bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Automated and Nonautomated Systems for Identification of
Burkholderia pseudomallei
Authors: Lowe Peter , Engler Catherine , Norton Robert
Published: November 2002

The identification of
Burkholderia pseudomallei
, the causative agent of melioidosis, is usually not difficult in laboratories in areas where it is endemic. With the increase in international travel and the threat of bioterrorism, it has become more likely that laboratories in areas where it is not endemic could encounter this organism. The increase in the use of and dependence upon automated identification systems makes accurate identification of uncommonly encountered organisms such as
B. pseudomallei
critically important. This study compares the manual API 20NE and 20E identification systems with the automated Vitek 1 and 2 systems. A total of 103
B. pseudomallei
isolates were tested and correctly identified in 98%, 99%, 99%, and 19% of cases, respectively. The failure of the Vitek 2 to correctly identify
B. pseudomallei
was largely due to differences in the biochemical reactions achieved compared to expected values in the database. It is suggested that this deficiency in the Vitek 2 may be due to the large number of uncertain results reported for these isolates. These results reduce the discriminating ability of the instrument to distinguish between uncommonly encountered isolates such as those of
B. pseudomallei
.
Molecular Epidemiology of Extended-Spectrum β-Lactamase-Producing, Fluoroquinolone-Resistant Isolates of
Klebsiella pneumoniae
in Taiwan
Authors: Yu W.-L. , Jones R. N. , Hollis R. J. , Messer S. A. , Biedenbach D. J. , Deshpande L. M. , Pfaller M. A.
Published: November 2002

Strains of extended-spectrum β-lactamase-producing
Klebsiella pneumoniae
(ESBL-KP) have emerged worldwide. Concomitant ciprofloxacin resistance with ESBL production in
K. pneumoniae
isolates would severely restrict treatment options. Among 39 (18.5%) of 211 ESBL-KP isolates resistant to ciprofloxacin (MIC, ≥4 μg/ml), 37 (95%) were high level resistant (MIC, ≥16 μg/ml). These isolates were also cross resistant to the newer fluoroquinolones, including levofloxacin, gatifloxacin, gemifloxacin, and garenoxacin (BMS 284756). Sitafloxacin was most active against these ciprofloxacin-resistant ESBL-KP isolates with MICs for 67% of the isolates being ≤2 μg/ml. The molecular epidemiology of these multiresistant isolates was investigated by automated ribotyping and pulsed-field gel electrophoresis (PFGE). Ribotyping identified 18 different strains among the 39 ciprofloxacin-resistant ESBL-KP isolates. The majority (67%) of these isolates were contained in six ribogroups which were further confirmed by PFGE. The distribution of the six major strains of ciprofloxacin-resistant ESBL-KP within Taiwan included one (ribogroup 255.3-PFGE type E) with a nationwide distribution and several institution-specific strains. Interhospital cooperation appears necessary, with strict infection control practices coupled with restriction of fluoroquinolone and extended-spectrum β-lactam use to control both the major epidemic strain and the more diverse strains observed within individual institutions.
Molecular Characterization of Cephalosporin-Resistant
Salmonella enterica
Serotype Newport Isolates from Animals in Pennsylvania
Authors: Rankin Shelley C. , Aceto Helen , Cassidy Jennifer , Holt Jeff , Young Sheri , Love Brenda , Tewari Deepanker , Munro Donald S. , Benson Charles E.
Published: November 2002

Multidrug-resistant (MDR) strains of
Salmonella enterica
serotype Newport have been described for many years. However, the recognition of Newport strains with resistance to cephalosporin antibiotics is more recent. Plasmid-mediated CMY-2 AmpC β-lactamases have been identified in
Salmonella
in the United States, and the
bla
CMY-2
gene has been shown to be present in
Salmonella
serotype Newport. This organism is currently undergoing epidemic spread in both animals and humans in the United States, and this is to our knowledge the first description of the molecular epidemiology of this
Salmonella
strain in animals. Forty-two isolates were included in this study. All isolates were characterized by pulsed-field gel electrophoresis, plasmid analysis, and antibiogram. Four pulsed-field profiles with
Xba
I were observed. Plasmid analyses showed that although the majority of isolates harbored a single plasmid of 140 kb, this plasmid was not identical in all strains. All isolates showed the presence of the
bla
CMY
gene by PCR. Integrons were detected in 16 of the 42 isolates; a fragment of approximately 1,000 bp, amplified with the
int
I-F and
aad
AI-R primers, confirmed the presence of the
aad
AI gene cassette within an integron in these 16 isolates. The potential for coselection of the
bla
CMY
gene, if located on an MDR replicon, may not be dependent on any particular antibiotic but rather may be the result of more general antimicrobial use. If this replicon is mobile, it is to be expected that similar MDR strains of additional
Salmonella
serotypes will be recognized in due course.
Novel Algorithm Identifies Species in a Polymycobacterial Sample by Fluorescence Capillary Electrophoresis-Based Single-Strand Conformation Polymorphism Analysis
Authors: Iwamoto Tomotada , Sonobe Toshiaki , Hayashi Kozaburo
Published: November 2002

An algorithm to directly identify multiple mycobacterial species in a sample by using fluorescence capillary electrophoresis (FCE)-based single-strand conformation polymorphism (SSCP) analysis was developed. Part of the 16S-23S ribosomal DNA internal transcribed spacer (ITS) region in 37 reference strains and 73 clinical isolates representing 19 mycobacterial species and
Mycobacterium tuberculosis
complex was PCR amplified with a fluorescence-labeled mycobacterium-specific primer, 6-carboxyfluorescein-labeled primer Sp1f, and 5-hexachlorofluorescein-tagged Sp2r. FCE-SSCP analysis was applied to both undigested PCR products and the corresponding
Hae
III-digested restriction fragments (RF) from each strain. The 23 resultant SSCP patterns distinguished all 19 species and
M. tuberculosis
complex. The technique is applicable for the detection of multiple mycobacterial species in a sample. It was demonstrated by analyzing two model mycobacterial communities consisting of five species with both rapidly and slowly growing species (model A) and four species commonly encountered in clinical practice (model B). The sensitivity study with spiked sputum samples with different amounts of
M. tuberculosis
H37Rv,
M. avium
, and
M. intracellulare
cells indicated that up to 25% of the amount of each mycobacterium could be detected relative to the two other species. Fifty-one sputum specimens analyzed by FCE-RF-SSCP were compared with the Amplicor assay (Roche Diagnostics GmbH). Species identified by both assays were always the same. Moreover, FCE-RF-SSCP could identify
M. abscessus
and
M. kansasii
, which are not targeted by Amplicor. FCE-RF-SSCP of sputum obtained from a patient with mixed
M. avium
and
M. intracellulare
infection gave SSCP patterns corresponding to these two species.
Development of a Real-Time Fluorescence PCR Assay for Rapid Detection of the Diphtheria Toxin Gene
Authors: Mothershed Elizabeth A. , Cassiday Pamela K. , Pierson Kevin , Mayer Leonard W. , Popovic Tanja
Published: November 2002

We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (
tox
) gene. When 23 toxigenic
Corynebacterium diphtheriae
strains, 9 nontoxigenic
C. diphtheriae
strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the
tox
gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the
tox
gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR
tox
-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the
tox
gene in clinical material.
Detection of Imported Malaria with the Cell-Dyn 4000 Hematology Analyzer
Authors: Wever Peter C. , Henskens Yvonne M. C. , Kager Piet A. , Dankert Jacob , van Gool Tom
Published: November 2002

The sensitivity and specificity of the Cell-Dyn 4000 hematology analyzer in the diagnosis of imported malaria were studied with samples from patients in an academic hospital setting. The performance of the Cell-Dyn 4000 hematology analyzer was compared with that of conventional diagnostic methods for malaria. The Cell-Dyn 4000 hematology analyzer detected hemozoin-containing depolarizing monocytes in 29 of 58 patients with malaria and 2 of 55 patients without malaria. The presence or absence of depolarizing monocytes in patients with malaria was related to duration of symptoms before presentation for malaria analysis. A second parameter, pseudoreticulocytosis due to nuclear material of intraerythrocytic malaria parasites, was detected by the Cell-Dyn 4000 hematology analyzer almost exclusively in
Plasmodium falciparum
malaria patients with parasitemia levels of ≥0.5%. Attention to these abnormalities in medical centers without tropical disease expertise may decrease a delay in the diagnosis of malaria.
Diversity among French
Bacillus anthracis
Isolates
Authors: Fouet Agnès , Smith Kimothy L. , Keys Chris , Vaissaire Josée , Le Doujet Claudine , Lévy Martine , Mock Michèle , Keim Paul
Published: November 2002

While outbreaks of animal anthrax zoonoses still regularly occur in France, little is known about the epidemiology links between them. We have used the eight-locus multilocus variable-number tandem repeat analysis typing technique against a collection of 50
Bacillus anthracis
isolates from France. There were eight distinct genotypes belonging to two dissimilar genetic clusters. Regional strain patterns were observed, with the B2 genotypes prevalent in southern France and the A1a genotypes found only in northern France.
Evaluation of a Plastic Nonvented Aerobic Blood Culture Bottle for Use with the BacT/ALERT Microbial Detection System
Authors: Snyder J. W. , Munier G. K. , Bostic G. D. , Bozigar P. S. , Hanna R.
Published: November 2002

The current BacT/ALERT SA (BTA SA) aerobic blood culture bottle is made from glass, does not require venting, and contains a liquid emulsion sensor (LES). Its performance has been shown to be equivalent to that of the vented standard aerobic culture bottle. A further-improved version of the BTA SA bottle, designated the BacT/ALERT plastic SA (BTA PSA) culture bottle, is made from clear plastic to prevent breakage, does not require venting, and contains a modified LES (LES 2) to reduce the possibility of false positives. The BTA PSA provides a practical alternative to the current glass version of this bottle. The plastic bottle is also comparable to the current glass bottle in transparency and growth performance and additionally minimizes the exposure to infectious agents due to glass bottle breakage.
Persistence of Pigment Production by Yeast Isolates Grown on CHROMagar Candida Medium
Authors: Hospenthal Duane R. , Murray Clinton K. , Beckius Miriam L. , Green Judith A. , Dooley David P.
Published: November 2002

We evaluated the persistence of pigmentation in yeast isolates grown on the chromogenic medium CHROMagar Candida over 7 days.
Candida
,
Cryptococcus
, and
Trichosporon
isolates were inoculated alone or mixed onto duplicate sets of plates and incubated at 30 and 35°C.
Candida albicans
and
Candida krusei
were readily identified throughout the reading period, but
Candida glabrata
was difficult to differentiate from other species until the 3- or 4-day time point.
Candida tropicalis
produced colonies similar to those of rare
Cryptococcus
and
Trichosporon
species, and mixed cultures were often difficult to identify as such.
Isolation of
Campylobacter fetus
subsp.
fetus
from a Patient with Cellulitis
Authors: Briedis Dalius J. , Khamessan Ali , McLaughlin Richard W. , Vali Hojatollah , Panaritou Maria , Chan Eddie C. S.
Published: November 2002

Campylobacter fetus
subsp.
fetus
is a gram-negative, slender, spirally curved bacterial pathogen. It has been isolated from human blood, spinal fluid, and abscesses, but cellulitis associated with bacteremia is rare. We report its isolation from a blood culture of a human patient with cellulitis as well as difficulties encountered in determining the identity of the subspecies of
C. fetus
.
A biomechanical review of factors affecting rowing performance
Authors: Baudouin A , Hawkins D
Published: November 2002

This review analyses rowing by linking the biological and mechanical systems that comprise the rowing system. Blade force was found to be the only propulsive force to counter the drag forces, consisting of both air drag and hydrodynamic drag, acting on the system. Vertical oscillations of the shell are shown to have minimal impact on system dynamics. The oar acts as the link between the force generated by the rower and the blade force and transmits this force to the rowing shell through the oarlock. Blade dynamics consist of both lift and drag mechanisms. The force on the oar handle is the result of a phased muscular activation of the rower. Oar handle force and movement are affected by the joint strength and torque-velocity characteristics of the rower. Maximising sustainable power requires a matching of the rigging setup and blade design to the rower’s joint torque-velocity characteristics. Coordination and synchrony between rowers in a multiple rower shell affects overall system velocity. Force-time profiles should be better understood to identify specific components of a rower’s biomechanics that can be modified to achieve greater force generation.
Have the attitudes of Australian squash players towards protective eyewear changed over the past decade?
Authors: Eime R M , Finch C F
Published: November 2002

Objective:To assess indicative trends in the use of protective eyewear by Australian squash players and their attitudes towards its use since 1989.

Methods:Data were extracted from three Australian surveys of squash players conducted in 1989, 1995, and 2000. Responses to directly similar attitudinal questions relating to protective eyewear use from each survey were compared. The proportion of players giving each response was calculated for each survey, along with 95% confidence intervals for the differences between the 2000 survey and those from the earlier surveys.

Results:Self reported use of protective eyewear ranged from 10.0% in 1989, to 8.6% in 1995, and 18.8% in 2000. However, only 8.9% of the players surveyed in 2000 actually wore appropriate standards approved/polycarbonate lens eyewear. This can be compared with 8.0% and 2.0% of players who reported wearing appropriate polycarbonate lens eyewear in the 1989 and 1995 surveys respectively. Compared with the 1995 survey, significantly more players in 2000 believed that more players should wear protective eyewear (95% confidence interval (CI) for difference 1 to 18). A significantly higher proportion of players also supported the compulsory use of protective eyewear by juniors in 2000 than in both 1989 (95% CI for difference 6 to 24) and 1995 (95% CI for difference 5 to 22). No other attitudes had significantly changed over the decade.

Conclusion:These data suggest that self reported use of protective eyewear has probably increased over the past decade. However, many players report wearing inappropriate eyewear. A transition from positive attitudes to appropriate eyewear behaviours is required before mandatory protective eyewear use can be effectively introduced.
Evaluating the level of injury in English professional football using a risk based assessment process
Authors: Drawer S , Fuller C W
Published: November 2002

Objectives:To show how epidemiological data can be presented and analysed in frequency based and risk based formats and how risk based information can simplify management decisions on injury prevention strategies in professional football.

Methods:The club physiotherapists at four English professional football clubs prospectively recorded players’ injuries over the period November 1994 to May 1997. The nature, location, and mechanism of each injury and the specific numbers of days that players were unavailable to train or play as a result of injuries were recorded. The rates of injury were evaluated on a risk matrix using the number of days and the estimated costs of absence as measures of injury consequences.

Results:There was a significant difference in the time lost through injury as a function of injury severity (p<0.001). Slight and minor injuries accounted for 51% of all injuries but represented only 17% of the risk from injury, whereas major injuries accounted for only 12% of the number of injuries and 47% of the risk. Player to player contact injuries accounted for 39% of the risk of injury, and football specific activities accounted for 47% of the risk. The risks of acute injury in professional football were three orders of magnitude greater than those in the construction, manufacturing, and service sectors of industry.

Conclusions:The risks associated with minor, moderate, and major acute injuries and osteoarthritis in lower limb joints of professional footballers were found to be unacceptable when evaluated against work based risk criteria used by the Health and Safety Executive. All stakeholders within professional football were shown to have an important contribution to make in reducing the overall level of risk to players through the provision of risk prevention strategies.
CRITICAL ANALYSIS OF ELECTRONIC SIMULATION OF FINANCIAL MARKET FLUCTUATIONS
Authors: FANCHIOTTI H. , GARCÍA CANAL C. A. , MARTÍNEZ N.
Published: November 2002

An interesting analog circuit for simulating a signal with fluctuations having a probability density function with a power tail has recently been proposed and constructed. The exponent of the power law can be fixed by tuning an appropriate circuit element. The proposal is to use the circuit as a simulator-generator of financial market fluctuations and as a tool for risk estimations and forecasts. We present a discussion of the stability conditions for multiplicative noise and an exhaustive analysis of the power law fluctuation generator in connection with the electronic components and their parameters. From our studies, one can conclude that the proposal is not adequate to provide a confident experimental scheme to follow the fluctuations of the financial market due to both electronic implementation and to difficulties in parameter tuning.
INVESTIGATION OF HETEROGENEOUS SCALING INTERVALS EXEMPLIFIED BY SUTURED QUARTZ GRAIN BOUNDARIES
Authors: SUTEANU CRISTIAN , KRUHL JÖRN H.
Published: November 2002

Quartz grain boundaries from metamorphic and igneous rocks may emphasize a complex geometry, characterized by self-similarity over one to two orders of magnitude. Their fractal analysis highlights scaling sub-domains, i.e. scale intervals with a particularly good correlation. Given the importance of these aspects for the deciphering of geological microstructures, the paper is dedicated to the detection and the objective depiction of the features of heterogeneous scaling intervals. A fractal analysis based on the divider method was followed by processing methods that (i) offer a global evaluation of the curve geometry from the point of view of the correlation sub-domains, and (ii) allow a local characterization of the curves in terms of scale, with special concern for the scaling intervals heterogeneity. The application of the proposed approach was exemplified both on natural and synthetic curves. On one hand, the grain boundary analysis highlighted scaling sub-domains most obviously in the case of microstructures that were subject to overprinting, due to successive processes. On the other hand, a pattern superposition in the case of the synthetic curves strongly emphasized scaling sub-domains, as compared to the unperturbed (recursively generated) curve geometry. These aspects were expressed quantitatively and highlighted in more detail on isocorrelation maps. The importance of a rigorous characterization of these sub-domains and, eventually, the detection of pattern overprinting phenomena in geological microstructures emphasize the relevance of such an approach.
FRACTAL DIMENSION AND SELF-SIMILARITY IN ASPARAGUS PLUMOSUS
Authors: CASTREJÓN PITA J. R. , SARMIENTO GALÁN A. , CASTREJÓN GARCÍA R.
Published: November 2002

We measure the fractal dimension of an African plant that is widely cultivated as an ornamental – the Asparagus plumosus. This plant presents self-similarity, remarkable in at least two different scalings. In the following, we present the results obtained by analyzing this plant via the box counting method for three different scalings. We show in a quantitative way that this species is a fractal.
Chemoresistant or aggressive lymphoma predicts for a poor outcome following reduced-intensity allogeneic progenitor cell transplantation: an analysis from the Lymphoma Working Party of the European Group for Blood and Bone Marrow Transplantation
Authors: Robinson Stephen P. , Goldstone Anthony H. , Mackinnon Stephen , Carella Angelo , Russell Nigel , de Elvira Carmen Ruiz , Taghipour Goli , Schmitz Norbert
Published: November 2002

We report the outcome of reduced-intensity allogeneic progenitor cell transplantation (alloPCT) for 188 patients with lymphoma from the Working Party Lymphoma of the European Group for Blood and Bone Marrow Transplantation (EBMT). The median age of the patients was 40 years, the median number of prior treatment courses was 3, and 48% of patients had undergone a prior autologous transplantation. Eighty-four percent of the patients received conditioning with fludarabine-based regimens and 10% with the BEAM (BCNU, etoposide, cytosine arabinoside, melphalan) protocol. Full donor chimerism was confirmed in 71% of 100 patients assessed. Acute graft-versus-host disease (GVHD) developed in 37% of patients and chronic GVHD in 17%. A disease response to donor leukocyte infusion (DLI) was seen in 10 of 14 patients. With a median follow-up of 283 days, the overall survival rates at 1 and 2 years were 62% and 50%, respectively. The 100-day and 1-year transplantation-related mortality (TRM) rates were 12.8% and 25.5%, respectively, and were significantly worse for older patients. The probability of disease progression at 1 year for patients with chemoresistant and chemosensitive disease were 75% and 25%, respectively (P = .001). The progression-free survival at 1 year was 46% and was significantly better for those with chemosensitive disease, Hodgkin disease (HD), and low-grade non-Hodgkin lymphoma (NHL). Patients with high-grade NHL, mantle cell lymphoma, or chemoresistant disease had a poor outcome. Reduced-intensity progenitor cell transplantation is associated with a reduced TRM and may control advanced HD and low-grade NHL. A longer period of follow-up is required to determine the benefit of DLI and the graft-versus-lymphoma effect.
Variants in the VCAM1 gene and risk for symptomatic stroke in sickle cell disease
Authors: VI James G. Taylor , Tang Delia C. , Savage Sharon A. , Leitman Susan F. , Heller Seth I. , Serjeant Graham R. , Rodgers Griffin P. , Chanock Stephen J.
Published: November 2002

Stroke is a major cause of morbidity and mortality in sickle cell (SS) disease. Genetic risk factors have been postulated to contribute to this clinical outcome. The human genome project has substantially increased the catalog of variations in genes, many of which could modify the risk for manifestations of disease outcome in a monogenic disease, namely SS. VCAM1 is a cell adhesion molecule postulated to play a critical role in the pathogenesis of SS disease. We identified a total of 33 single nucleotide polymorphisms (SNPs) by sequencing the entire coding region, 2134 bp upstream of the 5′ end of the published cDNA, 217 bp downstream of the 3′ end of the cDNA, and selected intronic regions of the VCAM1 locus. Allelic frequencies for selected SNPs were determined in a healthy population. We subsequently analyzed 4 nonsynonymous coding, 2 synonymous coding, and 4 common promoter SNPs in a genetic association study of clinically apparent stroke in SS disease conducted in a cohort derived from a single institution in Jamaica (51 symptomatic cases and 51 matched controls). Of the 10 candidate SNPs analyzed in this pilot study, the variant allele of the nonsynonymous SNP, VCAM1 G1238C, may be associated with protection from stroke (odds ratio [OR] 0.35, 95% confidence interval [CI] 0.15-0.83, P = .04). Further study is required to confirm the importance of this variant inVCAM1 as a clinically useful modifier of outcome in SS disease.
Expression of MUM1/IRF4 correlates with clinical outcome in patients with B-cell chronic lymphocytic leukemia
Authors: Chang Chung-Che , Lorek Jennifer , Sabath Daniel E. , Li Ying , Chitambar Christopher R. , Logan Brent , Kampalath Bal , Cleveland Ronald P.
Published: November 2002

In this study, we evaluated the prognostic significance of multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF4) expression in B-cell chronic lymphocytic leukemia (B-CLL). Our results demonstrated that the absence of MUM1/IRF4 expression showed the highest relative risk among the factors analyzed in determining the probability for death in patients with B-CLL using univariate and multivariate Cox regression analysis. Patients without MUM1/IRF4 expression had significantly worse overall survival than did those with MUM1/IRF4 expression (52% cumulative survival, 63 months vs not reached, Kaplan-Meier survival analysis; P < .03, log-rank test). Patients with MUM1/IRF4 expression were more likely to have disease at low Rai stage and interstitial/nodular marrow involvement. Furthermore, only 1 of 11 patients with MUM1/IRF4 expression and interstitial/nodular marrow involvement died during a 100-month follow-up. Our results suggest that B-CLL with expression of MUM1/IRF4, indicative of postgerminal center origin, has a more favorable clinical course and that MUM1/IRF4 is an important prognostic marker in B-CLL.
Low levels of ABCG2 expression in adult AML blast samples
Authors: Abbott Brian L. , Colapietro Anne-Marie , Barnes Yuxiao , Marini Frank , Andreeff Michael , Sorrentino Brian P.
Published: November 2002

Previous reports have suggested that the adenosine triphosphate–binding cassette protein ABCG2 (breast cancer resistance protein [BCRP], mitoxantrone resistance [MXR]) is associated with drug resistance in acute myeloid leukemia (AML). The aims of this study were to determine the level of ABCG2 mRNA expression necessary to produce drug resistance and to define the ABCG2 levels in normal bone marrow (BM), peripheral blood (PB), cord blood (CB), and adult AML blast cell populations. First, using transduced clonal cell lines expressing varying levels of ABCG2, we found that ABCG2 expression conferred resistance to mitoxantrone and topotecan, but not to idarubicin. Next, we developed a real-time reverse transcription–polymerase chain reaction assay for measuring ABCG2 mRNA expression levels in clinical samples. Normal BM and PB contained low levels of ABCG2 mRNA, while higher levels were measured in CB mononuclear cells, CD34+, and Ac133+populations, consistent with the known stem cell enrichment in these populations. Next, we studied the ABCG2 mRNA levels in 40 specimens from newly diagnosed adult AML patients. Only 7% of these samples contained ABCG2 mRNA levels within the range of our drug-resistant clone, although another 78% were higher than normal blood and bone marrow. Flow cytometry revealed very small subpopulations of ABCG2-expressing cells in the cases we examined. Our data suggest that high levels of ABCG2 mRNA expression in adult AML blast specimens are relatively uncommon and that ABCG2 expression may be limited to a small cell subpopulation in some cases.
Short-chain fatty acid derivatives induce fetal globin expression and erythropoiesis in vivo
Authors: Pace Betty S. , White Gary L. , Dover George J. , Boosalis Michael S. , Faller Douglas V. , Perrine Susan P.
Published: November 2002

Orally bioactive compounds that induce γ globin gene expression at tolerable doses are needed for optimal treatment of the β-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce γ globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce γ globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which γ globin can be induced. Selected short-chain fatty acid derivatives (SCFADs) were recently found to induce γ globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human β globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, γ globin mRNA increased 2-fold, reticulocytes increased 3- to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2- to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate γ globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the β hemoglobinopathies and useful for the oral treatment of other anemias.
CHK2-decreased protein expression and infrequent genetic alterations mainly occur in aggressive types of non-Hodgkin lymphomas
Authors: Tort Frederic , Hernàndez Silvia , Beà Silvia , Martı́nez Antonio , Esteller Manel , Herman James G. , Puig Xavier , Camacho Emma , Sánchez Montse , Nayach Iracema , Lopez-Guillermo Armando , Fernández Pedro L. , Colomer Dolors , Hernàndez Luis , Campo Elias
Published: November 2002

The CHK2 gene codifies for a serine/threonine kinase that plays a central role in DNA damage response pathways. To determine the potential role of CHK2 alterations in the pathogenesis of lymphoid neoplasms we have examined the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid samples. A heterozygous Ile157Thr substitution, also present in the germ line of the patient, was detected in a blastoid mantle cell lymphoma (MCL). CHK2 protein and mRNA expression levels were similar in all types of lymphomas and reactive samples, and these levels were independent of the proliferative activity of the tumors. However, 5 tumors, one typical MCL, 2 blastoid MCLs, and 2 large cell lymphomas, showed marked loss of protein expression, including 2 samples with complete absence of CHK2 protein. These 2 lymphomas showed the highest number of chromosomal imbalances detected by comparative genomic hybridization in the whole series of cases. However, no mutations, deletions, or hypermethylation of the promoter region were identified in any of these tumors. mRNA levels were similar in cases with low and normal protein expression, suggesting a posttranscriptional regulation of the protein in these tumors. CHK2 gene and protein alterations were not related to p53 and ATMgene status. In conclusion, CHK2 alterations are uncommon in malignant lymphomas but occur in a subset of aggressive tumors independently of p53 or ATM alterations. The high number of chromosomal imbalances in tumors with complete absence of CHK2 protein suggests a role of this gene in chromosomal instability in human lymphomas.
Abnormal T cell–dependent B-cell responses in SCID mice receiving allogeneic bone marrow in utero
Authors: Waldschmidt Thomas J. , Panoskaltsis-Mortari Angela , McElmurry Ronald T. , Tygrett Lorraine T. , Taylor Patricia A. , Blazar Bruce R.
Published: November 2002

In allogeneic hematopoietic stem cell transplant recipients, restoration of humoral immunity is delayed and can remain impaired for years. In many severe combined immune deficiency (SCID) patients given haploidentical bone marrow (BM), lesions in humoral immunity are exacerbated by poor engraftment of donor B cells. The nature of these defects is important to understand as they render patients susceptible to infection. Previous work in mice suggested that in utero transplantation (IUT) of allogeneic BM might offer several advantages for the correction of primary immune deficiencies. In SCID mice given fully allogeneic BM in utero, the lymphoid compartment was restored with minimal evidence of graft-versus-host disease (GVHD). The present report examines B-cell reconstitution and function in mice that have received allogeneic IUT. Results are compared with those of adult mice given total body irradiation (TBI) followed by transplantation with allogeneic BM. In addition to enumerating the various B-cell subsets present in BM, spleen, and peritoneal cavity (PC), B-cell competence was assessed by challenging mice with T cell–independent (TI) and T cell–dependent (TD) antigens. The results demonstrated that all B-cell subsets in the BM and periphery were restored in allogeneic IUT and TBI mice, as were antibody responses after TI challenge. Upon immunization with TD antigens, however, IUT and TBI mice exhibited suboptimal responses as measured by the capacity to isotype switch and generate germinal center (GC) B cells. Thus, although allogeneic BM transplantation results in complete recovery of the B-cell compartment, certain elements of the humoral response remain defective.
The chiaroscuro stem cell: a unified stem cell theory
Authors: Quesenberry Peter J. , Colvin Gerald A. , Lambert Jean-Francois
Published: November 2002

Hematopoiesis has been considered hierarchical in nature, but recent data suggest that the system is not hierarchical and is, in fact, quite functionally plastic. Existing data indicate that engraftment and progenitor phenotypes vary inversely with cell cycle transit and that gene expression also varies widely. These observations suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. This may, in turn, be dependent on shifting chromatin and gene expression with cell cycle transit. If the phenotype of these primitive marrow cells changes from engraftable stem cell to progenitor and back to engraftable stem cell with cycle transit, then this suggests that the identity of the engraftable stem cell may be partially masked in nonsynchronized marrow cell populations. A general model indicates a marrow cell that can continually change its surface receptor expression and thus responds to external stimuli differently at different points in the cell cycle.
Complement-mediated clearance of erythrocytes: mechanism and delineation of the regulatory roles of Crry and DAF
Authors: Molina Hector , Miwa Takashi , Zhou Lin , Hilliard Brendan , Mastellos Dimitrios , Maldonado Michael A. , Lambris John D. , Song Wen-Chao
Published: November 2002

The role of complement in the pathogenesis of autoimmune hemolytic anemia (AIHA) has been controversial and may depend on a number of factors, including the affinity and isotype of the pathogenic antibodies involved. We have recently shown that mouse erythrocytes deficient in the membrane C3 regulatory protein, complement receptor 1–related gene/protein y (Crry), but not decay-accelerating factor (DAF), were spontaneously eliminated in vivo by complement. Here, by generating a mouse deficient in both DAF and Crry, we further delineated the roles of Crry and DAF in regulating alternative and classical pathway C3 activation. By using immunoglobulin-, Fcγ receptor (FcγR)–, C3-, C4-, and C5-deficient mice, we also determined the mechanism by which membrane C3 regulator-deficient erythrocytes are cleared from the circulation. Finally, we evaluated the relative importance of the Fc receptor versus the complement pathway in disposing antibody-opsonized DAF/Crry–deficient erythrocytes. We conclude that (1) Crry plays a more dominant role than DAF in regulating the alternative pathway of complement, whereas DAF and Crry are equally effective in preventing antibody-induced runaway complement activation on mouse erythrocytes; (2) DAF/Crry–deficient erythrocytes are eliminated by the alternative pathway of complement via complement receptor–mediated erythrophagocytosis in the spleen; and (3) when opsonized with an immunoglobulin G2a (IgG2a) autoantibody, Crry/DAF–deficient erythrocytes are eliminated more rapidly by complement than by the Fc receptor pathway. These results shed new light on the relative activities of Crry and DAF and underscore the critical roles of membrane C3 regulators in preventing spontaneous and antibody-induced erythrocyte damage in vivo.
Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice
Authors: Piacibello Wanda , Bruno Stefania , Sanavio Fiorella , Droetto Sara , Gunetti Monica , Ailles Laurie , de Sio Francesca Santoni , Viale Andrea , Gammaitoni Loretta , Lombardo Angelo , Naldini Luigi , Aglietta Massimo
Published: November 2002

The ability of advanced-generation lentiviral vectors to transfer the green fluorescent protein (GFP) gene into human hematopoietic stem cells (HSCs) was studied in culture conditions that allowed expansion of transplantable human HSCs. Following 96 hours' exposure to flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), and interleukin-6 (IL-6) and overnight incubation with vector particles, cord blood (CB) CD34+ cells were further cultured for up to 4 weeks. CD34+ cell expansion was similar for both transduced and control cells. Transduction efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) was assessed by transplants into NOD/SCID mice. Mice that received transplants of transduced week 1 and week 4 expanded cells showed higher levels of human engraftment than mice receiving transplants of transduced nonexpanded cells (with transplants of 1 × 105 CD34+ cells, the percentages of CD45+ cells were 20.5 ± 4.5 [week 1, expanded] and 27.2 ± 8.2 [week 4, expanded] vs 11.7 ± 2.5 [nonexpanded]; n = 5). The GFP+/CD45+ cell fraction was similar in all cases (12.5% ± 2.9% and 12.2% ± 2.7% vs 12.7% ± 2.1%). Engraftment was multilineage, with GFP+/lineage+ cells. Clonality analysis performed on the bone marrow of mice receiving transduced and week 4 expanded cells suggested that more than one integrant likely contributed to the engraftment of GFP-expressing cells. Serial transplantations were performed with transduced week 4 expanded CB cells. Secondary engraftment levels were 10.7% ± 4.3% (n = 12); 19.7% ± 6.2% of human cells were GFP+. In tertiary transplants the percentage of CD45+ cells was lower (4.3% ± 1.7%; n = 10); 14.8% ± 5.9% of human cells were GFP+, and human engraftment was multilineage. These results show that lentiviral vectors efficiently transduce HSCs, which can undergo expansion and maintain proliferation and self-renewal ability.
Sphincter-Sparing Local Excision and Adjuvant Radiation for Anal-Rectal Melanoma
Authors: Ballo Matthew T. , Gershenwald Jeffrey E. , Zagars Gunar K. , Lee Jeffrey E. , Mansfield Paul F. , Strom Eric A. , Bedikian Agop Y. , Kim Kevin B.S. , Papadopoulos Nicholas E. , Prieto Victor G. , Ross Merrick I.
Published: November 2002

PURPOSE: To evaluate the outcome and toxicity of a sphincter-sparing treatment strategy in the management of patients with anal-rectal melanoma.

PATIENTS AND METHODS: Between 1989 and 2000, 23 patients with invasive anal-rectal melanoma were managed with sphincter-sparing surgical resection and adjuvant radiation. Surgery consisted of primary local excision, as well as dissection for patients with documented regional nodal disease. Adjuvant radiation was delivered using a hypofractionated regimen of 30 Gy in five fractions over 2.5 weeks. Adjuvant systemic therapy was delivered to nine patients: cytotoxic chemotherapy in seven and immunotherapy in two.

RESULTS: After a median follow-up of 32 months, 15 patients had relapsed and 15 patients had died. The actuarial 5-year overall, disease-specific, disease-free, and distant metastasis-free survival rates were 31%, 36%, 37%, and 35%, respectively. The actuarial 5-year local and regional nodal control rates were 74% and 84%, respectively. No patient had locoregional failure as the sole site of failure and no patient required salvage abdominoperineal resection (APR). By univariate analysis, patients with nodal disease at presentation had a decreased actuarial 5-year disease-specific (0% v 45%, P = .004), disease-free (0% v 45%, P < .001), and distant metastasis-free survival (0% v 42%, P < .001). The actuarial complication-free survival rate was 71%. Two patients developed mild scrotal edema (grade 1), and four patients developed moderate proctitis requiring prolonged medical management (grade 2).

CONCLUSION: Sphincter-sparing local excision and adjuvant radiation is well tolerated and can effectively control local-regional disease while avoiding the functional morbidity of APR.
Efficacy and Quality-of-Life Data Are Related in a Phase II Trial of Oral Chemotherapy in Previously Untreated Patients With Metastatic Colorectal Carcinoma
Authors: Hobday Timothy J. , Kugler John W. , Mahoney Michelle R. , Sargent Daniel J. , Sloan Jeff A. , Fitch Tom R. , Krook James E. , O’Connell Michael J. , Mailliard James A. , Tirona Maria Tria , Tschetter Loren K. , Cobau Charles D. , Goldberg Richard M.
Published: November 2002

PURPOSE: To evaluate quality of life (QOL) and tumor response after administration of an oral chemotherapy regimen in patients with previously untreated metastatic colorectal cancer.

PATIENTS AND METHODS: Seventy-eight patients received a mean number of 5.8 cycles of therapy. QOL data were analyzed at baseline, after every two cycles of therapy, and at the time of treatment discontinuation. The Uniscale and the European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire C30 were both utilized.

RESULTS: The confirmed response rate was 26% (95% confidence interval [CI], 17% to 37%). Median survival was 11.3 months (95% CI, 9.6 to 15.1 months). Global QOL scores were unchanged over the course of therapy by either tool. Only the physical function subscale score had worsened at the end of therapy. In an analysis of responding patients, significant and durable improvements in both global QOL measures as well as select subscale scores were observed. Diarrhea and physical function QOL scores had declined at the time of treatment discontinuation. Patients who did not respond to therapy had preserved QOL scores when they were evaluated after two cycles of therapy.

CONCLUSION: This oral treatment strategy preserved QOL in treated patients. Global QOL measures as well as several QOL subscale scores significantly improved in patients with a documented response to therapy. The profile of improved QOL components indicated that patient well-being was related to tumor response in specific and perceivable ways. Nonresponding patients reported preserved QOL during the first two cycles of therapy. QOL analysis was feasible and informative in this moderately sized multicenter phase II trial.
Hydrolysis of lactose by free and immobilized β‐galactosidase from Thermus sp. strain T2
Authors: Ladero M. , Perez M.T. , Santos A. , Garcia‐Ochoa F.
Published: November 2002

The hydrolysis of lactose by a β‐galactosidase from the thermophilic microorganism Thermus sp. strain T2, both in solution and immobilized on a commercial silica‐alumina, has been studied. The enzyme has been previously produced by Escherichia coli JM101 harboring the plasmid pBGT1, which contains the codifying gene under the promoters lppP and lacPQ. The enzyme was immobilized on the support activated with tris‐hydroxymethylphosphine (THP). Activity and stability of the free and the immobilized enzyme towards pH and temperature were tested. To study the activity at different pH and temperature values, lactose was used as substrate. To check the stability, the enzyme was incubated either in buffer BP or in a solution of lactose in buffer BM at different pH and temperatures, being the remaining activity tested by withdrawing samples and determining their activity toward ONPG at 70°C in buffer BP. Afterward, runs were performed to obtain kinetic models adequate for the description of the hydrolysis of lactose by the free and the immobilized enzyme. These data were fitted to the kinetic models proposed (all based on the Michaelis–Menten mechanism) by non‐linear regression, being the models and their parameters compared to determine the effect of the immobilization on the kinetic behavior of the enzyme. Both the free and the immobilized enzyme are competitively inhibited by galactose, while glucose inhibited only the action of the free enzyme, in an uncompetitive way. The immobilization step seems to eliminate the inhibition by glucose. Moreover, the immobilization reduced to a half the inhibitory action of galactose. In general, the immobilization reduced the activity of the enzyme, but increased its thermal stability. Finally, a comparison between the kinetic behavior of this thermophilic enzyme and enzymes of mesophile microorganisms previously studied by us (E. coli and K. fragilis) and by other authors (Aspergillus niger) is performed. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81:241–252, 2003.
Enclosed outdoor photobioreactors: Light regime, photosynthetic efficiency, scale‐up, and future prospects
Authors: Janssen Marcel , Tramper Johannes , Mur Luuc R. , Wijffels René H.
Published: November 2002

Enclosed outdoor photobioreactors need to be developed and designed for large‐scale production of phototrophic microorganisms. Both light regime and photosynthetic efficiency were analyzed in characteristic examples of state‐of‐the‐art pilot‐scale photobioreactors. In this study it is shown that productivity of photobioreactors is determined by the light regime inside the bioreactors. In addition to light regime, oxygen accumulation and shear stress limit productivity in certain designs. In short light‐path systems, high efficiencies, 10% to 20% based on photosynthetic active radiation (PAR 400 to 700 nm), can be reached at high biomass concentrations (>5 kg [dry weight] m−3). It is demonstrated, however, that these and other photobioreactor designs are poorly scalable (maximal unit size 0.1 to 10 m3), and/or not applicable for cultivation of monocultures. This is why a new photobioreactor design is proposed in which light capture is physically separated from photoautotrophic cultivation. This system can possibly be scaled to larger unit sizes, 10 to >100 m3, and the reactor liquid as a whole is mixed and aerated. It is deduced that high photosynthetic efficiencies, 15% on a PAR‐basis, can be achieved. Future designs from optical engineers should be used to collect, concentrate, and transport sunlight, followed by redistribution in a large‐scale photobioreactor. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 193–210, 2003.