Use of adhesion counts to help predict symptomatic infection and the ability of fluoroquinolones to penetrate bacterial biofilms on the bladder cells of spinal cord injured patients

There were three objectives to the present study: (1) compare the bladder infection rate and extent of biofilm formation for seven untreated spinal cord injured (SCI) patients and seven given prophylactic co-trimoxazole, (2) identify a level of bacterial adhesion to bladder cells which could be used to help predict symptomatic infection, and (3) determine from in vivo and in vitro studies whether fluoroquinolones were effective at penetrating bacterial biofilms. The results showed that the infection rate had not changed with the introduction of prophylaxis. However, the uropathogenic population had altered subsequent to the introduction of prophylaxis with E. coli being replaced by E. faecalis as the most common cause of infection. In 63% of the specimens from asymptomatic patients, the bacterial counts per cell were <20, while 81% of specimens from patients with at least one sign and one symptom of urinary tract infection (UTI) had > 20 adherent bacteria per bladder cell. Therefore, it is proposed that counts of > 20 bacteria adherent to sediment transitional epithelial bladder cells may be predictive of symptomatic UTI. Clinical data showed that fluoroquinolone therapy reduced the adhesion counts to <20 per cell in 63% of cases, while trimethoprim-sulfamethoxazole only did so in 44%. Further in vitro testing showed that ciprofloxacin (0.1, 0.5 and 1.0 micrograms/ml) partially or completely eradicated adherent biofilms from 92% of spinal cord injured patients' bladder cells, while ofloxacin did so in 71% cases and norfloxacin in 56%. These findings have important implications for the detection and treatment of bacteriuria in spinal cord injured patients.

Antimicrobic susceptibility and plasmid profile analysis as identity tests for multiple blood isolates of coagulase-negative staphylococci

We compared a disk diffusion antimicrobic susceptibility panel with plasmid DNA profiles as tests for identity of 106 isolates of coagulase-negative staphylococci cultured from the blood of 45 patients on multiple occasions. The antimicrobic panel included penicillin, oxacillin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, tobramycin, kanamycin, and gentamicin. Nineteen patterns of antimicrobic susceptibility were found. The most common pattern was present in 25% of the isolates, and at least one isolate from 31% of the patients had this pattern. Forty-seven distinct plasmid DNA profiles were found. The most common plasmid profile was present in 8.5% of the isolates, and at least one isolate from 15% of the patients had this profile. Twenty-eight patients had multiple isolates that were identical by plasmid profile analysis. Twenty-seven (96%) of these patients had isolates that were also identical by antimicrobic susceptibility. Nineteen patients had multiple isolates that were different by plasmid profile analysis. In 18 (95%) of these patients, the isolates were also different by antimicrobic susceptibility. Although plasmid DNA profile analysis is a more discriminating tool, these data confirm that a selected disk diffusion antimicrobic susceptibility panel may be used to screen multiple blood isolates of coagulase-negative staphylococci for identity or differences.

Development of a rapid colorimetric time-kill assay for determining the in vitro activity of ceftazidime and tobramycin in combination against Pseudomonas aeruginosa

It is standard clinical practice to use a combination of two or more antimicrobial agents to treat an infection caused by Pseudonionas aeruginosa. The antibiotic combinations are usually selected empirically with methods to determine the antimicrobial effect of the combination such as the time-kill assay rarely used as they are time-consuming and labour intensive to perforin. Here, we report a modified time-kill assay, based on the reduction of the tetrazolium salt, 2,3-bis[2-methyloxy-4-nitro-5-sulfopheny1]-2H-tetrazolium-5-carboxanilide (XTT), that allows simple, inexpensive and more rapid determination of the in vitro activity of antibiotic combinations against P aeruginosa. The assay was used to determine the in vitro activity of ceftazidime and tobramycin in combination against P. aertiginosa isolates from cystic fibrosis patients and the results obtained compared with those from conventional viable count time-kill assays. There was good agreement in interpretation of results obtained by the XTT and conventional viable count assays, with similar growth curves apparent and the most effective concentration combinations determined by both methods identical for all isolates tested. The XTT assay clearly indicated whether an antibiotic combination had a synergistic, indifferent or antagonistic effect and could, therefore, provide a useful method for rapidly determining the activity of a large number of antibiotic combinations against clinical isolates. (C) 2004 Elsevier B.V. All rights reserved.

A split-combination approach to merging knowledge bases in possibilistic logic

In this paper, we propose an adaptive approach to merging possibilistic knowledge bases that deploys multiple operators instead of a single operator in the merging process. The merging approach consists of two steps: one is called the splitting step and the other is called the combination step. The splitting step splits each knowledge base into two subbases and then in the second step, different classes of subbases are combined using different operators. Our approach is applied to knowledge bases which are self-consistent and the result of merging is also a consistent knowledge base. Two operators are proposed based on two different splitting methods. Both operators result in a possibilistic knowledge base which contains more information than that obtained by the t-conorm (such as the maximum) based merging methods. In the flat case, one of the operators provides a good alternative to syntax-based merging operators in classical logic.

Epidermal Growth Factor Receptor Activity Determines Response of Colorectal Cancer Cells to Gefitinib Alone and in Combination with Chemotherapy.

Purpose: Up to now, there have been no established predictive markers for response to epidermal growth factor receptor (EGFR/HER1/erbB1) inhibitors alone and in combination with chemotherapy in colorectal cancer. To identify markers that predict response to EGFR-based chemotherapy regimens, we analyzed the response of human colorectal cancer cell lines to the EGFR-tyrosine kinase inhibitor, gefitinib (Iressa, AstraZeneca, Wilmington, DE), as a single agent and in combination with oxaliplatin and 5-fluorouracil (5-FU). Experimental Design: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays and analyzed by ANOVA. Apoptosis was measured by flow cytometry, poly(ADP-ribose) polymerase, and caspase 3 cleavage. EGFR protein phosphorylation was detected by Western blotting. Results: Cell lines displaying high constitutive EGFR phosphorylation (a surrogate marker for EGFR activity) were more sensitive to gefitinib. Furthermore, in cell lines exhibiting low constitutive EGFR phosphorylation, an antagonistic interaction between gefitinib and oxaliplatin was observed, whereas in cell lines with high basal EGFR phosphorylation, the interaction was synergistic. In addition, oxaliplatin treatment increased EGFR phosphorylation in those cell lines in which oxaliplatin and gefitinib were synergistic but down-regulated EGFR phosphorylation in those lines in which oxaliplatin and gefitinib were antagonistic. In contrast to oxaliplatin, 5-FU treatment increased EGFR phosphorylation in all cell lines and this correlated with synergistic decreases in cell viability when 5-FU was combined with gefitinib. Conclusions: These results suggest that phospho-EGFR levels determine the sensitivity of colorectal cancer cells to gefitinib alone and that chemotherapy-mediated changes in phospho-EGFR levels determine the nature of interaction between gefitinib and chemotherapy.

PAD combination therapy (PS-341/bortezomib, doxorubicin and dexamethasone) for previously untreated patients with multiple myeloma

Summary Bortezomib (formerly PS-341) has significant activity in patients with relapsed multiple myeloma (MM), its efficacy is increased with the addition of dexamethasone and it demonstrates synergy with doxorubicin, thus providing the rationale for combination therapy with bortezomib, doxorubicin and dexamethasone (PAD). Patients with untreated MM received four 21-d cycles of PAD, comprising bortezomib 1·3 mg/m2 on days 1, 4, 8 and 11, along with dexamethasone 40 mg on days 1–4, 8–11 and 15–18 during cycle 1 and days 1–4 during cycles 2–4. During days 1–4, patients also received 0, 4·5 or 9 mg/m2 of doxorubicin at dose levels 1, 2, and 3 respectively. Following peripheral blood stem cell (PBSC) collection, patients received high-dose melphalan (MEL200) with PBSC transplantation (PBSCT). After PAD induction alone, 20 of 21 patients (95%) achieved at least a partial response (PR), including complete response (CR) in five patients (24%). Twenty of 21 had PBSC mobilized, and 18 of 20 received MEL200/PBSCT. In an intention-to-treat analysis, response rates were: CR 43%, near CR 14%, very good PR 24%, PR 14% and stable disease 5%. PAD was effective, did not prejudice subsequent PBSC collection, and should be further evaluated in prospective randomized trials.

Survival of Swiss-Webster mouse cerebellar granule neurons is promoted by a combination of potassium channel blockers

Cultured cerebellar granule neurons (CGN) are commonly used to assess neurotoxicity, but are routinely maintained in supraphysiological (25 mM) extracellular K⁺ concentrations [K⁺]_o. We investigated the effect of potassium channel blockade on survival of CGN derived from Swiss-Webster mice in supraphysiological (25 mM) and physiological (5.6 mM) [K⁺]_o. CGN were cultured for 5 days in 25 mM K⁺, then in 5.6 mM K⁺ or 25 mM K⁺ (control). Viability, assayed 24 h later by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction and by lactate dehydrogenase (LDH) release, was ∼50% in 5.6 mM K⁺ versus 25 mM K⁺ (p < .001). Potassium channel blockers, 2 mM 4-aminopyridine (4-AP), 2 mM tetraethylammonium (TEA) or 1 mM Ba²⁺, individually afforded limited protection in 5.6 mM K⁺. However, survival in 5.6 mM K⁺ with a combination of 4-AP, TEA and Ba²⁺ was similar to survival in 25 mM K⁺ without blockers (p < .001 versus 5.6 mM K⁺ alone). CGN survival in 25 mM K⁺ was attenuated 25% by 2 μM nifedipine (p > .001), but nifedipine did not attenuate neuroprotection by K⁺ channel blockers. Together, these results suggest that the survival of CGN depends on the K⁺ permeability of the membrane rather than the activity of a particular type of K⁺ channel, and that the mechanism of neuroprotection by K⁺ channel blockers is different from that of elevated [K⁺]_o.

A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods.

The coronavirus main protease, Mpro, is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compounds that was performed to identify potential Mpro inhibitors revealed etacrynic acid tert-butylamide (6a) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the phenyl ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-butyl moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (6b) as a promising lead for nonpeptidic active-site-directed Mpro inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compound 6b showed a Ki value of 35.3 M. Since the novel lead compound does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compound 6b.