Airway hyperresponsiveness to histamine in mycoplasmal infection: role of histamine N-methyltransferase.

To elucidate the modulatory role of histamine-degrading enzymes in airway constrictor responses in mycoplasmal infection, we studied hamster tracheal segments under isometric conditions in vitro. Nasal inoculation with Mycoplasma pneumoniae potentiated the contractile responses to histamine but not to methacholine. Pretreatment of tissues with the histamine N-methyltransferase inhibitor SKF 91488 abolished the infection-induced potentiation, whereas, the diamine oxidase inhibitor aminoguanidine had no effect. The histamine N-methyltransferase but not diamine oxidase activity in tracheal tissues was decreased in infected animals. These results suggest that M. pneumoniae causes airway hyperresponsiveness to histamine probably through a reduction of endogenous histamine N-methyltransferase activity.

The effects of serum on cellular uptake and phototoxicity of mono-L-aspartyl chlorin e6 (NPe6) in vitro.

Previous reports showed that the photosensitizer mono-L-aspartyl chlorin e6 (NPe6) binds to serum proteins. However, the influence of this binding on the cellular uptake and photodynamic therapy (PDT) phototoxicity of NPe6 is still undefined. In this paper, we studied how serum in medium affected the P388 cellular uptake and PDT phototoxicity of NPe6 in vitro. This was assessed by (1) detection of the red shift (654 nm Q band peak of absorption) induced by protein binding NPe6; (2) detection of intracellular concentration of NPe6 by HPLC and (3) measurements of the cell survival ratio after PDT by MTT assay. The 654 nm Q band peak of NPe6 shifted to 665 nm after binding of NPe6 and serum proteins. The protein-bound NPe6 cannot be uptaken by cells, thus there was no PDT phototoxicity. Nevertheless, phototoxicity recovered when the concentration of NPe6 excessed the serum protein binding ability or there was free serum protein in the medium. These data suggested that the cellular uptake of NPe6 is inhibited by serum components in the medium, and that only free NPe6 is accumulated by P388 cells even during relatively long incubations. The cytotoxicity of PDT mainly depends on the free NPe6 level in the medium.

Cladinose analogues of sixteen-membered macrolide antibiotics. I. Synthesis of 4-O-alkyl-L-cladinose analogues via glycosylation.

The synthesis and biological evaluation of sixteen-membered macrolides possessing a 4-O-alkyl-alpha-L-cladinosyl moiety as the neutral sugar are described. The nine novel derivatives have been synthesized by glycosylation with 1-thio sugars. The most active derivative of them showed prolonged antibacterial activity in rat plasma in vitro and improved pharmacokinetics.

Cladinose analogues of sixteen-membered macrolide antibiotics. VI. Synthesis of metabolically programmed, highly potent analogues of sixteen-membered macrolide antibiotics.

Five novel 3-hydroxyl derivatives of sixteen-membered macrolide possessing 4-O-acyl-alpha-L-cladinose as a neutral sugar moiety were synthesized by using a combination of structurally stable silyl acetal protection and selective hydrogenolysis of a 3"-methylthiomethyl ether to a 3"-OMe group. Several derivatives having n-butyryl, i-butyryl and n-valeryl substituent at the 4"-OH group exhibited significant antibacterial activity in vitro. One of them, 4"-O-n-butyryl-3"-O-methylleucomycin V, showed improved therapeutic effect in mice.

Cladinose analogues of sixteen-membered macrolide antibiotics. IV. Improved therapeutic effects of 4-O-acyl-L-cladinose analogues of sixteen-membered macrolide antibiotics.

Six derivatives of sixteen-membered macrolides possessing 4-O-acyl-alpha-L-cladinose as a neutral sugar were synthesized via 3"-methylthiomethyl ether intermediates in reasonable yield. Introduction of a methyl group on the 3"-hydroxyl group of midecamycin A1 was effective for enhancing its antibacterial activity. All these derivatives exhibited excellent therapeutic effects in mice, and some of them showed improved pharmacokinetics compared with the natural antibiotics (mycarose type) in mice. Facile synthesis of 9-O-acylated analogues are also described.

[Evaluation of once-daily administration of arbekacin. Experimental study and determination of pharmacokinetic properties in man].

Experimental and phase I clinical studies were performed to evaluate the efficacy and safety of once-daily administration of arbekacin (ABK). The results obtained were as follows: 1. ABK displayed dose-dependent, excellent antibacterial activity and post-antibiotic effects (PAE) against MRSA. 2. No significant difference was found between once-daily and divided administration regimens in protection against an experimental MRSA infection in mice. 3. There was no significant difference between once-daily and twice-daily administration of ABK in ototoxicity in guinea pigs or in nephrotoxicity in rats. 4. In the phase I clinical study using 200 mg single daily administration of ABK, no abnormal laboratory test results or symptoms were observed. 5. In the phase I clinical study of 5-day repeated administration of 200 mg/day of ABK, headache and increase in WBC sediment in the urine was noted in 1 volunteer; however these were not confirmed to be attributable to ABK. No abnormal laboratory test results were obtained other than increases in beta 2-microglobulin, NAG and gamma-GTP levels, each of which returned to normal after the completion of ABK administration. No abnormality was observed in the audiometry examination. 6. Maximum serum concentration (Cmax), serum half-life (T1/2 beta) and urinary recovery rate (0-48 hours) after single administration of 200 mg of ABK, were 13.20 micrograms/ml, 2.30 hours and 86.75%, respectively. There were no significant differences in pharmacokinetic parameters or urinary recovery rates between day 1 and day 5 in the 5-day repeated administration study. These findings suggest that once-daily administration regimen of ABK may be as effective and safe as divided administration regimen for the treatment of MRSA infection. Further clinical evaluation is required, however.

[Bactericidal activity of biapenem against various bacteria].

The bactericidal activity of biapenem (BIPM), a new carbapenem agent, against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens was compared with those of imipenem (IPM), panipenem (PAPM), meropenem (MEPM) and ceftazidime (CAZ). The bactericidal activity of BIPM against S. aureus was equal to those of IPM, PAPM and MEPM. Against E. coli and K. pneumoniae, BIPM showed higher bactericidal activity than IPM and PAPM. Against P. aeruginosa, BIPM showed excellent bactericidal activity campared with IPM. The killing speeds of BIPM and IPM were obviously the most rapid among four carbapenems. BIPM showed a strong bactericidal activity against 5 species of bacteria including P. aeruginosa.

[Effect of arbekacin on the production of toxic shock syndrome toxin 1 by methicillin-resistant Staphylococcus aureus].

The effect of arbekacin (ABK), vancokmycin (VCM) and teicoplanin (TEIC) on the production of toxic shock syndrome toxin-1 (TSST-1) by methicillin-resistant Staphylococcus aureus was examined. In logarithmic-phase cultures, ABK, VCM and TEIC inhibited TSST-1 production by 85, 10 and 25%, respectively, at the concentration of one-fourth the each MIC. In stationary-phase cultures, ABK inhibited TSST-1 production by 50% or 90% compared with the control at the concentration of 4.0 micrograms/ml or 5.0 micrograms/ml respectively. VCM and TEIC did not inhibit TSST-1 production at the concentration of 8.0 micrograms/ml or lower. In human blood cultures, TSST-1 production was inhibited by ABK by 50% at 0.04 microgram/ml (1/256 of Cmax), but not inhibited by VCM and TEIC at the concentration of 1/16 of Cmax or lower. It has been already known that ABK has higher bactericidal activity than VCM and TEIC. ABK combined the inhibition of TSST-1 production with high bactericidal activity in both bacterial growth phases, and therefore ABK should be considered for the treatment of TSST-1-mediated MRSA-infection.

[Combination effect of arbekacin and cefepime on mixed culture of MRSA and P. aeruginosa].

We investigated the in vitro combination effects of arbekacin (ABK), vancomycin (VCM) or teicoplanin (TEIC) and cefepime (CFPM) on methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa isolated from various clinical specimens. Using checkerboard titration technique by agar dilution, combinations of ABK, VCM or TEIC and CFPM exhibited synergistic effects on 25 MRSA strains. The similar effects were also observed on ABK-resistant MRSA. Combination of ABK and CFPM exhibited a good effect on P. aeruginosa, but combinations of VCM or TEIC and CFPM exhibited no synergistic effect on P. aeruginosa. In vitro bactericidal activities of ABK, VCM or TEIC and CFPM against mixed cultures of MRSA with P. aeruginosa were examined at concentration of each drug that corresponds to the serum concentration at 3 hours after the usual therapeutic dosage. VCM or TEIC alone showed bacteriostatic effects against MRSA, and no enhancements were observed when combined with CFPM. ABK alone showed good bactericidal activity against MRSA and combination with CFPM enhanced the bactericidal activity. Against P. aeruginosa, ABK or CFPM alone showed the bactericidal activity, and strong bactericidal activity was induced by the combination of ABK and CFPM. VCM and TEIC showed no bactericidal activities against P. aeruginosa. When CFPM combined with VCM or TEIC, the bactericidal activity of CFPM was not enhanced against P. aeruginosa.

Comparison of ciliostasis by mycoplasmas in mouse and chicken tracheal organ cultures.

The cilia-stopping effect of mycoplasmas of human and various animal origin in mouse and chicken tracheal organ cultures was studied. From the results in mouse tracheal organ cultures, the mycoplasma strains tested were divided into three groups: Mycoplasma pulmonis m53, M pulmonis JB, M. pulmonis OK, M. mycoides subsp. Mycoides PGl and M. Gallisepticum S6 showed a strong cilia-stopping effect; M. pulmonis PG22, M. mycoides subsp. capri PG3, M. meleagridis 19729, M. neurolyticum Type A and M. arthritidis PG6 showed a mild effect; and M. pneumoniae FH, M. salivarium Hup, M. hominis type 1-C and M. orale N-C human origin and Acholeplasma laidlawii PG8 showed a weak effect. On the other hand, in chicken tracheal organ cultures, only M. gallisepticum S6 showed a strong effect, M. meleagridis 19729 was affected to a lesser degree, and the other mycoplasma strains showed a weak or no effect. The results indicate that some murine and poultry mycoplasmas showed a cilia-stopping tendency in mouse and chicken tracheal organ cultures, respectively, while human mycoplasmas showed weak or little effect in both organ cultures. In mouse tracheal organ cultures, M. pulmonis m53 treated with heat, trypsin or formaldehyde, and the sterile filtrate of an m53 broth culture showed no cilia-stopping effect. The relationship of the pathogenicity of mycoplasmas for their natural hosts to that for cultured respiratory cells is discussed.

Attachment of Mycoplasma pulmonis to rat and mouse synovial cells cultured in vitro.

The attachment of Mycoplasma pulmonis m53 organisms to mouse and rat synovial cells was examined by using the organisms and the synovial cells treated in various ways. M. pulmonis treated with trypsin attached to the synovial cells, but the organisms treated with pronase, formaldehyde, glutaraldehyde, or heat did not. These findings suggest that the sites for binding M. pulmonis to the mouse and rat synovial cells are of polypeptide nature. Treatment of M. pulmonis with sialic acid and treatment of the synovial cell sheets with neuraminidase did not affect the attachment. The synovial cell surface for receptors M. pulmonis organisms would be different from those on respiratory cells or erythrocytes for M. pneumoniae or M. gallisepticum. Even nonviable organisms and M. pulmonis membranes attached to the mouse or rat synovial cells. The nature of the receptor of mouse synovial cells would be different from that of rat cells, since rat cells were affected by treatment with formaldehyde or glutaraldehyde, but mouse cells were not.

Activation of human vascular endothelial cells by IFN-gamma: acquisition of HLA class II expression, TSST-1-binding activity and accessory activity in T cell activation by the toxin.

Human umbilical vascular endothelial cells (HUVEC) stimulated with recombinant human IFN-gamma were investigated for expression of HLA class II molecules, toxic shock syndrome toxin-1 (TSST-1)-binding activity and accessory cell (AC) activity in TSST-1-induced T cell activation. HUVEC stimulated with recombinant human IFN-gamma ranging from 10 to 1,000 units/ml for 72 h express HLA class II molecules. Intensity of the expression was dependent on the concentration of IFN-gamma. HUVEC stimulated with 1,000 units/ml of IFN-gamma for 72 h exhibited 125I-TSST-1-binding that was blocked by the unlabeled toxin and monoclonal antibodies (mAb) to DR/DP. The activity was not removed by paraformaldehyde fixation. The IFN-gamma-stimulated HUVEC exhibited AC activity in TSST-1-induced IL-2 production by T cells from human peripheral blood mononuclear cells. The activity was blocked by mAb to DR. The above two activities were also observed in L cells transfected with DR2 genes but not in the unstimulated HUVEC and control L cells. In view of the fact that TSST-1 binds directly to HLA class II molecules and activates human T cells in association with HLA class II molecules on AC, it is likely that the acquisition of TSST-1-binding activity and AC activity in the toxin-induced T cell activation is mediated by the expression of HLA class II molecules. Vascular endothelial cells may play an important role in the development of pathological changes in TSS patients.

Modulatory effect of antibiotics on cytokine production by human monocytes in vitro.

Some antimicrobial agents have been reported to modify the host immune and inflammatory responses both in vivo and in vitro. Fosfomycin (FOM) and clarithromycin (CAM) have immunomodulatory activity on human lymphocyte function. In the present study, we examined the effects of FOM and CAM on cytokine synthesis by lipopolysaccharide (LPS)-stimulated human monocytes in comparison with that of dexamethasone in vitro. The three drugs demonstrated positive or negative effects on the synthesis of various cytokines by LPS-primed monocytes. They suppressed the synthesis of tumor necrosis factor alpha, interleukin 1 alpha (IL-1 alpha), IL-1 beta, the IL-1 receptor antagonist, and granulocyte-macrophage colony-stimulating factor in a concentration-dependent manner at concentrations between 1.6 and 40 micrograms/ml. On the contrary, the drugs showed different actions on the synthesis of IL-6 and IL-10. Namely, FOM enhanced both IL-6 and IL-10 synthesis, CAM enhanced only IL-10 synthesis, but dexamethasone deeply suppressed the synthesis of both cytokines. These data indicate that antibacterial agents may modify acute-phase inflammatory responses through their effects on cytokine synthesis by monocytes.

Mycoplasma pulmonis arthritis in congenitally athymic (nude) mice. Histopathological features.

Histopathological examinations were performed on arthritis joints and other organs of strain BALB/cA nu/nu and nu/+ mice intravenously injected with Mycoplasma pulmonis strain m53. In both groups of mice suffering from polyarthritis, acute inflammatory lesions with infiltration of polymorphonuclear leukocytes in the synovia and periarticular tissues were observed one to two weeks after injection. In nu/nu mice, the acute inflammation appeared repeatedly up to 20 weeks after inoculation, when the experiment was terminated, and furthermore, extensive synovial and periarticular necrosis were characteristically present after the 4th week. Only a small number of lymphocytes and plasma cells were in the lesions. In nu/+ mice, after the early acute inflammation of arthritis, relapses of the infiltration of polymorphonuclear leukocytes were also observed in some mice in and after the 10th week. In addition, infiltration of lymphocytes and plasma cells were substantial after the 15th week. Focal necrosis was sometimes found in the liver of nu/nu mice. Perivascular infiltration of small lymphocytes and plasma cells was found in the lungs, liver and kidney of nu/+ mice in and after the 15th week. Repair mechanisms of injured articular tissues in nu/nu mice were histopathologically poor, while those in nu/+ mice seemed to be progressive and quite similar to those reported by many investigators for mice with the thymus intact. The histopathological differences are discussed in respect to the thymus-dependent immune responses.

In vitro antiseptic susceptibility of clinical isolates from nosocomial infections.

To evaluate the susceptibility of a large number of strains to various antiseptics, we elaborated a simple, qualitative broth turbidity method in which we could quickly judge the efficacy visually. For this method, we prepared a modified neutralizer broth, consisting of trypticase soy broth containing 15% Tween 80, 1% soybean lecithin and 0.5% sodium thiosulfate. The susceptibilities of Serratia marcescens No. 26 to 4 antiseptics obtained from the turbidity method showed a good agreement with those obtained from the colony-counting method; the 4 antiseptics tested were povidone-iodine (PVP-I), chlorhexidine gluconate (CHG), benzalkonium chloride (BAC) and alkyldiaminoethylglycine hydrochloride (AEG). Both PVP-I and BAC had complete efficacy in 0.5 min against all isolates tested [100 isolates of S. marcescens, 103 of Klebsiella pneumoniae, 99 of Pseudomonas aeruginosa, 19 of Alcaligenes faecalis and 30 of A. xylosoxidans subsp. xylosoxydans (A. xylosoxydans)]. In contrast, the effectiveness of CHG was weak compared with PVP-I, BAC and AEG. Strong resistance against AEG was noted even after 3-min exposure in 1 isolate each of A. faecalis and A. xylosoxydans. It is concluded that the turbidity test is a simple and accurate method to evaluate susceptibility to various antiseptics and that it is suitable for a screening of a large number of strains. Among the 4 antiseptics tested, PVP-I and BAC showed a consistently high activity against all isolates, confirming PVP-I and BAC to be clinically useful antiseptics.