Antibiotic penetration through the blood-brain barrier in rats and the effect on this process of proteolytic enzymes]

Penetration of penicillin, ampicillin, oxacillin, tetracycline, streptomycin and gentamycin through the hemato-encephalic barrier in rats and effect of proteolytic enzymes on the above process were studied comparatively. The levels of penetration to the brain were highest for ampicillin, then followed penicillin, tetracycline, oxacillin, streptomycin and gentamycin. Preliminary intramuscular administration of trypsin and chimotrypsin (10 mg/kg) to the animals had no effect on permeability of the hemato-encephalic barrier for the antibiotics tested, except penicillin. Higher levels of the antibiotics in the brain tissue may be explained by higher antibiotic blood levels under the effect of proteolytic enzymes.     

Metal ion-tetracycline interactions in biological fluids. Part 4. Potential influence of ca2+ and mg2+ ions on the bioavailability of chlortetracycline and demethylchlortetracycline,as expected from their computer-simulated distributions in blood plasma

Coordination of tetracydines with calcium and magnesium was previously shown to exert a determining effect on the distribution of these antibiotics in blood plasma. In particular, it was clearly established by computer simulation that the free fraction of the drug is quite negligible with respect to its metal-bound fraction. The bioavailabiity of a tetracycline in blood plasma is thus expected to depend directly on the electrical charge of its predominant metal complexes in the biofluid. On account of the metal to ligand ratio corresponding to the usual therapeutic levels, bioavailability is critically sensitive to the property of the antibiotic to give rise to electrically charged binuclear species. The blocking of one of the two potential binding sites of the tetracycline molecule should thus result in a larger percentage of neutral complexes, hence in a better tissue penetration by the drug.The present work is devoted to the investigation of the coordination of 7-chlortetracycline (CTC) and 6-demethyl-7-chlortetracycline (DMC) with calcium and magnesium in blood plasma. The influence of the chloro substituent is discussed with respect to the objective defined above.     

Platelet activating factor induces transient blood‐brain barrier opening to facilitate edaravone penetration into the brain

The blood–brain barrier (BBB) greatly limits the efficacy of many neuroprotective drugs' delivery to the brain, so improving drug penetration through the BBB has been an important focus of research. Here we report that platelet activating factor (PAF) transiently opened BBB and facilitated neuroprotectant edaravone penetration into the brain. Intravenous infusion with PAF induced a transient BBB opening in rats, reflected by increased Evans blue leakage and mild edema formation, which ceased within 6 h. Furthermore, rat regional cerebral blood flow (rCBF) declined acutely during PAF infusion, but recovered slowly. More importantly, this transient BBB opening significantly increased the penetration of edaravone into the brain, evidenced by increased edaravone concentrations in tissue interstitial fluid collected by microdialysis and analyzed by Ultra‐performance liquid chromatograph combined with a hybrid quadrupole time‐of‐flight mass spectrometer (UPLC‐MS/MS). Similarly, incubation of rat brain microvessel endothelial cells monolayer with 1 μM PAF for 1 h significantly increased monolayer permeability to ¹²⁵I‐albumin, which recovered 1 h after PAF elimination. However, PAF incubation with rat brain microvessel endothelial cells for 1 h did not cause detectable cytotoxicity, and did not regulate intercellular adhesion molecule‐1, matrix‐metalloproteinase‐9 and P‐glycoprotein expression. In conclusion, PAF could induce transient and reversible BBB opening through abrupt rCBF decline, which significantly improved edaravone penetration into the brain.

     

Development of hematogenous pneumococcal meningitis in adult mice: the role of TNF-alpha

Bacterial penetration across the blood-brain barrier (BBB) into the central nervous system is the first step in development of meningitis. The role of tumor necrosis factor-alpha (TNF-alpha) in the penetration process was examined with peripheral infection of Streptococcus pneumoniae type 6. After intraperitoneal infection of S. pneumoniae type 6, the BBB opening was increased continuously from 6 h and the mice died of septic shock within 36 h due to bacterial overgrowth. The bacteria crossed the BBB and began to deposit in brain at 6 h post infection. There was strong staining of TNF-alpha on blood vessels of brain from 6 h to 24 h post infection. Anti-TNF-alpha antibody blocked both the BBB opening and the entrance of circulatory S. pneumoniae type 6 into brain, indicating that TNF-alpha played an important role in controlling the opening of BBB. Furthermore, an adult murine model of hematogenous pneumococcal meningitis was developed that is based on opening of the BBB by TNF-alpha and controlling the degree of bacteremia by cefazolin antibiotic. In conclusion, hematogenous meningitis developed as TNF-alpha initiated BBB opening, peripheral bacteria entered into the brain and formed bacterial emboli, and then progressed to meningitis.     

Determination of the penetration of 9-fluoropropyl-(+)-dihydrotetrabenazine across the blood-brain barrier in rats by microdialysis combined with liquid chromatography-tandem mass spectrometry

To evaluate the penetration of the blood-brain barrier by 9-fluoropropyl-(+)-dihydrotetrabenazine (AV-133), microdialysis probes were implanted simultaneously into rat blood and brain, and a liquid chromatography-tandem mass spectrometric method was developed and validated to monitor the AV-133 concentration in the microdialysates. The chromatographic separation was performed on an XTerra C(18) column (150 mm × 2.1 mm i.d., 5 μm particles) with gradient elution. The mass spectrometer was operated in positive mode using electrospray ionization. The analytes were measured using the multiple-reaction-monitoring mode. The calibration curves were linear over the range of 5.00-1000 ng/mL AV-133, with a coefficient of determination >0.995. The accuracies ranged from 99.5% to 105.0% and the precisions were <10% for AV-133. This method was used to determine the concentrations of AV-133 and its pharmacokinetics in the brains and blood of rats. The blood and brain concentration-time profiles for AV-133 were obtained, and the blood-brain barrier penetration was evaluated.     

African trypanosomes and brain infection – the unsolved question

African trypanosomes induce sleeping sickness. The parasites are transmitted during the blood meal of a tsetse fly and appear primarily in blood and lymph vessels, before they enter the central nervous system. During the latter stage, trypanosomes induce a deregulation of sleep–wake cycles and some additional neurological disorders. Historically, it was assumed that trypanosomes cross the blood–brain barrier and settle somewhere between the brain cells. The brain, however, is a strictly controlled and immune‐privileged area that is completely surrounded by a dense barrier that covers the blood vessels: this is the blood–brain barrier. It is known that some immune cells are able to cross this barrier, but this requires a sophisticated mechanism and highly specific cell–cell interactions that have not been observed for trypanosomes within the mammalian host. Interestingly, trypanosomes injected directly into the brain parenchyma did not induce an infection. Likewise, after an intraperitoneal infection of rats, Trypanosoma brucei brucei was not observed within the brain, but appeared readily within the cerebrospinal fluid (CSF) and the meninges. Therefore, the parasite did not cross the blood–brain barrier, but the blood–CSF barrier, which is formed by the choroid plexus, i.e. the part of the ventricles where CSF is produced from blood. While there is no question that trypanosomes are able to invade the brain to induce a deadly encephalopathy, controversy exists about the pathway involved. This review lists experimental results that support crossing of the blood–brain barrier and of the blood–CSF barrier and discuss the implications that either pathway would have on infection progress and on the survival strategy of the parasite. For reasons discussed below, we prefer the latter pathway and suggest the existence of an additional distinct meningeal stage, from which trypanosomes could invade the brain via the Virchow–Robin space thereby bypassing the blood–brain barrier. We also consider healthy carriers, i.e. people living symptomless with the disease for up to several decades, and discuss implications the proposed meningeal stage would have for new anti‐trypanosomal drug development. Considering the re‐infection of blood, a process called relapse, we discuss the likely involvement of the newly described glymphatic connection between the meningeal space and the lymphatic system, that seems also be important for other infectious diseases.     

Antibiotic interaction with proteolytic enzymes]

The effect of penicillin, tetracycline, aminoglucozide antibiotics and streptomycin on BAEE-esterase activity of trypsin was studied. It was found that benzylpenicillin in amounts of 50, 100 and 300 mg, ampicillin in an amount of 25 mg, methicillin in an amount of 12 mg and tetracycline in an amount of 2.5 mg as calculated per 1 mg of trypsin had no effect in vitro on the esterase activity of the enzyme. Neomycin, kanamycin and streptomycin in amounts of 5, 10, 100 or 300 mg per 1 mg of trypsin catalyzed splitting of BAEE by trypsin. When the antibiotics were added to the bile, its esterase activity increased. Preliminary intramuscular administration of trypsin and kanamycin to the rats had no effect on the ampicillin levels in the blood serum and brain and did not affect the permeability of the hemato-encephalic barrier as compared to the use of trypsin alone.     

Shigella toxicity in immunological tolerance

The toxic effect of killed and live Shigella sonnei cultures on normal mice and on mice, tolerant to Shigella O-antigen and to human erythrocytes of different blood groups (in the ABO system) was under study. The toxicity of shigellae, introduced intraperitoneally, has been found to depend on their viability, on their capacity for penetration into the blood, and on the split character of immunological tolerance to Shigella antigens.     

Clinical trial of berberine in acute watery diarrhoea.

Four hundred adults presenting with acute watery diarrhoea were entered into a randomised, placebo controlled, double blind clinical trial of berberine, tetracycline, and tetracycline and berberine to study the antisecretory and vibriostatic effects of berberine. Of 185 patients with cholera, those given tetracycline or tetracycline and berberine had considerably reduced volume and frequency of diarrhoeal stools, duration of diarrhoea, and volumes of required intravenous and oral rehydration fluid. Berberine did not produce an antisecretory effect. Analysis by factorial design equations, however, showed a reduction in diarrhoeal stools by one litre and a reduction in cyclic adenosine monophosphate concentrations in stools by 77% in the groups given berberine. Considerably fewer patients given tetracycline or tetracycline and berberine excreted vibrios in stools after 24 hours than those given berberine alone. Neither tetracycline nor berberine had any benefit over placebo in 215 patients with non-cholera diarrhoea.     

Comparative study of the physicochemical properties of the surface of Escherichia, their sensitivity to ampicillin and tetracycline and their capacity to absorb tetracycline]

Development of resistance to ampicillin and tetracycline in Escherichia resulted in an increase in the electrokinetic potential and a decrease in the level of hydration and isoelectric values of pH. The changes in the hydration level mainly depended on the accompanying dissociation. Studies on 3H-tetracycline binding revealed a low accumulation capacity of the resistant mutants. The rate of 3H-tetracycline binding did not depend on the changes in the physico-chemical parameters of the cell surface due to resistance and dissociation.     

Changes in virulence of Enterobacteriaceae in the presence of sub-inhibitory levels of antibiotics

The effect of subinhibitory concentrations of antimicrobial agents such as chloramphenicol, tetracycline, rifampicin and nalidixic acid on various factors of virulence in Shigella spp. and enteroinvasive colon bacilli was studied. It was shown that under such conditions changes took place in various events of the interaction with the epithelial cells, i.e. in adhesion, penetration, intracellular multiplication and transfers between contacting cells. The total effect of chloramphenicol resulted in increasing of the virulence while that of nalidixic acid, tetracycline and rifampicin resulted in its decreasing.     

Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli.

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.     

Synergy of clavine alkaloid ‘chanoclavine’ with tetracycline against multi-drug-resistant E. coli

The emergence of multi drug resistance (MDR) in Gram-negative bacteria (GNB) and lack of novel classes of antibacterial agents have raised an immediate need to identify antibacterial agents, which can reverse the phenomenon of MDR. The purpose of present study was to evaluate synergy potential and understanding the drug resistance reversal mechanism of chanoclavine isolated from Ipomoea muricata against the multi-drug-resistant clinical isolate of Escherichia coli (MDREC). Although chanoclavine did not show antibacterial activity of its own, but in combination, it could reduce the minimum inhibitory concentration (MIC) of tetracycline (TET) up to 16-folds. Chanoclavine was found to inhibit the efflux pumps which seem to be ATPase-dependent. In real-time expression analysis, chanoclavine showed down-regulation of different efflux pump genes and decreased the mutation prevention concentration of tetracycline. Further, in silico docking studies revealed significant binding affinity of chanoclavine with different proteins known to be involved in drug resistance. In in silico ADME/toxicity studies, chanoclavine was found safe with good intestinal absorption, aqueous solubility, medium blood–brain barrier (BBB), no CYP 2D6 inhibition, no hepatotoxicity, no skin irritancy, and non-mutagenic indicating towards drug likeliness of this molecule. Based on these observations, it is hypothesized that chanoclavine might be inhibiting the efflux of tetracycline from MDREC and thus enabling the more availability of tetracycline inside the cell for its action.     

Pharmacokinetics of nicotine in adult and infant mice

Experiments were done to compare the time-courses of the nicotine concentration in the blood, heart, and brain of infant and adult mice after small and large single doses of radioactive nicotine tartrate. In some experiments the nicotine receptors were blocked with mecamylamine or hexamethonium, and their effects on nicotine levels were measured. The nicotine-induced tremor was allowed visually, and its effects on the heart rate were measured by ECG. In adult mice the peak levels of brain nicotine occurred at 10 min, whereas in infant mice the brain nicotine levels were still rising at 20 min. In the latter the blood and heart nicotine levels were higher than the respective brain levels, and the nicotine level in the brain stem exceeded the hemisphere level. The results were reversed in adult mice. A remarkable accumulation of nicotine in the infant heart was measured. Pretreatment with mecamylamine lowered brain nicotine levels in adult mice, and in infant mice the nicotine levels in blood and heart were lowered as well. This pretreatment abolished the nicotine tremor and its effects on the heart rate similarly in both age groups. This suggests that the difference in nicotine levels after mecamylamine in infant and adult mice may not depend solely on possible differences in circulatory changes but can represent differences in "receptor population" as well. Hexamethonium did not abolish the central depressant effect of nicotine on the heart rate nor did it lower the brain nicotine levels. This supports the view that there is some correlation of the central effects of nicotine and its brain levels.     

Combined action of amphotericin B and methacyclin on Candida albicans

The combined effect of amphotericin B, a polyene antibiotic, and metacycline, a tetracycline antibiotic, on the cells of C. albicans was studied. The method of square titration followed by quantitative plating of the samples was used for estimation of the combination efficiency. An attempt was also made to investigate the characteristic features of metacycline penetration into the yeast cells under the effect of various doses of the polyene antibiotic. The capacity of metacycline for fluorescence in the yellow-green pectral region was employed for this purpose. It was shown that the drugs had a synergistic effect on C. albicans. The fluorescence research methods allowed one to demonstrate that even low subinhibitory doses of amphotericin B increased the permeability level of the cytoplasmic membrane and provided penetration of metacycline into the cytoplasm almost during the first hours of the contact. The time course of metacycline cumulation in the cells was followed up and the characteristic features of the antibiotic localization were analysed.     

Cryptococcus neoformans phospholipase B1 activates host cell Rac1 for traversal across the blood-brain barrier

Cryptococcus neoformans penetration into the central nervous system (CNS) requires traversal of the blood–brain barrier that is composed of a single layer of human brain microvascular endothelial cells (HBMEC), but the underlying mechanisms of C. neoformans traversal remain incompletely understood. C. neoformans transcytosis of HBMEC monolayer involves rearrangements of the host cell actin cytoskeleton and small GTP‐binding Rho family proteins such as Rac1 are shown to regulate host cell actin cytoskeleton. We, therefore, examined whether C. neoformans traversal of the blood–brain barrier involves host Rac1. While the levels of activated Rac1 (GTP‐Rac1) in HBMEC increased significantly upon incubation with C. neoformans strains, pharmacological inhibition and down‐modulation of Rac1 significantly decreased C. neoformans transcytosis of HBMEC monolayer. Also, Rac1 inhibition was efficient in preventing C. neoformans penetration into the brain. In addition, C. neoformans phospholipase B1 (Plb1) was shown to contribute to activating host cell Rac1, andSTAT3 was observed to associate with GTP‐Rac1 in HBMEC that were incubated with C. neoformans strain but not with its Δplb1 mutant. These findings demonstrate for the first time that C. neoformans Plb1 aids fungal traversal across the blood–brain barrier by activating host cell Rac1 and its association with STAT3, and suggest that pharmacological intervention of host–microbial interaction contributing to traversal of the blood–brain barrier may prevent C. neoformans penetration into the brain.     

The pharyngeal beta-hemolytic streptococcal carrier state among blood donors

459 blood donors aged 18-50 years were examined in 1987-1988 in Moscow. Among them, carrier state with respect to beta-hemolytic streptococci was detected in 107 donors (23.3%). The number of carriers gradually decreased with the increase of age of the examined donors. Group C streptococci occurred least of all (6.9%). Group A beta-hemolytic streptococci were isolated in 16.7% of the carriers. The isolation rate of streptococci from blood achieved its maximum in autumn and winter months and did not depend on preceding diseases, unhealthy working conditions, the rhesus factor and, with the exception of group A streptococci, the blood group. Among tonsillectomized donors carrier state with respect to beta-hemolytic streptococci occurred 2.2 times less frequently than among donors who had not undergone tonsillectomy. Carrier state with respect to beta-hemolytic streptococci was accompanied by higher levels of salivary sIgA antibodies to polysaccharide A, serum antibodies to polysaccharide A and circulating polysaccharide A. All beta-hemolytic streptococci were sensitive to erythromycin. All groups of streptococci showed the highest percentage of cultures resistant to gentamicin and tetracycline. In 100% of cases group A streptococci were sensitive to benzylpenicillin, methicillin, ampicillin, erythromycin and lincomycin.     

Preparing platelet concentrates from banked blood stored for 1-5 days by using tetracycline antibiotics

It was shown that storage of banked blood up to five days did not change number of platelets and their functional integrity was retained rather well. That made possible the development of a method for preparation of viable platelet concentrates (PC) from stored blood. This method is based on the reversibility of the tetracycline antibiotics inhibitory effect on platelet activation process. According to the results of our in vitro studies PC from stored blood seem to be suitable for clinical usage. This tetracycline method of PC preparation from stored blood may provide a more efficient utilization of available donor blood to meet the ever-expanding needs for PC transfusions.     

Molecular studies of diabetes reported at the Fourth European Congress of endocrinology

In his lecture at the Fourth European Congress of Endocrinology, C.R. Kahn considered the effects of knock-out of genes encoding the proteins involved into insulin signal transduction on the development of insulin-resistance and non-insulin-dependent diabetes mellitus. The latter were induced in animals by knockout of genes encoding insulin receptors and intracellular substrate proteins of the insulin receptor. Using special technology, the authors achieved selective knock-out of the insulin receptor gene in muscles and pancreatic beta-cells of mice. Non-insulin-dependent diabetes mellitus developed only after the knock-out of the insulin receptor gene in beta-cells and resulted from the inability of glucose to penetrate into beta-cells and stimulate insulin secretion. The insensitivity of muscles to insulin due to the lack of its receptor did not result in diabetes. In these animals insulin and glucose blood level did not differ from the control values, but blood lipid concentration was increased. For the cases of the reduction in the insulin-dependent penetration of glucose into muscles, these data may indirectly indicate a transition of energy metabolism in muscles from carbohydrate utilization to increased fat consumption as an energy source.     

Molecular cloning in plasmid pBR322 giving altered expression of the tetracycline resistance gene

The two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, a site located in the RNA polymerase promoter involved in the expression of tetracycline resistance. Although insertion of foreign DNA into this site did not always result in the complete loss of tetracycline resistance, Escherichia coli K12 strain chi 1776 harbouring recombinant plasmids exhibited reduced growth properties in liquid culture with tetracycline and could easily be differentiated from bacteria transformed by non-recombinant plasmids. The formation of plasmid multimers increased the resistance to tetracycline at the level of the induction period, presumably as a result of a gene dosage effect.