bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of Caspofungin and Voriconazole Combinations in Experimental Aspergillosis
Authors: MacCallum Donna M. , Whyte Julie A. , Odds Frank C.
Published: August 2005

Guinea pigs were infected with
Aspergillus fumigatus
at two challenge doses and treated for 7 days with a placebo, intraperitoneal caspofungin (1 mg/kg daily), oral voriconazole (1 mg/kg twice a day), or a combination of the caspofungin and voriconazole treatments. The combination therapy statistically significantly prolonged survival over that with the control at both challenge doses and achieved a statistically significant reduction in kidney burdens as measured by quantitative PCR. The same was true for animals given caspofungin alone at both levels of challenge and for animals treated with voriconazole alone at the lower challenge dose. However, the effects of combination therapy on prolongation of survival were greater than those of either monotherapy at both challenge doses, and the reduction in kidney burdens with combination therapy was significantly greater than that with caspofungin alone in the animals given the lower challenge dose. No synergistic interactive effects were seen for the two agents in checkerboard titration experiments in vitro. We conclude that therapy of experimental aspergillosis with caspofungin and voriconazole combined offers slight additional improvements in efficacy rather than effects of a clearly synergistic nature.
Genomic Approach to Identifying the Putative Target of and Mechanisms of Resistance to Mefloquine in Mycobacteria
Authors: Danelishvili Lia , Wu Martin , Young Lowell S. , Bermudez Luiz E.
Published: August 2005

The emergence of mycobacterial resistance to multiple antimicrobials
emphasizes the need for new compounds. The antimycobacterial activity
of mefloquine has been recently described.
Mycobacterium
avium
,
Mycobacterium smegmatis
, and
Mycobacterium
tuberculosis
are susceptible to mefloquine in vitro, and activity
was evidenced in vivo against
M. avium
. Attempts to obtain
resistant mutants by both in vitro and in vivo selection have failed.
To identify mycobacterial genes regulated in response to mefloquine, we
employed DNA microarray and green fluorescent protein (GFP) promoter
library techniques. Following mefloquine treatment, RNA was harvested
from
M. tuberculosis
H37Rv, labeled with
32
P, and
hybridized against a DNA array. Exposure to 4× MIC resulted in
a significant stress response, while exposure to a subinhibitory
concentration of mefloquine triggered the expression of genes coding
for enzymes involved in fatty acid synthesis, the metabolic pathway,
and transport across the membrane and other proteins of unknown
function. Evaluation of gene expression using an
M. avium
GFP
promoter library exposed to subinhibitory concentrations of mefloquine
revealed more than threefold upregulation of 24 genes. To complement
the microarray results, we constructed an
M. avium
genomic
library under the control of a strong sigma-70 (G13) promoter in
M.
smegmatis
. Resistant clones were selected in 32 μg/ml of
mefloquine (wild-type
M. avium
,
M. tuberculosis
, and
M. smegmatis
are inhibited by 8 μg/ml), and the
M.
avium
genes associated with
M. smegmatis
resistant to
mefloquine were sequenced. Two groups of genes were identified: one
affecting membrane transport and one gene that apparently is involved
in regulation of cellular
replication.
Multidrug-Resistant
Pseudomonas aeruginosa
Strain That Caused an Outbreak in a Neurosurgery Ward and Its
aac(6′)-Iae
Gene Cassette Encoding a Novel Aminoglycoside Acetyltransferase
Authors: Sekiguchi Jun-ichiro , Asagi Tsukasa , Miyoshi-Akiyama Tohru , Fujino Tomoko , Kobayashi Intetsu , Morita Koji , Kikuchi Yoshihiro , Kuratsuji Tadatoshi , Kirikae Teruo
Published: August 2005

We characterized multidrug-resistant
Pseudomonas aeruginosa
strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a
P. aeruginosa
strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, β-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of
P. aeruginosa
, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of
bla
IMP-1
, a novel aminoglycoside resistance gene, and the
aadA1
gene. The aminoglycoside resistance gene, designated
aac(6′)-Iae
, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6′)-Iq. Recombinant AAC(6′)-Iae protein showed aminoglycoside 6′-
N
-acetyltransferase activity by thin-layer chromatography.
Escherichia coli
expressing exogenous
aac(6′)-Iae
showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of
gyrA
and
parC
at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that
P. aeruginosa
IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the
aac(6′)-I
family and mutations in drug resistance genes.
BEL-1, a Novel Clavulanic Acid-Inhibited Extended-Spectrum β-Lactamase, and the Class 1 Integron In120 in
Pseudomonas aeruginosa
Authors: Poirel Laurent , Brinas Laura , Verlinde Annemie , Ide Louis , Nordmann Patrice
Published: August 2005

Screening by a double-disk synergy test identified a
Pseudomonas aeruginosa
isolate that produced a clavulanic acid-inhibited expanded-spectrum β-lactamase (ESBL). Cloning and sequencing identified a novel ESBL, BEL-1, weakly related to other Ambler class A ESBLs. β-Lactamase BEL-1 hydrolyzed significantly most expanded-spectrum cephalosporins and aztreonam, and its activity was inhibited by clavulanic acid, tazobactam, cefoxitin, moxalactam, and imipenem. This chromosome-encoded ESBL gene was embedded in a class 1 integron containing three other gene cassettes. In addition, this integron was bracketed by Tn
1404
transposon sequences at its right end and by
P. aeruginosa
-specific sequences at its left end.
Short-Course Regimens of Artesunate-Fosmidomycin in Treatment of Uncomplicated
Plasmodium falciparum
Malaria
Authors: Borrmann Steffen , Adegnika Ayola A. , Moussavou Félicien , Oyakhirome Sunny , Esser Gilbert , Matsiegui Pierre-Blaise , Ramharter Michael , Lundgren Ingrid , Kombila Maryvonne , Issifou Saadou , Hutchinson David , Wiesner Jochen , Jomaa Hassan , Kremsner Peter G.
Published: August 2005

Fosmidomycin is effective against malaria, but it needs to be given for ≥4 days when used alone. We conducted a study of 50 children with
Plasmodium falciparum
malaria to evaluate the safety and efficacy of consecutively shortened regimens of artesunate-fosmidomycin (1 to 2 mg/kg of body weight and 30 mg/kg of body weight, respectively; doses given every 12 hours). All dosing regimens were well tolerated. Artesunate-fosmidomycin acted rapidly, resulting in consolidated geometric mean parasite and fever clearance times of 24 h and 15 h, respectively. Treatment regimens of ≥2 days led to cure ratios of 100% by day 14 (39/39; 95% confidence interval [95% CI], 91% to 100%). Most importantly, the 3-day regimen achieved 100% cure on day 28 (10/10; 95% CI, 69% to 100%). Treatment with artesunate-fosmidomycin was associated with transient grade I or II neutropenia (absolute neutrophil counts of 750 to 1,200/μl and 400 to 749/μl, respectively) in six or two patients, respectively. Artesunate-fosmidomycin demonstrates the feasibility and potential value of short-course artemisinin-based combination chemotherapy with rapidly eliminated combination partners.
Analysis of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Subunit Structure/Function in the Context of Infectious Virions and Human Target Cells
Authors: Mulky Alok , Kappes John C.
Published: August 2005

The reverse transcriptase (RT) of all retroviruses is required for synthesis of the viral DNA genome. The human immunodeficiency virus type 1 (HIV-1) RT exists as a heterodimer made up of 51-kDa and 66-kDa subunits. The crystal structure and in vitro biochemical analyses indicate that the p66 subunit of RT is primarily responsible for the enzyme's polymerase and RNase H activities. Since both the p51 and p66 subunits are generated from the same coding region, as part of the Pr160
Gag-Pol
precursor protein, there are inherent limitations for studying subunit-specific function with intact provirus in a virologically relevant context. Our lab has recently described a novel system for studying the RT heterodimer (p51/p66) wherein a LTR-vpr-p51-IRES-p66 expression cassette provided in
trans
to an RT-deleted HIV-1 genome allows precise molecular analysis of the RT heterodimer. In this report, we describe in detail the specific approaches, alternative strategies, and pitfalls that may affect the application of this novel assay for analyzing RT subunit structure/function in infectious virions and human target cells. The ability to study HIV-1 RT subunit structure/function in a physiologically relevant context will advance our understanding of both RT and the process of reverse transcription. The study of antiretroviral drugs in a subunit-specific virologic context should provide new insights into drug resistance and viral fitness. Finally, we anticipate that this approach will help elucidate determinants that mediate p51-p66 subunit interactions, which is essential for structure-based drug design targeting RT heterodimerization.
Quantification of Different Human Alpha Interferon Subtypes and Pegylated Interferon Activities by Measuring MxA Promoter Activation
Authors: François Catherine , Bernard Isabelle , Castelain Sandrine , Charleston Bryan , Fray Martin D. , Capiod Jean-Claude , Duverlie Gilles
Published: August 2005

Alpha interferons (α-IFNs) are potent biologically active proteins synthesized and secreted by somatic cells during viral infection. Quantification of α-IFN concentrations in biological samples is used for diagnosis. More recently, recombinant IFNs have been used as antiviral, antiproliferative, and immunomodulatory therapeutic agents, and particularly for the treatment of chronic hepatitis C virus infection. For this purpose, IFN has recently been coupled to polyethylene glycol (PEG) to improve the pharmacokinetic properties. The measure of α-IFN in biological samples from treated patients could be useful to ensure compliance to therapy and the true IFN activity in relation to viral decay during follow-up. In particular, it could be used to monitor the PEG-IFN concentration in patients treated for hepatitis C virus infection. The most frequently used test is a bioassay based on the antiviral property of the IFN, but the assay is not highly reproducible. Here, we present a reporter test based on MxA promoter activation of chloramphenicol acetyltransferase expression (Mx-CAT). MxA is an antiviral protein induced and tightly regulated by α-IFN. The Mx-CAT assay showed good reproducibility of 15% and was suitable to quantify PEG-IFN and numerous other α-IFN subtypes as well, despite a differential MxA promoter activation in relation with the subtype. A good correlation was obtained with the reporter assay and a commercial enzyme-linked immunosorbent assay on samples from treated patients. This test could be useful for monitoring IFN therapy of chronically infected hepatitis C virus-infected patients treated with the standard IFN, PEG-IFN, and probably forthcoming recombinant IFNs.
Use of
Leishmania donovani
Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening
Authors: Ashutosh  , Gupta Suman , Ramesh  , Sundar Shyam , Goyal Neena
Published: August 2005

Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed
Leishmania donovani
field isolates that constitutively express the firefly luciferase reporter gene (
luc
) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed.
luc
expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.
Assessment of Effective Renal Plasma Flow, Enzymuria, and Cytokine Release in Healthy Volunteers Receiving a Single Dose of Amphotericin B Desoxycholate
Authors: Pai Manjunath P. , Norenberg Jeffrey P. , Telepak Robert A. , Sidney David S. , Yang Shu
Published: August 2005

The present study assessed potential subclinical markers of amphotericin B (AmB)-related nephrotoxicity and infusion-related reactions (IRR). Subjects were pretreated with diphenhydramine and acetaminophen and received a 500-ml bolus infusion of 0.9% sodium chloride prior to each effective renal plasma flow (ERPF) assessment. ERPF was measured before and after administration of a single 0.25-mg/kg intravenous AmB dose using technetium-99m mercaptoacetyltriglycine. Blood was collected before and 3 h after AmB infusion for tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) plasma concentrations. Overnight 12-h urine collections were performed before administration of AmB and for 2 nights after administration of AmB and analyzed for α and π glutathione-S-transferases (GSTα and GSTπ, respectively) andN-acetyl-β-d-glucosaminidase (NAG). Six men and six women with mean ± standard deviation (SD) ages of 24.8 ± 5.3 and 28.0 ± 8.5 years, respectively, were studied. Baseline serum creatinine values were within the normal range and were unaltered after administration of AmB. The mean ± SD decrease in ERPF after administration of AmB was significant (P< 0.05) in males (15.7 ± 8.1%) but not females (9.5 ± 14.0%). The GSTπ and GSTα indices increased significantly (P< 0.05) by two to fourfold and returned to baseline in males but were unaltered in females. NAG indices were unaffected by AmB. Six patients experienced an IRR that was associated with increased TNF-α (P< 0.05) but not IL-1β (P= 0.09). These results suggest a potential sex-related difference in AmB-induced nephrotoxicity and provide a rationale for use of ERPF, urine GST, and TNF-α as subclinical markers of polyene-induced toxicity.
Role of
embB
Codon 306 Mutations in
Mycobacterium tuberculosis
Revisited: a Novel Association with Broad Drug Resistance and IS
6110
Clustering Rather than Ethambutol Resistance
Authors: Hazbón Manzour Hernando , Bobadilla del Valle Miriam , Guerrero Marta Inírida , Varma-Basil Mandira , Filliol Ingrid , Cavatore Magali , Colangeli Roberto , Safi Hassan , Billman-Jacobe Helen , Lavender Caroline , Fyfe Janet , García-García Lourdes , Davidow Amy , Brimacombe Michael , León Clara Inés , Porras Tania , Bose Mridula , Chaves Fernando , Eisenach Kathleen D. , Sifuentes-Osornio José , Ponce de León Alfredo , Cave M. Donald , Alland David
Published: August 2005

Mutations at position 306 of
embB
(
embB
306) have been proposed as a marker for ethambutol resistance in
Mycobacterium tuberculosis
; however, recent reports of
embB
306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical
M. tuberculosis
isolates with different drug susceptibility patterns and of different geographical origins for associations between
embB
306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in
embB
306; however, only 55 of these mutants were ethambutol resistant. Mutations in
embB
306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (
P
< 0.001), and the association between
embB
306 mutations and resistance to increasing numbers of drugs was highly significant (
P
< 0.001 for trend). We examined the association between
embB
306 mutations and IS
6110
clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in
embB
306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44;
P
= 0.004). In a multivariate model that also included mutations in
katG
315,
katG
463,
gyrA
95, and
kasA
269, only mutations in
embB
306 (odds ratio, 2.14;
P
= 0.008) and
katG
315 (odds ratio, 1.99;
P
= 0.015) were found to be independently associated with clustering. In conclusion,
embB
306 mutations do not cause classical ethambutol resistance but may predispose
M. tuberculosis
isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.
Mycobacterium smegmatis
Erm(38) Is a Reluctant Dimethyltransferase
Authors: Madsen Christian Toft , Jakobsen Lene , Douthwaite Stephen
Published: August 2005

The waxy cell walls of mycobacteria provide intrinsic tolerance to a broad range of antibiotics, and this effect is augmented by specific resistance determinants. The inducible determinant
erm
(38) in the nontuberculous species
Mycobacterium smegmatis
confers high resistance to lincosamides and some macrolides, without increasing resistance to streptogramin B antibiotics. This is an uncharacteristic resistance pattern falling between the type I and type II macrolide, lincosamide, and streptogramin B (MLS
B
) phenotypes that are conferred, respectively, by Erm monomethyltransferases and dimethyltransferases. Erm dimethyltransferases are typically found in pathogenic bacteria and confer resistance to all MLS
B
drugs by addition of two methyl groups to nucleotide A2058 in 23S rRNA. We show here by mass spectrometry analysis of the mycobacterial rRNA that Erm(38) is indeed an A2058-specific dimethyltransferase. The activity of Erm(38) is lethargic, however, and only a meager proportion of the rRNA molecules become dimethylated in
M. smegmatis
, while most of the rRNAs are either monomethylated or remain unmethylated. The methylation pattern produced by Erm(38) clarifies the phenotype of
M. smegmatis
, as it is adequate to confer resistance to lincosamides and 14-member ring macrolides such as erythromycin, but it is insufficient to raise the level of resistance to streptogramin B drugs above the already high intrinsic tolerance displayed by this species.
Accumulation of Mutations in both
gyrB
and
parE
Genes Is Associated with High-Level Resistance to Novobiocin in
Staphylococcus aureus
Authors: Fujimoto-Nakamura Mika , Ito Hideaki , Oyamada Yoshihiro , Nishino Takeshi , Yamagishi Jun-ichi
Published: August 2005

Coumarin-resistant mutants of
Staphylococcus aureus
were isolated by three-step selection with novobiocin at different concentrations. Sequencing analysis of the
gyrB
and
parE
genes of the first-, second-, and third-step mutants revealed that successive point mutations first occurred specifically in the
gyrB
gene, followed by a point mutation in the
parE
gene and then an additional point mutation in the
gyrB
gene. These findings demonstrate that DNA gyrase is the primary target and that topoisomerase IV is the secondary target for novobiocin and that the accumulation of point mutations in both the
gyrB
and the
parE
genes is associated with high-level resistance to novobiocin in
S. aureus
. Moreover, our results show that the amino acid substitutions (Asp-89 to Gly and Ser-128 to Leu) found in GyrB are associated with resistance to novobiocin but not to coumermycin A1, suggesting that the interactions of novobiocin and coumermycin A1 with GyrB differ at the molecular level.
Molecular Basis for Increased Susceptibility of Isolates with Atazanavir Resistance-Conferring Substitution I50L to Other Protease Inhibitors
Authors: Yanchunas Joseph , Langley David R. , Tao Li , Rose Ronald E. , Friborg Jacques , Colonno Richard J. , Doyle Michael L.
Published: August 2005

Protease inhibitors (PIs) are highly effective drugs against the human immunodeficiency virus (HIV), yet long-term therapeutic use is limited by emergence of HIV type 1 (HIV-1) protease substitutions that confer cross-resistance to multiple protease inhibitor drugs. Atazanavir is a highly potent HIV protease inhibitor with a distinct resistance profile that includes effectiveness against most HIV-1 isolates resistant to one or two PIs. The signature resistance substitution for atazanavir is I50L, and it is frequently (53%) accompanied by a compensatory A71V substitution that helps restore viability and increases atazanavir resistance levels. We measured the binding affinities of wild-type (WT) and I50L/A71V HIV-1 proteases to atazanavir and other currently approved PIs (ritonavir, lopinavir, saquinavir, nelfinavir, indinavir, and amprenavir) by isothermal titration calorimetry. Remarkably, we find that all of the PIs have 2- to 10-fold increased affinities for I50L/A71V protease, except for atazanavir. The results are also manifested by thermal stability measures of affinity for WT and I50L/A71V proteases. Additional biophysical and enzyme kinetics experiments show I50L/A71V protease is a stable enzyme with catalytic activity that is slightly reduced (34%) relative to the WT. Computational modeling reveals that the unique resistance phenotype of I50L/A71V protease likely originates from bulky tert-butyl groups at P2 and P2′ (specific to atazanavir) that sterically clash with methyl groups on residue L50. The results of this study provide a molecular understanding of the novel hypersusceptibility of atazanavir-resistant I50L/A71V-containing clinical isolates to other currently approved PIs.
High-Throughput Human Immunodeficiency Virus Type 1 (HIV-1) Full Replication Assay That Includes HIV-1 Vif as an Antiviral Target
Authors: Cao Joan , Isaacson Jason , Patick Amy K. , Blair Wade S.
Published: August 2005

Antiviral screens have proved useful for the identification of novel human immunodeficiency virus type 1 (HIV-1) inhibitors. In this study, we describe an HIV-1 full replication (HIV-1 Rep) assay that incorporates all of the targets required for replication in T-cell lines, including the HIV-1 Vif gene. The HIV-1 Rep assay was designed to exhibit optimal sensitivity to late-stage as well as early-stage inhibitors to maximize the likelihood of identification of novel target antiviral compounds in a screen. In addition, the flexibility of the HIV-1 Rep assay allows the rapid evaluation of antiviral compounds against different virus strains in different T-cell lines without significant modification of the assay format. We demonstrate that the HIV-1 Rep assay exhibits characteristics (e.g., a favorable
Z
′ value) compatible with high-throughput screening in a 384-well format. The utility of the HIV-1 Rep assay was demonstrated in a high-throughput screen of >10
6
compounds. To our knowledge, this study represents the first example of an HIV-1 antiviral screen that includes Vif as a functional target and was executed on an industrial scale.
Structure-Function Characterization and Optimization of a Plant-Derived Antibacterial Peptide
Authors: Suarez Mougli , Haenni Marisa , Canarelli Stéphane , Fisch Florian , Chodanowski Pierre , Servis Catherine , Michielin Olivier , Freitag Ruth , Moreillon Philippe , Mermod Nicolas
Published: August 2005

Crushed seeds of theMoringa oleiferatree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such asPseudomonas aeruginosaandStreptococcus pyogeneswithout displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.
Protection from Anthrax Toxin-Mediated Killing of Macrophages by the Combined Effects of Furin Inhibitors and Chloroquine
Authors: Komiyama Tomoko , Swanson Joel A. , Fuller Robert S.
Published: August 2005

Cell surface proteolytic processing of anthrax protective antigen by furin or other furin-related proteases is required for its oligomerization, endocytosis, and function as a translocon for anthrax lethal and edema factors. Countering toxin lethality is essential to developing effective chemotherapies for anthrax infections that have proceeded beyond the stage at which antibiotics are effective. The primary target for toxin is the macrophage, which can be killed by lethal factor via both necrotic and apoptotic pathways. Here we show that three high-affinity inhibitors of furin efficiently blocked killing of murine J774A.1 macrophages by recombinant protective antigen plus lethal factor: RRD-eglin and RRDG-eglin, developed by engineering the protein protease inhibitor eglin c, and the peptide boronic acid inhibitor acetyl-Arg-Glu-Lys-boroArg pinanediol. Inhibition of killing was dose dependent and correlated with prevention of protective antigen processing. Previous studies have shown that weak bases, such as chloroquine, which neutralize acidic compartments, also interfere with toxin-dependent killing. Here we show that combining furin inhibitors and chloroquine strongly augments the inhibition of toxin-dependent killing, suggesting that combined use of antifurin drugs and chloroquine might provide enhanced therapeutic benefits. Reversible furin inhibitors protected against anthrax toxin killing for at least 5 h, but by 8 h, toxin-dependent killing resumed even though furin inhibitors were still active. An irreversible chloromethylketone inhibitor did not exhibit this loss of protection.
Interleukin-8-Derived Peptide Has Antibacterial Activity
Authors: Björstad Åse , Fu Huamei , Karlsson Anna , Dahlgren Claes , Bylund Johan
Published: August 2005

Chemokines are inflammatory mediators with effects on diverse processes associated with the immune response. Some of the proteins belonging to the CXC chemokine subfamily, one of four groups in the family, possess inherent antibacterial activity against a wide range of bacteria. The CXC chemokine interleukin-8 (IL-8) has not been ascribed any direct antibacterial activity, but the fact that several of the amino acids in the carboxy-terminal part of the protein are identical or similar to those in a bactericidal cecropin-like peptide [Hp(2-20)] from
Helicobacter pylori
suggests that processing of the cytokine might generate peptide fragments with antibacterial properties. Synthetic peptides representing the carboxy-terminal part of IL-8 were investigated for antibacterial activities. These fragments possessed an antibacterial activity absent in the full-length IL-8. The antibacterial effects were reduced at increasing salt concentrations whereas the activity was increased when the pH was lowered. The IL-8-derived peptide shared structural similarity with and was also functionally additive to the Hp(2-20) peptide. The IL-8-derived peptide lacked the proinflammatory effects of the full-length protein. We also showed that acid hydrolysis of IL-8 generated a major peptide fragment corresponding to the antibacterial carboxyl terminus of the protein. The results presented are of special interest when put in the context of the suggested importance of antimicrobial peptides for microbial colonization of the gastric mucosa.
Oxazolidinones Inhibit Cellular Proliferation via Inhibition of Mitochondrial Protein Synthesis
Authors: Nagiec Eva E. , Wu Luping , Swaney Steve M. , Chosay John G. , Ross Daniel E. , Brieland Joan K. , Leach Karen L.
Published: August 2005

The oxazolidinones are a relatively new structural class of antibacterial agents that act by inhibiting bacterial protein synthesis. The oxazolidinones inhibit mitochondrial protein synthesis, as shown by [
35
S]methionine incorporation into intact rat heart mitochondria. Treatment of K562 human erythroleukemia cells with the oxazolidinone eperezolid resulted in a time- and concentration-dependent inhibition of cell proliferation. The cells remained viable, but an increase in doubling time was observed with eperezolid treatment. Inhibition was reversible, since washing and refeeding of cells in the absence of compound resulted in a resumption of growth. The growth-inhibitory effect of the oxazolidinones did not appear to be cell type specific, and inhibition of CHO and HEK cells also was demonstrated. Treatment of cells resulted in a decrease in mitochondrial cytochrome oxidase subunit I levels, consistent with an inhibition of mitochondrial protein synthesis. Eperezolid caused no growth inhibition of rho zero (ρ
0
) cells, which contain no mitochondrial DNA; however, the growth of the parent 143B cells was inhibited. These results provide a direct demonstration that the inhibitory effect of eperezolid in mammalian cells is the result of mitochondrial protein synthesis inhibition.
Tigecycline MIC Testing by Broth Dilution Requires Use of Fresh Medium or Addition of the Biocatalytic Oxygen-Reducing Reagent Oxyrase To Standardize the Test Method
Authors: Bradford Patricia A. , Petersen Peter J. , Young Mairead , Jones C. Hal , Tischler Mark , O'Connell John
Published: August 2005

Tigecycline is a broad-spectrum glycylcycline antibiotic with activity against not only susceptible gram-positive and gram-negative pathogens but also strains that are resistant to many other antibiotics. In the process of determining quality control (QC) limits for the American Type Culture Collection reference strains for tigecycline, a number of inconsistencies in MICs were encountered which appeared to be related to the age of the Mueller-Hinton broth (MHB) medium used in the MIC testing. The objective of this study was to determine the cause of the discrepant MIC results between fresh and aged MHB. The MICs of tigecycline were determined in MHB that was either prepared fresh (<12 h old), prepared and stored at 4°C, stored at room temperature, stored anaerobically, or supplemented with the biocatalytic oxygen-reducing reagent Oxyrase. When tested in fresh media, tigecycline was 2 to 3 dilutions more active against the CLSI-recommended QC strains compared to aged media (MICs of 0.03 to 0.25 and 0.12 to 0.5 μg/ml, respectively). Media aged under anaerobic conditions prior to testing or supplemented with Oxyrase resulted in MICs similar to those obtained in fresh medium (MICs of 0.03 to 0.12 and 0.03 to 0.25 μg/ml, respectively). Time-kill kinetics demonstrated a >3 log
10
difference in viable growth when tigecycline was tested in fresh or Oxyrase-supplemented MHB compared to aged MHB. High-pressure liquid chromatography analysis revealed the accumulation of an early peak (oxidative by-product of tigecycline) to be 3.5% in fresh media and 25.1% in aged media after 24 h and that addition of Oxyrase prevented the accumulation of this oxidized by-product. These results suggested that the activity of tigecycline was affected by the amount of dissolved oxygen in the media. The use of fresh MHB or supplementation with Oxyrase resulted in a more standardized test method for performing MIC tests with tigecycline.
Targeting of the Virulence Factor Acetohydroxyacid Synthase by Sulfonylureas Results in Inhibition of Intramacrophagic Multiplication of
Brucella suis
Authors: Boigegrain Rose-Anne , Liautard Jean-Pierre , Köhler Stephan
Published: August 2005

The acetohydroxyacid synthase (AHAS) of
Brucella suis
can be effectively targeted by the sulfonylureas chlorimuron ethyl and metsulfuron methyl. Growth in minimal medium was inhibited, and multiplication in human macrophages was totally abolished with 100 μM of sulfonylureas. Metsulfuron methyl-resistant mutants showed reduced viability in macrophages and reduced AHAS activity.
In Vitro Activity and Killing Effect of Uperin 3.6 against Gram-Positive Cocci Isolated from Immunocompromised Patients
Authors: Giacometti Andrea , Cirioni Oscar , Kamysz Wojciech , Silvestri Carmela , Licci Alberto , D'Amato Giuseppina , Nadolski Piotr , Riva Alessandra , Lukasiak Jerzy , Scalise Giorgio
Published: August 2005

The in vitro activity of uperin 3.6, alone or combined with six antibiotics, against gram-positive cocci, including
Rhodococcus equi
, methicillin-resistant staphylococci, and vancomycin-resistant enterococci, was investigated. All isolates were
inhibited at concentrations of 1 to 16 mg/liter. Synergy was demonstrated when uperin 3.6 was combined with clarithromycin and
doxycycline.
The
vanB2
Gene Cluster of the Majority of Vancomycin-Resistant
Enterococcus faecium
Isolates from Taiwan Is Associated with the
pbp5
Gene and Is Carried by Tn
5382
Containing a Novel Insertion Sequence
Authors: Lu Jang-Jih , Chang Tein-Yao , Perng Cherng-Lih , Lee Shih-Yi
Published: August 2005

Thirty-two
vanB2 Enterococcus faecium
isolates were found to harbor Tn
5382
. Twenty-four isolates had a 1,419-bp sequence inserted within the open reading frame (ORF) C of Tn
5382
. This 1,419-bp sequence contained a 638-bp ORF with a 72% amino acid sequence homology with the transposase gene of IS
150
. Thirty isolates had the
pbp5
gene linked to Tn
5382
.
Chromosome- and Plasmid-Encoded β-Lactamases in
Capnocytophaga
spp
Authors: Handal Trude , Giraud-Morin Chantal , Caugant Dominique A. , Madinier Isabelle , Olsen Ingar , Fosse Thierry
Published: August 2005

Chromosome- and plasmid-encoded CfxA2 and CfxA3 β-lactamases were detected in
Capnocytophaga
spp. from oral sources in France, Norway, and the United States. Unidentified chromosome-encoded β-lactamases were present in
Capnocytophaga sputigena
. Nucleotide sequence analysis of the CfxA3-encoding plasmid from
C. ochracea
revealed an unreported insertion sequence (IS
Coc1
) upstream of the
cfxA
gene.
Use of Pharmacokinetic-Pharmacodynamic Target Attainment Analyses To Support Phase 2 and 3 Dosing Strategies for Doripenem
Authors: Bhavnani Sujata M. , Hammel Jeffrey P. , Cirincione Brenda B. , Wikler Matthew A. , Ambrose Paul G.
Published: August 2005

A doripenem population pharmacokinetic model and Monte Carlo simulations were utilized for dose regimen decision support for future clinical development. Simulation results predict that 500 mg of doripenem administered over 1 h every 8 h would be effective against bacterial strains with MICs less than 2 μg/ml and that less susceptible strains could be treated with prolonged infusions.
Cloning and Characterization of a Novel Trimethoprim-Resistant Dihydrofolate Reductase from a Nosocomial Isolate of
Staphylococcus aureus
CM.S2 (IMCJ1454)
Authors: Sekiguchi Jun-ichiro , Tharavichitkul Prasit , Miyoshi-Akiyama Tohru , Chupia Vena , Fujino Tomoko , Araake Minako , Irie Atsushi , Morita Koji , Kuratsuji Tadatoshi , Kirikae Teruo
Published: August 2005

A novel gene,
dfrG
, encoding a trimethoprim (TMP)-resistant dihydrofolate reductase (DHFR, designated S3DHFR) was cloned from a clinical isolate of methicillin-resistant
Staphylococcus aureus. Escherichia coli
expressing
dfrG
was highly resistant to TMP. Recombinant S3DHFR exhibited DHFR activity that was not inhibited by TMP.
In Vitro Antifungal Susceptibilities ofSporothrix schenckiiin Two Growth Phases
Authors: Trilles Luciana , Fernández-Torres Belkys , dos Santos Lazéra Márcia , Wanke Bodo , de Oliveira Schubach Armando , de Almeida Paes Rodrigo , Inza Isabel , Guarro Josep
Published: August 2005

We have determined the antifungal susceptibilities of 34 clinical isolates of the dimorphic fungusSporothrix schenckiito 11 drugs using a microdilution method. In general, the type of growth phase (mycelial or yeast) and the temperature of incubation (30 or 35°C) exerted a significant influence on the MICs.
Upregulation of tissue kallikrein, kinin B1receptor, and kinin B2receptor in mast and giant cells infiltrating oesophageal squamous cell carcinoma
Authors: Dlamini Z , Bhoola K D
Published: August 2005

Background:The mitogenic kinin peptides formed by the serine protease, tissue kallikrein (TK1), stimulate the proliferation of tumour cells and, by increasing vascular permeability, enhance metastasis. Oesophageal mucosal epithelial cells are derived from the epithelial cell germ layer, which expresses the kallikrein–kinin cascade.

Aim:To determine the cellular distribution of active TK1, prokallikrein, and the kinin B1and B2receptors in oesophageal carcinoma by immunocytochemistry and in situ hybridisation (ISH).

Methods:Fifty oesophageal specimens (33 biopsies and 17 resections) and 10 control specimens adjacent to tumour or normal oesophageal biopsies were studied. Specific antibodies were used to determine the cellular localisation of TK1, prokallikrein, and the kinin B1and B2receptors in normal and oesophageal specimens by standard immunohistochemical techniques. The intensity of immunolabelling was quantified by image analysis. Antisense probes for TK1 and the kinin B1and B2receptors were also used to localise mRNA.

Results:TK1 (active and prokallikrein) was expressed in the mucosa of normal and tumour oesophageal epithelium. In general, expression was highest in activated mast cells, followed by giant tumour cells. Immunolabelling results were confirmed by ISH experiments.

Conclusions:This is the first demonstration that TK1 and kinin B1and B2receptors are expressed in oesophageal carcinoma. Because TK1 released from tumour cells enzymatically generates mitogenic kinins from its endogenous substrate, kininogen, it is possible that third generation kinin receptor antagonists, which have been shown to be cytotoxic to cancer cells, may be useful therapeutic agents in this disease.
Differences between serum and plasma for intact parathyroid hormone measurement in patients with chronic renal failure in routine clinical practice
Authors: Twomey P J , Whitlock T , Pledger D R
Published: August 2005

Background:Parathyroid hormone (PTH) is important in the evaluation of patients with calcium metabolism disorders and/or chronic renal disease.

Aims:To assess the differences between serum and plasma PTH measurements using the Advia Centaur.

Methods:Twenty six paired serum and edetate samples from patients with chronic renal failure were analysed using the Advia Centaur.

Results:The EDTA results ranged from 2.3 to 76.1 pmol/litre and the Deming regression equation was: serum  =  0.8927 EDTA − 0.447. The percentage difference plot had a mean difference of 13.8% (95% confidence interval, 2.2% to 25.4%; significant). The available time to separation and freezing ranged from 10 to 231 (median, 85) minutes. The correlation coefficient for the percentage difference against the time to separation and the percentage difference against the mean PTH concentration were −0.13 and −0.07, respectively.

Conclusions:These results go beyond the previous controlled research conditions by showing that such differences between serum and edentate plasma exist in routine clinical practice. They also show that intra-individual PTH differences as large as 25.0% can exist on the same day between serum and edetate plasma. This may partly explain some of the variability of PTH concentrations found in some patients with chronic renal failure.
High frequency of germinal centre derivation in diffuse large B cell lymphoma from Asian patients
Authors: Shia A K H , Gan G-G , Jairaman S , Peh S-C
Published: August 2005

Background:Recent reports have divided diffuse large B cell lymphoma (DLBCL) into germinal centre B cell-like and activated B cell-like subgroups with implicated differences in prognosis.

Aims:To delineate the germinal centre B cell derivation group from an Asian series of DLBCLs.

Methods:Fifty four cases were analysed by polymerase chain reaction to detect the t(14;18) translocation and immunohistochemistry for BCL2, CD10, BCL6, and E2F1 expression.

Results:Eighteen of 54 cases hadbcl2gene rearrangement, 36 of 52 expressed BCL2, 29 of 52 expressed BCL6, 20 of 53 expressed CD10, and 18 of 53 expressed E2F1. There was a significant association betweenbcl2gene rearrangement and the expression of both BCL2 and CD10. Using the minimally acceptable criteria of t(14;18) rearrangement and/or CD10 expression, 26 of 54 cases were probably germinal centre derived, in agreement with other reports. A higher proportion of cases had t(14;18) translocation, suggesting that they may be derived from transformed follicular lymphomas. E2F1 positivity did not correlate with the known germinal centre markers, even though it has recently been suggested that it may be a new GC marker.

Conclusions:It may be possible to stratify patients for treatment using markers for specific lineages of B cell differentiation.
ALK positive inflammatory myofibroblastic tumour of the pineal region
Authors: Clarke A J , Jacques T S , Galloway M J , Thom M , Kitchen N D , Plant G T
Published: August 2005

Inflammatory myofibroblastic tumours (IMTs) are an uncommon spindle cell neoplasm with a dense inflammatory infiltrate, usually encountered in children. IMTs of the central nervous system are extremely rare. This report describes the case of an IMT in a 61 year old man, in the pineal region. The tumour was completely excised, and immunohistochemistry demonstrated anaplastic lymphoma kinase 1 expression. There was no tumour recurrence during 18 months of follow up. Our case extends both the age range and sites of occurrence of this rare tumour.
CD56: a useful tool for the diagnosis of small cell lung carcinomas on biopsies with extensive crush artefact
Authors: Kontogianni K , Nicholson A G , Butcher D , Sheppard M N
Published: August 2005

Background/Aims:The diagnosis of small cell lung carcinoma (SCLC) on bronchial biopsy is often problematical as a result of intense crush artefact. Several antibodies are now available to help in the diagnosis of SCLC and their value was assessed in this clinical situation.

Methods/Results:Twenty cases of SCLC and 10 control cases (one non-Hodgkin lymphoma, three non-small cell carcinomas, one follicular reactive hyperplasia, and five chronic non-specific inflammations) with extensive crush artefact were stained using antibodies to CD56, MNF116, thyroid transcription factor 1 (TTF-1), and CD45. All SCLCs showed strong positive staining for CD56 in 75–100% of recognisable tumour cells, even in areas where there was extensive crush artefact. Eighteen of 20 cases were positive for TTF-1 and 16 of 20 were positive for MNF116 in the tumour cells, but both of these antibodies showed little or no staining in areas of crush artefact. Control cases comprising lymphoid cells were positive for CD45 in areas of crush artefact, but all cases of SCLC were negative.

Conclusion:CD56, along with markers for cytokeratins—TTF-1, and CD45—are useful in the diagnosis of SCLC in biopsies with extensive crush artefact and can help confirm the diagnosis in cases where features are equivocal.
Increased E2F-1 expression via tumour cell proliferation and decreased apoptosis are correlated with adverse prognosis in patients with squamous cell carcinoma of the oesophagus
Authors: Yamazaki K , Hasegawa M , Ohoka I , Hanami K , Asoh A , Nagao T , Sugano I , Ishida Y
Published: August 2005

Background:The retinoblastoma (Rb) pathway, which governs cell cycle progression, is frequently genetically altered in cancer, causing deregulated expression of the E2F-1 transcription factor, which promotes DNA synthesis and cell cycle progression. Recent studies show that E2F-1 also participates in apoptosis induction in a p53 dependent or independent manner. Despite its crucial role and paradoxical effects on cell turnover, the function of E2F-1 in human cancer is unclear.

Aims:To evaluate E2F-1 expression using immunohistochemistry in 43 surgically resected oesophageal squamous cell carcinoma (OSCC) specimens.

Methods:This study analysed the association of E2F-1 with tumour cell proliferation and apoptosis and the upstream regulators modulating these processes, and its impact on patient outcome. Tumour cell proliferation and apoptosis were assessed as percentage of MIB-1 positive or apoptotic cells (MIB-1 labelling index (MI) and apoptotic index (AI)), respectively.

Results:Entire specimens showed abnormal expression of one or more upstream regulators of pRb/E2F-1. Although E2F-1 positivity was not associated with the expression of upstream regulators, it showed a linear and positive correlation with MI but not AI. Patients with high MI, low AI, or high E2F-1 positivity had significantly shorter recurrence free survival. By multivariate analysis, high MI and low AI were independently associated with recurrence free survival, but E2F-1 was not.

Conclusions:Increased cell proliferation and decreased apoptosis are associated with adverse prognosis in patients with OSCC. Although E2F-1 remains a controversial prognostic factor, its expression was closely associated with tumour cell proliferation and might influence clinical outcome, mainly via cell cycle progression.
How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies
Authors: Krenacs T , Bagdi E , Stelkovics E , Bereczki L , Krenacs L
Published: August 2005

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only ∼30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.