bluuebunny/crossref_metadata_2025 huggingface dataset:
SU‐EE‐A4‐01: Regional Change in Brain Perfusion, in Irradiated Normal Tissue: Correlation Study Between Perfusion MRI and Spatial Distribution of Radiation Dose Delivered
Authors: Spencer D , Igna C , Kay I , Chan A , Kiss Z , Frayne R ,   Alberta Radiosurgery Center, Calgary, AB, CA
Published: June 2005

Purpose: When irradiating the normal brain, one of the principal causes of complications is damage to the cerebral vasculature, particularly the micro‐vasculature. This is of particular concern when treating diseases which are slow‐growing or are not malignant. While there is some data relating loss of perfusion to radiation dose in other tissues such as lung, there is very little data for the brain. We intend to determine the relationship between the change due to irradiation in hemodynamic measures, such as relative regional Mean Transit Time (rrMTT) and relative regional Cerebral Blood Volume (rrCBV), and the radiation dose delivered to the normal brain tissue. Method and Materials: We acquired data in the form of perfusion weighted images (PWI) for two patients. We used a 3.0 T magnet at the Seaman Family MRI Centre at the Foothills Medical Centre and a single‐shot echo‐planar imaging (EPI) sequence following the injection of a paramagnetic contrast agent (Gd‐DTPA‐Magnevist; Berlex, Wayne, NJ). These images have been processed to yield rrCBV and rrMTT. The patients had previously been treated with surgery, but had received no chemotherapy. Results: Our preliminary results show that with a follow‐up time of 4 months after receiving approximately 5000 cGy/25 fractions, in normal brain tissue the rrMTT is reduced by approximately 2% (1.8 ± 0.5) and the rrCBV is reduced by almost 12% (11.8 ± 2.2). The tumor showed reductions in both rrMTT (1.2 ± 1.5)% and rrCBV (19.9 ± 3.5)%. It is expected that these changes will increase with longer term follow‐up. Conclusion: Perfusion Weighted MR Imaging can be used to assess the change in hemodynamic measures in the normal brain tissue after radiotherapy.
SU‐FF‐T‐111: Fast Nonlinear Optimization with Simple Bounds for IMRT Planning
Authors: Cheong K , Suh T , Romeijn H , Li J , Dempsey J
Published: June 2005

Purpose: The advent of image‐guided adaptive conformal radiotherapy demands the development of very efficient methods of intensity modulated radiation therapy (IMRT) fluence map optimization (FMO). To enable adaptive image guidance with daily IMRT optimization the ability to generate high quality treatment plans “on‐the‐fly” to correct for changes in patient anatomy during the course of radiotherapy is needed. To this end, we have tested the feasibility of employing analytic convex nonlinear objective functions in a FMO model with simple nonnegative bounds on beamlet fluence. Method and Materials: The nonlinear FMO model is based on previously studied voxel‐based dose‐penalty functions that were approximated in linear and quadratic programming models. Well‐known gradient and quasi‐Newton algorithms were employed along with an Armijo line search that requires sufficient objective function decrease. A simple projected‐direction interior‐point method was used to enforce fluence non‐negativity. The algorithms were implemented in an in‐house treatment‐planning system. We tested the following methods: steepest‐descent, BFGS, symmetric‐rank‐1, and combinations thereof. The algorithms were applied to 10 head‐and‐neck cases. The numbers of beamlets were between 900 and 2,100 with a 3‐mm isotropic dose grid. Results: The tested methods were all found to be at least as fast as a commercial linear and quadratic programming solver (CPLEX, ILog, Inc.). While no single method was best for all cases, the steepest‐descent method typically converged the fastest. Conclusion: Nonlinear optimization methods can efficiently solve the IMRT FMO problem in well under a minute with high quality for clinical cases. A simple steepest‐descent algorithm was highly successful when combined with methods to enforce simple bounds and to perform a robust line search. Quasi‐Newton methods have been successful in solving many large‐scale nonlinear programming problems, but provide little advantage for the IMRT FMO problem. In the future, we will test the algorithm's robustness with a larger population of cases.
MO‐A‐T‐6B‐01: Proton Beam Radiotherapy — The State of the Art
Authors: Bortfeld T , Paganetti H , Kooy H
Published: June 2005

The number of proton therapy centers has doubled every 10 years since the beginning of proton therapy in 1954. Today there are 22 proton centers in clinical operation worldwide. In the past 5 years proton and heavy charged particle therapy have received a particular boost in Japan and in Europe, but also in the USA, where there will soon be 5 clinical proton therapy centers. The increased interest in and application of proton therapy motivates this continuing education course. We assume that the audience has a basic understanding of the principles of proton radiotherapy but we will refresh memories by giving a brief review of the principles. The main purpose of the course is to give an update of relatively recent developments in proton beam radiotherapy. The first part of the presentation will deal with the rationale of proton therapy. We will give a brief summary of the physical characteristics of proton‐tissue interactions leading to the typical Bragg peak. We will review recent clinical studies describing the application of protons to new treatment sites, and compare protons with other treatment modalities. The radio‐biological effectiveness (RBE) of proton therapy will be addressed next. In‐vivo and in‐vitro experiments to determine the RBE will be summarized. The proton RBE will also be compared to the RBE of carbon ions. Next, we will discuss technological aspects proton therapy, starting with beam shaping devices in the “nozzle”. We will review passive double scattering approaches such as the ones integrated in the NPTC and Loma Linda facilities. We will also review active pencil beam scanning techniques. The use of rotating gantries will be contrasted with fixed beamlines. We will then talk about cyclotrons vs. synchrotrons for proton acceleration. The clinical workflow will be presented for the Northeast Proton Therapy Center (NPTC) in Boston as an example. In this context, we will specifically address important questions of patient setup and immobilization. Recent studies on economical aspects and cost per treatment will be discussed as well. That protons beams stop in matter is their unique advantage (compared to conventional radiotherapy), but this also poses a challenge for proton treatment planning because the stopping position is somewhat uncertain. If possible, beam angles are selected that avoid stopping protons proximal to critical structures. In other cases “patch fields” are used. How protons are affected by organ motion and how the effects can be reduced will also be discussed. Finally, the potential of intensity‐modulated proton therapy in making treatment plans more conformal and more robust will be presented.

Educational objectives

1. To be able to put the physical and clinical potential of proton therapy and its cost into perspective, especially in comparison with photon IMRT.

2. To name the main technical components of a proton therapy system and the design alternatives.

3. To name the characteristics and at least 2 main advantages of intensity‐modulated proton therapy (IMPT)
Performance analysis of adaptive equalization for coherent acoustic communications in the time-varying ocean environment
Authors: Preisig James C.
Published: June 2005

Equations are derived for analyzing the performance of channel estimate based equalizers. The performance is characterized in terms of the mean squared soft decision error (σs2) of each equalizer. This error is decomposed into two components. These are the minimum achievable error (σo2) and the excess error (σε2). The former is the soft decision error that would be realized by the equalizer if the filter coefficient calculation were based upon perfect knowledge of the channel impulse response and statistics of the interfering noise field. The latter is the additional soft decision error that is realized due to errors in the estimates of these channel parameters. These expressions accurately predict the equalizer errors observed in the processing of experimental data by a channel estimate based decision feedback equalizer (DFE) and a passive time-reversal equalizer. Further expressions are presented that allow equalizer performance to be predicted given the scattering function of the acoustic channel. The analysis using these expressions yields insights into the features of surface scattering that most significantly impact equalizer performance in shallow water environments and motivates the implementation of a DFE that is robust with respect to channel estimation errors.
Extracting the frequencies of the pinna spectral notches in measured head related impulse responses
Authors: Raykar Vikas C. , Duraiswami Ramani , Yegnanarayana B.
Published: June 2005

The head related impulse response (HRIR) characterizes the auditory cues created by scattering of sound off a person’s anatomy. The experimentally measured HRIR depends on several factors such as reflections from body parts (torso, shoulder, and knees), head diffraction, and reflection/diffraction effects due to the pinna. Structural models (Algazi et al., 2002; Brown and Duda, 1998) seek to establish direct relationships between the features in the HRIR and the anatomy. While there is evidence that particular features in the HRIR can be explained by anthropometry, the creation of such models from experimental data is hampered by the fact that the extraction of the features in the HRIR is not automatic. One of the prominent features observed in the HRIR, and one that has been shown to be important for elevation perception, are the deep spectral notches attributed to the pinna. In this paper we propose a method to robustly extract the frequencies of the pinna spectral notches from the measured HRIR, distinguishing them from other confounding features. The method also extracts the resonances described by Shaw (1997). The techniques are applied to the publicly available CIPIC HRIR database (Algazi et al., 2001c). The extracted notch frequencies are related to the physical dimensions and shape of the pinna.
Enhancement of temporal periodicity cues in cochlear implants: Effects on prosodic perception and vowel identification
Authors: Green Tim , Faulkner Andrew , Rosen Stuart , Macherey Olivier
Published: June 2005

Standard continuous interleaved sampling processing, and a modified processing strategy designed to enhance temporal cues to voice pitch, were compared on tests of intonation perception, and vowel perception, both in implant users and in acoustic simulations. In standard processing, 400Hz low-pass envelopes modulated either pulse trains (implant users) or noise carriers (simulations). In the modified strategy, slow-rate envelope modulations, which convey dynamic spectral variation crucial for speech understanding, were extracted by low-pass filtering (32Hz). In addition, during voiced speech, higher-rate temporal modulation in each channel was provided by 100% amplitude-modulation by a sawtooth-like wave form whose periodicity followed the fundamental frequency (F0) of the input. Channel levels were determined by the product of the lower- and higher-rate modulation components. Both in acoustic simulations and in implant users, the ability to use intonation information to identify sentences as question or statement was significantly better with modified processing. However, while there was no difference in vowel recognition in the acoustic simulation, implant users performed worse with modified processing both in vowel recognition and in formant frequency discrimination. It appears that, while enhancing pitch perception, modified processing harmed the transmission of spectral information.
Effect of variability in level on forward masking and on increment detection
Authors: Jesteadt Walt , Schairer Kim S. , Neff Donna L.
Published: June 2005

In the first of four experiments, all with the same four subjects, varying the level of a forward masker from interval to interval in a two-interval forced-choice (2IFC) adaptive procedure had little effect on threshold. In the second experiment, the signal level was fixed and performance was measured in units of d′. Varying the level of the forward masker again had little effect. Analyses of trial-by-trial data indicated that subjects did not vote for the interval with the higher-level masker, as would an energy detector. Performance was better on trials where the masker level in the interval with the signal was lower and was relatively independent of masker level in the nonsignal interval. In the third experiment, these results were replicated for a wider range of masker variability and with maskers lower in frequency than the signal. In the fourth experiment, the same range of variability from interval to interval was imposed on the level of the pedestal in an increment-detection task. Results were similar to those observed in forward masking. The results suggest that decision processes involved in both forward masking and increment detection are similar and that neither is based on energy detection. Template matching remains a viable alternative.
Auditory brainstem responses in the Eastern Screech Owl: An estimate of auditory thresholds
Authors: Brittan-Powell Elizabeth F. , Lohr Bernard , Hahn D. Caldwell , Dooling Robert J.
Published: June 2005

The auditory brainstem response (ABR), a measure of neural synchrony, was used to estimate auditory sensitivity in the eastern screech owl (Megascops asio). The typical screech owl ABR waveform showed two to three prominent peaks occurring within 5ms of stimulus onset. As sound pressure levels increased, the ABR peak amplitude increased and latency decreased. With an increasing stimulus presentation rate, ABR peak amplitude decreased and latency increased. Generally, changes in the ABR waveform to stimulus intensity and repetition rate are consistent with the pattern found in several avian families. The ABR audiogram shows that screech owls hear best between 1.5 and 6.4kHz with the most acute sensitivity between 4–5.7kHz. The shape of the average screech owl ABR audiogram is similar to the shape of the behaviorally measured audiogram of the barn owl, except at the highest frequencies. Our data also show differences in overall auditory sensitivity between the color morphs of screech owls.
Sound propagation and energy relations in churches
Authors: Cirillo Ettore , Martellotta Francesco
Published: June 2005

The results of an acoustic survey carried out in a group of Italian churches differing in style, typology, and location were used in order to study how the acoustic energy varies inside this kind of space. The effect of different architectural elements on sound propagation was investigated by means of three-dimensional impulse responses measured using a B-format microphone with sweep signals. Side chapels, columns, and trussed roofs appeared to scatter the reflections, so that the purely diffuse exponential sound decay begins after a time interval which grows with the source–receiver distance and with the complexity of the church. The results of the measurements were then compared with predictions given by existing theoretical models to check their accuracy. In particular a model previously proposed by the authors for a specific type of Romanesque churches was further refined taking into account the new findings and making some simplifications. Its application to the wider sample of churches under analysis showed that strength, clarity, and center time can be predicted with reasonable accuracy.
Different Anti-
Candida
Activities of Two Human Lactoferrin-Derived Peptides, Lfpep and Kaliocin-1
Authors: Viejo-Díaz Mónica , Andrés María T. , Fierro José F.
Published: June 2005

The synthetic peptides Lfpep and kaliocin-1 include the sequences from positions 18 to 40 and 153 to 183 of human lactoferrin, respectively. Lfpep is a cationic peptide with bactericidal and giardicidal effects, whereas kaliocin-1 is a novel bactericidal peptide that corresponds to a highly homologous sequence present in the transferrin family of proteins. Both peptides presented fungicidal activity against
Candida
spp., including fluconazole- and amphotericin B-resistant clinical isolates. Lfpep exhibited higher antifungal activity (8- to 30-fold) and salt resistance than kaliocin-1. The killing activity of Lfpep was mediated by its permeabilizing activity on
Candida albicans
cells, whereas kaliocin-1 was unable to disrupt the cytoplasmic membrane, as indicated by its inability to allow permeation of propidium iodide and the small amount of K
+
released. The amino acid sequence of kaliocin-1 includes the “multidimensional antimicrobial signature” conserved in disulfide-containing antimicrobial peptides and a striking similarity to brevinin-1Sa, an antimicrobial peptide from frog skin secretions, exhibiting a “
Rana
box”-like sequence. These features may be of interest in the design of new antifungals.
Pharmacology and Pharmacokinetics of the Antiviral Agent β-d-2′,3′-Dideoxy-3′-Oxa-5-Fluorocytidine in Cells and Rhesus Monkeys
Authors: Hernandez-Santiago Brenda I. , Chen Huachun , Asif Ghazia , Beltran Thierry , Mao Shuli , Hurwitz Selwyn J. , Grier Jason , McClure Harold M. , Chu Chung K. , Liotta Dennis C. , Schinazi Raymond F.
Published: June 2005

β-d-2′,3′-Dideoxy-3′-oxa-5-fluorocytidine (d-FDOC) is an effective inhibitor of human immunodeficiency virus 1 (HIV-1) and HIV-2, simian immunodeficiency virus, and hepatitis B virus (HBV) in vitro. The purpose of this study was to evaluate the intracellular metabolism ofd-FDOC in human hepatoma (HepG2), human T-cell lymphoma (CEM), and primary human peripheral blood mononuclear (PBM) cells by using tritiated compound. By 24 h, the levels ofd-FDOC-triphosphate (d-FDOC-TP) were 2.8 ± 0.4, 6.7 ± 2.3, and 2.0 ± 0.1 pmol/106cells in HepG2, CEM, and primary human PBM cells, respectively. Intracellulard-FDOC-TP concentrations remained greater than the 50% inhibitory concentration for HIV-1 reverse transcriptase for up to 24 h after removal of the drug from cell cultures. In addition tod-FDOC-monophosphate (d-FDOC-MP), -diphosphate (d-FDOC-DP), and -TP,d-FDOC-DP-ethanolamine andd-FDOC-DP-choline were detected in all cell extracts as major intracellular metabolites.d-FDOC was not a substrate forEscherichia colithymidine phosphorylase. No toxicity was observed in mice givend-FDOC intraperitoneally for 6 days up to a dose of 100 mg/kg per day. Pharmacokinetic studies in rhesus monkeys indicated thatd-FDOC has at1/2of 2.1 h in plasma and an oral bioavailability of 38%. The nucleoside was excreted unchanged primary in the urine, and no metabolites were detected in plasma or urine. These results suggest that further safety and pharmacological studies are warranted to assess the potential of this nucleoside for the treatment of HIV- and HBV-infected individuals.
Proteus mirabilis
Bloodstream Infections: Risk Factors and Treatment Outcome Related to the Expression of Extended-Spectrum β-Lactamases
Authors: Endimiani Andrea , Luzzaro Francesco , Brigante Gioconda , Perilli Mariagrazia , Lombardi Gianluigi , Amicosante Gianfranco , Rossolini Gian Maria , Toniolo Antonio
Published: June 2005

Bloodstream infection (BSI) due to
Proteus mirabilis
strains is a relatively uncommon clinical entity, and its significance has received little attention. This study was initiated to evaluate risk factors and treatment outcome of BSI episodes due to
P. mirabilis
producing extended-spectrum β-lactamases (ESBLs). Twenty-five BSI episodes caused by
P. mirabilis
occurred at our hospital (Ospedale di Circolo e Fondazione Macchi, Varese, Italy) over a 7.5-year period. Phenotypic and molecular methods were used to assess ESBL production. Clinical records of BSI patients were examined retrospectively. Demographic data, underlying diseases (according to McCabe and Jackson classification and Charlson weighted index), risk factors, and treatment outcome were investigated by comparing cases due to ESBL-positive strains to cases due to ESBL-negative strains. Eleven isolates were found to express ESBLs (TEM-52 or TEM-92). The remaining 14 isolates were ESBL negative and were uniformly susceptible to extended-spectrum cephalosporins and monobactams. Comparison of the two groups showed that previous hospitalization in a nursing home (
P
= 0.04) and use of bladder catheter (
P
= 0.01) were significant risk factors for infections due to ESBL-positive strains. In addition, cases due to ESBL-positive strains showed a significantly higher mortality attributable to BSI (
P
= 0.04). BSI cases due to ESBL-negative isolates uniformly responded to therapy, whereas 5/11 cases due to ESBL-positive isolates failed to respond (
P
< 0.01). Use of carbapenems was associated with complete response independently of ESBL production. Therapeutic failure and mortality may occur in BSI episodes caused by ESBL-positive
P. mirabilis
isolates. Thus, recognition of ESBL-positive strains appears to be critical for the clinical management of patients with systemic
P. mirabilis
infections.
Sequential Actions of the Two Component Peptides of the Lantibiotic Lacticin 3147 Explain Its Antimicrobial Activity at Nanomolar Concentrations
Authors: Morgan Sheila M. , O'Connor Paula M. , Cotter Paul D. , Ross R. Paul , Hill Colin
Published: June 2005

Lacticin 3147 is a two-peptide (LtnA1 and LtnA2) lantibiotic produced by
Lactococcus lactis
subsp.
lactis
DPC3147 and has inhibitory activity against all gram-positive microorganisms tested. In this study the specific activities of the component peptides (alone or in combination) were determined by using
L. lactis
subsp.
cremoris
HP as the target strain. Lacticin 3147 exhibited an MIC
50
of 7 nM for each component peptide (in combination), suggesting a peptide stoichiometry of 1:1. Interestingly, the LtnA1 peptide demonstrated independent inhibitory activity, with an MIC
50
of 200 nM against
L. lactis
HP. In parallel studies, the single peptide bacteriocin nisin exhibited an MIC
50
of 50 nM against the same target strain. Sequential peptide addition (with an intermediate washing step) demonstrated that LtnA1 must be added before LtnA2 rather than vice versa to observe inhibitory activity. The nanomolar activity of the lacticin peptides suggests the involvement of a docking molecule, speculated to be lipid II. Taken together with the recently determined structure of lacticin 3147 (N. I. Martin, T. Sprules, M. R. Carpenter, P. D. Cotter, C. Hill, R. P. Ross, and J. C. Vederas, Biochemistry, 43:3049-3056, 2004), these data support the hypothesis that the mode of action for lacticin 3147 involves a lipid II binding step (by the mersacidin-like LtnA1 peptide, which would explain its independent inhibitory activity), followed by insertion of the more linear LtnA2 peptide into the target membrane, resulting in pore formation and ultimate cell death.
Microplate Alamar Blue Assay for
Staphylococcus epidermidis
Biofilm Susceptibility Testing
Authors: Pettit Robin K. , Weber Christine A. , Kean Melissa J. , Hoffmann Holger , Pettit George R. , Tan Rui , Franks Kelly S. , Horton Marilyn L.
Published: June 2005

Biofilms are at the root of many infections largely because they are much more antibiotic resistant than their planktonic counterparts. Antibiotics that target the biofilm phenotype are desperately needed, but there is still no standard method to assess biofilm drug susceptibility.
Staphylococcus epidermidis
ATCC 35984 biofilms treated with eight different approved antibiotics and five different experimental compounds were exposed to the oxidation reduction indicator Alamar blue for 60 min, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration was defined as ≤50% reduction and a purplish well 60 min after the addition of Alamar blue. All of the approved antibiotics had biofilm MICs (MBICs) of >512 μg/ml (most >4,096 μg/ml), and four of the experimental compounds had MBICs of ≤128 μg/ml. The experimental aaptamine derivative hystatin 3 was used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2
H
-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for
S. epidermidis
ATCC 35984, ATCC 12228, and two clinical isolates. For all four strains, Alamar blue results correlated well with XTT (
r
= 0.83 to 0.97) and with CFU/ml results (
r
= 0.85 to 0.94). Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. If the described method of microplate Alamar blue biofilm susceptibility testing, which is simple, reproducible, cost-effective, nontoxic, and amenable to high throughput, is applicable to other important biofilm forming species, it should greatly facilitate the discovery of biofilm specific agents.
Emergence of a Novel Mutation in the FLLA Region of Hepatitis B Virus during Lamivudine Therapy
Authors: Pai S. Balakrishna , Bozdayi A. Mithat , Pai Rekha B. , Beker Tolunay , Sarioglu Mustafa , Turkyilmaz Ahmet R. , Grier Jason , Yurdaydin Cihan , Schinazi Raymond F.
Published: June 2005

The emergence of resistance to lamivudine has been one of the major stumbling blocks to successful treatment and control of hepatitis B virus (HBV) infections. The major mechanism of resistance has been attributed to the alteration in the YMDD motif of the HBV polymerase due to an amino acid change of rtM204 to V/I and an accompanying rtL180M conversion. A novel mutation pattern in a patient having clinical breakthrough under lamivudine therapy was discovered. The mutant had a rtL180C/M204I genotype and was detected after 2 years of therapy with lamivudine. To characterize this novel variant, site-directed mutagenesis was performed using a vector construct containing the HBV genome. Transient transfection studies in human hepatoma cells with HBV carrying the new mutant demonstrated that the rtL180C/M204I mutant was resistant to lamivudine up to 10 μM. The resistance profile was comparable to that of the previously reported rtL180 M/M204I-containing virus. These observations were further confirmed by generation of stable cultures transfected with the mutant virus.
Putative VanRS-Like Two-Component Regulatory System Associated with the Inducible Glycopeptide Resistance Cluster of
Paenibacillus popilliae
Authors: Fraimow Henry , Knob Christopher , Herrero Inmaculada A. , Patel Robin
Published: June 2005

Paenibacillus popilliae
contains
vanF
encoding a putative
d
-Ala:
d
-lactate (
d
-Lac) ligase, VanF, as part of the
vanY

F

Z

F

H

F

FX

F

cluster that is similar in structure to the enterococcal
vanA
and
vanB
clusters. Using growth curves, we demonstrated that vancomycin resistance in
P. popilliae
is inducible. Using degenerate oligonucleotides targeted at bacterial cell wall ligases, we identified a second ligase gene with features of a
d
-Ala:
d
-Ala ligase in both
P. popilliae
and the related, vancomycin-susceptible,
Paenibacillus lentimorbus
. The 3,380-bp region upstream of
vanY

F

Z

F

H

F

FX

F

in
P. popilliae
ATCC 14706 was sequenced and found to contain genes encoding a putative two-component regulator, VanR
F
S
F
, similar to VanRS but more closely related to a family of two-component regulators linked to VanY-like carboxypeptidases in several glycopeptide-susceptible
Bacillus
species. This upstream region also included a transposase similar to a transposase found in
Bacillus halodurans
and, in some strains, a 99-bp insertion of unknown function with 95% nucleotide identity to a portion of the Tn
1546
transposase gene. Analysis of glycopeptide resistance-associated clusters from soil and/or insect-dwelling organisms may provide important clues to the molecular evolution of acquired glycopeptide resistance elements in human pathogens.
Biological Activity of an Intravenous Preparation of Human Vaccinia Immune Globulin in Mouse Models of Vaccinia Virus Infection
Authors: Shearer Jeffry D. , Siemann Linda , Gerkovich Mary , House Robert V.
Published: June 2005

The biological activity of a new intravenous (i.v.) preparation of human vaccinia immune globulin (VIGIV) was evaluated in two mouse models of vaccinia virus (VV) infection. In a mouse tail lesion model, female CD-1 mice were inoculated i.v. with 7 × 10
4
PFU of VV to produce >10 lesions per tail 8 days later. In a mouse lethality model, female severe combined immunodeficient (SCID) mice were inoculated i.v. with 3 × 10
4
PFU of VV to produce 100% mortality within 45 days. The ability of VIGIV to reduce tail lesion formation in CD-1 mice and mortality in SCID mice was determined by (i) pretreatment of a lethal VV dose with VIGIV prior to i.v. inoculation into SCID mice and (ii) i.v. administration of VIGIV to CD-1 and SCID mice the day before and up to 8 days after VV infection. VIGIV reduced the proportion of CD-1 mice with >10 tail lesions in a dose-related manner when VIGIV was given 1 day before and up to 1 day after VV inoculation. The pretreatment of VV with VIGIV prolonged survival and decreased mortality. VIGIV (100 and 400 mg/kg) prolonged survival when given up to 4 days after VV inoculation, and the 400-mg/kg dose reduced the mortality rate by 80% when given the day before or immediately after VV inoculation. The biological activity of VIGIV was demonstrated in both the immunocompetent and immunocompromised murine models. The timing of treatment relative to VV inoculation appeared to be important for the demonstration of VIGIV's biological activity.
Antistaphylococcal Effect Related to the Area under the Curve/MIC Ratio in an In Vitro Dynamic Model: Predicted Breakpoints versus Clinically Achievable Values for Seven Fluoroquinolones
Authors: Firsov Alexander A. , Lubenko Irene Y. , Vostrov Sergey N. , Portnoy Yury A. , Zinner Stephen H.
Published: June 2005

Prediction of the relative efficacies of different fluoroquinolones is often based on the ratios of the clinically achievable area under the concentration-time curve (AUC) to the MIC, usually with incorporation of the MIC
50
or the MIC
90
and with the assumption of antibiotic-independent patterns of the AUC/MIC-response relationships. To ascertain whether this assumption is correct, the pharmacodynamics of seven pharmacokinetically different quinolones against two clinical isolates of
Staphylococcus aureus
were studied by using an in vitro model. Two differentially susceptible clinical isolates of
S. aureus
were exposed to two 12-h doses of ciprofloxacin (CIP) and one dose of gatifloxacin (GAT), gemifloxacin (GEM), grepafloxacin (GRX), levofloxacin (LVX), moxifloxacin (MXF), and trovafloxacin (TVA) over similar AUC/MIC ranges from 58 to 932 h. A specific bacterial strain-independent AUC/MIC relationship with the antimicrobial effect (

I
E

) was associated with each quinolone. Based on the

I
E

-log AUC/MIC relationships, breakpoints (BPs) that are equivalent to a CIP AUC/MIC ratio of 125 h were predicted for GRX, MXF, and TVA (75 to 78 h), GAT and GEM (95 to 103 h) and LVX (115 h). With GRX and LVX, the predicted BPs were close to those established in clinical settings (no clinical data on other quinolones are available in the literature). To determine if the predicted AUC/MIC BPs are achievable at clinical doses, i.e., at the therapeutic AUCs (AUC
ther
s), the AUC
ther
/MIC
50
ratios were studied. These ratios exceeded the BPs for GAT, GEM, GRX, MXF, TVA, and LVX (750 mg) but not for CIP and LVX (500 mg). AUC/MIC ratios above the BPs can be considered of therapeutic potential for the quinolones. The highest ratios of AUC
ther
/MIC
50
to BP were achieved with TVA, MXF, and GEM (2.5 to 3.0); intermediate ratios (1.5 to 1.6) were achieved with GAT and GRX; and minimal ratios (0.3 to 1.2) were achieved with CIP and LVX.
The L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Counteracts Enhanced Excision of Zidovudine Monophosphate Associated with Thymidine Analog Resistance Mutations
Authors: Miranda Luis R. , Götte Matthias , Kuritzkes Daniel R.
Published: June 2005

Thymidine analog mutations (TAMs) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) confer resistance to zidovudine (AZT) by increasing the rate of ATP-dependent phosphorolysis of the terminal nucleotide monophosphate (primer unblocking). By contrast, the L74V mutation, which confers resistance to didanosine, sensitizes HIV-1 to AZT and partially restores AZT susceptibility when present together with one or more TAMs. To compare rates of primer unblocking in RTs carrying different clusters of TAMs and to explore the biochemical mechanism by which L74V affects AZT susceptibility, ATP-mediated rescue of AZT-blocked DNA synthesis was assayed using a series of purified recombinant RTs. Rates of primer unblocking were higher in the 67N/70R/219Q RT than in the 41L/210W/215Y enzyme and were similar to rates observed with an RT carrying six TAMs (41L/67N/70R/210W/215Y/219Q). The presence of 74V in an otherwise wild-type RT reduced the rate of primer unblocking to a degree similar to that observed with the M184V mutation for lamivudine resistance, which also sensitizes HIV-1 to AZT. Introduction of 74V into RTs carrying TAMs partially counteracted the effect of TAMs on the rate of primer unblocking. The effect of 74V was less marked than that of the 184V mutation in the 67N/70R/219Q and 41L/210W/215Y RTs but similar in the RT carrying six TAMs. These results demonstrate that L74V enhances AZT susceptibility by reducing the extent of its removal by ATP-dependent phosphorolysis and provides further evidence for a common mechanism by which mutations conferring resistance to didanosine and lamivudine sensitize HIV-1 to AZT.
Impaired Rescue of Chain-Terminated DNA Synthesis Associated with the L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Frankel Fernando A. , Marchand Bruno , Turner Dan , Götte Matthias , Wainberg Mark A.
Published: June 2005

The L74V and M184V mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type 1 (HIV-1) are frequently associated with resistance to the nucleoside reverse transcriptase inhibitors abacavir, didanosine, and lamivudine. Yet viruses containing any of these mutations often display hypersusceptibility to zidovudine (ZDV). Two distinct mechanisms have been described to explain HIV-1 drug resistance. One of these involves diminished rates of incorporation of the nucleotide analogue by mutated RT, while the other mechanism involves increased rates of phosphorolytic excision of the drug-terminated primer. To understand the biochemical mechanisms responsible for the hypersensitization of L74V-containing viruses to ZDV, we studied the efficiency of excision of ZDV-monophosphate (ZDV-MP)-terminated primers by recombinant wild-type and mutated HIV-1 RTs in cell-free assays. We observed that the L74V mutation in RT caused reductions in ATP-dependent removal of ZDV-MP from newly synthesized viral DNA. In addition, we determined that the L74V and M184V mutations did not affect the ratio between the populations of RT-DNA/DNA complexes found at pre- and posttranslocational stages; however, they might have affected proper alignment between incorporated chain terminator and pyrophosphate donor, substrate orientation, affinity for ATP, and/or primer-template substrate. Finally, we confirmed previous findings that L74V-containing viruses display diminished replication capacity and that this is associated with reduced levels of synthesis of early reverse-transcribed viral DNA molecules.
Antimicrobial Activity of Novel Dendrimeric Peptides Obtained by Phage Display Selection and Rational Modification
Authors: Pini Alessandro , Giuliani Andrea , Falciani Chiara , Runci Ylenia , Ricci Claudia , Lelli Barbara , Malossi Monica , Neri Paolo , Rossolini Gian Maria , Bracci Luisa
Published: June 2005

A large 10-mer phage peptide library was panned against whole
Escherichia coli
cells, and an antimicrobial peptide (QEKIRVRLSA) was selected. The peptide was synthesized in monomeric and dendrimeric tetrabranched form (multiple antigen peptide [MAP]), which generally allows a dramatic increase of peptide stability to peptidases and proteases. The antibacterial activity of the dendrimeric peptide against
E. coli
was much higher than that of the monomeric form. Modification of the original sequence, by residue substitution or sequence shortening, produced three different MAPs, M4 (QAKIRVRLSA), M5 (KIRVRLSA), and M6 (QKKIRVRLSA) with enhanced stability to natural degradation and antimicrobial activity against a large panel of gram-negative bacteria. The MICs of the most potent peptide, M6, were as low as 4 to 8 μg/ml against recent clinical isolates of multidrug-resistant
Pseudomonas aeruginosa
and members of the
Enterobacteriaceae
. The same dendrimeric peptides showed high stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells, making them promising candidates for the development of new antibacterial drugs.
Correlation between Ratio of Serum Doxycycline Concentration to MIC and Rapid Decline of Antibody Levels during Treatment of Q Fever Endocarditis
Authors: Rolain Jean-Marc , Boulos Areen , Mallet Marie-Noëlle , Raoult Didier
Published: June 2005

Endocarditis is the major clinical manifestation of chronic Q fever. Although doxycycline along with hydroxychloroquine remains the mainstay of medical therapy for Q fever endocarditis, there are wide variations in the rapidity of the patient's decline of antibody levels during such therapy. We undertook a retrospective examination of whether there was any correlation between the ratio of serum concentration to MIC of doxycycline and response to treatment in patients with Q fever endocarditis. Included herein are 16 patients from whom
Coxiella burnetii
was isolated from cardiac valve materials. Serology and measurement of doxycycline and hydroxychloroquine serum levels were performed and recorded after 1 year of treatment. The MIC of doxycycline for
C. burnetii
isolates was determined using the shell vial assay in a real-time quantitative PCR assay. At the completion of a yearlong therapy with doxycycline-hydroxychloroquine, all those that showed a low decline of antibody levels (
n
= 6) (i.e., <2-fold decrease in antibody titer to phase I
C. burnetii
antigen) had a ratio of serum doxycycline concentration to MIC between 0.5 and 1. In contrast, those having a ratio of ≥1 showed a rapid decline of phase I antibody levels (
n
= 9;
P
< 0.05). The only patient who died had a serum doxycycline-to-MIC ratio of <0.5, and the isolate of
C. burnetii
cultured from this patient was resistant to doxycycline (MIC = 8 μg/ml). The ratio of serum doxycycline concentration to MIC should be monitored during the course of therapy in patients with Q fever endocarditis.
Alteration of Fatty Acid and Sterol Metabolism in Miltefosine-Resistant
Leishmania donovani
Promastigotes and Consequences for Drug-Membrane Interactions
Authors: Rakotomanga M. , Saint-Pierre-Chazalet M. , Loiseau P. M.
Published: June 2005

Miltefosine (hexadecylphosphocholine [HePC]) is the first orally active drug approved for the treatment of visceral leishmaniasis. In order to investigate the biochemical modifications occurring in HePC-resistant (HePC-R)
Leishmania donovani
promastigotes, taking into account the lipid nature of HePC, we investigated their fatty acid and sterol metabolisms. We found that the content of unsaturated phospholipid alkyl chains was lower in HePC-R parasite plasma membranes than in those of the wild type, suggesting a lower fluidity of HePC-R parasite membranes. We also demonstrated that HePC insertion within an external monolayer was more difficult when the proportion of unsaturated phospholipids decreased, rendering the HePC interaction with the external monolayer of HePC-R parasites more difficult. Furthermore, HePC-R parasite membranes displayed a higher content of short alkyl chain fatty acids, suggesting a partial inactivation of the fatty acid elongation enzyme system in HePC-R parasites. Sterol biosynthesis was found to be modified in HePC-R parasites, since the 24-alkylated sterol content was halved in HePC-R parasites; however, this modification was not related to HePC sensitivity. In conclusion, HePC resistance affects three lipid biochemical pathways: fatty acid elongation, the desaturase system responsible for fatty acid alkyl chain unsaturation, and the C-24-alkylation of sterols.
Reduced Susceptibility of
Staphylococcus aureus
to Vancomycin and Platelet Microbicidal Protein Correlates with Defective Autolysis and Loss of Accessory Gene Regulator (
agr
) Function
Authors: Sakoulas George , Eliopoulos George M. , Fowler Vance G. , Moellering Robert C. , Novick Richard P. , Lucindo Natalie , Yeaman Michael R. , Bayer Arnold S.
Published: June 2005

Loss of
agr
function, vancomycin exposure, and abnormal autolysis have been linked with both development of the GISA phenotype and low-level resistance in vitro to thrombin-induced platelet microbicidal proteins (tPMPs). We examined the potential in vitro interrelationships among these parameters in well-characterized, isogenic laboratory-derived and clinical
Staphylococcus aureus
isolates. The laboratory-derived
S. aureus
strains included RN6607 (
agr
II-positive parent) and RN6607V (vancomycin-passaged variant; hetero-GISA), RN9120 (RN6607
agr
::
tetM
;
agr
II knockout parent), RN9120V (vancomycin-passaged variant), and RN9120-GISA (vancomycin passaged, GISA). Two serial isolates from a vancomycin-treated patient with recalcitrant, methicillin-resistant
S. aureus
(MRSA) endocarditis were also studied: A5937 (
agr
II-positive initial isolate) and A5940 (
agr
II-defective/hetero-GISA isolate obtained after prolonged vancomycin administration). In vitro tPMP susceptibility phenotypes were assessed after exposure of strains to either 1 or 2 μg/ml. Triton X-100- and vancomycin-induced lysis profiles were determined spectrophotometrically. For
agr
II-intact strain RN6607, vancomycin exposure in vitro was associated with modest increases in vancomycin MICs and reduced killing by tPMP, but no change in lysis profiles. In contrast, vancomycin exposure of
agr
II-negative RN9120 yielded a hetero-GISA phenotype and was associated with defects in lysis and reduced in vitro killing by tPMP. In the clinical isolates, loss of
agr
II function during prolonged vancomycin therapy was accompanied by emergence of the hetero-GISA phenotype and reduced tPMP killing, with no significant change in lysis profiles. An association was identified between loss of
agr
II function and the emergence of hetero-GISA phenotype during either in vitro or in vivo vancomycin exposure. In vitro, these events were associated with defective lysis and reduced susceptibility to tPMP. The precise mechanism(s) underlying these findings is the subject of current investigations.
Population Structure of
Enterococcus faecium
Causing Bacteremia in a Spanish University Hospital: Setting the Scene for a Future Increase in Vancomycin Resistance?
Authors: Coque Teresa M. , Willems Rob J. L. , Fortún Jesús , Top Janetta , Diz Sergio , Loza Elena , Cantón Rafael , Baquero Fernando
Published: June 2005

Over an 8-year period (1995 to 2002), 86
Enterococcus faecium
blood isolates from 84 patients, of which 54 were ampicillin resistant (AREF) and 32 were ampicillin susceptible (ASEF), were studied in a university hospital (1,200 beds; serving a population of 600,000) in Spain, a country characterized by a near-absence of resistance to vancomycin and very high rates of ampicillin resistance among enterococci. Clonal relatedness by pulsed-field gel electrophoresis (PFGE), antibiotic susceptibility, presence of the virulence/epidemicity genes
esp
Efm
and
hyl
Efm
, and identification of
purK
alleles were studied. A group of isolates was also analyzed by amplified fragment length polymorphism (AFLP) and multilocus sequence typing. Medical charts (30 variables collected) were reviewed for 60/84 patients. ASEF showed high clonal diversity (32 PFGE types, 11
purK
alleles, 4 AFLP genogroups), did not harbor putative virulence genes, and had no specific association with hospital acquisition. AREF isolates belonged to a clonal complex (CC) of genetically related strains (
purK
-
1
, AFLP genogroup C), occasionally harboring putative virulence traits, and were from patients with particular risk factors. Within this CC, previously associated with vancomycin-resistant
E. faecium
isolates causing outbreaks worldwide (W. L. Homan et al., J. Clin. Microbiol.
40:
1963-1971, 2002), a great genetic diversity of antibiotic resistance and virulence/epidemicity profiles was found. Associations between
esp
and a >7-day hospital stay and between
purK
-
1
, hospital location, and nosocomial acquisition were noted (
P
< 0.001). These findings reflect the importance of local environmental differences in the evolution of this CC, suggesting that the emergence of vancomycin resistance among AREF strains in Spain may be a question of time.
Differential Gene Expression in Benznidazole-Resistant
Trypanosoma cruzi
Parasites
Authors: Villarreal Diana , Nirdé Philippe , Hide Mallorie , Barnabé Christian , Tibayrenc Michel
Published: June 2005

We analyzed the differential gene expression among representative
Trypanosoma cruzi
stocks in relation to benznidazole exposures using a random differentially expressed sequences (RADES) technique. Studies were carried out with drug pressure both at the natural susceptibility level of the wild-type parasite (50% inhibitory concentration for the wild type) and at different resistance levels. The pattern of differential gene expression performed with resistant stocks was compared to the population structure of this parasite, established by random amplified polymorphic DNA analysis and multilocus enzyme electrophoresis. A RADES band polymorphism was observed, and over- or underexpression was linked to the resistance level of the stock. The analysis of RADES bands suggested that different products may be involved in benznidazole resistance mechanisms. No significant association was found between phylogenetic clustering and benznidazole susceptibility. Benznidazole resistance may involve several mechanisms, depending on the level of drug exposure.
Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene
Authors: Chou Sunwen , Van Wechel Laura C. , Lichy Heather M. , Marousek Gail I.
Published: June 2005

A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and
pol
gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC
50
). Changes in the IC
50
conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and
pol
del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and
pol
del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate.