Effects of drugs on water permeability of erythrocyte membranes

The method of NMR-relaxation with the manganese doping has been applied to study changes of water permeability of red blood cell membranes affected by various concentrations of chlorhexidine digluconate and dimephosphone. It is shown that both investigated substances suppress the water permeability of the red blood cell membrane in a dose-dependent manner. Half-maximum inhibitory effect of studied substances was reached at the concentrations of 9 μM of chlorhexidine and 400 μM of dimephosphone.     

Oxidative and phagocytic functions of macrophages during infections induced in mice by Mycobacterium intracellulare and Listeria monocytogenes

The oxidative metabolism (chemiluminescence and H2O2 release) and phagocytic activity of mouse peritoneal macrophages during chronic infections induced by Mycobacterium intracellulare and more acute infections due to Listeria monocytogenes were studied. In M. intracellulare infections, macrophage chemiluminescence in response to phorbol myristate acetate (PMA) was greatest at around 2 weeks, with a 1 week lag phase after infection, while the PMA-triggered H2O2 release was markedly enhanced even 1 d after challenge, and remained high thereafter for up to 10 weeks. The pattern of changes in the phagocytic activity of host macrophages in response to latex beads during this infection resembled the pattern seen with macrophage H2O2 release. In the L. monocytogenes infections, the PMA-triggered chemiluminescence of the host macrophages increased 4 d (in a sublethal infection) and 2 d (in a lethal infection) after bacterial challenge, whereas the PMA-triggered H2O2 release was markedly enhanced as early as 1 d after infection and the elevated level persisted until either the bacteria were eliminated or the animals died. The patterns of changes in phagocytic activity of the host macrophages during L. monocytogenes infection at sublethal and lethal doses differed. In the former, phagocytosis was most active in the early phase of infection, with a peak around day 2, followed by a rapid decrease; in the latter, the phagocytic ability increased more slowly, and remained elevated until the animals died. The results suggest that the macrophages induced by M. intracellulare are in a more activated state than are those induced by L. monocytogenes.     

Luminol-dependent chemiluminescence of the primary focus at different infecting doses of Staphylococcus aureus

Guinea pigs were infected intramuscularly with different doses of S. aureus. At different periods after infection the chemiluminescence (CL) of the primary focus in the animals infected with 10(10) colony-forming units (CFU) of S. aureus (abscess formation on day 3, mortality rate equal to 70%) was poor and increased only on days 6-9; in animals injected with 10(9) CFU (abscess formation on day 6, mortality rate equal to 16%) the CL increased on day 3 and lasted till day 20; in the animals infected with 10(8) CFU (the appearance of infiltration, no mortality) the reaction increased on day 1, then decreased; and in those infected with 10(2) CFU (no local manifestations of the process) the reaction was low at all periods. CL peak was recorded early mainly due to the neutrophil reaction, while a later CL peak (in cases of the favorable outcome of the infection) was explained by the macrophagal reaction. Thus, for the favorable outcome of the infection (in the presence of inflammation) the early increase of the CL of neutrophils migrating to the focus is characteristic, while the resolution of the local process is manifested by CL decrease and the prevalence of the macrophagal component of the reaction.     

The microbial factor in the chemiluminescence of the peripheral blood neutrophils of patients with suppurative surgical infection

The luminol-dependent chemiluminescence of neutrophils in the peripheral blood of 30 healthy adults and 39 patients with the local and generalized forms of purulent infection was studied. Nonstimulated chemiluminescence and the index of chemiluminescence stimulation in the presence of opsonized Staphylococcus aureus added in vitro were determined. The former characteristic was found to be directly and the latter one, inversely related to the concentration of S. aureus, Escherichia coli and Candida albicans, but not E. epidermidis, Pseudomonas aeruginosa or Citrobacter, in the primary focus. At the microbial concentration exceeding 10(4) cells/g of tissue, the former characteristic was essentially higher than the level of chemiluminescence in healthy persons. With the improvement of the general state of the patients and in the absence of microorganisms in the wound as the result of complex treatment this characteristic decreased to values comparable with the reaction of neutrophils in healthy persons.     

Mechanism of staphylococcal resistance to non-oxidative antimicrobial action of neutrophils: importance of pH and ionic strength in determining the bactericidal action of cathepsin G

The staphylococcalcidal action of highly purified, enzymically inactive human lysosomal cathepsin G was studied. The bactericidal action of cathepsin G was optimal at pH 7.5 and was inhibited by NaCl; concentrations greater than 0.15 M NaCl completely inhibited killing of Staphylococcus aureus. Under optimal conditions (pH, temperature and NaCl concentration) the ED50 (effective dose) of cathepsin G against S. aureus strain 8325-4 was about 3.1 micrograms ml-1. Polymeric teichoic acid may serve as a binding site for cathepsin G by promoting electrostatic interactions since a mutant lacking this surface component exhibited enhanced resistance to the lethal action of cathepsin G, compared to the teichoic-acid-positive parental strain. These results suggest that (i) the ability of cathepsin G to kill intraphagosomal staphylococci may be regulated in part by the ionic strength of the environment and the pH of the maturing phagolysosome, and (ii) that strategies which retard acidification of the developing phagolysosome would promote the staphylococcalcidal action of cathepsin G.     

Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of free radical formation at key stages of apoptosis

Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 μM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 μM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 μM.     

Immunolocation of lipoteichoic acid on group B streptococcal surface

Abstract The effect of intraperitoneal (i.p.) infection with rat cytomegalovirus on the effector functions of peritoneal macrophages was investigated. There was an influx of polymorphonuclear leukocytes into the peritoneum on day 1 followed by an influx of macrophages on day 4. The macrophages harvested on day 4 showed enhanced levels of chemiluminescence emitted during phagocytosis of zymozan particles, and enhanced capacity to kill Staphylococcus aureus . Thereafter, the chemiluminescence level and the bactericidal capacity decreased, remaining low up to 6 months post-infection. In addition, macrophages harvested from animals on day 7 showed increased phagocytosis of sheep red blood cells.     

Functional characterization of lipase in the pathogenesis of Staphylococcus aureus

During infection, Staphylococcus aureus produces multiple enzymes that enable it to invade and destroy host tissues and metastasize to other sites. One such enzyme, lipase, has been recognized for its relationship in the virulence of S. aureus. However, a direct involvement of lipase in the pathogenesis of S. aureus remains to be demonstrated. Our prior study indicated that anti-lipase serum inhibits biofilm formation in S. aureus clinical strains. The aim of this study was to further characterize the roles of lipase in the pathogenesis in S. aureus. We found that deletion of the lipase-coding gene reduced biofilm formation relative to the wild-type strain. This was shown by culture in 96-well plates coated with collagen to resemble the in vivo infection process. Intraperitoneal inoculation of mice with a lipase mutant strain showed defective formation of peritoneal abscesses, and bacterial loads in different organs were much lower compared with the wild-type. Importantly, active immunization with recombinant lipase protected mice against a lethal challenge with S. aureus. Altogether, our data provide evidence that S. aureus lipase plays important roles in the pathogenesis of S. aureus.     

Antimicrobial potentiality of a phenothiazine group of antipsychotic drug-prochlorperazine

The antipsychotic drug, prochlorperazine (Pcp), was tested for its antimicrobial efficacy against 103 strains belonging to both gram positive and gram negative bacteria. The drug was found to possess maximum activity against Staphylococcus aureus, Vibrio cholerae and Shigella spp. Pcp was moderately active against E. coli but most of the strains belonging to Bacillus spp, Klebsiella spp, Salmonella spp and Lactobacillus spp were found to be resistant to this drug. The drug was tested for its mode of antibacterial activity against Shigella dysenteriae 1 and it was found to be bacteriostatic in action. In in vivo studies, Pcp offered significant protection to Swiss albino mice at concentrations of 0.75 micro g/g (P < 0.01) and 1.5 microg/g (P < 0.001) body weight when challenged with 50 median lethal dose of Salmonella typhimurium NCTC 74. Thus the result depicts that prochlorperazine may emerge as a strong antimicrobial drug to replace the conventional antibiotics and to overcome the problem of drug resistance.     

Role of ATP in specific binding of 125I-insulin to cytoplasmic receptors of the liver and muscle membranes of controls and diabetic rats

A study was made of the action of various concentrations of ATP on insulin ability to bind to the receptors of the liver and muscle membranes in control and streptozocin-induced diabetes animals. Specific binding of 125I-insulin to the receptors of the liver and muscle membranes was shown to rise in animals with streptozocin-induced diabetes as compared to control. This effect was most pronounced in the muscle membranes. Preincubation of the membranes with ATP did not affect insulin binding to the liver and muscle receptors of control animals. However, hormone binding to the liver receptors of diabetic rats was drastically suppressed by ATP (10(-3) M). Less ATP concentrations (10(12) M) produced an additional inhibitory action which was not marked. ATP led to decreased insulin binding to the muscle receptors of diabetic rats only at extremely low concentrations (10(-12) M). The data obtained may be of importance for regulation of membrane phosphorylation in the states characteristic of insulin resistance.     

Cytopathic effect of the tularemia microbe on a culture of peritoneal macrophages

Morphological analysis of the process of interaction of tularemia microbe strains differing by virulence with macrophages demonstrated that all these strains produced a lethal effect on macrophages obtained from the animales sensitive to the infection. The macrophages obtained from the animals were but little sensitive to tularemia and were resistant to the action of the causative agent of this infection. The data obtained led to a supposition on the presence in the tularemia causative agent of a factor responsible for its lethal action on the macrophages.     

Stimulation of the respiratory burst of the neutrophils from healthy subjects by different Staphylococcus aureus concentrations

Preopsonized live and heat-killed S. aureus stimulated, without the washing of serum, the luminol-dependent chemiluminescence of human neutrophils obtained from healthy donors. The intensity of chemiluminescence was evaluated by the index of stimulation with staphylococci, with due consideration for their concentration. With the microbe/phagocyte ratio equal to 10:1, these indices had the maximum values when both live and killed staphylococci were used. At high concentrations of staphylococci, especially live ones, all indices were low (those for live staphylococci had negative values) and uniform. As the concentration of the antigen decreased, individual features in the reaction of each donor became apparent. With the microbe/phagocyte ratio equal to 100:1, stimulation with live and killed staphylococci induced the identical fluorescence of neutrophils. The capacity of nonopsonized staphylococci for inducing chemiluminescence was poorly pronounced. For this reason, the test system using S. aureus at low concentrations was proposed for the prognostication of this infection, while the ratio 100:1 can be used for the evaluation of the opsonin-phagocytic system in case of a developed purulent process.     

A comparison of fertility control and lethal control of bovine tuberculosis in badgers: the impact of perturbation induced transmission.

In this paper we use mathematical modelling to consider the broad advantages and disadvantages of fertility control over lethal control for bovine tuberculosis in badger populations. We use a deliberately simple model, attempting to capture only the key transmission processes. The model is parametrized with reference to the long-term Woodchester Park study. Estimates of mortality rate from this study suggest no significant extra mortality risk for animals with evidence of infection as indicated by the presence of anti-Mycobacterium bovis antibodies or M. bovis isolation. We find that large reductions in prevalence are sometimes the consequence of only moderate reductions in population numbers. If we assume that the act of control does not in itself affect transmission rates, then as far as eradication is concerned, both fertility control and mortality control operate through the same epidemiological mechanism, the removal of susceptibles: if one is in principle capable of keeping a population low enough to be infection free then so is the other. It is necessary to continue either form of control at regular intervals to maintain a constant level of infection in the long term. If control were to be stopped, return to precontrol levels of badger population and infection prevalence would be expected within a few years. Fertility control is less effective in reducing population density than lethal control since it can only act, at maximum, to remove one age cohort per year. It is also less effective in reducing transmission as it can only ever remove susceptibles, while lethal control also removes infectious badgers. However, if the social disturbance caused by lethal control does in fact increase contact rates for the remaining infectious badgers, the relative efficacies of the two strategies become a great deal less clear. While we have no quantitative data on the extent to which social perturbation does act to promote transmission, model simulations show that it is possible to develop plausible scenarios in which the lethal control may actually act to increase the absolute numbers of animals infected, while reducing the number of uninfected animals to very low numbers.     

The effect of different infectious agents on the intensification of hematopoiesis during immunosuppression

The action of Staphylococcus aureus, Escherichia coli and Herpes simplex virus on the hemopoiesis of mice with cytostatic myelosuppression was studied. The study revealed that the infection of the animals simultaneously with the action of cyclophosphamide considerably activated the processes leading to the restoration of hemopoiesis due to an increase in the mitotic activity of hemopoietic cells, the accelerated differentiation of hemopoietic precursor cells which could survive the cytostatic action and an increase in the functional activity of the hemopoiesis-producing microenvironment.     

MyD88-Dependent Signaling Influences Fibrosis and Alternative Macrophage Activation during Staphylococcus aureus Biofilm Infection

Bacterial biofilms represent a significant therapeutic challenge based on their ability to evade host immune and antibiotic-mediated clearance. Recent studies have implicated IL-1β in biofilm containment, whereas Toll-like receptors (TLRs) had no effect. This is intriguing, since both the IL-1 receptor (IL-1R) and most TLRs impinge on MyD88-dependent signaling pathways, yet the role of this key adaptor in modulating the host response to biofilm growth is unknown. Therefore, we examined the course of S. aureus catheter-associated biofilm infection in MyD88 knockout (KO) mice. MyD88 KO animals displayed significantly increased bacterial burdens on catheters and surrounding tissues during early infection, which coincided with enhanced dissemination to the heart and kidney compared to wild type (WT) mice. The expression of several proinflammatory mediators, including IL-6, IFN-γ, and CXCL1 was significantly reduced in MyD88 KO mice, primarily at the later stages of infection. Interestingly, immunofluorescence staining of biofilm-infected tissues revealed increased fibrosis in MyD88 KO mice concomitant with enhanced recruitment of alternatively activated M2 macrophages. Taken in the context of previous studies with IL-1β, TLR2, and TLR9 KO mice, the current report reveals that MyD88 signaling is a major effector pathway regulating fibrosis and macrophage polarization during biofilm formation. Together these findings represent a novel example of the divergence between TLR and MyD88 action in the context of S. aureus biofilm infection.     

Binding of [3H]des-Arg9-BK to rabbit anterior mesenteric vein

Binding studies of [3H]des-Arg9-BK have been performed on pieces of rabbit anterior mesenteric veins. Kinetic studies have permitted us to evaluate an affinity constant of 1.04 X 10(-7) M, which is not so different from the apparent affinity constant determined by bioassay (1.6 X 10(-7) M). Furthermore, inhibition of the binding of [3H]des-Arg9-BK with various kinins results in an order of potency of kinins very similar to that observed in the bioassay. Taken together, these results suggest that we are dealing with binding sites which might be the same as those subserving the biological action of des-Arg9-BK (pharmacological receptors). The preincubation of tissues in Krebs' solution brings about an increase of the specific binding from 0.06 pmol/mg of wet weight at time 0 to 0.75 pmol after 24 h; cycloheximide inhibits this increase for at least 6 h. Veins taken from animals treated with LPS, which have shown an increase in sensitivity compared with veins extracted from untreated animals, have a higher number of specific binding sites for [3H]des-Arg9-BK. The results support the hypothesis that the increased response of tissues to des-Arg9-BK is due to the de novo synthesis of receptors for kinins in some experimental and pathological conditions.     

Staphylococcus aureus pneumonia in hamsters with elastase-induced emphysema--the virulence enhancing activity of mucin

Staphylococcus aureus pneumonia was studied in hamsters with elastase-induced emphysema and in saline-treated controls. Emphysematous animals cleared endotracheally administered inocula of S. aureus in saline as rapidly as controls. After infection with S. aureus in 1% mucin, emphysematous animals had impaired clearance compared with controls; after infection with S. aureus in 5% mucin, emphysematous animals had decreased survival at 96 hr compared to controls (6/24 vs 15/24, P less than 0.01 Fisher's exact test). Bronchoalveolar lavage of uninfected elastase-treated hamsters yielded twice as many cells per animal as uninfected controls (P less than 0.0001, paired t test), and the cells contained a higher percentage of polymorphonuclear leukocytes (37.8% vs 3.8%, P less than 0.0001). Lavage cells from both groups of animals were equally efficient per cell at killing opsonized S. aureus in an in vitro bactericidal assay. Hamsters with elastase-induced emphysema were resistant to infection with S. aureus alone despite marked structural abnormalities in the lung, possibly due in part to increased numbers of resident phagocytic cells. After infection with S. aureus in mucin as a virulence enhancing factor emphysematous animals had impaired clearance and decreased survival compared to controls.     

Characteristics of luminol chemiluminescence induced by the catalytic action of myeloperoxidase

The effects of pH, luminol myeloperoxidase and hydrogen peroxide concentrations on the intensity of luminol chemiluminescence induced by myeloperoxidase catalysis were investigated. It was found that the intensity of luminescence is proportional to the enzyme concentration (up to 8.10(-8) M) and reaches the saturation level at higher enzyme concentrations. The dependence of chemiluminescence intensity on [H2O2] is bell-shaped: at H2O2 concentrations above 1.10(-4) M the luminescence is inhibited with a maximum at neutral values of pH. Luminol at concentrations above 5.10(-5) M inhibits this process. It was demonstrated that the effects of singlet oxygen, superoxide and hydroxyl radicals on the chemiluminescence reaction are insignificant. Luminol oxidation in the course of the myeloperoxidase reaction is induced by hypochlorite.     

Regulation of morphologicaland functional properties of astroglial cells by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H2O2 at 1 x 10(-8) - 5 x 10(-7) M concentrations acts as a regulator of morphological and functional properties of astrocytes by inducing their reactivation that is manifested as a cell body hypertrophy and an increase of proliferative activity and of menadione-dependent production of superoxide (O2- ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 microM on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O2- under menadione action. Using lipopolysaccharide B as a functional stimulator it has been shown that H2O2 modifies signaling pathways leading to the increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-enhanced chemiluminescence of astrocytes under menadione action to the level of control values.     

Comparative Activities of Cattle and Swine Platelet Microbicidal Proteins

The bactericidal activities of cattle and swine platelet microbicidal proteins (PMPs) with their comparison with human PMP were studied. Activities of PMP were tested against Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus lysodeikticus and Escherichia coli. B. subtilis and B. cereus were high susceptible to PMP at very low concentrations. Of the gram-positive cocci studied, M. lysodeikticus and S. aureus were the most, and S. epidermidis the least, susceptible. E. coli was found to be relatively resistant to the lethal action of all PMP. The findings of this study confirm that the existence of antimicrobial peptides is conserved among mammalian platelets.