C-glycosidic analogues of lipid A and lipid X: synthesis and biological activities.

The synthesis of a series of novel analogues of lipid A, the lipophilic terminal of lipopolysaccharides (LPS), and lipid X, the reducing monosaccharide unit in lipid A, is reported. In these compounds, the native 1-O-phosphate group has been replaced by a "bioisosteric" CH2COOH substituent. The new N,O-acylated monosaccharide C-glycosides were obtained by Wittig reaction of suitably protected glucosamine derivatives. These lipid X analogues were recognized as substrates by the enzyme lipid A synthase and could be coupled with UDP-lipid X to afford the corresponding disaccharide analogues of the lipid A precursor on preparative scale. All compounds were characterized by NMR, MS, and elemental analysis, and were tested for their ability to enhance nonspecific resistance to infection in mice and also for endotoxicity. The results clearly show that the new compounds express biological activities similar to those of their O-phosphorylated natural counterparts. Furthermore, these compounds exhibit a better therapeutic index in mouse models than the standard LPS obtained from Salmonella abortus equi.

Acyclic analogues of lipid A: synthesis and biological activities.

The synthesis of a series of novel acyclic analogues of lipid A, the lipophilic terminal of lipopolysaccharide (LPS), is reported. In these compounds, the reducing glucose unit of lipid A has been replaced by an acyclic analogue unit (abbreviated as AAU) consisting of a spacer (of varying length), an (R)-3-hydroxytetradecanamido moiety (of varying configuration at the carbon of attachment), and a CO2H group. The AAU has been attached to the anomeric carbon of the nonreducing glucose unit of lipid A, either through glycosidic linkage or through an acyl linkage. Further, amide isosteres of these acyclic analogues have been prepared using suitably protected 2,3-diamino-2,3-dideoxyglucose instead of 2-amino-2-deoxyglucose. All the compounds were well characterized and were tested for their ability to induce TNF-alpha in mouse bone marrow-derived macrophages, to enhance nonspecific resistance to infection in mice and to induce endotoxic shock in mice. The results showed a dramatic dependence, for the first time, on the length of the spacer and on the configuration of the carbon bearing the amido group in the AAU part of the analogues.

Differing complement-mediated opsonic activity of rabbit interstitial fluids from autologous serum.

Lack of appropriate methods for withdrawing extravascular or interstitial fluid from an animal host has limited in vitro study on the role of complement in the local defence of the extravascular space. In the present study, we obtained fluids from membrane diffusion chambers (porosity 0.22 micron) implanted into the kidneys, peritoneal cavity and soft tissues in rabbits. The complement-mediated opsonic activity (CMOA) of these fluids for Staphylococcus aureus ATCC 502A and Escherichia coli 01 was then compared to that of autologous sera. Soft tissue and renal interstitial fluids were as opsonic for E. coli as autologous sera but were however, poor opsonins for S. aureus. The peritoneal fluid was marginally effective in opsonization of both bacterial strains. While chelation of the fluids with MgEGTA (to block the classical pathway) did not diminish CMOA for E. coli, it reduced the CMOA for S. aureus by half. Conversely, heat-inactivation of the fluids and serum eliminated the opsonic activity for E. coli but only decreased the opsonic activity for S. aureus by half. Following a 24 h in vivo growth of E. coli in the implanted chambers, the CMOA was drastically reduced. Concomitant to the reduction in functional complement in the fluids, E. coli recovered from the chambers were found coated, though not maximally, with C3b as evidenced by studies with fluorescent antibody. The differences in opsonic content of extravascular fluids observed here might explain why certain sites of the body may be more vulnerable to attack by some bacterial species which are not effectively opsonized and therefore phagocytized.

Differing antimicrobial potency and specificity of peripheral blood and autologous exudative polymorphonuclear leucocytes.

Little is known about the antimicrobial potency and specificity of polymorphonuclear leucocytes which actually appear at the sites of bacterial invasion in tissues. In the present work we have compared inflammatory leucocytes induced by intraperitoneal injection of casein in rabbits with autologous peripheral blood cells in killing Escherichia coli serotype 01 and Staphylococcus aureus 502A. The results indicate that inflammatory leucocytes differ significantly from their virgin blood ancestors. While the blood leucocytes were only able to suppress the growth of the gram-negative bacteria, autologous exudative cells killed more than 95% of the test organisms within 1 h of incubation at 37 degrees C. The enhanced microbicidal activity of the inflammatory cells however, was only specific for the gram-negative bacteria, as evidenced by the failure of leucocytes to kill Staph. aureus to the same extent as the peripheral blood cells. In association with the enhanced gram-negative microbicidal activity the inflammatory cells produced chemiluminescence and released two to three times more O2-anions than the peripheral cells. We interpret these observations to mean that chemotactic factors such as casein activate inflammatory cells to increase their oxidative metabolism. Since microbicidal action of leucocytes is thought to proceed in part through oxygen-dependent reactions, the inflammatory leucocytes would be expected to effectively kill bacteria that are highly susceptible to these lethal oxygen metabolites. It cannot therefore be assumed that assessment of the functional capacity of the virgin peripheral blood PMNs would provide information on the functional characteristics of activated leucocytes which actually migrate to and accumulate at inflammatory sites.

Blood level studies in chickens, turkey poults and swine with tiamulin, a new antibiotic.

Tiamulin concentrations have been determined in the serum of chickens, turkeys and swine after a single oral administration of various doses. The compound peaked between 2 and 4 hours after drug supply. In the highest dose used (50 mg/kg body weight), mean concentrations of 3.5, 2.9, 4.5 microgram/ml for chickens, turkeys and swine, respectively, could be obtained. In poultry, the levels declined between 12 and 24 hours to values not useful for calculation. In swine at 24 hours after administration measurable values could still be detected.

In vitro activity of tiamulin (81.723 HFU), a new pleuromulin derivative, against clinically significant anaerobes.

The susceptibility of more than 40 strains of Gram-negative and Gram-positive anaerobes to tiamulin (Sandoz 81.723 hfu), a new pleuromulin (pleuromutilin) derivative, was determined by broth dilution and agar dilution tests. The influences of density of the inoculum upon MICs was studied by a specially designed pour plate-technique. Bacteroides fragilis, B. vulgatus, B. splanchnicus, B. oralis, B. asaccharolyticus, B. melaninogenicus, Fusobacterium fusiforme (F. nucleatum), Sphaerophorus necrophorus, Clostridium perfringens, C. fallax, Propionibacterium acnes and several species of Peptococcaceae showed broth dilution MICs of 0.03 similar to 1 microgram/ml. Members of B. thetaiotaomicron, B. distasonis and S. freundii (F. mortiferum) were inhibited by 8 similar to 32 microgram/ml and 2 strains of S. varius had a broth dilution MIC of 256 microgram/ml. With most strains, the agar dilution MICs were 2 similar to 4 similar to 8 times the broth dilution MICs. In pour plate-tests, the MICs were not considerably influenced influenced by varying initial concentrations of viable cells. With most anaerobes, the MBCs of tiamulin were more than 100-fold higher than the MICs. The results obtained indicated that, apart from S. varius, B. thetaiotaomicron, B. distasonis and S. freundii (F. mortiferum), members of 16 other anaerobic species including B. fragilis were without exception sensitive to tiamulin.

[The symptomatic therapy of cervical hard-substance defects with dentin-adhesive-composite systems]. / Symptomatische Therapie zervikaler Hartsubstanzdefekte mit Dentinadhäsiv-Komposit-Systemen.

For covering of hypersensitive, morphologically disturbed and non-esthetic cervical (wedge-shaped) defects of hard dental tissues, dentine adhesive composite systems are suitable for a careful and less invasive preparation. Presumption is a smooth and at probing hard-touched defect surface. A clinical 2 year study (15 patients with 143 cervical lesions) using 5 different dentine adhesive composite systems resulted in superiority of the hybrid composite P-30 in view of low filling losses (after 1 year: 1 of 28 fillings after 2 years: 2 of 27 fillings), the acceptable bond zone morphology (after 1 and 2 years clinical intact bond zones), and a constant volume behaviour (after 1 and 2 years no clinical changes of volume). The micromorphological evaluation correlated in a lot of cases with the clinical findings after 2 years. It has to be expected that improved dentine adhesive composite systems together with changed setting mechanisms of dentine adhesives will be able to eliminate filling losses in the treated indication.

[In vitro and in vivo activity of tiamulin against leptospires (author's transl)]. / Wirksamkeit von Tiamulin gegen Leptospiren in vitro und in vivo.

The minimal growth inhibiting concentration of tiamulin, a derivative of the diterpen antibiotic pleuromutilin, was evaluated in vitro against 11 different serogroups of leptospira interrogans by twofold serial dilution technique, in comparison to tetracyclin, dihydrostreptomycin and tylosin. The range of the MIC values of tiamulin is between 0.07 and 2.5 microgram/ml and thus comparable to the activities of the standard antibiotics tested (see table 1). The chemotherapeutic efficacy (ED50) of the compound was examined in two experimental leptospiral infections of the Syrian hamster, in comparison to tetracyclin. Both compounds were administered orally for 3 days. In the L. canicola infection, the ED50 values were 103.8 mg/kg and 306.3 mg/kg body-weight for tiamulin and tetracyclin, respectively. In the L. grippotyphosa infection, the ED50 values amounted to 35.16 and 277.5 mg/kg bodyweight for tiamulin and tetracyclin, respectively. Based on these values, tiamulin in comparison to tetracyclin showed 3-8 fold higher efficacy in vivo after oral administration.

In vivo efficacy of 81.723 hfu, a new pleuromutilin derivative against experimentally induced airsacculitis in chicks and turkey poults.

The efficacy of the pleuromutilin derivative 81.723 hfu was tested in chicks and turkey poults by experimentally infecting them with Mycoplasma gallisepticum. The data were treated to obtain the mean effective doses, and these were compared to those obtained with tylosin tartrate. The compounds were administered either by ingluvial catheter or via the drinking water. Therapy was started on the day of inoculation or 7 days thereafter, respectively. The experiments showed that the compound 81.723 hfu was significantly more active in chickens than tylosin tartrate by both methods of administration of drinking water. In turkey poults the pleuromutilin derivative and tylosin tartrate had comparable activity.

Concentrations of various antibiotics in serum and fluids accumulated in diffusion chambers implanted in various sites in rabbits.

Diffusion chambers with Millipore membranes were implanted in soft tissue, kidneys, and peritoneal and pleural cavities of rabbits. Single doses of azlocillin, cefazolin, and gentamicin were injected intramuscularly and ampicillin was administered orally 2--5 weeks after implantation. The concentrations of the respective drugs in simultaneously collected samples of fluid from each diffusion chamber were measured and compared with concentrations found at the same time in serum. All chambers were tolerated well, and the method proved to be effective for collecting data on the distribution of drugs throughout the body.

[Clinical and histopathological findings in mycoplasmal polyarthritis of rats. I. Course of infection during the first 8 days (author's transl)]. / Klinische und histopathologische Befunde bei der Mykoplasma-Polyarthritis der Ratte. I. Der Ablauf der Infektion in den ersten 8 Tagen

Polyarthritis in rats could be induced by intraperitoneal infection with M. arthritidis. The mycoplasma culture alone or in combination with infusorial earth was used as inoculum. The histopathological changes consisted of initial lesions in the transitional zone of the articular capsule which could be attributed to disturbances of vascular permeability. After two days suppurative arthritis occurred, which was accompanied by massive inflammatory lesions of the articular capsule and of the periarticular tissues. Towards the end of the observation period erosions of cartilage, pannus formation and periarticular foci of purulent necrobiosis were found. Especially the synovial membrane was subjected to profound changes and dense cellular infiltration. The cell infiltration, mesenchymal proliferation and changes of vascular permeability showed variations in timing and degree depending on the modi of infection. The histological examinations of organs indicated generalization of the infection.

[Clinical morphology of bond zone in bonding agent composite systems]. / Klinische Bindungszonenorphologie von Haftvermittler-Komposite-Systemen.

Three composites--Colténe DRS, P-10, Silux--and their bonding agents (enamel bond systems, Scotchbond) have been investigated. To this purpose the usual examination of "margin close" according to Lutz in 1977 was modified in "morphology of bond zone" (bm). After one year the phosphonated bonding agents has shown better results dependent on composites than enamel bond systems. A year later the "bons zoné's morphology" of various composites did not differ from another because P-10 underlied a heavy occlusal wear. In spite of that the phosphonated bond to enamel has proven better results than to dentin. Replication technique was applied in some chosen cases with clinical acceptable bond zones.

[Clinical and histopathological findings in mycoplasmal polyarthritis of rats. III. Course of infection during the weeks 7-30 and 54-61 (author's transl)]. / Klinische und histopathologische Befunde bei der Mykoplasma-Polyarthritis der Ratte. III. Der Krankheitsablauf in der 7.-30. und 54.-61. Woche

Clinical, microbiological and histological findings of the late phase of experimentally induced Mycoplasma-polyarthritis in rats are described. Investigations were carried out between the 7.-30. week and between the 54-61. week p.i. M. arthritidis could be reisolated from affected joints regularly up to the 7th week p.i. Thereafter until 12th week only occasional recovery was possible. The healing process of affected joints dominated signs of arthritis which could be confirmed histologically as a purulent process. The alterations of carpal and tarsal joints were divided into purulent, chronic deforming and chronic minor lesions. The findings derived from investigation of joints and parenchymatous organs during the course of the disease are discussed.

[Clinical and histopathological findings in mycoplasmal polyarthritis of rats. II. Course of disease between the 2. and 6. week (authors transl)]. / Klinische und histopathologische Befunde bei der Mykoplasma-Polyarthritis der Ratte. II. Der Krankheitsablauf in der 2. bis 6. Woche

Between the 9th and 42nd day p.i. the clinical features and microbiological findings of rats infected intraperitoneally with mycoplasma arthritidis were recorded and a histological investigation was carried out. From the 12th day p.i. arthritic lesions clinically showed a gradual regression. Until the 42nd day p.i. mycoplasmas and purulent exudate were present within the joint cavity. During the course of infection a gradual increase of mesenchymal cell activity was found, leading to the formation of considerable amounts of secondary synovialis. Between the epiphysis the pannus formed tissue laces, which after the 3rd week p. i. showed sequestration. During the further course of disease an increased number of focal plasma cell accumulations was observed within the joint capsule. Partial erosions of hyaline cartilage, especially near its edge, could frequently be seen. Within the cellular exudate of the joint cavity the number of macrophages steadily increased whereas remmants of exudate were mainly located in the recesses of the joint capsule. In the periarticular tissue, cellular infiltration, mesenchymal cell proliferation and purulent necrotic foci were present. In the supporting osseous tissues osteomyelitis, abscesses, periosteal new bone formation and the occurrence of mosaic bone could be observed. The lesions found in spleen and lymph-nodes indicated the persistence of a systemic reaction within the RES.

Measurements of antimicrobial drug concentrations in renal interstitial fluid using the diffusion chamber technique.

A technique is described to obtain renal interstitial fluid (RIF) from rabbits after implantation of diffusion chambers with permeable membranes of 0.45 mu porosity in both kidneys. Pharmacokinetic studies were conducted two to three weeks after implantation. No difference in gentamicin concentrations, as measured microbiologically, was seen between RIF withdrawn from the left and right kidney chambers at the same points in time. Simultaneous drug concentrations were determined in RIF and serum of rabbits after oral administration of ampicillin or nalidixic acid and after intramuscular injection of gentamicin. Ampicillin concentrations in RIF peaked at two hours with about one fourth of the peak concentration measured in serum at one hour. These curves crossed at 3.45 hours. In RIF, the maximum concentration of gentamicin found at two hours was again approximately one fourth of the serum peak level determined at half an hour. The gentamicin curves crossed at 3.15 hours. No levels of nalidixic acid could be detected microbiologically in serum and RIF. In collected urine, however, concentrations of this drug could be measured for several sampling periods. Our results show that this diffusion chamber technique can be useful in the pharmacokinetic examination of drugs, also with respect to their distribution in the kidneys.

Opsonic activity of the alternative complement pathway in infected human intra-abdominal fluid.

The complement-mediated opsonic activity (CMOA) in intra-abdominal exudates collected during laparotomy from peritonitis patients was examined by an in vitro phagocytic bactericidal assay. It was found that infected intra-abdominal exudates barely promoted detectable killing of Escherichia coli 01 by polymorphonuclear leukocytes. Only the minority of bacteria recovered by differential centrifugation from the infected exudates had C3 on their surfaces. Such bacteria were not optimally opsonized in vivo, whereas they became fully coated with C3 during a brief incubation in vitro in normal human serum. The low level of CMOA in the peritoneal fluid paralleled a depletion of complement in the peripheral blood. Thus, in cases complicated by sepsis with gram-negative bacteria, the CMOA in the blood was extremely low. These data suggest that the high susceptibility of the peritoneum to infection by gut flora, despite the normal phagocytic response, may be partly explained by the low local level of functional complement which is inadequate for optimal opsonization of the bacteria.

Membrane-disorganizing property of polymyxin B nonapeptide.

The possibility of improving the antibacterial activities of drugs normally excluded by Gram-negative bacteria with polymyxin B nonapeptide (PMBN) has been explored. In vitro, PMBN rendered clindamycin, erythromycin, novobiocin, rifampicin and vancomycin very active against a number of Gram-negative enteric bacteria. The drug also sensitized the previously resistant bacterial strains to human, mouse or guinea pig serum. However, parenterally administered PMBN failed to influence bacterial growth in chambers implanted into mice and guinea pigs. It was also ineffective in experimental septicaemia at a dose of up to 200 mg/kg or when combined with antibiotics with which it interacted synergistically in vitro.

Protection of mice from mortality caused by living and heat-killed bacteria by SDZ MRL 953.

Protective effects of SDZ MRL 953, a monosaccharidic lipid A analog with a reduced toxicity, were investigated in models of experimental septic shock caused by injections of LPS, and inoculations of heat-killed or live bacteria. Female B6D2F1 mice were challenged with a combination of galactosamine (800 mg/kg) plus various doses of heat-killed isolates of Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Staphylococcus aureus or LPS from Salmonella abortus equi. In some experiments, isolates of living bacteria at sublethal inocula were also combined with galactosamine. More than 90% of the animals died within 24 hr when the challenge was performed either simultaneously with or up to 4 hr after an intraperitoneal administration of galactosamine. No death was observed when galactosamine was omitted or administered after the microbial or LPS challenge. Pretreatment of the animals with SDZ MRL 953 (1-10 mg/kg) rendered the animals resistant to the lethal effects of both bacterial and LPS challenge in a time- and dose-dependent manner. The levels of TNF-alpha in control mice rose to greater than 600 pg/ml 2 hr postbacterial or LPS challenge, but were below detection in animals pretreated with SDZ MRL 953. Protection against both the infection and the toxicity of heat-killed bacteria or LPS was also achieved when murine anti-TNF-alpha monoclonal antibody was administered prophylactically. Together, these data suggest that SDZ MRL 953 enhances the resistance of mice against the toxicity of heat-killed gram-negative bacteria and S. aureus, and attenuates host responses to living bacteria which may lead to irreversible shock and death.