure was given to young adults. Preliminary results on the enhancing effect of low dose radiation on cytotoxic T cell response in vitro are also discussed.     

Response to antibiotics of Proteus strains isolated from different types of clinical material

The data on the study of the antibiotic response to 42 Proteus strains isolated from different sources in the hospitals of Kharkov are presented. The isolates belonged to P. mirabilis and P. vulgaris. Many strains were resistant to gentamicin, ampicillin and carbenicillin irrespective of the isolation source. 58.0 and 90.3 per cent of the strains isolated from patients with intestinal infections, 66.6 and 100 per cent of the strains isolated from patients with otitis, 33.3 and 66.6 per cent of the strains isolated from patients with bronchopulmonary affections and 100 and 100 per cent of the strains isolated from patients with urological diseases were resistant to gentamicin and carbenicillin, respectively. As for ampicillin, the respective figures were 74.2, 66.6, 66.6 and 100 per cent. All the strains of P. vulgaris isolated from patients with otitis, urological diseases and bronchopulmonary affections were resistant to ampicillin. The MIC of carbenicillin for all the strains except 4 indole-positive strains of P. vulgaris isolated from the faeces and bronchial excreta was much higher than the borderline values.     

Antibiotic resistance and its transmissibility in strains of opportunistic enterobacteria

Sensitivity of 39 opportunistic Enterobacteria strains was studied with respect to tetracycline, kanamycin, ampicillin, levomycetin, streptomycin and nevigramon. The strains were isolated from children with acute intestinal infections of obscure etiology. 38 isolates were resistant to one or more drugs. The highest number of the strains was resistant to kanamycin, ampicillin and levomycetin, i.e. 81.5, 84.2 and 84.2 per cent of the cultures respectively. Nevigramon proved to be the most effective in vitro (76.4 per cent of the sensitive strains). The ability of the strains to transfer the antibiotic resistance markers on conjugation was tested. Transmission of R plasmids was shown in 75 per cent of the isolates.     

Isolation, growth on prebiotics and probiotic potential of novel bifidobacteria from pigs

Bifidobacteria were isolated from the faeces of pigs of various ages and examined for their potential use as probiotics in combination with di- and oligosaccharides. Ninty-six per cent of the isolates were found to have characteristics in common with Bifidobacterium boum, B. thermophilum and B. choerinum. B. thermophilum was most commonly isolated from sows, whereas most of the other strains were isolated from piglets. A few strains of each species were able to grow in the presence of air. A microplate assay was modified to allow comparison of growth on different substrates. Di- and oligosaccharides considered to promote bifidobacteria were screened for their ability to support growth of selected isolates in vitro. Growth on these substrates varied within and between species. Of the fructose oligosaccharides tested, Actilight P supported the best growth of the widest range of strains. The strains which grew best on the disaccharide lactulose were related to B. choerinum and some of these strains grew on xylo-oligosaccharides. It seems that prebiotic di- and oligosaccharides may have both a species and intra-species/strain selective effect. B. choerinum appeared to be well adapted to the gut of pre-weaned piglets.     

Candida albicans and HIV-1 Infection

Candida albicans virulence is in part mediated by fibronectin (FN) interaction. We compared the adherence level to FN (using Becton Dickinson FN-coated plates) of several strains of yeast isolated from HIV-1 infected or uninfected subjects affected by candidiasis (30 strains from HIV+ subjects and 18 from HIV- subjects). More adhesive strains were found in HIV+ patients than in HIV- subjects. In particular a mean increase of 120 per cent as regards the total number of adhesive cells and 230 per cent as regards the adhesive cells producing germ tubes was detected in the former group of strains as compared to the latter ( p < 0.001 in both cases). The enhancement of FN expression induced by HIV-1 infection, as we have previously demonstrated, can increase interest in the adherence to FN of C. albicans strains isolated from AIDS-affected patients. Moreover, we also underline the important role played by HIV Nef protein in increasing the C. albicans aggressiveness. In fact a significant inhibitory effect of Nef on the phagocytosis of this yeast by macrophages has been demonstrated and the oxidative processes of these cells seem to be down-regulated by this protein.     

Action of aureolic acid and dactinomycin group antibiotics on human brain tumors in culture]

The cytotoxic effect of antitumour antibiotics, such as dactinomycin, mithramycin, variamycin and olivomycin on the cells of the human brain tumours (multiform glyoblastoma, arachnoidendotelioma and astrocytoma) grown by the method of the primary plasmic culture was studied. Dactinomycin was superior to the antibiotics of the aureolic acid group in the rate and level of the cytotoxic effect on the tumour cells: 76 per cent of the above tumours were sensitive to dactinomycin, 56 per cent to mithramycin and 52 per cent to variamycin and olivomycin. Among the total number of the tumours sensitive to the drugs the number of the highly sensitive tumours amounted to 57.9 per cent for dactinomycin and 30.8--38.5 per cent for the antibiotics of the aureolic acid group. Definite differences in the efficiency of the antibiotics of the aureolic acid group with respect to different types of the brain tumours were observed.     

The role of the BCG dose and the mouse strain in the inhibition of development of neoplasms susceptible and nonsusceptible to the action of natural cytotoxic cells

The susceptibility of Ehrlich Ascites Carcinoma (EAC) cells to the action of natural cytotoxic cells of DBA/2 and Balb/c mice in vitro was established. Leukaemia L 1210 cells proved insensitive to the in vitro action of natural cytotoxic cells of DBA/2 mice, but not to those of Balb/c ones. BCG, one of the inductors of cytotoxic NK cells, when administered to DBA/2 or Balb/c mice before introduction of EAC cells inhibited the growth of this tumour but did not retard the development of leukaemia L 1210 in DBA/2 mice. The change in the number of peritoneal exsudate cells (PEC) in DBA/2 mice after intraperitoneal injection of BCG was demonstrated to be dependent on the dose and the time elapsed after bacilli introduction. The antitumour action of BCG does not depend on changes in the number of PEC caused by the bacilli. Both large (3.0 mg) and small (0.02 mg) doses of BCG inhibit the development of EAC in Balb/c mice ("sensitive" to BCG), notwithstanding the time of administration of the bacilli. In DBA/2 mice ("resistant" to BCG) development of EAC can be inhibited only by the large dose of BCG since small one is sometimes ineffective.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Study of the activity of a new glycopeptide antibiotic eremomycin combined with tobramycin against Staphylococci in vitro

Eremomycin is an original natural antibiotic with glycopeptide structure isolated at the Institute of New Antibiotics, the USSR Academy of Medical Sciences. Activity of eremomycin alone or in combination with tobramycin was studied with using 25 clinical strains of staphylococci. 56 and 88 per cent of the strains were respectively resistant to gentamicin and kanamycin, two aminoglycoside antibiotics. All the staphylococcal strains were sensitive to eremomycin in concentrations of 0.12 to 1 microgram/ml. The MIC of tobramycin for 10 (40 per cent) sensitive strains ranged within 0.25-2 micrograms/ml. For 60 per cent of the strains the MIC was equal to or higher than 16 micrograms/ml. When eremomycin was used in combination with tobramycin the antibacterial effect with respect to 17 strains (68 per cent) increased. In 32 per cent of the strains the effect was synergistic and in 36 per cent of the strains it was additive. Indifference and antagonism were detected with respect to 7 (28 per cent) and 1 (4% per cent) strains respectively. No significant difference was shown in manifestation of the synergistic-additive nature of eremomycin and tobramycin interaction with respect to the tobramycin sensitive and resistant strains.     

Drug resistance of opportunistic enterobacteria isolated from various sources]

Resistance of 159 strains of opportunistic enterobacteria to 9 antibacterial drugs was studied. The strains were isolated from man and cattle. It was shown that the overwhelming majority of the isolates (93 per cent) were polyresistant irrespective of the genus. There was a high frequency of the strains resistant to the widely used antibiotics such as chloramphenicol (73 per cent), ampicillin (73.6 per cent) and rifampicin (95.6 per cent) and sulfanilamides (99.3 per cent). Gentamicin and nalidixic acid proved to be the most active against the cultures: 11.9 and 10 per cent of the resistant strains, respectively. The strains of enterobacteria isolated from different sources had a sensitivity to the antibiotics. Multiple antibiotic resistance to at least 5 drugs, variability of the spectra and high resistance were more characteristic of the isolates from the animals. The necessity of a rational use of antibacterial drugs in veterinary is indicated.     

Possible dependence of the results of phage typing Ps. aeruginosa strains on the presence of R plasmids

The plasmid composition of 209 strains of Ps. aeruginosa was determined. The strains were isolated from patients, animals and environment in different geographical areas. The number of the plasmid-containing strains averaged 26.8 per cent. The molecular weights of the plasmids varied from less than 10 to more than 150 MD. 41 conjugative plasmids were transmitted to the recipients of Ps. aeruginosa RAO 303. 66 per cent of them had a restrictive effect on the development of phages used in genetic studies, epidemiological phage typing (Lindberg Collection), and medical practice. This resulted in the changing of the phage type of the host strain. Similar results were obtained in the studies with 10 standard R plasmids representing different incompatibility groups. No relation between the spectrum of the phage restriction, group specificity and the other properties of the plasmids was observed. About 50 per cent of the plasmids markedly lowered the sensitivity level of Ps. aeruginosa RAO 303 to the therapeutic pyocyanic phage. The systems of restriction and modification of DNA coded with plasmids were not detected. A possible changing of the phage type of Ps. aeruginosa strains under the effect of R plasmids should be considered in epidemiological assays and respective treatment measures.     

Antibiotic sensitivity and the characteristics of transmissive R plasmids in Shigella belonging to various species]

Sensitivity of 241 Shigella strains isolated from patients at various regions of the USSR in 1975--1978 was tested with respect to 14 antibiotics by the method of serial dilutions. 90.5 per cent of the isolates proved to be resistant to the antibacterial drugs and the greater part of 75.9 per cent of them had multiple resistance. The resistance of the Shigella was most pronounced and frequent with respect to tetracycline, streptomycin, levomycetin, as well as ampicillin and carbenicillin. Gentamicin, cephaloridin, polymyxin M, kanamycin, monomycin, neomycin and rifampicin were highly active against the Shigella. More than 50 per cent of the isolates were sensitive to levomycetin, ampicillin and carbenicillin. Differences in the frequency of the resistant strains and the spectrum of the antibiotic resistance of different Shigella subgroups (species) were observed. The study of 173 multiple resistant Shigella strains showed that about 67 per cent of the strains had a capacity for transduction of the resistance markers into the recipient cells of E. coli. The conjugative R-plasmids were most frequent in Sh. boydii and Sh. sonnei (95 and 95 per cent respectively), less frequent in Sh. flexneri and Sh. newcastle (68 and 53 per cent respectively) and least frequent in the mannitol negative Shigella (25 per cent). The capacity for transduction of R-plasmids in the strains carrying the determinants of resistance to 2 or 3 antibiotics was higher than in the strains carrying the determinant of resistance to one antibiotic. The clinical Shigella strains tested mainly had transmissive R-plasmids of fi--character (79 per cent).     

Antibiotic sensitivity of Proteus, Pseudomonas pyocyanea and staphyloccoci isolated from scleroma and ozena patients]

Antibiotic sensitivity of 292 strains of Proteus, 60 strains of Ps, aeruginosa, 309 strains of S. aureus and 88 strains of S. epidermidis isolated from the upper respiratory tract of patients with scleroma and ozena was studied. The cultures of Pr. mirabilis were sensitive to aminoglucosides (54.9-96.2 per cent) and Pr. morganii were sensitive to levomycetin (81.5 per cent) and neomycin (92.6 per cnet). Sensitivity of Pr. vulgaris and Pr. morganii was reliably higher (p less than 0.001) than that of Pr. mirabilis. The strains of Pr. morganii were less sensitive to monomycin (P less than 0.001) and streptomycin (p less than 0.01) as compared to the cultures of other Proteus species tested. The strains of Ps. aeruginosa were sensitive only to gentamicin (90 per cent) and neomycin (81.1 per cent). Most of the strains of S. aureus (85.4-100 per cent) were sensitive to oleadomycin, erythromycin, olemorphocycline, tetraolean, oxacillin, methicillin ceporin, lincomycin, ristomycin, kanamycin, monomycin and gentamicin. Benzylpenicillin (90.8 per cent of the sensitive strains), ampicillin (67.1 per cent), tetracycline (66.7 per cent), levomycetin (68.6 per cent) and streptomycin (38.1 per cent) were less effective. Antibacterial therapy in cases with scleroma and ozena should be directed not only against causative agents of the diseases but also against the microbes developing due to disbacteriosis. Combination of parenteral and local use of the antibiotics in the treatment of chronic clebsiellesis decreased the isolation rate of Proteus and Ps. aeruginosa in the patients.     

EXPERIMENTALLY INDUCED CHANGES IN THE BASE COMPOSITION OF THE RIBONUCLEIC ACIDS OF ISOLATED NERVE CELLS AND THEIR OLIGODENDROGLIAL CELLS

The effect of tricyano-amino-propene, a dimer of malononitrile, on the base composition of the RNA in isolated Deiters' nerve cells and their oligodendroglial cells has been studied using a microelectrophoretic method. Tri-a-p in a dose of 20 mg/kg has the effect of increasing the RNA and protein content per nerve cell by 25 per cent and decreasing the glia RNA by 45 per cent. The RNA base composition of the nerve cells from the control animals differs from that of their glial cells. The guanine of the nerve cell is significantly higher than that of the glia, but the content of cytosine is higher in the glia than in the RNA of nerve cell. The cytosine of nerve cells decreased significantly after tri-a-p administration. In the glial cells the cytosine showed a 20 per cent increase, and the guanine a 25 per cent decrease. Tri-a-p sharpened the difference in RNA composition already existing between the control nerve cells and their glial cells by almost 300 per cent for the guanine and by 400 per cent for the cytosine. The chemical and functional relationship between the nerve cell and its oligodendroglial cells is discussed.     

Comparative characteristics of the antibioticograms and biological properties of the staphylococci isolated from patients with various eye diseases]

A total of 1488 examinations of the conjunctival content of patients with various ophthalmic diseases were performed within 5 years (1973--1977). 989 (66.4 per cent) cultures were isolated. Staphylococci were isolated in 79.2 per cent of the patients irrespective of the diagnosis. The plasma-coagulating strains were isolated in 43.6 per cent of the patients with acute conjunctivitis. The plasma-negative staphylococci were more frequent in chronic conjunctivitis (83.5 per cent). A total of 188 strains were studied according to 18 tests. Determination of the antibiotic sensitivity showed that the number of the benzylpenicillin and tetracycline resistant strains among the plasma-coagulating staphylococci was higher in the patients with acute ophthalmic diseases. In chronic conjunctivitis no differences in the antibiotic sensitivity of the plasma-coagulating and plasma-negative strains were noted. Similar antibiotic sensitivity of all plasma-negative staphylococci irrespective of the diagnosis was found. High biological activity of the plasma-negative staphylococci was shown: 74.1 per cent of the isolates possessed phosphatase, 59.8 per cent possessed protease and 88.4 per cent a lysozyme-like enzyme. The strains isolated in chronic conjunctivitis produced protease more frequently that those isolated in acute ophthalmic diseases.     

Effectiveness of gentamicin sulfate in suppurative-inflammatory processes of varying localization]

Antibiotic sensitivity of 486 strains of grampositive and gramnegative organisms isolated from patients with purulent infections was studied in vitro. Gentamicin was shown to be highly active as compared to kanamycin and other antibiotics against the main causative agents of purulent inflammatory infections including multiresistant E. coli, Proteus, Ps. aeruginosa, Staphylococcus. High efficiency of gentamicin in therapy of peritonitis, septic conditions, purulent postoperative wounds, infections of the urinary tract, pneumonia, etc. (197 patients) was shown. Positive results were obtained in 87.4 per cent of the cases. Side effects, such as albuminuria, hyperthermic reaction, rash, pruritis were registered in 3 per cent of the patients.     

Different progress of MDCK cell death after infection by two different influenza virus isolates

The effect of influenza strains A (H₃N₂) and B, isolated during the seasons of 1994 and 1995 in the Czech Republic, on MDCK cells was studied. Various concentrations of virus and conditions of nutrition were used during the cell culture. The virus replication and consequently fragmentation of genomic DNA together with cytotoxicity were investigated in the absence and presence of 10 per cent calf serum. Virus replication, regardless of type A or B, caused earlier DNA fragmentation in comparison to non-infected cells in tissue culture. The results showed that the influenza B strain had a greater cytotoxic effect on MDCK cells than influenza A. A higher infection dose of influenza A virus accelerated the onset of apoptosis; conversely, a higher infection dose of influenza B virus delayed the onset of apoptosis. The absence of serum enhanced the progress of influenza-induced apoptosis in conditions in vitro. © 1997 John Wiley & Sons, Ltd.     

Antibiotic sensitivity of Vibrio cholerae 01 isolated from environmental objects]

The sensitivity of 252 Vibrio cholerae-O1 strains isolated from environmental objects to antibiotics of various groups was assayed by the method of serial dilutions on solid media. The biological characteristics of the isolates are presented. The Vibrio cholerae isolates with serological variation were the most frequent (36.6 per cent), so are the cultures detected by their sensitivity to the specific phages (87.5 per cent). It was found that changes in some biological properties of the strains did not coincide with the changes in the antibiotic sensitivity. The isolates were highly sensitive to tetracycline, chloramphenicol, gentamicin, erythromycin and rifampicin and less sensitive to novobiocin and the other aminoglycosides. The sensitivity to the beta-lactams was the lowest. The resistance determinants were detected in single strains (6.3 per cent), the kanamycin and novobiocin resistance determinants being detected in 15 out of the 16 strains tested. The study showed that the cultures of Vibrio cholerae-O1 isolated from the environmental objects generally preserved their sensitivity to the diverse group antibiotics.     

Level and nature of the drug resistance of Shigella flexneri]

Characteristics of antibiotic resistance of 300 strains of Shigella flexner 2a isolated from patients within 1976--1977 in the regions where these bacteria were very rare for a long period of time were studied. It was shown that most of the isolates were resistant to chloramphenicol (88.1 per cent), tetracycline (94.8 per cent), streptomycin (89.1 per cent), polymyxin M (82.4 per cent) and others. 46.5--61.6 per cent of the isolates were resistant to neomycin antibiotics. A high level of the resistance was also noted: the bactericidal effect was registered in 52.0-74.0 per cent of the cultures at a dose of 500--1000 microgram/ml. 91.4 per cent of the strains possessed multiple dug resistance, 78.8 per cent of them being simultaneously resistant to 4--7 drugs. Transmissive R-plasmids were found in 68.8 per cent of the isolates. After exposure to acridine dyes the plasmid nature of the resistance was confirmed in 72.3 per cent of the cultures. Variability of the r-determinant sets in r-plasmids was noted. Strains (64.9 per cent) carrying r-determinants Tc, Cm, Sm and Tc, Cm were more frequent. Strains with one transmissive r-determinant were usually solitary.