Pathogenicity of human anti-platelet factor 4 (PF4)/heparin in vivo: generation of mouse anti-PF4/heparin and induction of thrombocytopenia by heparin

Acinetobacter baumannii strain A148, a clinical isolate resistant to imipenem (MIC = 32 mg l-1), synthesized two beta-lactamases with pIs 6.3 and > 9.2. The pI 6.3 enzyme hydrolyzed the penicillins, including isoxazoylpenicillins, first-, second- and, to a lesser extent, third-generation cephalosporins. It was inhibited by chloride ions and by the penem beta-lactamase inhibitor BRL 42715. Clavulanate was a weak inhibitor and EDTA did not affect the beta-lactamase activity. This enzyme also hydrolyzed imipenem with a catalytic efficiency (Kcat/Km) of 1500 mM-1 s-1. Moreover, this purified beta-lactamase produced a positive microbiological clover-leaf test with imipenem. Therefore, the pI 6.3 beta-lactamase was considered to be involved in the imipenem resistance of A. baumannii strain A148.     

Antimicrobial susceptibility of Capnocytophaga

Capnocytophaga (Bacteroides ochraceus, Center for Disease Control biogroup DF-1) is associated with sepsis in granulocytopenic patients and is isolated in large numbers from the affected periodontal pockets in patients with juvenile periodontosis. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of 17 antimicrobial agents for 13 strains of Capnocytophaga organisms were determined. In addition, the ratio of the MBC to the MIC for each antimicrobial agent was determined for each strain. At concentrations of 1 microgram/ml or less, penicillin, ampicillin, carbenicillin, erythromycin, and clindamycin killed 90% of the strains. At concentrations of 3.12 microgram/ml or less, tetracycline, metronidazole, cefoxitin, and chloramphenicol killed 90% of the strains. None of the aminoglycosides tested demonstrated antibacterial activity at 50 microgram/ml. Penicillin, ampicillin, carbenicillin, and cefoxitin exhibited MBC/MIC ratios of 4 or less with all strains. Erythromycin, tetracycline, and metronidazole exhibited MBC/MIC ratios of 4 or less for 12 of 13 strains. The MICs of cephalothin and cefazolin for 90% of the strains were 25 and 50 microgram/ml, respectively. The MBC/MIC ratios for these drugs were 4 or less for 12 of 13 and 7 of 13 strains, respectively. The MIC of cefamandole for 90% of the strains was 3.12 microgram/ml; however, only nine strains had an MBC/MIC ratio of 4 or less.     

Basic and clinical studies on tazobactam/piperacillin in pediatric field

A drug susceptibility test of the combination drug TAZ/PIPC, which consists of a newly developed beta-lactamase inhibitor, tazobactam (TAZ), and one of penicillin antibiotics, piperacillin (PIPC), with combination ratio of 1:4 in potency, was conducted with stock strains and clinical isolates. The clinical efficacy and safety of its injection was also evaluated in children with a variety of infectious diseases. The results were as follows: 1. In susceptibility test, 114 strains from 4 species of stock strains were treated with 8 drugs, that is, TAZ/PIPC, PIPC, penicillin G (PCG), ampicillin (ABPC), cefotiam (CTM), cefotaxime (CTX), ceftazidime (CAZ), and sulbactam/cefoperazone (SBT/CPZ). Of three clinically isolated species from patients, Staphylococcus aureus (S. aureus) was treated with TAZ/PIPC, PIPC, methicillin (DMPPC), CTM, CTX, and SBT/CPZ, and the others were treated with the same drugs except for DMPPC. The MICs were measured for these bacterial strains inoculated at the concentration of 10(6) CFU/ml. The MIC90 values of TAZ/PIPC against 45 strains of Streptococcus pyogenes (S. pyogenes), one of the stock cultures of Gram-positive cocci, were 0.05 microgram/ml and similar to those of PIPC, CTM, CAZ, and SBT/CPZ. The MICs of TAZ/PIPC for 28 strains of Streptococcus agalactiae (S. agalactiae) were 0.39 microgram/ml and similar to those of PIPC, CTM, CAZ, and SBT/CPZ. As for Gram-negative bacilli, the MIC90 of TAZ/PIPC against 10 strains of Bordetella pertussis (B. pertussis) were 0.10 microgram/ml and similar to those of PIPC. The MIC90 of TAZ/PIPC against 31 strains of Haemophilus influenzae (H. influenzae) were 0.05 microgram/ml and similar to those of PIPC, CTX, and SBT/CPZ. Regarding Gram-positive cocci isolated from patients received this combination drug, the MIC90 of TAZ/PIPC against 2 strains of S. aureus, a non beta-lactamase producing strain and a low-beta-lactamase producing strain, were 0.78 microgram/ml and 3.1 micrograms/ml, respectively; the former value was similar to those of PIPC, DMPPC, CTM, and CTX, and the latter was similar to those of PIPC, DMPPC, CTX, and SBT/CPZ. Of 4 strains of Streptococcus pneumoniae, 2 strains were inhibited at 0.05 microgram/ml, and the others at 1.56 micrograms/ml; both values were similar to those of PIPC, SBT/CPZ. As for Gram-negative bacilli, 6 of 7 strains of H. influenzae did not produce beta-lactamase and 1 strain was a high producer. The MICs of TAZ/PIPC against beta-lactamase nonproducing strains were < or = 0.025 microgram/ml in 5 strains and 0.39 microgram/ml in 1 strain, and the values were similar to those of PIPC and SBT/CPZ. While the MIC of TAZ/PIPC against the high beta-lactamase producing strain was 0.78 microgram/ml; similar to that of SBT/CPZ and smaller than that of PIPC. 2. The results of clinical effects on 7 diseases in 33 cases were as follows: TAZ/PIPC was clinically judged "excellent" in 17 (51.5%); good in 14 (42.4%); fair in 2 (6.1%). No case with no response was seen in this study, and the total efficacy rate of "excellent" and "good" was 93.9%. 3. Bacteriological effects were evaluated in 17 strains of 4 species, and all of them were eradicated. 4. Adverse reactions were judged in 35, which consisted of 33 in which the clinical effects were evaluated and 2 dropped from this study. Of these cases, diarrhea was observed in 4 (11.4%). 5. Laboratory tests revealed an increase in platelets in 1 of 32 cases (3.1%), and eosinophilia in 2 of 29 cases (6.9%). Biochemical profile showed an increase in GPT alone and abnormal increases in both GOT and GPT in 1 each out of 21 cases.     

Drug resistance of Helicobacter pylori strains isolated from patients with inflammation of stomach mucous membrane and peptic ulcer of stomach and duodenum

Twelve strains of H. pylori were tested. They were isolated from biosamples from 23 patients with stomach inflammation (16 persons) and stomach (3) and duodenum peptic ulcer. By application of solid medium dilution of MIC50 and MIC90-16 antibiotics and antimicrobials were tested. Values of MIC50 and MIC90 for antibiotics from the tetracycline group were, respectively 0.25-0.5 microgram/ml and 0.12-1.0 microgram/ml. Similar results were obtained with macrolide antibiotics. All strains were sensitive to penicillin (MIC 0.03-0.12 microgram/ml, ampicillin (MIC 0.06-0.5 microgram/ml) and amoxicillin and rifampicin (MIC 0.07-0.3 microgram/ml). Ten out of 12 investigated strains were resistant to metronidazole (MIC90 = 30 micrograms/ml). Results of this study may be important for etiotropic treatment of infections with H. pylori.     

The beta-lactamases of Moraxella (Branhamella) catarrhalis isolated from Danish children

Two distinct beta-lactamases have been isolated from Moraxella catarrhalis: the stronger acting BRO-1 enzyme and the weaker acting BRO-2. Several reports have noted an effect of penicillin and ampicillin on infections caused by M. catarrhalis in spite of the presence of beta-lactamase production. The purpose of this work was to charaterize the beta-lactamases of M. catarrhalis isolated from Danish children regarding type and susceptibility, and to relate these findings to the eradication of beta-lactamases-producing strains by use of antibiotic treatment with penicillin or ampicillin. MICs for penicillin V, ampicillin, cefuroxime and amoxicillin/clavulanic acid (2:1) were determined in 70 strains of M. catarrhalis: 46 strains from children with lower respiratory tract infection (LRTI) and 24 strains from respiratory healthy children, beta-lactamase production was found in 59 strains. The BRO-1 enzyme was identified by isoelectric focusing in 55 strains (93.2%) and BRO-2 in 3 strains (5.1%); in 1 strain no isoelectric bands were produced. All strains were susceptible to cefuroxime and amoxicillin/clavulanic acid, and non-beta-lactamase-producing strains were susceptible to penicillin and ampicillin. For the beta-lactamase-producing strains, MIC50 of penicillin was 8.0 micrograms/ml, while MIC50 of ampicillin was 1.0 microgram/ml and MIC90 of ampicillin was 2.0 micrograms/ml. M. catarrhalis was more often eradicated from the children who received antibiotic treatment with penicillin or ampicillin than from those who did not receive any treatment, indicating an in vivo effect of penicillin and ampicillin in spite of the beta-lactamase production.     

Reducing saturated fat intake is associated with increased levels of LDL receptors on mononuclear cells in healthy men and women

1. Previous studies in our laboratory have shown that the synthetic xanthine analogue denbufylline, a selective type 4 phosphodiesterase (PDE-4) inhibitor, is a potent activator of the hypothalamo-pituitary-adrenal (HPA) axis when given orally or intraperitoneally (i.p.) to adult male rats. This paper describes the results of experiments in which well established in vivo and in vitro methods were used to compare the effects of denbufylline on HPA function with those of two other selective PDE-4 inhibitors, rolipram and BRL 61063 (1,3-dicyclopropylmethyl-8-amino-xanthine). For comparison, parallel measurements of the immunoreactive- (ir-) luteinising hormone (LH) were made where appropriate. 2. When injected intraperitoneally, rolipram (40 and 200 micrograms kg-1, P < 0.005), denbufylline (0.07-0.6 microgram kg-1, P < 0.05) and BRL 61063 (30 micrograms kg-1, P < 0.005) each produced marked rises in the serum ir-corticosterone concentrations. However, lower doses of rolipram (1.6 and 8 micrograms kg-1) and BRL 61063 (0.25-6 micrograms kg-1) were without effect (P > 0.05). By contrast, intracerebroventricular (i.c.v.) injection of rolipram (8 ng-1 micrograms kg-1) or denbufylline (50 ng-1 microgram kg-1) failed to influence the serum ir-corticosterone concentration. BRL 61063 (8-120 ng kg-1, i.c.v.) was also ineffective in this regard although at a higher dose (1 microgram kg-1, i.c.v.) it produced a small but significant (P < 0.05) increase in ir-corticosterone release. Denbufylline also increased the serum ir-LH concentration when given peripherally (0.2-0.6 microgram kg-1, i.p., P < 0.05) or centrally (100 ng kg-1, i.c.v., P < 0.05) but rolipram (1.6-200 micrograms kg-1, i.p. or 8 ng-1 microgram kg-1, i.c.v.) and BRL 61063 (0.25-30 micrograms kg-1, i.p. or 1 ng-1 microgram kg-1, i.c.v.) did not (P > 0.05). 3. In vitro rolipram (10 microM, P < 0.01), denbufylline (1 mM, P < 0.001) and BRL 61063 (1 and 10 microM, P < 0.05) stimulated the release of corticotrophin releasing hormone (ir-CRH-41) but lower concentrations of the drugs were without effect as also was BRL 61063 at 100 microM (P > 0.05); the rank order of potency was thus BRL 61063 > rolipram > denbufylline. The adenylyl cyclase activator forskolin (100 microM, P < 0.01) also stimulated the release of ir-CRH-41, producing effects which were additive with those of rolipram and denbufylline but not with those of BRL 61063. The secretory responses to forskolin (100 microM) were accompanied by a highly significant increase in the cyclic AMP content of the hypothalamic tissue (P < 0.005). Rolipram (10 microM) also significantly (P < 0.05) elevated the hypothalamic cyclic AMP but denbufylline (10 mM) and BRL 61063 (10 microM) did not. However, all three PDE-inhibitors potentiated the rise in cyclic AMP induced by forskolin (P < 0.05). None of the drugs tested, alone or in combination, modified the release of arginine vasopressin (ir-AVP) from the hypothalamus. 4. Rolipram (100 microM), denbufylline (100 microM) and BRL 61063 (100 microM) stimulated the release of corticotrophin (ir-ACTH) from pituitary tissue in vitro (P < 0.05) but in lower concentrations they were without significant effect. In addition, rolipram (10 microM, P < 0.05), denbufylline (0.1 microM, P < 0.05) and BRL 61063 (10 microM, P < 0.05) potentiated the significant (P < 0.05) rises in ir-ACTH secretion induced by forskolin (100 microM). Forskolin (100 microM) also produced a highly significant increase (P < 0.01) in the tissue cyclic AMP content which was further potentiated by rolipram (10 microM), denbufylline (10 microM) and BRL 61063 (10 microM) which, alone did not affect the cyclic AMP content of the tissue. 5. Since both denbufylline and BRL 61063 possess significant adenosine A1 receptor blocking activity, further studies examined the potential influence of these receptors on the secretion in vitro of CRH-41, AVP and ACTH. The release of ir-CRH-41 was increased significantly by adenosine deaminase (ADA, 5microml-1, P<0.05) and the A1-receptor antagonist, 1,3-dicyclopropyl-8-cyclopentylxanthine (DPCPX, 0.1-10nM, P<0.05). The responses to ADA were abolished by the A1 receptor agonist N6-cyclo-hexyladenosine (CHA, 100nM, P<0.05) which alone had no significant effect on ir-CRH-41 release. ADA (0.1-10microml-1) and DPCPX (1nM) had weak stimulant and inhibitory effects, respectively, on the release of ir-ACTH from the pituitary gland while CHA (0.1-10nM) was without effect. Ligand binding studies with [3H]-DPCPX as a probe demonstrated the presence of specific high affinity A1 binding sites in the hypothalamus (Kd=0.7nM; Bmax=367+/-32fmolmg-1 protein) and in the hippocampus (Kd=1nM; Bmax=1165 +/-145fmolmg-1 protein). In both tissues binding of the ligand was displaced by CHA (IC50=1nM (hypothalamus) and 2nM (hippocampus)), BRL 61063 (IC50=80nM (hypothalamus) and 100nM (hippocampus)) and denbufylline (IC50=5microM (hypothalamus) and 9microM(hippocampus)) but not by rolipram. 6.The results suggest that rolipram, denblufylline and BRL 61063 stimulate the HPA axis in the rat, acting at the levels of both the hypothalamus and the pituitary gland. Their actions may be explained, at least in part, by inhibition of PDE-4 but additional actions including blockade of hypothalamic adenosine A1 receptors by denbufylline and BRL 61063 cannot be excluded.     

Antimicrobial susceptibility testing of 59 strains of Campylobacter fetus subsp. fetus

The susceptibilities of 59 Campylobacter fetus subsp. fetus isolates to eight antibiotics were studied by the agar dilution, E-test, and disk diffusion methods. None of the isolates were beta-lactamase producers. All were susceptible to ampicillin, gentamicin, imipenem, and meropenem as determined by the three methods, with MICs at which 90% of the isolates are inhibited (MIC90s) (determined by agar dilution) of 2, 1, < or = 0.06, and 0.12 microgram/ml, respectively. Twenty-seven percent of the isolates were resistant to tetracycline, with complete agreement between the agar dilution and disk diffusion results. The MIC90s determined by agar dilution were 2 micrograms/ml for erythromycin, 1 microgram/ml for ciprofloxacin, and 8 micrograms/ml for cefotaxime.     

Resistance of bovine and porcine Pasteurella to florfenicol and other antibiotics

The resistance pattern of 221 (89 bovine, 132 porcine) pasteurella strains isolated in 1996 against 16 antibiotics or chemotherapeutics was determined by agar diffusion. Pasteurella haemolytica showed a higher level of resistance compared to Pasteurella multocida; porcine strains were more resistant than bovine strains. Over 90% of porcine Pasteurella multocida were sensible to penicillin G, ampicillin, cephalothin, polymyxin B, enrofloxacin, chloramphenicol and florfenicol. In addition, bovine strains were at least 90% sensible to oxacillin, erythromycin, gentamycin and sulfmethoxazole-trimethoprim. More than 90% of porcine Pasteurella haemolytica were classified as sensible to polymyxin B, enrofloxacin und florfenicol; bovine strains to cephalothin, neomycin und chloramphenicol as well. In 1996, 2 years after the chloramphenicol ban for food rendering animals, only 6.3% of bovine pasteurella strains proved to be resistant against chloramphenicol compared to a 16.27% fraction in 1994. The mean MIC-values of florfenicol against pasteurella spp. were nearly the same in bovine and porcine isolates with 0.53 microgram/ml and 0.52 microgram/ml respectively. Pasteurella haemolytica, however, showed higher MIC-values (0.68 microgram/ml in bovine, 0.70 microgram/ml in porcine isolates) than Pasteurella multocida with 0.47 microgram/ml in bovine and 0.51 microgram/ml in porcine strains. No isolate had a MIC of florfenicol greater than 1.0 microgram/ml, all pasteurella strains were classified sensible to florfenicol.     

Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (1996)

The bacteria isolated from the patients with lower respiratory tract infections were collected by institutions located throughout Japan, since 1981. Ikemoto et al. have been investigating susceptibilities of these isolates to various antibacterial agents and antibiotics, and characteristics of the patients and isolates from them each year. Results obtained from these investigations are discussed. In 16 institutions around the entire Japan, 557 strains of presumably etiological bacteria were isolated mainly from the sputa of 449 patients with lower respiratory tract infections during the period from October 1996 to September 1997. MICs of various antibacterial agents and antibiotics were determined against 98 strains of Staphylococcus aureus, 93 strains of Streptococcus pneumoniae, 84 strains of Haemophilus influenzae, 84 strains of Pseudomonas aeruginosa (non-mucoid strains), 17 strains of Pseudomonas aeruginosa (mucoid strains), 31 strains of Moraxella subgenus Branhamella catarrhalis, 21 strains of Klebsiella pneumoniae etc., and the drug susceptibilities of these strains were assessed except for those strains that died during transportation. 1) S. aureus S. aureus strains for which MICs of oxacillin (MPIPC) were higher than 4 micrograms/ml (methicillin-resistant S. aureus) accounted for 67.3%. The frequency of the drug resistant bacteria increased comparing to the previous year's 52.7%. Arbekacin (ABK) and vancomycin (VCM) showed the highest activities against both S. aureus and MRSA with MIC80s of 1 microgram/ml. 2) S. pneumoniae Imipenem (IPM) and panipenem (PAPM) of carbapenems showed the most potent activities with MIC80s of 0.063 microgram/ml. Faropenem (FRPM) showed the next potent activity with MIC80 of 0.125 microgram/ml. The other drugs except erythromycin (EM), clindamycin (CLDM) and tetracycline (TC) were active against S. pneumoniae tested with MIC80s of 8 micrograms/ml or below. 3) H. influenzae The activities of all drugs were potent against H. influenzae tested with MIC80s of 4 micrograms/ml or below. Cefotiam (CTM), cefmenoxime (CMX), cefditoren (CDTR) and ofloxacin (OFLX) showed the most potent activities with MIC80s of 0.063 microgram/ml. 4) P. aeruginosa (mucoid strains) Tobramycin (TOB) showed the most potent activity against P. aeruginosa (mucoid strains) with MIC80 of 1 microgram/ml. Ceftazidime (CAZ), cefsulodin (CFS), IPM, gentamicin (GM), ABK and ciprofloxacin (CPFX) showed the next potent activities, with MIC80s of 2 micrograms/ml. The MIC80s of the other drugs ranged from 4 micrograms/ml to 16 micrograms/ml. 5) P. aeruginosa (non-mucoid strains) TOB and CPFX showed the most potent activities against P. aeruginosa (non-mucoid strains) with MIC80s of 1 microgram/ml. The MIC80s of piperacillin (PIPC) and cefoperazone (CPZ) were 16 micrograms/ml in 1995, and they were 64 micrograms/ml in 1996. 6) K. pneumoniae All drugs except ampicillin (ABPC) were active against K. pneumoniae. CMX, cefpirome (CPR), cefozopran (CZOP) and carumonam (CRMN) showed the most potent activities against K. pneumoniae with MIC80s of 0.125 microgram/ml. The MIC80s of the other drugs ranged from 0.25 microgram/ml to 2 micrograms/ml. 7) M.(B) catarrhalis Against M.(B.) catarrhalis, all drugs showed good activities with MICs of 4 micrograms/ml or below. IPM and minocycline (MINO) showed the most potent activities with MICs of 0.063 microgram/ml. Also, we investigated year to year changes in the characteristics of patients, their respiratory infectious diseases, and the etiology. Patients' backgrounds were examined for 557 isolates from 449 cases. The examination of age distribution indicated that the proportion of patients with ages over 60 years was 71.0% of all the patients showing a slight increase over that in 1994. Proportions of diagnosed diseases were as follows: Bacterial pneumonia and chronic bronchitis were the most frequent with 35.9% and 30.3% respectively. They were followed by bronchiectasis with a proportion of 10.     

Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (1995). I. Susceptibility distribution]

The frequencies of isolation and susceptibilities to antimicrobial agents were investigated on 704 bacterial strains isolated from patients with urinary tract infections (UTIs) in 11 hospitals during the period of June 1995 to May 1996. Of the above bacterial isolates, Gram-positive bacteria accounted for 29.8% and a majority of them were Enterococcus faecalis. Gram-negative bacteria accounted for 70.2% and most of them were Escherichia coli. Susceptibilities of several isolated bacteria to antimicrobial agents were as followed; 1. Enterococcus faecalis Ampicillin (ABPC) and imipenem (IPM) showed the highest activities against E. faecalis isolated from patients with UTIs. The MIC90S of them were 1 microgram/ml. Vancomycin (VCM) and piperacillin (PIPC) were also active with the MIC90S of 2 micrograms/ml and 4 micrograms/ml, respectively. The others had low activities with the MIC90S of 16 micrograms/ml or above. 2. Staphylococcus aureus including MRSA VCM showed the highest activities against S. aureus isolated from patients with UTIs. Its MIC90 was 1 microgram/ml against both S. aureus and MRSA. Arbekacin (ABK) was also active with the MIC90 of 2 micrograms/ml. The other except minocycline (MINO) had very low activities with the MIC90S of 64 micrograms/ml or above. 3. Staphylococcus epidermidis ABK and MINO showed the strongest activities against S. epidermidis isolated from patients with UTIs. The MIC90S of them were 0.25 microgram/ml. VCM was also active with the MIC90 of 1 microgram/ml. The MIC90S of cephems ranged from 2 micrograms/ml to 16 micrograms/ml in 1994, but they ranged from 8 micrograms/ml to 128 micrograms/ml in 1995. These results indicated that some resistances existed among S. epidermidis to cephems. 4. Streptococcus agalactiae All drugs except gentamicin (GM) were active against S. agalactiae. ABPC, cefmenoxime (CMX), IPM, erythromycin (EM), clindamycin (CLDM) and clarithromycin (CAM) showed the highest activities. The MICs for all strains were lower than 0.125 microgram/ml. The MIC90S of the others were 2 micrograms/ml or below. 5. Citrobacter freundii IPM showed the highest activity against C. freundii isolated from patients UTIs. Its MIC90 was 1 microgram/ml. GM was also active with the MIC90 of 2 micrograms/ml. Cefpirome (CPR), cefozopran (CZOP) and amikacin (AMK) were also active with the MIC90S of 4 micrograms/ml. Penicillins and cephems except CMX, CPR and CZOP showed low activities with MIC90S of 256 micrograms/ml or above. 6. Enterobacter cloacae IPM showed the highest activity against E. cloacae. The MICs for all strains were equal to or lower than 1 microgram/ml. MINO and tosufloxacin (TFLX) were also active with the MIC90S of 8 micrograms/ml. Penicillins and cephems except CPR and CZOP showed lower activities with the MIC90S of 256 micrograms/ml or above. 7. Escherichia coli. Most of the antimicrobial agents were active against E. coli. Particularly CPR, CZOP and IPM showed the highest activities against E. coli. The MICs for all strains were equal to or lower than 0.5 microgram/ml. CMX and TFLX were also active with the MIC90S of 0.125 microgram/ml or below. Penicillins were slightly active with MIC90S of 128 micrograms/ml or above. 8. Klebsiella pneumoniae K. pneumoniae was susceptible to all drugs except penicillins, with MIC90S of 2 micrograms/ml or below. Carumonam (CRMN) had the strongest activity against K. pneumoniae, the MICs for all strains were equal to or lower than 0.125 microgram/ml. Comparing with the result of 1994, the sensitivities of K. pneumoniae against all drugs had obviously changed into a better state. For example, the MIC90S of cephems ranged from 0.25 microgram/ml to 16 micrograms/ml in 1994, but they were all lower than 2 micrograms/ml in 1995. 9. Proteus mirabilis P. mirabilis was susceptible to a majority of drugs. CMX, ceftazidime (CAZ), cefixime (CFIX), and CRMN showed the highest activities against P. mirabilis isolated from patients with UTIs. MICs of CRMN for all     

The N-7-substituted acyclic nucleoside analog 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine is a potent and selective inhibitor of herpesvirus replication

2-Amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) represents the first antivirally active nucleoside analog with the side chain attached to the N-7 position of the purine ring. Compound S2242 strongly inhibits the in vitro replication of both herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) (50% effective concentration [EC50], 0.1 to 0.2 microgram/ml), varicella-zoster virus (EC50, 0.01 to 0.02 microgram/ml) and thymidine kinase (TK)-deficient strains of HSV (EC50, 0.4 microgram/ml) and varicella-zoster virus (EC50, 0.2 to 0.5 microgram/ml). Potent activity was also observed against murine cytomegalovirus (EC50, 1 microgram/ml), human cytomegalovirus (HCMV) (EC50, 0.04 to 0.1 microgram/ml), and human herpesvirus 6 (EC50, 0.0005 microgram/ml). Compound S2242 (i) was not cytotoxic to confluent Vero, HeLa, or human fibroblast cells at concentrations of > 100 micrograms/ml, (ii) proved somewhat more cytostatic to Vero, HEL, HeLa, and C127I cells than ganciclovir, and (iii) was markedly more cytostatic than ganciclovir to the growth of the human lymphocytic cell lines HSB-2 and CEM degrees. In contrast to ganciclovir, (i) compound S2242 proved not to be cytocidal to murine mammary carcinoma (FM3A) cells transfected with the HSV-1 or HSV-2 TK gene, (ii) exogenously added thymidine had only a limited effect on its anti-HSV-1 activity, and (iii) the compound was not phosphorylated by HSV-1-encoded TK derived from HSV-1 TK-transfected FM3A cells, indicating that the compound is not activated by a virally encoded TK. Compound S2242 inhibited (i) the expression of late HCHV antigens at an EC50 of 0.07 microgram/ml (0.6 microgram/ml for ganciclovir) and (ii) HCMV DNA synthesis at an EC50 of 0.1 microgram/ml (0.32 microgram/ml for ganciclovir), i.e., values that are close to the EC50S for inhibition of HCMV-induced cytopathogenicity. Neither ganciclovir nor S2242 had any effect on the expression of immediate-early HCMV antigens, which occurs before viral DNA synthesis. In time-of-addition experiments, S2242 behaved like ganciclovir and acyclovir; i.e., the addition of the drugs could be delayed until the onset of viral DNA synthesis.     

Antiovulatory antagonists of LHRH related to antide

We report 104 analogues of the potent antiovulatory antagonist of LHRH, N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Nic)-D-Lys(Nic)-Leu-Ilys-Pro-D-Ala- NH2, Antide. We replaced the Nic group in Antide with other acyl substituents to modulate size, hydrophilicity or basicity of the molecule, we also replaced the Lys residues with shorter basic amino acids, and made cyclic 5/6 analogues as well as position 5 or 6 dimers. We substituted Ilys8 with other alkyl groups and acyl derivatives. When injected in 0.1% DMSO in water in a typical antiovulatory (AO) assay. Antide gives six rats ovulating out of eight (6/8) at 2 micrograms, 4/8 at 4 micrograms, and in the histamine release assay (HRA). ED50 is > 300 micrograms/ml; [Lys(N-Isobutyl)8]Antide gave 2/8 at 2 micrograms/rat; [Lys (8-Qis)5]Antide gave 1/8 at 1 microgram, and 0/8 at 2 micrograms, and in the HRA ED50. 22 micrograms/ml; [D-Lys(8-Qis)5]Antide gave 4/8 at 1 microgram and 0/8 at 2 micrograms, and in the HRA, ED50 was 27 micrograms/ml; [Lys(8-Qic)8] gave 5/8 at 1 microgram 1/8 at 2 micrograms/ [Lys(2-Pyc)6]Antide gave 3/8 at 1 microgram, and 0/8 at 2 micrograms, and in the HRA ED50 was 116 micrograms/ml; [D-Lys (2-Pyc)5]Antide gave 5/8 at 1 microgram and in the HRA, ED50 was 100- > 300 micrograms/ml; [Lys(2-Pyc)5.D-Lys(2-Pyc)6]Antide gave 2/8 at 1 microgram. The substitutions of the Nic groups of Antide at Lys5 or D-Lys6 with 8-Qis or with 2-Pyc groups seem to give highly potent antiovulatory antagonists of LHRH and constitute significant new leads to generate potent antiovulatory compounds endowed with moderate or low histamine release.     

Antifungal susceptibility of clinically isolated Candida albicans by broth microdilution method

In this study we investigated the antifungal susceptibility of 285 strains of Candida albicans isolates at Kinki University Hospital from March 1995 to December 1996. The antifungal agents tested were fluconazole, miconazole, intraconazole, amphotericin B and flucytosine. The susceptibility testing were performed according to the broth microdilution method standardized by National Committee for Clinical Laboratory Standards (M27-T). Most isolates of C. albicans showed relatively a low MIC value and the MIC90S were calculated at 1 microgram/ml; fluconazole, 0.125 microgram/mg; miconazole, 0.06 microgram/ml; itraconazole, 1 microgram/ml; amphotericin B, 0.25 microgram/ml; flucytosine. There was only one strain that showed high resistance against fluconazole and it showed cross-resistance against miconazole and itraconazole. There were two flucytosine resistant strains. The MICs of amphotericin B were tightly clustered and resistant strain were not observed.     

Application of pbp1A PCR in identification of penicillin-resistant Streptococcus pneumoniae

A seminested PCR assay, based on the amplification of the pneumococcal pbp1A gene, was developed for the detection of penicillin resistance in clinical isolates of Streptococcus pneumoniae. The assay was able to differentiate between intermediate (MICs = 0.25 to 0.5 microgram/ml) and higher-level (MICs = >/=1 microgram/ml) resistance. Two species-specific primers, 1A-1 and 1A-2, which amplified a 1,043-bp region of the pbp1A penicillin-binding region, were used for pneumococcal detection. Two resistance primers, 1A-R1 and 1A-R2, were designed to bind to altered areas of the pbp1A gene which, together with the downstream primer 1A-2, amplify DNA from isolates with penicillin MICs of >/=0.25 and >/=1 microgram/ml, respectively. A total of 183 clinical isolates were tested with the pbp1A assay. For 98.3% (180 of 183) of these isolates, the PCR results obtained were in agreement with the MIC data. The positive and negative predictive values of the assay were 100 and 91%, respectively, for detecting strains for which the MICs were >/=0.25 microgram/ml and were both 100% for strains for which the MICs were >/=1 microgram/ml.     

Sesquiterpene lactones. Part XV. New cytostatic active sesquiterpene lactone from herb of Anthemis nobilis L

From fresh and dried herb (without inflorescences) of Anthemis nobilis L. a new sesquiterpene lactone C20H26O6 was isolated. This compound showed a cytotoxic activity at a level of ED50 HeLa -- 0-56 microgram/ml (1-5 x 10(-6) M), and ED50 KB -- 1.23 microgram/ml (3-5 x 10(-6) M), what qualifies it for further in vivo studies.     

A possible mechanism for initiation of lipid peroxidation by ascorbate in rat liver microsomes

RRR-alpha-tocopheryl succinate (VES) was studied for effects on murine EL-4 cell proliferation and production of interleukin-2 (IL-2) and transforming growth factor-beta (TGF-beta). VES was biphasic in its actions: 0.1 microgram/ml enhanced EL-4 cell proliferation, whereas 10-20 microgram/ml inhibited cellular proliferation. Cell-conditioned media (CM) from EL-4 cells treated with 0.2 ng/ml phorbol myristate acetate (PMA) + 0.1 microgram/ml VES contained increased amounts of IL-2, as determined by the murine cytotoxic T cell IL-2-dependent CTLL-2 bioassay. VES at 0.1 microgram/ml or 0.1 microgram/ml VES + 0.2 ng/ml PMA induced the expression of IL-2 mRNA by EL-4 cells three to nine hours after treatment. CM from EL-4 cells treated with VES at 10-20 microgram/ml exhibited potent antiproliferative activity when tested in the TGF-beta-responsive mink lung cell (Mv1Lu) bioassay and showed reduced inhibitory effects when tested on TGF-beta receptor-negative mink lung (DRA-27) cells. CM from control-treated EL-4 cells exhibited no antiproliferative activity. The VES-induced antiproliferative activity was characterized as TGF-beta by neutralization analyses and immunoprecipitation of metabolically labeled proteins with TGF-beta-specific reagents. VES treatment of EL-4 cells had no effect on TGF-beta 1 mRNA expression while downregulating TGF-beta 3 mRNA expression. In summary, these studies showed that 0.1 microgram/ml VES enhanced cellular proliferation, in part, via increased IL-2 production, whereas 10-20 micrograms/ml VES inhibited cellular proliferation, in part, via the secretion of biologically active TGF-beta.     

Clinical pharmacokinetic and pharmacodynamic studies with the nonclassical antifolate thymidylate synthase inhibitor 3, 4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolone dihydrochloride (AG337) given by 24-hour continuous intravenous infusion

3,4-Dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolon e dihydrochloride (AG337) is a nonclassical inhibitor of thymidylate synthase (TS) designed to avoid potential resistance mechanisms that can limit the activity of classical antifolate antimetabolites. A clinical pharmacokinetic and pharmacodynamic study of AG337 given as a 24-h i.v. infusion was performed. Thirteen patients received 27 courses over the dose range 75-1350 mg/m2. Plasma AG337 concentrations were achieved which, in preclinical models, were associated with antitumor effects. AG337 clearance was saturable, and the pharmacokinetics of the drug at doses above 300 mg/m2 was best described by a one-compartment model with saturable elimination (median Km = 6.5 microgram/ml; range, 4.1-13 microgram/ml; median Vmax = 2.0 microgram/ml/h/m2; range, 0.96-5.6 microgram/ml/h/m2). Following the end of the infusion, AG337 was cleared rapidly (t1/2, 53-193 min), and levels were less than 0.2 microgram/ml in all patients by 48 h. Plasma protein binding was 96-98%, and the urinary excretion of AG337 as unchanged drug did not exceed 30% of the dose administered. Measurements of plasma deoxyuridine (dUrd) concentrations showed that doses of 600 mg/m2 and above of AG337 produced a consistent elevation in plasma dUrd levels (60-290%), suggesting that TS inhibition was being achieved in patients. However, in all cases dUrd concentrations had returned to pretreatment levels 24 h after the end of the infusion, suggesting that TS inhibition was not maintained. Local toxicity, probably due to the infusate pH, was the only significant adverse effect observed. These studies have shown that cytotoxic AG337 plasma concentrations can be readily achieved without acute toxicity and that these concentrations are associated with elevations in plasma dUrd levels. The lack of prolonged dUrd elevations indicates that extended administration should be explored using central line or p.o. administration to avoid local toxicity.     

Comparative studies on activities of antimicrobial agents against causative organisms isolated from urinary tract infections (1993). I. Susceptibility distribution

The frequencies of isolation and susceptibilities to antimicrobial agents were investigated on 657 bacterial strains isolated from patients with urinary tract infections in 10 hospitals during the period of June 1993 to May 1994. Of the above total bacterial isolates, Gram-positive bacteria accounted for 28.3% and a majority of them were Enterococcus faecalis. Gram-negative bacteria accounted for 71.7% and most of them were Escherichia coli. 1. Enterococcus faecalis Ampicillin (ABPC), imipenem (IPM) and vancomycin (VCM) showed the highest activities against E. faecalis isolated from patients with urinary tract infections. The MIC90s of them were 2 micrograms/ml. Piperacillin (PIPC) was also active with the MIC90 of 8 micrograms/ml. The others were not so active with the MIC90s of 32 micrograms/ml or above. 2. Staphylococcus aureus including MRSA VCM showed the highest activities against S. aureus isolated from patients with urinary tract infections. Its MIC90 was 1 microgram/ml. Arbekacin (ABK) was also active with the MIC90 of 2 micrograms/ml. The others were not so active with the MIC90s of 32 micrograms/ml or above. 3. Staphylococcus epidermidis VCM showed the strongest activity against S. epidermidis isolated from patients with urinary tract infections. Its MIC90 was 1 microgram/ml. ABK was also active with the MIC90 of 4 micrograms/ml. The others except ABPC were not so active with the MIC90s of 32 micrograms/ml or above. 4. Streptococcus agalactiae Most of the agents were active against S. agalactiae isolated from patients with urinary tract infections. Penicillins, cephems, erythromycin (EM), and clindamycin (CLDM) showed the highest activities. The MIC90s of them were 0.25 microgram/ml or below. Amikacin (AMK) and minocycline (MINO) showed somewhat low activities with the MIC90s of 16 micrograms/ml. 5. Citrobacter freundii IPM showed the highest activities against C. freundii isolated from patients with urinary tract infections. Its MIC90 was 2 micrograms/ml. Cefozopran (CZOP) and gentamicin (GM) were also active with the MIC90s of 8 micrograms/ml. Penicillins and cephems generally were not so active. 6. Enterobacter cloacae IPM and GM showed the highest activities against E. cloacae. The MIC90s of them were 1 microgram/ml. CZOP and tosufloxacin (TFLX) were also active with the MIC90s of 8 micrograms/ml. Penicillins and cephems except CZOP showed lower activities with the MIC90s of 64 micrograms/ml or above. 7. Escherichia coli Most of antimicrobial agents were active against E. coli. Flomoxef (FMOX), CZOP, IPM, CPFX and TFLX showed the highest activities against E. coli. The MIC90s of them were 0.125 microgram/ml or below. Cefmenoxime (CMX), ceftazidime (CAZ), cefuzonam (CZON), latamoxef (LMOX), carumonam (CRMN), norfloxacin (NFLX) and ofloxacin (OFLX) were also active with the MIC90s of 0.25 microgram/ml. Penicillins and MINO were not so active with the MIC90s of 32 micrograms/ml or above. 8. Klebsiella pneumoniae CZOP, IPM and CRMN showed the highest activities against K. pneumoniae. The MIC90s of them were 0.125 microgram/ml or below. CAZ, CZON, CFIX, CPFX and TFLX were also active the MIC90s of 0.25 microgram/ml. Penicillins were not so active with the MIC90s of 128 micrograms/ml or above. 9. Proteus mirabilis P. mirabilis was susceptible to a majority of drugs. CMX, CAZ, CZON, LMOX, CFIX, CRMN and CPFX showed the highest activities against P. mirabilis isolated from patients with urinary tract infections. The MIC90s of them were 0.125 microgram/ml or below. MINO was not so active with the MIC90 of 256 micrograms/ml or above. 10. Pseudomonas aeruginosa Most of the agents were not so active against P. aeruginosa. IPM showed MIC90 of 8 micrograms/ml.     

Effects of macrophage colony-stimulating factor (M-CSF) on lipopolysaccharide (LPS)-induced mediator production from monocytes in vitro

M-CSF is a macrophage-lineage-specific growth factor that causes proliferation and differentiation of progenitor cells in the bone marrow. To investigate the effects of M-CSF on more matured cells, human monocytes were cultured in the presence or absence of M-CSF for 6 days. Addition of M-CSF at more than 10(2) U/ml resulted in higher viability and caused morphological differentiation to large macrophage-like cells. LPS-induced mediator production was also compared between M-CSF-treated and control cell. Monocytes were incubated with or without M-CSF for 3 days, and were stimulated with 1 microgram/ml of LPS for 2 days. IL-1 beta was not detected in the both culture supernatants, and PGE2 production was not influenced by M-CSF. However, amounts of G-CSF, GM-CSF, IL-6, and TNF-alpha produced in response to 1 microgram/ml of LPS were 1.5 to 2 times greater from monocytes treated with 10(4) U/ml of M-CSF than from control cells. The priming effect of M-CSF on LPS-induced cytokine production was found to require 3-day preincubation, and reached a maximum at the concentration of 10(4) U/ml. M-CSF-treated cells responded to a 10 times lower concentration of LPS than control cells in terms of cytokine production. M-CSF was also shown by flowcytometric analysis to influence the expression of CD14, a receptor for LPS, which might render monocytes more sensitive to LPS.     

Characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-N-acetyl glucosamine-based polymer matrix

Identification of tumor-associated antigens (TAAs) and their class I MHC-restricted epitopes now allows for the rational design of peptide-based cancer vaccines. A biocompatible system capable of sustained release of biologically relevant levels of cytokine and TAA peptide could provide a more effective microenvironment for antigen presentation. Our goal was to test a sustained-release cytokine/TAA peptide-based formulation using a highly purified polysaccharide [poly-N-acetyl glucosamine (p-GlcNAc)] polymer. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 microgram) and MART-1(27-35) peptide (128 microgram in DMSO) were formulated into p-GlcNAc. Peptide release was assayed in vitro using interleukin 2 production from previously characterized MART-1(27-35)-specific Jurkat T cells (JRT22). GM-CSF release was assayed via ELISA and proliferation of M-07e (GM-CSF-dependent) cells. Local bioavailability of MART-1(27-35) peptide for uptake and presentation by antigen-presenting cells was demonstrated for up to 6 days (>0.5 microgram/ml). More than 1.0 microgram/ml GM-CSF was concomitantly released over the same period. Biocompatibility and local tissue response to p-GlcNAc releasing murine GM-CSF was determined in C57BL/6 mice via s.c. injection using murine GM-CSF (0. 2 microgram/ml) in 200 microliter of a 2.5% polymer gel. Significant lymphocytic and eosinophilic infiltration was observed 2-7 days after injection with polymer containing murine GM-CSF. The results of our studies show that this biocompatible system is capable of a sustained concomitant release of biologically active peptide and cytokine into the local microenvironment. These findings support further studies to validate a p-GlcNAc delivery system vehicle for a cytokine/TAA peptide-based cancer vaccine.