Effect of prodigiozan, levamisole and methyluracil on delayed hypersensitivity against a background of immunosuppressor use

Immunostimulants, such as prodigiosan, levamisol and methyluracil, as well as their combinations with imuran, prednisolone or cyclophosphamide were studied for their effect on the delayed type hypersensitivity (DTH) induced by dinitrofluorobenzene alcohol solutions applied in challenge doses to the floor of the auricle of mice. It was shown that the immunostimulants did not affect the DTH in intact mice. In mice treated with imuran the DTH was significantly increased only by prodigiosan. Prednisolone used for a prolonged period before and after the sensitization and cyclophosphamide administered 24 hours before the sensitization increased the DTH. According to the literature data it was connected with T-suppressor inhibition. With the use of cyclophosphamide it was also connected with B-suppressor inhibition. Under such conditions the DTH was decreased to the control level by prodigiosan after the use of prednisolone or by levamisol after the use of cyclophosphamide. This was probably associated with increasing of the suppressor effect of the macrophages and activation of the T-suppressor effect by levamisol. Methyluracil had no effect on the DTH.     

Immunocorrecting and protective effect of proteolytic enzymes on antibody formation in mice in staphylococcal infection and antibiotic treatment

The impact of proteolytic enzymes on the humoral immune response, survival rate and mean survival time of mice, infected with S. aureus culture and receiving antibiotics was studied. Infection with staphylococcal suppressed the formation of antibodies to sheep red blood cells. Ampicillin made this immunosuppression even more pronounced, while gentamicin produced practically no effect on the degree of immunosuppression in the infected animals. Proteolytic enzymes terrilytin and terridecase exhibited immunocorrecting properties when used in combination with antibiotics. Terridecase, the immobilized form of the enzyme proved to have the highest activity. In experimental generalized staphylococcal infection all preparations under study produced a protective effect. The maximum effect was noted after the use of ampicillin in combination with terridecase.     

The evaluation of the possibility of using prodigiozan for isolating extracts that stimulate proliferative processes in the resected liver]

The experiments were performed on 126 white male rats. The inclusion of 3H-thymidine in nuclear DNA of the liver was studied on h 24 and 48 after 70% resection of the liver versus prodigiosan injection or combination of prodigiosan with 70% resection of the liver. Liver extracts stimulating proliferation (ESP) obtained under the above schedules were studied on the model of 30% liver resection. It is shown that prodigiosan (0.25 mg/kg) injected to intact rats and rats with 70% resected liver initiated ESP production promoting the inclusion of 3H-thymidine in nuclear DNA of the liver after 30% resection of the liver.     

Effect of dilute solutions of biologically active substances on cell membranes

The article presents data on changes in physicochemical properties of different biological membranes (plasmatic, microsomal, synaptosomes) under the action of biologically active substances, which are different in their chemical structure and the mechanism of action (natural and synthetic antioxidants, thyrotropin - releasing hormone, phorbol esters), in the wide range of concentrations (10^?22?10^?3 M). Dose dependences of the effect of biologically active substances on the activity of membrane-bound enzymes, lipid peroxidation, the structural state of the various regions of the lipid bilayer of membranes have been obtained and analyzed in terms of their formal generality of polymodality, number and position of the maxima, a sign change of the effect. An attempt to explain the mechanism of each of the observed peaks in these curves has been made. The maximum in the range of relatively high “physiological” concentrations (10^?3–10^?7 M) is associated with introduction of biologically active substances into biomembranes. In this study maxima in the range of ultra-low doses (10^?11–10^?16 M) and “apparent” concentrations (10^?18 M), where the presence of biologically active substance molecule in a reaction volume is probabilistic in nature, are explained by physicochemical properties of diluted biologically active substances solutions. This conclusion is based on our data on the changes in IR spectra of aqueous solutions of biologically active substances and the results obtained by academician A.I. Konovalov et al. concerning the physicochemical properties of dilute solutions of biologically active substances (conductivity, surface tension, charge), due to the formation of so-called “nanoassociates” from biologically active substance molecule and numerous number of water molecules. The nanoassociates formation and biological effect disappear if the low concentration solutions are kept in a special shielded permalloy container protecting its contents from external electromagnetic field. Thus, nanoassociates are the material carriers of the unique ability of the ultra-low doses of biologically active substances to exhibit biological effects.     

Protease inhibitors interfere with the transforming growth factor-beta-dependent but not the transforming growth factor-beta-independent pathway of tumor cell-mediated immunosuppression.

Tumor cells have been reported to exert inhibitory effects on the activation of T lymphocytes in vitro. We show that the IL-2-stimulated proliferation of a Th cell line is suppressed when the T cells are cocultured with human glioblastoma and melanoma cell lines. The use of two Th cell clones that differ in their responsiveness to growth-inhibition by transforming growth factor-beta (TGF-beta) and the analysis of tumor cell-derived supernatants as well as of TGF-beta 1/TGF-beta 2 gene expression allowed to distinguish two pathways of tumor-induced immunosuppression. Glioblastoma cells exert their immunosuppressive effects by producing biologically active TGF-beta 2, whereas the immunosuppressive state induced by melanoma cells is TGF-beta-independent and requires direct contact between tumor cell and T cell. The TGF-beta-dependent immunosuppression is down-regulated by various protease inhibitors and up-regulated by estradiol via modulation of the production of biologically active TGF-beta 2 by glioblastoma cells leaving total activatable TGF-beta 2 unaffected. No such modulation is functional for the TGF-beta-independent pathway of immunosuppression. We conclude that the production of active TGF-beta by tumor cells is regulated at a posttranslational level by the coordinated action of several proteolytic enzymes.     

Immunosuppressive properties of sera and urine dialysates from kidney-graft recipients treated with azathioprine,prednisolone, and niridazole

Niridazole, an antischistosomal agent, was given to renal transplant recipients in addition to azathioprine and prednisolone, as there is experimental evidence that this combination of drugs is highly immunosuppressive. Sera obtained from kidney-graft recipients during the first two weeks after transplantation were examined for their ability to inhibit the one-way mixed lymphocyte reaction (MLR). Sera from seven patients receiving azathioprine, prednisolone, and niridazole (triple-drug treatment), five patients receiving azathioprine and prednisolone, and two other patients treated with niridazole alone for schistosomiasis produced MLR inhibition by comparison with pretreatment (control) sera.A mean of 78% inhibition was observed with sera taken after one day''s treatment with the three-drug combination, whereas this level of in-vitro immunosuppression occurred only after eight days of treatment with azathioprine and prednisolone. Niridazole alone produced an effect similar to azathioprine and prednisolone. Concentrated dialysate of urine from a patient receiving triple-drug treatment not only inhibited the MLR but also significantly prolonged the survival of heterotopic heart allografts in rats, whereas dialysate from the same patient after niridazole had been stopped gave less MLR inhibition and failed to prolong heart allograft survival.Since niridazole thus increased the in-vitro and in-vivo immunosuppressive action of azathioprine and prednisolone, we suggest that this triple-drug combination might be useful for preventing early acute kidney graft rejection.     

Bactericidal substances in cytoplasm of leucocytes. An attempt to separate lysozyme by gel filtration

A substance with a bactericidal effect onEscherichia coli was isolated from the cytoplasm of polymorphonuclear leucocytes. It consists of a mixture of different active substances and therefore a gel filtration using s Sephadex G-100 column was used to separate lysozyme from other active substances. The extract was separated by this procedure into five peaks, out of which peak I. possesed a significant bactericidal activity, peak II. and III. had a weak one, whereas the lysozyme activity was present in peak III., IV. and V.     

Effect of prodigiosin and its combination with immunodepressants on the graft versus host reaction in mice

The local (lymph node) graft-versus-host reaction (GVHR) in F1 (CBA X C57BL/6) mice and the lethal GVHR in C57BL/6 mice were induced by transfer of lymph node cells of CBA mice with skin allotransplants from C57BL/6 mice. Prednisolone in combination with asathioprin (imuran) administered to CBA mice inhibited the GVHR. Prodigiosan used alone was not active, while in combination with immunodepressants it increased their inhibitory effect. Adhesive cells with a suppressive activity were detected in the spleen of mice treated with prodigiosan. Such cells were capable of suppressing the capacity of syngeneic lymphocytes for inducing the GVHR.     

Effect of Fatty Acids on the Activity of Bacteriolytic Enzymes

The ability of fatty acids to sensitize gram-negative and gram-positive bacterial cells to the action of bacteriolytic enzymes was studied. By synergetic effects between bacteriolytic enzymes and fatty acids isolated from Myxococcus such bacteria, which were otherwise resistant to the enzymes, could be lysed. Isobranched and unbranched acids with 11–15 carbon atoms were active and could sensitize Bacillus megaterium and Aerobacter aerogenes to the action of bacteriolytic enzymes from myxobacteria and to lysozyme. The sensitizing activity of tetradecanoic acid was enhanced with increasing concentration even after the solution was saturated. Neither ethylene diaminetetraacetate (0.1 and 1 mM) nor Triton X-100 (1 0/00) could sensitize resistant bacteria to the action of bacteriolytic enzymes. However, they were active in combination and they could also increase the effect of tetradecanoic acid.     

Measurement of ultraviolet radiation-induced suppression of recall contact and delayed-type hypersensitivity in humans

This article describes methodology used for assessment of ultraviolet radiation-induced suppression of recall responses in humans. Nickel allergy is common in the general population and patch testing of nickel-allergic volunteers provides a convenient model of contact hypersensitivity. Similarly, Mantoux-positive volunteers, recruited from within hospital staff, are used as a model for delayed-type hypersensitivity. Use of secondary rather than primary immune responses allows placement of multiple test sites on each volunteer. Further, each volunteer acts as his or her own unirradiated control. This enables UV immunosuppression to be studied with relatively few human volunteers, and makes determination of UV immunosuppression dose responses feasible in human subjects. The method can also be used for assessment of the level of immune protection afforded by agents such as sunscreens or biologically active substances.     

Possible mechanisms of the radiomodifying action of exogenous hypoxia and electromagnetic radiation in the SHF range

It was shown that biologically active substances formed in the liver tissue under the effect of hypoxia and SHF electromagnetic radiation produce a radioprotective effect when administered 15 min before exposure and have no protective effect when administered after irradiation.     

Immunological changes in the thymus epithelial tissue of mice during accidental involution

Using sera containing antibodies to antigens of epithelial reticular cells it is possible to detect by immunofluorescence early stages of organ accidental involution and to trace parenchyma demasking that results from lymphocyte migration upon injection of prednisolone, azathioprin or their combinations. Prior to the drug administration only certain short thin processes of epithelial cells masked by a great number of lymphoid elements are detectable in the cortical zone of the thymus. Injection of 10-40 mg/kg prednisolone, 100-200 mg/kg azathioprin or their combinations causes stable different-degree demasking of the epithelial reticulum seen during immunofluorescence as a network of fluorescent processes. Administration of drug combinations also causes marked attenuation of the reaction, probably resulting from dystrophic changes in thymic epithelial cells.     

Conjugation and modeled structure/function analysis of lysozyme on glycine esterified cotton cellulose-fibers

The antimicrobial activity of lysozyme covalently bound to glycine-derivatized cotton cellulose was assessed in a 96-well format. Lysozyme was immobilized on glycine-bound cotton through a carbodiimide reaction. The attachment to cotton fibers was made through both a single glycine and a glycine dipeptide esterified to cotton cellulose. Higher levels of lysozyme incorporation were evident in the diglycine-linked cotton cellulose samples. The antibacterial activity of the lysozyme-conjugated cotton cellulose against Bacillus subtilis was assessed as a suspension of pulverized cotton fibers in microtiter wells. Inhibition of B. subtilis growth was observed to be optimal within a range of 0.14-0.3 mM (equivalent to 4-20 mg of lysozyme-bound cotton/mL) of lysozyme. Enhancement of activity over soluble lysozyme may result from the solid-phase protection afforded by the cellulose linkage of the glycoprotein against proteolytic lysis. Computational models of lysozyme based on its crystal structure attached through aspartate, glutamate, and COOH-terminal residues to cellopentaose-(3) Gly-O-6-glycyl-glycine ester were constructed. The models demonstrate no steric constraints to the active-site cleft from the glycine-conjugated cellulose chain when lysozyme is bound at the carboxylates of Asp-87, Glu-7, Asp-119, Asp-18, and COOH-terminal Leu-129. The more robust antibacterial activity of the enzyme when bonded to cotton fibers suggests good potential for biologically active enzymes on cotton-based fabrics.     

Use of sodium nucleinate and its combinations with bacterial polysaccharides for preventing the nephrotoxic action of aminoglycoside antibiotics

Possible prevention of the nephrotoxic effect of different doses of aminoglycoside antibiotics, such as monomycin, kanamycin and gentamicin was studied experimentally on chinchilla rabbits. Substances increasing the cell resistance (sodium nucleinate, prodigiosan and pyrogenal alone and sodium nucleinate combinations with the bacterial polysaccharides) were used. It was shown that sodium nucleinate prevented nephrotoxicity of the aminoglycosides in the doses 2.5 times higher than the therapeutic ones. The combined use of sodium nucleinate with pyrogenal or prodigiosan was most effective. It prevented the nephrotoxic effect of the antibiotics in the doses 5 times higher than the therapeutic ones.     

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.     

Experimental effect of lysozyme on macrophage metabolism and resistance to infection

The peritoneal macrophages of mice treated with lysozyme were studied by cytochemical assay. In single and repeated doses of 0.5-5 mg/kg lysozyme induced an increase in macrophage metabolism. This was evident from an increased activity of succinate dehydrogenase, NADP X N-DH and the enzymes catalyzing glycolysis typical of these cells (lactate dehydrogenase and alpha-glycerophosphate). The changes in the activity of the enzymatic systems were most pronounced in minute and less mature macrophages after repeated administrations of the drugs. In a dose of 50 mg/kg lysozyme somewhat decreased the activity of a number of the enzymes. In the doses optimal for the macrophage activity lysozyme had a low effect on the infection resistance and slightly increased the cephotaxim efficiency in experimental staphylococcal infection. This may be mainly due to the immunomodulating effect of lysozyme and its low effect on the large macrophages having the bactericidal effect.     

Effects of additional vitamin E and selenium supply on antioxidative defence mechanisms in the kidney of rats treated with high doses of glucocorticoid

The aim of this work was to determine the effects of dietary vitamin E and selenium (Se) on lipid peroxidation as thiobarbituric acid reactive substances (TBARS) and on the antioxidative defence mechanisms in the kidney of rats treated with high-doses of prednisolone. Two hundred and fifty adult male Wistar rats were randomly divided into five groups. The rats were fed a normal diet, but groups 3, 4, and 5 received a daily supplement in their drinking water of 20 mg vitamin E, 0.3 mg Se, and a combination of vitamin E and Se, respectively, for 30 days. For 3 days subsequently, the control group (group 1) was treated with a placebo, and the remaining four groups were injected intramuscularly with 100 mg kg(-1) body weight (bw) prednisolone. After the last administration of prednisolone, 10 rats from each group were killed at 4, 8, 12, 24, and 48 h and the activities of glutathione peroxidase (GSH-Px) and catalase (CAT) enzymes, and the levels of glutathione (GSH) and TBARS in their kidneys were measured. GSH-Px and CAT enzyme activities and GSH levels in the prednisolone treatment group (group 2) began to decrease gradually at 4 h, falling respectively to 48 and 65% of the control levels by 24 h, and recovering to the control levels at 48 h. In contrast, prednisolone administration caused an increase in TBARS in the kidneys, reaching up to twice the levels of the control group at 24 h. However, supplementation with vitamin E and Se had a preventive effect on the elevation of kidney TBARS and improved the diminished activities of the antioxidative enzymes and the levels of GSH. Therefore, the present study demonstrates the effectiveness of vitamin E and Se in reducing kidney damage in glucocorticoid-treated rats and suggests that reductions in increased TBARS due to prednisolone may be an important factor in the action of vitamin E and Se.     

Use of prodigiozan for preventing acute respiratory viral diseases in a children's collective]

The epidemiological efficacy of 0.02 per cent solution of prodigiosan, a bacterial polysaccharide was used for the treatment of children in an area with acute respiratory infections, such as influenza and parainfluenza. The drug was administered intranasally by means of a dosing sprayer in the amounts of 0.2 ml once in 4 days for 4 months. Among the children treated with prodigiosan the rate of the acute respiratory viral infections was 2 times lower and the average duration of the disease was 2.4 times lower as compared to the control group. After 4 months of the drug use the average value of the "skin autoflora" test was much lower than that in the control group which testified to an increase in the non-specific immunobiological reactivity of the children under the effect of prodigiosan.     

The use of enzymes for searching for biologically active compounds

Possible use of extracellular staphylococcal DNAase in screening substances with potential antibacterial activity was studied on quinoxalin as an example. For screening substances with antiviral activity the possible use of the influenza virus neuraminidase was studied on fluoren as an example. Close correlation between the biological activity of quinoxalin derivatives and the ability to inhibit DNAase was revealed. The most active inhibitors of the enzyme were dioxidin and other biologically active analogs of quinoxalin 1,4-di-N-oxide. The use of the extracellular nuclease as a biochemical model permitted to establish the structure/function dependence with respect to the quinoxalin derivatives. The effect of the fluoren derivatives on activity of the influenza virus neuramididase was studied. It was shown that florenal, an antiviral drug inhibited the virus specific enzyme by 80 to 90 per cent and had no effect on catalytic activity of bacterial neuraminidase. Biologically inactive and slightly active derivatives of the compounds did not inhibit the influenza virus enzyme. At the same time some of them lowered the activity of the bacterial enzyme.     

Development of multifunctional foods

Foods contain various biologically active substances with extent physiological effects. Among them, some substances, such as dietary fibers, polyunsaturated fatty acids, and antioxidants, exert multiple activities. In addition, combinational uses of some components, such as sesamin and α-tocopherol, enhance their biological effects each other. The expression of multiple effect leads to the production of multifunctional foods which prevent the occurrence of various diseases. Furthermore, synergic effects between biologically active substances allow us to decrease the dose of each component, which lead to the improvement of safety and reduction of production cost of multifunctional foods. To use these active components for human health, studies on their adsorption and transportation are important, since distribution of some components, such as tocotrienols, is limited to some tissues. In addition, extent safety studies are essential to use artificially synthesized substances such as 10t, 12c derivative of conjugated linoleic acid for human health.